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CONTRIBUTORS

Michael J. Adang
Department of Entomology, and Department of Biochemistry and Molecular Biology,
University of Georgia, Athens, Georgia, USA
Md. Shohidul Alam
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience,
The University of Queensland, St. Lucia, Queensland, Australia
James A. Baum
Monsanto Company, Chesterfield, Missouri, USA
Niraj S. Bende
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience,
The University of Queensland, St. Lucia, Queensland, Australia
Colin Berry
Cardiff School of Biosciences, Cardiff University, Cardiff, United Kingdom
Neil Crickmore
School of Life Sciences, University of Sussex, Falmer, Brighton, United Kingdom
Rhoel R. Dinglasan
Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg
School of Public Health, Baltimore, Maryland, USA
Andrea J. Dowling
Biosciences, University of Exeter, Cornwall, United Kingdom
Richard H. ffrench-Constant
Biosciences, University of Exeter, Cornwall, United Kingdom
Volker Herzig
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience,
The University of Queensland, St. Lucia, Queensland, Australia
Juan Luis Jurat-Fuentes
Department of Entomology and Plant Pathology, University of Tennessee, Knoxville,
Tennessee, USA
Robert M. Kennedy
Vestaron Corporation, Kalamazoo, Michigan, USA
Glenn F. King
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience,
The University of Queensland, St. Lucia, Queensland, Australia
Paul J. Linser
The University of Florida Whitney Laboratory, St. Augustine, Florida, USA

vii
viii Contributors

Thomas Meade
Dow AgroSciences, LLC., Indianapolis, Indiana, USA
Kenneth E. Narva
Dow AgroSciences, LLC., Indianapolis, Indiana, USA
Leda Regis
Centro de Pesquisas Aggeu Magalhaes-Fiocruz, Recife-Pernambuco, Brazil
James K. Roberts
Monsanto Company, Chesterfield, Missouri, USA
Maria Helena Neves Lobo Silva Filha
Centro de Pesquisas Aggeu Magalhaes-Fiocruz, Recife-Pernambuco, Brazil
Nicholas P. Storer
Dow AgroSciences, LLC., Indianapolis, Indiana, USA
H. William Tedford
Vestaron Corporation, Kalamazoo, Michigan, USA
Yidong Wu
College of Plant Protection, Nanjing Agricultural University, Nanjing, China
PREFACE

The idea for this volume on Insect midgut and insecticidal proteins was
conceived from the realization that not a single source of reviews covers the
insect midgut and insecticidal proteins isolated from bacteria or arthropods.
This volume benefits anyone researching to find solutions for insect pest
control in agriculture and in public health.
The first chapter reviews Insect gut structure, function, development
and target of biological toxins. The insect midgut is the first barrier or a
target for ingested toxophores (small-molecule insecticides or insecticidal
proteins). For insecticidal proteins from the bacteria, Bacillus, Lysinibacillus
and Photorhabdus, the midgut provides several target sites by which these
proteins manifest their toxic action. However, for many target sites in other
tissues, the midgut can be a barrier for efficient delivery, like peptides from
spider venom (Chapter 8). Although a lot of the information reviewed
here is from mosquito and Drosophila midguts, these approaches and under-
standing can help draw parallels and differences between phytophagous
insects (agriculturally important) versus hematophagus insects (medical
importance). Additional proteomic studies on the midgut to identify and
characterize putative target sites would be beneficial for developing or
discovering alternate mechanisms of action. Linser and Dinglasan have pro-
vided an excellent review of the insect midgut with a discussion of possible
target sites.
Chapters 25 review various aspects of insecticidal proteins from Bacillus
and Lysinibacillus. In Chapter 2, Adang et al. review the diversity of insec-
ticidal proteins (three domain crystal (Cry), Cytolytic (Cyt), Binary Cry and
other parasporal toxins) from Bacillus. They review the mode of action of
these proteins, providing similarities and differences in the receptors used
for manifesting toxicity. The identification and characterization of toxin
receptors is important not only to create opportunities for discovering newer
toxins but also to modify known toxins to target insect pests that are less or
non-susceptible. Moreover, such investigations allow the development of
strategies to overcome or delay the development of resistance to insecticidal
proteins.
In Chapter 3, Filha et al. review the Binary (Bin) proteins from
Lysinibacillus sphaericus (Ls) that are mosquitocidal. The authors discuss the
structure, function and mechanisms by which these proteins cause toxicity

ix
x Preface

in mosquito larvae. Unlike genes encoding insecticidal proteins from Bacillus

species, which are now used as transgenes in crops for creating insect pest
resistance, the Ls bacteria have been used as biolarvicides.
The potential of using Bacillus thuringiensis (Bt) as bioinsecticides was rec-
ognized in the early twentieth century, and subsequently many Bt products
from Bt strains were developed for commercial use. However, these prod-
ucts suffered from a lack of stability in the sprayed environment and reduced
efficacies. It was not until the first genes encoding Cry insecticidal proteins
were cloned that research to their use as transgenes was initiated. Between
1995 and 1996, the first transgenic crop (potato, corn and cotton) carrying a
Cry gene for controlling an insect species was developed. Since then, there
has been a rapid adoption of transgenic crops worldwide, increasing from 1.7
million hectares in 1996 to just over 175 million hectares in 2013. This trend
for reliance on transgenic crops will continue to grow until newer, better
and more effective approaches to prevent damage from insect pests are dis-
covered and developed.
In Chapter 4, Narva et al. review the discovery and use of genes
encoding insecticidal Bt Cry proteins for developing transgenic crops that
provide control of insect pests. In this chapter, the use of multiple Cry genes
(gene stacking or pyramiding) is also reviewed to describe approaches to not
only broaden the spectrum of insect pests controlled within a crop but also
providing an approach to delay the development of insect resistance to a sin-
gle Bt gene product. The authors not only review the various Bt genes that
have been used for developing transgenic crops but also provide an overview
of approaches used for transferring genes into crops, selection of transgenic
events and what needs to be done to register and the deployment of such
transgenic crops in different geographic regions. Every time a new mecha-
nism for insect pest control is developed, it comes with the possibility of the
target insect developing resistance, making the product less efficacious. The
authors provide a brief overview of insect resistance management strategies,
which is reviewed more extensively in Chapter 6 by Wu.
In Chapter 5, Baum and Roberts review yet another approach that relies
on knocking down or down regulating genes encoding proteins essential for
target insect pest survival. The use of double-stranded RNAi (dsRNAi) has
been very effectively used in non-arthropods and plants to knock down
genes to understand gene function in specific pathways. This approach
has now been used for inactivating specific genes critical to the survival
of insect pests. This approach is an alternative to the use of chemical
Preface xi

insecticides for interfering with the function of target site proteins. How-
ever, the use of dsRNAi provides a much higher level of selective toxicity
to insect pests and serves as an attractive approach. Although there are no
commercial products harnessing this approach as yet, it will not be long
before such products are commercially available.
In the last chapter (Chapter 6) related to Bt insecticidal proteins, Wu
reviews resistance development and resistance management strategies for
transgenic crops carrying Bt genes. The development of resistance is inev-
itable, and the challenge faced is how strategies can be deployed to delay the
targeted insect pests from developing resistance to the insecticidal proteins in
host transgenic crops. In this respect, it is also important to understand the
mechanisms and target site receptors/proteins these insect toxins use for
manifesting insecticidal activity and the mechanisms that lead to resistance
development. This aspect of resistance ties very well with the review in
Chapter 1 on mode of action of Bt proteins.
Chapters 7 and 8 review alternate sources of insecticidal proteins or pep-
tides. The discovery of insecticidal proteins from the bacteria Photorhabdus
and Xenorhabdus created a lot of interest among academic labs and industry
to understand the structurefunction and mode of action of these very large
(molecular size) and complex proteins. This is reviewed in Chapter 7.
Although genes encoding these proteins or their peptides have not been used
as transgenes in crops to control specific insect pests, the information gen-
erated can be leveraged with new approaches and capabilities to possibly
make use of such genes (modified or unmodified). ffrench-Constant and
Dowling have provided an extensive review of the many proteins from
the two bacteria, high-resolution structures and possible mechanisms of
action of the insecticidal proteins.
In Chapter 8 on Methods for deployment of spider venom peptides as
bioinsecticides, the authors describe a novel source of peptides from spider
venom that show very interesting and selective toxic activities in insects.
Most of these act on neuropeptide targets and provide a challenging oppor-
tunity as how to make use of these peptides as biopesticides or use knowl-
edge of their structures to invent new small-molecule toxophores that can
interact at the same target sites used by the peptides from the spider venom.
Chapters in this volume were chosen to provide a single comprehensive
review of structure and function of the insect midgut and the insecticidal
proteins and genes that have been used as alternatives to chemical insecti-
cides for controlling insect pests of agricultural and medical importance.
xii Preface

Discovery of newer insect control approaches and their use will be an

important component of increasing crop yields in an ever-shrinking arable
land and continued insect transmission of many human diseases in an
increasing world population that is projected to increase to 9 billion by 2050.

TARLOCHAN S. DHADIALLA
SARJEET S. GILL
 CHAPTER ONE

Insect Gut Structure, Function,

Development and Target of
Biological Toxins
Paul J. Linser*, Rhoel R. Dinglasan
*The University of Florida Whitney Laboratory, St. Augustine, Florida, USA

Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public
Health, Baltimore, Maryland, USA

Contents
1. Introduction 1
2. Mosquito Larval Alimentary Canal 3
3. Other Insects 27
3.1 Lepidopteran larvae (caterpillars) 28
3.2 Coleopterans (beetles and their larvae) 30
3.3 Hemipterans (aphids) 31
4. Conclusions and Comment 32
References 33

Abstract
Insects as vectors of disease to humans and domesticated animals and as direct agri-
cultural pests are a source of tremendous economic and health-related challenge.
The eating habits of insects can provide the bases for disease transmission or the out-
right destruction of crops. The alimentary canal of insects is a common target and often
barrier for pest control strategies. Recent advances in technology have made it possible
to develop ever better understanding of the structure/function of the insect gut and
hence provide new and better targets for developing novel methods for limiting the
burdens that insects can present to humanity. In this review, we focus attention on
recent developments in our understanding of insect gut structure/function with partic-
ular emphasis on a few of the most challenging groups of insects: mosquitoes (dip-
terans), caterpillars (lepidopterans), beetles (coleopterans) and aphids (hemipterans).

1. INTRODUCTION
The alimentary canal of any higher organism is part of that organisms
first order environmental contact. Consequently insects have evolved highly

Advances in Insect Physiology, Volume 47 # 2014 Elsevier Ltd 1

ISSN 0065-2806 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800197-4.00001-4
2 Paul J. Linser and Rhoel R. Dinglasan

specialised capacities to live in many varied ecological niches ranging from

aquatic to terrestrial to airborne. In all cases, gut function is crucial for sur-
vival and hence is specifically adapted to the life style of the insect. Details
associated with the ingestion of biological substrate (food), digestion of that
material into useable small molecules and finally the absorption of the liber-
ated nutrients into the cells, tissues and hemolymph of the animal are com-
plex and varied from specie to specie. The purpose of this review is to address
structural details of the insect alimentary canal with commentary on the
structural interface for targeting of the gut with biological toxins. The
importance of developmental changes and lifestyle differences between life
stages will also be addressed. It is beyond the scope of any single review to
address specific details for the wide variety of insects and their specialisations
of the gut. Therefore, we have selected a few representative model systems
for discussion.
The importance of insects to life on earth including human existence is
indisputable. For us as co-inhabitants of the planet, insects have particular
relevance in their capacity to interfere with aspects of our health and
wellbeing. Many insects have evolved complex relationships with organisms
and viruses that can cause human disease. Hematophagy has evolved in
arthropods over 20  (Black and Kondratieff, 2005). The propensity to take
blood meals from vertebrates in general has been accompanied by the devel-
opment of the capacity to harbour and transmit disease microbes and viruses.
This reality creates numerous challenges for human beings ranging from
negative impacts on domesticated animal stocks as well as the vectoring
of human pathogens directly. Therefore, one of the most important groups
of insects for the purposes of this review is mosquitoes, which transmit some
of the deadliest known human pathogens. The morbidity and mortality
brought about by hematophagy of mosquitoes results in incalculable losses
of life and human potential. Our efforts to control mosquito populations
with various pesticides and integrated strategies are continuously thwarted
by the capacity of mosquitoes to adapt and evolve rapidly under selective
pressure. Hence, a deep understanding of mosquito biology is essential
for the development of new disease control strategies. The gut of the mos-
quito (Dipterans) in both larval and adult stages is a productive target for
control strategies and hence a point of emphasis in this review.
Human development and the use of agriculture has provided a basis for
the expansion of our species from hunter-gatherers dependent on the whim
of Mother Nature to the truly dominating natural force on our planet. Agri-
cultural development has been continuously challenged by opportunistic
Insect Gut Structure, Function, Development and Target of Biological Toxins 3

and natural competitors for the crops in the field. Relevant to this review of
course are a range of insect pests which consume or damage crops in a
variety of ways including the transmission of plant diseases. The impact of
pest insects on the world economy and the security of the human food sup-
ply is gigantic. Hence, we will also review the structural biology of certain
groups of agricultural threats: Lepidopteran and coleopteran larvae and
hemipteran life stages that impact crops. Of course, there are many and
diverse insects that will not be covered in this review but we hope to present
structural considerations that can be instructive and of fairly generalised
relevance.

2. MOSQUITO LARVAL ALIMENTARY CANAL

For the purpose of this review, we will not go into detailed discussion of
structure/function analyses that have been reviewed in great detail previously.
There are superb resources for examining the depth of analyses performed
with the foundational techniques of traditional microscopy and biochemistry
(e.g. Billingsley, 1990; Billingsley and Lehane, 1996; Lehane and Billingsley,
1996). Herein, we will focus on a broad structural view associated with fairly
recent applications of newer techniques for structure/function analysis.
Development of the insect alimentary canal has been investigated
exhaustively and excellent reviews and reference texts are available (e.g.
Klowden, 2007). A generalised summary of the embryological origins of
the cells of the gut is shown in Fig. 1.1. Posterior and anterior invaginations
of the embryonic ectoderm give rise to the anus and mouth respectively.
Masses of endodermal cells emerge from the invaginating epithelium and
give rise to the endodermal tube that will eventually connect forming the
midgut. The invaginating ectodermal cells will become the hindgut and
foregut. Fusion of the epithelial primordia eventually produces the continu-
ity of the alimentary canal and all of its subdivisions (Klowden, 2007).
Dipterans such as mosquitoes are holometabolous. This term means that
they exhibit very distinct larval developmental stages, pupation and the
emergence of an adult imago that does not resemble the larval stages
(Klowden, 2007). Similarly lepidopterans that includes butterflies and moths
such as Manduca sext and coleopterans (beetles) have very distinct larval and
adult stages such that casual observation might lead one to believe the dif-
ferent life stages are actually different organisms. Differences in organismal
structures are quite severe such that environmental niches of dissimilar stages
of a given organism can be vastly different representing very distinct selective
4 Paul J. Linser and Rhoel R. Dinglasan

Figure 1.1 Embryonic development of the main components of the alimentary canal in
insects. Invaginations of the ectoderm at the anterior and posterior poles give rise to the
foregut and hindgut, respectively. The midgut forms from endodermal cells adjacent to the
invaginations, proliferating and migrating to enclose the central yolk. The ectodermal and
endodermal tubes eventually fuse to form a contiguous alimentary canal. The sequence of
developmental steps progress from A through D. Redrawn by Gabriela Marie Ferguson
after several sources including Johansen and Butt (1941).

pressures. Mosquitoes are an excellent example of this phenomenon in that

larvae are completely aquatic whereas adults are winged and live aloft or rest-
ing on terrestrial surfaces. The alimentary canal of larval mosquitoes (and
others) is nearly completely autolysed and replaced during pupation so that
the adult digestive apparatus is largely built anew.
These differences in environmental niche and the associated structural
adaptations naturally produce significant distinctions in supplying control
agents to a targeted pest or disease vector species. The genomic era that
has captured us all has shown that the huge physical differences that distin-
guish embryonic, larval, pupal and adult stages of holometabolous insects are
the product of surprisingly subtle modifications in gene expression rather
than the exposure of batteries of stage-specific genes (Goltsev et al., 2009;
Marinotti et al., 2006).
In contrast, hemimetabolous insects (hemipterans, e.g. aphids) show
much less dramatic structural remodelling during development from nymph
stages to adults. The alimentary canal though varied in structure between
species is retained and expanded as the insect matures.
In general considerations, the insect alimentary canal is a contiguous epi-
thelial tube with the typical anterior oral opening (mouth) and posterior anus
of higher metazoans. Figure 1.2A shows a scanning electron micrograph
Insect Gut Structure, Function, Development and Target of Biological Toxins 5

Figure 1.2 Architecture of the larval mosquito alimentary canal. Panel A: a scanning
electronmicrograph of a fourth instar larva of Aedes aegypti that has been dissected
to reveal the full length of the midgut amidst the exoskeleton and integument. Panel
B: a cross section of the gut epithelium in the region of the posterior midgut (PMG)
showing that it is a single cell thick epithelial tube but with varying morphological
characteristics of the individual cells. Panel C: a diagrammatic rendering of the larval
mosquito alimentary canal with labels applied for orientation. Numbers 18 indicate
abdominal segments. GC, gastric caecum; AMG, anterior midgut; CMG, central midgut;
PMG, posterior midgut; MT, malpighian tubules; HG, hindgut; Py, pyloris; Ai, anterior
intestine; Rc, rectum; Ac, anal canal; Ph, pharynx; Oe, oesophagus; Ca, cardia; pm,
peritrophic membrane; cm, caecal membrane; lm, gut lumen. The approximate pH of
the lumen of the gut is shown at the bottom of the cartoon. Taken from Linser et al.
(2007) with permission.

(SEM) of a fourth instar Aedes aegypti larvae (Linser et al., 2007). In this
image, the cuticular exoskeleton has been peeled back revealing the gross
architecture of nearly the entire alimentary canal. Figure 1.2B shows a
cross-sectional histological view that highlights one of the important char-
acteristics of the insect gut: it is a tubular organ system made up of a single cell
thick epithelium of highly polarised cells in terms of structure (i.e. apical,
lateral and basal structural distinctions) which presumably reflect distinctions
in function as well. Figure 1.2C provides a cartoon rendering of the major
organisational features or functional zones of the gut. A major subdivision of
the alimentary canal not shown in this cartoon is the pair of salivary glands
(SGs) that extend laterally from the oesophagus but these will enter the
6 Paul J. Linser and Rhoel R. Dinglasan

discussion later. Also, the structural components of the mouth and oral cav-
ity leading to the pharynx are not covered herein but the reader can find
many details in the literature (e.g. Clements, 1992). Figure 1.2C also depicts
one of the key structure/function relationships in many larval insect
alimentary canals: the lumen of the gut exhibits a range of pH values that
can reach extreme levels of basicity (Boudko et al., 2001; Corena et al.,
2004; Dadd, 1975; Terra et al., 1996; Zhuang et al., 1999). In mosquito lar-
vae, the anterior midgut (AMG) lumen pH can be as high as 11 (Boudko
et al., 2001; Corena et al., 2004; Dadd, 1975; Terra et al., 1996; Zhuang
et al., 1999). In certain other insect larvae such as caterpillars (e.g. Manduca
sexta) the luminal pH may exceed 12 (Cioffi, 1979; Harvey et al., 1983;
Wieczorek, 1992). The evolution of a digestive strategy that employs
extremely high pH is a subject of considerable interest both from the detailed
physiology of the system to the impact such pH extremes have on the imple-
mentation of control strategies that target gut function such as the bacterial
toxins from Bacillus thuringiensis and B. thuringensis israeliensis (BT and BTI
respectively; Gill et al., 1992; Chapter 2). This will be a recurring theme
within this review and volume.
The gross architecture of the larval mosquito gut is depicted in greater
detail in Fig. 1.3. We will address certain details for most of the specialised
functional zones. The first zone in this figure is the pair of bi-lobed (anterior
and posterior lobe) SGs. Although relatively little research has been done on
larval SGs, much is known about adult SGs as they are part of the infection
pathway for the transmission of viral and protozoan pathogens (Black and
Kondratieff, 2005). The SGs of larvae do in fact produce some of the earliest

Figure 1.3 Figure shows a detailed cartoon of key structural elements of the larval mos-
quito alimentary canal from the foregut including the salivary glands (SGs) to the rectum
(RC). Abbreviations are as in Fig. 1.2 except that the CMG is called the TR (transition
region) in this figure. Note the extent and location of the ectoperitrophic space
(ECTO), the cuticular lining of the foregut and hindgut and the variable distribution
and size of brush border membranes (microvilli) on the apical aspects of the gut cells
at the various regions of functional specialisation.
Insect Gut Structure, Function, Development and Target of Biological Toxins 7

effectors of digestion as defined by micro array-based transcriptomic analyses

(Neira Oveido et al., 2009) as is true for SGs of adult mosquitoes and other
organisms (e.g. Chagas et al., 2013). The structure implies a cascade of func-
tionalities from the inline organisation of the two lobes of each gland. The
dynamics and contents of larval saliva are areas with little basic information
but may affect the effectiveness of control agents that encounter the saliva
once consumed by the larva.
Posterior to the point in the oesophagus at which the SGs connect is a
complex structure (much simplified in Fig. 1.3) called the cardia that is the
junction between the foregut and the midgut. Epithelial layers of the foregut
and midgut overlap for a short distance creating a crypt in which the
peritrophic matrix (PM) is secreted and assembled. The PM of larval mos-
quitoes is referred to as a Type II PM and is constitutively and continuously
synthesised by the complex arrangement of cells of the cardia (Clements,
1992). This acellular material, sometimes referred to as the peritrophic
membrane, is similar in function to dialysis tubing and even looks very sim-
ilar to dialysis tubing at the macroscopic level of examination. The PM pro-
vides a physical barrier between the ingested bolus of food and the actual
epithelial cells of the midgut. It also provides a barrier to macromolecular
complexes that may be secreted by gut cells or sloughed by cells. The
PM is composed of a complex mixture of proteins, and chitin microfibrils
in a proteo glycan matrix (Hegedus et al., 2009; Lehane, 1997). The tubular
PM lines the midgut and is eventually passed from the rectum in various
stages of disintegration and possibly reabsorption during faecal elimination.
The PM is a microporous barrier and the porosity limits diffusion through
the membrane to specific size limits (Edwards and Jacobs-Lorena, 2000;
Hegedus et al., 2009; Lehane, 1997). This barrier function may affect the
access of certain toxic materials and biological materials to the gut cells.
The cuticular lining of the oral cavity and foregut ends as the PM manifests
at the anterior extreme of the midgut. From this point posteriorly, the mid-
gut epithelial cells are separated from the food bolus by the PM. This sep-
aration defines a specific compartment, which is termed the ectoperitrophic
space (Clements, 1992; Smith et al., 2007). This fluid-filled compartment is
very dynamic and provides the medium from which ions, solutes and nutri-
ents are trafficked into and out of the midgut cells (Terra, 1990; Terra et al.,
1996). Any macromolecules, solutes or even biological control toxins that
will contact the epithelial cells directly will do so from this active space.
The water movement within the ectoperitrophic space is also dynamic
and tracer studies have shown that there is a net movement of water from
8 Paul J. Linser and Rhoel R. Dinglasan

the posterior midgut through this compartment to absorption in more ante-

rior positions (Terra, 1990). We will address more details of the PM and the
ectoperitrophic space in a later discussion.
Posterior to the cardia, the gut tube of mosquito larvae flares laterally into
eight diverticuli termed the gastric caeca (GC). Such lateral pouches off the
main structure of the gut tube are common in many insects but not all. The
pouches define an interior space surrounded by epithelial cells. The interior
space is also set apart from the ectoperitrophic space by the existence of
another acellular membrane similar to the PM, which is termed the caecal
membrane (CM). Like the PM, the CM has a barrier function and presum-
ably allows for traffic into and out of the caecal cavity on a size-exclusion
basis and perhaps other forms of selectivity (Edwards and Jacobs-Lorena,
2000). Various types of transport physiology studies, as well as micro array
analyses of gene expression, indicate that caecal cells are involved in both the
secretion of various gene products including certain digestive enzymes as
well as the uptake of specific solutes and small-molecule nutrients (e.g.
amino acids, Harvey et al., 2009; Volkman and Peters, 1989a,b). It is appar-
ent that control agents that would target the cells within the caecal cavity
would have to pass through both the PM as well as the CM. The use of fluo-
rescently labelled plant lectins which discriminate the glycoconjugate pat-
terns on a variety of structural macromolecules show that the CM and
PM are not biochemically identical (Linser et al., 2008) which is also inferred
by ultrastructural details (Hegedus et al., 2012; Lehane, 1997). The caecal
cavity is fluid filled but that fluid is viscous and also exhibits strong labelling
with certain fluorescent lectins. The caecal fluid is likely composed of a rich
mixture of proteins, glycoproteins and proteoglycans (Linser et al., 2008).
Any molecules such as small molecule nutrients or ingested toxins must tra-
verse the CM and the caecal fluid to make contact with the caecal
epithelial cells.
The endodermal epithelial cells that comprise the caeca exhibit striking
structural characteristics. As a transporting epithelium, the caecal cells possess
extensive expansion of the plasma membrane both on the apical and basal
aspects of the cells. Two major cell types have been described, each
possessing extensive microvilli patterns at the apical surface. What have been
termed ion-transporting cells exhibit very long and densely packed
microvilli, usually containing long tubular mitochondria within each micro-
villus (Seron et al., 2004). The second principal cell type, which has been
called a resorbing/secreting cell, also possesses extensive microvilli at
the apical surface that usually lack internal (microvillar) mitochondria
Insect Gut Structure, Function, Development and Target of Biological Toxins 9

(Seron et al., 2004). The basal aspect of both aforementioned types of caecal
cell exhibit varied but extensive plasma membrane infoldings (labyrinth),
again indicative of extensive expansion of the cell surface area. Figure 1.4
shows a representative transmission electron microscopic (TEM) image of
caecal cells from Aedes aegypti larva with these specific characteristics evident.
In addition to the two main types of caecal cell described above and numer-
ous times in the classical literature, a third type of epithlelial cell has been
noted. These cells typically occur at the posterior extreme of each caecal div-
erticulus and thus have been referred to as CAP cells (Seron et al., 2004).
CAP cells show very small or no microvilli on the apical surface and gen-
erally appear to be less broad in the apical to basal dimension. In subsequent

Figure 1.4 Gastric caeca cells as seen with transmission electron microscopy. Two dif-
ferent cells are shown: a lightly staining ion transporting cell (on the right) has micro-
villi (shown in cross section) containing mitochondria. The darker staining of the
resorptive cell (left) is due to the presence of extensive rough endoplasmic reticulum.
The scale bar represents 5 m. The inset is a high-magnification electron micrograph of
a transverse section from an ion-transporting cell with microvilli that contain mitochon-
dria. Portasomes (arrowheads) are prominent on the cytoplasmic face of the membrane.
Scale bar represents 100 nm. From Zhuang et al. (1999) with permission.
10 Paul J. Linser and Rhoel R. Dinglasan

sections, we will explore some of what is known about the structure/func-

tion relationships of these three main types of caecal cell. Additionally, as is
true in most of the regions of the gut throughout larval development, smaller
cells (typically adjacent to the basal side of the cell layer) that have been ter-
med regenerative cells dot the epithelium. These are thought to be simple
diploid precursor cells that will eventually either divide to produce new gut
cells or undergo endoreplication of the chromosomal DNA to produce the
typically polyploid mature gut cells (Ray et al., 2009). Finally, there are
scattered and relatively small enteroendocrine cells within the caecal epithe-
lium (as well as other regions of the gut) which are presumed to mediate
chemical and neurological signalling in the gut (Brown and Lea, 1988).
The connection of the caecal diverticuli to the gut tube proper (the cae-
cal neck) occurs via cells with specific structure and functional qualities. The
cells of the caeca that are internal relative to the CM are distinguishable from
those in this neck region (i.e. outside of the CM relative to the main lumen
of the gut tube). Once we have completed a general description of gut cell
structure, we will return to structure/function data as determined by recent
investigations of the disposition of specific gene products and functionalities.
The posterior wall of the caecal neck adjoins the AMG and the architec-
ture of the epithelial cells undergoes a significant change in character. The
largest of the AMG cells, which are the majority of cells in his region of
the gut tube, have few and small apical microvilli in stark contrast with the
GC cells as well as the PMG cells (Clements, 1992; Zhuang et al., 1999). This
of course suggests less of an absorptive function for these cells in relation to the
other gut compartments, the GC or PMG cells. This also implies that the api-
cal surface area of AMG cells is much lower than that of the GC and PMG
cells, which possess extensive microvillar-based extension of the plasma mem-
brane. The basal membranes of the AMG cells are similarly expansive to those
of other gut cells. Numerous intracellular vesicles and labyrinthine extensions
of the basal membranes fill much of the cytoplasm of the AMG cells (Volkman
and Peters, 1989a,b; Zhuang et al., 1999). Most AMG cells possess large poly-
ploid nuclei. As in the GC, there are scattered, smaller diploid cells that are
either regenerative stem cells or neuroendocrine cells.
Figure 1.5 (Clark et al., 2005) shows an SEM image of a fourth instar
Aedes aegypti larval gut. The alimentary canal from the GC (at top) to the
malpighian tubules (MT) at the junction with the hindgut was dissected.
The tube from AMG through PMG was slit anterior to posterior and then
the epithelium curled upon itself such that the internal surface is now dis-
played as the outer surface of this preparation. Note that the upper half of
Insect Gut Structure, Function, Development and Target of Biological Toxins 11

Figure 1.5 A scanning electronmicrograph of isolated larval Aedes aegypti midgut

showing the different structurally distinct regions. The gut tube was cut along the
length of the tube, and the tube then curled upon itself thus exposing the inner surfaces
of the AMG and TR and the PMG. The GC are still intact and so the outer surface of the GC
are shown at the top of the figure. The MTs are at the bottom. Note that, what the inner
surface of the AMG, TR and PMG exhibit is a very different gross architecture. From Clark
et al. (2005) with permission. Scale bar represents 600 m.

the tube as shown here, which represents the AMG, has a very smooth
appearance. The lower portion, the PMG, shows a more granular surface.
At higher magnification or by viewing cross-sectioned material from the
AMG and PMG it is evident that the difference in appearance of the everted
tube is that the AMG cells have few and very short microvilli, whereas the
major cells of the PMG have apical arrays of microvilli that are tightly packed
and appear to be a solid cap when viewed by low magnification SEM (Clark
et al., 2005). At the region where the AMG and PMG meet, there is a tran-
sitional region that is several cells broad from anterior to posterior along the
12 Paul J. Linser and Rhoel R. Dinglasan

long axis of the gut. The apical surfaces of cells in the transitional region
show an increase in the numbers and dimensions of microvilli (Clark
et al., 2005). Other distinctions have been described between the AMG,
transitional region and the PMG and we will return to these shortly.
The PMG is broader than the AMG and the epithelial cells are also larger
than those of the AMG. The apical surface is tremendously extended by
microvilli that are tightly packed. Ultrastructural analyses show basal mem-
brane infoldings, copious numbers of intracellular vesicles and extensive
mitochondrial profiles, which are indicative of cells that are very metabol-
ically active and involved in absorptive and secretory processes (Billingsley,
1990; Clements, 1992; Zhuang et al., 1999).
The hindgut is composed of the pyloris and MTs, ileum (anterior intes-
tine), rectum and anal canal that are all of ectodermal origin in distinction
with the endodermal midgut. The PM, which originates at the cardia at
the posterior end of the foregut, begins to lose its integrity as it enters the
pyloris of the hindgut (HG). The HG has a cuticular lining that encompasses
the PM and the food bolus as it continues its journey toward excretion. The
cells of the pyloris are thin epithelial cells and the funnel-shaped region of the
HG has a posterior band of muscle forming a sphincter (the pyloric sphinc-
ter). At the most anterior extreme of the pyloris, the epithelial cells are quite
small and contiguous with the stem cells of the posterior imaginal ring
(Clements, 1992; Klowden, 2007). The five MTs are tubes with an opening
into the pyloris and a closed terminus at the distal end of each. Two types of
cells are described in the MTs: principal cells and stellate cells, which have
differential embryological origins (Davies and Terhzaz, 2009; Dow, 2009).
Principal cells are large with extensive apical microvilli and each cell can
extend nearly around the circumference of the MT lumen, which is zigzag
shaped due to the apical intrusion of the principal cells. Principal cells possess
a large polytene nucleus and in later stages of larval development accumu-
lations of membrane-bound inclusions or concretion bodies (Bradley and
Snyder, 1989). The second type of cell in the MT is called the stellate cell
and their shape is indeed star like. Stellate cells localise between some of the
principal cells and can be difficult to detect with simple microscopy as they
can appear to be the interconnections of the lateral membranes of principal
cells. Recent physiological and immunohistochemical analyses have pro-
vided insights into the structural relationships between principal cell and
stellate cells (see later discussion of this section). Stellate cells possess much
smaller nuclei than principal cells and it is not clear whether or not they are
diploid or polyploid. The function of the MTs is generally held to be similar
Insect Gut Structure, Function, Development and Target of Biological Toxins 13

to that of the vertebrate kidney in ion regulation and the formation of the
primary urine (Beyenbach et al., 2009; Bradley, 1987).
The ileum or anterior intestine is a thin-walled epithelium within a
prominent muscular tube. Relatively little is known of the functional spe-
cialties of the ileum other than it serves as the continuation of the pathway
for the movement of the excreta. The muscular tube surrounding the ileum
is at least partly responsible for pushing faecal material through the rectum
and into the anal canal.
The rectum is a complex region of the gut and exhibits substantial archi-
tectural variations between genera of mosquitoes and insects in general. Sim-
ply stated it is an essential organ in the regulation of ionic balance in the
animal and the retention or elimination of specific solutes. The cells of
the insect rectum have at times been described as among the most complex
cells in biology (Berridge and Oschman, 1972). The rectum of mosquito
larvae varies in general architecture. The tubular nature of the alimentary
canal continues into the ectodermal rectum. The length and architecture
of the rectum varies between species but, generally, the rectum is a
cuticle-lined epithelium that can exhibit longitudinal folds. The two sub-
families of mosquitoes, the Culicinae and the Anophelinae are somewhat
different in terms of rectum structure (Bradley, 1987; Smith et al., 2007,
2008, 2010; White et al., 2013). Additionally, the osmolarity of the aquatic
environment in which larvae develop can be reflected in sometimes-gross
variations on the architectural details of the rectum. Culicinae species, such
as Aedes aegypti, which select low ionic strength aquatic habitats exhibit a
single compartment rectum. In contrast, Culicinae, such as Aedes
campestris, that are tolerant of much higher osmolarities possess a rectum with
distinct anterior and posterior compartments (Bradley, 1987; Smith et al.,
2007, 2008, 2010; White et al., 2013). Anophelinae such as Anopheles
gambiae (salt intolerant) and Anopheles merus (salt tolerant) possess rectal struc-
tures that are somewhat intermediate between the partitioned (anterior and
posterior) rectum of salt-tolerant culicinae and the single-compartment rec-
tum of salt intolerant species. Specifically, Anophelinae possess two distinct
populations of cells but lack a clear compartmental divide between them
(Bradley, 1987; Smith et al., 2007, 2008, 2010; White et al., 2013). The
two populations are functionally and structurally distinct and have been
named the dorsal anterior rectum (or DAR) cells and the remaining non-
DAR cells (Smith et al., 2007). We will return to a discussion of the struc-
ture/function details of these cells later. In general, whether discussing Cul-
icinae or Anophelinae larval rectum cells, the cells are characterised by
14 Paul J. Linser and Rhoel R. Dinglasan

extreme modifications of the plasma membrane on both the apical and

basal sides. Meredith and Phillips (1973) described apical extension of the
plasma membrane that takes the form of tightly packed parallel channels
of membrane similar to tightly packed microvilli as described in the GC
and the PMG. However, in the rectum, the parallel membrane channels
do not end in free microvillar tips but rather in a surface that is embedded
in a characteristic cuticular layer (Meredith and Phillips, 1973). The mem-
brane channels or stacks are associated with numerous mitochondria and
portasomes indicative of a very intense role in ion transport (Meredith
and Phillips, 1973). The basal side of rectal cells can exhibit labyrinthine
membrane infoldings that are much less organised into parallel arrays than
the apical membranes but nonetheless very extensive. In Culicinae with
divided anterior and posterior lobes, anterior cells look less extended in
terms of plasma membrane amplification than do the posterior cells. In
the Anophelinae, the DAR and non-DAR cells exhibit structural distinc-
tions similar to those seen in the anterior and posterior cells of the Culicinae
(Smith et al., 2008, 2010).
Throughout the epithelial tube that is the alimentary canal of insect lar-
vae, we have described gross structural aspects and some details of the cell
apical and basal plasma membranes. We have ignored the lateral membranes
thus far. We will not dwell on the lateral membranes other than to state
that as in all epithelia, cellcell junctions exist in various arrangements,
which of course hold the epithelia together as a sheet and also serve to influ-
ence the apical-to-basal movement or diffusion of molecules. A great deal of
research has gone into the analyses of junctional complexes and the differ-
ences between vertebrate and insect model systems (e.g. Matter and Balda,
2003). Suffice it to say here that the lateral membranes of larval mosquito
alimentary canal epithelial cells exhibit varied junctional complexes such
that movement of molecules between cells is regulated but not always
completely blocked (Neira Oviedo et al., 2009).
Figure 1.3 shows a cartoon rendering of the gross details of the larval
mosquito alimentary canal. There are several highlights to recall as we con-
tinue into the next discussion of cell biology and cell polarity. The disposi-
tion of extracellular material that separates the food bolus from physical
contact with the gut cells varies: in the foregut it is cuticular; from the cardia
to the termination of the midgut at the pyloris there is a type II PM; from
the beginning of the hindgut, the PM becomes surrounded by cuticular
extracellular matrix; the gastric caeca possess a distinct additional barrier
called the CM. The PM defines a fluid-filled environment called the
Insect Gut Structure, Function, Development and Target of Biological Toxins 15

ectoperitrophic space, which is in contact with the lumen of the gut and the
lumen of the caeca. Various subdivisions of the gut are associated with char-
acteristic structural specialisation of the several cell types located therein. In
relation to the accessibility and effectiveness of orally administered biological
toxins (a focus of this volume), gut structure is of obvious importance. In
addition, certain details of the cell biology that have come to light in recent
years have either already become demonstrably important to new control
strategies or clearly have that potential. At this point, we are going to review
a number of fairly recent investigations that have provided insight into the
structure/function of the important cell types of the alimentary canal.
Material ingested by mosquito larvae will encounter the secretions of the
SGs (i.e. saliva) as one of the earliest steps in digestion. In addition to aiding
digestion, the saliva contains numerous gene products associated with
immune surveillance and the inactivation of potential pathogens. Trans-
criptomic and proteomic analyses of the Anopheles gambiae larval SGs rev-
ealed the production and secretion of such immune effectors as defensins,
lysozyme and TIL-domain proteins (Neira Oviedo et al., 2009). Hence,
ingestion of materials that might inactivate such components of the saliva
may well render the larvae more susceptible to biological toxins or organ-
isms. Methods for generally inhibiting SG function would also be reasonable
targets for the development of novel control strategies. The anterior and
posterior lobes of the SGs are biochemically distinguishable but nothing
is known about the actual compartmentalisation of specific salivary compo-
nent synthesis and secretion. A clearer understanding of SG structure/func-
tion and cell biology will be a valuable pursuit in the future.
The glycocalyx-type extracellular matrix linings of the alimentary canal
provide a range of functions such as facilitating the one-way movement of
the food bolus from the mouth to the anus, physical protection of
delicate cell surfaces from what can be abrasive particulates, barriers to
full blown biological invasion from microbiota in the food bolus, size-
exclusion barriers to macromolecular diffusion and even selective perme-
ability and toxin sequestration. The lining of the foregut and the hindgut
is cuticular and exhibits structural qualities of cuticle. The PM and the
CM are also chitin-containing acellular barrier matrices with diverse func-
tions (Hegedus et al., 2009; Lehane, 1997; Rudin and Hecker, 1989). The
CM and PM are distinguishable from each other as well as the cuticular lin-
ings of the foregut and hindgut on several structural and biochemical bases.
For example, lectin labelling shows that the CM and PM are readily labelled
with several lectins including wheat germ agglutinin but the cuticular
16 Paul J. Linser and Rhoel R. Dinglasan

exoskeleton and the linings of the foregut and hindgut are distinguished
from the PM and CM with ricinus communis 1 (RCA; Linser et al.,
2008; Neira Oviedo et al., 2009). Figure 1.6 shows such a comparison
between Dolichus biflorus Agglutinin (DBA) and RCA1 in a longitudinal sec-
tion of a fourth instar Anopheles gambiae larva. The green fluorescence (DBA)
highlights the PM and CM vividly and in contrast to the red signal (RCA1),
which stains the exoskeleton cuticle and the linings of the foregut and hind-
gut. Indeed one can see that in the pyloris, the red cuticular lining of the
hindgut surrounds the PM and that the PM begins to be compacted at
the junction with the ileum. Furthermore, the origin of the PM (in green)
is visible in the folded layering of the cardia while the foregut cuticle is visible
within the innermost channel of the termination of the oesophagus (as col-
ours are not presented in the print version of this volume, please visit the
on-line version for full colour details). These chitin-containing extra cellular

Figure 1.6 Figure shows longitudinal sections of paraffin embedded Anopheles

gambiae fourth instar larvae at low (upper montage) and high (lower three panels) mag-
nification with the anterior (head) to the right. Labelling was with TRITC-conjugated
Ricinus communis Agglutinin I (red), FITC-conjugated Dolichos biflorus Agglutinin (green)
and DRAQ5 for DNA (blue). For the purpose of this discussion, note that the green DBA
staining labels the peritrophic membrane (PM) and the caecal membrane (CM), and the
red RCA labels cuticular structures including the exoskeleton and the lining of the fore-
gut adjacent to the beginnings of the PM in the cardia and at the posterior end of the
PM at the beginning of the hindgut at the level of the ileum-pyloris junction (arrows).
Also note that the rectum is lined by red, RCA + cuticle. From Linser et al. (2008) with
permission.
Insect Gut Structure, Function, Development and Target of Biological Toxins 17

matrices (ECMs) are structural barriers to any ingested material and hence
need to be penetrated by any biological toxins or control organisms. Lectins,
as used herein, are capable of discriminating specific details of glyc-
oconjugate structure in terms of specific sugar structure, chemical linkages
and other details. The analyses described here serve to show that the detailed
glycobiology of the ECMs associated with gut possess distinct biochemical
signatures. The impact of that varied biochemistry is largely untested.
The structure of specific cell types within the gut provides insight into
their functional roles. As described earlier, the apical surface of the large,
principal epithelial cell types of the GC and PMG possess extensive arrays
of microvilli. This implies an absorptive role. AgAPN1, a GPI-anchored
plasma membrane glycoprotein, first identified in adult Anopheles gambiae
is a peptidase presumably involved in the final stages of protein digestion,
liberating amino acids for absorption (Dinglasan et al., 2007). AgAPN1 is
also a putative point of attachment for the malaria parasite Plasmodium
falciparum (Armistead et al., 2014; Dinglasan et al., 2007; Mathias et al.,
2013). Antibodies to this protein specifically label the microvillar arrays
on the PMG cells and on a specific subset of the GC cells. Figure 1.7 shows
AgAPN1 labelling of the PMG and the GC cells that form the neck region of
each GC lobe. Only GC cells that are exterior to the CM label for this pro-
tein. In contrast, AgAPN2, another distinct cell surface aminopeptidase
thought to be a binding site for the Cry11Ba toxin of Bacillus thuringiensis
(Zhang et al., 2008) is also found on the microvilli of PMG cells and GC
cells. But, in this case, only the GC cells that lie within the GC, internal
to the CM, exhibit the protein. Additionally, the CAP cells (see earlier dis-
cussion) at the posterior extreme of each caecum contrast with the surround-
ing neighbouring GC cells by lacking AgAPN2 (Fig. 1.8; Harvey et al.,
2010; Linser et al., 2007).
As mentioned earlier, one of the striking qualities of the larval mosquito
alimentary canal is the extreme pH of portions of the gut lumen (Fig. 1.2).
The evolutionary selective pressure for extremely high pH in the gut lumen
is often associated with the high content of plant material in the diets of many
insect larvae including caterpillars and mosquito larvae (Terra et al., 1996).
This may have some truth but it should be noted that even in the Tsetse
(Glossinidae) which derives all of its biological energy for its entire life cycle
from blood meals, the gut pH can exceed 10 (Liniger et al., 2003). Regard-
less of the physiological ramifications of an alkaline digestive system, the
mechanisms which drive the pH gradient along the length of the mosquito
larva gut from nearly neutral at the level of the GC, to pH 10.5 or even
18 Paul J. Linser and Rhoel R. Dinglasan

Figure 1.7 Figure shows the distribution of two integral membrane amino peptidases,
AgAPN1 (panel A) and AgAPN2 (panel B) in larval Anopheles gambiae gut sections. In (A),
AgAPN1 (blue) is seen on the brush border membranes (BBM) of the posterior midgut
(PMG) cells (short arrow to left) and the BBM of the neck of the gastric caeca (GC) (short
arrow to right and in high mag images at bottom of panel). Note that FITC-conjugated
Vicia Villosa Lectin (green) was used to highlight the cuticular structures, the peritrophic
membrane (PM) and the caecal membrane (CM) (long arrows) making the limitation of
AgAPN1 to the neck cells of the GC evident. In (B), AgAPN2 (green) is compared to Na+/
K+-ATPase (red) and the cytoplasmic marker carbonic anhydrase-9 (CA9). The short
arrows indicate labelling for AgAPN2 on the BBM of the PMG and on the BBM of the
GC cells internal to the CM. From Linser et al. (2008) with permission.
Insect Gut Structure, Function, Development and Target of Biological Toxins 19

Figure 1.8 Figure shows the GC of fourth instar Anopheles gambiae at high magnifica-
tions to highlight the distribution of AgAPN2 (green) relative to the basal membrane
marker Na+/K+-ATPase (red) and the cytoplasmic CA9 (blue). Panels A and B show
the anterior portion of a caecum at the junction with the AMG. Note that AgAPN2 is
prominently localised to the BBM of the anterior GC cells. Panels C and D show the
posterior portion on the same caecum. Note the complete loss of staining for both
Na+/K+-ATPase and AgAPN2 (arrow) at the posterior extreme of the caecum, which is
the CAP cells. The inset in D shows only CA9 staining at the same magnification as
shown in A and B to provide reference. From Linser et al. (2008) and Harvey et al.
(2010) with permission.

higher in the AMG, to pH 8 in the PMG and slightly acidic pH in the rec-
tum are a study in functional cell polarity (Filippova et al., 1998; Zhuang
et al., 1999). To clarify, the major process that drives the up and down
pH gradient is the pumping of protons via a proton pump called
vacuolar-ATPase (V-ATPase; Filippova et al., 1998; Zhuang et al., 1999).
V-ATPase and its roles in epithelial physiology have been studied exhaus-
tively in numerous model systems. For the purpose of our structural discus-
sion here, pharmacological studies have demonstrated a central role in
establishing and maintaining the highly alkaline environment within the
gut. The V-ATPase is actually a macromolecular complex of several proteins
20 Paul J. Linser and Rhoel R. Dinglasan

and can be visualised with TEM as a sub-plasmalemmal knob termed a por-

tasome. Typically, portasomes are associated with closely juxtaposed mito-
chondria and indeed the function of the proton pump is dependent on a
substantial supply of ATP. Figure 1.4 shows TEMs of GC cells from larval
Aedes aegypti. The two principal cell types of the GC are shown and the high-
magnification inset reveals the studded plasma membrane of microvilli in
which the central cytoplasmic domain of the microvillus is dominated by
a mitochondrion (Meredith and Phillips, 1973; Volkman and Peters,
1989a; Zhuang et al., 1999). The internal pH of the GC and that of the adja-
cent AMG region is neutral to slightly alkaline. However, as the point of
reference moves posteriorly in the AMG, the luminal pH rapidly rises to
1011. The several log units of decrease in luminal proton concentration
is accompanied by a shift in epithelial cell polarity, at least in reference to
the position of the proton pumping V-ATPase. In the GC where the pH
is near neutrality, the proton pump is situated on the apical side of the epi-
thelial cell and hence is moving protons into the lumen. In the AMG, the
proton pump is now localised to the basal side of the epithelial cells and
hence involved in pulling protons out of the lumen in a transcellular path-
way (Filippova et al., 1998; Zhuang et al., 1999). At the region of the PMG,
the disposition of the V-ATPase reverses again such that the apical microvilli
of the PMG cells possess high levels of V-ATPase in contrast to the basal
plasma lemma, which shows little or none. This shifting pattern of
V-ATPase (portasome) localisation from apical to basal and then back again
in the course from anterior to posterior midgut has been verified both by
immunochemical approaches (Fig. 1.9) as well as TEM (Filippova et al.,
1998; Zhuang et al., 1999). In stark contrast to the V-ATPase paradigm,
sodiumpotassium ATPase (NaK-ATPase), which as the name implies
pumps sodium and potassium (exchange) is expressed in opposite polarity
to V-ATPase (Patrick et al., 2006). That is, in the GC NaK-ATPase is
located on the basal side of most GC cells, in the AMG it is apical and in
the PMG it returns to basal. The opposite cell polarity regarding these
two ATPases is common in other areas of the gut but not universal
(Patrick et al., 2006; Smith et al., 2007). Physiological modelling of the sys-
tem has incorporated these important ion regulatory membrane proteins as
well as numerous others in recent years. This complexity highlights the
regional specialisation of the plasma membrane and such information is use-
ful in developing novel targeting strategies for insect control.
The region of the gut epithelium where the cells undergo the remarkable
flip-flop in functional polarisation of the plasma membrane has been termed
Insect Gut Structure, Function, Development and Target of Biological Toxins 21

Figure 1.9 Figure demonstrates the apical to basal back to apical transitions in the
polarised distribution of V-ATPase in the Aedes aegypti larval midgut. Panels AC show
regions of the GC, AMG and PMG, respectively. Green/yellow labelling shows the
localisation of V-ATPase on the apical BBM of the GC (A) and PMG (C) and on the basal
membrane of the AMG (B). Red labelling identifies the basal side of the epithelium in all
cases as it depicts the external layer of muscle labelled with TRITC-conjugated Phalloidin.
Blue is DAPI staining of cell nuclei. Panels DF show the same series of gut regions at
higher magnification. From Zhuang et al. (1999) with permission.

the transitional region (TR; Clark et al., 2005; Smith et al., 2007). The cells
of the TR exhibit a graded shift in several parameters including the numbers
and size of microvilli, susceptibility to Cry4Ba toxin damage, the patterning
of cell nuclei, and the flip-flop polarised location of the V-ATPase and NaK-
ATPase (Clark et al., 2005; Smith et al., 2007). Figure 1.10 shows an analysis
of this region of the Anopheles gambiae larval midgut with immunohisto-
chemical markers for NaK-ATPase. Panel C provides a view of the shift
in functional polarity with NaK-ATPase on the basal membrane infoldings
in the PMG (to the right) and the shift of this protein to the apical side in the
AMG cells (to the left; Smith et al., 2007). Also shown in this figure is the
distribution of another important enzyme in the regulation of gut and cel-
lular pH, carbonic anhydrase 9 (CA9) which is 1 of 12 mosquito carbonic
anhydrase genes/proteins. This pH regulator is expressed by cells of the GC
and the rectum as a cytoplasmic protein but is also secreted by the GC cells
22 Paul J. Linser and Rhoel R. Dinglasan

Figure 1.10 Figure shows the entire length of the larval Anopheles gambiae alimentary
canal from the cardia at the extreme to the rectum at far right in panel A. CA9 is labelled
in green and Na+/K+-ATPase in red. The arrow in (A) indicates the transition region at
which Na+/K+-ATPase shifts from basal in the AMG to apical in the PMG. Panels BD
show the transition region at higher magnification. CA9 is expressed by several cell
types but is secreted by GC cells into the ectoperitrophic space. In the transition region,
CA9 is also found in cell nuclei (arrows, B and D). Panel C isolates the Na+/K+-ATPase
signal and thus reveals the shift in epithelial cell polarity from basal (PMG, solid arrow)
to apical (AMG, hollow arrow) of this very important physiological function. Panel
D overlays the red and green images with a blue (DRAQ5, DNA) signal. From Smith
et al. (2007) with permission.

into the ectoperitrophic space and hence part of the digestive milieu and a
component that will be encountered by ingested microbiota and toxins.
The PMG of larval mosquitoes is the largest region of the alimentary
canal in terms of cell surface exposure to ingested materials. The apical brush
border of the principal PMG cells is massive and difficult to estimate in terms
of gross quantity. Its extensive nature has made it possible to engineer rather
Insect Gut Structure, Function, Development and Target of Biological Toxins 23

simple methodologies for isolating highly enriched membrane vesicle prep-

arations termed the BBMV (brush border membrane vesicle; Harvey et al.,
2009). This preparation has facilitated numerous analyses of the biochemical
constituents of the apical plasma membrane from several perspectives
including the purification of toxin receptor proteins (Zhang et al., 2008).
Proteomic analyses have provided numerous potential targets for novel con-
trols. Among proteins of the brush border membrane (BBM) are nutrient
amino acid transporters, cadherins, alkaline phosphatase, sodiumproton
antiporters and of course V-ATPase (Harvey et al., 2009 and elsewhere
in this volume). The structure/function of the BBM of the PMG is yet
to be explored to its fullest but to date many opportunities for physiological
targeting or membrane binding of disruptive molecules has been realised
with undoubtedly more to come.
The MTs of the insect alimentary canal are unique in structure and func-
tion and have received a great deal of attention over many years (Fig. 1.11).
MTs represent a major component of the insects machinery for regulating
ionic homeostasis and have been loosely equated to the vertebrate kidney
(Beyenbach, 2003; Dow, 2009). As mentioned earlier, there are five MTs
extending from the HG. The odd number of biological structures in a
bilaterian is in itself unusual. Additionally, the MTs are the exception to
the rule that the alimentary canal is completely regenerated in the transition
from larval to adult imago. The MTs of the adult are essentially the same cells
as were present in the fourth instar larva. There are two main cell types in the
MTs: the principal cells and the stellate cells. The principal cells make up the
vast majority of the substance of the MTs and are large cells with large poly-
tene nuclei. The tubular structure of the MTs is characterised by an irregular
lumen that has a zigzag space defined by the apical microvillar arrays of the
principal cells. The BBM of the principal cells is characterised by microvilli
filled with mitochondria and plasmalemmal V-ATPase (Harvey et al., 2009;
Patrick et al., 2006). Principal cells also contain large and variable numbers of
membrane bound inclusions that contain organometallo concretions made
up of complexed metal cations (Bradley and Snyder, 1989). Numerous
transport proteins including the bicarbonate transporter NDAE1 are
expressed on the basal/lateral membranes of principal cells. From the prox-
imal to the distal extreme of a MT, the distribution of principal cell transport
proteins and other markers is not uniform. A gradient of expression patterns
has been described indicating that MTs of mosquitoes as well as other insects
are regionally specialised along the long axis of each MT (Dow, 2009;
Rheault et al., 2007). Stellate cells are far less numerous (20% of the total
24 Paul J. Linser and Rhoel R. Dinglasan

Figure 1.11 Figure reveals several details of MT cell biology in the hindgut region of
Anopheles gambiae larva sections. Panel A shows immunolabelling for the bicarbonate
transporter AgNDAE1 (red), V-ATPase (green) and CA9 (blue). The MT is characterised by
basal membrane staining of the principal cells for AgNDAE1 and internal and apical
staining for V-ATPase. Panel A also shows a partial view of the rectum and that CA9
is characteristic of the DAR cells whereas V-ATPase is also present in and on the non-
DAR cells. Panels B and C show a high magnification view of a cross section through
an MT displaying the basal labelling for AgNDAE1 and the apical labelling for
V-ATPase. The arrow in (A) indicates a stellate cell which shows little signal in this label-
ling combination. Panels DH show high magnification views of MT whole mounts
labelled for proton antiporter NHA2 (red), Na+/K+-ATPase (blue) and Griffonia
simplicifolia I lectin (green) and in D&H, DAPI for nuclei (aqua). NHA2, Na+/K+-ATPase
and GSL-I all label the stellate cells of the MT. In the merge images G and H, the polarity
of the markers is revealed. NHA2 and GSL1 co-localise (long arrow in H) to the apical
aspect of the stellate cell whereas Na+/K+-ATPase is basal. Figures AC from Linser
et al. (2012) and DH from Xiang et al. (2012) with permission.
Insect Gut Structure, Function, Development and Target of Biological Toxins 25

number of cells) and much smaller than principal cells. Stellate cells are first
evident amongst the principal cells after the first (proximal to the hindgut)
10% of the length of each MT. Like the principal cells, stellate cells exhibit
specific patterns of plasma lemmal proteins on both the apical and basal/
lateral membranes (Beyenbach, 2003; Linser et al., 2012; Xiang et al.,
2012). Models for the physiological roles of both principal cells and stellate
cells have been proposed and supported by many varied studies (Beyenbach,
2003; Linser et al., 2012; Xiang et al., 2012). An interesting and controver-
sial characteristic of MTs is that a substantial series of investigations supports
the movement of certain ions such as Cl between cells, which indicates a
certain specialisation of cellcell junctions that can provide a regulated peri-
cellular pathway (Beyenbach, 2003). In general, MTs monitor the makeup
of the hemolymph and actively produce the primary urine, providing one of
the key homeostatic functions within the larva.
The final component of the larval alimentary canal to be discussed herein
is the rectum. As mentioned earlier, the rectum is contiguous with the ante-
rior components of the alimentary canal by the ileum or posterior intestine.
The rectum is a major regulator of excretion, elimination and retention of
solutes critical for homeostasis. Mosquito larvae have adapted to many
aquatic environments that can range in ionic strength from very low salinity
fresh water to salt concentrations that exceed that of sea water (Smith et al.,
2010; White et al., 2013). One rather obvious structural variation that cor-
relates with salt tolerance is the structure of the rectum. Figure 1.12 shows
the three major generalised structures of rectum from the simple bulb made
up of seemingly a single type of cell found in culicine mosquitoes with low
salt tolerance, to the bi-lobed rectum of salt-tolerant culicines to the region-
ally specialised two-cell type form in anopheline mosquito larvae (Smith
et al., 2007, 2008). The salt tolerance of mosquito larvae has been investi-
gated extensively and species that are capable of short term adaptation and
those that have very narrow ranges of salinity are well documented in the
classical literature (e.g. Bradley, 1987; Clements, 1992). Recent investiga-
tions have also shown that certain species of anopheline mosquitoes exhibit
dynamic structural changes in the rectum when placed into changing con-
ditions of salinity (Smith et al., 2008; White et al., 2013). It appears that the
balance of functionalities compartmentalised within the two rectum cell
types, DAR and non-DAR can change in response to ionic fluctuations.
An interpretation of this dynamic is that the associations between transport
mechanisms compartmentalised within the cell types can be modified to
produce different potentials for vectorial and linked ion transport (Smith
26 Paul J. Linser and Rhoel R. Dinglasan

Figure 1.12 Figure depicts the gross structure of the three types of larval mosquito rec-
tum as seen in fresh-water culicenes, salt-tolerant culicenes and all anophelines. Fresh-
water culicenes such as Aedes aegypti have a uniform rectum with one primary cell type
as seen in this whole mount immunolabelled for basal NaK-ATPase (left). Salt-tolerant
culicenes such as Ochlerotatus taeniorhynchus have a rectum with distinct anterior and
posterior regions. Anophelines possess a rectum that is distinguished by an anterior
patch of distinct cells termed dorsal anterior rectum (DAR) cells and a posterior majority
of non-DAR cells. AR, anterior rectum; PR, posterior rectum. Image modified from Smith
et al. (2008).

et al., 2008; White et al., 2013). Another aspect of rectum structure/func-

tion is that the PM that protects the midgut from microbial attack loses
integrity in the hindgut. The cuticular lining of the rectum is very intimately
associated with the epithelial cells and the gross structure of the rectum cre-
ates channels and crypts between folds. These pockets are typically teaming
with bacteria in very close association with the rectum cuticle. The micro-
bial flora that is resident in the insect gut has become an increasingly atten-
tion getting topic and may hold details of the biological balance in insect
systems as it does in other complex metazoans (e.g. Engel and Moran, 2013).
The entire alimentary canal of the mosquito larva is surrounded by mus-
cles in several forms from dense sheaths to glass-sponge-like baskets and net-
works. Figure 1.13 is representative of the circular and longitudinal
musculature that produces waves of contraction in both anterior to posterior
and posterior to anterior directions (Seron et al., 2004; Terra, 1990) The
muscle contractions are influenced by neurochemical activity and involved
in some aspects of food digestion (Krajniak, 2005). Although the anterior to
posterior movement of the food bolus is largely driven by ingestion at one
end and defecation at the other (Terra, 1990), the contraction of the various
muscular investment of the gut plays several roles including the mixing of
Insect Gut Structure, Function, Development and Target of Biological Toxins 27

Figure 1.13 Figure shows whole mount preparations of isolated midgut from fourth
instar larvae of Aedes aegypti (AC) and Anopheles gambiae (DF) immunolabelled for
the peripheral membrane protein carbonic anhydrase CA-10 (green) and Phalloidin to
reveal muscle (red). The arrows in (A) and the merged images show that a specific subset
of the gut musculature labels for CA-10 whereas other muscles of the gut do not. Thus the
musculature of the gut exhibits more structure/function variability than simple longitudi-
nal versus circumferential. From Seron et al. (2004) with permission.

components in the ectoperitrophic space and the GC compartment (Smith

et al., 2008; White et al., 2013).
A final note on the mosquito larval gut epithelium; the epithelium in
adult mosquitoes, by an unknown process, is either porous to or transports
across whole protein molecules such as ingested vertebrate immunoglobu-
lins (Beyenbach et al., 2009; Jeffers and Roe, 2008). Also, as noted above in
the discussion on MTs, there is an apparent pathway between epithelial cells
through which small molecules are selectively passed. Therefore, it seems
possible that some molecules such as toxins that can pass through the type
II PM of larvae might gain rapid access to the hemolymph and thus the basal
aspect of the epithelial cells.

3. OTHER INSECTS
The general architecture of the alimentary canal of insects varies con-
siderably depending on adaptive specialisations. A useful summary is
28 Paul J. Linser and Rhoel R. Dinglasan

presented by Engel and Moran (2013). The embryonic development of the

insect gut has numerous common features and in both larvae of holometab-
olous insects and in adults of the same and nymphs and adults of hemime-
tabolous insects the macroscopic features may look very different but the
same derivation of foregut and hindgut from ectoderm and the midgut from
endoderm is the rule. Compartmentalisation of digestion, pH regulation,
ion balance and the flow and recycling of water varies considerably and evo-
lutionary opportunities have resulted in great diversity. The hypothetical
pressures that have driven the evolutionary flow of compartmentalisation
of function have been reviewed several times (e.g. Terra et al., 1996). Three
groups of insects with agricultural impact that we will discuss are the lepi-
dopterans (larvae), coleopterans (beetles and their larvae) and hemipterans
(aphids).

3.1. Lepidopteran larvae (caterpillars)

Larvae of butterflies and moths have long been challenging pests to agricul-
ture. Due to this fact and the additional fact that many are large and readily
manipulated for biological research has led to a very substantial body of
research for well over 100 years. Most texts on insect biology and physiology
present caterpillars such as the larvae of the tobacco horn worm (Manduca
sexta), and the silk worm (Bombyx mori), as favoured model systems. The
gross architecture of lepidopteran larvae alimentary canal has of course
the same general layout for most insects: foregut, midgut and, hindgut.
Prominent diverticuli (gastric caeca) are absent and the midgut is by a large
margin the most substantial part of the system. Individual caterpillars can
yield gram quantities of dissected midgut tissue. The midgut is a pleated tube
with cellular distinctions between the anterior, middle and posterior midgut
(e.g. Cioffi, 1979; Fig. 1.14 [Dow, 1992]). The tube also contains a type II
PM. Perhaps the most distinguishing characteristic of the lepidopteran larval
midgut occurs at the cellular level with the existence of a specialised cell type
known as a goblet cell (Dow, 1992; Harvey et al., 1983). Figure 1.14 also
shows the gross cell biology of the lepidopteran gut epithelium. There
are two principal cell types thought to be involved in digestion, ion balance,
pH modulation and water flow. The first is a typical columnar epithelial cell
with a very extensive apical microvillar array or BBM. These cells also
exhibit varying degrees of basal membrane expansion, a large polyploid
nucleus and varying vesicular content. The columnar cells are structurally
similar to insect gut epithelial cells in general. In contrast, interspersed
Insect Gut Structure, Function, Development and Target of Biological Toxins 29

Figure 1.14 Structure of the lepidopteran midgut. Regional specialisation into three
zones is visible at both coarse and progressively finer resolution. Approximate magni-
fications are shown. At 500 , the two forms of goblet cell are evident in the regions of
the gut epithelium. Taken from Dow (1992) with permission.

among the columnar cells are the very distinct goblet cells. These cells are so
named because of their architecture, which resembles a goblet with a prom-
inent internal cavity. Goblet cells of the AMG are somewhat different from
those of the PMG in that in the AMG goblet cells the goblet cavity is prox-
imal to the basal aspect of the cell whereas in the PMG goblet cells the cavity
is adjacent to the apical extreme of the cell (Fig. 1.14). The inner surface of
the goblet cell plasma membrane adjacent to the cavity is studded with
potasomes as described earlier in the mosquito system. The lepidopteran lar-
val gut, due to its large size and its tractability as an experimental system,
made it possible for numerous investigators to track down the nature of
the portasome and to demonstrate the role that the V-ATPase plays in insect
epithelial physiology (Harvey et al., 1983; Klein et al., 1991) and as a target
for novel control strategies (Baum Chapter 5 this volume). The activity of
this cell-surface proton pump is the driving force of another striking char-
acteristic of the lepidopteran gut: a luminal pH that can be as high as 12 and
transepithelial potential of nearly 240 mV (Harvey et al., 1983; Wieczorek,
30 Paul J. Linser and Rhoel R. Dinglasan

1992). These are physiological extremes found nowhere else in nature. Cer-
tainly part of capacity for lepidopterans to generate these remarkable phys-
iological conditions is contributed by the unique structure/function of the
epithelium and in particular the goblet cells (Harvey et al., 1983;
Wieczorek, 1992).
Lepidopterans do possess MTs although in the vicinity of the rectum
they differ from mosquitoes. The distal ends of the MTs in lepidopterans
(and coleopterans) are embedded in an extracellular matrix that holds them
in a fixed juxtapositional relationship with the basal surface of the rectum
(Azuma et al., 2012; Fermino et al., 2010; Ramsay, 1976). This association
between the tissues is called the cryptonephric rectal complex (Azuma et al.,
2012; Fermino et al., 2010; Ramsay, 1976). Most other insects have free dis-
tal ends that can actually be moved about the hemocoel by muscular con-
tractions in the body wall. The physiological details of this fixed association
with the rectum are poorly understood but is the subject of numerous inves-
tigations (Azuma et al., 2012; Fermino et al., 2010; Ramsay, 1976).

3.2. Coleopterans (beetles and their larvae)

As the most successful group of insects in terms of the numbers of species,
coleopterans have physical and physiological adaptations for uncounted eco-
logical niches and dietary specificities. Both larvae and adults can be agricul-
tural pests and so control strategies can target the life stages differentially. The
alimentary canal of coleopterans possesses the same basic elements as do
other insects: foregut, midgut, hindgut with the same embryological origins
(ectoderm, endoderm, ectoderm, respectively). The structural details vary
greatly depending on food source and digestive strategies with tremendous
variability in the presence, absence and/or nature of gastric caeca the num-
bers and extent of MTs and the relative size of the hindgut. Some exhibit
alkaline extremes in regions of the gut and others do not (Terra et al.,
1996). Some produce Type I peritrophic matices even in the larval stages
(Ryerse et al., 1994).We will limit most of our comments to one species
of very significant agricultural importance, the corn rootworm (Diabrotica
sp.) The larvae of the western corn rootworm (Dibrotica virgifera virgifera)
are of worldwide importance due to feeding damage to corn roots and
the resulting losses in corn yield (Chu et al., 2013; Sayed et al., 2007; also
refer to Chapters 4 and 5). Larvae feed on the roots of the corn plant whereas
the adults feed on the reproductive components of the flowering plant.
Many species of pestiferous beetles exhibit the capacity for rapid
Insect Gut Structure, Function, Development and Target of Biological Toxins 31

development of resistance to conventional pesticides as has been true for

Dibrotica (Al-Deeb and Wilde, 2005; see also Chapter 6). Hence, control
strategies are constantly evolving. A common strategy in use today is depen-
dent on genetically modified crops, such as corn, which produce specific
bacteriological toxins discussed in great length in Chapters 4 and 6. The corn
rootworm is sensitive to certain Cry toxins of Bacillus thuringiensis (Bt) as dis-
cussed in Chapter 4. In 2003, genetically modified corn (maize) which pro-
duces Bt toxins was commercialised in the United States (EPA, 2003) for the
primary purpose of controlling the western corn rootworm. The general
pathobiology of Bt in a variety of susceptible insects will be discussed in
Chapter 2 but suffice it to say here that the conditions in the gut of Dibrotica
v.v. are appropriate for the cell-lytic action of Cry3Bb1. Ingestion of such
genetically modified (GM) maize results in intoxication and death of
Dibrotica v.v. The widespread use of Bt-maize has been successful in terms
of reducing crop loss. But the selective forces brought in to play by the
use of this and other toxic strategies have begun to create resistant
rootworms (Frank et al., 2011; Petzold-Maxwell et al., 2012; Chapter 6).
This has necessitated crop rotation and pesticide co-applications to support
the continued utility of these GM crops. Additionally, the use of GM crops
that can reduce the need for pesticides has generated a general reduction in
such pesticide use. This in itself has produced circumstances in which other
pestiferous insects that are not sensitive to the engineered GM toxins have
been able to flourish and create new challenges.

3.3. Hemipterans (aphids)

Aphids are hemimetabolous insects. Therefore, they do not produce larval
stages but rather nymphs that closely resemble the adult form. Aphids rarely
reproduce sexually but rather undergo parthenogenic production of off-
spring by the females. Aphids feed on plant fluids typically by virtue of pierc-
ing mouth parts (proboscis with stylets) that enter the plant and suck phloem
fluids from the plant (Pelton, 1938). The sugar-rich sap of the plant is very
low in other essential nutrients for the aphid including amino acids and so it
is necessary for aphids to intake large quantities of fluid and then excrete the
excess sugary material in a fluid called honey dew. This sugary fluid on the
surface of the plant can be a source of food for other insects or microbial
plant pests (Dedryver et al., 2010). Aphids as agricultural pests have become
a problem of greater proportions in recent years due to the various events
that have reduced the use of chemical pesticides such as the use of GM crops,
32 Paul J. Linser and Rhoel R. Dinglasan

which in themselves do not impact aphid fluid-based feeding (Chougule and

Bonning, 2012). Due to the feeding strategy of aphids, they have the poten-
tial to transmit other pathogens from one plant to another as they feed. Sim-
ilar to the potential disease transmission that has been realised in
hematophagus insects, sap-sucking aphids can transmit numerous viral dis-
eases of plants (Brault et al., 2010; Ng and Perry, 2004). In some instances, a
dirty proboscis and stylets is adequate to move a pathogen from one plant
to another (non-circulative transmission). In many viral pathologies, the
transmission event requires intermediate stages within the aphid (circulative
transmission; reviewed in Bragard et al., 2013). In this form of transmission,
the alimentary canal of the aphid plays a key role. As the fluid meal is taken
into the gut, the virus must eventually exit the gut, enter the hemoceol,
make its way to the SGs and then make its way into the saliva. This is para-
lleled by the processes of pathogen transmission in hematophagus insects. In
some cases, the virus particles have evolved specific methods of access
through the various cellular barriers in the path to the saliva that do not
require actual viral replication within cells of the aphid (Bragard et al.,
2013). Other virus/aphid relationships do in fact involve replication of
the virus within the cells of the gut and eventual release into the hemoceol
(Bragard et al., 2013). Some plant rhabdoviruses can be persistent in the
aphid for life and can even be transmitted vertically to offspring
(Hogenhout et al., 2008). The expansion of aphids as agricultural pests par-
ticularly for GM crops expressing Bt toxins has lead to efforts to engineer
into the Bt transgene, peptide motifs that will render the toxins effective
on specific aphids (Chougule and Bonning, 2012; Chougule et al., 2012).
For Cyt toxins to be effective in any insect, there needs to be a specific bind-
ing interaction between the toxin and the BBM of insect gut epithelial cells.
Recent manipulation of the Cyt2Aa toxin amino acid sequence in which a
binding peptide was added seems to hold promise (Chougule and Bonning,
2012; Chougule et al., 2012).

4. CONCLUSIONS AND COMMENT

The insect alimentary canal is structurally complex and varied from
species to species. As a target for developing arthropod control strategies,
the gut is both an important and major barrier for insect control agents
and hence an important target for intervention. The specific cell biology
of the gut also provides novel targets for the development of gut-function-
disrupting agents. With the recent and ongoing expansion of technological
Insect Gut Structure, Function, Development and Target of Biological Toxins 33

approaches to defining fundamental biological structure/function relation-

ships, it is ever more possible to identify unique but vital functional molec-
ular target sites. The era of omic biology in which we are currently
immersed provides the investigator with unprecedented opportunities to
discover and integrate knowledge of fundamental biology. Greater under-
standing of the cellular, biochemical and molecular structure and processes
of insect gut will help develop rational strategies for intervening with target
sites important for the survival of the pest stage, as well as exploiting existing
macromolecules for delivering toxins, which may otherwise may not be
effective.

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 CHAPTER TWO

Diversity of Bacillus thuringiensis

Crystal Toxins and Mechanism
of Action
Michael J. Adang*,, Neil Crickmore{, Juan Luis Jurat-Fuentes}
*Department of Entomology, University of Georgia, Athens, Georgia, USA

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA
{
School of Life Sciences, University of Sussex, Falmer, Brighton, United Kingdom
}
Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, Tennessee, USA

Contents
1. Introduction 40
2. General Characteristics of B. thuringiensis Crystal Toxins 42
2.1 Definition and classification of crystal toxins 42
2.2 The diversity of Cry toxins 43
2.3 The parasporin toxins 47
2.4 The ricin domain 48
2.5 Toxin discovery 49
3. Cry Toxin Structure: Function 49
3.1 Overview of Cry structure 49
3.2 Cry domain I 50
3.3 Cry domain II 51
3.4 Cry domain III 53
3.5 Cry intoxication process 55
3.6 Cry toxin solubilization and proteolytic processing 56
4. Midgut Cry-Binding Proteins and Receptor Function 57
4.1 Aminopeptidase 58
4.2 Cadherin 59
4.3 Alkaline phosphatase 60
4.4 ABC transporter 61
4.5 Other Cry-binding (receptor) proteins and molecules 62
5. Models of Cry Toxin Action 63
6. Cytolytic Toxins 68
Acknowledgements 70
References 70

Advances in Insect Physiology, Volume 47 # 2014 Elsevier Ltd 39

ISSN 0065-2806 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800197-4.00002-6
40 Michael J. Adang et al.

Abstract
Parasporal crystals produced by Bacillus thuringiensis (Bt) bacteria are the main virulence
factors underlying Bt toxicity to insects. Parasporal crystals are composed primarily of Cry
and Cyt proteins that act on the midgut of susceptible insects. Cry proteins are an
important component of Bt biopesticides and are vital tools for insect control via expres-
sion in transgenic crop plants. Some members of the Cry group are more distantly
related including ETX/MTX and binary type toxins. Cry toxin structure and action
involves critical steps in toxin activation, binding to receptors such as cadherin and then
aminopeptidase or alkaline phosphatase probably in a sequential binding manner.
Specific Cry toxinreceptor interactions are a focus of this review. Recently, the impor-
tance of midgut ATP-binding cassette proteins to Cry intoxication of insects has been
demonstrated. Mechanistic details involved in sequential binding and pore formation
models are examined. The Cyt toxin of Bt subspecies israelensis is an important and inter-
esting component in Crymidgut interactions in mosquitoes. For some Cry toxins, Cyt
serves as a receptor for docking to midgut membrane. Recent engineering work has
demonstrated that Cyt can be re-targeted generating novel toxins for insect control.
Overall, we review the remarkable progress made in the past 20 years in discovering
novel Cry toxins and in elucidating complex mechanisms of Cry and Cyt toxin action;
subjects relevant to the long-term control of insects that damage crops and vector
human disease.

1. INTRODUCTION
The Gram-positive bacterium Bacillus thuringiensis (Bt) is characterized
by the proteinaceous crystals that it synthesizes in the mother cell during
sporulation (Aronson et al., 1986; Bulla et al., 1980). The history of the dis-
covery and development of Bt has been extensively reviewed (Beegle and
Yamamoto, 1992; Burges, 2001; Jurat-Fuentes and Jackson, 2012). Tens
of thousands of Bt strains have been isolated and most Bt strains are active
against larval stages of insects. Products based on Bt have been registered
as pesticides in the United States since 1961. As with other biological pes-
ticides, Bt offers a number of advantages over synthetic pesticides, including
lack of polluting residues, high specificity to target insects and safety to non-
target organisms. Consequently, the broadest uses of Bt are on food crops
and in forestry where safety and specific action are desirable. Disadvantages
of Bt are its high specificity and low persistence. While Bt is the most suc-
cessful biopesticide for insect control it remains a small part, about 2%, of the
total insecticide market. The widest usage of Bt for insect control is through
Diversity and Mechanisms of Bt Crystal Toxin Action 41

transgenic plants producing Bt insecticidal proteins, particularly corn and

cotton ( James, 2009).
The presence of a parasporal crystal is the phenotypic trait of Bt and is
used to separate this bacterium from other Bacillus species (Vilas-Boas
et al., 2007). The main components of the crystal are the delta-endotoxins,
that act as the primary virulence factor for this pathogen (Raymond et al.,
2010), although other proteins are also present in the crystal and have a role
in toxin and/or crystal structure (Diaz-Mendoza et al., 2012; Staples et al.,
2001). Toxins found in the crystal are classified into two families known as
Cry and Cyt (H ofte and Whiteley, 1989). The Cry (from crystal) toxins
represent a large family currently consisting of around 300 different mem-
bers (Crickmore et al., 2014). The Cyt toxins are characterized by possessing
a general cytolytic activity in vitro, although they show a primarily dipteran-
specific activity in vivo (Soberon et al., 2013a). A third family of protein
toxinsthe Vegetative Insecticidal Proteins (Vips)are not classified as
crystal toxins since they are secreted from vegetatively growing cells rather
than included in the crystal during sporulation (de Maagd et al., 2003).
The characteristics of Bt crystal toxins will be discussed in Section 2
beginning with how Cry toxins are defined, named and classified into
groups. We discuss conserved features of three-domain Cry toxins and
the structurally distinct ETX/MTX and binary-like toxins that have Cry
designations. We briefly describe Vip toxins with respect to their relation-
ship to Cry toxins. Also in Section 2, we introduce parasporins, which typ-
ically have no known insect target, but have toxicity to specific human cell
lines (Mizuki et al., 1999). Recent advances and innovations in toxin discov-
ery are presented in Section 2. Recent reviews of mechanisms of three-
domain Cry toxin action (Pardo-Lopez et al., 2012; Vachon et al., 2012)
have integrated complex events which occur in insect midgut after Cry
protoxin processing and upon contact with the target midgut membrane.
In Section 3, we discuss Cry toxin structure in the context of the respective
role of each structural domain in Cry toxin action. We also discuss the
importance of Cry solubilization from crystals and proteolytic processing
to host specificity. Receptor molecules on insect midgut such as cadherin,
aminopeptidase and alkaline phosphatase (ALP) have been extensively
reviewed (Bravo et al., 2011; Pigott and Ellar, 2007), particularly in the con-
text of models of Cry toxin action. In Section 4, we discuss those receptor
molecules with an emphasis on combinations of Cry toxins and insect
systems where receptor function has been established. We also include in
the discussion evidence demonstrating a critical role in toxin action for
42 Michael J. Adang et al.

ATP-binding cassette (ABC) proteins (Heckel, 2012), and the relevance of

glycolipids and other midgut molecules to toxin action. Models of Cry toxin
action describing events occurring between Cry-binding midgut and
pore formation are extant in recent literature (Bravo et al., 2004, 2011). In
Section 5, we examine some of the mechanistic details involved in the
sequential binding and pore formation models of toxin action. The Cyt
toxin of Bt subsp. israelensis is a major crystal component and is critical to
mosquitocidal toxicity (Ben-Dov, 2014). Since an excellent recent review
summarizes the mechanism of Cyt toxin action (Soberon et al., 2013b),
we will briefly describe Cyt pore formation, its role as a Cry receptor
and recent engineering work that re-targets Cyt toxin resulting in a
novel toxin for pest control (Chougule et al., 2013). An excellent review
on mechanism of Bt Cry toxin action was previously published in
Advances in Insect Physiology (Knowles, 1994) and we focus in this
review to provide an update on the remarkable progress made in the past
20 years on the subject.

2. GENERAL CHARACTERISTICS OF B. THURINGIENSIS

CRYSTAL TOXINS
2.1. Definition and classification of crystal toxins
Cry toxins are officially defined as proteins that have significant sequence
similarity to existing toxins within the nomenclature or be a B. thuringiensis
parasporal inclusion protein that exhibits pesticide activity, or some experi-
mentally verifiable toxic effect, to a target organism (Crickmore et al.,
1998). Naming of toxins is based solely on amino acid sequence identity
and does not take into account their toxicity; thus, toxins that are active against
the same order of insect will not necessarily share any similarity in their names.
A toxins name consists of four levels, e.g., Cry41Ab1, the first number is the
primary level and all toxins sharing this first number (41 in the example above)
will share significant sequence identityat least 45%. Toxins sharing primary,
secondary and tertiary level descriptors will have increasing sequence identity.
Toxins that differ only in the quaternary level descriptor (e.g. Cry41Ab1 and
Cry41Ab2) will have at least 95% sequence identity. A different quaternary
level descriptor is given to all newly characterized toxins, and as a result some
toxins are actually identical to others in the nomenclature, but have been
assigned different names. The definition of a Cry toxin as given above does
allow toxins that are neither produced by B. thuringiensis nor found in the crys-
tal, to be given a Cry name. Examples include Cry16Aa that was cloned from
Diversity and Mechanisms of Bt Crystal Toxin Action 43

Clostridium bifermentans (Barloy et al., 1996) and Cry1Ia that is secreted rather
than being part of a crystal (Varani et al., 2013). In both these cases, the pro-
teins share significant sequence similarity to existing toxins and so are classified
as Cry toxins. For proteins that are found in the crystal, but share only weak
similarity to existing toxins, more evidence is required in order to classify
them. Normally, this would be a demonstration that either the purified toxin
or a recombinant form of the toxin has toxic activity to some target organism
or cell. In the case of a recombinant toxin, the protein would not actually have
to be produced by the parent strain. Indeed commercially important toxins,
such as Cry2Ab, are often encoded by cryptic genes (Crickmore et al., 1994).
The mere presence of a protein in the crystal of a B. thuringiensis strain is not
sufficient for that protein to be classified as a Cry toxin. The same criteria are
used when classifying Cyt toxins.

2.2. The diversity of Cry toxins

There are currently around 75 primary subgroups of Cry toxinsi.e., with
different primary ranks in the nomenclature (Cry1, Cry2, Cry3, etc.).
Figure 2.1 shows a cartoon illustrating the different lengths of these toxins.
Their lengths vary from 369 (Cry34) to 1344 amino acids (Cry43). In their
1989 review, H ofte and Whiteley identified five conserved sequence blocks
in all the Cry toxins, which are also shown in Fig. 2.1. Note that not all of the
toxins contain these blocks and that some only contain a subset of them.
Based on experimentally derived structures, and molecular modelling, it
is believed that the toxins that contain all, or some, of these conserved blocks
are likely to possess the same basic three-domain fold (Pardo-Lopez et al.,
2013). Figure 2.2 provides a more complete list of currently classified toxins,
excluding those that share the same, quaternary rank. Of the 294 toxins in
this list, 262 (89%) are predicted to have the three-domain fold and are
coloured blue in this figure. It is likely that the majority of Cry toxins pro-
duced by Bt will be in this three-domain class, although the proportion of
these toxins in Fig. 2.2 is likely to be inflated due to the fact that many of
them were isolated by PCR techniques based on conserved sequences in
existing toxins. Two other classes of Cry toxins have been previously iden-
tified: the ETX/MTX-like toxins and the Binary (Bin) like ones. There are
11 toxins in the former group (coloured orange in Fig. 2.2) which show
sequence similarity to the Clostridium perfringens epsilon toxin (Bokori-
Brown et al., 2011). The mosquitocidal MTX2 toxin from Lysinibacillus
sphaericus is also related to this class (Berry, 2012). They are structurally dis-
tinct from the three-domain Cry toxins in that they adopt an elongated, and
Figure 2.1 Graphical representation of the diversity of Bt Cry toxins. The length of each
toxin is drawn to scale and the five conserved blocks described in Schnepf et al. (1998)
are shown as coloured inserts.
Figure 2.2 List of Bt toxins. The toxins are grouped according to their primary ranking
within the nomenclature. The background colour represents likely protein tertiary struc-
tural groupings and those toxins named with red text indicate the parasporin class.
46 Michael J. Adang et al.

predominately -sheet-based structure. However, like the three-domain

toxins, they are believed to act via forming pores in the membranes of
the target cells (Bokori-Brown et al., 2011). The Bin-like toxins are so
named since they resemble the two components of the mosquitocidal binary
toxin from L. sphaericus. While, neither the molecular structure nor the
mechanism of action, of these toxins is particularly clear, there is some evi-
dence that they form pores but also indications that one of the components
acts intracellularly (Berry, 2012). There are 13 toxins in this class shown in
Fig. 2.2 (coloured purple), although interestingly none appear to exist as
homologous pairs as found with BinA and BinB from L. sphaericus. Cry49
has a binary partnership with the three-domain toxin Cry48 ( Jones et al.,
2007) for example, whilst Cry35 forms a binary partnership with Cry34
(Schnepf et al., 2005)a toxin that does not fit into any of the three classes
mentioned above but has similarities to the fungal aegerolysins (Berne et al.,
2009). It is not known whether or not the Bin-like Cry36 has a binary part-
ner. Other toxins appear to exist as binary pairs including the ETX/MTX-
like Cry23 and Cry37 (Donovan et al., 2000) despite neither resembling the
Bin toxins. Two further ETX/MTX-like toxinsCry15 (Naimov et al.,
2008) and Cry33 (Kim et al., 2003)are believed to form binary partner-
ships with other proteins, but these partners have not been characterized suf-
ficiently well enough to give them Cry designations. Other Cry toxins do
not fit into these three main classes (three-domain, ETX/MTX and Bin-
like) but like the aegerolysin-like Cry34 mentioned above, do have some
resemblance to other known toxins or proteins. These relationships are
shown in Fig. 2.3 in which the differently coloured filled circles represent
the same groups of toxins identified in Fig. 2.2. The red open circles repre-
sent toxins with a shared characteristic such as the binary toxins discussed
above, with the blue open circles representing related proteins found in
other species. An example of such a relationship is Cry46 which shares sim-
ilarities with the hydralysin (Sher et al., 2005) pore-forming toxins. Simi-
larly, the Vip1, Vip4 and Cry37 toxins show similarity to the 1b
component of the Clostridial iota toxin (Sakurai et al., 2009) with Vip2
resembling the 1a component. Two of the other toxins, Cry6 and Cry22
do not resemble any other known toxin, although they do show sequence
similarity to a structural maintenance of chromosome and a cell wall anchor
protein, respectively. No homologues have been identified for the Vip3 or
Cry55 toxins. The Cyt toxins have a homologous fungal toxin, volva toxin
(Weng et al., 2004).
Diversity and Mechanisms of Bt Crystal Toxin Action 47

Figure 2.3 Venn diagram showing structural or functional relationships between toxins.
The individually coloured circles represent the different structural groups identified
in Fig. 2.2. Overlapping open blue circles indicate sequence similarity with non-Bt toxins
or proteins. The open red circles indicate which of the structural groups contain mem-
bers that share that particular characteristic namely binary toxin, parasporin or ricin
domain containing.

2.3. The parasporin toxins

In an attempt to understand, why so many Bt strains exist without a known
insect target, a large screen was set up in the mid-1990s to look for activ-
ities against non-insect species. From this screen came a number of toxins
which were non-haemolytic but had activity against one or more human
cells (Mizuki et al., 1999). Many of these toxins were specifically active
against cancer cell lines. Currently, there are six classes of toxin that have
this activity; whilst they have been separately termed Parasporins they have
also been allocated Cry toxin names (Akiba et al., 2009). In Fig. 2.1, the
Parasporin toxins have been highlighted in red text. It can be seen from this
figure that Cry31 (Parasporin 1), Cry41 (Parasporin 3) and Cry63
(Parasporin 6) are all three-domain toxins, whilst Cry45 (Parasporin 4)
and Cry 64 (Parasporin 5) are both ETX/MTX-type toxins. The final
48 Michael J. Adang et al.

toxin in this group Cry46 (Parasporin 2), as mentioned above, does not
resemble any other Cry toxin but does have some similarity to hydralysin.
Although Parasporin 1 appears to be a typical three-domain toxin, its
mechanism of action is believed to be more complex than simple pore for-
mation (Katayama et al., 2007). In particular, the toxin induces an increase
in intracellular Ca2+ which is then associated with apoptosis as assessed by
increases in Caspase-3 and PARP cleavage. Less is known about the two
other three-domain toxins (Parasporins 3 and 6), although the cell swelling
observed after toxin administration is consistent with pore formation
(Nagamatsu et al., 2010; Yamashita et al., 2005). Parasporin 2 (Cry46) does
not share significant sequence similarity with other known Cry toxins but
its structure (Akiba et al., 2009) suggests that it ought to belong to the
ETX/MTX family. Its mechanism of action is believed to be through pore
formation after the toxin is targeted to lipid rafts on the cell surface where it
interacts with glycosylphosphatidyl inositol (GPI)-anchored proteins
(Kitada et al., 2009). Parasporin 4 appears to act as a cholesterol-
independent -pore-forming toxin that does not induce apoptosis
(Okumura et al., 2011). Nothing is currently known about the mechanism
of action of Parasporin 5.

2.4. The ricin domain

Figure 2.3 indicates that a number of toxins contain the so-called ricin
domain (pfam00652, conserved domain 00161), a beta-trefoil-putative car-
bohydrate-binding, domain presumed to have arisen through gene triplica-
tion and containing the (Q-X-W)3 conserved motif (Hazes, 1996).
A number of the Parasporin toxins (Cry41A, Cry41B and putative
parasporin Cry42) contain this domain leading to speculation that this
lectin-like feature could be responsible for specific binding, and thus toxic-
ity, to certain human cell lines. However, removal of the ricin domain either
proteolytically or genetically did not appear to affect the activity of at least
one of these Parasporins against human cells (Krishnan, 2013; Yamashita
et al., 2005). Other toxins that contain this domain include Cry35A (Ellis
et al., 2002) and Cyt1Ca (Manasherob et al., 2006), and in neither of these
cases is there any evidence that the domain is involved in toxicity. Despite
the fact that the beta-trefoil domain does have a role to play in the mech-
anism of action of other toxins such as ricin and pierisin (Matsushima-Hibiya
et al., 2003), it has a ubiquitous presence in many genomes and may not
always confer function.
Diversity and Mechanisms of Bt Crystal Toxin Action 49

2.5. Toxin discovery

The discovery of new toxin genes increased dramatically as innovations in
molecular biology, most notably PCR and next-generation sequencing,
became widespread. Originally, discovery started with the characterization
of a toxin followed by the rather lengthy procedure of identifying and cloning
its gene. As more genes were cloned similarities between them could be used
to design probes, and later PCR primers, to pull out new genes (H ofte and
Whiteley, 1989). The emphasis changed from characterizing interesting
toxins to identifying strains with useful activities and then cloning and
expressing putative toxin genes in order to find one with good toxicity. In
recent years, the emphasis has changed once more towards the identification
of novel genes through sequencing either the entire genome of the bacterium,
or just the megaplasmids on which the toxin genes are normally found. To
support this approach, computational pipelines have been developed to iden-
tify putative genes from genomic sequencing data (Ye et al., 2012). Whilst this
mass sequencing approach has many advantages there is a need to be cautious,
the fact that Bt strains often contain multiple copies of similar toxin genes can
cause problems with both contig assembly and with database annotation
(Guan et al., 2012). Whilst genomic sequencing can readily identify putative
toxin genes that are related to known ones, it suffers from the same problem as
previous PCR-based methods in being unable to identify completely novel
toxin genes. One solution to this is to revert back to first analysing the protein
and then finding the gene, but now making use of more advanced proteomic
techniques to identify putative toxins from the Bt crystal (Lee et al., 2006) or
via expression at particular growth phases (Huang et al., 2012).

3. CRY TOXIN STRUCTURE: FUNCTION

3.1. Overview of Cry structure
The majority of Cry toxins in the 73 subgroups have similar predicted three-
domain structures despite differences in primary amino acid sequence and
overall length of protoxin (Pardo-Lopez et al., 2013). The presence of some
of the five conserved blocks (Hofte and Whiteley, 1989) is a constant feature
of the three-domain Cry toxins (Pardo-Lopez et al., 2013). This conclusion is
supported by three-dimensional structures resolved first for Cry3Aa (Li et al.,
1991) by X-ray crystallography and then for Cry1Aa (Grochulski et al., 1995),
Cry2Aa (Morse et al., 2001), Cry3Bb (Galitsky et al., 2001), Cry4Aa
(Boonserm et al., 2006), Cry4Ba (Boonserm et al., 2005), Cry8Ea1 (Guo
50 Michael J. Adang et al.

Figure 2.4 Views of Cry1Aa toxins from the Resource for Structural Bioinformatics Protein
Data Bank (http://www.rcsb.org/pdb/) of (id: 1CIY) (Grochulski et al., 1995) modified using
PyMOL Version 1.7 (http://www.pymol.org/). Domain amino acid sequence position infor-
mation was obtained from NCBI data base (GenBank: AAP40639.1) and (Grochulski et al.,
1995). Overall views of Cry1Aa toxin are shown (Panels A and B). Domain I is red, domain
II is pink and domain III is orange. Domain I with the names of -helices and domain II with
named -helices and loops are shown in Panels C and D, respectively.

et al., 2009a), and nematicidal Cry5B toxin. Figure 2.4 shows views of the
Cry1Aa molecule generated from three-dimensional structure data
(Grochulski et al., 1995). The structure of domain I is a bundle of 78
-helices with a centrally located hydrophobic -helix 5. Based on observa-
tions that most of the -helices of domain I are long enough to span a hydro-
phobic cellular membrane and that domain I has similarities to pore-forming
domains in other bacterial toxins, it was hypothesized and later proven correct
that domain I is involved in membrane insertion and pore formation (Li et al.,
1991). Domain II, a three -sheet structure, is involved in receptor binding,
oligomerization, and membrane insertion. Domain III participates in receptor
binding and possibly membrane insertion.

3.2. Cry domain I

The functional roles of the three Cry structural domains were implied by
the details of Cry toxin structure (Li et al., 1991), comparisons with other bac-
terial toxins and then methodically investigated by functional analyses using
Diversity and Mechanisms of Bt Crystal Toxin Action 51

wild-type and mutated Cry toxins. The importance of domain I -helical

bundles and specific amino acid residues to membrane insertion and pore for-
mation were suggested by mutations such as A92D, R93D and Y153D (Chen
et al., 1995; Wu and Aronson, 1992) in Cry1Ab that caused loss of toxicity
without changing binding to brush border membrane. How Cry toxins
transition from soluble to membrane-bound forms and the nature of the final
form generating pores has been the subject of many functional analyses. Mem-
brane partitioning of domain I involves intramolecular movements and inser-
tion of a hydrophobic hairpin formed by -helices 4 and 5. By restricting
intramolecular movements of domain I helices, and Domains I and II relative
to each other, researchers (Schwartz et al., 1997a) demonstrated that channel
formation requires domain I swinging away from domain II and insertion of
-helices 4 and 5 into the membrane. According to the umbrella model based
on the crystal structure of Cry3Aa (Li et al., 1991) toxin, helices lay on the sur-
face of the cell membrane much like ribs of an umbrella with 45 helices
inserting and forming the membrane pore. The hydrophobicity of the 45
pair, which is consistent with their insertion into membrane, has been com-
pared to the pore-forming colicin toxins. Also, the central 45 helices alone
in an antiparallel conformation have pore-forming ability in membranes (Gazit
et al., 1998). Mutational alterations of 4 and the central 5 helix yield inactive
Cry toxins, while mutations in other helices do not result in significant effects on
insect toxicity (Aronson et al., 1995; Kumar and Aronson, 1999; Wu and
Aronson, 1992). Additionally, mutated 4 residues that impair Cry1Aa pore
formation in planar lipid bilayers (Masson et al., 1999) and in brush border
membrane vesicles from midgut of larval tobacco hornworm, Manduca sexta
(Girard et al., 2009) support a pore-lining function of helix 4. Similar types
of mutations in helix 5 support its participation in channel formation (Florez
et al., 2012). Interestingly, a fortuitous V171C exchange in -helix 5 in Cry1Ab
toxin had positive effects increasing translocation into midgut membranes
and conferring a 25-fold increase in toxicity to the gypsy moth, Lymantria dispar,
albeit a similar increase of toxicity to M. sexta was not observed (Alzate et al.,
2010). Site-directed mutagenesis of residues involved in coiled-coil structures
in helices -3 and 6 of Cry1Ab resulted in mutant toxins capable of binding
but unable to form oligomers or pores, supporting the relevance of these struc-
tures to oligomerization ( Jimenez-Juarez et al., 2007).

3.3. Cry domain II

Domain II consists of three antiparallel -sheets arranged in a triangular
-prism with projecting loop regions 8, loop 1, 2 and 3. Similar -prism
52 Michael J. Adang et al.

folding patterns have been noted in numerous carbohydrate-binding pro-

teins (Sharma et al., 2007); including vitelline from hens egg white and
the plant lectins jacalin (Sankaranarayanan et al., 1996), KM+ (Rosa et al.,
1999), Maclura pomiferan agglutinin (Lee et al., 1989) and hundreds of others
which are referred to as -prism fold lectins (Sharma et al., 2007). With
respect to Cry toxins, the three apical loops at the base of domain II are
thought to be flexible and they vary considerably in length and amino acid
sequence. The domain II loops are involved in recognition of midgut recep-
tors (Pigott and Ellar, 2007; Smedley and Ellar, 1996), irreversible binding
(Rajamohan et al., 1995) and toxicity to specific hosts. In general, toxins
with high sequence similarity in the loops of domain II share at least some
binding sites on midgut brush border (Hernandez and Ferre, 2005; Jurat-
Fuentes and Adang, 2001) and shared binding sites are a factor in cross-
resistance to Cry toxins in pest insect species (Heckel et al., 2007).
The location of Cry toxin domain II loops deduced from solving a Cry
crystal structure (Li et al., 1991), provided researchers critical information
needed for designing experiments to investigate functional properties of
Crymidgut interactions. The following comments on structuralfunctional
analyses will focus on the action of Cry1A toxins in lepidopteran larvae, with
comparisons to Cry toxin action in other target insects. A primary approach
to investigating the role of domain II loops in toxin function has been to
mutagenize loops by deleting or substituting amino acid residues. Synthetic
peptides corresponding to loop regions have also been valuable tools
through their use as competitors to probe toxinmidgut interactions. Studies
of Cry1A toxin action in lepidopteran larvae show that loops 2, 3 and -8
of Cry1A domain II are involved in receptor recognition and toxicity (Lee
et al., 2000, 2001; Lu et al., 1994; Rajamohan et al., 1995, 1996a,b). Loop 1
of Cry1A toxins has not been implicated in receptor recognition to the
extent of other domain II loops. Toxicity assays showed that the G312 res-
idue in Cry1Ac loop 1 is important for toxicity, but relatively non-toxic
mutants are not significantly changed in binding affinity (Smedley and
Ellar, 1996). In contrast, mutations in loop 2 residues of Cry1Ab have been
particularly valuable for investigating toxin interaction with midgut mem-
brane and isolated receptors due to the dominant role of loop 2 residues in
toxin binding and toxicity. For example, mutations in Cry1Ab at Phe371
(deletion of Phe371 or F371A) and at Gly372 (G3724A) reduced toxicity
to M. sexta larvae, yet while the toxins had similar reversible binding,
irreversible binding to midgut brush border membrane vesicles (BBMV)
was reduced (Rajamohan et al., 1995). These results suggested the
Diversity and Mechanisms of Bt Crystal Toxin Action 53

involvement of loop 2 in insertion of toxin into brush border membrane. As

cadherins, aminopeptidase and ALP were identified as Cry1A receptors in
lepidopteran larvae those proteins were integrated into functional analyses
of Cry toxins (Pigott and Ellar, 2007). The -8 and loop 2 residues
of Cry1Ab determine recognition of binding epitopes in cadherin BtR1
of M. sexta (Gomez et al., 2002; Gomez et al., 2003). Similarly, loop 2 of
Cry1Aa is thought to recognize BtR175 cadherin of silkworm, Bombyx mori
(Obata et al., 2009). According to a proposed model (Gomez et al., 2002),
recognition of receptor epitopes by domain II loops involves hydropathic
interactions between residues in the apical loops and epitopes on receptors.
The apical residues of domain II loops have even been replaced with anti-
body complementarity determining regions creating toxins called Cry bod-
ies that have insecticidal activity (Pigott et al., 2008). Recently, a model was
proposed where there are no critical residues needed for functional binding
sites on loops, but rather conformational flexibility of loops involved in
binding provides for optimal toxincadherin receptor interactions and insect
toxicity (Fujii et al., 2013).

3.4. Cry domain III

Domain III of Cry toxins has a complex role in toxin action that is less
defined than the roles of domains I and II. The structure of domain III is
a -sandwich comprised of two antiparallel -sheets compressed into a jelly
roll topology (Li et al., 1991). Each -sheet has five strands and the two long
loops near the end of the -sheets interface with helices 6 and 7 of domain
I (Grochulski et al., 1995). The structure of Cry domain III is similar to the
cellulose-binding domain of Cellulomonas fimi -1,4-glucanase (Burton
et al., 1999). Conserved blocks 3, 4 and 5 of H ofte and Whiteley (1989)
are located in domain III. Block 4, which is composed of four alternating
arginines in the second -strand of the bottom sheet of domain III, is
directed towards the other domains and has a suggested role in toxin stability
(Grochulski et al., 1995) plus more specific functions. Mutations replacing
Block 4 arginines had an effect on toxin-induced ion channel function with
the suggestion that alternating arginines stabilize toxin organization within
membrane (Chen et al., 1993; Masson et al., 2002b; Schwartz et al., 1997c).
Domain III also is involved in receptor binding as revealed by analyses of Cry
toxins mutated at predicted surface-exposed loops connecting -strands and
by analyses of hybrid Cry toxins that have domain III from another toxin.
Aronson et al. (1995) targeted the unique loop region of Cry1Ac by
54 Michael J. Adang et al.

mutagenizing residues S503 and S504 and attributed reduced M. sexta and
tobacco budworm, Heliothis virescens toxicity as being due to reduced initial
binding. How Cry1Ac binds molecules is unique among characterized
toxins, as the loop forms a lectin-like pocket and binds carbohydrates
(Burton et al., 1999; Jenkins et al., 1999). More specifically, this Cry1Ac
loop region participates in recognition of M. sexta midgut and in binding
to N-acetylgalactosamine (GalNAc) attached to aminopeptidase
(de Maagd et al., 1999b). It is also notable that the pattern of Cry1Ac
GalNAc recognition applies to the midgut ALPs of H. virescens and cotton
bollworm, Helicoverpa armigera ( Jurat-Fuentes and Adang, 2004; Sarkar et al.,
2009; Sengupta et al., 2013) and that mutations in the GalNAc binding
region reduced toxicity. Recently, the GalNAc binding pocket on Cry1Ac
was disrupted by amino acid replacements resulting in reduced binding to
monomeric ALP and insecticidal activity. The results of Pardo-Lopez
et al. (2006) indicate that GalNAc binding to Cry1Ac induces a slight con-
formational change that enhances membrane insertion of an oligomeric pre-
pore structure. The relevance of Cry1Ac domain III discussed above to
binding and toxicity is also buttressed by studies showing that domain III
of Cry1Ac mediates specificity in lepidopteran species including L. dispar
(Lee et al., 1995), H. virescens and the cabbage looper, Trichoplusia ni
(Ge et al., 1991). Construction of Cry1Cry1Ac hybrid toxins where
Cry1Ac is attached C-terminal to domains III from another Cry1 toxin
in some cases resulted in Cry toxins more toxic to H. virescens (Karlova
et al., 2005). Studies of Cry1C, an important toxin for its activity against
armyworm, Spodoptera species, identified domain III as a critical determinant
in insecticidal specificity. Similar to the Cry1Ac domain III loop discussed
above, Cry1C has a predicted loop connecting two -strands on the outer
-sheet (Herrero et al., 2004). The importance of this region in Cry1C was
suggested by hybrid toxin G27 (domains I and II from Cry1Ea and domain
III from Cry1Ca) when two residues were substituted and toxicity to
Spodoptera exigua, but not M. sexta larvae was reduced (de Maagd et al.,
1999a). Recently, Herrero et al. (2004) showed that mutation of residues
in the Cry1Ca domain III loop reduced toxicity to beet armyworm,
S. exigua, larvae and binding to BBMV, but did not alter oligomerization.
Transfer of toxicity against Spodoptera species from Cry1C to Cry1Ac was
achieved by transfer of Cry1C domain III (Bosch et al., 1994). A novel
domain III exchange involved the transfer of Cry1Ab domain III onto
Cry3Aa domains III in coleopteran activity that did not compete for bind-
ing with Cry3Aa-like proteins (Walters et al., 2010). This result supports an
Diversity and Mechanisms of Bt Crystal Toxin Action 55

important binding role for the lepidopteran-active Cry1Ab domain III that
is functional in the coleopteran western corn rootworm, Diabrotica virgifera.

3.5. Cry intoxication process

The production of Cry toxins represents a large commitment of resources,
supporting the importance of these proteins for the evolutionary success of
B. thuringiensis as an effective insect pathogen (Raymond et al., 2010). The
conserved three-dimensional structure of most Cry toxins first described for
Cry3Aa (Li et al., 1991) supports a common mode of action, with primary
and secondary Cry protein structures being responsible for specificity
(de Maagd et al., 2001). Figure 2.5 shows details of Cry toxin mode of

Figure 2.5 Representation of the current models of Cry toxin action in the insect midgut
epithelium. After ingestion by susceptible larvae, toxin crystals are solubilized in the mid-
gut fluids to yield the Cry protoxin form, which is processed to an activated Cry toxin form
equivalent to the Cry toxin ingested by insects feeding on transgenic Bt crops. The Cry
toxin core then traverses the peritrophic matrix, which is able to retain some of the toxin
(darker toxin molecules in the figure). Once reaching the brush border membrane of the
midgut epithelium, the Cry toxin binds with high affinity to cadherin, which results in acti-
vation of intracellular cell death pathways (represented by G protein, adenylate cyclase
and cAMP in the figure), and/or according to the sequential binding model, further pro-
teolysis of the toxin monomer to result in formation of a pre-pore oligomer. This pre-pore
oligomer is proposed to bind to alkaline phosphatase (ALP) or aminopeptidase (APN) to
insert in the membrane forming a pore that leads to osmotic cell death. The insertion of
toxin monomer and formation of a pore by oligomerization of inserted monomers is also
presented as an alternative step. Disruption of the midgut epithelium barrier allows for
bacterial invasion of the haemocoel, leading to septicaemia and death of the insect.
56 Michael J. Adang et al.

action. It is well established that insecticidal Cry toxins target enterocytes in

the insect host gut to compromise the gut epithelial barrier, which is key to
facilitate access of B. thuringiensis to the haemocoel. The collapsing integrity
of the gut epithelium during Cry intoxication also results in altered gut phys-
iological conditions, which together with interaction with the midgut brush
border membrane favours germination of the B. thuringiensis spore (Du and
Nickerson, 1996). Ultimately, vegetative B. thuringiensis cells grow and mul-
tiply in the haemolymph ( Johnston and Crickmore, 2009) resulting in
septicaemia and insect death.

3.6. Cry toxin solubilization and proteolytic processing

The first step in the Cry intoxication process after ingestion by a susceptible
host is the solubilization of the crystal by cleavage of interchain disulfide
bonds to release Cry protoxins. Multiple factors affect solubility of Cry
protoxins in the insect host gut, including gut physicochemical conditions
( Jaquet et al., 1987); crystal composition (Aronson et al., 1991); intramolec-
ular processing (Carroll et al., 1997); and in some Cry proteins, the action of
helper proteins (Naimov et al., 2011). The functional association between
protoxin solubilization and host gut physicochemical conditions governs
specificity in some cases, and it has been suggested to depend on the exis-
tence of typical disulfide bonds (Du et al., 1994) and predominant amino
acids (Grochulski et al., 1995) in Cry proteins.
Solubilized protoxin molecules are then processed by endogenous Bt pro-
teases and/or proteases in the host gut fluids to an active toxin core that is
mostly resistant to further proteolysis. Typically, this toxin activation step
is modelled in vitro by digesting Cry protoxins with trypsin or chymotrypsin
(Andrews et al., 1985; Bietlot et al., 1989), the most common proteolytic
enzymes in insect gut fluids (Terra and Ferreira, 1994). However, a number
of reports suggest that Cry toxins activated with digestive enzymes from tar-
get insects may display distinct properties compared to trypsin or
chymotrypsin-activated Cry toxins. For instance, increased toxin pore for-
mation activity compared to activation with trypsin was reported when
Cry3 toxins were activated in the presence of brush border membrane pro-
teins of Colorado potato beetle, Leptinotarsa decemlineata (Rausell et al., 2004),
or when Cry9Ca (Brunet et al., 2010b) or Cry1Ab (Gomez et al., 2014) were
activated with digestive fluids from M. sexta larvae. In another example,
Cry1Ie toxin oligomers formed after activation with trypsin were signifi-
cantly less toxic than the monomeric toxin form (Guo et al., 2009b), which
would be expected to oligomerize in the insect gut environment during the
Diversity and Mechanisms of Bt Crystal Toxin Action 57

bioassays. The mechanisms explaining these differences are largely unknown,

although a role for insect protease inhibitors in regulating Cry toxin activa-
tion (Brunet et al., 2010b) and insect lipids in promoting toxin oligomeriza-
tion (Ma et al., 2012) have been proposed. Importantly, these observations
may suggest that biologically relevant information may only be obtained
when using Cry toxin activation with insect digestive fluids.
During the activation process, most well-studied Cry protoxins are
sequentially digested to a 65- to 55-kDa toxin core by removal of about
500 and 43 amino acids from the C- and N-termini, respectively. In other
cases, like the mosquitocidal Cry4A and Cry11Aa and the scarab-specific
Cry8Da toxins, activation generates two peptide fragments that remain asso-
ciated to form a toxin complex (Yamagiwa et al., 1999; Yamaguchi et al.,
2010). The processing at the N-terminus has been shown to be crucial to
subsequent steps in the Cry intoxication process (Bravo et al., 2002) and
is the only region processed during activation of the smaller (6775 kDa)
Cry protoxins. Interestingly, in most Cry protoxins, this N-terminus is
tightly associated to 20-kilobase (kb) DNA fragments (Bietlot et al.,
1993) that may contain cry toxin genes (Xia et al., 2005). These DNA frag-
ments not only appear to dictate sequential proteolysis of the Cry protoxin
(Clairmont et al., 1998) but may also have a critical role in toxin binding
specificity and membrane insertion (Ai et al., 2013; Guo et al., 2011).
The rate of Cry protoxin processing is important to determine suscep-
tibility, as supported by enhanced activity in engineered Cry toxins with
increased activation rates compared to wild-type toxins (Walters et al.,
2008). Moreover, resistance to diverse Cry toxins has been associated with
altered toxin processing or reduced rate of activation in a number of insects
(Ferre and Van Rie, 2002). In this regard, endogenous Bt proteases may con-
tribute to accelerate protoxin processing in vivo to increase insect suscepti-
bility (Oppert, 1999). Activated Cry toxins then have to pass through the
peritrophic matrix, a process that may reduce the amounts of active toxin
interaction with the target tissue (midgut) and thus affecting susceptibility
(Hayakawa et al., 2004; Rees et al., 2009). This protective role exerted
by the peritrophic matrix can be overcome by Bt endogenous or exogenous
chitinases (Kramer and Muthukrishnan, 1997).

4. MIDGUT CRY-BINDING PROTEINS AND RECEPTOR

FUNCTION
Specific binding of Cry toxins to molecules located on the brush bor-
der membrane of the midgut cells is a major factor in determining the host
58 Michael J. Adang et al.

range of Cry toxins. This feature of Crymidgut interaction was revealed

using BBMV prepared from midgut and labelled Cry toxins in binding
assays. Major challenges of Bt researchers over the past two decades were
the identification of molecules that specifically bind Cry toxins and then
deducing functional relevance of this interaction to Cry toxin action. The
proteins and glycoconjugates reported as involved in Cry toxin action are
described in more detail in recent reviews (Bravo et al., 2011; Jurat-
Fuentes and Jackson, 2012; Pigott and Ellar, 2007).

4.1. Aminopeptidase
Aminopeptidase-N (APN) (Knight et al., 1994, 1995; Sangadala et al., 1994)
and cadherin (Vadlamudi et al., 1995) were the first proteins identified as
putative Cry receptors in insects. Having two distinct proteins types as puta-
tive receptor raised questions as to whether or not both proteins were
involved in Cry toxin action; an issue that is resolved in some of the pro-
posed intoxication models, as discussed below. APNs are tethered to the
midgut brush border by GPI anchors (Garczynski and Adang, 1995) from
where they cleave N-terminal amino acids from peptides, a step necessary
for amino acid co-transport into epithelial cells (Terra et al., 1996). Phylo-
genetic analyses of lepidopteran APNs cluster these proteins into seven clas-
ses (Crava et al., 2010; Hughes, 2014). Among APNs for which expression
data are available, members of the APN1 class are the most highly expressed
in midgut tissue (Hughes, 2014). Interactions between Cry1 toxins and mid-
gut APNs are known to involve recognition of epitopes on the primary pro-
tein structure, or in the case of Cry1Ac, an attached glycan with a terminal
GalNAc moiety. APNs that bind Cry1Ac via GalNAc are identified in
M. sexta (Masson et al., 1995), H. virescens (Gill et al., 1995; Luo et al.,
1997), L. dispar ( Jenkins et al., 2000) and H. armigera (Sarkar et al., 2009).
Cry1Ac binding to APNs in M. sexta and L. dispar has sugar-dependent
and -independent components ( Jenkins et al., 2000; Masson et al., 1995)
leading to proposal of a bivalent binding model involving sequential inter-
actions with domain III and then domain II loop residues ( Jenkins et al.,
2000). Cry1Ac also recognizes a 106-kDa APN in H. virescens in a
GalNAc-independent manner (Banks et al., 2001). Similarly, and although
only known to recognize receptor proteins at amino acid epitopes, the
Cry1Aa toxin binds through domains II and III to a 117-kDa APN in
B. mori (Atsumi et al., 2005). A small patch of seven amino acids near the
N-terminus of a cotton leafworm, Spodoptera litura APN serves as epitope
for recognition by loops 2 and 3 of Cry1C domain II (Kauer et al., 2014).
Diversity and Mechanisms of Bt Crystal Toxin Action 59

In the 1990s, evidence for APN function as Cry1 receptors was provided
by the ability of an APN preparation to enhance Cry1-induced pore forma-
tion in membrane vesicles (Luo et al., 1997; Sangadala et al., 1994) and ion
channels in membrane bilayers (Schwartz et al., 1997c). This approach was
particularly challenging due to the difficulty of purifying midgut APNs with
the lipid moiety remaining on the GPI anchor (Garczynski and Adang,
1995). In vivo evidence of Cry receptor was suggested by work of Gill
and Ellar (2002) who expressed M. sexta APN in transgenic Drosophila ren-
dering larvae susceptible to Cry1Ac. Silencing midgut APNs in S. litura, sug-
arcane borer (Diatraea saccharalis) and H. armigera by RNA interference
(RNAi) established their role as Cry1C (Rajagopal et al., 2002), Cry1Ab
(Yang et al., 2010), and Cry1Ac (Sivakumar et al., 2007) receptors, respec-
tively. Mutation or reduced expression of specific APNs has been correlated
with resistance to Cry1 toxins in D. saccharalis (Yang et al., 2010), S. exigua
(Herrero et al., 2005) and H. armigera (Zhang et al., 2009). In a greenhouse-
derived strain of T. ni, Cry1Ac resistance was associated with differential
alteration of two APNs; with APN1 being downregulated and APN6 being
upregulated in resistant larvae (Tiewsiri and Wang, 2011). Also, the lack of
110-kDa APN1 protein in resistant larvae correlated with reduced transcript
levels and was conferred by a trans-regulatory mechanism (Tiewsiri and
Wang, 2011).
The role of APNs in the action of mosquitocidal Cry toxins has received
considerable attention. Diverse APNs have been identified as putative
receptors of Cry11Aa and Cry11Ba in yellow fever mosquito, Aedes aegypti
(Chen et al., 2009b, 2013; Likitvivatanavong et al., 2011), and Cry11Ba in
malaria mosquitoes, Anopheles albimanus and An. gambiae (Abdullah et al.,
2006; Zhang et al., 2008). The specific APNs in Aedes and Anopheles that
bind Cry11Aa and Cry11Ba, respectively, do so with high affinity (nM
range). Interestingly, in bioassays, the presence of partial APN fragments
enhanced Cry larval mortality (Chen et al., 2013; Zhang et al., 2010), an
unexpected observation from a functional receptor that may suggest inter-
actions between APN and toxin are reversible. Silencing of APN expression
in Ae. aegypti by RNAi resulted in increased tolerance to Cry4Ba toxin
(Saengwiman et al., 2011), supporting a functional toxin receptor role for
this APN.

4.2. Cadherin
Cadherin-like proteins are widely accepted as functional Cry toxin recep-
tors. Contrary to the generally observed localization for cadherin proteins
60 Michael J. Adang et al.

to regions of cellcell interactions, cadherin proteins binding Cry toxins

localize mostly to the brush border membrane of midgut cells (Aimanova
et al., 2006; Chen et al., 2005; Hara et al., 2003; Valaitis, 2011). Cadherins
have also been found to be present in the basement membrane anchoring
midgut epithelial cells of M. sexta and L. dispar larvae (Chen et al., 2005;
Valaitis, 2011). A cadherin called BT-R1 (Bt receptor 1) was first identified
as a Cry toxin receptor in M. sexta larvae (Vadlamudi et al., 1995). Cry1A
toxins were shown to bind Bt-R1 with high affinity and expression of
Bt-R1 in cultured insect cells conferred sensitivity to Cry1A toxins (Hua
et al., 2004; Keeton and Bulla, 1997). Functional evidence of a Cry1A
receptor role for cadherins has been reported for B. mori (Nagamatsu
et al., 1999), O. nubilalis (Flannagan et al., 2005) and H. virescens ( Jurat-
Fuentes and Adang, 2006). Cadherin mutations associated with resistance
to Cry1A proteins are well documented for a number of insects species,
including pink bollworm, Pectinophora gossypiella (Morin et al., 2003),
H. armigera (Xu et al., 2005) and H. virescens (Gahan et al., 2001). Gene
silencing of a cadherin from S. exigua identified that protein as a putative
receptor of Cry1Ca toxin, extending the role of cadherin as a Cry receptor
in lepidopteran larvae outside the Cry1A class of toxins (Park and Kim,
2013; Ren et al., 2013). Cadherin proteins are also reported as functional
receptors of Cry3Aa and Cry3Bb toxins in Coleoptera (Fabrick et al.,
2009; Hua et al., 2014). In Diptera, cadherins are identified as putative
receptors of Cry11Aa and Cry11Ba in Ae. aegypti and Cry11Ba in An.
gambiae (Chen et al., 2009a; Hua et al., 2013; Likitvivatanavong et al.,
2011). Although Cry4Ba binds to cadherins in Ae. aegypti (Bayyareddy
et al., 2009; Hua et al., 2008) and An. gambiae (Hua et al., 2008), the binding
affinities are lower than for Cry11 binding, which may explain why silencing
of cadherin expression by RNAi in Aedes larvae increased larval tolerance to
Cry11Aa but had no effect on Cry4Ba toxicity (Rodriguez-Almazan et al.,
2012). Cadherin has been associated with resistance to Bt subsp. israelensis in
a laboratory-selected strain of Ae. aegypti, but the cadherin is not a known
Cry-binding protein (Bonin et al., 2009).

4.3. Alkaline phosphatase

Membrane-bound midgut ALPs are a major group of Cry-binding proteins
identified in Lepidoptera, Coleoptera and Diptera larvae; and in many cases,
receptor function has been established. Interactions between Cry1Ac and
ALP were recognized when incubation with Cry1Ac reduced ALP
Diversity and Mechanisms of Bt Crystal Toxin Action 61

enzymatic activity in midgut proteins from H. virescens and M. sexta larvae

(English and Readdy, 1989; Sangadala et al., 1994). Binding of Cry proteins
to ALPs in BBMVs from M. sexta (McNall and Adang, 2003), H. virescens
(Krishnamoorthy et al., 2007) and Ae. aegypti (Bayyareddy et al., 2009) were
detected by proteomic analyses. As noted above for APN, binding of
Cry1Ac to ALP from H. virescens and H. armigera larvae involves interactions
with GalNAc ( Jurat-Fuentes and Adang, 2004; Ning et al., 2010; Sengupta
et al., 2013). In the case of Cry1Ab, initial interaction with ALP in M. sexta
through -16 in domain III is critical to binding to ALP and toxicity to lar-
vae (Arenas et al., 2010). RNAi silencing of APN and ALP genes in M. sexta
larvae showed that binding to ALP is more important for Cry1Ab toxicity
than to APN; in contrast to Cry1Ac which relies more on APN (Flores-
Escobar et al., 2013). Correlations between reduced ALP expression and
resistance to Cry1 toxins in strains of H. virescens, H. armigera and the fall
armyworm (S. frugiperda) further support an in vivo role of ALPs in Cry
intoxication of lepidopteran larvae ( Jurat-Fuentes et al., 2011). In Diptera,
mosquitocidal Cry11Aa and Cry11Ba toxins bind ALP in Aedes and Anoph-
eles BBMV, and this interaction is relevant to in vivo toxicity, as feeding
toxin-binding regions of ALP with Cry toxins to larvae reduced toxicity
(Fernandez et al., 2006; Hua et al., 2009). Evidence for receptor function-
ality in vitro has been provided from expression of an Aedes ALP on the sur-
face of insect cells conferring susceptibility to Cry4Ba toxin (Dechklar et al.,
2011). Recently, ALP1 in Ae. aegypti was established as a functional receptor
for Cry11Aa and Cry4Ba by RNAi silencing ( Jimenez et al., 2012). Recep-
tor ALPs were under expressed in a Bt var. israelensis-resistant strain of Ae.
aegypti (Tetreau et al., 2012). In Coleoptera, midgut ALPs binding Cry3Aa
in the yellow mealworm, Tenebrio molitor (Zuniga-Navarrete et al., 2013), or
Cry1Ba6 in cotton boll weevil, Anthonomus grandis (Martins et al., 2010),
have been identified, with confirmation of receptor function yet to be deter-
mined (Martins et al., 2010).

4.4. ABC transporter

The ABC family of proteins in insects are related to the multi-drug resistance
proteins in animals (Heckel, 2012). The comparative genomics of the ABC
gene family in arthropods and the relationship of ABC proteins to xenobi-
otics and insecticide transport were recently reviewed (Dermauw and Van
Leeuwen, 2014). Initial evidence for a role of ABCC2 proteins in the Cry1
mode of action was provided by genetic linkage between an ABCC2
62 Michael J. Adang et al.

mutation and resistance to Cry1A toxins in strains of H. virescens (Gahan

et al., 2010). Isolation of resistant loci into distinct strains allowed for the
demonstration that mutations in cadherin explained lack of Cry1Aa binding
( Jurat-Fuentes et al., 2004) in these H. virescens strains, an inactivating muta-
tion in the ABCC2 gene was associated to lack of Cry1Ab and Cry1Ac bind-
ing (Gahan et al., 2010). Resistance to Cry1Ac was also mapped to the
ABCC2 locus in diamondback moth, Plutella xylostella, and T. ni providing
further support for the function of this protein in Cry1A toxin action (Baxter
et al., 2011). Direct confirmation of the importance of ABC transporters to
Cry1A toxin action was obtained from studies of the silkworm, B. mori. In
this insect, a single tyrosine insertion on an outer lumen-facing loop of an
ABCC2 protein was linked to resistance against the Cry1Ab toxin, and germ
line transformation of this insertion conferred resistance to this toxin in sus-
ceptible silkworms (Atsumi et al., 2012). Moreover, the importance of the
tyrosine insertion and functional role of the ABCC2 protein in susceptibility
to Cry1Ab, Cry1Ac, Cry1Fa and even relatively unrelated Cry8Ca in
B. mori was demonstrated by comparing expression of mutated and wild-
type ABCC2 genes in cultured insect cells (Tanaka et al., 2013). Although
these studies illustrate a critical role for ABCC2 proteins in Cry toxin action,
there is no experimentally tested mechanistic description of ABCC2-Cry
toxin interactions available.

4.5. Other Cry-binding (receptor) proteins and molecules

Glycolipids from M. sexta, a glycoconjugate from L. dispar, members of the
polycalin protein family, an ADAM metalloprotease in L. decemlineata, a
sodium solute transporter in flour beetle (Tribolium castaneum), amylases in
An. albimanus and An. gambiae and an -glucosidase in An. gambiae have been
reported as Cry toxin-binding molecules and putative receptors. Initial evi-
dence for interactions between lipids and Cry toxins was provided by studies
of Cry1A protoxin and activated toxin binding to glycolipids from pupae of
the blowfly Calliphora vicina (Dennis et al., 1986). Although toxin binding to
glycolipids was detected, this evidence was not widely considered, probably
because fly larvae are not usually susceptible to Cry1A toxins. Functional
evidence for glycolipids as Cry toxin receptors emerged from studies of resis-
tance to Cry5Ba in the nematode, Caenorhabditis elegans (Griffitts et al.,
2005). The same study included evidence for specific Cry1A toxin binding
to glycolipids extracted from M. sexta midgut tissue, supporting a role for
lipids in Cry intoxication. A role for glycolipids in formation of Cry toxin
Diversity and Mechanisms of Bt Crystal Toxin Action 63

oligomers through interaction with domain II of the toxin was proposed

(Ma et al., 2012). However, the functional relevance of lipidCry toxin
interactions to toxicity has not been experimentally established.
In the gypsy moth (L. dispar), a 270 kDa glycoconjugate binds Cry1Aa,
Cry1Ab and Cry1Ba toxins (Valaitis et al., 2001), although the biological
relevance of this interactions is unknown. A 252 kDa member of the
polycalin family in B. mori (P252) was shown to bind Cry1A toxins in a
GalNAc-independent manner (Hossain et al., 2004; Pandian et al., 2008).
This protein exists in the insect as an oligomer that forms a complex with
Cry1A toxins, but it does not interfere with activity against B. mori larvae
(Pandian et al., 2010). Interactions between Cry toxins and members of
the polycalin protein family have also been reported in H. armigera
(Angelucci et al., 2008; Ma et al., 2012). Taken together, these observations
may reflect the importance of interactions between Cry toxins and lipids or
proteins in the midgut fluids to subsequent binding to receptors on midgut
cells, a possibility that needs further experimental study.
Diverse proteins have been reported to participate in Cry3A toxicity in
L. decemlineata. In this system, proteolysis of Cry3Aa by BBMV proteins
reduces toxin pore formation (Rausell et al., 2007), yet interactions of
Cry3Aa domain II loop 1 with an ADAM metalloprotease are important
for effective toxin pore formation (Ochoa-Campuzano et al., 2007). More
recently, a prohibitin protein has been proposed to participate in recruiting
of the toxin to the ADAM protease (Ochoa-Campuzano et al., 2013).
Unfortunately, the critical function of prohibitin prevented the use of silenc-
ing approaches to test its relevance for Cry intoxication. Interestingly,
prohibitin has been also described to bind Cry4Ba in Ae. Aegypti
(Bayyareddy et al., 2009), although both Cry4Ba and Cry11Aa toxins were
also reported to bind a 70-kDa -amylase in An. albimanus (Fernandez-Luna
et al., 2010). The functional role of these in vitro interactions for in vivo tox-
icity needs to be experimentally tested.

5. MODELS OF CRY TOXIN ACTION

Although extensively investigated and reviewed ( Jurat-Fuentes and
Jackson, 2012), mechanistic details for interactions between midgut brush
border proteins and Cry toxins that are conducive to toxicity remain con-
troversial (Vachon et al., 2012). It is well established that activated Cry toxins
must recognize sites on insect midgut cell proteins to exert toxicity and that
this binding step determines specificity, yet it is not sufficient to predict
64 Michael J. Adang et al.

susceptibility (Garczynski et al., 1991; Wolfersberger, 1990). Since Cry

toxin binding is mostly localized to the brush border membrane of the mid-
gut epithelium where binding proteins are located (Bravo et al., 1992; Chen
et al., 2005), vesicle preparations representing an enriched fraction for this
tissue (BBMV) have been used as in vitro model to study interactions
between Cry toxins and midgut cells. While diverse proteins, lipids and
glycoconjugates have been proposed to contain Cry toxin-binding sites
and represent functional receptors (Pigott and Ellar, 2007), most data avail-
able are focused on cadherin, APN, and ALP proteins. Interestingly, Cry1A
and Cry3Aa protoxins bind specifically to midgut cadherin proteins in target
insects (Fabrick and Tabashnik, 2007; Fabrick et al., 2009; Gomez et al.,
2014), although the relevance of this binding toxicity in vivo is unclear con-
sidering the fast rate of activation of protoxins in the midgut lumen after
solubilization.
The sequential binding model developed for Cry1A toxinreceptor
interactions (Pardo-Lopez et al., 2013) suggests initial reversible binding
of Cry1A-activated toxins to abundant APN and ALP proteins, favouring
higher affinity toxin interactions with less abundant cadherin proteins
(Pacheco et al., 2009a). This binding of toxin to the extracellular cadherin
region most proximal to the cell membrane (Pigott and Ellar, 2007) is pro-
posed to promote proteolytic removal of helix 1 and result in formation of a
pre-pore toxin oligomer that displays high affinity for APN and ALP pro-
teins (Bravo et al., 2004; Gomez et al., 2002). Binding to APN and ALP
proteins facilitates concentration of toxin oligomers on specialized mem-
brane regions called lipid rafts (Zhuang et al., 2002), facilitating insertion
and formation of a toxin pore that leads to cell death by osmotic shock.
In agreement with this model, reversible (initial binding to APN and
ALP, binding to cadherin, and toxin oligomer binding to APN and ALP)
and irreversible (toxin insertion on the membrane) binding components
have been described for Cry toxins, with irreversible binding being directly
correlated to toxicity (Liang et al., 1995). Further evidence supporting this
model comes from reports of Cry toxin oligomer formation in vitro in the
presence of cadherin peptides (Fabrick et al., 2009; Pacheco et al., 2009b;
Peng et al., 2010) and augmented toxicity associated with increased produc-
tion of Cry toxin oligomers (Gao et al., 2011; Pacheco et al., 2009b). Lack or
low levels of susceptibility to Cry1A toxins in heterologous systems
expressing cadherin (Aimanova et al., 2006; Hua et al., 2004; Jurat-
Fuentes and Adang, 2006) proteins are in agreement with the dependence
on sequential receptor interactions for Cry toxicity. Unfortunately,
Diversity and Mechanisms of Bt Crystal Toxin Action 65

alternative reports of heterologous cadherin or APN expression previously

suggested as evidence against sequential binding (Vachon et al., 2012), did
not report quantitative data or monitored only changes in cell morphology,
which complicates accurate assessment of cytotoxicity. Only a report of het-
erologous expression of APN from M. sexta in Drosophila melanogaster larvae
resulted in high susceptibility to Cry1Ac could be considered as evidence for
the sequential binding model, yet in this case, the existence of cadherin and
ALP Drosophila orthologs capable of interacting with the toxin was
not tested.
Nevertheless, there are some reported observations that are difficult to
explain with the sequential binding model (Vachon et al., 2012), some
of which may be a consequence of the Cry1AM. sexta model system used
for its development, potentially limiting extended applicability. For
instance, the model does not consider the role of membrane lipids or
ABC transporters, which have been proposed as relevant Cry toxin recep-
tors (Gahan et al., 2010; Griffitts et al., 2005; Ma et al., 2012). Moreover,
modified Cry1A toxins with cadherin-independent oligomerization
(Soberon et al., 2007) did not overcome resistance due to alterations in
cadherin receptors and were less potent against susceptible insects than
wild-type toxins (Tabashnik et al., 2011). This reduced potency compared
to wild-type toxin may be explained by the pre-pore oligomer formed by
modified Cry1A toxins differing from the oligomer formed by wild-type
toxin (Gomez et al., 2014). However, lack of direct correlation between
amounts of oligomer formed in vitro and toxicity in vivo in some cases
(Gomez et al., 2014; Gomez et al., 2002) question the relevance of these
pre-pore oligomers for toxicity. In this regard, Cry toxin oligomeric struc-
tures are also formed in solution (Masson et al., 2002a; Walters et al., 1994)
and form functional pores in model lipid membranes in the absence of recep-
tor proteins (Gomez et al., 2014; Vie et al., 2001). The functional relevance
of pre-pore Cry toxin oligomers for toxin insertion in the membrane is fur-
ther challenged by toxin mutants unable to form oligomers but capable of
binding irreversibly, suggesting insertion, in BBMV from susceptible insects
(Tigue et al., 2001). The observation that these mutants could insert as
monomers but not cause toxicity supports the possibility that oligomeriza-
tion may occur after insertion of toxin monomers in the membrane (Vachon
et al., 2012).
Two alternative models for Cry toxin pore formation on the midgut cell
membrane have been proposed (Knowles, 1994). In the penknife model
(Hodgman and Ellar, 1990), only the hydrophobic 5 and 6 helices in
66 Michael J. Adang et al.

domain I insert into the membrane, while the rest of the domain lays flat on
the membrane surface. Alternatively, most available evidence supports the
umbrella model, in which a conformational change in the toxin molecule
upon binding to receptors results in insertion of a hydrophobic hairpin com-
posed of 4 and 5 helices (Li et al., 2001; Schwartz et al., 1997c). The posi-
tion of domains II and III of the Cry toxins during insertion is still a matter of
debate. While protease protection studies (Aronson, 2000; Aronson et al.,
1999) and mutagenesis (Dean et al., 1996; Nair and Dean, 2008) support that
the whole toxin molecule is confined to the membrane, there is also evi-
dence suggesting that the toxin remains associated to membrane receptors
after pore formation (Fortier et al., 2007) and at least residues of domain
III may be exposed to the solvent (Pardo-Lopez et al., 2006). In contrast
to the pre-pore oligomers presented by the sequential binding model, pro-
posals modelling pore formation support that association between inserted
toxin monomers results in a tetrameric ion channel with a diameter of
approximately 15 A (Groulx et al., 2010, 2011). However, trimeric pores
have been proposed from sequence analysis (Torres et al., 2008) and lipid
membrane experiments (Ounjai et al., 2007). Evidence supporting the pos-
sibility that Cry toxin pores may arrange from association of diverse Cry
toxin monomers (heterooligomers) has been presented (Carmona et al.,
2011), which may help explain synergistic and inhibitory effects among
Cry toxins (Ibargutxi et al., 2008). More importantly, the significance of
heterooligomeric pore formation for binding competition studies needs
to be considered and tested experimentally.
The 4 helix lining the Cry toxin pore seems to control the passage of
ions through the pore (Kumar and Aronson, 1999; Masson et al., 1999),
although there are also reports suggesting a role in controlling pore perme-
ability by alternative domain I helices (Alcantara et al., 2001; Arnold et al.,
2001). Pore properties as well as formation are also dependent on midgut
epithelium components (Peyronnet et al., 2001; Schwartz et al., 1997b)
and ionic composition (Brunet et al., 2010a; Fortier et al., 2005). Variable
pore properties depending on the environment may reflect adaptation of
Cry toxins to diverse functional environments (Schnepf et al., 1998).
An alternative model for Cry intoxication disregards pore formation and
highlights the activation of intracellular oncotic cell death pathways as
responsible for enterocyte death (Zhang et al., 2008). However, evidence
supporting this model is limited to Cry1Ab and studies with insect cell cul-
tures expressing a cadherin from M. sexta (Zhang et al., 2005). In these cells,
binding of Cry1Ab toxin was associated with cell death and increased cAMP
Diversity and Mechanisms of Bt Crystal Toxin Action 67

production. It is important to note that the ovarian Hi5 cell line used for
these analyses is susceptible to the Cry1Ac toxin in the absence of expression
of proposed midgut toxin receptors, suggesting that they may display a
mechanism of susceptibility that differs from cells in the insect midgut epi-
thelium. Evidence supporting a role for intracellular pathways in Cry intox-
ication of midgut cells was provided by a deletion of 55 amino acids in the
cytoplasmic domain of a cadherin being genetically linked with non-
recessive resistance to Cry1Ac in H. armigera (Zhang et al., 2012). However,
only a 20% reduction in susceptibility to Cry1Ac was detected in Sf9 cells
expressing the wild type versus the cadherin with the cytoplasmic deletion,
challenging the relative importance of cadherin-mediated intracellular sig-
nalling in Cry susceptibility.
Although speculative, a third possibility would consider effects of both
pore formation and intracellular cell death pathways (Pigott and Ellar,
2007). Thus, there are examples of bacterial pore-forming toxins that induce
host cell death through oncosis (Zhou et al., 2009). The observation that
modified Cry toxins that do not depend on binding to cadherin for oligo-
merization display lower activity against susceptible insects (Soberon et al.,
2007), may suggest that intracellular signalling activated by wild type, but
not by modified Cry toxins binding to cadherin may contribute to toxicity.
Consequently, it is plausible that intracellular cell death may occur in the
presence of lower Cry toxin concentrations, while higher toxin concentra-
tions would promote increased toxin insertion and formation of pores. Fur-
ther work to establish functional connections between pore formation and
intracellular signalling for cytotoxicity are needed to clarify the molecular
events resulting in midgut cell death by Cry toxins.
In the case of binary Cry toxins, low sequence homology suggests dif-
ferences in the intoxication model when compared with the three-domain
Cry toxins. Most data available are focused on the Cry34/35 complex (Ellis
et al., 2002). In this case, while the Cry35 protein displays lectin folds sug-
gestive of receptor binding and the Cry34 protein has homology to pro-
teins involved in intracellular signalling (Schnepf et al., 2005), they
probably exert their toxicity through pore formation (Masson et al.,
2004). Data from binding assays support that Cry34 toxin greatly enhances
Cry35 binding to D. virgifera BBMV proteins (Li et al., 2013). Interestingly,
comparisons between results from homologous competition tests in the
presence or absence of Cry34 suggest lower binding affinity for the
Cry34/35 complex. Binding of Cry35 to D. virgifera BBMV proteins in
the absence or presence of Cry34 was to binding sites not recognized
68 Michael J. Adang et al.

by Cry3Aa, Cry8Ba or Cry6Aa toxins (Li et al., 2013). The identity of the
specific Cry34/35 or Cry35 receptors is unknown.

6. CYTOLYTIC TOXINS
The Cyt (cytolytic) toxins were originally discovered in Bt subspecies
israelensis (Goldberg and Margalit, 1977) and have been recently reviewed
(Ben-Dov, 2014; Soberon et al., 2013b). These proteins seem to be specific
to some Bt subspecies (Tyrell et al., 1981). The first feature observed for the
28 kDa protein from Bt subsp. israelensis was its cytolytic activity against cul-
tured mammalian and insects cells and toxicity when injected in mice, while
it did not display activity against larvae of the cabbage butterfly, Pieris brassicae
(Thomas and Ellar, 1983a). Against mosquitoes, the 28-kDa protein was less
active than native Bt subsp. israelensis crystals (Chilcott and Ellar, 1988;
Davidson and Yamamoto, 1984; Yamamoto et al., 1983), and it synergized
activity of other Bt subsp. israelensis crystal proteins (Chilcott and Ellar, 1988;
Wu and Chang, 1985). Currently, there are also cyt genes that have been
described in Bt strains targeting lepidopteran or coleopteran insects
(Guerchicoff et al., 1997), adding to, three cyt toxin gene families (cyt1,
cyt2 and cyt3) that include 11 holotype toxins in the current nomenclature
(Crickmore et al., 2014).
The three-dimensional Cyt structures resolved to date support a con-
served structural model including two -helix hairpins flanking a
-sheet core containing seven to eight -strands (Cohen et al., 2008,
2011; Li et al., 1996). This highly conserved structure is also revealed in
sequence alignments, which identify highly conserved blocks (Butko,
2003). Mutagenic studies identified -sheet residues as critical for toxicity,
while mutations of residues on the helical domains did not affect toxicity,
suggesting a critical role for the -sheet core.
Ingested Cyt1A and Cyt2A protoxins are processed by digestive prote-
ases at the same sites in the N- and C-termini to a stable toxin core of 25 and
23 kDa, respectively (Koni and Ellar, 1994). Activated Cyt toxins display
high affinity for membrane lipids containing unsaturated acyl chains (Gill
et al., 1987; Thomas and Ellar, 1983b), which are abundant in midgut brush
border membrane of Diptera (Li et al., 1996). A putative phospholipid-
binding site pocket homologous to Erwinia virulence factor (Evf ) was
described for Cyt2Ba (Rigden, 2009). Two non-exclusive mechanisms
for Cyt1A-induced cytolysis have been proposed: pore formation and
detergent-like membrane disruption (Butko, 2003). Pore formation by
Diversity and Mechanisms of Bt Crystal Toxin Action 69

Cyt toxins is supported by evidence from studies with planar bilayers

(Knowles et al., 1989) and erythrocytes (Promdonkoy and Ellar, 2003),
and by the importance of pre-pore oligomerization for toxicity (Lopez-
Diaz et al., 2013). According to this model, binding to membrane lipids leads
to a conformational change in the toxin resulting in movement of the
amphiphilic helices to expose the hydrophobic face of the -sheet for mem-
brane insertion (Li et al., 1996). Three of the -strands are long enough to
insert spanning the cell membrane (Promdonkoy and Ellar, 2005), which is
followed by oligomerization to form a -barrel pore conducive to cell death
by osmotic shock (Du et al., 1999; Li et al., 1996). Recent reports suggest the
existence of pre-pore oligomers (Lopez-Diaz et al., 2013), yet the relevance
of these oligomers for toxicity has been questioned (Canton et al., 2014). In
contrast to pore formation, the detergent model advocates that cell death
occurs through localization of the toxin to the membrane surface to induce
detergent-like defects in lipid packing, which result in leakage of intracel-
lular molecules (Butko et al., 1996). In support of this model, large Cyt toxin
aggregates, rather than smaller oligomers capable of forming pores, are
observed during binding to cell membranes (Chow et al., 1989), membrane
permeation (Rodriguez-Almazan et al., 2011) and lipid vesicles exposed to
Cyt1A fragment into smaller forms as it would be expected from a
detergent-like action (Manceva et al., 2005). It is generally accepted that
both models may occur in vivo depending on toxin concentration, with
lower concentrations favouring oligomeric pores and higher concentrations
leading to membrane breaks (Butko, 2003).
Synergistic effects between Cyt and other bacterial toxins have fre-
quently been reported, even to overcome resistance. For instance, a Cyt1A
overcame resistance to Cry4 toxins (Wirth et al., 1997) and delayed evolu-
tion of resistance to Cry11Aa (Wirth et al., 2005) in Culex quinquefasciatus.
Synergism was also observed for Bin toxins in strains of Cx. pipiens and Cx.
quinquefasciatus (Thiery and Hamon, 1998) and for Mtx1 toxicity in Cx.
quinquefasciatus (Zhang et al., 2006). The Cyt synergistic effect for Cry toxins
is proposed to involve Cyt1A binding to domain II of Cry toxins (Lailak
et al., 2013) in solution or on the membrane plane to promote formation
of a Cry toxin pre-pore oligomer (Perez et al., 2007). Formation of this
Cry oligomer is independent of the Cyt oligomerization, binding or inser-
tion (Lopez-Diaz et al., 2013), which may help explain why synergism
between Cyt1A and Cry3A toxin against the cottonwood leaf beetle,
Chrysomela scripta, does not correspond with binding of Cry3A to
membrane-bound Cyt1A (Federici and Bauer, 1998). Because binding to
70 Michael J. Adang et al.

lipids limits specificity of Cyt toxins for insecticidal applications, Cyt2Aa

toxin has been engineered to contain a pea aphid gut-binding peptide
resulting in a novel toxin with specific activity against major aphid pests
(Chougule et al., 2013). Lack of competition for midgut sites between
the gut-binding peptide of the engineered Cyt toxin and Cry toxins suggests
the potential use of both toxins for sustainable pest control.

ACKNOWLEDGEMENTS
This work was supported by USDA NIFA award number 2010-65105-20590 to M. J. A.
(University of Georgia) and Biotechnology Risk Assessment Grant Program competitive
grant no. 2010-33522-21700 from USDA NIFA to J. L. J-F. (University of Tennessee).
We thank Dr. Leara Rhodes (University of Georgia) for editing an earlier version of this
manuscript. Ruchir Mishra is thanked for generating and assembling the three-
dimensional Cry images.

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 CHAPTER THREE

Lysinibacillus sphaericus: Toxins

and Mode of Action, Applications
for Mosquito Control and
Resistance Management
Maria Helena Neves Lobo Silva Filha*, Colin Berry, Lda Regis*
*Centro de Pesquisas Aggeu Magalhaes-Fiocruz, Recife-Pernambuco, Brazil

Cardiff School of Biosciences, Cardiff University, Cardiff, United Kingdom

Contents
1. Introduction 90
1.1 Background 90
1.2 General features and strains 91
1.3 The relevance of L. sphaericus as a mosquito-control agent 93
2. Toxins and Mode of Action 95
2.1 Spectrum of action 95
2.2 Binary toxin 97
2.3 Cry48/Cry49 104
2.4 Mosquitocidal toxin 1 104
2.5 Other Mtx toxins 107
2.6 Sphaericolysin 108
2.7 S-layer proteins 109
2.8 Safety issues 109
3. Receptors of the Binary Toxin 112
3.1 Binding of the binary toxin to larvae midgut 112
3.2 Receptors 114
3.3 Comparative analysis of the Cqm1 and Aam1 -glucosidases 119
4. Applications for Mosquito Control 121
4.1 Field trials 121
4.2 Factors affecting field performance 125
4.3 Trials against the vectors of lymphatic filariasis 126
4.4 Recent large-scale trials 127
4.5 Operational use in mosquito-control programmes 129
5. Resistance 130
5.1 Factors involved in the selection of resistance 130
5.2 Laboratory and field reports 131
5.3 Mechanisms and inheritance of resistance 135

Advances in Insect Physiology, Volume 47 # 2014 Elsevier Ltd 89

ISSN 0065-2806 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800197-4.00003-8
90 Maria Helena Neves Lobo Silva Filha et al.

5.4 Resistance alleles of the cpm1/cqm1 gene 135

5.5 Diagnosis and field survey of resistance 139
5.6 Biological cost of resistance 141
6. Management of Resistance 143
6.1 Integrated mosquito-control programmes 143
6.2 Factors involved in the prevention of resistance 144
6.3 Candidates for managing Bin-toxin resistance 145
Acknowledgements 150
References 150

Abstract
Lysinibacillus sphaericus (Ls) strains that produce insecticidal proteins show high activity
against mosquito larvae. The most active of these is the binary (Bin) toxin that acts fol-
lowing ingestion and, after midgut processing and binding to specific receptors, pro-
vokes cytopathological effects and leads to larval death. Bin toxin displays specific action
against some species of medical importance (e.g. Culex and Anopheles) and it is safe to
non-target organisms. These features have led to the production of biolarvicides based
on this bacterium and its effectiveness to control mosquito larvae has been widely
related in the literature. The field utilisation of Ls has also shown that resistance could
be selected among exposed populations and the mechanisms and genes involved in
this process have been described. Management strategies can be successfully
employed to avoid resistance and Ls can be used within integrated programmes as
a selective and efficient agent to control mosquitoes.

1. INTRODUCTION
1.1. Background
The utilisation of entomopathogenic bacteria for insect control started in the
1960s with the discovery and development of Bacillus thuringiensis (Bt) vari-
eties that produced insecticidal proteins active against agricultural insect
pests. The B. thuringiensis serovar. israelensis (Bti) discovered by Goldberg
and Margalit (1978) was the first serotype identified as active against Diptera
larvae (de Barjac, 1978). This entomopathogenic bacterium enjoyed a rapid
development from the characterisation of its properties to field utilisation
(Becker, 1997; Guillet et al., 1990; Margalit and Dean, 1985), mainly
because of the serious resistance problems encountered by synthetic insec-
ticides in vector-control programmes during that period. The second
mosquitocidal bacterium Lysinibacillus sphaericus (Ls), previously designated
as B. sphaericus, was identified by Neide in 1904 (Neide, 1904). Character-
isation of this species as a mosquito pathogen was initiated by Kellen, much
Microbial Toxins for Mosquito Control 91

later, when a toxic strain was isolated from cadavers of Culiseta incidens larvae
(Kellen et al., 1965). The Kellen (K) strain displayed a low level of toxicity
and did not attract much interest for its development as a control agent. The
discovery by Singer (1973), of the SSII-1 strain, that displayed a higher activ-
ity than the K strain renewed the interest in this bacterium and motivated the
search for new strains. Later, strains with high activity were discovered
(Singer, 1977; Weiser, 1984; Wickremesingue and Mendis, 1980) that
led to the development of the use of Ls as a mosquito-control agent.
Insecticidal factors produced by Ls were identified in strains isolated
worldwide, and these isolates were classified according to their toxicity to
mosquitoes. Early studies showed that the high activity of some strains
was associated with the production of crystalline inclusions during the bac-
terial sporulation (Fig. 3.1) (de Barjac and Charles, 1983; Kalfon et al., 1984;
Payne and Davidson, 1984; Yousten and Davidson, 1982). The crystals are
synthesised during stage III of sporulation, and once formed, they remain
associated with the spore within the exosporium (Kalfon et al., 1984;
Yousten and Davidson, 1982). A study showed that mutant strains that were
blocked from the early stages of sporulation did not produce crystals and lost
their toxicity toward larvae, which confirmed the essential role played by the
crystals for the mosquitocidal activity of these strains (Charles et al., 1988).
The active crystals contain the binary (Bin) protoxin, which is the major
insecticidal protein produced by Ls (Baumann et al., 1985).

1.2. General features and strains

Ls is a Gram positive, aerobic, sporulating and cosmopolitan bacterium. This
species is a saprophytic organism that occurs in several habitats, including soil
and aquatic environments. The most important morphological characteristic
is the presence of spherical terminal spores, and the most marked phenotypic
features are its inability to grow under anaerobic conditions or to use sugars
as source of energy (White and Lotay, 1980). Instead, this species uses carbon
compounds, such as organic acids and other amino acids. The reclassification
of this organism as Ls was due to some differential phenotypic features,
including the capacity to produce peptidoglycans containing lysine and
aspartate (Ahmed et al., 2007).
In terms of classification, Ls is quite a heterogeneous species (Nakamura,
2000), and in view of its application to mosquito control, the strains can be
divided into toxic and non-toxic strains that share 79% DNA homology
(Krych et al., 1980). This chapter addresses Ls strains classified in the
92 Maria Helena Neves Lobo Silva Filha et al.

Figure 3.1 Micrography of Lysinibacillus sphaericus strain 2297 at the end of sporulation.
(A) Spore and crystal are in the left- and right side of the exosporium, respectively.
(B) Crystal lattice. Taken from Charles et al. (2010).

DNA homology group IIA, as will be described below. The highly toxic
strains isolated to date, can produce different insecticidal factors as the Bin
protoxin from crystalline inclusions and other toxins, such as the
mosquitocidal toxins (Mtxs) and the Cry48/Cry49 toxin, as will be
described in Section 2.
General classifications of Ls strains have been performed based on
different systems. The DNA homology of the strains (Krych et al., 1980)
and serotyping based on the flagellar antigen (de Barjac, 1990; de Barjac
Microbial Toxins for Mosquito Control 93

et al., 1985) are the most commonly used methods. Other approaches have
also been proposed to classify this diverse group, such as bacteriophage typ-
ing (Yousten, 1984b; Yousten et al., 1980), numerical classification using the
taxonomy of phenotypic features (Alexander and Priest, 1990), analysis of
the cellular fatty acids (Frachon et al., 1991), ribotyping (Aquino de
Muro et al., 1992), and the profiling of randomly amplified polymorphic
DNA (Woodburn et al., 1995). The toxic strains were found to belong
to few groups according to all the classifications used. DNA homology anal-
ysis placed the strains into five groups (IV) that probably represent distinct
species, and those that displayed some level of mosquito activity were clus-
tered in subgroup IIA. The flagellar antigen analysis generated approxi-
mately 49 serotypes, and nine serotypes host the highly toxic strains. The
serotype 5a5b contains active strains that produce the Bin toxin and includes
1593 and 2362, which are the most commonly strains used to date for the
production of larvicides. Other high-toxicity strains producing the Bin
toxin from DNA group IIA include the IAB881, IAB59, 2297 and
IAB872 strains, belonging to serotypes 3, 6, 25 and 48, respectively.
Table 3.1 shows some representative examples of strains and their classifica-
tion based on previously published data (Charles et al., 1996). Other highly
toxic strains have also been isolated and employed for the production of
biolarvicides. The C3-41 strain (serotype 5a5b) isolated in China, for
instance, has been extensively used in control programmes in that country
(Yuan et al., 1999). The screening of new strains for improved larvicidal
activity and optimal performance for large-scale production continues to
sustain the development of new products (Hire et al., 2010; Poophati
et al., 2013; Prabhu et al., 2013; Sun et al., 1996).

1.3. The relevance of L. sphaericus as a mosquito-control agent

Ls occupies an important position among the set of insecticidal agents that
are currently available for mosquito control. This bacterium produces insec-
ticidal factors that display high activity against culicids of medical relevance
that include Culex and Anopheles species. The production of biolarvicides
based on Ls has been successfully achieved since the 1980s (Lacey, 2007;
Lecadet, 1996), and its high insecticidal potency allied to a selective spec-
trum (Section 2) are important features that allowed the development of
commercial products. In addition, the development of Ls biolarvicides at
the industrial level was supported by the successful production and applica-
tion of Bt.
94 Maria Helena Neves Lobo Silva Filha et al.

Table 3.1 Examples of Lysinibacillus sphaericus strains and their larvicidal properties to
mosquito larvae
Genes encoding
mosquitocidal proteinsb
Flagellar DNA Larvicidal Cry48/
Strain Origin serotype group activitya Crystal Mtxs Cry49
Kellen USA 1a IIA Low
1, 2, 3 Nd
K
SSII-1 India 2a2b IIA Medium
1, 2, 3 Nd
c
IAB881 Ghana 2a2b Nd Medium + 1, (2 and 3 +
Nd)
LP1-G China 3 IIA Medium + 1, (2 and 3 +
Nd)
1593M Indonesia 5a5b IIA High + 1, 2 (3 Nd)

2362 Nigeria 5a5b IIA High + 1, 2, 3

1691 El 5a5b IIA High + 1, 2, 3 Nd
Salvador
IAB59 Ghana 6 Nd High + 1, 2, 3 +
2297 Sri Lanka 25 IIA High + 1, 2, 3

IAB872 Ghana 48 Nd High + 1, (2 and 3 Nd
Nd)
C3-41 China 5a5b IIA High + 1 (pseudo)

2, 3, 4
a
Based on criteria defined by Charles et al. (1996).
+ Presence,
absence.
b
c
Not determined.
Modified and extended from Charles et al. (1996)

The experiences of Ls larvicide utilisation provided a solid background

for its adoption in mosquito-control programmes worldwide (Section 4).
These larvicides were first introduced for Cx. pipiens control in many areas
of France as early as 1987 to reduce the nuisance caused by this species
(Thiery et al., 1996). An initiative by the World Health Organisation
(WHO) created a multi-centre study for the evaluation of the field effec-
tiveness of Ls to control Cx. quinquefasciatus larvae in urban areas of tropical
countries, with particular attention to the role of this mosquito as the vector
of Wuchereria bancrofti the causative agent of filariasis (WHO, 1993). These
pioneer trials, along with others, supported the broad utilisation of these
Microbial Toxins for Mosquito Control 95

larvicides. The control of Culex spp. has gained more importance recently
in regard to their role as vectors of emergent arboviruses, such as the West
Nile Virus that has provoked important epidemics in human populations
(Kramer et al., 2008; Petersen and Fischer, 2012). The control of anophe-
lines is a challenge, and previous studies have shown that some relevant spe-
cies involved in Plasmodium transmission are susceptible to Ls. The control
of Anopheles (An.) stephensi and An. sinensis in India and China, respectively,
showed the operational viability of Ls to control this group of mosquitoes
(Kumar et al., 1994; Thiery et al., 1996; Yuan et al., 2000). In addition to its
application for vector control in urban areas, the selective activity and bio-
compatibility of Ls is of great utility when the target species breed in envi-
ronmentally sensitive areas. The utilisation of Ls also raises concerns about
the selection of resistance. High levels of resistance achieved due to its
utilisation have been reported, and the major findings on this issue are pres-
ented in Section 5 along with the strategies that can be introduced for the
management of resistance in Section 6. These strategies can ensure the
effectiveness of Ls when used in the scope of integrated control programmes
and can overcome the potential onset of resistance. Different aspects of Ls
and its properties as an entomopathogenic bacterium have been covered by
previous reviews and book chapters that can provide additional information
(Baumann et al., 1991; Becker, 2000; Berry, 2012; Charles and Nielsen-
LeRoux, 2000; Charles et al., 1996, 2010; Delecluse et al., 2000; Lacey,
2007; Porter et al., 1993; Regis and Nielsen-LeRoux, 2000).

2. TOXINS AND MODE OF ACTION

2.1. Spectrum of action
Ls strains in DNA group IIA may produce a range of toxins as detailed in this
section, including those with activity against mosquitoes, which are the Bin,
the Mtx and the Cry48Aa/49Aa toxins. The profile of toxins produced by
individual strains is varied and contributes to the designation of bacteria
as either high- or low-toxicity strains with respect to their activity against
mosquito larvae. The strains with the highest activity are characterised by
the presence of the Bin protoxin that is produced as parasporal crystals during
sporulation (de Barjac et al., 1980; Payne and Davidson, 1984; Yousten,
1984a). Strains lacking Bin crystals display low toxicity and some of them
produce Mtx toxins, however, the latter undergo degradation during their
production in the vegetative phase and they do not contribute to provide a
high activity to the strains. The performance of strain 1593 that produces Bin
96 Maria Helena Neves Lobo Silva Filha et al.

and Mtx toxins to larvae is, for instance, 3000-fold superior compared to the
SSII-1 strain that produces Mtx toxins only (Myers et al., 1979). Additional
studies on different strains have shown that Bin accounts for most activity
recorded for the sporulated cultures and this is the main active ingredient
of biolarvicides based on Ls, as reviewed by Charles et al. (1996). According
to the Insect Resistance Action Committee (www.irac-online.org), the
insecticidal toxins from Ls are classified into the mode of action group
11 (Moa11), along with Bti, and those agents are defined as bacterial
disruptors of insect midgut membranes. The midgut of mosquito larvae
is the central site for the action of these toxins, since they act following inges-
tion, are processed under specific conditions in this environment and they
act on specific receptors located on the epithelium, to cause mortality of lar-
vae. More details of insect midgut are provided in Chapter 1.
Mosquitoes are the principal targets of the Ls toxins and this is reflected in
the activity spectrum of the individual toxins. However, Ls toxicity to
Phlebotomus sandflies has been reported for high-toxicity strains 1593 and
2362 that may result in larval death and reduced fecundity of surviving
insects (Penner and Wilamowski, 1996; Robert et al., 1997, 1998;
Wahba, 2000). Strain 2362 also showed low toxicity against Lutzomyia
sandflies (Wermelinger et al., 2000). In addition, larvicidal effects of Ls
extracts against the nematode Trichostrongylus colubriformis have also been
reported (Bone and Tinelli, 1987) and some toxicity was seen against the
crustacean Palaemonetes pugio (Key and Scott, 1992).
Within the mosquitoes, there is differential toxicity to the species studied.
The most susceptible are Culex spp, in particular, those from the Cx. pipiens
complex, but one exception in this genus is Cx. cinereus larvae (Nicolas and
Dossou-Yovo, 1987). Anophelines including species of medical impor-
tance such as An. gambiae, An. stephensi, An. albimanus, An. quadrimaculatus,
An. darlingi and An. nuneztovari are also susceptible to the Bin toxin
(Arredondo-Jimenez et al., 1990; Davidson, 1989; Karch et al., 1992; Lacey
et al., 1988b; Rodrigues et al., 1998, 1999; Young et al., 1990). Aedes or
Ochlerotatus show a variable scenario including susceptible species such as
Oc. atropalpus, Ae. vexans and Oc. trivittatus, as well as Ae. aegypti larvae that
are refractory to Bin toxin (Berry et al., 1993; Delecluse et al., 2000;
Nielsen-Leroux and Charles, 1992). The lethal concentration (LC) of Ls
for these larvae is between 100- and 1000-fold higher than the respective
LC for Cx. pipiens larvae (Thiery and de Barjac, 1989). The screening of Ls
activity has also demonstrated susceptible larvae from Psorophora and Mansonia
species. On the other hand, Simulium larvae that are susceptible to Bti cannot be
Microbial Toxins for Mosquito Control 97

targeted by the Bin toxin. Table 3.2 presents a non-exhaustive list of mosquito
susceptibilities to Ls strains. The most common species targeted by Ls in field-
control trials or programmes are described in Section 4.

2.2. Binary toxin

The Bin toxin, comprising the BinA and BinB proteins, is the best
characterised of the toxins from Ls. It is produced during early sporulation
by highly mosquitocidal strains (El-Bendary et al., 2005; Kalfon et al., 1984)
and is deposited as a parasporal crystalline inclusion within the exosporium
(Davidson and Myers, 1981; Kalfon et al., 1984; Yousten and Davidson,
1982). In these strains, Bin contributes to the majority of the toxicity and
this fact, in turn, is a factor in the relative ease with which mosquitoes
can develop resistance to Ls (see Section 5). Bin toxin acts in the midgut
and the major steps of its mode of action in culicid larvae are: ingestion
of crystals; dissolution of the crystal matrix under the alkaline pH conditions
and release of Bin protoxin in the midgut lumen; processing of the Bin
protoxin into active toxin; binding of the active toxin to specific receptors
available on the midgut epithelium; occurrence of cytopathological effects
on the midgut are followed by larval death, which is provoked by

Table 3.2 Sensitivity of mosquito species to Lysinibacillus sphaericus

Susceptibility (strain
Species tested) References
Culex pipiens High (1593, 2013-4); Thiery and de Barjac (1989), Wraight
moderate (SSII-1) et al. (1987)
Cx. High (1593); moderate Cheong and Yap (1985), Mulligan
quinquefasciatus (SSII-1) et al. (1978), Wraight et al. (1987)
Cx. nigripalpus High (1593, 1404, SSII-1) Ramoska et al. (1977)
Cx. salinarius High (1593) Wraight et al. (1987)
Cx. restuans High (2013-4) Wraight et al. (1987)
Cx. tarsalis High (1593) Mulligan et al. (1978)
Cx. cinereus Refractory (2362) Nicolas and Dossou-Yovo (1987)
Culiseta High (1593) Wraight et al. (1987)
melanura
Mansonia High Cheong and Yap (1985)
uniformis
Continued
98 Maria Helena Neves Lobo Silva Filha et al.

Table 3.2 Sensitivity of mosquito species to Lysinibacillus sphaericuscont'd

Susceptibility (strain
Species tested) References
Psorophora High (1593); moderate Ramoska et al. (1977)
columbiae (SSII-1)
Anopheles High (multiple strains) Davidson (1989), Thiery and de Barjac
stephensi (1989)
An. gambiae High (2362) Davidson (1989), Nicolas et al. (1987)
An. albimanus High (2362) Davidson (1989)
An. High (2362) Davidson (1989), Young et al. (1990)
quadrimaculatus
An. darlingi High (2362) Rodrigues et al. (1998, 1999)
An. High (2362) Rodrigues et al. (1998, 1999)
nuneztovari
An. braziliensis High (2362) Rodrigues et al. (1998, 1999)
An. Moderate Cheong and Yap (1985)
balabacensis
Ochlerotatus High Mulligan et al. (1978)
nigromaculis
Oc. atropalpus High Berry et al. (1993)
Oc. intrudens High (1593) Wraight et al. (1987)
Oc. triseriatus Moderate (SSII-1); low Wraight et al. (1987)
(1593)
Oc. canadensis Moderate (SSII-1) Wraight et al. (1987)
Oc. fitchii Moderate (1593) Wraight et al. (1987)
Oc. stimulans Moderate (1593); low Wraight et al. (1987)
(SSII-1)
Oc. Moderate (1593, 1404, Ramoska et al. (1977)
taeniorhynchus SSII-1)
Aedes vexans Moderate Wraight et al. (1987)
Ae. aegypti Refractory (1593); low Thiery and de Barjac (1989), Wraight
(SSII-1 vegetative cells) et al. (1987)
Toxorhynchites Refractory (2362, 2297) Lacey et al. (1988b)
rutilus
Microbial Toxins for Mosquito Control 99

mechanisms that are still under investigation. Recently, activity of Bin toxins
against human cancer cells has also been reported (Luo et al., 2014).
The analysis of Bin proteins isolated from parasporal crystals was reported
in the mid-1980s (Baumann et al., 1985; Narasu and Gopinathan, 1986) and
initially it appeared that they were derived from a larger precursor protein
(Broadwell and Baumann, 1986). Subsequent cloning of the gene encoding
BinA (Berry and Hindley, 1987; Hindley and Berry, 1987) and BinB
(Baumann et al., 1987, 1988) showed that, in fact, the two components were
produced from a single operon as independent proteins of approximately
42 and 51 kDa, respectively. The proteins are produced in approximately
equimolar amounts and form a co-crystal in sporulating Ls whereas the indi-
vidual components expressed in recombinant Ls did not form crystals
(Charles et al., 1993). The combination of BinA + BinB forms crystals in
Ls and Bt strains but not in recombinant B. subtilis (Baumann and
Baumann, 1991; Broadwell et al., 1990a; Charles et al., 1993; Yuan
et al., 1999) suggesting that the former, insect pathogenic bacteria, encode
a factor that facilitates crystallisation and that is absent from B. subtilis. Bin
protein synthesis is enhanced by recombinant co-expression of the P20 pro-
tein from Bt (Park et al., 2007) but a region downstream of the bin operon in
Ls strain 2297 reduces Bin synthesis (Park et al., 2009). The activity of the
Bin toxin appears to be synergistic with the Cyt1Aa protein from Bt when
the two are co-expressed in acrystaliferous Bt (Li et al., 2000) but expression
of Cyt1Ab in Ls did not show synergy although it did help to overcome Bin
resistance in Culex larvae (Thiery et al., 1998). Other studies have shown
synergy of Bt Cyt and Cry toxins with Ls against wild-type or Bin-resistant
Culex, which may indicate synergy with Bin toxins although the use of Ls
cells in these assays may also indicate synergy with other toxins that they pro-
duce (see below) (Wirth et al., 2000a,c, 2001a, 2004).
Circular dichroism analysis has suggested that BinA and BinB are predom-
inantly composed of beta sheet (Hire et al., 2009; Kale et al., 2013;
Srisucharitpanit et al., 2012) although BinB in wild-type and truncated forms
has also been reported to contain considerable alpha helix (Tangsongcharoen
et al., 2011). Crystallization of BinB protein (Chiou et al., 1999;
Srisucharitpanit et al., 2013) and BinA/BinB co-crystals (Smith et al., 2004)
have been described and the structure of BinB has recently been published
(Srisucharitpanit et al., 2014). This protein has an N-terminal domain with
a beta-trefoil architecture found in lectins and a C-terminal region rich in
extended beta sheets that shows structural similarity to aerolysin beta pore for-
ming toxins. These results may suggest a role for the N-terminal region in
100 Maria Helena Neves Lobo Silva Filha et al.

receptor binding and roles for the C-terminal region and the related BinA
structure in formation of a beta pore. Association of the two proteins with
each other and the membrane may result in conformational changes to the
structures (Boonserm et al., 2006; Kale et al., 2013).
As described, the ingestion of the Bin proteins by mosquito larvae results
in their solubilisation in the alkaline environment of the gut (Charles, 1987)
and activation of the protoxin forms by proteolytic cleavage mediated by gut
proteinases (Aly et al., 1989; Broadwell and Baumann, 1987; Brownbridge
and Margalit, 1987; Davidson et al., 1987, 1990). These will activate both
subunits BinA (51 kDa) and BinB (42 kDa) into smaller polypeptides of
43 and 39 kDa, respectively. After proteolysis, the 43 kDa BinB derivative
results from the removal of 21 and 53 residues from the N- and C-termini,
respectively (Clark and Baumann, 1990). For the BinA active fragment of
39 kDa, cleavage of 10 and 17 amino acids occur in these respective posi-
tions (Broadwell et al., 1990c). The correct processing of the Bin subunits
and their presence in equimolar amounts are essential conditions that assure
the optimal activity of this Bin toxin (Broadwell et al., 1990b; Davidson
et al., 1990; Nicolas et al., 1993; Oei et al., 1990). Similar patterns of
protoxin cleavage occur on exposure to digestive enzymes from non-
susceptible larvae, indicating that the protoxin processing is not the origin
of insect specificity (Nicolas et al., 1990). In solution, NaOH solubilised
BinA/BinB crystal proteins may associate into a BinA2BinB2 heterotetramer
but this association may be lost on trypsin activation (Smith et al., 2005) with
activated proteins showing weak interactions between the two proteins
(Kale et al., 2013), although formation of oligomeric complexes between
activated toxins has also been suggested (Srisucharitpanit et al., 2012).
The solubilised and active toxin binds regionally to the larval midgut in
the gastric caecum and posterior midgut (Davidson, 1988, 1989; Mulla et al.,
1984a; Oei et al., 1992) leading to toxicity. In Culex larvae, the BinB com-
ponent of the toxin is responsible for receptor binding and the BinA com-
ponent subsequently binds to BinB or the BinB/receptor complex (Charles
et al., 1997; Oei et al., 1992). The situation in An. gambiae appears a little
more complex with a possible role for BinA in binding as well (Charles
et al., 1997). The receptor for Bin binding has been identified in Cx. pipiens
as a midgut-bound -glucosidase (Silva-Filha et al., 1999). Orthologs of this
protein have been identified in other mosquito species including An. gambiae
(Opota et al., 2008) and the refractory Ae. aegypti (Ferreira et al., 2010) and
differences in this receptor are believed to be the crucial factor in determin-
ing sensitivity (Section 3). Changes in the receptor are also known to cause
resistance to the Bin toxin (Section 5).
Microbial Toxins for Mosquito Control 101

Toxin binding to the midgut receptors is essential, and soon after the
ingestion of Bin crystals, cytopathological alterations can be observed.
The pathogenesis associated with Ls treatment was first described based
on the study on the action of strain SSII-1, which produces only Mtx toxins,
on Cx. quinquefasciatus larvae (Davidson, 1979). Subsequently, studies
showed the cytopathological alterations in midgut cells of larvae treated with
different strains all of which produced the Bin toxin as their major toxin
(Charles, 1987; de Melo et al., 2008; Silva Filha and Peixoto, 2003;
Singh and Gill, 1988). For Cx. pipiens, the major alterations observed in
the midgut cells are the intense disruption of microvilli, intense cytoplasmic
vacuolisations (or cytolysosomes) with broken membranes, pronounced
swelling of mitochondria and break-down of the endoplasmatic reticulum.
Ultra-structural effects investigated using an in vitro processed form of Bin
toxin to treat Cx. pipiens-cultured cells, showed similar effects as those seen
in midgut cells from larvae (Davidson and Titus, 1987). The study of Singh
and Gill (1988) also recorded damage in neural and muscles tissues that were
detected later than the major effects that were primarily observed in the mid-
gut cells. Treatment of resistant Cx. pipiens larvae that lacked the midgut
receptors with high concentration of Bin toxin showed minor alterations
that were comparable with those observed for Ae. aegypti, a refractory species
that does not have functional receptors in their midgut (Charles, 1987; de
Melo et al., 2008). Physiological studies of the Bin action are not available
except for one that shows an inhibition of the oxygen uptake of mitochon-
dria and in the activity of the enzyme choline acetyl transferase in larvae
treated with the Bin toxin (Narasu and Gopinathan, 1988).
The mode of action of the Bin toxin, following receptor binding, remains
somewhat unclear. Many reports have suggested that to exhibit toxicity, both
BinA and BinB components are absolutely required (Broadwell et al., 1990b;
Charles et al., 1993; Nicolas et al., 1993; Oei et al., 1990), with optimal activity
reported when components are present in approximately equimolar amounts
(Davidson et al., 1990). Nevertheless, toxicity of BinA in purified form (Hire
et al., 2009) or produced in B. subtilis (de la Torre et al., 1989) or in Bt (Nicolas
et al., 1993) has been reported by some authors. When the Cx. pipiens Bin
receptor Cpm1 was expressed in Madin Darby canine kidney cells, patch-
clamp experiments showed that toxin binding is followed by the induction
of currents that are likely to be due to the opening of pores (Pauchet et al.,
2005). Experiments with Culex cells in culture (Cokmus et al., 1997) and arti-
ficial membranes (Schwartz et al., 2001), also suggest that the toxin may be able
to form pores and indicated that BinA was better able to form pores than BinB,
consistent with the model whereby BinB is the receptor-binding component
102 Maria Helena Neves Lobo Silva Filha et al.

and BinA forms a pore. In contrast, a separate report described the ability of
BinB to interact with artificial membranes and form pores in the absence of
BinA (Boonserm et al., 2006). This pore formation was proposed to be
through membrane insertion of beta sheet rather than alpha helical structures.
Thus, an alternative model for Bin toxicity may involve receptor binding and
pore formation by BinB coupled with an unknown role for BinA or by a
BinA/BinB complex and this may be supported by the crystal structure of
BinB (Srisucharitpanit et al., 2014).
In addition to the possibility of pore formation, a further effect is char-
acteristic of Bin intoxication. The vacuolisation of target cells is seem
(Charles, 1987; Davidson, 1988; Pauchet et al., 2005) accompanied by
the uptake of labelled toxins into vesicles (Davidson, 1988), a phenomenon
that only occurs when both BinA and BinB components are present
together (Oei et al., 1992). A detailed study of this phenomenon was carried
out using Madin Darby cells expressing the Bin receptor Cpm1 (Opota
et al., 2011). This investigation showed the opening of cationic pores in
the membrane and demonstrated that the large vacuoles formed in target
cells were autophagic. These structures were transient but, having dis-
appeared from the cells, these vacuoles then reappeared following cell divi-
sion: a novel phenomenon termed post-mitotic vacuolation. The uptake of
Bin into the cells, along with their receptor, was shown to be via recycling
endosomes; structures that are distinct from the large transient autophagic
vesicles. Thus, Bin intoxication induces autophagy, while Bin uptake in sep-
arate structures protects it from degradation by targeting to recycling path-
ways. The overall significance of these events for toxicity remains to be
clarified but Bin trafficking may allow it access to tissues beyond the midgut.
The BinA and BinB proteins are related to each other and to a family of
Bin-like proteins including Cry49 from Ls (see below), Cry35 and Cry36 from
Bt, and sequences of unknown function from B. cereus group strains (e.g. acces-
sion number ZP_17404242) and Chlorobium phaeobacteroides (accession num-
ber Y_911930) (Baumann et al., 1988; Jones et al., 2007). Cry36 acts alone to
cause insect mortality, whereas Cry35 requires the 14 kDa Cry34 protein for
toxicity and Cry49 requires the three-domain family toxin Cry48 for its func-
tion. The various interactions that this family of proteins may require for
toxicity, further complicates our understanding of their modes of action.
The Bin toxin proteins themselves are highly conserved. Strains isolated
from around the world produce Bin toxins (Priest et al., 1997) but only six
variants of BinA (in which nine amino acids are altered) and four variants of
BinB (in which six amino acids are altered) have been described (Hire et al.,
2009; Humphreys and Berry, 1998; Priest et al., 1997). Two variants, Bin1
Microbial Toxins for Mosquito Control 103

and Bin2 have been shown to share the same receptor (Silva-Filha et al.,
2004) and cross-resistance between variants is seen (Yuan et al., 2003). Nev-
ertheless, Bin toxin variants can show differential activity against mosquito
targets. BinA variants were shown to alter the activity against the marginal
target Ae. aegypti and to alter the progression of growth and mortality for Cx.
quinquefasciatus and these effects were localised to amino acids 99 and 104 in
this protein (Berry et al., 1993). Reciprocal exchange of the amino acid at
position 93 of the BinA protein between the BinA2 variant, which is highly
active against Cx. pipiens larvae and the BinA4 variant, which is non-toxic to
this insect, showed that this residue was also a key determinant of activity
(Yuan et al., 2001). Deletion experiments have defined the essential core
regions of the Bin toxins. BinA can be truncated by 17 residues at both
the N- and C-termini without loss of toxicity. BinB can be truncated by
34 residues at the N-terminus and 53 residues at the C-terminus without
loss of toxicity (Broadwell et al., 1990c; Clark and Baumann, 1990, 1991;
Limpanawat et al., 2009; Oei et al., 1990; Sebo et al., 1990). Analysis of
the binding of non-toxic variants suggested that the N-terminal region of
BinB may have a role in interaction with the receptor and its
C-terminus, along with both the N- and C-termini of BinA may be
involved in the interaction of the two proteins (Oei et al., 1992). Predictions
of structural disorder within the BinA and BinB proteins have suggested that
the N- and C-termini may be flexible, consistent with a role in protein
protein interactions (Kale et al., 2013). More detailed analysis of the
N-terminal region of BinB and receptor binding, confirmed the importance
of residues 33158 in this interaction and, particularly, the sequences Ile-
Arg-Phe (residues 8587) and Phe-Gln-Phe (residues 147149) (Romao
et al., 2011). Mutagenesis studies on BinB indicated that individual substi-
tution of Pro35, Glu36, Phe41 and Tyr42, resulted in reduced activity but all
were able to interact with BinA (Singkhamanan et al., 2013). Pro35 and
Phe41 to alanine substitutions could bind to the larval midgut at a compa-
rable level to the wild-type BinB but binding of the Tyr42 to alanine mutant
was reduced and the Pro35Ala replacement decreased penetration of the
membrane. Block mutation of BinB from residues 113150 showed the
protein to have some tolerance to mutation in this region (Singkhamanan
et al., 2010). Alanine replacement of individual amino acids Phe149 and
Tyr150 resulted in loss of toxicity and loss of midgut binding for the latter
mutant. Toxicity could be rescued by replacement of these two residues
with other aromatic residues. In BinB, the substitution of Cys67 or
Cys161 reduced BinB interaction with BinA and eliminated toxicity while
replacement of Cys241 had no effect (Boonyos et al., 2010). In similar
104 Maria Helena Neves Lobo Silva Filha et al.

experiments with BinA, alanine or serine replacement drastically reduces

(Cys195) or abolishes (Cys31, Cys47) activity (Promdonkoy et al., 2008).
These three cysteine residues in each protein were shown not to be involved
in disulphide bonding and the nature of their roles is not clear at present.
Substitution of charged residues with alanine in BinA produced a loss
(Arg97) or reduction in activity (Glu98, Arg101, Glu114) but no change
in the ability of BinA to interact with BinB (Sanitt et al., 2008). Substitution
of Arg312 has also been shown to eliminate toxicity (Elangovan et al., 2000).
Replacement of tryptophan residues at positions 222 and 226 in BinA pro-
duces proteins that are still able to permeabilise liposomes but which have
lost biological activity (Kunthic et al., 2011). It has been shown that alanine
mutants in both BinA and BinB can eliminate toxicity and that, for each pro-
tein, a non-toxic variant mutated close to the N-terminus and a non-toxic
variant mutated close to the C-terminus, can complement each other to pro-
duce a toxic combination (Shanmugavelu et al., 1998).

2.3. Cry48/Cry49
While the Bin toxin is the major sporulation-associated toxin in most Ls
strains, the ability of the spores of a small number of strains to overcome
Bin resistance in mosquitoes, indicated the presence of another toxin in
these strains and an approximately 49 kDa protein was identified as a candi-
date (Pei et al., 2002; Shi et al., 2001). The genes encoding the Cry49 pro-
tein and a second crystal protein (Cry48) related to the three-domain toxins
of Bt has been cloned and expressed ( Jones et al., 2007) and show a very
narrow target range, so far active only against Culex mosquitoes ( Jones
et al., 2008). It is of interest that the two proteins form a novel type of
Bin toxin since both components are necessary for activity and no other
three-domain protein has ever been shown to have a requirement for
another protein for its activity ( Jones et al., 2007). Consistent with the pres-
ence of a Bin-type toxin and a three-domain toxin, the cytopathology of
Culex cells exposed to the Cry48/Cry49 toxin shows features of both toxin
types, including the vacuolation observed on Bin intoxication (de Melo
et al., 2009) (Fig. 3.2). Similar effects were seen when Bin susceptible larvae
were treated with the synergistic combination of Bin and Cry11Aa toxins.

2.4. Mosquitocidal toxin 1

The mosquitocidal toxin 1 (Mtx1) was first discovered in Ls strain SSII-1, a
strain that shows low toxicity and lacks the Bin toxin (Thanabalu et al.,
Figure 3.2 Cytopathological effects of Cry48Aa/Cry49Aa toxin to Culex quinquefasciatus.
Transverse ultrathin sections of the posterior midgut from fourth instar larvae treated
with Cry48Aa/Cry49Aa toxin from Lysinibacillus sphaericus IAB59 strain. (A) Cells from
a non-treated larva rich in microvilli (Mv) and mitochondria (Mt); (B) cells from
1 h-treated larva showing mitochondrial vacuolation (Mt); (C) cells from 1 h-treated larva
presenting electron-dense granules (*), cytoplasmic vacuoles (V) and small vesicles from
endoplasmic reticulum breakdown; mitochondria (Mt); (D) cells from 1 h-treated larva
with preserved microvilli (Mv) and mitochondrial swelling (Mt) at the apical side of
the cell; (E) cells from 6 h-treated larva showing evident microvilli damage (Mv) and
mitochondrial vacuolation (Mt). (F) Cells from 6 h-treated larva presenting large
cytoplasmic vacuoles (V) and small ribosome-coated vesicles (arrows); mitochondria
(Mt). Scale bars 1 m. Taken from de Melo et al. (2009).
106 Maria Helena Neves Lobo Silva Filha et al.

1991). The toxin is present in many high- and low-toxicity strains but is lac-
king in some strains that produce Bin toxin such as LP1-G (Liu et al., 1993;
Priest et al., 1997) and is present as a disrupted pseudogene in others (e.g.
strain C3-41) (Hu et al., 2008). The toxin is active against both Cx.
quinquefasciatus and Ae. aegypti larvae but has no effect on the mosquito
Toxorhynchites splendens (Thanabalu, 1992; Thanabalu et al., 1992a). It also
has low-level toxicity against Chironomus riparus (Partridge and Berry,
2002). Mtx1 acts synergistically with Mtx2 from Ls (Rungrod et al.,
2009) and Cry11 but is antagonistic with Cyt1Aa (Wirth et al., 2007). In
combination with other toxins from mosquitocidal Bt, synergy can also
be seen and mosquitoes resistant to individual Bt toxins may show some
cross-resistance to Mtx1 (Wirth et al., 2014).
The gene encoding Mtx1 is preceded by an inverted repeat region, char-
acteristic of a binding site for a regulatory protein (Thanabalu, 1992). The
production of a reporter protein driven by the mtx1 promoter was found to
be higher in B. subtilis than in Ls, suggesting regulation in the latter species
(Ahmed et al., 1995) and it has been speculated that the gene upstream of
mtx1 may encode a BglG family regulator of Mtx1 production (Berry,
2012). The toxicity of purified Mtx1 is high, showing equivalent potency
to the Bin toxin (Thanabalu et al., 1992a) and the low-level toxicity of
Ls strain SSII-1 is thought to be due to low levels of Mtx1 production
and to the degradation of Mtx1 by the producing bacterium (Thanabalu
and Porter, 1995). The proteinase responsible for this degradation has been
identified (Wati et al., 1997; Yang et al., 2007b) and is known as sphericase/
sfericase (Almog et al., 2003; Yoshida et al., 1977). Some success in
protecting the toxin by expression in proteinase negative strains of Ls has
been reported (Thanabalu and Porter, 1995) but the broad specificity of
sfericase has blocked attempts to stabilise the toxin by mutagenesis (Yang
et al., 2007b).
Produced as a 100 kDa protein, Mtx1 is processed by gut enzymes to an
approximately 27 kDa moiety, with regional sequence similarity to ADP-
ribosylating toxins and a C-terminal, 70 kDa moiety containing lectin-like
beta-trefoil repeat sequences (Hazes and Read, 1995; Thanabalu et al.,
1992a). This cleavage produces the two subunits that enable Mtx1 to func-
tion as a two component, AB toxin in which the 70 kDa protein is expected
to act as the receptor-binding unit, probably mediated by the lectin-like
motifs. This is expected to allow the enzymatic subunit (27 kDa) to enter
the susceptible cells where it will modify target proteins by ADP-
ribosylation. The C-terminal 70 kDa protein is able to cause morphological
Microbial Toxins for Mosquito Control 107

changes in Cx. quinquefasciatus and Ae. aegypti cells in culture but not to An.
gambiae cells or human HeLa cells (Thanabalu et al., 1993). However, both
components are required for toxicity to insects. The ADP-ribosyl transferase
subunit has been shown to promote ADP-ribosylation of Mtx1 components
and proteins of 38 and 42 kDa from Cx. quinquefasciatus cell extracts
(Thanabalu et al., 1993). Following activation with chymotrypsin, the
kinetics of the Mtx1 ADP-ribosyl transferase have been elucidated with
respect to the NAD+ substrate using soybean trypsin inhibitor as an artificial
substrate and ribosylation is seen to occur at arginine residues (Carpusca
et al., 2006; Schirmer et al., 2002a). The ADP-ribosylation activity of the
catalytic subunit can be inhibited by the 70 kDa protein, which binds
non-covalently, mediated by residues Asp273 and Asp275, and a
C-terminal region (Carpusca et al., 2004). Furthermore, transfection of
the catalytically active subunit into HeLa cells produced cytotoxic effects
(Schirmer et al., 2002a), suggesting that specificity of Mtx1 is due to the
70 kDa component. Expression of the enzymatic subunit alone in Escherichia
coli proved problematic due to toxicity of the protein to host cells. This was
shown to be due to ADP-ribosylation of the bacterial elongation factor Tu
by the toxin (Schirmer et al., 2002b).
The structure of the catalytic subunit (with a small sequence from the
N-terminus of the 70 kDa protein) has been solved (Reinert et al., 2006)
as has the structure of the full-length protoxin (Treiber et al., 2008). In
the latter form, the putative receptor-binding moiety (70 kDa subunit)
can be seen with the four lectin domains curled around the catalytic domain
and the authors propose a mechanism for the toxin whereby proteolytic acti-
vation occurs at the exposed site between the two subunits; the binding sub-
unit then interacts with glycolipid via the lectin-like domains and is
endocytosed with the catalytic unit non-covalently attached. At low pH,
the Mtx1 forms a heptamer, membrane insertion of N-terminal segments
occurs, leading to translocation of the catalytic core into the cytosol. This
translocation also separates the enzyme from the 70 kDa protein, thereby
freeing it from inhibition.

2.5. Other Mtx toxins

A further family of related proteins, synthesised during the vegetative phase,
has also been identified in Ls strains. The best studied member of this family,
Mtx2, was the second insecticidal toxin to be isolated from the low-toxicity
strain SSII-1 (Thanabalu and Porter, 1996). It is unrelated in structure or
108 Maria Helena Neves Lobo Silva Filha et al.

function to Mtx1 but is related to Mtx3, later identified in SSII-1 (Liu et al.,
1996), and Mtx4 identified in the genome of the high-toxicity strain C3-41
(Hu et al., 2008), a strain that also encodes Mtx2 and Mtx3 along with a
pseudogene related to this family of proteins, indicating probable gene
duplication in this family of genes (Berry, 2012). In addition, the proteins
are related to Clostridium epsilon toxin and Cry15, Cry23, Cry33 and
Cry38 from Bt (de Maagd et al., 2003) in the Pfam ETX_MTX2 superfamily
of proteins. The similarity to epsilon toxin suggests that these Mtx toxins act
by pore formation. Mtx3 is active against Cx. quinquefasciatus larvae and is
highly conserved, with only one conservative amino acid replacement
reported (Liu et al., 1996). Most studies on this family have focused on
Mtx2. This protein shows less conservation than Mtx3. Although a series
of individual mutations in the mtx2 genes did not alter their toxicity to
Cx. quinquefasciatus, one mutant, from lysine to threonine at residue 224,
abolished activity. Double mutants at positions 224 and 279 significantly
affected activity against this insect and Ae. aegypti so that specificity can,
effectively, be switched between the two species (Chan et al., 1996). The
Mtx2 family of proteins features a putative N-terminal signal sequence,
although their secretion has not been confirmed. Mtx2 can be truncated
by up to 23 amino acids at its N-terminus (including this signal sequence)
without loss of activity but deletion of only five residues from the
C-terminus produced inactive toxin (Phannachet et al., 2010). Mtx2 shows
instability on exposure to conditioned media or the Ls proteinase sfericase
(Yang et al., 2007b) so that, like Mtx1, it appears to be a rather short-lived
toxin. Mtx2 is synergistic with Cyt1Aa and Cry11Aa (Wirth et al., 2007)
while interactions with other Bt toxins is somewhat complex with some
cross-resistance exhibited (Wirth et al., 2014).

2.6. Sphaericolysin
Sphaericolysin is a cholesterol dependent cytolysin that was discovered in Ls
strain A3-2 that was isolated from the crop of Myrmeleon bore (ant lion) larvae
(Nishiwaki et al., 2007). The protein shows injection toxicity against the
cockroach, Blattella germanica, and, to a lesser extent, against the common
cutworm, Spodoptera litura. Sphaericolysin induces pores in erythrocyte
membranes and inserts to form a complex with an external diameter of
around 35 nm. The residue Tyr187, equivalent to Tyr159 in perfringolysin
is important in pore formation and the Tyr to alanine mutant markedly
reduced haemolytic activity as did dosing with cholesterol. A cholesterol
binding motif is located close to the C-terminus of the protein and is
Microbial Toxins for Mosquito Control 109

assumed to have a role in membrane interaction. The sequence of

sphaericolysin includes an N-terminal signal peptide and secretion of the
protein has been demonstrated in vivo in S. litura larvae (Nishiwaki
et al., 2007).
The sphaericolysin family of proteins appears to be well conserved in Ls
strains, even outside the DNA group 11A that is normally associated with
insecticidal activity. A gene with 100% sequence identity to that from strain
A3-2 has been found in the group IIB type-strain 7055 (C. Berry,
unpublished) and a closely related (91.4% identical) protein has been iden-
tified in the Ls strain B354, belonging to DNA homology group V (From
et al., 2008). Evidence for the presence of related genes in DNA group IV
was also shown. The strain B354 protein was shown to cause haemolysis and
osmotic protection assays suggested formation of a pore of 57 nm.
Very closely related proteins are common amongst other Gram positive
bacteria including the B. cereus group and Paenibacillus alvei (Fig. 3.3). The
crystal structure of one of these homologs, anthrolysin, has been solved
(PDB accession number 3CQF) and, based on this, a model of the
sphaericolysin structure can be built (Fig. 3.4).

2.7. S-layer proteins

A recent report has indicated that the SlpC S-layer proteins, produced dur-
ing the vegetative stage of growth of Ls strains OT4b25, OT4b26, III(3)7
and 2362, can show insecticidal activity against Cx. quinquefasciatus mosqui-
toes (Lozano et al., 2011). This is in contrast to a previous report that the
S-layer protein of strain 2362 is non-toxic (Bowditch et al., 1989). SlpC
from Ls strain C3-41 also shows no toxicity and has been the subject of
extensive study (Hu et al., 2008; Li et al., 2013). Previously, the insecticidal
activity of a Bt S-layer protein has been reported (Pena et al., 2006).
Although the toxic variants of the Ls SlpC were compared to other SlpC
proteins from Ls in terms of % identities (>94%), specific comparisons
and nucleotide accession numbers were not published (Lozano et al.,
2011) so that identification of potential residues responsible for toxicity is
not possible at this time.

2.8. Safety issues

The safety of Ls to non-target organisms is one of the most prominent fea-
tures of this entomopathogenic bacterium. It is innocuous for most organ-
isms and this brings the advantage that the lack of negative effects on natural
predators allows the possibility of co-introducing other biological agents in
110 Maria Helena Neves Lobo Silva Filha et al.
Microbial Toxins for Mosquito Control 111

breeding sites to achieve an additional level of control. The known insec-

ticidal toxins from Ls strains have narrow spectra of action, and screening
of other possible target species for these toxins confirmed the high specificity
within the Diptera group, which is limited to culicid species, further
restricted with respect to some Aedes and Ochlerotatus species (Table 3.2).
The safety for other invertebrate species, especially those insects and other
organisms in co-habitation with mosquito larvae in the aquatic environ-
ment, has been recorded through laboratory evaluations as well as field trials.
Among the groups of organisms that are not affected by standard doses of Ls
are associated fauna of invertebrates and vertebrates of breeding sites, which
includes macroinvertebrates, insects belonging to Odonata, Coleoptera,
Diptera orders, crustaceans, amphibians and fish (Brown et al., 2004;
Davidson et al., 1977, 1984; Lacey and Mulla, 1990; Lacey and Siegel,
2000; Mulla et al., 1984b; Mulligan et al., 1978; Rodcharoen et al., 1991;
Sternberg et al., 2012; Yousten, 1984a; Yousten et al., 1991, 1992).
A more extended field study on wetlands, evaluated the possible impact
of Ls treatments for mosquito control on invertebrates associated with this
environment (Merritt et al., 2005). Quantitative parameters to measure
abundance and diversity throughout a 3-year period indicated the lack of
effects due to exposure in the groups analysed. The effects of Ls on mammals
were assessed in the early days of its development and studies based on spe-
cific procedures confirmed the safety of this microbial agent for the group
(Shadduck et al., 1980; Siegel and Shadduck, 1990).
Another safety concern is the fate of Ls spores in the environment. Lab-
oratory and field studies showed that spores can recycle in specific condi-
tions, for instance, in the larval cadavers but this feature has been seen
rather as an advantage, because it can provide extended control in some
breeding sites (Charles and Nicolas, 1986; Matanmi et al., 1990; Nicolas
et al., 1987). It has been also demonstrated that recycling does not occur

Figure 3.3 Alignment of sphaericolysin and related proteins. Identical residues are
shaded green or cyan, spaces introduced to optimise the alignment are shaded grey.
The sequences shown are Ls A3-2 (sphaericolysin from Lysinibacillus sphaericus strain
A3-2, accession number BAF62176), Ls B354 (sphaericolysin from L. sphaericus strain
B354, accession number EU043116), Bcer (anthrolysin from Bacillus cereus accession
number ZP_03231124), BtkAlv (alveolysin from B. thuringiensis subsp. kurstaki accession
number ZP_04117355), P alv (P. alvei alveolysin accession number ZP_10866820),
B14905 (perfringolysin O precursor from Bacillus sp B14905 accession number
ZP_01724867). Alignment generated using ClustalX (Thompson et al., 1994).
112 Maria Helena Neves Lobo Silva Filha et al.

Figure 3.4 Modelled structure of sphaericolysin. The structure of sphaericolysin from

Lysinibacillus sphaericus strain A3-2 was generated using Swiss model (Schwede
et al., 2003) based on the structure of anthrolysin O from Bacillus anthracis (sequence
identical to the B. cereus anthrolysin shown in Fig. 3.3, PDB accession number 3CQF,
Bourdeau et al., 2009). Model coloured according to secondary structure: alpha helix
(brown), beta sheet (yellow), other (grey). The putative cholesterol binding loop is
shown in green and residue Tyr 187 in blue.

in a number of environments such as treated water and dried environments

that are re-flooded, which do not ensure the conditions needed for the spore
germination (Davidson et al., 1984). Investigation of the fate of spores in
non-target invertebrates shows that they are eliminated and suggested that
their growth was not maintained in these organisms (Yousten et al.,
1991, 1992, 1995). The accumulation of spores in the soil and aquatic envi-
ronments can also be a concern associated with the intensive use of these
biolarvicides. (Guidi et al., 2011) performed a quantitative study of the fate
of spores after Ls application in catch basins and concluded that the number
of spores did not increase significantly during the trial and the indigenous
microbiota was not affected.
Safety studies of the Mtx and Cry48/Cry49 toxins have been limited
since these toxin factors have not been effectively used as major active prin-
ciples in the commercial preparations. However, laboratory studies have
shown their narrow spectra of action, in particular, Cry48/Cry49, and it is
likely that there will be no safety concerns related to their potential utilisation.

3. RECEPTORS OF THE BINARY TOXIN

3.1. Binding of the binary toxin to larvae midgut
The Bin toxin displays the highest activity among the set of Ls toxins. After
the ingestion of Bin crystals and successful midgut processing as described in
Microbial Toxins for Mosquito Control 113

Section 2, the activated form of the toxin recognises and binds to the midgut
epithelium of susceptible larvae. The cytopathological effects and the lethal
action observed in Bin-treated larvae depend, essentially, on the ability of
this toxin to bind to the midgut cells. Treatment of larvae with labelled-
toxin and further analysis of the midgut showed the interaction of the
Bin toxin along the epithelium, and different patterns were described
(Davidson, 1988, 1989; Oei et al., 1992). For Cx. pipiens, the most suscep-
tible species, Bin displays a marked and regionalised binding to the gastric
caeca and posterior midgut (Fig. 3.5). For anopheline larvae such as An.
gambiae, An. stephensi, An. albimanus and An. quadrimaculatus, the binding
to their midguts was variable and less defined than that found for Cx. pipiens.
For Ae. aegypti, Bin refractory larvae, this interaction was very weak, com-
pared to the Bin binding for the other species. The midgut binding patterns
observed are directly correlated to the in vivo susceptibility for the species
studied.
Quantitative assays that were performed to measure the Bin binding
affinity for the proteins from the midgut, confirmed this association.
In vitro binding assays between radiolabelled toxin and midgut brush-border
membrane fractions (BBMF) enriched with proteins from the microvilli of
apical cells, showed that Bin toxin binds with high affinity to BBMF from
Cx. pipiens and Cx. quinquefasciatus larvae (Nielsen-Leroux and Charles,
1992; Nielsen-Leroux et al., 1995, 1997, 2002; Oliveira et al., 2004;
Silva-Filha et al., 1997, 2004, 2008). Binding is saturable, reversible and

Figure 3.5 Binding of fluorescent-labelled binary toxin regionalized in the gastric caeca
(GC) and posterior midgut (PMG) of Culex quinquefasciatus larvae. Figure kindly provided
by Colin Berry, Cardiff University (UK).
114 Maria Helena Neves Lobo Silva Filha et al.

the values of the dissociation constants recorded in different studies

(Kd 520 nM) indicated the high affinity of the complex formed by the
toxin receptor in these species (Fig. 3.6). Specific binding of Bin toxin
was also detected for the BBMF of An. gambiae and An. stephensi, however,
the plateau of saturation and affinity (Kd 30110 nM) did not reach the
same levels detected for BBMF samples from the Cx. pipiens complex
(Silva-Filha et al., 1997). The LC of Bin toxin for these species is, overall,
10-fold higher than that for Cx. pipiens (Davidson, 1989). The level of
specific binding detected for the BBMF of Ae. aegypti is equivalent to the
non-specific binding detected, and the data obtained do not fit to the
ligandreceptor model (Nielsen-Leroux and Charles, 1992). The Bin LC
for this species is between 100 and 1000-fold higher, compared to that
for Cx. pipiens. The availability and binding capacity of midgut molecules
that act as receptors for the Bin toxin is likely to be the main factor that deter-
mines the status of susceptibility among related mosquito species (Table 3.3).
The findings regarding the mechanism of resistance to the Bin toxin have
reinforced the central role of the midgut receptors for the Bin toxin mode
of action (see Section 5).

3.2. Receptors
Midgut-bound proteins from insect larvae play an essential role as receptors
for insecticidal proteins from entomopathogenic bacteria. Aminopeptidases
(APNs), cadherins (CADs), alkaline phosphatases (ALPs) and -amylase
have been characterised as receptors to the Cry toxins from Bt strains in
lepidopteran and mosquito larvae (Abdullah et al., 2006; Chen et al.,
2009a,b; Fernandez et al., 2006; Fernandez-Luna et al., 2010; Hua et al.,
2008, 2009; Jurat-Fuentes and Adang, 2004; Knight et al., 1994; Luo
et al., 1997; Vadlamudi et al., 1995; Valaitis et al., 2001). More information
on receptors of Bt toxins is presented in Chapter 2. The receptors of
the Bin toxin from Ls are -glucosidases and they were characterised
in three species as follows: Cpm1 for Cx. pipiens maltase 1 (Darboux
et al., 2001; Silva-Filha et al., 1999), Cqm1 for Cx. quinquefasciatus maltase
1 (Romao et al., 2006) and Agm3 for An. gambiae maltase 3 (Opota et al.,
2008). Aedes aegypti has also a gene that encodes an orthologue of the Bin
toxin receptor, Aam1 protein (Ae. aegypti maltase 1) that displays 74% iden-
tity to the Cqm1 -glucosidase; however, Aam1 is not able to bind to Bin
toxin (Ferreira et al., 2010; Nene et al., 2007; Opota et al., 2008; Romao
et al., 2006).
Microbial Toxins for Mosquito Control 115

7
Bound 125l-toxin (pmol / mg)
Specific
6

4
Non-specific
3

0
0 50 100 150 200
125
[Free l -toxin] (nM)

7
Bound 125l-toxin (pmol / mg)

3
2

0
0 50 100 150 200
[Free 125l -toxin] (nM)

Figure 3.6 In vitro saturation binding assays between binary toxin and brush-border
membrane fractions (BBMF) from fourth instar Culex quinquefasciatus larvae from a sus-
ceptible colony (A) and from CqRL1/2362, a Lysinibacillus sphaericus-resistant colony (B).
Taken from Oliveira et al. (2004).
Table 3.3 Mosquito susceptibility to Lysinibacillus sphaericus and the capacity of the Binary (Bin) toxin to interact with larval midgut
Characteristic Culex pipiens a Cx. quinquefasciatus a Anopheles gambiae b An. stephensi b Aedes aegypti c

Larval susceptibility High High Moderate Moderate Refractory

d
Bin binding to midgut Regionalized ND Diffuse Diffuse Non-specific
Bin-specific binding to BBMFe Yes Yes Yes Yes No
Bin binding affinity or Kd (nM) 620 515 30 110 Nd
No. of receptors Bmax (pmol/mg) 38 610 5 4 Nd
Cytopathological effects Yes Yes Yes Yes Minor
a
Charles (1987), Davidson (1988, 1989), de Melo et al. (2008), Nielsen-Leroux and Charles (1992), Nielsen-Leroux et al. (1995), Nielsen-Leroux et al. (1997), Oei et al.
(1992), Silva-Filha et al. (2004), Silva-Filha et al. (2008), Silva Filha and Peixoto (2003) and Singh and Gill (1988).
b
Charles (1987), Davidson (1988, 1989) and Silva-Filha et al. (1997).
c
Charles (1987), Davidson (1988, 1989), Ferreira et al. (2010) and Nielsen-Leroux and Charles (1992).
d
Not determined.
e
Brush-border membrane fractions.
Microbial Toxins for Mosquito Control 117

Those -glucosidases (EC 3.2.1.20) belong to the -amylase family that

is composed of several enzymes that have the ability to hydrolase starch
( Janecek, 1997). This was the first amylase sensu lato identified as a receptor
of insect toxins, followed by the report of an -amylase identified as the
receptor of Cry4Ba and Cry11Aa toxins in An. albimanus larvae
(Fernandez-Luna et al., 2010). The primary role of the -glucosidases is
the digestion of carbohydrates through their ability to hydrolyse -1-4 links
between glucose residues of carbohydrates (Krasikov et al., 2001). The stud-
ies of these enzymes in mosquitoes are still scarce despite their essential role
for the digestion of carbohydrates. A recent study showed that the genes
coding for -glucosidases from Cx. quinquefasciatus, An. gambiae and Ae.
aegypti are organised in two clusters, and each one contains three to five
genes (Gabrisko, 2013). Some -glucosidases have been characterised in
the salivary glands, since these enzymes are fundamental for the hydrolysis
of sucrose, a main component of the adult diet (Marinotti and James,
1990; Marinotti et al., 1996). The -glucosidases have also importance in
the digestion of carbohydrates the activity of which is mainly located in
the posterior midgut as characterised in An. aquasalis and An. gambiae
(Souza-Neto et al., 2007; Zheng et al., 1995). In whole extracts and BBMF
from Cx. quinquefasciatus larvae, four -glucosidases were detected through
in-gel catalytic assays, using 4-methylumbelliferyl -D-glucopyranoside as a
substrate (Romao et al., 2006). Among them, only Cqm1 is recognised by a
polyclonal antibody raised against this protein and this suggests that the
remaining -glucosidases did not share a high level of identity with
Cqm1 (Romao et al., 2006).
The Cpm1 -glucosidase was the first receptor characterised for the Bin
toxin in Cx. pipiens larvae and it shares 97% and 66% identity with the Cqm1
and Agm3 orthologs, respectively. The genes encoding these proteins are
organised in a similar manner in three exons and two introns that are posi-
tioned in the same regions (Darboux et al., 2001; Opota et al., 2008; Romao
et al., 2006). The open reading frames of the Cpm1/Cqm1 and Agm3 genes
encode proteins of 580 and 588 residues, respectively (Table 3.4). Among
the conserved features observed in the protein sequences, there are four con-
served -glucosidase motifs, some predicted glycosylation sites and a con-
served signalling sequence for a glycosylphosphatidylinositol (GPI) anchor
at the C-terminal end.
Most studies of these proteins were based on their expression using a
Spodoptera frugiperda (Sf9) cell line that proved to be an adequate system
for their characterisation (Darboux et al., 2002, 2007; Opota et al., 2008).
118 Maria Helena Neves Lobo Silva Filha et al.

Table 3.4 Features of four ortholog -glucosidases from mosquito larvae

Cx. Anopheles Aedes
Culex pipiens quinquefasciatus gambiae aegypti
Cpm1a Cqm1b Agm3c Aam1d
Gene ORF (bp) 1740 1740 1764 1746
Protein no. of residues 580 580 588 582
Protein identity to 100 97 66.4 74.2
Cpm1 (%)
Protein-predicted mass 66 66 67 66
(kDa)
Protein observed mass 66 6466 67 73
( kDa)
No. of predicted 3 3 3 6
glycosylation sites
In vivo glycosylatione Ndf No Nd Yes
Location along the Caeca and Midgut Posterior Midgut
midgut posterior cells cells
-Glucosidase activity Yes Yes Nd Yes
Binding to the Bin Yes Yes Yes No
toxin
a
Darboux et al. (2002, 2007) and Silva-Filha et al. (1999).
b
Romao et al. (2006).
c
Opota et al. (2008).
d
Ferreira et al. (2010), Opota et al. (2008) and Romao et al. (2006).
e
Glycosylation was verified using PNGaseF.
f
Not determined.

The expression of Cpm1 in Madin and Darby kidney canine (MDKC) cells
was also successfully achieved. Both catalytic activity and Bin binding capac-
ity were observed, in addition, this recombinant receptor was able to medi-
ate the cytopathological effects derived from the treatment with Bin toxin
(Pauchet et al., 2005). The expression of Cpm1/Cqm1 as membrane-bound
proteins is essential for the activity of Bin toxin on the larva and they are
bound to the epithelium through a GPI anchor. These proteins can be sol-
ubilized from the midgut through the action of the enzyme pho-
sphatidylinositol phospholipase C that specifically releases GPI-bound
proteins from the cell surface (Darboux et al., 2002; Ferreira et al., 2010;
Silva-Filha et al., 1999). Mutations in the cpm1/cqm1 genes that prevent
Microbial Toxins for Mosquito Control 119

the expression of these molecules as GPI-bound proteins are the most

important resistance mechanism shown in Cx. pipiens larvae (Section 5).
A second essential feature of Cpm1/Cqm1 proteins as receptors of the
Bin toxin is their conformational state. The characterisation of these recep-
tors was performed using proteins from larvae or recombinant proteins,
under their native state, since denatured samples failed to bind the Bin toxin
(Romao et al., 2006; Silva-Filha et al., 1999). The analysis of Cqm1 showed
that its apparent molecular mass differs between 66 and 85 kDa under dena-
turing and semi-denaturing conditions, respectively, suggesting that the
native protein assumes a different conformation (Ferreira et al., 2010).
The direct binding of the Bin toxin to one class of receptors, also con-
trasts with the mode of action of Cry toxins that seem to rely on the sequen-
tial binding to two midgut receptors (Arenas et al., 2010; Bravo et al., 2004,
2007). In this model, the Cry toxins bind primarily to CADs, and this step
is essential to confer a conformational change to oligomers that are then
able to bind with high affinity to GPI-anchored receptors such as APNs
and/or ALPs. Taking into account the complexity of the interactions
between the Cry toxins and their receptors, the Bin toxin binding to its
single class of receptors can be considered a vulnerable step in mode of
action of Ls.

3.3. Comparative analysis of the Cqm1 and Aam1

-glucosidases
The comparative studies of the orthologs Cqm1 and Aam1 provided data to
understand the molecular basis of Bin toxin action on culicid larvae and, in
particular, the refractoriness of Ae. aegypti larvae. Aam1 has 74% identity and
83% similarity to Cqm1, higher levels than those found for the Agm3 pro-
tein that is the receptor for the Bin toxin in An. gambiae, a moderately sus-
ceptible species. The open reading frame of the aam1 gene is organised in a
similar fashion, however, the entire gene is significantly bigger compared to
the 1.8 kb observed for the cpm1/cqm1/agm3 orthologs, due to the great
size of the two introns found in the aam1 sequence (Nene et al., 2007). Pre-
vious studies ruled out some factors that could be the reason for the refrac-
toriness of Ae. aegypti. The processing of Bin toxin in Ae. aegypti midgut
occurs in a similar manner as in Cx. pipiens and this is not the reason for
the refractoriness of the latter species (Nielsen-Leroux and Charles,
1992). Subsequently, it was demonstrated that Aam1 is produced as a
GPI-anchored protein and this protein is expressed during larval and adult
stages. In midgut brush-border membranes from Ae. aegypti larvae, Aam1 is
120 Maria Helena Neves Lobo Silva Filha et al.

Figure 3.7 Immunoblotting of midgut brush-border proteins from Culex quin-

quefasciatus (Cq) and Aedes aegypti (Aa) larvae. Protein samples (2.520 g) were sep-
arated on SDS-PAGE, transferred to nitrocellulose membrane and submitted to
immunodetection with an anti-Cqm1 antibody. MW, molecular weight markers
in kDa. Taken from Ferreira et al. (2010).

expressed in a comparable level to that observed for Cqm1 (Fig. 3.7), how-
ever, this protein does not display capacity to bind the Bin toxin (Ferreira
et al., 2010). One major difference between Cqm1 and Aam1 is
N-glycosylation and, although both sequences present predicted sites
(Table 3.4), only Aam1 was found to have carbohydrates added that signif-
icantly increase its size, while the glycosylation of Cqm1, if present, could
not be detected (Ferreira et al., 2010). Cqm1 treatment with the endo-
glycosidase PNGase F does not abolish its capacity to bind to BinB,
suggesting that this interaction does not involve glycans. On the other hand
the hypothesis that glycans from Aam1 could hide or prevent the access of
the Bin toxin to the binding site was refuted because the removal of carbo-
hydrates by deglycosylation with PNGase F did not provoke any effect in
this aspect (Ferreira et al., 2010). The differential role of this major post-
translational modification in Aam1 remains unknown. Further investigation
using wild and mutant Cqm1 and Aam1 proteins expressed in Sf9 cells
showed that non-conserved residues of Cqm1 are needed for the BinB sub-
unit binding (Ferreira et al., 2014). This study showed that a segment of
Cqm1 N-terminus is responsible for binding to BinB subunit and the
replacement of the doublet 159GG160, located in a loop region, by the
respective residues (KL) from Aam1 protein abolished binding, showing that
they are required for this interaction. This loop from the Aam1 ortholog
Microbial Toxins for Mosquito Control 121

shows other divergent residues and the insertion of five amino acids that
could be implicated in the lack of capacity of this protein to bind BinB.
Those residues showed to be essential for binding, probably by making pos-
sible the access of Bin toxin to amino acids that may act as binding sites
themselves. Additional investigation of these proteins will certainly be
important to understand the molecular basis of Bin toxin selectivity in the
insect midgut.
The studies of the molecules involved in the mode of action and selec-
tivity of the Bin toxin has been restricted to the midgut receptors. Despite
the role of -glucosidases as the major target site of the Bin toxin, it is pos-
sible that novel molecules and pathways could be involved in the mode of
action and selectivity of Ls, as demonstrated by recent studies in the action of
Cry toxins to mosquitoes and lepidopterans. The immune defence involving
the mitogen-activated protein kinase p38 (MAPK p38) pathway seems to
participate in the response to pore-forming toxins and it is responsible for
protecting Ae. aegypti larvae from the action of Cry11Aa toxin (Cancino-
Rodezno et al., 2010; Porta et al., 2011). Silencing of genes from this path-
way rendered larvae hypersensitive to that toxin indicating its importance to
modulate the Cry toxin action. Mutations in the ABC transporters were
directly linked to the resistance of lepidopteran larvae to Bt Cry1A toxins
also proving their involvement in the mode of action (Atsumi et al.,
2012; Gahan et al., 2010). The role of other molecules in the mode of action
of Bin toxin, therefore, remains a field to be explored.

4. APPLICATIONS FOR MOSQUITO CONTROL

4.1. Field trials
Following the discovery and characterisation of potent Ls strains, such as
1593, 2362 and 2297, field trials against several mosquito species have been
conducted in countries located at different latitudes, including India,
Malaysia, Thailand, USA, Australia, Ivory Coast, Burkina Faso, Tanzania,
Mali and other African countries, as reviewed by Lacey (2007). Small-
and medium-scale tests targeting mosquito larvae in the genera Culex,
Anopheles, Aedes, Ochlerotatus, Mansonia and Psorophora breeding in a large
variety of site types provided evidence of persistent larvicidal activity and
the potential for spore recycling in polluted aquatic habitats. These findings
attracted the interest of the scientific community in areas like entomology,
microbiology and tropical health, stimulating further field studies, especially
against those mosquito species breeding in environments characterised by
122 Maria Helena Neves Lobo Silva Filha et al.

organically enriched water. As a result, fundamental information about the

activity of this bacterium, its efficacy and its persistence in Culex breeding
sites under natural conditions has been produced. In the following years, fur-
ther field experiments against the vectors of lymphatic filariasis and malaria
were conducted in many places in Africa (Barbazan et al., 1997; Hougard
et al., 1993; Karch et al., 1991; Ragoonanansingh et al., 1992; Seyoum
and Abate, 1997; Skovmand and Sanogo, 1999) and in the South and Cen-
tral Americas (Arredondo-Jimenez et al., 1990; Berrocal et al., 2000; Berti
and Gonzales, 2004; Blanco-Castro et al., 2000; Consoli et al., 1997;
Montero Lago et al., 1991; Regis et al., 1995, 1996, 2000a,b; Rivera
et al., 1997; Rodrigues et al., 2008; Silva-Filha et al., 2001; Suarez and
Morales, 1999).
These small- and medium-scale field trials to evaluate Ls-based products
in aquatic habitats under different climatic conditions helped to build a sig-
nificant body of knowledge on appropriate formulations, doses, methods for
product application and for measuring the impact of treatments on mosquito
populations. Table 3.5 presents a set of field evaluations of experimental or
commercial products against mosquito larvae in a wide range of breeding
sites. Cx. quinquefasciatus and Cx. pipiens are undoubtedly the most important
target species because Ls displays the highest activity to this group and is able
to show a prolonged activity in their breeding sites (Davidson et al., 1984;
Des Rochers and Garcia, 1984; Mulla et al., 1984a; Nicolas et al., 1987). The
abundance of these species in urban areas is due to the availability of breeding
sites with organically polluted water, which offer optimal conditions for
their development. Waste water lagoons resulting from human activities
(e.g. dairies) can also be excellent breeding sites for some Culex spp., and
such environments can be responsible for a huge production of mosquitoes
( Jones et al., 1990; Matanmi et al., 1990; Mulla et al., 1984a, 1988a). Anoph-
eles is the second most important group that has been successfully targeted by
Ls. Control strategies to fight different species that are Plasmodium vectors
have adopted Ls-based larvicides (Table 3.5). Some important anopheline
vector species (e.g. An. gambiae, An. stephensi, An. darlingi and An.
nuneztovari) are susceptible to Ls, and field trials have been conducted in a
diversity of scenarios in malaria-endemic regions. The control of anophe-
lines is often complex due to factors such as their ecological features, the
diversity and extension of breeding sites, and constraints to establishing
larvicide applications in those areas. Nevertheless, Ls has been an important
ally in Anopheles control programmes, especially when it is integrated with
other strategies. Psorophora, Mansonia, Aedes and Ochlerotatus are also targets
Microbial Toxins for Mosquito Control 123

Table 3.5 Field trials using Lysinibacillus sphaericus-based products to control mosquito
larvae
Targeted species Environment References
Culex quinquefasciatus Containers Kuppusamy et al. (1987),
Lacey et al. (1984)
Sod-lined potholes Lacey et al. (1988b)
Polluted sites, polluted Hougard (1990), Karch et al.
ponds, cesspools, cesspits, (1991, 1990), Kumar et al.
catch basins, settling basins (1996), Mulla et al. (1997,
1999), Nicolas et al. (1987),
Paing et al. (1987), Siegel
and Novak (1997, 1999),
Skovmand and Bauduin
(1997), Skovmand and
Sanogo (1999), Su and
Mulla (1999), Su (2008)
Diverse urban habitats Andrade et al. (2007),
Barbazan et al. (1997),
Hougard et al. (1993), Mulla
et al. (2001), Regis et al.
(1995, 1996, 2000b), Silva-
Filha et al. (2001),
Skovmand et al. (2009)
Dairy wastewater Jones et al. (1990), Mulla
et al. (1984a)
Cx. quinquefasciatus, Cx. Polluted pools Barbazan et al. (1998),
annulirostris, Anopheles Brown et al. (2004),
funestus, An. albimanus Montero Lago et al. (1991),
Ragoonanansingh et al.
(1992)
Cx. quinquefasciatus, Cx. Experimental containers Su and Mulla (1999)
stigmatosoma, Cx. tarsalis
Cx. quinquefasciatus, Cx. Diverse urban habitats Yadav et al. (1997)
tritaeniorhynchus, Cx.
vishnui
Cx. pipiens Artificial pools, settling Berry et al. (1987), Karch
basins et al. (1990)
Cx. pipiens, Cx. restuans Catch basins Siegel and Novak (1997)
Continued
124 Maria Helena Neves Lobo Silva Filha et al.

Table 3.5 Field trials using Lysinibacillus sphaericus-based products to control mosquito
larvaecont'd
Targeted species Environment References
Cx. pipiens, Ochlerotatus Waste water lagoons Berry et al. (1987)
trivittatus,
Cx. peus, Cx. Dairy wastewater Matanmi et al. (1990), Mulla
stigmatosoma et al. (1988a)
Cx. nigripalpus Wastewater Lacey et al. (1988a)
Cx. restuans Woodland pools Lacey et al. (1988b)
Culex spp Sod-line ponds Lord (1991)
An. gambiae Irrigation ponds, sunlit Fillinger et al. (2003), Karch
ponds, rain puddles et al. (1991), Skovmand and
Bauduin (1997), Skovmand
and Sanogo (1999)
An. stephensis Artificial breeding sites Kumar et al. (1994)
An. arabiensis Pools, rice fields water Romi et al. (2003), Shililu
ditches et al. (2003)
An. quadrimaculatus, Rice fields Dennett et al. (2001),
Psorophora columbiae, An. Dennett and Meisch (2000),
crucians Lacey et al. (1986, 1988b)
An. albimanus Lake habitats Rivera et al. (1997)
An. albimanus, Culex spp Experimental potholes Arredondo-Jimenez et al.
(1990)
An. culicifacies Rice fields Sundararaj and Reuben
(1991)
An. darlingi Artificial breeding sites Berrocal et al. (2000),
Galardo et al. (2013),
Rodrigues et al. (2008)
An. nuneztovari Field pools Rojas et al. (2001)
An. marajoara, An. Field pools Moreno et al. (2010)
triannulatus, An.
braziliensis
An. aquasalis Field ponds Berti and Gonzales (2004),
Moser et al. (2002, 2012)
Mansonia spp, Ma. Rice ditches and rice Floore and Wardz (2009),
indiana, Ma. uniformis, ponds Pradeepkumar et al. (1988),
Ma. dyari Yap et al. (1991)
Microbial Toxins for Mosquito Control 125

Table 3.5 Field trials using Lysinibacillus sphaericus-based products to control mosquito
larvaecont'd
Targeted species Environment References
Ps. columbiae Rice field plots, flood- Bowles et al. (1990), Groves
water habitats and Meisch (1996), Lacey
et al. (1986), Lord (1991)
Ps. columbiae, Oc. Irrigated fields Mulla et al. (1985), Mulla
nigromaculis et al. (1988b)
Oc. nigromaculis Irrigated pastures Mulla et al. (1988b)
Oc. triseriatus, Cx. Tires Siegel and Novak (1997,
restuans, Cx. pipiens 1999), Siegel et al. (2001)
Aedes vexans Riparian woodland Becker (2003)
Culiseta incidens Tires Kramer (1990)

of Ls-based larvicides, and there are successful examples of trials for control-
ling these mosquitoes in their typical habitats, which are commonly situated
in rural areas, such as irrigated fields, pastures, rice fields and other flooded
environments (Table 3.5). Numerous trials to control mosquitoes based on
Ls larvicides have been conducted; however, it is not possible to establish a
common methodology to employ this agent, and each programme should be
designed according to the targets biology and ecology, the biotic and abiotic
conditions, and other factors that play a role in the proliferation of that
species.

4.2. Factors affecting field performance

Since the first few years of studies, a cast of biotic and abiotic factors
influencing the larvicidal activity and its persistence in the environment
was identified, and this knowledge was essential to plan strategies for the
operational use of microbial larvicides. Relevant factors limiting or facilitat-
ing the toxic action of bacteria are the intrinsic susceptibility of the mosquito
species, the feeding behaviour of larvae, climatic variables (e.g. temperature,
rainfall and solar radiation), habitat conditions (e.g. water temperature,
organic content, larval density, density of non-target organisms, water flow
and sunlight exposure), features of the formulation used (e.g. presentation,
solar protection and dispersion) and methods of application. Among the
numerous factors that can negatively affect the performance of Ls products
in the field, exposure to solar radiation might be the most critical. Although
126 Maria Helena Neves Lobo Silva Filha et al.

Ls is less susceptible to this factor than Bti (Silapanuntakul et al., 1983), both
produce insecticidal proteins and, thus, can suffer degradation due to solar
radiation (Burke et al., 1983; Karch and Charles, 1987). Prolonged Ls per-
sistence is often observed in septic tanks and other breeding sites that are
protected from the sunlight (Silva-Filha et al., 2001). In a similar manner,
Bti can achieve enhanced performance under shadowed conditions
(Araujo et al., 2007; Melo-Santos et al., 2009). Sedimentation of the
crystal-spore complex also reduces the activity of Ls because Culex and
Anopheles larvae feed at the surface of the breeding sites. Some studies
showed a correlation between the crystal spores settling and the loss of tox-
icity in experimental sites (Davidson et al., 1984; Lacey and Lacey, 1990;
Skovmand and Guillet, 2000). Water flow, typically found in breeding sites
such as catch basins or water ditches in urban areas, has a negative impact on
the persistence of Ls because the current carries away the active ingredient.
Ls persistence for long periods is most likely the major feature that con-
tributes to its field effectiveness, particularly in urban areas (Davidson et al.,
1984; Mulla et al., 1984a; Mulligan et al., 1980; Silapanuntakul et al., 1983).
This persistence occurs because Ls has the ability to recycle in mosquito
cadavers or in the soil of breeding sites. This process involves a new cycle
of spore germination, vegetative development and the production of new
batches of spores and crystals that can be released in the environment and
sustain the larvicidal activity. The recycling has been demonstrated under
laboratory and field conditions and can take place in some mosquito species
(Becker et al., 1995; Correa and Yousten, 1995; Des Rochers and Garcia,
1984; Hertlein et al., 1979; Karch and Coz, 1986; Menon et al., 1982;
Nicolas et al., 1987). Laboratory studies have shown that de novo production
of spores is approximately 20-fold greater than the amount that was origi-
nally ingested by larvae (Charles and Nicolas, 1986). Recycling can provide
effective results if the favourable conditions required for bacterial germina-
tion occur (mainly in the mosquito cadavers) and if the newly produced
spore crystals are located in the feeding zone of larvae. The availability of
spore crystals on the bottom of breeding sites cannot contribute to activity
against larvae (Singer, 1980).

4.3. Trials against the vectors of lymphatic filariasis

In a context of the control of lymphatic filariasis transmission, the World
Health Organization-Special Programme for Research and Training in
Tropical Diseases (WHO-TDR) organised in 1990 an informal consultation
Microbial Toxins for Mosquito Control 127

to develop a strategy for large-scale field trials aiming to evaluate the efficacy
of Ls against Culex populations under different ecological conditions in
Brazil, Cameroun, India, Sri Lanka and Tanzania (WHO, 1993). The
projects were conducted in four steps, according to the protocol developed
by TDR, which also considered the effects of vector control on the trans-
mission of Wuchereria bancrofti: a baseline collection of data (i), a preparation
phase (ii), the implementation of an 18 month-larviciding period (iii), and a
6-month follow-up phase after larviciding stopped (iv). Entomological and
parasitological data gathered from all the phases showed a remarkable impact
of Ls treatment of breeding sites on reducing the vector-biting rate and gen-
erating a significant decline in human exposure to filarial infective larvae
(Barbazan et al., 1997; Becker, 2000; Maxwell et al., 1999; Regis et al.,
2000b). The output of these projects, together with previous knowledge
and experience from field trials, contributed to constructing a solid basis
for the design of large-scale implementation of Ls-based larvicides under dif-
ferent ecological conditions. The large-scale use of Ls also resulted in the
emergence of resistance in larvae of the Cx. pipiens complex (see
Section 5). However, given the known advantages of Ls over conventional
larvicides, that discovery promptly led to a research effort to elucidate the
resistance process and mechanisms. As a consequence, solutions to avoid,
delay or manage resistance were soon made available (see Section 6). Since
then, Ls has been applied in association with Bti in rotation or as a mixture,
or associated with other control agents, and the susceptibility of mosquito
larvae to the Bin toxin should be duly monitored.

4.4. Recent large-scale trials

The discovery of resistance to Ls does not seem to have lessened the interest
in the use of this entomopathogen for the control of mosquito larvae, as
suggested by the many large-scale field tests reported in recent years
(Table 3.6). It is important to emphasise that the use of Ls and Bti mixture,
recommended as a resistance-management strategy, gives more advantages
to the use of these biolarvicides in vector-control programmes: this mixture
greatly reduces the risk of developing resistance to Ls, broadens the spectrum
of target species, including Ae. aegypti, and takes advantage of the synergy
between the toxins of the two species, beyond the separate performance
of each one (Wirth et al., 2004). Recent field trials have been conducted,
mostly against malaria vectors, as in Tanzania, Kenya, Ivory Coast, Burkina
Faso, Benin and Brazil, but also against Culex and Aedes species in other
128 Maria Helena Neves Lobo Silva Filha et al.

Table 3.6 Examples of field trials using combined Lysinibacillus sphaericus and Bacillus
thuringiensis serovar. israelensis strategies for mosquito control
Country Targeted species References
Kenya, Mbita Anopheles gambiae, An. funestus Fillinger and Lindsay
(2006)
Gambia, Farafeni An. gambiae, An. arabiensis, An. melas Majambere et al.
Town (2007)
Turkey, Antalya Culex pipiens Cetin et al. (2007)
Colombia, Cali Cx. quinquefasciatus, Aedes aegypti Giraldo-Calderon
et al. (2008)
Cote dIvoire, An. funestus, An. gambiae Tchicaya et al. (2009)
Tiemelekro
Tanzania, Dar es An. gambiae, An. funestus, An. coustani, Geissbuhler et al.
Salaam Cx. quinquefasciatus (2009)
Kenya, Highland An. gambiae, An. funestus, An. arabiensis Fillinger et al. (2009)
Valley
Poland, Wroclaw Anopheles spp. Rydzanicz et al.
(2009)
Benin Cx. quinquefasciatus and other Lingenfelser et al.
mosquito species (2010)
Kenya, Malindi An. gambiae, Cx. quinquefasciatus Mwangangi et al.
(2011)
USA, Stratford Cx. pipiens, Cx. restuans, Ae. japonicus Anderson et al. (2011)
CN
USA, California Cx. tarsalis, Ochlerotatus melanimon Dritz et al. (2011)
Switzerland, Cx. pipiens, Ae. albopictus Guidi et al. (2013a)
Chiasso, Ticino
Tanzania, Dar es An. gambiae, An. funestus Maheu-Giroux and
Salaam Castro (2013)
Brazil, Amapa An. darlingi Galardo et al. (2013)

countries (Table 3.6). A common finding in these field trials is that all the
tested species of malaria vectors were highly susceptible to Ls, in each loca-
tion that testing was conducted. Furthermore, an evaluation of the impact of
the biolarvicide on the vector population and malaria transmission was per-
formed in most of these works. As an example, Fillinger and Lindsay (2006)
Microbial Toxins for Mosquito Control 129

have demonstrated that microbial larvicides (Bti and Ls) reduced the malarial
vector mosquito larvae and adult females by more than 90% in West Kenya.
In a location in Tanzania, one year of community-based larvicide applica-
tion reduced transmission by the primary malaria vector, An. gambiae, by
31% (Fillinger et al., 2008). As described, the first field trials of Ls against
mosquitoes were conducted, mainly in Africa, against Culex and Anopheles
species in the 1980s (Table 3.5). Currently, there appears to be a renewed
interest in the use of entomopathogenic bacteria to control malaria vectors,
which can be attributed to factors related to the fast urbanisation of the
African population and relatively easy access to most breeding sites in urban
areas (Fillinger et al., 2008), to the exophagic behaviour that has been
exhibited by An. gambiae in such areas (Fillinger and Lindsay, 2011;
Geissbuhler et al., 2007; Killeen et al., 2007), and to the insecticide resistance,
mainly to pyrethroids used in insecticide-treated nets, that has emerged for
the primary malaria vectors in many regions of Africa (Cuamba et al.,
2010; Munhenga et al., 2008; Ranson et al., 2011). In view of this scenario,
there is a strong belief that integrated vector management targeting both larval
and adult mosquitoes is the future for malaria control (Townson et al., 2005).

4.5. Operational use in mosquito-control programmes

Presently, Ls is almost always used in association with Bti as a mixture or in
rotation and integrated with other environmentally friendly control mea-
sures. These treatments are used in some countries to combat vector-borne
diseases or the nuisance caused by mosquitoes. Some examples of opera-
tional use will be presented below. In Germany, the KABS (German
Mosquito Control Organisation) has conducted a comprehensive
programme in a combined control strategy to control Ae. vexans and other
species of mosquitoes in the Rhine Valley, that has remained active since
1985 (Becker, 1997). The KABS was the first to adopt the use of Ls, as a
complement to the routine use of Bti to fight the nuisance caused by
mosquitoes, especially Culex, during the tourist season. There was no record
of resistance to these entomopathogens or of negative impacts on non-target
fauna, which were carefully monitored (Becker, 1998). In Tanzania, the
microbial larvicides Bti and Ls have been used operationally since 2006 in
the context of the Urban Malaria Control Programme in Dar es Salaam
(Fillinger et al., 2008; Geissbuhler et al., 2009). In Brazil, Recife city,
with 1.5 million inhabitants, is an endemic area for Bancroftian filariasis
transmitted by Cx. quinquefasciatus, with a 6% prevalence estimated two
130 Maria Helena Neves Lobo Silva Filha et al.

decades ago (Maciel et al., 1996). The Recife City Halls Department of
Health has been running a Filariasis Control Programme, integrating vector
control and mass treatment of humans with diethylcarbamazine since 2003.
Vector-control actions, including Ls treatments, were established in some
critical areas (Cartaxo et al., 2011; Silva-Filha et al., 2008) and were progres-
sively expanded; since 2006, the whole city (94 districts) has been treated.
A conjugate product containing Ls and Bti crystals was also applied in a city
district in 20102011, in an attempt to develop a suitable approach to con-
trol both Cx. quinquefasciatus and Ae. aegypti in urban areas using a single
product that assures effectiveness and low potential for the development
of resistance (C. M. F. Oliveira, personal communication). Another pro-
gramme has been conducted in the Pinheiros River, which crosses the met-
ropolitan area of Sao Paulo (Brazil). This rivers banks are a major source of
Cx. quinquefasciatus in the city, and larvicidal treatment to reduce mosquito
populations is conducted on 22.4 km of the river. From July 2003 to July
2006, 30 treatments were applied, using an Ls-based product in rotation
with a Bti-based larvicide. The larvicides were applied using a boat and
the frequency of treatments is based on surveillance of mosquito immature
stages. No resistance was found in the Cx. quinquefasciatus local population
(Andrade et al., 2007; Silva-Filha et al., 2008). In the United States, Ls and
Bti-based products, as well as conjugate products, have been used in many
counties to fight several species of mosquitoes since the introduction of these
products for vector control.

5. RESISTANCE
5.1. Factors involved in the selection of resistance
The production of biolarvicides based on Ls strains with high larvicidal
action supported their large-scale utilisation in many countries, especially
to fight larvae from the Cx. pipiens complex and anophelines. Field
utilisation showed the effectiveness of this agent but also revealed the onset
of resistance. Some aspects that have contributed to this phenomenon are
mentioned below. The environmental conditions of subtropical and tropical
countries promote mosquito proliferation throughout the year and require
continuous treatment cycles, which increase the selection pressure imposed
on these populations (Barbazan et al., 1997, 1998; Hougard and Back, 1992;
Hougard et al., 1993; Mulla et al., 2001; Regis et al., 1995, 2000b; Silva-
Filha et al., 2001; Skovmand and Bauduin, 1997; Skovmand et al., 2009;
Yuan et al., 2000). The persistence of Ls in some breeding sites (e.g. septic
Microbial Toxins for Mosquito Control 131

tanks) due to its capacity for recycling (Becker et al., 1995; Charles and
Nicolas, 1986; Karch and Coz, 1986) also contributes to a prolonged expo-
sure of larvae to the insecticidal crystals. The adoption of larvicides as the
major tool in control programmes is a strategy that increases the selection
pressure, while the adoption of integrated strategies can significantly reduce
the breeding site area that must be covered by larvicide application. Finally,
the intrinsic mode of action of Ls, based on the action of one major toxin
that targets a single class of receptors (Nielsen-Leroux and Charles, 1992), is
critical for the selection of resistance as reviewed by Wirth (2010) and
Ferreira and Silva-Filha (2013).

5.2. Laboratory and field reports

Resistance associated with the Bin toxin from Ls biolarvicides has been
reported in mosquito colonies artificially selected in the laboratory as well
as in field populations exposed to this agent (Table 3.7). The first resistant
laboratory colony (GEO) was established from Cx. pipiens field samples from
California (EUA), and it achieved a high level of resistance ratio
(RR  100,000) after 12 generations of selection pressure (Wirth et al.,
2000b). The samples employed for the foundation of this colony had already
showed a reduced susceptibility to Ls, which could be the reason for the high
resistance achieved in such a short period. Another laboratory selection per-
formed with populations from California, the L-SEL colony, showed a RR
of 37-fold after 80 generations (Rodcharoen and Mulla, 1994), a much
slower rate. Two laboratory colonies of Cx. quinquefasciatus from Brazil
(CqRL1/2362) and China (RLCq1/C3-41) were selected on a similar basis
with Ls strains 2362 and C3-41, respectively. A high-resistance level
(RR > 100,000) was achieved in both cases after 46 and 13 generations of
selection, respectively (Pei et al., 2002). In a second colony (CqRL2/
IAB59) selected with the IAB59 strain in Brazil, the resistance to Bin
evolved more slowly (Amorim et al., 2007), likely due to the presence of
the Cry48Aa/Cry49Aa toxin produced by this strain ( Jones et al., 2007).
This colony showed an RR  46,000 towards the IAB59 strain after 70 gen-
erations, and a strong cross-resistance (RR  70,000) to the Bin toxin was
detected, as expected. The selection of a colony with the IAB59 strain in
China, RLCq2/IAB59, showed a high level of resistance (RR > 100,000)
that was detected after 18 generations (Pei et al., 2002).
Field resistance was first detected in a Cx. pipiens population from
Southern France that was submitted to Ls exposure for approximately
Table 3.7 Reports of Culex pipiens and Culex quinquefasciatus resistance to Lysinibacillus sphaericus selected under laboratory (Lab) conditions
or after field exposure
Binding to
Sample Country Origin RRa receptorsb r allelesc Inheritanced References
GEO USA Lab >100,000 No cpm1GEO R/A Darboux et al. (2002), Nielsen-Leroux et al.
(1995), Wirth et al. (2000b)
L-SEL USA Lab 37 Nd Nd Nd Rodcharoen and Mulla (1994)
CqRL1/2362 Brazil Lab >100,000 No cqm1REC R/A Oliveira et al. (2004), Pei et al. (2002),
Romao et al. (2006)
CqRL2/ Brazil Lab  40,000 No cqm1REC R/A Amorim et al. (2007), Oliveira et al. (2004),
IAB59 and Pei et al. (2002)
RLCq1/C3- China Lab >100,000 No cqm1R R/A Amorim et al. (2007), Guo et al. (2013),
41 Oliveira et al. (2004), Pei et al. (2002)
RLCq2/ China Lab >100,000 Nd Nd Nd Pei et al. (2002)
IAB59
SPHAE France Field >20,000 Yes Nd R/S Nielsen-Leroux et al. (1997), Nielsen-Leroux
et al. (2002)
Kochi India Field  150* No Nd Nd Rao et al. (1995)
Coque Brazil Field  10 Yes Nd Nd Silva-Filha et al. (1995)
RFCq1 China Field >20,000 Nd Nd Nd Yuan et al. (2000)
BP France Field >10,000 No cpm1BP, R/S Darboux et al. (2007), Nielsen-Leroux et al.
cpm1BP-del (2002)
TUNIS Tunisia Field  750* Yes Nd R/S Nielsen-Leroux et al. (2002)
Nonthaburi Thailand Field >125,000 Nd Nd Nd Mulla et al. (2003)
a
Resistance ratio between the lethal concentration of L. sphaericus for the test sample and that obtained for a susceptible reference colony. *These field samples were
submitted to further laboratory selection and achieved higher levels of resistance (>10,000).
b
Detection of binary toxin binding to midgut microvilli proteins from larvae.
c
Resistance alleles.
d
Inheritance of resistance: R/A (recessive and autosomal), R/S (recessive and sex-linked), Nd (not determined).
134 Maria Helena Neves Lobo Silva Filha et al.

five years (Sinegre et al., 1994). This finding, although based on larval sam-
ples from a few breeding sites, revealed for the first time high levels of resis-
tance (RR > 20,000) in field populations. This result raised concerns about
the intensive use of Ls that were confirmed by subsequent resistance reports.
Cx. quinquefasciatus or Cx. pipiens populations from India (Rao et al., 1995),
China (Yuan et al., 2000), Tunisia (Nielsen-Leroux et al., 2002), Thailand
(Mulla et al., 2003), and a second population (BP) from France (Chevillon
et al., 2001; Nielsen-Leroux et al., 2002), exposed to treatments within con-
trol programmes, showed RRs that were, in most cases, higher than 10,000-
fold (Table 3.7). The Ls strains employed in these field-control programmes
were 2362 and 1593, in addition to the C3-41 strain isolated and widely used
for the production of this biolarvicide in China (Yuan et al., 1999). In Brazil,
a Cx. quinquefasciatus population from Coque (Recife) exposed to Ls during
a 2-year period showed a low resistance level (RR  10-fold) (Silva-Filha
et al., 1995). The utilisation of Ls for the control of Cx. quinquefasciatus in
two other urban areas of Agua Fria (Recife) and Rio Pinheiros (Sao Paulo)
in Brazil did not result in the selection of resistance (Silva-Filha et al., 2008).
Factors recorded in those areas, such as the interruption of treatments for
some periods, migration of individuals and rotations with Bti treatment,
might have contributed to the decrease of the selection pressure. Neverthe-
less, the other reports have undoubtedly indicated that high levels of resis-
tance can be achieved in the field, and this resistance remains a central issue
concerning Ls utilisation.
The reports described above indicate that different factors may play an
important role in the development of resistance. The initial susceptibility
of the population to the larvicides used and the frequency of resistance alleles
can be crucial to the evolution of resistance (Andow et al., 2000; Gould et al.,
1997; Tabashnik et al., 2003, 2006). Laboratory Bin-resistant colonies
showed heterogeneous responses when they were subjected to selection
pressure, suggesting that their genetic background could be a determining
factor. Baseline studies of the susceptibility of Cx. pipiens larvae to Ls are
limited; however, those performed with Bti showed variation. The evalua-
tion of approximately 50 Cx. pipiens populations without historic Bti expo-
sure have shown RRs to this agent from less than 3 to 12.5-fold and indicates
pre-existing natural variations (Vasquez et al., 2009; Wirth et al., 2001b).
Generally, the susceptibility of the target populations is not assessed before
the introduction of insecticides in a control programme, although such data
can be critical to detect the development of resistance. For this reason, the
evaluation of larval susceptibility through bioassays and the screening of alleles
associated with Ls resistance in Culex larvae should be routinely performed.
Microbial Toxins for Mosquito Control 135

5.3. Mechanisms and inheritance of resistance

Laboratory-selected and field-derived colonies have been used to character-
ise resistance mechanisms to the Bin toxin. Most of the studies have focused
on the detection of functional receptors in the larval midgut because these
molecules are essential for the Bin toxins mode of action (Section 2). Quan-
titative in vitro binding assays showed that the Bin toxin fails to interact with
BBMF from larvae of the laboratory-selected colonies GEO (EUA),
CqRL1/2362 (Brazil) and CqRL/C3-41 (China) (Darboux et al., 2002;
Guo et al., 2013; Oliveira et al., 2004). For the field-derived colonies BP
from France (Darboux et al., 2007) and Kochi from India (S. Poophathi
and C. Nielsen-LeRoux, unpublished data), this mechanism was also behind
the resistance. In contrast, larvae from the SPHAE (France) and TUNIS
(Tunisia) field-derived colonies showed abundant and functional receptors
(Nielsen-Leroux et al., 2002), as was recorded for the susceptible colonies
that were used as references (Nielsen-Leroux et al., 1997, 2002). It is likely
that another mechanism related to the post-binding step is responsible for
the high-resistance levels observed in those larvae, but it remains unknown.
Larvae from the field population of Coque (Brazil) displayed capacity to bind
the Bin toxin; however, the resistance level (RR  10) was most likely quite
low to be correlated to a loss in the capacity of binding (Silva-Filha et al.,
1995). The available data show that the lack of receptors for the Bin toxin
in the larval midgut is the major mechanism reported for resistance to Ls.
The availability of those resistant colonies allowed studies of the inheritance
of resistance to be performed through crosses and back-crosses, and the data
are summarised in Table 3.7. Monofactorial and recessive inheritance was
detected in all the cases studied, and some alleles of the cpm1/cqm1 genes that
presented mutations associated with resistance due to failures in Bin binding
to receptors have been identified in three laboratory-selected colonies
(GEO, CqRL1/2362 and CqRL/C3-41) and in one field population
(BP). For the SPHAE and TUNIS populations, it has been demonstrated
that one gene, sex-linked and also recessively inherited, is responsible for
the high level of resistance displayed by these larvae (Nielsen-Leroux
et al., 1997, 2002). Further studies of these colonies might reveal new path-
ways involved in the mode of action of Ls.

5.4. Resistance alleles of the cpm1/cqm1 gene

The availability of laboratory colonies with high levels of resistance provided
a robust model for the characterisation of mechanisms and genes involved in
the resistance to the Bin toxin. The identification of the gene coding for the
136 Maria Helena Neves Lobo Silva Filha et al.

Cpm1 receptor in Cx. pipiens (Darboux et al., 2001) opened perspectives for
the investigation of the molecular basis of resistance. To date, seven alleles
from cpm1/cqm1 genes associated with Bin resistance were characterised and
the first allele was described in larvae from the GEO colony (Darboux et al.,
2002). The allele cpm1GEO showed a mutation that provoked the loss of the
GPI anchor and prevented the expression of a full Cpm1 midgut-bound
protein. As a consequence, this truncated protein is absent in the epithelial
cell membrane of the midgut and, therefore, cannot act as a receptor for the
Bin toxin. This mechanism consistently explains the high level of resistance
observed for GEO larvae, which is associated with the lack of functional
receptors on the midgut (Nielsen-Leroux et al., 1995; Wirth et al.,
2000b). This pioneer study was followed by the identification of six other
resistance alleles of the cpm1/cqm1 gene in populations from different coun-
tries. Each allele is characterised by a particular mutation and all of them code
for transcripts of truncated or non-functional proteins, which disrupt the Bin
toxin binding to the midgut cells. The mutation in the r alleles associated to
Bin resistance that have been identified to date are shown in Fig. 3.8 and
their features are described below.
The cpm1GEO allele is characterised by a nonsense mutation T1706A that
changes the Leu-569 to a premature stop codon, in the proximity of the puta-
tive GPI signature located in the C-terminus of the Cpm1 protein. This muta-
tion prevents GPI anchoring, while the -glucosidase activity and the toxin
binding capacity of this molecule remains intact, as demonstrated by the
respective recombinant proteins expressed in Sf9 cells (Darboux et al.,
2002). This mutant protein lacks only eleven amino acid residues compared
to the wild-type Cpm1 protein that is composed of 580 amino acids. A second
independent allele was identified in the CqRL1/2362 Cx. quinquefasciatus col-
ony from Brazil that was also characterised by a high level of resistance and lack
of midgut receptors for Bin toxin (Oliveira et al., 2004; Pei et al., 2002). In
larvae from this colony the cqm1REC allele had a deletion of 19 nucleotides (nt)
located from nucleotides 12761294 of its sequence (Romao et al., 2006),
resulting in the reading frame shift that creates a premature stop codon at posi-
tion 1362. The potential truncated protein coded by this transcript would
have only 437 residues and it remains unknown if this protein is produced
in these larvae, since immunodetection assays failed to detect its presence
(Romao et al., 2006). These authors hypothesised that the premature stop
codon could be recognised by the ubiquitous nonsense-mediated decay path-
way of mRNA degradation (Gonzalez et al., 2001; Wagner and Lykke-
Andersen, 2002; Wilkinson and Shyui, 2002) causing the removal of this
transcript.
Microbial Toxins for Mosquito Control 137

Figure 3.8 Nucleotide and deduced amino acid sequence of the open reading frame
from the cqm1 gene, which codes the Cqm1 receptor in Culex quinquefasciatus larvae
(GenBank accession number DQ333335). Seven polymorphisms associated to resistance
to binary toxin that were independently identified in different alleles of the cpm1/cqm1
genes, are represented in this sequence for illustration purpose only. (1) cqm1R allele
shows a deletion of a cytosine (asterisk, boxed) at position 445 that creates a stop codon
downstream (tga, boxed); (2) cpm1BP shows a nonsense mutation that creates a stop
codon (Gln396Stop) at this position (arrow); (3) cpm1BP-del has an alternative splicing
event responsible for the deletion of 66 residues (bold and underlined); (4) cqm1REC
has a 19-nt deletion (bold and underlined); (5) cqm1REC-D25 has a 25-nt deletion
encompassing the 19-nt from the previous deletion (bold and underlined) and six sub-
sequent bases (bold and boxed); (6) cqm1REC-D16 has a 16-nt deletion (bold and italics);
the three last deletions (19-, 25-, 16-nt) create the stop codon located at the same posi-
tion 1362 (tga, boxed); (7) cqm1GEO has a nonsense mutation (T1706A) that creates a
stop codon (Leu569Stop) at this position (arrow).
138 Maria Helena Neves Lobo Silva Filha et al.

During a screening of the cqm1REC in field populations from Recife city

(Brazil), two new alleles containing mutations potentially associated with
resistance were found (Chalegre et al., 2012). The cqm1REC-D16 allele
showed a 16-nt deletion (13061321) that was found in a few individuals
from non-treated populations. The second allele named cqm1REC-D25 had
a 25-nt deletion, which encompassed the 19-nt deletion from the cqm1REC
and six additional nucleotides downstream of this sequence (12761300),
and it was found in one larva from a Ls-treated population. The 19-,
16- and 25-nt deletions are closely located in the sequence and their changes
in the sequence reading frame generated the same premature stop codon
located at position 1362.
Two r alleles detected in the BP population from Southern France that
showed a high level of resistance to Ls, were also associated with a lack of
functional midgut receptors (Chevillon et al., 2001; Nielsen-Leroux
et al., 2002). Darboux et al. (2007) confirmed the co-existence of these
alleles in that population and identified their mutations. The cpm1BP allele
shows a nonsense mutation Gln396Stop that produces a protein without
the last 184 residues including the signature for the GPI anchor. Similar
to other cases, it is expected that these truncated proteins are secreted as sol-
uble proteins, instead of being produced as midgut membrane proteins. The
second allele found among BP larvae, the cpm1BP-del, has a 198-nt internal
deletion provoked by the insertion of a transposon that is responsible for
generating an alternative splicing event. This allele codes for a transcript
of a protein with a deletion of 66 amino acids located between the
Val422 and Gln487, and it retains the predicted GPI-anchor in its
C-terminus. The respective recombinant Cpm1BP-del protein expressed
in Sf9 cells showed that it correct locates to the cell membrane but this pro-
tein is unable to bind the Bin toxin. It is likely that the loss of these amino
acids residues provokes an alteration of the protein conformation and pre-
vents its binding to the Bin toxin. The frequency of these alleles verified in a
sample of 108 larvae was similar: 50% carried a copy of each allele, 25% were
homozygous for the cpm1BP, and the remaining individuals were homozy-
gous for the cpm1BP-del.
The molecular basis of the resistance behind the CqRL/C3-41
laboratory-selected colony from China was recently identified (Guo
et al., 2013). Cloning and analysis of the cqm1 gene from the CqRL/C3-
41 larvae revealed the cqm1R allele characterised by the deletion of a cytosine
at position 445. This single-deletion changes the reading frame of the sub-
sequent 39 residues and a stop codon is created at position 582 in this
Microbial Toxins for Mosquito Control 139

sequence. The transcript codes a truncated protein composed of 194 residues

and homozygous larvae for this allele are resistant due to absence of full-
length Cqm1 midgut-bound molecules.
The characterisation of these alleles indicates that cpm1/cqm1 is a highly
polymorphic gene and five (cqm1REC, cqm1REC-D16, cqm1REC-D25 from
Brazil; cpm1BP and cpm1BP-del from France), among the seven mutations
recorded, are located in the same region of the gene indicating that this
seems to be a hot-spot for such events. In addition, these mutations can have
a high impact because they are responsible for provoking full refractoriness
due to the lack of membrane-bound receptors. Mechanisms of target site
alteration to synthetic insecticides, for instance, are often associated to muta-
tions in their genes that provoke a reduction in their capacity for binding the
active principle (Du et al., 2013; Rinkevich et al., 2013), and they do not
provoke the total loss of activity. This has serious implications because
the Cpm1/Cqm1 receptors are essential for the action of the Bin toxin,
and the selection of individuals with such polymorphisms in their genes
can lead to operational failures.

5.5. Diagnosis and field survey of resistance

The investigation of Ls resistance is commonly performed through suscepti-
bility bioassays that are employed to compare the doseresponse of larvae from
unknown samples to the response of larvae from a susceptible sample, used as a
reference. Bioassays allow the determination of the LCs of the given Ls sample
tested for 50% (LC50) and 90% (LC90) of the larvae, exposed for 48 h (WHO,
1985). The ratio between the LC for the test sample and that for the reference
colony, named RR, informs the level of resistance detected. It is difficult to
establish the RR value that truly corresponds to initial stages of resistance and,
often, when the RR of a given sample to Ls and Bt toxins is equal or greater
than 10-fold, it is referred to as a low level of resistance. This parameter could
be greatly improved if baseline data for the susceptibility of the populations
under analysis were available. Populations can exhibit natural variations to
the larvicides prior to their utilisation that are not necessarily a consequence
of the selection pressure (Robertson et al., 1995).
Bioassay is also the major tool used to evaluate larval susceptibility to Ls,
however, studies on the genetics of resistance to this agent have shown that
this trait is recessively inherited, at least for the samples analysed to date
(Table 3.7). In this case, heterozygous individuals carrying r alleles can hardly
be detected in the bioassays because they will display a susceptible
140 Maria Helena Neves Lobo Silva Filha et al.

phenotype. Nevertheless, in most cases the r alleles have not been identified
and their screening is performed using techniques based on crosses and
followed by the analysis of the progeny susceptibility, such as the F2 and
F1 screens that have been employed to characterise r alleles to Bt toxins
in pest insects (Andow et al., 2000; Gould et al., 1997; Xu et al., 2009;
Yang et al., 2006, 2007a; Zhao et al., 2010). These approaches although effi-
cient, are indirect methods to detect r alleles. On the other hand, the iden-
tification of such alleles allows the development of molecular methods for
their direct detection. Polymerase chain reaction (PCR) methods have been
specifically designed to target specific alleles or to amplify strategic gene seg-
ments that host mutations associated to resistance. Molecular methods for
the detection of mutations in the voltage-gated sodium channel genes that
confer knock-down resistance to pyrethroids have been employed for the
screening of resistance in mosquito populations (Saavedra-Rodriguez
et al., 2007; Sarkar et al., 2009; Singh et al., 2009; Tripet et al., 2006).
For Ls, the identification of alleles of the cpm1/cqm1 gene associated with
resistance opened perspectives for their molecular detection in field
populations. The cqm1REC primarily identified in the CqRL1/2362 Cx.
quinquefasciatus laboratory-selected colony, was detected by allele specific
PCR in all field populations surveyed from Recife city (Brazil), including
six non-treated and one Ls-treated populations. Its frequency in non-treated
samples ranged from 0.001 to 0.017 and the average of five populations was
0.003 (Chalegre et al., 2009, 2012). In three evaluations performed in the
treated area, cqm1REC showed an average frequency of 0.048, which was sig-
nificantly higher than those recorded for the non-treated ones (Chalegre
et al., 2009, 2012). Bioassay analysis performed with these populations
did not reveal RR values significantly between the treated and the non-
treated areas but the early detection of an increased number of individuals
carrying this allele in the treated area is likely to be correlated to the selection
pressure imposed by the Ls exposure. This method can be strategic for the
surveillance of resistance, especially for recessive r alleles. The two other
alleles cqm1REC-D16 and cqm1REC-D25, that were identified in the Cx. quin-
quefasciatus field samples from Recife city, showed a frequency between
0.001 and 0.006 in five populations and a more limited distribution in
the samples compared to the cqm1REC (Chalegre et al., 2012). The study sug-
gests that the cqm1REC is the most important allele associated with resistance
in these populations and it can be used as a marker for the surveillance in that
specific area. To date, the other r alleles to Ls recorded in the literature have
not been tracked in the Cx. pipiens populations.
Microbial Toxins for Mosquito Control 141

5.6. Biological cost of resistance

The biological performance of individuals from three laboratory-selected
resistant colonies has been evaluated. The L-SEL Cx. quinquefasciatus colony
from California with a RR of 37-fold (Rodcharoen and Mulla, 1994)
showed lower fecundity and fertility compared with their susceptible coun-
terparts (Rodcharoen and Mulla, 1997). A marked reduction was observed
in those parameters and, for instance, the number of larvae hatched per raft
from the resistant females was less than 50%, compared to the susceptible
colony. The evaluation of the CqRL1/2362 colony from Brazil with a
high-resistance level (RR > 100,000) showed alteration in those parameters,
and, although statistically significant, those were minor reductions. The
fecundity, which was found to be the most affected parameter for instance,
was only 8% lower than that recorded for the susceptible colony (de Oliveira
et al., 2003). Individuals from the CqRL1/2362 colony also exhibit a longer
developmental time (from egg to egg) and females required more time to lay
the first batch of eggs. A third study performed with the CqRL2/IAB59 col-
ony, selected with the IAB59 strain (Amorim et al., 2007), did not show
significant differences in the fertility, fecundity or pupal weight of the resis-
tant individuals compared to their susceptible counterparts (Amorim
et al., 2010).
Data from these studies indicate that Ls resistance is likely to be associated
with discrete biological costs rather than to a dramatic impact on the fitness
of the resistant individuals from these colonies, at least under laboratory con-
ditions. It is worth noting that, in some cases, the resistance to Bt toxins
might be associated to high biological costs that can prevent the maintenance
of the insect colonies in the laboratory (Anilkumar et al., 2008; Tabashnik
et al., 1994). This does not seem to be case of the Ls-selected colonies that
were maintained under laboratory conditions after a considerable number of
generations (M.H. Silva-Filha, personal communication).
One direct consequence of Ls resistance in these insects seems to be the
replacement of the midgut-bound Cqm1 -glucosidase by a soluble form of
this protein that could display, or not, its catalytic activity. In some cases, it is
possible that the soluble form of the protein is not expressed neither, as
described bellow. The mutant protein from GEO larvae, if expressed for
instance, would retain its -glucosidase activity (Darboux et al., 2002).
On the other hand, the Cqm1 mutant proteins encoded by the resistance
alleles from BP and CqRL/C3-41 larvae did not display -glucosidase activ-
ity when they were expressed in heterologous hosts (Darboux et al., 2007;
142 Maria Helena Neves Lobo Silva Filha et al.

A
CqSF CqRL1/2362

L B L B

83

62

48

32

B
CqSF CqRL1/2362

L B L B

83

62

48

32

Figure 3.9 Alpha-glucosidases from susceptible (CqSF) and Lysinibacillus sphaericus-

resistant (CqRL1/2362) Culex quinquefasciatus larvae. (A) In-gel -glucosidase assay per-
formed with whole crude extracts (L) and midgut microvilli proteins (B) from larvae.
Bands indicating cleavage of the substrate were visualised with a UV transilluminator.
(B) Immunoblotting of the samples shown in (A) with the anti-Cqm1 antiserum showing
the Cqm1 protein indicated by arrows. Molecular weight markers (kDa) are shown on
the left. Taken from Romo et al. (2006).

Guo et al., 2013). The analysis of larvae from the CqRL1/2362 resistant col-
ony showed that the truncated Cqm1 mutant protein does not seem to be
expressed in larvae (Romao et al., 2006). These results suggest that if these
proteins are expressed in larvae it is not certain that they could play their role
in digestion. Biochemical and bioinformatic analyses (Gabrisko, 2013;
Romao et al., 2006) have provided evidence that Cx. quinquefasciatus dis-
plays a set of -glucosidases and the comparative pattern in susceptible
and resistant larvae from the CqRL1/2362 colony are similar, except for
the Cqm1 that is missing in resistant larvae (Fig. 3.9). Maybe the expression
of other -glucosidases in these larvae can compensate the lack of Cqm1 and
Microbial Toxins for Mosquito Control 143

this could be an explanation for the minor level of biological cost observed in
these colonies, although, this hypothesis has not been proved. The specific
role played by Cqm1 and the other -glucosidases in digestion in this species
is still unknown and this would be useful to understand the impact of those
mutations more clearly in the biological fitness of these insects.
The CqRL1/2362 colony, for instance, has been maintained for more than
200 generations under laboratory conditions and the Cqm1 -glucosidase,
which is lacking, does not seem to be essential. A study performed with
the progeny from the cross between resistant CqRL1/2362 and susceptible
individuals in a 1:1 ratio, showed that the resistance alleles displayed a stable
frequency of 0.5 during 10 generations while this colony was kept in the
absence of Ls treatments (Amorim et al., 2010). The biological fitness of
SPHAE and GEO field-derived colonies was not investigated, but these
are also examples of a highly resistant colonies successfully established and
maintained under laboratory conditions during several years (M.H. Silva-
Filha, personal communication). This is consistent with the hypothesis that
some resistance alleles would not be necessarily associated with crucial
adverse effects on biological fitness and they could be maintained in the
populations in the absence of selection pressure (Ffrench-Constant, 2007).

6. MANAGEMENT OF RESISTANCE
6.1. Integrated mosquito-control programmes
The major goal of integrated vector-control approach is to achieve the
reduction of population density, based on the adoption of different methods,
in a cost-effective manner (WHO, 1983, 2004). The utilisation of multiple
strategies to impact the density of mosquitoes, contributes to the decrease of
insecticide utilisation and, thus, for a lower selection pressure. The appear-
ance of resistance has often been associated with the intensive use of larvi-
cides and/or adulticides, as the major intervention adopted to control
insects. This model has predominated since the introduction of synthetic
insecticides and it has greatly contributed to the disseminated resistance
reported worldwide which affects, in particularly, dipteran insects that have
been targeted by control programmes (Brogdon and McAllister, 1998;
Georghiou and Lagunes-Tejeda, 1991; Hemingway et al., 2004; WHO,
1986). In developing countries, the situation is more critical due to lack
of resources and structure that are necessary to set up sustainable
mosquito-control programmes. The choice of suitable insecticides and
144 Maria Helena Neves Lobo Silva Filha et al.

the monitoring of their efficacy are important aspects required for the success
of interventions (Becker et al., 2003). After the large-scale utilisation of Ls
for Cx. quinquefasciatus control, resistance was recorded in exposed
populations in different countries (Section 5), highlighting the need to man-
age the resistance to this control agent.

6.2. Factors involved in the prevention of resistance

The mode of action of all Ls larvicides available, to date, relies on the Bin
toxin that targets one specific class of midgut molecules, and this character-
istic greatly favours the selection of resistance. Therefore, efforts to eliminate
mosquito breeding sources and to reduce the utilisation of larvicides are
important strategies to reduce the selection pressure. The adoption of con-
trol agents with different modes of action, in a system of rotation or mix-
tures, should be designed according to the local conditions. The Bin
toxin is essentially distinct from other insecticidal compounds and it is safe
to other organisms, thus, other control agents can be used in association with
Ls. Monitoring the susceptibility of populations exposed to larvicides is cru-
cial to detect early stages of resistance selection and to introduce manage-
ment strategies. As described in Section 5, it is important to evaluate
larval susceptibility to the products employed in a control programme
through bioassays and also to adopt methods that determine the frequency
of resistance alleles in populations.
Hence, when resistance to Ls is detected in an exposed population, its use
should be stopped and alternative larvicides with different modes of action
can be utilised to revert the resistance developed. Mosquitoes are r-strategist
organisms and they are able to recover rapidly after suspension of control
interventions. Therefore, the interruption of treatments per se allows for
the immigration of susceptible individuals from the surrounding areas and
also from untreated breeding sites, which leads to the dilution of the resis-
tance alleles. The reversal of Ls resistance in a field population from China
was recorded 6 months after stopping treatment, and the RR decreased from
22,672- to 5.78-fold (Yuan et al., 2000). The low-level of resistance (10-
fold) observed in a treated population from Coque (Brazil) was replaced
by full-susceptibility to Ls, 11 months after treatment suspension in that area
(Silva-Filha and Regis, 1997). The reversal of Ls resistance is also facilitated
by the recessive inheritance of the gene involved (Amorim et al., 2007,
2010; Chevillon et al., 2001; Nielsen-Leroux et al., 1995, 1997, 2002;
Oliveira et al., 2004).
Microbial Toxins for Mosquito Control 145

6.3. Candidates for managing Bin-toxin resistance

Ls strains such as 2362, 1593, C3-41, IAB881, IAB872 display a profile of
cross-resistance, because they all produce the Bin toxins, even if their Bin
toxins belong to different classes (Nielsen-LeRoux et al., 2001; Regis and
Nielsen-LeRoux, 2000; Rodcharoen and Mulla, 1996; Silva-Filha et al.,
2004). One exception is the LP1-G strain that produces the Bin4 toxin type
that has low toxicity due to replacement of a leucine by a serine at position
93 of the BinA subunit (Humphreys and Berry, 1998; Priest et al., 1997).
Alternatively, some strains can produce other classes of toxins, such as the
Mtx toxins and Cry48Aa/Cry49Aa that can be used to overcome Bin resis-
tance. These toxins do not confer a high level of activity of their respective
native strains (see Section 2), but they have an important potential when
they are expressed and delivered under optimal conditions. Their ability
to overcome resistance to Bin toxin will be discussed further in this section.
Among the potential agents to be used in association with Ls, Bti-based
biolarvicides are considered the most promising and in addition, they are
commercially available. Bti is a proven alternative for the management of
Ls resistance because its toxins and their modes of action are unrelated to
the Bin toxin. The most common protoxins found in Bti crystals are Cry4Aa
(125 kDa), Cry4Ba (135 kDa), Cry11Aa (68 kDa) and Cyt1Aa (28 kDa)
(Berry et al., 2002). Some Bti strains can produce other dipteran-active
toxins such as Cry10Aa (58 kDa) and Cyt2Ba (30 kDa) (Guerchicoff
et al., 1997; Thorne et al., 1986). Bti does not display cross-resistance to
Bin toxin and, in addition, its mode of action does not favour the selection
of resistance. Some factors behind these features are the multiple composi-
tion of Cry and Cyt protoxins available in the Bti crystal; the synergistic role
played by the Cyt1Aa toxin as a surrogate receptor for the Cry toxins (Perez
et al., 2005, 2007); the participation of other midgut molecules that are also
receptors for the Cry toxins (Likitvivatanavong et al., 2011). These features
endorse Bti as an excellent candidate to be used for the management of Ls
resistance and, to date, there are no records of resistance in mosquito field
populations exposed to this agent (Ferreira and Silva-Filha, 2013; Wirth,
2010). Cry toxins from other Diptera-active Bt strains also do not display
cross-resistance to Bin toxin as will be described in the following topics.

6.3.1 Bacillus thuringiensis mosquitocidal toxins

Cx. pipiens larvae resistant to the Bin toxin are susceptible to Bti, as has been
shown by studies based on resistant colonies and populations from United
146 Maria Helena Neves Lobo Silva Filha et al.

States, Brazil, India, China and Thailand (Amorim et al., 2007; Nielsen-
Leroux et al., 1995; Rao et al., 1995; Silva-Filha et al., 1995; Su and
Mulla, 2004; Yuan et al., 2003). Individual Cry and Cyt toxins from Bti
or other mosquitocidal Bt strains, such as serovar. medellin (Btmed) and
serovar. jegathesan (Btjeg) are able to overcome Bin toxin resistance. The syn-
ergistic interactions among these toxins and Bin toxin have been demon-
strated using mixtures of toxins produced in the respective native strains,
or by their combined expression in recombinant Bti or Ls strains as
described below.
The synergistic effect of Cyt toxins on Bin toxicity was first investigated
using the Cyt1Ab1 from Btmed. In this case, a recombinant Ls 2297 strain
that produced both Bin and Cyt1Ab1 exhibited a significant activity against
the larvae from GEO- and SPHAE-resistant colonies (Thiery et al., 1998).
Mixtures of Cyt1Aa or Cyt2Ba, from Bti, with the Bin toxin were shown to
be effective against Ae. aegypti larvae that is a refractory species to Bin toxin
(Wirth et al., 2000a) and to Cx. pipiens larvae that were resistant to the Bin
toxin (Wirth et al., 2000c, 2001a, 2004). A laboratory selection trial of Cx.
quinquefasciatus larvae using a mixture of Bin and Cyt1 toxins in a 3:1 ratio
during 20 generations showed a RR of 1.4 while a RR > 1000 was observed
for their counterparts selected with the Bin toxin only (Wirth et al., 2005),
corroborating the role of the Cyt1Aa toxin to delay Bin toxin resistance.
The synergy of the whole Bti crystal, or its individual Cry toxins, with
Bin toxin has also been demonstrated. The introduction of Bti toxins in Ls
strains has been attempted, before the advent of Bin toxin resistance, aiming
at the improving the spectrum of Ls toxicity since this is more restricted than
Bti (Bar et al., 1991; Trisrisook et al., 1990). Later, working with Bin resis-
tance, a study evaluated a set of mixtures of Bin with Bti and its toxins that
showed an enhanced activity to Bin-resistant larvae and the potential of
these mixtures to overcome the resistance to the Bin toxin (Wirth et al.,
2004). Evaluation of the synergism of six different mixtures of wild Ls
and Bti crystals to susceptible Cx. quinquefasciatus showed that the highest syn-
ergism factor found was observed using a 3:1 ratio of Ls to Bti (Sreshty et al.,
2011). This combination also displayed faster cytopathological effects on the
midgut epithelium and muscles.
The production of recombinant Ls strains containing Bti toxins and/or
the Cry11Ba toxin from Btjeg showed an enhanced activity to larvae from
Bin-resistant colonies and partially restored their susceptibility to the Bin
toxin (Poncet et al., 1994, 1997; Servant et al., 1999). However, it is impor-
tant to note that the introduction of Bt genes in Ls strains has been often
Microbial Toxins for Mosquito Control 147

marked by the low expression and/or instability of the respective foreign

proteins (Federici et al., 2010; Gammon et al., 2006). Elucidation of the
molecular factors involved in this issue could lead to a significant advance-
ment for the construction of improved strains.
The integration of Bin toxin into Bti was also performed but there are
fewer reports of this, perhaps due to the fact that Bti displays a worse field
performance compared to Ls, especially in Culex and Anopheles spp breeding
sites (Nicolas et al., 1987; Silapanuntakul et al., 1983). Nevertheless, Bti
recombinant constructs were obtained and they successfully produced Bti
and Bin toxins with improved toxicity. A recombinant Bti, producing an
association of Cyt1Aa, Cry11Ba and the Bin toxin, showed improved tox-
icity to susceptible Cx. quinquefasciatus larvae (Park et al., 2003). Another
construct expressing the complete set of Bti toxins and the Bin toxin showed
improved toxicity to Cx. quinquefasciatus, compared to the toxicity of each of
the strains used, and it also suppressed Bin resistance (Park et al., 2005). The
integration of Bt toxins and Bin toxins is effective to delay or avoid the selec-
tion of Bin toxin resistance and recombinant bacteria have potential as tools
to produce a set of toxins for mosquito control. The expression of Bt and Ls
insecticidal toxins has also been attempted in other microorganisms found in
the aquatic environment in order to achieve a combined expression of their
toxins and a suitable delivery in these larval habitats (Tanapongpipat et al.,
2003; Tandeau de Marsac et al., 1987; Thanabalu et al., 1992b; Yap et al.,
1994). Products based in such recombinant bacteria have not been used in
the field and it remains a promising perspective in view of the substantial
advancements achieved, as previously reviewed (Federici et al., 2003,
2007, 2010; Park et al., 2005).
To date, the management of Ls resistance has been based on rotation or
mixtures with Bti, since this agent is commercially available and its field
effectiveness has been proved worldwide (Becker, 1997, 2000; Regis
et al., 2001). The choice between the use of Ls and Bti in rotation or mix-
tures depends on operational issues of the control programme, and also if the
strategy is applied for the reversal of the resistance, or for preventing its
appearance. Both rotation and mixtures seem to be effective in the first sce-
nario, but mixtures seem more efficient to delay the onset of resistance
(Mulla et al., 2003). Laboratory experiments using Cx. quinquefasciatus col-
onies subjected to mixtures of Ls and Bti proved to be more efficient to delay
resistance, compared to the scheme of rotation (Zahiri and Mulla, 2003;
Zahiri et al., 2002). Association of Ls and Bti products has been used for mos-
quito control and, recently, commercial formulations containing the
148 Maria Helena Neves Lobo Silva Filha et al.

mixtures of crystals produced by each agent in a single product have been

developed (Anderson et al., 2011; Kahindi et al., 2008; Mwangangi et al.,
2011). These products aim to target a wider range of mosquito species
and breeding sites. Trials have been carried out to control Culex and Aedes
species that colonise breeding sites from urban areas (Anderson et al., 2011;
Eritja, 2013; Guidi et al., 2013a), or other species that occur in wetlands
from environmentally sensitive areas (Dritz et al., 2011). These multi-toxin
products showed promising results and can be effectively used in mosquito-
control programmes ensuring the environmentally safe action and a low
potential for resistance selection.

6.3.2 Other L. sphaericus mosquitocidal toxins

Other insecticidal factors produced by Ls strains have proved to be suitable in
principle for the management of Bin resistance due to their lack of cross-
resistance with this toxin. The Mtx toxins do not provide an important con-
tribution for the activity exhibited by their native strains, as previously
described (Section 2). On the other hand when Mtx1 and Mtx2 are produced
as recombinant proteins in E. coli they revealed a capacity to synergise the
activity of Bin toxin towards Cx. quinquefasciatus (Wirth et al., 2007). Low
amounts of Mtx1 and Mtx2 combined with the Bin toxin (1:1:8 ratio) pro-
voked a significant reduction of the resistance levels to Bin toxin in a selected
colony. Evaluation of Mtx1 showed a high activity against larvae from one
susceptible and two highly Bin-resistant colonies selected in China, indicating
its distinct mode of action and target sites (Wei et al., 2007).
The discovery of the Cry48Aa/Cry49Aa Bin toxin in strain IAB59 was
supported by the earlier reports of the low cross-resistance displayed by this
strain to the Bin toxin (Amorim et al., 2007; Nielsen-LeRoux et al., 2001;
Pei et al., 2002; Yuan et al., 2003). Similarly to the Mtx toxins, Cry48Aa/
Cry49Aa does not contribute to an enhanced activity of IAB59 final spor-
ulated cultures due to the low expression of Cry48Aa, which is required in
an equimolar ratio with Cry49Aa for optimal toxicity ( Jones et al., 2007).
The adequate production of these factors as recombinant proteins and the
administration of their mixtures at an equimolar ratio show a remarkable
toxicity to both susceptible and to Bin-resistant Cx. quinquefasciatus larvae
( Jones et al., 2007, 2008). The Cry48Aa/Cry49Aa combination probably
targets midgut molecules that are distinct from the receptors identified for
the Bin toxin in view of its activity in Bin-resistant Cx. quinquefasciatus larvae
lacking the Cqm1 receptor (de Melo et al., 2009). This toxin is an also an
interesting candidate for the development of multi-toxin biolarvicides in
Microbial Toxins for Mosquito Control 149

view of its unique composition as described in Section 2. Studies of the mor-

phological alterations provoked by this toxin on the midgut cells of suscep-
tible and Bin-resistant larvae showed a complex combination of effects that
are similar to those of a mixture of Bin and Cry11Aa toxins (de Melo
et al., 2009).

6.3.3 Other control agents

In a broader view for the management of Ls resistance, the integration of
control agents available, other than toxins from Bt and Ls presented above,
should also be taken in account (Hurst et al., 2006; Keiser et al., 2005; Lacey
and Lacey, 1990; Lingenfelser et al., 2010; Skovmand et al., 2011; Tietze
et al., 1994). Biological control agents can be used with Ls, in view of its
safety for other organisms that occur in the aquatic environment of mosquito
breeding sites. Lacey reviewed this aspect and presented studies of the com-
bined use of Ls with predators (fish, aquatic insects), entomopathogenic
fungi and parasites (nematodes), as viable choices to be adopted (Lacey,
2007). In practise, the use of these agents has been much more limited by
other factors related to their production, storage, transport and liberation
but they can be successfully associated with Ls. Other control agents such
as synthetic insecticides, insect growth regulators and spinosins can be con-
sidered to be use with Ls and Bti due to their unrelated modes of action

Environmental management
Use of L. sphaericus within
integrated programme Other mosquito-control agents
Personnal protection

Evaluation of field effectiveness

Monitoring insect susceptibility In vivo bioassays
Detection of resistance alleles

Bti, Bti + L. sphaericus

Other agents to prevent Bin
Synthetic insecticides, IGRs
resistance
Spinosins

Cry and Cyt (Bti, Btjeg, Btmed)

Toxins able to overcome Bin resistance Mtxs
Cry48Cry49

Figure 3.10 Strategies for preventing the resistance to the Bin toxin from Lysinibacillus
sphaericus (Ls) and agents/toxins that can be used for its management. Insect growth
regulator (IGR); Bacillus thuringiensis serovar. israelensis (Bti), serovar. jegathesan (Btjeg),
serovar. medellin (Btmed); mosquitocidal toxins (Mtxs).
150 Maria Helena Neves Lobo Silva Filha et al.

(Cetin et al., 2005; Giraldo-Calderon et al., 2008; Guidi et al., 2013b; Liu
et al., 2004; Marcombe et al., 2011; Pridgeon et al., 2008; Tetreau et al.,
2013). Spinosins, for instance, are also bacterial larvicides that have been
recently registered in some countries for mosquito control (Hertlein
et al., 2010). They show a relatively narrow toxicity spectrum, and have
recently been introduced with promising results, alone or combination with
other biolarvicides (Anderson et al., 2011; Cetin et al., 2005; Harbison et al.,
2013; Jiang and Mulla, 2009; Kumar et al., 2011; Marcombe et al., 2011;
Marina et al., 2012). The rational utilisation of Ls in the context of an inte-
grated programme using suitable control strategies and including monitoring
of the mosquito susceptibility can ensure its effectiveness for the control
interventions and can prevent the onset of resistance. The major aspects
concerning the management of resistance to Ls presented in this section
are summarised in Fig. 3.10.

ACKNOWLEDGEMENTS
We thank all colleagues who provided suggestions for this work; publishers for the
permissions for using figures presented in this chapter; Claudia Maria Fontes de Oliveira,
Const^ancia Junqueira Ayres, Maria Alice Varjal de Melo-Santos and Ros^angela Maria
Barbosa from the Department of Entomology (CPqAM-FIOCRUZ) for the
encouragement for writing this chapter.

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 CHAPTER FOUR

Discovery and Development

of Insect-Resistant Crops Using
Genes from Bacillus thuringiensis
Kenneth E. Narva, Nicholas P. Storer, Thomas Meade
Dow AgroSciences, LLC., Indianapolis, Indiana, USA

Contents
1. Introduction 178
2. Bt-Based Biopesticides 179
2.1 History of use of Bt for insect control 179
2.2 Biopesticides based on Bt 180
2.3 Molecular eraFirst cloned Bt insecticidal protein genes 181
2.4 Transconjugation, recombinant strains and alternative delivery systems
for Bt-based biopesticides 182
3. Discovery, Characterization and Development of Insecticidal Protein Genes
as Crop Traits 184
3.1 Diversity of Bt insecticidal proteins 184
3.2 Biological activity of Bt insecticidal proteins 185
3.3 Bt insecticidal protein structure and function: Cry proteins 187
3.4 Cry protein mechanism of action 188
3.5 Bt insecticidal protein structure and function: Cyt proteins 190
3.6 Bt insecticidal protein receptors 191
3.7 Mechanisms of resistance to Bt insecticidal proteins 191
4. Discovery and Development of Bt Crops 193
4.1 The discovery and development process 193
4.2 Gene discovery 194
4.3 First demonstrated success of Bt Cry GE plants 196
4.4 Transformation technologies 197
4.5 Introgression and testing 198
5. Regulation 198
5.1 Product identification and characterization 201
5.2 Human health assessment 201
5.3 Environmental effects 203
5.4 Considerations for stacks 206
5.5 Continued regulatory oversight of commercialized GE events 206

Advances in Insect Physiology, Volume 47 # 2014 Elsevier Ltd 177

ISSN 0065-2806 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800197-4.00004-X
178 Kenneth E. Narva et al.

6. Insect Resistance Management 207

7. Bt CropsA Snapshot of Today 210
7.1 Commercialized Bt proteins 210
7.2 Global adoption of Bt crops 214
7.3 Commercialized products 215
8. Bt CropsProspects for the Future 230
8.1 Novel Bt proteins 230
9. Conclusions 232
Acknowledgements 233
References 233

Abstract
Bacillus thuringiensis (Bt) is a ubiquitous, spore-forming soil bacterium that is well known
for production of insecticidal proteins that are active on a wide range of pest insects. The
potential of Bt to be used as an insecticide was recognized in the early twentieth century
and since that time many Bt-based biopesticides have been commercialized. The
advent of modern molecular biology tools made it possible to engineer plants to
express the genes coding for Bt insecticidal proteins as a safe, convenient and highly
effective means to protect plants from insect damage. The first Bt crop was commer-
cialized in 1995, and today Bt corn, cotton and soybean are cultivated on ca. 76 million
hectares in 27 countries. First generation products containing single Bt genes were
followed by broader spectrum products containing multiple Bt genes with the most
recent generation of products contain multiple Bt genes encoding proteins that target
the same pest(s) but with differences in their mechanism of action (i.e. gene pyramids)
as a means of increasing product durability. Developing Bt crops is a long and expensive
process that by recent estimates averages 13 years at a cost of $136 million. The process
of obtaining approvals by government regulatory agencies is among the most critical in
the later stages of the development process and represents ca. 25% of the total cost in
bringing a Bt crop to the market. Multiple factors drive the search for novel insect resis-
tance (IR) traits and Bt remains a significant focus of new IR trait discovery.

1. INTRODUCTION
Bacillus thuringiensis (Bt) is a ubiquitous, spore-forming soil bacterium
that is well known for production of parasporal crystalline inclusions during
the stationary phase of cell growth. These parasporal inclusions are comprised
of insecticidal proteins known as -endotoxins, including those classified as
Cry (crystalline) or Cyt (cytolytic) proteins. The parasporal crystalline
inclusions produced by Bt are composed of a diversity of proteins across dis-
tinct phylogenetic groups of sequences (Crickmore et al., 1998) and (http://
www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). Collectively, Cry
Bt Crops 179

proteins are active on a wide range of insects including those among the
orders of Lepidoptera, Diptera and Coleoptera (van Frankenhuyzen,
2009). Bt also produces soluble insecticidal proteins during the cell vegetative
growth phase before the onset of sporulation that are named Vips (vegetative
insecticidal proteins) (Estruch et al., 1996; Warren, 1997; http://www.
lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/).
Various subspecies of Bt have historically been developed for use as foliar
applied biopesticides (Sanahuja et al., 2011) and have a long history of safe
use (Siegel, 2001). With the advent of modern molecular biology tools, it has
become possible to engineer plants to express the genes coding for Bt insec-
ticidal proteins as a safe, convenient and highly effective means to protect
plants from insect damage. The development of insect resistant crops has
rapidly progressed since the commercial introduction of Bt potato in
1995 and Bt corn and cotton in 1996 (http://www.epa.gov/oppbppd1/
biopesticides/pips/pip_list.htm). Today insect resistance (IR) traits based
on Bt proteins have achieved a high rate of world-wide adoption ( James,
2013). A current challenge for Bt trait seed producers is protecting the
long-term durability of Bt trait technology. Innovative insect resistance
management (IRM) strategies include the use of genetically engineered
(GE) crops containing combinations of Bt genes encoding novel insecticidal
proteins (i.e. pyramids). This chapter provides an overview of the history of
Bt biopesticides leading to Bt crop development, the success of Bt-based IR
traits and future prospects for Bt as a source of IR trait technology.

2. Bt-BASED BIOPESTICIDES
2.1. History of use of Bt for insect control
Bt has a long history of safe use as a biopesticide for insect control (Siegel,
2001). For an elegant review of the early historical events in the discovery
and development of insecticidal bacteria with significant attention directed
at Bt see Federici (2005). The bacterium that became known as Bt was first
reported in Japanese literature by Ishiwata (1901) during study of bacterial
disease of silkworms. Later, Berliner (1915) described a similar Bacillus bac-
terium that killed flour moths and named the organism B. thuringiensis for the
Thuringia region in Germany where the bacterial disease was discovered.
Research into the utility of Bt as an insecticide followed (Mattes, 1927)
and activity in field trials against the European corn borer, Ostrinia nubilalis
(Hubner), was reported in 1930 (Husz, 1930). This work led to the devel-
opment of a Bt product known as Sporeine that was commercialized in
180 Kenneth E. Narva et al.

the late 1930s (Federici, 2005). The potential for Bt to be used as an insec-
ticide became more widely appreciated years after these early studies. Pub-
lications by Hannay (1953) on the Bt parasporal crystal bodies and
demonstration that the parasporal crystals were capable of killing silkworms
(Angus, 1954)set the stage for an increase in research focused on developing
Bt as an insect control agent.

2.2. Biopesticides based on Bt

Advances in the applied science of Bt were aided by systematic characteri-
zation of the insecticidal properties of Bt strains. A system for naming strains
based on flagellar serotype (de Barjac and Bonnefoi, 1962, 1968) and estab-
lishment of standardized bioassay techniques based on B. thuringiensis HD-1
(Dulmage, 1981) provided the basis for characterizing strains and comparing
insecticidal properties among Bt isolates. This led to the development of suc-
cessful commercial Bt products in the 1960s, most notably Dipel (Abbot
Laboratories) and Thuricide (Sandoz Corporation), both of which were
based on the HD-1 isolate of Bt subspecies kurstaki (serotype H 3a3b)
(Federici, 2005). These products controlled lepidopertan pests important
in agriculture and forestry such as the cabbage looper, Trichoplusia ni
(Hubner), corn earworm or bollworm, Helicoverpa zea (Boddie), the tobacco
budworm, Heliothis virescens (F.), the diamondback moth, Plutella xylostella
(L.), the gypsy moth, Lymantria dispar (L.) and the spruce budworm,
Choristoneura fumiferana (Clemens).
The success of Dipel and Thuricide led to the development in the United
States of 177 registered products containing viable Bt between the years
1961 and 1995. Bt-based biopesticide products have an excellent mamma-
lian safety record based on laboratory studies and extensive field experience
(Siegel, 2001). Examples of Bt-based biopesticide products are shown in
Table 4.1. For a listing of currently registered Bt biopestides, refer to the
United States Environmental Agency website (http://www.epa.gov/
pesticides/biopesticides/).
Efforts to increase Bt strain productivity through optimized fermentation
and formulation processes drove the development of improved products that
replaced earlier product offerings (Kaur, 2000). Further, the discovery of Bt
strains with activity on different orders of insects provided the opportunity
to expand the range of pests controlled by Bt biopesticides. While many of
the most successful products for control of lepidopteran pests were based on
Bt kurstaki strains, novel Bt subspecies were discovered with activity against
other insect orders. Importantly, Bt subspecies israelinsis (H 14) (Goldberg
Bt Crops 181

Table 4.1 Commercialized Bt biopesticides

Trade name Bt subsp. strain Producer Specificity
Bactospeine kurstaki HD-1 Abbott Lepidoptera
Biobit kurstaki HD-1 Abbott Lepidoptera
Dipel kurstaki HD-1 Abbott Lepidoptera
Florbac aizawai Abbott Lepidoptera
Costar kurstaki SA-12 Thermo trilogy Lepidoptera

Del n kurstaki SA-11 Thermo trilogy Lepidoptera
Javelin kurstaki SA-11 Thermo trilogy Lepidoptera
Thuricide kurstaki HD-1 Thermo trilogy Lepidoptera
Tekar israelensis Thermo trilogy Diptera
Bactimos israelensis Abbott Diptera
Vectolex GC B. sphaericus Abbott Diptera
Acrobe israelensis American cyanamide Diptera
Novodor tenebrionis Abbott Coleoptera
Trident tenebrionis Thermo trilogy Coleoptera
From Kaur (2000).

and Margalit, 1977); recently reviewed by Ben-Dov (2014) was found to be

active on mosquitoes and black flies, while Bt subspecies morrisoni (H 8a8b,
variety tenebrioinis) was active on the larvae of coleopteran species (Krieg
et al., 1983). The expanded range of pests controlled by various Bt subspecies
suggested that additional new strains could be found with unique pesticidal
properties. This prompted a vigorous world-wide effort to discover novel
strains with new insecticidal activity profiles (see for example Feitelson
et al., 1992; Jung et al., 1998; Wasano and Ohba, 1998). Efforts to discover
Bt isolates with novel biological activity and characterization of the insecti-
cidal proteins that are responsible for strain activity continue today (Arrieta
and Espinoza, 2006; Bravo et al., 1998; Noguera and Ibarra, 2010; Vidal-
Quist et al., 2009).

2.3. Molecular eraFirst cloned Bt insecticidal protein genes

Plasmid-based DNA cloning became a routine laboratory procedure in the
late 1970s (Bolivar et al., 1977), making it possible to isolate and study
recombinant genes and proteins. The fact that parasporal protein inclusions
182 Kenneth E. Narva et al.

were known to be responsible for the insecticidal activity of Bt led

researchers to use molecular biology techniques to search for the genes
encoding these proteins. Schnepf and Whiteley (1981) cloned the first Bt
gene encoding an insecticidal protein from the Bt strain in Dipel, Bt kurstaki
HD-1. Under the revised nomenclature system for Bt insecticidal proteins
(Crickmore et al., 1998) this gene later became known as cry1Aa1. Further
molecular biology work demonstrated that genes coding for different insec-
ticidal proteins were located on distinct restriction endonuclease fragments
of DNA from Bt HD-1 (Kronstad et al., 1983). These results were impor-
tant in establishing that Bt strains most often contain multiple genes coding
for insecticidal proteins. The characterization of the genes encoding Cry
proteins in Bt kurstaki HD-1 (now designated Cry1Aa, Cry1Ab, Cry1Ac
and Cry2Aa) was followed quickly by the isolation of genes coding for
many additional Cry proteins including cry3Aa (see for example Herrnstadt
et al., 1987; Hofte and Whiteley, 1989; Schnepf et al., 1998; Sekar et al.,
1987). Cry3Aa is notable as the first example of a Bt protein with activity on
a coleopteran pest, the Colorado potato beetle, Leptinotarsa decemlineata
(Say). Characterization of recombinant Cry proteins in E. coli or
acrystalliferous Bt strains using shuttle plasmids (Arantes and Lereclus,
1991; Lecadet et al., 1992) provided a means to investigate the genetic basis
for different strain-level insecticidal activity. This set the stage to develop
new pest control technology based on recombinant Bt insecticidal proteins.

2.4. Transconjugation, recombinant strains and alternative

delivery systems for Bt-based biopesticides
Several approaches have been used to develop Bt biopesticides improved for
properties such as increased toxicity, expanded range of target pests, or for
delaying the development of resistant insect populations by combining
insecticidal proteins that target the same pests but differ in their mechanism
of action, such as by acting at different binding sites. Ecogen Corporation
developed methods for conjugal transfer of Cry protein encoding native
Bt plasmids, e.g. Bt strain 2424 that expresses both cry1A and cry3A genes
for control of lepidopteran and coleopteran pests (Carlton and Gawron-
Burke, 1993). However, strain construction by plasmid conjugation is lim-
ited by factors including plasmid incompatibility, location of cry genes on
large, non-transmissible plasmids and segregational loss of plasmids in trans-
conjugant strains. Ecogen addressed these challenges by developing recom-
binant DNA technology and site-specific recombination systems to
introduce cry genes into Bt recipient host strains and subsequently eliminate
Bt Crops 183

the antibiotic selectable marker resistant genes that might cause environ-
mental safety concerns (Baum et al., 1998). Several examples of recombinant
Bt strains are listed in Table 4.2.
Mycogen Corporation used a different approach to produce novel bio-
pesticides based on over-expression of cry genes in recombinant Pseudomonas
fluorescens (Gaertner et al., 1993). This gene expression system used recom-
binant DNA technology to express Cry proteins at high levels under high-
density cell culture fermentation conditions. The recombinant bacteria were
fixed in a proprietary treatment that rendered cells non-viable without
impacting the activity of the insecticidal proteins. The fixed,

Table 4.2 Bt biopesticides based on novel/recombinant strains

Product Bt subsp. strain or genes Producer Specificity
Transconjugant
strains Bt subsp. strain
Agree aizawai Thermo trilogy Lepidoptera
Condor kurstaki Ecogen Lepidoptera
Cutlass kurstaki Ecogen Lepidoptera
Design aizawai Ecogen Lepidoptera
Foil kurstaki Ecogen Lepidoptera/
Coleoptera
Recombinant
strains genes
Raven cry1Ac (x2), cry3A + cry3Bb Ecogen Lepidoptera/
(recombinant) Coleoptera
CRYMAX cry1Ac (x3), cry2A + cry1C Ecogen Lepidoptera
(recombinant)
Lepinox cry1Aa, cry1Ac (x2), cry2A Ecogen Lepidoptera
+ cry1F-1Ac (recombinant)
Maatch kurstaki cry1A and aizawai Mycogen Lepidoptera
cry1C
M/C aizawai cry1C Mycogen Lepidoptera
M-Peril kurstaki cry1Ac Mycogen Lepidoptera
MVP kurstaki cry1Ac Mycogen Lepidoptera
MTRAK cry3Aa Mycogen Coleoptera
184 Kenneth E. Narva et al.

bioencapsulated proteins were more persistent under environmental field

conditions. These were the first recombinant biopesticides approved for
field tests and commercialization by the United States Environmental Pro-
tection Agency in 1991 (http://www.epa.gov/pesticides/biopesticides/).
Mycogen Corporation marketed products based on Cry1Ac for control
of Lepidoptera (MVP) and Cry3Aa for control of Colorado potato beetle
(MTRAK), along with combinations of Cry1Ac and Cry1C (Maatch)
for broad spectrum control of Lepidoptera.
Crop Genetics International used yet another approach for delivering Bt
biopesticides in recombinant endophytic bacteria (Dimock et al., 1993).
The probability of endophytes surviving outside the plant host are low,
thereby providing a level of biological containment. Lampel et al. (1994)
engineered Clavibacter xyli subspecies cynodontis (CXC), a bacterial endo-
phyte that inhabits the xylem of Bermuda grass, to express a chromosomally
integrated cry1Ac gene. C. xyli can colonize other grasses including maize.
Colonized maize expressing Cry1Ac showed reduced feeding damage by
O. nubilalis though the level of protection to insect feeding damage did
not translate to increased grain yield (Tomasino et al., 1995).

3. DISCOVERY, CHARACTERIZATION AND

DEVELOPMENT OF INSECTICIDAL PROTEIN GENES
AS CROP TRAITS
3.1. Diversity of Bt insecticidal proteins
Bt produces a variety of crystalline and soluble insecticidal proteins that com-
prise various primary sequence homology groups (Schnepf et al., 1998). To
date, over 750 unique Bt proteins ranging in size from ca. 14 kDa to over
140 kDa have been described that are classified into at least 73 distinct
homology groups. Most Bt insecticidal proteins fall within three main phy-
logenetic groups: Cry, Cyt or Vip (Crickmore et al., 1998); (http://www.
lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html).
The Cry class of pesticidal proteins contains the largest number of
sequences, many of which share conserved amino acid sequence and structural
similarity. The Cry family also includes binary, two component insecticidal
proteins, some of which share similarity to the Lysinibacillus sphaericus Bin pro-
teins, as well as proteins related to the Mtx families of toxins (Berry, 2012) and
parasporins with cytotoxicity to human cancer cells (Ohba et al., 2009).
The Cyt family comprises a group of generally cytolytic proteins with no
sequence homology to the Cry proteins (http://www.lifesci.sussex.ac.uk/
Bt Crops 185

home/Neil_Crickmore/Bt/intro.html). Cyt proteins can synergize Cry

proteins (Ben-Dov, 2014; Chang et al., 1993; Wu et al., 1994) in a manner
that depends on the binding interaction of Cyt and Cry proteins (Perez et al.,
2005, 2007). Cyt proteins share varying levels of sequence homology with
proteins originating from a variety of microbial pathogens (Soberon
et al., 2013).
Vips are soluble proteins produced during the logarithmic phase of Bt
growth. To date, four main groups, Vip1, Vip 2, Vip3 and Vip4, have been
described. The soluble Vips, Vip1Aa1 and Vip2Aa1, are approximately 100
and 52 kDa molecular weight, respectively, and act together as a binary
toxin (Warren, 1997). Vip1Aa is homologous to the CdtB toxin component
of Clostridium difficile, the Ib component of Clostridium perfringens iota toxin
and the protective antigen of B. anthracis. Vip2Aa is an ADP-ribosylase with
a high degree of sequence and structural similarity to the enzymatic domains
of CdtA of C. difficile and iota toxin of C. perfringens (de Maagd et al., 2003;
Han et al., 1999). Vip3 proteins are approximately 80 kDa proteins that are
active on lepidopteran pests. The biological activity of Vip4 has not been
published.
The number and diversity of genes encoding Bt insecticidal proteins
continues to rapidly expand as researchers world-wide search for new Bt iso-
lates with novel biological activity (Fig. 4.1).

3.2. Biological activity of Bt insecticidal proteins

A highly valued benefit of Bt insecticidal proteins is the relatively narrow
spectrum of activity against susceptible insects. Bt insecticidal proteins are
highly active on insect larvae but have little or no activity on adult insects
(Betz et al., 2000).
Insecticidal activity of Bt Cry proteins across insect orders was recently
reviewed by van Frankenhuyzen (2009, 2013). These reviews are based on
over 25 years of published data on biological specificity of Cry and Cyt pro-
teins. Much of these data are incorporated into the Bt Toxin Specificity
Database (http://www.glfc.cfs.nrcan.gc.ca/bacillus). Information contained
in the Bt Toxin Specificity Database is focused on spore free preparations of
crystals or insecticidal proteins that were obtained through expression of
cloned genes or purified from strains expressing a single insecticidal protein.
As the number of Bt insecticidal protein sequences has grown, many differ-
ent pests were found to be susceptible to Bt proteins including orders not
previously tested such as Hymenoptera, Hemiptera and Rhadbditida
186 Kenneth E. Narva et al.

120

Genes
100
Holotypes

80
Bt genes (n)

60

40

20

0
19
19
19 6
19
19 8
19 9
19 0
19
19 2
19
19 4
19
19 6
19 7
19
20
20 0
20 1
20 2
20
20 4
20
20 6
20
20
20 9
20 0
20 1
20
85
8
87
8
8
9
91
9
93
9
95
9
9
98
99
0
0
0
03
0
05
0
07
08
0
1
1
12
13
Figure 4.1 Discovery of Bt genes recorded on the Bt Toxin Nomenclature Website
maintained by the Bt toxin nomenclature committee (Crickmore et al., 2014). The total
number of new Cry, Cyt and Vip genes recognized by the committee in a given year is
shown as Genes. The total number of new gene classes (as defined by the committee)
recognized in a given year is shown as Holotypes.

(nematodes). Data in the Bt Toxin Specificity Database also reveal cross-

order activity in 13 primary rank families across three classes of insecticidal
proteins (Cry, Cyt and Vip) (van Frankenhuyzen, 2013). Cross-order activ-
ity is an important consideration in selecting Bt proteins for commercializa-
tion because it necessitates the appropriate design of studies to characterize
risk associated with activity outside the primary insect specificity range. The
data also reflect that variation in factors such as assay conditions, methods of
protein preparation and quantitation, pre-ingestion protein activation and
insect population differences or life stage, to name a few, make comparison
of protein insecticidal potency difficult. This highlights the need for stan-
dardized assays for estimating insecticidal protein expression levels and
potency, factors important to IRM.
The class of Bt Vips have been more recently discovered (Estruch et al.,
1996; Warren, 1997). Vip1Aa1 and Vip2Aa1 act together as a binary toxin
that is highly potent against the western corn rootworm (WCR), Diabrotica
virgifera virgifera LeConte, coleopteran pest that feeds on corn roots. Mem-
bers of the Vip3 group of proteins have received more attention owing to
excellent activity on economically important lepidopteran pests such as the
black cutworm, Agrotis ipsilon (Hufnagel), H. zea, H. virescens, the fall army-
worm, Spodoptera frugiperda ( J. E. Smith) and the beet armyworm, Spodoptera
Bt Crops 187

exigua (Hubner) (Estruch et al., 1996; Fang et al., 2007; Hernandez-

Martinez et al., 2013; Lee et al., 2003). The Vip3 proteins are very different
in primary sequence compared to the lepidopteran-active Cry protein
group, and binding studies suggest a different mechanism of action com-
pared to three domain Cry proteins (Bergamasco et al., 2013; Lee et al.,
2003, 2006; Sena et al., 2009). The novel mechanism of action for Vip3 pro-
teins makes this group attractive for commercial applications when com-
bined with Cry proteins as gene pyramids for IRM.
Cyt toxins (reviewed by Butko, 2003; Soberon et al., 2013; Chapter 3)
are a subclass of Bt insecticidal crystal proteins that are named for their gen-
eral cytolytic activity. Cyt proteins show selective toxicity against mosqui-
toes and blackflies. However, examples of coleopteran-active Cyt proteins
are Cyt1Aa activity against Chrysomela scripta F. (Federici and Bauer, 1998)
and the ability of Cyt1Ba (Payne et al., 1995) and Cyt2Ca1 (Rupar et al.,
2000) to kill WCR larvae.

3.3. Bt insecticidal protein structure and function: Cry proteins

In terms of structurefunction relationships, the most well studied Bt proteins
are members of the three domain Cry -endotoxins. These proteins range in
size from approximately 70130 kDa. Many Cry proteins are produced as
protoxins requiring activation by proteolytic removal of the C-terminal crys-
tallization domain to produce the core insecticidal protein (Schnepf et al.,
1998). Primary protein sequence analysis reveals five conserved sequence
blocks and a high degree of sequence variability between conserved blocks
three and five (Hofte et al., 1988; Schnepf et al., 1998). In contrast, the
C-terminal crystallization domain sequences tend to be highly conserved
among subclasses. The correlation of bioactivity spectrum with sequence
variability among the activated forms of different Bt -endotoxins led to early
hypotheses that the hypervariable regions between conserved blocks three
and five are responsible for differences in insect specificity.
The first three-dimensional Bt crystal structures determined were of
Cry3Aa1 (Li et al., 1991) and Cry1Aa1 (Grochulski et al., 1995;
Fig. 4.2). The Cry1 and Cry3 structures are remarkably similar and are com-
prised of three distinct domains with the following features (for reviews see
de Maagd et al., 2003; Pigott and Ellar, 2007). Domain 1 is a bundle of seven
alpha helices where helix five is surrounded by six amphipathic helices. This
domain has been implicated in pore formation and shares homology with
other pore forming proteins including hemolysins and colicins. Domain 2
188 Kenneth E. Narva et al.

Figure 4.2 Protein crystal structures of representative Bt insecticidal proteins. (A) Three
dimensional structure of Cry1Aa1 (PDB code: 1CIY), a three domain Cry protein.
(B) Three-dimensional structure of the cytolytic crystal protein Cyt2Aa (PDB code: 1CBY).

is comprised of three anti-parallel beta sheets. This domain shares homology

with certain carbohydrate-binding proteins including vitelline and jacaline.
The loops of this domain play important roles in binding insect midgut
receptors. Domain 3 is a beta sandwich of two anti-parallel beta sheets.
Structurally this domain is related to carbohydrate-binding domains of pro-
teins such as glucanases, galactose oxidase, sialidase and others. This domain
binds certain classes of receptor proteins and perhaps participates in pore for-
mation. Conserved Bt sequence blocks two and three map near the
N-terminus and C-terminus of domain 2, respectively. Hence, these con-
served sequence blocks 2 and 3 are approximate boundary regions between
the three functional domains. For greater detail of the structure and function
relationships of these toxins, the reader is referred to Chapter 3.
Several other Cry protein structures have been determined (Table 4.3),
including diverse structures for Cyt1Aa (Cohen et al., 2011), Cyt2Aa1
(Li et al., 1996; Fig. 4.2) and binary (Cry34Ab1/Cry35Ab1) proteins (see
Table 4.3 for PDB accession numbers).

3.4. Cry protein mechanism of action

Cry proteins intoxicate insects by disrupting midgut epithelial tissues follow-
ing oral ingestion (see Chapter 3 for greater detail). The mode of action of
Cry involves pore formation. The mechanism of action, i.e. the molecular
Bt Crops 189

Table 4.3 Bt insecticidal protein crystal structures available in the Protein Data Bank
(PDB) (Website: http://www.rcsb.org/pdb/home/home.do)
PDB
Protein accession Structure Citation Year
Cyt2A1 1CBY Non-three domain Li et al. (1996) 1996
Cry1Aa1 1CIY Three domain Grochulski et al. (1995) 1995
Cry3Aa1 1DLC Three domain Li et al. (1991) 1991
Cry2Aa 1I5P Three domain Morse et al. (2001)
Cry3Bb1 1JI6 Three domain Galitsky et al. (2001) 2001
Cry4Ba 1W99 Three domain Boonserm et al. (2005) 2005
Cry4Aa 2C9K Three domain Boonserm et al. (2006) 2006
Cyt2Ba 2RCI Non-Three domain Cohen et al. (2008) 2008
Cry8Ea1 3EB7 Three domain Guo et al. (2009) 2009
Cyt1Aa 3RON Non-Three domain Cohen et al. (2011) 2011
Cry5Ba1 4D8M Three domain Hui et al. (2012) 2012
Cry34Ab1 4JOX Non-Three domain, unpublished 2014
binary with Cry35Ab1
Cry35Ab1 4JP0 Non-Three domain, unpublished 2014
binary with Cry34Ab1

events that lead to pore formation, can be summarized as follows. Cry pro-
teins are often produced as protoxins that are first solubilized in the insect
midgut and then proteolytically processed to yield smaller, activated poly-
peptides. The activated Cry proteins then bind to specific receptors on
the surface of insect midgut epithelial cells. Receptor binding is followed
by assembly of activated Cry proteins into pores that result in colloid osmotic
lysis of midgut cells due to an influx of solutes from the midgut lumen. Cell
lysis leads to disruption of the midgut epithelium and, ultimately, death of
the insect larva. This is often considered the classical model for Bt mech-
anism of action (Fig. 4.3). However, many details of this model remain unre-
solved. Two models have been researched in recent years that propose more
detailed mechanistic steps leading to insect death. These models are the
sequential binding model leading to pore formation (reviewed in
Soberon et al., 2009; Soberon et al., 2010) that builds on the classical pore
formation model and the signalling pathway model wherein Bt protein
190 Kenneth E. Narva et al.

Crystal produced during Bt sporulation

Ingestion

Insecticidal protein solubilized in

the insect midgut

Proteolysis
Insecticidal protein activated by
midgut proteases

Binding
Activated insecticidal proteins bind receptors
on the surface of midgut epithelial cells

Membrane insertion
Pore formation
Increased permeability
Loss of membrane function

Damaged midgut epithelium

Insect dealth
Figure 4.3 Schematic representation of the steps leading to pore formation and insect
death according the classical model of Bt mechanism of action (Vachon et al., 2012).

binding to receptors is proposed to activate signalling pathways that lead to

necrosis and cell death (Zhang et al., 2005, 2006). Critical review of Bt pro-
tein mechanism of action data continues to support the classical pore forma-
tion model as a sufficient description of how Cry proteins function, as the
molecular events following receptor binding that lead to pore formation in
insect midgut cell membranes remain poorly understood (Vachon
et al., 2012).

3.5. Bt insecticidal protein structure and function: Cyt proteins

(The reader is referred to Chapter 3 for more detailed review of Cyt protein
structurefunction.) Cyt2Aa1 (Fig. 4.2) exemplifies the general fold of the
Cyt group of proteins with known structures (Cohen et al., 2008, 2011; Li
et al., 1996). Cyt proteins have a structure wherein two outer layers of alpha
helix hairpins surround a beta sheet. Cyt proteins function through interac-
tions with non-saturated membrane lipids including phosphatidylcholine,
phospahtidylehtanolamine and sphingomylin (Ben-Dov, 2014; Thomas
and Ellar, 1983). Cyt proteins are proposed to exert their insecticidal effect
Bt Crops 191

by formation of multimeric pores or by a less-specific detergent mechanism

(Butko, 2003).

3.6. Bt insecticidal protein receptors

Despite the lack of a full understanding of Bt insecticidal protein mechanism
of action, considerable information is available on the role of insect midgut
receptors that bind Cry proteins (Gomez et al., 2007; Heckel et al., 2007;
Likitvivatanavong et al., 2011a,b; Pigott and Ellar, 2007; Chapter 3). The
demonstration of high-affinity binding sites on midgut membranes has led
to the characterization of a number of functional Cry protein receptors. In
Lepidoptera these membrane receptors include cadherin-like proteins, ami-
nopeptidases (APNs), alkaline phosphatases (ALPs),and ABC transporters.
Several coleopteran midgut proteins other than cadherins have been
demonstrated to function as Bt Cry protein receptors. These include a
sodium solute symporter for Cry3Aa in Tribolium castaneum (Herbst) that
contains cadherin repeats (Contreras et al., 2013). ADAM (A Disintegrin
And Metalloprotease) was demonstrated to be a functional receptor for
Cry3Aa in L. decemlineata (Ochoa-Campuzano et al., 2007). ADAMs belong
to the metzincin subgroup of the zinc protease superfamily. ADAMs are
modular transmembrane proteases implicated in the control of membrane
adhesion. Cry3Aa domain 2 loop 1 was shown to be involved in ADAM
recognition by competition with a synthetic peptide.
Lastly, in the nematode Caenorhabditis elegans (Maupas), glycolipids were
identified as receptors for Bt Cry5Ba (Griffitts et al., 2005). C. elegans
mutants resistant to Cry5Ba were determined to have lost glycolipid carbo-
hydrates. It was further shown that Cry5Ba binds glycolipids and that bind-
ing is dependent on carbohydrates for toxicity in vivo.

3.7. Mechanisms of resistance to Bt insecticidal proteins

(The reader is referred to Chapter 8 for a detailed review of resistance to Bt
proteins.) Field selection for insect populations resistant to Bt insecticidal
proteins is a concern for the long-term durability of commercialized Bt
products. As a result, significant research has been directed at characterizing
Bt-resistant insect colonies selected in laboratory experiments to understand
the genetic and molecular basis of Bt resistance. Resistance to Bt insecticidal
proteins could possibly occur at any step in the mechanism of action outlined
in Fig. 4.3. Among the different Bt-resistant insects several different mech-
anisms of resistance have been characterized (reviewed in Heckel et al.,
192 Kenneth E. Narva et al.

2007; Pardo-Lopez et al., 2013) including altered activation of Cry proteins

by midgut proteases (Keller et al., 1996; Li et al., 2004; Oppert et al., 1997),
protein sequestration by glycolipids (Ma et al., 2012) or esterases (Gunning
et al., 2005), elevated immune response (Hernandez-Martinez et al., 2010;
Rahman et al., 2004) or by reduced Bt insecticidal protein binding to insect
midgut membranes.
The most common type of resistance to Bt insecticidal proteins, referred
to as Mode 1 resistance (Tabashnik et al., 1998), is characterized by a high
level of resistance (>500-fold) to a Cry toxin, recessive inheritance and
reduced Cry protein binding to insect midgut brush border membranes.
Among the insect colonies resistant to Bt insecticidal proteins are multiple
examples of reduced binding resulting from mutations in receptor molecules
or reduced transcription of receptor genes (Heckel et al., 2007; Pardo-Lopez
et al., 2013). Different resistant insect species are known to have receptor
mutations in cadherin, APN or the ABCC2 transporter (Baxter et al.,
2011; Gahan et al., 2001, 2010; Herrero et al., 2005; Jurat-Fuentes
et al., 2004).
The first report of genetic linkage to cadherin-mediated resistance in
Lepidoptera was in the Cry1A-resistant H. virescens YHD2 strain. Cadherin
in this strain is interrupted by a retrotransposon resulting in high levels of
resistance to Cry1Ac (Gahan et al., 2001; Jurat-Fuentes et al., 2004). The
second example of cadherin-mediated resistance was in a Cry1Ac-resistant
strain of the pink bollworm, Pectinophora gossypiella (Saunders), a pest of cot-
ton (Morin et al., 2003). This strain harboured three mutant alleles of a
cadherin encoding gene linked with resistance to Bt toxin Cry1Ac. The
mutations all disrupted cadherin gene alleles upstream of the Cry protein
binding region. In H. armigera, strain GYBT a deletion in a gene coding
for cadherin resulted in high levels of resistance to activated Cry1Ac
(Xu et al., 2005). Last, Cry1Ab-resistant sugarcane borer, Diatraea saccharalis
(F.), with high levels of resistance to Cry1Ab, exhibited reduced levels of
cadherin. RNAi was used to validate the role of cadherin in reduced suscep-
tibility to Cry1Ab in D. saccharalis (Yang et al., 2011).
The first report implicating GPI-anchored APN in Cry protein resis-
tance was in S. frugiperda where resistance to Cry1C correlated with a lack
of APN expression (Herrero et al., 2005). These results are consistent with
RNAi down regulation of Spodoptera litura (F.) APN, resulting in tolerance
to Cry1C (Rajagopal et al., 2002). It was later demonstrated in H. armigera
that a deletion in APN1 conferred resistance to Cry1Ac (Zhang et al., 2009).
In resistant strains of the O. nubilalis, two mutations in the APN-P gene were
Bt Crops 193

identified by expressed sequence tag analysis (Khajuria et al., 2011). Lastly,

Cry1Ac resistance in T. ni was found not to result from mutations in APN,
but rather that downregulation of APN at the transcriptional level by a trans-
regulatory mechanism resulted in Cry protein resistance. Down-regulation
of APN was genetically linked to the Cry-resistance phenotype but was not
caused by mutations in APN1.
The discovery of ABCC2 as a resistance determinant for Bt insecticidal
proteins is more recent. In laboratory selected H. virescens, Cry protein resis-
tance was genetically linked to mutant alleles of ABCC2 with a 22-base pair
deletion (Gahan et al., 2010). In P. xylostella and T. ni, resistance to Cry1Ac
mapped to a single homologous locus for ABCC2 (Baxter et al., 2011).
Together these results suggest parallel evolutionary responses that raise ques-
tions on how ABCC2 interacts with other mechanisms of resistance to Bt.

4. DISCOVERY AND DEVELOPMENT OF Bt CROPS

4.1. The discovery and development process
The discovery and development process employed by the major developers
of Bt crops has been the subject of recent reviews (Mumm, 2013; Privalle
et al., 2012). Company websites are also a good source of information on
current products and the innovation in their respective discovery and devel-
opment pipelines.
Details of how each company manages its pipeline vary but all use a stag-
ing system that is similar to that illustrated in Fig. 4.4. The genetic basis for
the desired trait is identified in the Discovery stage. In the Proof of Concept
stage, genes are tested in plants to assess their potential to deliver the desired
trait phenotype. Successful candidates are advanced to the Early Development
stage which marks the start of the effort to produce a specific GE event for
commercialization. This is also the stage in which studies are initiated that
will be included in regulatory submissions to government agencies. Testing
of events under more diverse environmental conditions and in more genetic
backgrounds occurs in the Advanced Development stage with the goal of iden-
tifying a single event for commercialization. Regulatory studies are com-
pleted in this stage and regulatory packages are submitted to government
agencies. In the Pre-Launch stage, plans are made for commercial introduc-
tion of the final product pending authorization by the relevant government
regulatory agencies.
The best estimates of the time and cost of discovering and developing a
Bt crop comes from a 2011 study conducted by the consulting firm Phillips
194 Kenneth E. Narva et al.

24 years 24 years 12 years 12 years 13 years

Proof of Early Late

Discovery Pre-launch
concept development development

Identification of Demonstration Transformation to Selection of an Bulk-up of

the gene(s) that the gene(s) produce an event event for seed for
responsible for a confer the for commercialization, commercial
trait. desired commercialization introgression into sale and
phenotype in the and initiation of commercial regulatory
crop of interest. regulatory studies. germplasm and approvals.
regulatory
submissions.
Figure 4.4 Generalized discovery and development staging system for a Bt crop. The
websites of Bt crop developers are typically a good source of information on their spe-
cific discovery and development staging systems as well as the innovation that is in their
pipelines.

McDougall for the industry association CropLife International based on data

provided by major developers of biotech crops (i.e. BASF Corporation,
Bayer CropScience, Dow AgroSciences, DuPont/Pioneer Hi-Bred,
Monsanto Company and Syngenta AG) (McDougall, 2011). For new bio-
tech crops introduced between 2008 and 2012, the average time required to
move through a pipeline from discovery to commercialization was 13.1 years
at an average cost of $136 million. Discovery followed by Proof of Concept
were the most expensive stages ($31 and $28 million, respectively) but the
collective costs of meeting regulatory requirements was $35.1 million rep-
resenting 25.8% of the total cost of bringing a biotech product to market.

4.2. Gene discovery

In the years following the isolation of the first gene coding for Bt Cry1Aa1 in
1981 (Schnepf and Whiteley, 1981), significant effort has been aimed at the
discovery of new Bt strains and new genes coding for Bt insecticidal proteins.
Today this research is driven by the wide range of potential applications of Bt
biopesticides and Bt trait technology along with the rapidly increasing adop-
tion of Bt crops ( James, 2013).
The industrial process for Bt insecticidal protein gene discovery begins
with the conception of a commercially important product idea to improve
upon existing technology or address an unmet need for pest control. Product
attributes considered important for insect control are pest spectrum,
Bt Crops 195

insecticidal protein potency and the low likelihood of cross resistance to

other Bt products on the market. Lack of cross resistance is important for
sustaining the utility of Bt products. The Bt discovery process often follows
an insecticidal activity-driven approach beginning with Bt strain character-
ization. Bt strains are cultured under varying conditions and characterized
for insecticidal activity on economically important insects. Bt strains with
novel or superior insecticidal properties are chosen as a source of genes
encoding insecticidal proteins.
Early Bt gene discovery efforts used standard biochemical fractionation
and recombinant DNA technology to identify, characterize and clone the
genes encoding insecticidal proteins. These techniques were based on com-
mon molecular biology methods such as DNA restriction fragment length
polymorphism (RFLP) to identify novel genes in the genomes of highly
active Bt strains (see for example Kronstad and Whiteley, 1986). Albeit a rel-
atively time consuming and low throughput process, early Bt gene discovery
work focused on Bt strains that had been well characterized for biological
activity, and as a result, genes encoding commercially important proteins
such as Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry3Aa were discovered.
These proteins are still being used in commercial GE, Bt products today,
although the rising pressure of field-evolved resistance to Bt crops expressing
these and other Cry proteins is threatening the long-term utility of some Bt
proteins in certain agricultural systems (Tabashnik et al., 2013).
Advances in molecular biology techniques and tools accelerated the rate
of Bt insecticidal gene discovery during the 1990s. Polymerase chain reac-
tion (PCR) represented a major advance in the ability to characterize DNA
(Saiki et al., 1988). Methods to apply PCR for Bt cry gene identification were
first reported by Carozzi et al. (1991). This led to the further development of
methods for the rapid genotyping of Bt strains for cry gene content (reviewed
in Porcar and Juarez-Perez, 2003). PCR-based methods can partially predict
Bt strain insecticidal activity based on known Cry proteins, and can also
detect novel cry sequence variants when coupled with multiplexed primer
reactions, restriction fragment length polymorphisms or DNA sequencing
of PCR amplicons. The use of cry-specific primers on DNA microarrays
has also been used to rapidly characterize cry genes in native Bt isolates
(Letowski et al., 2005).
Next-generation DNA sequencing technology represents the most
recent advance in the ability to rapidly discover new genes coding for Bt
insecticidal proteins. The affordability and high-throughput data generation
of next-generation sequencing platforms promise to enable sequencing of
196 Kenneth E. Narva et al.

many more Bt genomes in the very near future. As of March 7, 2014, there
are 12 completed genomes publicly available on NCBI (http://www.ncbi.
nlm.nih.gov/genome/genomes/486). The challenges of data analysis and
identification of genes encoding new Bt insecticidal proteins are being
addressed (Ye et al., 2012). Recently, a pangenomic study of Bt was reported
(Fang et al., 2011) in which chromosomes and plasmids encoding Cry pro-
teins were sequenced to a high degree of coverage for seven Bt strains. The
pangenomic approach, which does not intend to assemble all genomes to
completion, but rather to interrogate sequence space across multiple Bt
strains, coupled with advances in the scale and throughput of insect bioas-
says, represents a powerful approach to identify genes encoding novel Bt
insecticidal proteins. One can surmise that the growing number of new pro-
tein sequences in the Bt nomenclature database are at least in part due to the
impact of next-generation sequencing (Fig. 4.1).

4.3. First demonstrated success of Bt Cry GE plants

While Bt biopesticides are environmentally safe, disadvantages of the tech-
nology include the relatively short timeframe of effectiveness under envi-
ronmental conditions, the need for repeated application over time, and
the inability to impact insects with specialized feeding behaviour such as
those that feed on plant sap or below ground on plant roots. The develop-
ment of biolistic and Agrobacterium-mediated plant transformation technol-
ogy created the possibility to deliver and express Bt genes encoding insect
control proteins within the plant for the duration of the plant growth cycle.
Early attempts to express Bt cry genes in plants resulted in poor expression of
Cry1A proteins and yet plant tolerance to insect feeding was achieved.
Fischhoff et al. (1987) transformed tomato with truncated Cry1Ac resulting
GE plants resistant to H. zea feeding damage. Vaeck et al. (1987) transformed
tobacco with Cry1A resulting in GE plants resistant to Manduca sexta (L.).
Because Cry proteins were expressed to very high levels in Bt strains, atten-
tion was given to the differences in gene structure and codon usage between
Bt and the target host plants as a cause for low Cry protein expression
observed in GE plants. Perlak et al. (1991) were successful in increasing
Cry protein levels up to 100-fold by creating synthetic Cry-encoding trans-
genes with a codon usage biased toward that favoured by plants and lacking
mRNA destabilizing sequences such as polyadenylation signal sequences,
ATTTA sequences and A+T rich regions. Partially modified or fully mod-
ified transgenes encoding Cry1Ab or Cry1Ac resulted in higher expression
Bt Crops 197

and a higher proportion of GE tobacco or tomato plants tolerant to M. sexta

damage. Similar success was achieved with a modified Cry3Aa gene
expressed in GE potato that was resistant to L. decemlineata (Adang et al.,
1993). Further gene expression improvements have been realized by
increasing promoter strength, better polyadenylation termination and
enhanced expression by including introns in the 50 untranslated region of
the mRNA (Koziel et al., 1993). These early successes in generating GE
plants expressing Bt proteins set the stage for an industry-wide trend among
seed producers to produce GE Bt crops.

4.4. Transformation technologies

The ability to successfully transform a plant depends on several factors
including the availability of target tissues that are competent for propa-
gation or regeneration, an efficient method for delivery of DNA, the
ability to select for transformed cells and the ability to recover fertile, GE
plants (Hansen and Wright, 1999). Many different plant tissue types are
amenable to transformation including immature embryos, embryogenic sus-
pension cultures, embryogenic shoot tips, immature cotyledonary-nodes,
hypocotyls and leaf tissue (Lee et al., 2013). The selection of a tissue type
for use in a transformation system depends on many factors including sim-
plicity and accessibility (e.g. free from patent restrictions), but in the end it is
critical that fertile, GE plants are produced.
Agrobacterium-mediated transformation and particle bombardment are
the two most commonly used methods of DNA delivery. Agrobacterium-
mediated transformation uses the gene-transfer machinery of the bacterium
to introduce a specific piece of DNA (i.e. T-DNA) into the host cell which
ultimately integrates into the genome. Agrobacterium-mediated transforma-
tion can be used to deliver DNA to both dicots and monocots, can deliver
relatively large pieces of DNA, and typically a small number of T-DNA cop-
ies are integrated into the host genome at a single location in the genome
(Hansen and Wright, 1999; Smith and Hood, 1995). (Note: the unique inte-
gration of DNA into the host genome is called an event.)
Particle bombardment and other physical delivery approaches do not rely
on a biological mechanism for the delivery of DNA. Instead, particles of var-
ious materials are coated with DNA and physically introduced into target
cells. The particles used for DNA introduction are typically gold or tungsten
but silicon fibre whiskers have also been used (Hansen and Wright, 1999;
Petolino and Arnold, 2009). Unlike Agrobacterium-mediated transformation,
198 Kenneth E. Narva et al.

particle bombardment often creates complex events containing multiple

copies and/or fragments of DNA and insertion of the DNA into multiple
genomic regions (Finer and Dhillon, 2007).
Selectable marker genes (genes allowing transformed cells, tissues or
plants to be differentiated from non-transformed ones) are an important
component of plant transformation systems. Positive selectable marker genes
are most commonly used in the production of GE crops and include anti-
biotic resistance (e.g. the nptII gene which confers resistance to the antibi-
otics kanamycin and neomycin), herbicide tolerance (e.g. the pat gene
which confers tolerance to the herbicide glufosinate) or other genes (e.g.
the pmi gene which enables plants to use mannose as a carbon source in tissue
culture systems) which enhance survival of plant cells containing and
expressing them (Rosellini, 2012). In some cases, plants are transformed
with the selectable marker on a separate piece of DNA so that the plants pro-
duced in these systems have the genes of interest and the selectable marker
genes integrated as two-independent events. In these cases, the selectable
marker can be removed from the commercial product through traditional
breeding processes. However, in most cases, the selectable marker gene is
integrated with the genes of interest and is therefore contained in the com-
mercialized event. In cases where the selectable marker gene confers herbi-
cide tolerance, its presence in the commercial product is desirable.

4.5. Introgression and testing

Germplasm that is amenable to the transformation and tissue culture regen-
eration process is typically not the high-performing germplasm used in
todays intensive production agriculture. It is necessary to introduce a GE
event into elite germplasm via process of breeding and selection. The use
of molecular markers can dramatically enhance the speed and effectiveness
of this introduction by minimizing the transfer of alleles from the GE donor
line and maximizing the recovery of alleles from the elite germplasm
(Mumm, 2013). Throughout the process of introgression, a Bt event is eval-
uated in increasingly diverse germplasm and environments for performance
of the IR trait and the germplasm.

5. REGULATION
GE crops undergo comprehensive regulatory reviews for human
health and environmental safety by agencies throughout the world. Indeed,
GE crops and food receive far greater regulatory and scientific scrutiny than
Bt Crops 199

any conventional counterpart (Fedoroff, 2011). While most regulatory

authorities profess to regulate the products of genetic engineering rather
than the process itself, oversight is usually triggered by the employment
of recombinant DNA techniques to introduce new traits into a crop. Com-
mercial launch of a GE crop requires authorization for commercial cultiva-
tion in the country (or countries) of production, and import, food and feed
approvals in that countrys trading partners.
When evaluating the safety of GE crops, regulatory systems cover two
broad areas of consideration. First, regulators examine the potential for harm
arising from the intended direct effect(s) of the genetic modification, in the
present discussion being the expression of the Bt proteins that provide pro-
tection from targeted insect pest feeding damage. This assessment generally
considers environmental effects such as toxicity to beneficial organisms (e.g.
predators, parasitoids and pollinators) that feed in or on the Bt crop and soil
fauna and flora. The assessment of direct effects also includes assessment of
the safety of the Bt protein in food and feed, including toxicity and potential
to be an allergen. Second, regulators examine the crops and food for any
potential unintended effects on human health or the environment arising
from the genetic transformation itself or unintended indirect effects of the
added gene(s) and trait(s). The potential for unintended effects is considered
to arise from effects of the transformation process itself on the crop genome,
such as gene disruption in the region where the transgene is inserted, and
from pleiotropic effects of the transgenic protein(s) on plant metabolic pro-
cesses. Either of these could lead theoretically to altered crop composition or
agronomic properties.
When developing the environmental, food and feed safety profile of a
GE crop, developers must demonstrate to the regulatory agencies that the
GE crop does not have any new or altered risks relative to its non-GE coun-
terpart in respects other than those that derive directly from the action of the
inserted gene(s) and trait(s) (Codex 2008). In the case of an insect-protection
event, the developers must show that the crop is compositionally and agro-
nomically similar to its non-GE counterparts in the absence of the target
pest(s) and that the only differences observed are related to the action of
the trait to reduce pest injury. With the demonstration that the GE and
non-GE counterparts are compositionally and agronomically equivalent
with no harmful unintentional changes, the risk assessment conducted by
the regulator can focus on the specific trait(s) added.
Table 4.4 lists the types of information that are generally provided to reg-
ulators in support of the safety assessment of a GE insect-protected crop.
Table 4.4 List of studies typically conducted in support of safety assessments
for human health and the environment of GE crops
Food/feed/ Environmental/
Test material Study type import cultivation
Crop Molecular characterization
Crop Inheritance
Crop/protein Detection methods
(ELISA, PCR)
Crop Protein expression (Leaf, pollen, stalk,
(Grain, seed root at various
after harvest) growth stages)
Crop Field efficacy
Crop Compositional analysis
Crop Agronomic properties
Protein Acute oral toxicity (mouse
gavage)
Protein Homology to known toxins
Protein Protein biochemistry
(digestive stability,
thermolability as indicators
of potential allergenicity)
Protein Homology to known
allergens
Crop Animal feeding with grain
Protein Soil degradation
Protein Spectrum of Cry protein
activity
Protein Non-target organism
hazard testing
Crop Field non-target organism (If exposure and
surveys hazard data suggest
potential effects under
field conditions)
Protein Endangered and threatened
species assessment
Crop Weediness potential
Protein Potential effects of gene
flow (if wild relatives
present in area of proposed
release)
Bt Crops 201

5.1. Product identification and characterization

Studies are conducted to characterize the transformation event being devel-
oped for commercialization. These studies include an analysis of the genetic
insert to ensure that the intended genetic elements are present and intact and
that unintended elements (like the backbone DNA sequence of a plasmid for
Agrobacterium-mediated transformation) are absent. Production of mRNA
and the gene product are also characterized. Studies of the inheritance of
the transgene across generations ensure that it is inherited in the expected
manner (typically Mendelian segregation). Both DNA and protein detection
methods are developed to enable identification of plants and plant tissues
containing the transgene(s) (Bt gene, selectable marker gene) and accompa-
nying regulatory elements. Finally, the efficacy of the product under field
conditions is characterized to ensure that the intended phenotype is present.

5.2. Human health assessment

The human health assessment of insect-protected GE crops includes char-
acterization of both the introduced protein and the food/feed derived from
the crop, including where appropriate processed products. The protein
safety assessment includes information on the source of the protein and his-
tory of safe exposure of the protein in its natural state (including toxicity and
allergenicity) and its insecticidal mode of action in the target pest. In the case
of insecticidal proteins derived from Bt strains, there is a considerable body of
evidence of safe history, dating back to the organisms discovery a hundred
years ago and its development as a biological insecticide over 60 years ago
(Sanahuja et al., 2011). Bt is a very common soil and phylloplane micro-
organism to which humans and animals have always been exposed with
no known adverse effects. Furthermore, the mechanism of action of the
insecticidal proteins has been characterized to involve binding to specific
receptors in the midgut of sensitive insects, receptors that are not present
in mammalian digestive tracts. Indeed, Bt proteins are generally very
selective in their toxicity to specific orders of insects or insects within a spe-
cific order even though other insects within or outside that order may also
have related receptor proteins. Additionally, Bt proteins are rapidly degraded
by digestive enzymes and the acidic condition of human stomachs
(Mendelsohn et al., 2003).
Bioinformatic approaches are used to investigate any amino acid
sequence homology to known toxins or allergens. The assessment of the
allergenic potential of the protein considers not only sequence homology
202 Kenneth E. Narva et al.

to known allergens, but also biochemical properties, such as digestibility and

heat lability that may be characteristics of some allergens, to understand if the
introduced protein may be a novel allergen. If there is significant homology
to a known allergen, additional testing can be conducted to understand
whether the introduced protein may elicit the allergenic response in sensi-
tive individuals (Ladics et al., 2011). Bt is not known to be a source of allergic
responses despite its ubiquity, and therefore these proteins have very low
potential to be allergens.
Finally, acute oral toxicity of the novel protein is assessed through gavage
with a large quantity of the protein in a model organism, usually a mouse.
Through all these tests, only proteins with no evidence of toxicity or aller-
genicity are developed for use in GE crops.
To complete the dataset required for the human health risk assessment
for the GE protein, data are provided on the expression levels of the proteins
in the harvested grain. For crops where the consumption is of processed
products, further analysis of protein levels after processing can be provided
(Hammond and Jez, 2011). Since the expressed proteins are not associated
with any hazard to human health, the expression data provide additional
assurance that there will be no harmful effects when the food is consumed.
In addition to information on the human health and food safety of the
insecticidal proteins, regulators also review information on unintended
effects to the crop of the transformation itself. Extensive data are provided
on the nutritional profile of the GE crop, its nearest non-GE isoline and a
broader set of varieties of the crop grown under diverse agricultural condi-
tions. This compositional analysis includes quantification of lipids (including
the fatty-acid profile), proteins (including the amino acid profile), carbohy-
drates, vitamins, minerals and anti-nutrients. Feeding studies using the grain
in rapidly growing animals such as broiler chickens and, in some cases, rats
provide further information on the food safety and nutritional value of
the crop.
Most of the required regulatory data generated for the human health and
food safety assessments address extremely remote risks that are no greater
than for any variety of a crop developed through conventional breeding
and crop improvement techniques (Herman and Price, 2013). For example,
the probability of introducing to the human diet a novel allergen is
extremely low given the very small proportion of proteins that are allergens
and that these relatively few proteins are clustered in a small number of pro-
tein families (Radauer et al., 2008). Similarly, in more than 140 studies of the
composition of GE crops, not a single crop has shown evidence of a harmful
Bt Crops 203

change in nutritional value or anti-nuturients (Herman and Price, 2013).

The variation in composition of crops developed through conventional crop
improvement techniques is many times greater, due to the introduction of
multiple new alleles and genes, most of which are uncharacterized, using
conventional techniques, compared with the one well-characterized-
specific intended change introduced through genetic engineering (DiLeo
et al., 2014; Herman and Price, 2013; Herman et al., 2009; Ricroch,
2013; Ricroch et al., 2011).

5.3. Environmental effects

While environmental regulations and frameworks differ among countries
according to their local laws and environmental protection goals, regulatory
requirements relating to environmental release of GE crops tend to be sim-
ilar across countries that permit commercial cultivation of these crops. Reg-
ulators seek to ensure that the environmental effects of a GE cropping system
are not more harmful to the environment than the conventional cropping
system that they would supplant. Under some regulatory regimes, agencies
also consider the economic, human health and environmental benefits of the
technology.
As with the human health and food safety studies, environmental safety
studies cover both the direct effects of the GE protein itself and the effects of
any unintended changes to the crop. For insect-protected GE crops, the data
generated include sensitivity of representative non-target organisms that
may occur in or around agricultural production fields, focusing on beneficial
species such as predators, parasitoids and pollinators. Such studies may also
include charismatic species, such as monarch butterflies. Hazard testing for
non-target organisms resulting from exposure to a new transgene product is
often accomplished following the tiered-testing paradigm (Romeis et al.,
2011). Under this approach, the non-target organism of concern (or a sur-
rogate that is functionally or phylogenetically similar to the organism of con-
cern), is tested in a bioassay with the purified transgene product at a
concentration many fold higher than the highest estimated exposure in
the field (Tier 1). If the test population is not affected at this high concen-
tration, or if the effects are moderate (for example, less than 50% mortality)
then there is a high likelihood that exposure to the transgene product will
not have significant effects under field conditions. If, however, effects are
seen at this high concentration, further bioassays are conducted using more
realistic exposure levels, perhaps using the tissues from the GE plant rather
204 Kenneth E. Narva et al.

than purified transgene product (Tier 2). Again, if the test population is not
affected at realistic exposure levels, of if effects observed would be acceptable
(for example less than would occur with alternative pest control tools), addi-
tional testing is not warranted. If significant effects are seen in Tier 2, addi-
tional testing can be conducted with whole plants in a green house or field
cages (Tier 3). Such tests allow more realistic spatial processes to function
that may more accurately reflect actual exposure under field conditions.
Finally, if these lower tier studies indicate potential for unacceptable harm,
a field study may be warranted whereby natural populations are monitored
under the same conditions as the proposed environmental release (Tier 4).
Progressing through the tiers increases the ecological relevance of the study
to the actual proposed release but decreases the ability to detect effects due to
greater variability in the test system. Methods or guidance for testing many
non-target organisms at several of the tiers are available in published litera-
ture (e.g. Romeis et al., 2011) or from regulatory agencies (e.g. U.S.
Environmental Protection Agency, 2007).
Complementing such hazard testing, exposure analysis is accomplished
by measuring the expression of the GE protein in representative tissues of
the crop that are fed upon by herbivores. This can include leaf, stalk, pollen,
flowers and fruits, depending on the tissues that are consumed. Expression is
measured at several time points in the life cycle of the plant to provide a
comprehensive assessment of the potential exposure of non-target organ-
isms. Data are also generated on the environmental fate of the GE protein,
typically examining the rate of degradation of the protein in agricultural soils
(Shan, 2011).
It is reasonable to expect that some non-target species may be sensitive to
the GE protein, especially those that are phylogenetically related to the target
pest species. For example, larvae of the monarch butterfly and some other
Lepidoptera are known to be sensitive to Bt proteins in the Cry1 class, which
are targeted at lepidopteran pests. Similarly, larvae of certain Chrysomelidae
are known to be sensitive to Bt proteins in the Cry3 class. The risk to such
organisms is characterized by integrating their estimated sensitivity to high
end estimate of exposure levels. Usually, conservative assumptions are made
that over-estimate the sensitivity and over-estimate the exposure. If this
characterization indicates that there is not a very low likelihood of a harmful
effect to the population of the non-target organism, field studies may be
warranted to investigate whether the estimated effects actually occur under
field conditions.
Bt Crops 205

When conducting non-target organism studies with GE crops, it is

important to use relevant comparators to understand the significance of
any effect. Typically the comparators are the nearest non-GE isoline that
is managed in accordance with conventional pest management practices.
Additional comparators may include the isoline that is not treated with
insecticides (providing a worst-case evaluation of the effects of the GE crop)
and additional varieties of the crop that are typically grown. These provide
estimates of the typical differences among varieties of a crop and therefore
the context to assess the biological significance of any effects measured with
the GE crop.
In addition to non-target organisms, environmental assessments of GE
crops include assessment of its agronomic properties when grown according
to normal agricultural practices. Such properties may include the growth
habit of the crop (e.g. time to flower, crop height and yield), observations
of susceptibility to pests (other than the targeted pests), diseases and other
environmental stressors. Such data are interpreted for any indications that
the transformation may have increased the potential of the crop to become
a weed for example through increased persistence, ecological competitive-
ness or ability to spread outside of agricultural areas (Raybould et al., 2012).
For crop species that are grown in the same area as sexually compatible
wild relatives, regulators will typically consider whether gene flow from
the crop to the wild relatives may occur, and what the consequences of such
gene flow could be on the population of the wild relative. In the case of insect
protection traits, the assessment might include an assessment of whether addi-
tion of the trait may reduce feeding on the wild relatives by insects such that
plant becomes more invasive. Generally, however, insect feeding is not an
important limiter of wild plant populations, and the addition of an insecticidal
trait would not have a biologically significant effect. Furthermore, hybrids of
wild plant populations and crops are generally less fit than native wild plants
due to the agronomic properties of the crop that have been bred for gener-
ations to make them suitable for cultivation and harvest.
In some regulatory systems, it is necessary to perform environmental risk
assessments for GE crops when the requested approval is not for commercial
environmental release, but instead for importation of grain that is for food and
feed use. In these situations, the assessment specifically considers the potential
for inadvertent environmental release of the GE crop. Because in these sit-
uations there is very low exposure potential, a conclusion about acceptability
of risk may usually be reached with very limited environmental data.
206 Kenneth E. Narva et al.

5.4. Considerations for stacks

Traditional breeding, which combines characterized and uncharacterized
traits, has generated products with a long history of safe use. Human and
animal diets have always included multiple food combinations, with no
documented adverse health effects from interactions. Different crops are
grown in adjacent spaces, and crop varieties have been crossed to generate
new genomic combinations, with the recognized principle that combina-
tion is not inherently unsafe.
There is no reason to expect GE traits or genes to interact in a different
manner compared with native traits or genes. Combined event products
(breeding stacks) contain two or more biotechnology-derived events
combined through conventional breeding. Where the individual events
have been determined to be as safe as the conventional counterparts and
no trait interaction is expected, the combined event can be considered
equally safe as food and feed (although many regulatory frameworks require
confirmatory data for example on efficacy or crop composition). When
seeking cultivation approval for a combination of two or more Bt proteins,
additional information on impacts to target and non-target species may be
required by the cultivating country. Where no trait interaction is antici-
pated, analysis of existing data from individual events can be used to assess
the effects of the combined event on target and non-target species. If labo-
ratory tests indicate trait interaction (e.g. synergism or antagonism), or an
interaction is expected, additional testing of the protein combination may
be warranted, similar to the non-target organism data generated for single
events discussed above. Combined insecticidal events may also be subject
to product-specific oversight relating to IRM.

5.5. Continued regulatory oversight of commercialized

GE events
Upon completion of regulatory review in the country or countries where a
GE crop is to be cultivated, regulatory agencies will issue a decision on its
permissibility for unconfined environmental released. With similar
approvals from any countries that typically import the crop, GE crops
may be commercially released. Depending on the regulatory framework
and agencies involved, the decision to permit commercialization can take
different forms. For example, when the USDA deregulates a product,
the regulators have no further oversight of the product. On the other hand,
EPA, which registers the Bt proteins expressed by GE crops, continues
Bt Crops 207

regulatory oversight. The EPA may require on-going studies on the envi-
ronmental effects of a Bt crop when grown on a commercial scale. Such
studies generally are confirmatory in nature, providing additional data on
exposure and effects of the Bt proteins. The European Food Safety Author-
ity requires technology providers to conduct on-going general surveillance
for changes in the agricultural ecosystem that may be attributable to the
release of GE crop.
However, such post-market monitoring is rarely scientifically justified.
The regulatory risk assessment prior to launch is in most cases sufficiently
thorough that unanticipated effects are known not to occur. General surveil-
lance is not hypothesis-driven, and collection of environmental data pro-
vides no information as to the cause of any changes, and whether such
changes are harmful or undesirable. Without a testable hypothesis, general
surveillance has little utility and is unlikely to identify environmental effects
resulting from the GE crop. Post market monitoring (PMM) is only
warranted when pre-market risk assessment identifies potentially unaccept-
able risks, and these risks can only be tested using large scale studies. In these
rare instances, post market monitoring can help determine actual levels of
harm and the efficacy of mitigation measures under the field conditions
reflective of commercialization. For additional information on PMM and
policy considerations, see FAO Expert Consultation on Genetically
Modified Organisms in Crop Production and Their Effects on the
Environment (2005).
Several regulatory agencies around the world require the technology
provider to implement resistance management programs that are designed
to slow the adaptation of target pest populations to GE Bt crops thereby
extending their utility and their benefits to the environment. Even where
these programs are not required, technology providers nevertheless will
implement measures to protect the durability of the products (Head and
Greenplate, 2012; MacIntosh, 2010).

6. INSECT RESISTANCE MANAGEMENT

The potential for targeted insect populations to evolve resistance to Bt
crops was recognized prior to when the first commercial crops were released
(Alstad and Andow, 1995; Gould, 1998; Roush and Shelton, 1997;
Tabashnik et al., 1990). These concerns have led to the development of pro-
active resistance management programs that are designed to delay the onset
of resistance and slow its spread. Today, such programs are in place for all Bt
208 Kenneth E. Narva et al.

crops in all geographies where they are grown. Resistance management pro-
grams are focused on the primary pest species that are of greatest importance
to the continued value of the Bt crop.
The primary tactic to delay resistance is the use of refuges, or host plants
that do not contain Bt genes and allow the persistence of susceptible pests.
Susceptible individuals are thereby able to mate with any resistant individuals
that emerge from the Bt crops and maintain susceptible alleles in the
population.
To be fully effective in delaying resistance development in a field pop-
ulation, refuges should produce sufficient insects to overwhelm any resistant
insectsa ratio of 500 susceptible to 1 resistant has been used as a rule of
thumb (U.S. Environmental Protection Agency, 2001b). The refuge should
be in sufficiently close proximity to Bt fields that normal insect dispersal will
promote mating between refuge-produced and Bt-produced insects. Adult
insect emergence from the refuge should occur at the same time as emer-
gence of resistant insects from Bt crops.
Different forms of refuge are used in resistance management programs.
Natural refuges can be composed of crop or non-crop host plants, often of
different species from the Bt crop, but nevertheless of sufficient abundance,
proximity and temporal overlap to promote mating of susceptible insects
with resistant insects from the Bt crop. Natural refuges consisting of crop
and non-crop hosts of H. virescens and H. zea provide the refuge for Bt cotton
in the south and southeastern United States (Gould et al., 2002; Gustafson
et al., 2006; Jackson et al., 2004) and for H. armigera in China (Qiao et al.,
2010). Structured refuges are specifically grown in association with Bt crops,
and consist of non-Bt varieties, usually of the same species as the Bt crop. The
recommended amount and layout of the refuge vary by pest species and
crop. For example, for O. nubilalis in the U.S. Corn Belt and single-gene
Bt maize hybrids, non-Bt maize must be on an area that is at least 20% of
the area of the Bt crop and the refuge must be planted within mile
(800 m) of each Bt field (U.S. Environmental Protection Agency,
2001a). For D. saccharalis in the Argentina corn belt and single-gene Bt
maize, the recommendation is for 10% refuge within 800 m of the Bt maize
field. For WCR in the U.S. Corn Belt and pyramided Bt maize, 5% refuge is
required which must be planted within or adjacent to the Bt maize field.
Recently, refuge provided as seed blends with Bt seeds that produce two
or more Bt proteins against each key target pest have been released to sim-
plify the refuge planting and management by growers and to ensure that the
required refuge is present (Onstad et al., 2011).
Bt Crops 209

Population genetics theory and simulation models indicate that refuges

are extremely effective for Bt crops that provide a high dose against the
key target pest(s) and when resistance alleles are initially rare (Alstad and
Andow, 1995; Gould, 1998). In these cases, the Bt crop kills nearly all sus-
ceptible larvae and 95% or more of larvae that are heterozygous for resistance
alleles. When resistance alleles are rare, most of the resistance alleles are car-
ried by heterozygotes and so removed by the high-dose Bt crop, greatly
delaying resistance.
An alternative (or additional) strategy to the high dose to remove hetero-
zygous insects is to pyramid more than one Bt protein active against the same
target pest. If each protein differs in their target insect midgut receptors, one
protein can kill insects that are heterozygous or homozygous for resistance
alleles to the other protein. This provides dramatic delays in resistance devel-
opment (Storer et al., 2012c) provided that resistance is not already devel-
oping to one of the component proteins (Tabashnik and Gould, 2012).
Understanding the receptors involved in the mode of action of each Bt pro-
tein, or at least understanding differences in binding sites as well as other
direct or indirect indicators of cross-resistance potential, therefore can be
important in designing appropriate resistance management programs. This
also applies in the situation where crops containing different Bt proteins
active against the same pest are deployed in the same agricultural environ-
ment, a situation which did not apply when the first Bt crops were developed
but is now the norm.
The design of refuge-based resistance management must balance the bio-
logical risks of resistance (which depend on the properties of the Bt crop, the
adoption of the product by farmers, the genetics of resistance and the eco-
logical interactions between the target pests and their host crops) with eco-
nomic and practical realities of crop production. Resistance management
programs are intended to delay but not prevent resistance, and the length
of the delay sought must also reflect the continued development of new pest
management tools including GE crops producing novel insecticidal mech-
anisms. Refuges, to be effective, must allow survival and development of
pest insects. These insects cause yield loss and economic costs. For example,
Marra et al. (2012) estimated that for every 1% decline in expected maize
yield in the United States, maize prices are expected to increase 4.2%. Prac-
tical considerations also have to be taken into account (MacIntosh, 2010).
The larger the refuge required, the smaller the benefit to growers using
the technology. It should be expected that a larger refuge would lower
grower acceptance of the product, and for those growers who do plant it,
210 Kenneth E. Narva et al.

compliance with the refuge is likely to be lower. Grower compliance with

requirements for 50% refuge for single-gene Bt maize in the southern
United States (cotton-growing region) (U.S. Environmental Protection
Agency, 2011) is much lower than for a 20% refuge in the northern United
States. There the intended durability benefits of larger refuges may not be
fully realized. Most models indicate that blended refuge with pyramided trait
products, while less durable than a separate refuge with which there is 100%
compliance, provides superior durability compared with a larger structured
refuge with single trait products (Carroll et al., 2012; Ives et al., 2011).

7. Bt CROPSA SNAPSHOT OF TODAY

7.1. Commercialized Bt proteins
GE corn and cotton comprise the majority of Bt crops cultivated in the
Americas. The Bt proteins in current GE products target lepidopteran pests
of corn, cotton, or soy and coleopteran pests of corn. GE potato expressing
Cry3Aa was developed for control of L. decemlineata but is not marketed today.
A relatively small number of the known Bt insecticidal proteins have
been developed for insect resistant GE crops. The Cry proteins developed
for control of foliage-feeding Lepidoptera include several well characterized
or modified three domain Cry proteins. These are Cry1Ab, Cry1Ac,
Cry1Fa2, Cry2Ab, Cry2Ae and Cry1A.105. Vip3Aa has also been devel-
oped for control of lepidopteran pests in both corn and cotton.

7.1.1 Cry1Ab
Cry1Ab is one of the most well studied 3-domain Cry proteins. The cry1Ab1
gene and encoded protein were first cloned and characterized by Wabiko
et al. (1986) from Bt subspecies Berliner. Cry1Ab is produced as a protoxin
that is activated by proteases in the insect midgut and functions by the clas-
sical pore formation mechanism of action described in Section 3.4. Cry1Ab
is noted for broad spectrum lepidopteran activity that includes economically
important stalk boring pests such as Ostrinia spp. and Diatraea spp. (van
Frankenhuyzen, 2009). Much is known regarding functional aspects and
mechanisms of IR to Cry1Ab and cross-resistance with other Cry proteins
(Heckel et al., 2007; Schnepf et al., 1998).

7.1.2 Cry1Ac
Cry1Ac from Bt kurstaki strain HD73 was first described by Adang et al.
(1985). Cry1Ac is another example of a classical three domain insecticidal
Bt Crops 211

protein (Derbyshire et al. unpublished; PDB 4ARX, 4ARY) with a long

history of study (Schnepf et al., 1998). Cry1Ac is one of the most potent
Cry proteins against a range of Lepidoptera: Noctuidae, resulting in wide-
spread deployment in GE crops, especially cotton. Cry1Ac was recently
approved for commercialization in soybean in Latin America for control
of lepidopteran pests including velvetbean caterpillar, Anticarsia gemmatalis
Hubner and soybean looper, Chrysodeixis includens (Walker) (http://www.
ctnbio.gov.br/index.php/content/view/15558.html).

7.1.3 Cry1Fa2
Cry1Fa2 was discovered by Payne and Sick (1993). Cry1Fa2 displays
sequence characteristics of classical three domain Cry proteins. The lepidop-
teran pest spectrum of Cry1Fa2 is quite broad and includes high potency
against Spodoptera spp. (van Frankenhuyzen, 2009), an attribute that makes
Cry1Fa2 valuable in combination with Cry1A proteins with low potency on
Spodoptera spp.

7.1.4 Cry1A.105
Developed by Monsanto Co., Cry1A.105 is a chimeric protein comprising
parts of four domains from other Cry proteins previously used in GE
plants. The amino acid sequences of domains 1 and 2 are identical with
the respective domains from Cry1Ab and Cry1Ac proteins, domain 3 is
almost identical to the Cry1F protein, and the C-terminal domain is iden-
tical to the Cry1Ac protein (http://cera-gmc.org/index.php?actiongm_
crop_database&modeShowProd&dataMON89034). As a result, the
Cry1A.105 chimeric protein combines most of the insecticidal properties
displayed by the Cry1A and Cry1F proteins.

7.1.5 Cry2Ab
Cry2Ab is a three domain protein that diverges in primary sequence and
predicted structure from Cry1A proteins. Cry2Ab was the first dual spectrum
Cry protein described with activity against both lepidopteran and dipteran
insects (Widner and Whiteley, 1989). Cry2Ab is valued for broad spectrum
lepidopteran activity and the ability to control resistant insect populations
(summarized in Schnepf et al., 1998). Based on lack of shared midgut binding
sites (Hernandez-Rodriguez et al., 2008), Cry2Ab proteins are candidates for
pyramiding with Cry1 proteins in insect resistant crops. Cry2Ab is often
targeted to chloroplasts in order to increase expression levels and reduce
negative plant phenotypes in GE plants (Corbin and Romano, 2006).
212 Kenneth E. Narva et al.

7.1.6 Cry2Ae
Cry2Ae is related in sequence to Cry2Ab. Cry2Ae represents a different
mechanism of action from Cry1 proteins based on lack of shared binding
sites (Caccia et al., 2010; Gouffon et al., 2011). Cry2Ae, like Cry2Ab, is
a candidate for combining with Cry1 proteins in pyramids for resistance
management.

7.1.7 Vip3Aa
Vip3Aa is a soluble insecticidal protein expressed during the vegetative
growth phase of Bt growth, prior to sporulation (Estruch et al., 1996).
Vip3Aa is broadly active on lepidopteran pests of corn and cotton. Vip3Aa
is different in sequence and mechanism of action compared with Cry1 or
Cry2A proteins (Lee et al., 2006). As a result, Vip3Aa is useful in combina-
tion with Cry1 or Cry2 proteins to slow the development of resistant insect
populations.
Proteins developed in GE corn for control of WCR are mCry3Aa,
eCry3A.1ab, Cry3Bb and Cry34Ab1/35Ab1. These proteins and other
GE approaches for the control of WCR were recently reviewed by
Narva et al. (2013).

7.1.8 mCry3Aa (modified Cry3Aa)

Cry3Aa, the first reported coleopteran-active Bt insecticidal protein
(Herrnstadt et al., 1987; Sekar et al., 1987) is ineffective against Diabrotica
spp. (Herrnstadt et al., 1987; MacIntosh et al., 1990; Slaney et al., 1992).
To improve activity on WCR, Walters et al. (2008) engineered Cry3Aa
to contain a chymotrypsin/cathespsin G protease site at a location in domain
1 known to enhance Cry3Aa proteolytic activation (Carroll et al., 1989,
1997). This modification enhanced Cry3Aa cleavage in WCR and
improved binding to WCR midgut membranes. The mCry3Aa insecticidal
activity against WCR larvae was superior to native Cry3Aa.

7.1.9 eCry3.1Ab
The eCry3.1Ab protein is a hybrid resulting from exchange of the domain 3
variable region from a lepidopteran-active toxin, Cry1Ab with domain 3
from Cry3Aa (Walters et al., 2010). The resulting protein, eCry3.1Ab,
has higher activity against WCR than Cry3Aa. Another interesting feature
of Cry3A.1Ab is that it binds WCR midgut brush border membrane vesicles
Bt Crops 213

(BBMVs) at sites independent of mCry3Aa binding. The lack of competitive

binding between mCry3A and eCry3.1Ab suggests that these proteins might
act by independent mechanisms on WCR midguts.

7.1.10 Cry3Bb1
Cry3Bb1 is active on coleopteran pests such as L. decemlineata (Donovan
et al., 1992). Cry3Bb1 is a three domain Bt protein with structural similarity
to many other Cry proteins (Galitsky et al., 2001) and functions by forming
ion channels in membranes (Von Tersch et al., 1994). The version of
Cry3Bb1 expressed in events MON863 and MON88017 from Monsanto
is a modified protein with six amino acid residue changes compared with
the native sequence (Vaughn et al., 2005).

7.1.11 Cry34Ab1/Cry35Ab1
The second major class of Bt toxins developed for protection of WCR injury
in maize are the binary Bt crystal proteins Cry34Ab1 and Cry35Ab1
(Cry34Ab1/Cry35Ab1) that function together as oral intoxicants of
WCR larvae (Ellis et al., 2002; Herman et al., 2002). Cry34Ab1/Cry35Ab1
are structurally different from the Cry3-type proteins described above.
Cry34Ab1 is one example of a family of 14 kDa proteins that have no pro-
tein sequence homology beyond the Bt Cry34 group (Schnepf et al., 2005),
whereas Cry35Ab1 is a member of a family of 44 kDa Bt proteins that share
low sequence homology to Bt Cry36Aa1, Bacillus sphaericus mosquitocidal
binary proteins BinA and BinB and B. sphaericus mosquitocidal binary pro-
tein, Cry49Aa1 ( Jones et al., 2007). Crystal structures for Cry34Ab1 and
Cry35Ab1 were recently solved (Cry34Ab1 PDB accession 4JOX;
Cry35Ab1 PDB accession 4JP0; Kelker et al., 2014). Cry34Ab1/Cry35Ab1
appears to function by disrupting the WCR midgut epithelium. Consistent
with this observation, Cry34Ab1/Cry35Ab1 formed ion channels in
artificial lipid membranes (Masson et al., 2004). Li et al. (2013) recently
demonstrated Cry34Ab1/Cry35Ab1-specific binding to WCR midgut
BBMV and a lack of competitive binding between Cry34Ab1/Cry35Ab1
and the coleopteran-active proteins Cry3Aa, Cry6Aa and Cry8Ba. Lastly,
Cry34Ab1/Cry35Ab1 are not cross resistant with Cry3Bb1 insect resistant
traits; Gassmann et al. (2011) recently demonstrated that field-derived WCR
populations with reduced susceptibility to Cry3Bb1 corn are susceptible to
Cry34Ab1/Cry35Ab1 maize.
214 Kenneth E. Narva et al.

7.2. Global adoption of Bt crops

Bt potato was the first Bt crop commercialized in the United States (1995)
followed by Bt corn and cotton in 1996 (Betz et al., 2000). Bt crops have
been rapidly adopted by growers around the world and in 2013 were grown
on nearly 76 million hectares in 27 countries by 18 million farmers ( James,
2013). While our focus on describing Bt crops will be on traits commercial-
ized in the United States, in recent years developing countries have planted
more biotech crops than industrialized countries ( James, 2013). In fact,
recently commercialized products like PowerCore maize and Intacta soy-
bean were developed primarily to meet the needs of growers in Latin
America.
Economists Graham Brookes and Peter Barfoot have, since 2005, pro-
vided the most comprehensive assessments of the global economic and envi-
ronmental impacts of biotech crops including Bt crops (Barfoot and
Brookes, 2014; Brookes and Barfoot, 2014). They calculate the aggregate
impact on global farm income in 2012 for Bt corn and cotton at $6.7 billion
and $5.3 billion respectively (Brookes and Barfoot, 2014). Economic gains
are tied to increased yields due to effective pest control and decreased costs of
production related to expenses associated with insecticide use. Reductions
in insecticide use are most often associated with Bt cotton which in 2012 was
estimated at 16.8 million kg (a 40% reduction of insecticides targeted at lep-
idopteran pests) but the reductions in insecticide use associated with Bt corn
are also impressive (2012: 7.6 million kg representing an 86.5 % reduction of
insecticides targeted at lepidopteran and coleopteran pests) (Barfoot and
Brookes, 2014). They also calculate the reduced greenhouse gas emissions
associated with reduced insecticide applications in Bt crops (and the increase
in reduced or no-till production systems associated herbicide tolerant crops)
to be the equivalent of taking 940,000 cars off the road (Barfoot and
Brookes, 2014).
A benefit associated specifically with Bt corn is the reduction in myco-
toxin contamination in corn kernels attributed to reduced insect damage
(Wu, 2006). Insect feeding on the ear provides a point of entry for fungi
(primarily Fusarium and Aspergillus spp.) known to produce these potent
toxins (and carcinogens) and the insects themselves may be important vec-
tors of these fungi (Abbas et al., 2013). In a recent review by Abbas et al.
(2013), a strong association between Bt corn and reductions in fumonisins
(mycotoxins produced by Fusarium spp.) was apparent while the association
with reductions in aflatoxin (produced by Aspergillus spp.) was less clear.
Bt Crops 215

7.3. Commercialized products

Information on the status of deregulated biotech traits is maintained by sev-
eral organizations including the Biotechnology Industry Organization
(BIO) (http://www.biotradestatus.com/), the Center for Environmental
Risk Assessment (CERA, 2012) and the International Service for the Acqui-
sition of Agri-Biotech Applications (ISAAA, http://www.isaaa.org/
gmapprovaldatabase/). In all cases, the information contained in these online
resources is a summary of publicly available information provided by various
governmental regulatory authorities. Tables 4.5, 4.7 and 4.9 are summaries
of information obtained from these sites for Bt events that have been
approved for cultivation in the United States and which, to the best of
our knowledge, have been commercialized.

7.3.1 Bt corn
The first Bt corn events were approved for cultivation in the United States in
1995 and commercialized in 1996 targeting control of O. nubilalis and other
stalk boring Lepidoptera such as southwestern corn borer, Diatraea grand-
iosella Dyer and D. sacharralis (Archer et al., 2001; Buntin et al., 2004).
O. nubilalis had not been a target for insecticide applications so the rapid
adoption of Bt corn was based on the yield improvements created by con-
trolling pest populations that were below the economic threshold justifying
insecticide applications (Catangui, 2003; Shelton et al., 2002).
Events expressing Cry1Ab (176, MON810 and Bt11), Cry1Ac
(DBT418), Cry9C (CBH351), Cry1Fa (TC1507) and Vip3Aa (MIR162)
have all been commercialized (Table 4.5) and 4 (MON810, Bt11,
TC1507 and MIR162) remain in the market today (Table 4.6). Events
176 (Cry1Ab) and DBT418 (Cry1Ac) were discontinued based on the supe-
rior performance of MON810 and Bt11 (Buntin et al., 2004; Walker et al.,
2000). The removal of event 176 from commercial production was hastened
when it became the focus of concerns over the potential for impact on sus-
ceptible, non-target Lepidoptera consuming plant tissue onto which pollen
from Bt corn had fallen (Hellmich et al., 2001) (Sears et al., 2001). StarLink
(event CBH-351 expressing Cry9C) was approved for cultivation and for
use in animal feed and industrial non-food uses. It was removed from com-
mercial use when traces of the product were found in the food supply
(Fox, 2001).
MON810 and Bt11, the single Bt gene events expressing Cry1Ab in the
market today, provide a high level of control of O. nubilalis and other stalk
Table 4.5 Corn events approved for cultivation and commercialized
Year approved Non-IR
Developer Event name OECD unique identifier Bt protein(s) Pest spectrum (cultivationUSA) genesa
Syngenta 176 SYN-EV176-9 Cry1Ab Lepidoptera 1995 pat
Monsanto MON810 MON-00810-6 Cry1Ab Lepidoptera 1996 nptII
Syngenta Bt11 SYN-BT011-1 Cry1Ab Lepidoptera 1996 pat
Dekalb Genetics Corporation DBT418 DKB-89614-9 Cry1Ac Lepidoptera 1997 bar
b
Aventis CropScience CBH-351 ACS-ZM004-3 Cry9C Lepidoptera 1998 pat
Dow AgroSciences TC1507 DAS-01507-1 Cry1Fa Lepidoptera 2001 pat
DuPont Pioneer
Monsanto MON863 MON-00863-5 Cry3Bb1 Coleoptera 2003 nptII
Dow AgroSciences DAS-59122-7 DAS-59122-7 Cry34Ab1 Coleoptera 2005 pat
DuPont Pioneer Cry35Ab1
Monsanto MON88017 MON-88017-3 Cry3Bb1 Coleoptera 2005 cp4 epsps
Syngenta MIR604 SYN-IR604-5 mCry3A Coleoptera 2007 pmi
Monsanto MON89034 MON-89034-3 Cry1A.105 Lepidoptera 2008
Cry2Ab
Syngenta MIR162 SYN-IR162-4 Vip3Aa20 Lepidoptera 2010 pmi
Syngenta 5307 SYN-05307-1 eCry3.1Ab Coleoptera 2012 pmi
a
Non-IR genes pat: a selectable marker which confers tolerance to the herbicide glufosinate ammonium in plant tissue. nptII: a selectable marker which confers the ability
to metabolize the antibiotics neomycin and kanamycin in plant tissue. bar: a selectable marker which confers tolerance to the herbicide glufosinate ammonium in plant
tissue. cp4 epsps: a selectable marker confers tolerance to the herbicide glyphosate in plant tissue. pmi: a selectable marker which confers the ability to utilize mannose as a
carbon source in plant tissue.
b
Approved for environmental release and use as animal feed only.
Bt Crops 217

boring Lepidoptera and moderate levels of control of H. zea (Buntin et al.,

2004; Farinos et al., 2004; He et al., 2003). TC1507, a single-gene event
expressing Cry1Fa, also provides a high level of control of O. nubilalis
and other stalk boring Lepidoptera (Farinos et al., 2012) but differs from
the Cry1Ab events in that it also provides protection against Spodoptera spe-
cies including S. frugiperda (Hardke et al., 2011), black cutworm (Kullik
et al., 2011) and western bean cutworm (Striacosta albicosta (Smith))
(Eichenseer et al., 2008; Fig. 4.5A). While TC1507 does provide suppres-
sion of H. zea, it does not provide the same level of protection as MON810
(Buntin, 2008). MIR162, a single-gene event expressing Vip3Aa, does not
provide protection against O. nubilalis but provides a high level of control
against other stalk boring Lepidoptera, fall armyworm, black cutworm,
western bean cutworm (Syngenta Seeds) and the highest level of control
of single-gene events on H. zea (Burkness et al., 2010).
Cry1A.105 and Cry2Ab are lepidopteran-active Bt proteins expressed in
event MON89034 (Table 4.5). This combination creates a trait with a broad
spectrum and strong performance in controlling many important lepidop-
teran pests (Reay-Jones et al., 2009; Rule et al., 2014; Shelton et al.,
2013; Siebert et al., 2012). It also provides a pyramid of two modes-of-
action on certain pests thereby decreasing the risk of resistance (Ghimire
et al., 2011; Storer et al., 2012a). This reduced risk for resistance develop-
ment makes practical the use of a blended refuge (i.e. refuge-in-a-bag or
RIB); where a bag of Bt-traited seed contains a small percentage of seed
without the Bt trait (Storer et al., 2012b). The built-in nature of a blended
refuge insures that a refuge is deployed thereby increasing the durability of
the Bt trait. Enhanced spectrum and the ability to deploy blended refuges by
virtue of mechanism of action pyramids are factors that drive the combina-
tion of Bt traits by breeding (Table 4.6) (Rule et al., 2014; Siebert et al.,
2012; Storer et al., 2012b).
The first Bt event targeting corn rootworm (Diabrotica spp.) was com-
mercialized in 2003 (MON863 expressing Cry3Bb) and today there are five
events in the market targeting control of this pest complex (Tables 4.5 and
4.6; Fig. 4.5). Products containing these events are sold primarily in North
America where their superior performance compared with insecticide treat-
ments led to rapid and widespread adoption (Petzold-Maxwell et al., 2013).
Increased protection from damage under high-rootworm pressure and the
increased durability associated with pyramiding mechanisms-of-action has
led to the development of products like SmartStax, which combines events
expressing Cry3Bb (MON88017) and the binary insecticidal protein
Table 4.6 Bt corn products currently sold in the United States
Continued
Table 4.6 Bt corn products currently sold in the United Statescont'd
Continued
Table 4.6 Bt corn products currently sold in the United Statescont'd
a
Non-IR events NK603: glyphosate tolerance trait. MON87460: DroughtGard drought tolerance trait. GA21: glyphosate tolerance trait. 3272: Enogen ethanol production trait.
Shaded boxes indicate that the event is present in the product. See Table 4.5 for Bt genes and pest spectrum associated with Bt events.
224 Kenneth E. Narva et al.

Figure 4.5 Insect-protected Bt corn. (A) Contrast of corn root systems after heavy pres-
sure from larval western corn rootworm. Top row is non-Bt plants showing severe root
damage. Bottom row is plants from the same genetic background as the plants in the
top row but containing event DAS-59122-7. (B) Contrast of ears after heavy pressure
from larval western bean cutworm. Top row is ears from non-Bt plants showing severe
damage. Bottom row is ears from the same genetic background as the plants in the top
row but containing event TC1507. Photo credit: Bader Rutter.

Cry34Ab/Cry35Ab (DAS-59122-7) (Head et al., 2014). Recent reports of

the decrease in effectiveness of events expressing Cry3Bb (MON863 and
MON88017) and mCry3A (MIR604) contrasted with the continued effec-
tiveness of DAS-59122-7 are additional evidence that Cry3s and Cry34/
Cry35 represent distinct modes-of-action and validate the value of the
SmartStax pyramid (Gassmann, 2012; Gassmann et al., 2014). Another pyr-
amid for rootworm control that is entering the market is Duracade; the
combination of mCry3A (MIR604) and eCry3.1Ab (5307) (Table 4.6).
The Handy Bt Trait Table (DiFonzo and Cullen, 2013) is a useful and fre-
quently updated guide to the Bt corn products available in the United States.

7.3.2 Bt cotton
Bt cotton was commercially introduced in the United States in 1996 and was
rapidly adopted due in large part to its effectiveness in controlling
H. virescens, a pest that had developed resistance to virtually all insecticides
available at the time (Blanco, 2012). Bt cotton was also introduced in
Australia in 1996 where the primary target was H. armigera (Downes and
Bt Crops 225

Mahon, 2012). In 2013, Bt cotton was grown in 15 countries and high-

adoption rates in the major cotton producing countries speak to the value
of the technology ( James, 2013; Naranjo, 2010; Fig. 4.6).
Since the introduction of BollGard cotton (MON531 expressing
Cry1Ac), events expressing Cry1Ab, Cry1Fa, Cry2Ab, Cry2Ae and
Vip3Aa have been commercialized and, with the exception of Cry1Ac,
have been introduced as pyramids (Tables 4.7 and 4.8). Control of H. vir-
escens with Cry1Ac-expressing events has been consistently strong with no
evidence of resistance development in the United States (Blanco, 2012). Bt
cotton expressing Cry1Ac is also highly effective at controlling pink boll-
worm and in the United States; it has been a pillar of the pink bollworm
eradication program (Tabashnik et al., 2012). Increased durability provided
by different mechanisms-of action on key pests as well as increased consis-
tency in the control of bollworm was seen with the introduction of
pyramided varieties like BollGardII and WideStrike (2003 and 2005,
respectively), but under high-bollworm pressure the yield of Bt cotton
varieties is enhanced by insecticide applications (Luttrell and Jackson,
2012). The recent addition of Vip3Aa to the pyramid of Cry1Ac and
Cry1Fa (Table 4.8) should bring additional levels of performance against
bollworm and Spodoptera species (Bommireddy et al., 2011).

7.3.3 Bt soybean
MON87701 is the only Bt event that has been commercialized in soybean
(Table 4.9). Monsanto commercialized this event in Brazil under the trade
name Intacta RR2 PRO which is a breeding stack with MON89788, an
event conferring the Roundup Ready 2 Yield trait. Published studies
show that events expressing Cry1Ac in soybean are efficacious against
important North and South American soybean pests including
C. includens, A. gemmatalis, Crocidosema aporema (Walsingham), Rachiplusia
nu (Guenee) and the yellow woolybear, Spilosoma virginica (F.) (Macrae
et al., 2005; McPherson and MacRae, 2009). A more recent study by
Bernardi et al. (2014) using soybeans containing the events in Intacta
RR2 PRO showed that the relatively low intrinsic potency of Cry1Ac
on Spodoptera translated to poor control of S. frugiperda, Spodoptera cosmioides
Walker and Spodoptera eridania (Stoll); economically important pests of soy-
beans in Brazil.
Foliage feeding Lepidoptera frequently reach economically damaging
levels in South American soybean production, so Bt soybean potentially
delivers a clear benefit to growers (de Freitas Bueno et al., 2011). The value
226 Kenneth E. Narva et al.

Figure 4.6 Insect-protected Bt cotton. Contrast of yield after heavy lepidopteran pest
pressure. Rows on the left are plants containing events 281-24-236 and 3006-210-23 (i.e.
WideStrike) showing a large number of open bolls and harvestable lint. Rows on the
right are plants of the same genetic background as plants on the left but without the Bt
events. Photo credit: Eileen Crosby and Jim Steadman, Bader Rutter.

of controlling these pests in North American soybean production is less clear

as the levels and timing of defoliation typically created by lepidopteran pests
do not negatively impact yield (Hammond et al., 2000). In the case of Bt
soybean, McPherson and MacRae (2009) conducted 3 years of replicated
field trials comparing several Cry1Ac-expressing soybean events with
non-GE checks. While they observed significant reductions in larval counts
and foliar damage on Bt compared with non-Bt soybean lines, they never
translated to increased yields.

7.3.4 Bt potato
Bt potato expressing Cry3Aa was developed by Monsanto and commercial-
ized by Monsanto subsidiary NatureMark in 1995. Multiple events were
deregulated and commercialized as NewLeaf potatoes (expressing
Cry3Aa), NewLeaf Plus potatoes (expressing Cry3Aa and the full length
replicase gene of potato leafroll virus) conferring high levels of control of
the Colorado potato beetle and potato leafroll virus (Lawson et al., 2001)
and NewLeaf Y potatoes (expressing Cry3Aa and potato potyvirus
Y coat protein) (Table 4.9).
Cry3Aa-expressing potatoes provided control of L. decemlineata that was
superior to the commercial insecticides available during the time this
Table 4.7 Cotton events approved for cultivation and commercialized
Year approved Non-IR
Developer Event name OECD unique identifier Bt protein(s) Pest spectrum (cultivationUSA) genesa
Monsanto MON531 MON-000531-6 Cry1Ac Lepidoptera 1995 nptII
aad
Calgene Inc. 31807 Cry1Ac Lepidoptera 1998 bxn
31808 nptII
Dow AgroSciences 281-24-236b DAS-24236-5 Cry1Fa Lepidoptera 2004 pat
b
Dow AgroSciences 3006-210-23 DAS-21023-5 Cry1Ac Lepidoptera 2004 pat
c
Monsanto MON15985 MON-15985-7 Cry1Ac Lepidoptera 2005 nptII
Cry2Ab aad
uidA
Bayer CropSciences GHB119b BCS-GH005-8 Cry2Ae Lepidoptera 2011 bar
b
Bayer CropSciences T304-40 BCS-GH004-7 Cry1Ab Lepidoptera 2011 bar
b
Syngenta COT 102 SYN-IR102-7 Vip3Aa Lepidoptera 2011 aph4
a
Non-IR genes nptII: a selectable marker which confers the ability to metabolize the antibiotics neomycin and kanamycin in plant tissue. aad: a selectable marker used in
the creation of the gene construct and is not expressed in plants. bxn: confers tolerance to the herbicide bromoxinil in plants. pat: a selectable marker which confers
tolerance to the herbicide glufosinate ammonium in plant tissue. uidA(GUS): a scorable marker enabling visual identification of transformed plants. bar: a selectable
marker which confers tolerance to the herbicide glufosinate ammonium in plant tissue. aph4: a selectable marker which confers the ability to metabolize the antibiotic
hygromycin in plant tissue.
b
Commercialized only as a breeding stack.
c
Created via biolistic transformation of germplasm containing Event MON531 (Cry1Ac) with Cry2Ab2.
Table 4.8 Bt cotton products currently sold in the United States

a
Herbicide tolerance events MON88913: glyphosate tolerance trait. LLCotton25: glufosinate ammonium tolerance trait. GBH614: glyphosate tolerance trait.
Shaded boxes indicate that the event is present in the product. See Table 4.7 for Bt genes and pest spectrum associated with Bt events.
Table 4.9 Soybean and potato events approved for cultivation and commercialized
OECD unique Year approved Non-IR
Crop Developer Event name identifier Bt protein(s) Pest spectrum (cultivationUSA) genesa
Potato Monsanto BT6 NMK-89812-3 Cry3A Coleoptera 1995 nptII
BT10 NMK-89175-5
BT12 NMK-89601-8
BT16 NMK-89167-6
BT17 NMK-89593-9
BT18 NMK-89906-7
BT23 NMK-89675-1
Potato Monsanto ATBT04-6 NMK-89761-6 Cry3A Coleoptera 1996 nptII
ATBT04-30 NMK-89613-2
ATBT04-36 NMK-89279-1
SPBT02-5 NMK-89576-1
Potato Monsanto RBMT21-129 NMK-89684-1 Cry3A Coleoptera 1998 nptII
RBMT21-350 NMK-89185-6 aad
RBMT22-082 NMK-89896-6 plrv orf1
plrv orf2
Potato Monsanto RBMT15-101 NMK-89653-6 Cry3A Coleoptera 1999 nptII
SEMT15-02 NMK-89935-9 aad
SEMT15-15 NMK-89930-4 pvy CP
Soybean Monsanto MON87701b MON-87701-2 Cry1Ac Lepidoptera 2011
a
Non-IR genes: nptII: a selectable marker which confers the abilty to metabolize the antibiotics neomycin and kanamycin in plant tissue. aad: a selectable marker used in
the creation of the gene construct and is not expressed in plants. plrv orf1: a viral protein which confers resistance to the potato leafroll virus. plrv orf2: a viral protein which
confers resistance to the potato leafroll virus. pvy CP: a viral coat protein which confers resistance to potato potyvirus Y.
b
Commercialized only as a breeding stack.
230 Kenneth E. Narva et al.

product was being developed (Reed et al., 2001). Imidaclprid, a

neonicitinoid insecticide, was introduced about the same time as NewLeaf
potatoes and provided growers with an effective alternative for control of
both L. decemlineata and aphid vectors of plant viruses (Boiteau et al.,
1997). The availability of this alternative coupled with the decision of large,
institutional potato consumers to not accept NewLeaf potatoes led
NatureMark to discontinue the product line in 2001.

8. Bt CROPSPROSPECTS FOR THE FUTURE

8.1. Novel Bt proteins
The large number of known Bt insecticidal proteins will continue to be eval-
uated for agricultural applications as new information is generated on the
spectrum, potency and potential utility in pyramids to control resistant
insects. Looking forward, it is highly probable that next-generation
sequencing will accelerate the discovery of many more insecticidal proteins
from Bt and its subspecies, as well as related bacilli (e.g. B. popilliae Zhang
et al., 1997) and other bacterial genera, e.g. Clostridium bifermentans
(Barloy et al., 1996), where new examples of Cry-type proteins are likely
to be present.
In addition to identification of new, naturally occurring insecticidal pro-
teins, there are several approaches to improve insecticidal properties of exis-
ting proteins through knowledge-driven protein engineering (reviewed in
part in Pardo-Lopez et al., 2009). Some of these approaches have delivered
novel Cry proteins that are used in GE crops. A brief review of approaches to
engineer Bt proteins for improved insecticidal attributes follows.

8.1.1 Protease activation

Bt Cry proteins are converted to an active form by insect midgut proteases.
Proteolytic removal of the C-terminal crystallization domain is necessary for
activation of many Cry proteins (Schnepf et al., 1998). Continuing to
understand the ability of different insects to activate Bt proteins will be
important to engineer new Cry protein variants to control pests of economic
concern. As mentioned previously, mCry3Aa for control of WCR is an
engineered version of Cry3Aa with a chymotrypsin/cathepsin G protease
recognition site inserted in domain 1. The mCry3Aa protein has consistently
improved activity against WCR compared with the parent Cry3Aa protein
(Walters et al., 2008). For Cry1A proteins, removal of the amino-terminal
-helix in Cry1a proteins can sometimes overcome IR (Soberon et al.,
Bt Crops 231

2007; Tabashnik et al., 2011), though this technology has yet to be devel-
oped for commercial application.

8.1.2 Site directed mutagenesis

Site directed mutagenesis has been used as a method to probe structure
function relationships in Cry proteins (see reviews by Dean et al., 1996;
Schnepf et al., 1998). Most mutations result in deleterious effects on insec-
ticidal activity. Notable exceptions are point mutations in Cry3Aa domain 2
that improved activity against T. molitor (Wu et al., 2000) and rational design
of Cry1Aa loop modifications to generate a protein more similar to Cry4Ba
and thereby convert Cry1Aa from a lepidopteran-active protein to a
mosquito-active protein (Liu and Dean, 2006).
Protein structure information, when available, greatly aids the rational
design of improved pesticidal proteins by site-specific mutagenesis. One
commercially successful example of structure-based protein engineering is
Cry3Bb1. Cry3Bb1 contains six amino acid residue changes compared with
the native Cry3Bb1 sequence (Vaughn et al., 2005). The engineered version
of Cry3Bb1 is more active on WCR than the parent Cry3Bb1 protein.
A more recent example of structure-based protein engineering was the
insertion of a 12 amino acid pea aphid gut-binding peptide in loop regions
of the Bt cytolytic toxin, Cyt2Aa, that resulted in enhanced binding and tox-
icity against both the pea aphid, Acyrthosiphon pisum (Harris) and the green
peach aphid, Myzus persicae (Sulzer) (Chougule et al., 2013).

8.1.3 Gene shuffling

DNA shuffling (Stemmer, 1994) is an in vitro approach to protein evolution
with many applications. When applied to Bt insecticidal protein improve-
ment, this approach depends on innovative high-throughput screens to
be successful. Two recent studies used similar approaches of DNA shuffling
combined with phage display screening by membrane binding to enhance
the insecticidal activity of Cry8Ka1 against cotton boll weevil Anthonomus
grandis grandis Boheman (Oliveira et al., 2011) and Cry1Ia against sugarcane
giant borer Telchin licus licus (Craveiro et al., 2010).

8.1.4 Domain 3 exchange

Three domain Cry proteins appear to have co-evolved domains 1 and 2
together, and in some cases domain 3 as well. However, phylogenetic analysis
suggests evidence for evolutionary exchange of domain 3 by homologous
recombination between some Cry proteins (reviewed in de Maagd et al.,
232 Kenneth E. Narva et al.

2001). Domain 3 exchange is a proven route to generate Cry proteins with

broader spectrum of biological activity or higher insecticidal potency. The
first published example of domain 3 exchange by homologous recombina-
tion generated a Cry1Aa/Cry1Ac hybrid protein with improved activity
on H. virescens (Caramori et al., 1991). Later, de Maagd et al. (1996b) created
Cry1 hybrid proteins containing Cry1C domain 3 to improve activity on
S. exigua. A Cry1Ab/Cry1C hybrid improved S. exigua activity 10-fold over
the parent Cry1C protein (de Maagd et al., 1996a). As mentioned previously,
Cry1A.105 is a commercialized chimera of Cry1Ab domains 1 and 2 fused to
Cry1Fa domain 3. Domain 3 swapping was also used to improve coleopteran
activity for Cry1Ba/Cry1Ia hybrid proteins (Naimov et al., 2001). Last,
Cry3A.1Ab combines Cry3Aa domains 1 and 2 with domain 3 from Cry1Ab
for improved activity on WCR (Walters et al., 2010).

9. CONCLUSIONS
It is safe to say that the introduction of Bt crops revolutionized agri-
cultural pest control. Success in the discovery of Bt genes for use in crops
leveraged a long history of development of Bt as a biopesticide. Application
of all of the tools of modern biotechnology was required to genetically engi-
neer crops capable of producing these proteins, and new approaches in many
fields, including plant breeding and regulatory science, were developed to
bring products to the market. The result is crops with a previously unknown
capacity to resist pest damage leading to significant economic, environmen-
tal and societal benefits ( James, 2013).
Nearly 20 years after the introduction of the first Bt crops, the future of
this technology remains bright. Insuring that farmers and society can con-
tinue to reap its benefits remains a priority for technology providers as is
evidenced by the development of pyramided products designed to slow
the inevitable evolutionary response of pest populations to the selection
pressure imposed by this highly effective control. Evidence of the
on-going commitment to commercializing mechanism of action pyramids
is seen in recently announced products in the late stages of development
including: DuPont Pioneers corn event DP4114, a molecular stack of
the Bt proteins contained in Herculex XTRA, which will only be com-
mercialized as a pyramid with other modes of action; Monsantos corn event
MON87411, a pyramid of Cry3Bb and a novel RNAi mechanism of
action, that will be commercialized as a pyramid with Dow AgroSciences
Bt Crops 233

DAS-59122-7; and Dow AgroSciences soybean event DAS-81419-2, a

pyramid of Cry1Fa and Cry1Ac for control of lepidopteran pests of soybean.
Publicly disclosed pipelines of the major technology providers list IR
corn, soybean and cotton efforts in virtually all stages reflecting their
long-term investments in bringing new IR traits technologies to the market.
Given the success of Bt crops to date, and the untapped potential for discov-
ering Bt proteins with novel modes of action and pest spectra, we expect to
see new Bt-based IR traits well into the future.

ACKNOWLEDGEMENTS
We would like to thank E. Bouquet, M. Garcia, T. Hey, S. Jayne, R. Maciak and A. Mel for
their review and critique of earlier versions of this chapter. We would also like to thank the
editors of this volume for their constructive comments and patience.

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 CHAPTER FIVE

Progress Towards RNAi-Mediated

Insect Pest Management
James A. Baum, James K. Roberts
Monsanto Company, Chesterfield, Missouri, USA

Contents
1. Introduction 250
2. Environmental RNAi 252
3. Insect Sensitivity to Environmental RNAi 253
3.1 Coleoptera 253
3.2 Diptera 262
3.3 Lepidoptera 264
3.4 Hemiptera 267
3.5 Other agricultural pests 269
4. Barriers to Delivery and Efficacy in Recalcitrant Species 270
5. Commercial Development of RNAi Actives 276
5.1 Next-generation rootworm-resistant corn 276
5.2 Topical application 278
6. Safety Considerations 280
7. Insect Resistance Management 282
8. Concluding Remarks 284
Acknowledgements 285
References 285

Abstract
Gene suppression via RNA interference (RNAi) provides an alternative strategy for insect
pest management. The ingestion by insects of double-stranded RNAs targeting essen-
tial insect genes can trigger RNAi and lead to growth inhibition, developmental aber-
rations, reduced fecundity, and mortality. This RNAi response is particularly acute in
certain coleopteran species, most notably the western corn rootworm, a devastating
pest impacting corn production in the United States. The development of next-
generation rootworm-protected corn hybrids includes an RNAi-based trait that provides
a mode of action distinct from those of Bacillus thuringiensis insecticidal protein-based
traits currently used for rootworm pest management. Unfortunately, many insect spe-
cies including important lepidoptera and hemiptera pests appear largely recalcitrant in
their response to environmental RNA, suggesting biological barriers that thus far limit
the utility of RNAi for agricultural pest management. This review will highlight recent

Advances in Insect Physiology, Volume 47 # 2014 Elsevier Ltd 249

ISSN 0065-2806 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800197-4.00005-1
250 James A. Baum and James K. Roberts

efforts to understand the barriers to RNA delivery in recalcitrant insect species, describe
recent advances in the commercial development of insect-protected crops and biolog-
ical insecticides utilizing RNAi, and discuss this strategy in the context of an integrated
pest management approach.

1. INTRODUCTION
Eukaryotic cells possess a conserved pathway by which exogenously
applied and endogenously expressed double-stranded (ds) RNAs direct
the degradation of complementary endogenous messenger RNA (mRNA)
transcripts within a cell resulting in sequence-specific gene suppression. This
phenomenon is referred to as RNA interference (RNAi) (Fire et al., 1998;
Hannon, 2002). In plants and animals, RNAi provides one line of defence
against RNA viruses and foreign dsRNA molecules. Small endogenous
RNAs known as micro RNAs are also processed by a related pathway to
regulate tissue-specific patterns of gene expression primarily via translational
regulation (Bartel, 2009). Long non-coding RNAs also play a prominent
role in the epigenetic regulation of gene expression (Lee, 2012). It is now
clear that far more of the genome is transcribed than previously thought
(Djebali et al., 2012) and that RNA, in addition to being the obligate mes-
senger and facilitator of protein synthesis in the cell, is also a central player in
the regulation of eukaryotic gene expression.
The general mechanism of dsRNA-mediated degradation of mRNA
transcripts is understood (Tomari and Zamore, 2005). Long dsRNAs are
a substrate for RNAse III-like proteins referred to as Dicer or Dicer-like
proteins. Dicer appears to preferentially initiate dsRNA cleavage at the ends
of the dsRNA, making successive cleavages to generate 21- to 24-bp silenc-
ing (si) RNA duplexes (Elbashir et al., 2001). The resulting siRNA duplexes
are loaded into a multiprotein complex called the RNA-induced silencing
complex (RISC) where the passenger (sense) strand is removed and the
guide (antisense) strand remains to target mRNA for silencing. The guide
strand in the RISC enables WatsonCrick base pairing of the complex to
complementary mRNA transcripts and enzymatic cleavage of the target
mRNA by a class of proteins referred to as Argonaute proteins, thereby
preventing mRNA translation. Accordingly, this mechanism of gene sup-
pression is highly sequence specific. The ability to selectively down-regulate
genes via RNAi has proved to be valuable, particularly in insects for which
genetic tools are not readily available to study gene function (Belles, 2010).
RNAi-Mediated Insect Pest Management 251

The surprising observation that ingested dsRNAs can trigger gene suppres-
sion in the nematode, Caenorhabditis elegans, (Timmons and Fire, 1998;
Timmons et al., 2001) offered hope that the oral delivery of dsRNA could
modulate gene expression in other invertebrates, including insects, for the
purpose of pest management.
The ability of herbivorous insects to adapt to insecticide use in agricul-
tural systems presents an ongoing challenge for pest management. Over
20 years ago, more than 500 species of arthropods were documented with
resistance to one or more pesticides (Georghiou et al., 1991); an updated
dataset can be found at http://www.pesticideresistance.com/index.php.
The development of insecticides with new modes of action (MOAs) is a pri-
ority, but so is the implementation of resistance management strategies to
prolong the use of existing insecticides for use in agriculture and public
health (http://www.irac-online.org/). Insect-protected crops, expressing
insecticidal proteins derived from the bacterium, Bacillus thuringiensis (Bt),
represent a significant fraction of the >170 M ha of transgenic crops culti-
vated worldwide ( James, 2013) and provide excellent control of many eco-
nomically important insect pest species. Consequentially, such crops also
impose strong selective pressure on insects to adapt. As is the case for
synthetic- and biological insecticides, alternative MOAs for insect-protected
crops are needed, either because some insect species are not sensitive to Bt
insecticidal proteins or because some have evolved field resistance to effica-
cious Bt proteins (Storer et al., 2010). To that end, RNAi-mediated insect
control represents a significant opportunity.
In 2007, papers from Baum et al. and Mao et al. demonstrated that trans-
genic plants expressing insect-derived dsRNAs could impact the growth and
development of insect herbivores. Corn plants expressing a dsRNA hairpin
that targets the vacuolar ATPase A subunit gene in western corn rootworm
(WCR), Diabrotica virgifera virgifera, caused severe rootworm stunting and
exhibited significant protection from rootworm feeding damage, consistent
with artificial diet feeding assays demonstrating the insecticidal activity of
such dsRNA species (Baum et al., 2007). Arabidopsis plants expressing a
dsRNA hairpin that targets a cytochrome P450 monooxygenase gene in
the cotton bollworm, Helicoverpa armigera, led to decreased bollworm
tolerance to the cotton sesquiterpene aldehyde, gossypol (Mao et al.,
2007), consistent with the proposed role of this enzyme in gossypol
detoxification. The former example has advanced towards commercial
development as a component of next-generation rootworm-protected corn
hybrids (Kupferschmidt, 2013) and will be discussed further in this review.
252 James A. Baum and James K. Roberts

Numerous studies have since explored the sensitivity of diverse insect

species to ingested dsRNAs. In addition, several useful reviews have been
published on the subject of RNAi and insects to which we refer the reader
(e.g. Aronstein et al., 2011; Belles, 2010; Burand and Hunter, 2013; Gu and
Knipple, 2013; Huvenne and Smagghe, 2010; Li et al., 2013b; Terenius
et al., 2011; Yu et al., 2013; Zhang et al., 2012a, 2013a). Rather than reca-
pitulate these publications, this review will focus on the development and
use of RNAi strategies for insect control in agriculture, highlight efforts
to understand the barriers to RNA delivery in recalcitrant insect species,
describe recent advances in the commercial development of insect-
protected crops and biological insecticides utilizing RNAi, and discuss this
strategy in the context of an integrated pest management (IPM) approach.

2. ENVIRONMENTAL RNAi
Definitions have been proposed to discuss the various aspects of RNAi
in plants and animals (Huvenne and Smagghe, 2010; Whangbo and Hunter,
2008). Cell-autonomous RNAi refers to the RNAi response that individual
cells carry out when encountering dsRNA, a response that is executed by a
broadly conserved or core RNAi machinery found in eukaryotic cells. Non-
cell autonomous RNAi includes the phenomenon of systemic RNAithe
movement of a silencing signal, presumably siRNA and/or dsRNA, from
cell to cell and from one part of an organism to another. Non-cell autono-
mous RNAi also includes the phenomenon of environmental RNAi which,
as its name suggests, refers to the triggering of RNAi by environmental
exposure to dsRNA by soaking or feeding. Environmental RNAi may or
may not be followed by systemic movement of the silencing signal, perhaps
a key step in determining the biological activity of a dsRNA.
Components of the core RNAi machinery are readily identified
in insect species whose genomes have been sequenced (e.g. Honeybee
Genome Sequencing Consortium, 2006; International Silkworm Genome
Consortium, 2008; The International Aphid Genomics Consortium,
2010; Tomoyasu et al., 2008; Tribolium Genome Sequence Consortium,
2008) and evidence for functional RNAi reported in a wide range of insect
species encompassing the orders Coleoptera, Diptera, Dictyoptera,
Hemiptera, Hymenoptera, Isoptera, Lepidoptera, Neuroptera, and Orthop-
tera. In order to be useful as an insect control agent in agriculture, environ-
mental RNAi must first be operational: dsRNA must be delivered to the
insect either by ingestion or by penetration of the insect cuticle in order
RNAi-Mediated Insect Pest Management 253

to trigger an RNAi response in recipient cells. For transgenic plant applica-

tions and for most topical spray applications, delivery via ingestion is likely
the dominant route of entry. An overall picture of the responsiveness of dif-
ferent insect species and orders to ingested dsRNAs is emerging (for reviews,
see Huvenne and Smagghe, 2010; Li et al., 2013b; Terenius et al., 2011). In
feeding studies, dsRNAs are supplied by droplet feeding, incorporated into
an artificial diet, or applied to leaf/plant tissue suitable for insect feeding.
While the dose of dsRNA consumed is not known in many instances, this
variable is less important than the concentration of dietary dsRNA presented
to the insect: the effective concentration of a dsRNA active ultimately deter-
mines its utility for insect control. In reviewing these studies, we determined
or estimated, whenever possible, the dietary concentration of dsRNA tested
in parts per million (ppm) or parts per billion (ppb) in order to normalize the
results and compare among studies. A summary of published studies evalu-
ating the effects of ingested dsRNAs or siRNAs on insect species is presented
in Table 5.1.

3. INSECT SENSITIVITY TO ENVIRONMENTAL RNAi

3.1. Coleoptera
A number of coleopteran species, including the WCR (D. virgifera virgifera),
southern corn rootworm, Diabrotica undecimpunctata howardi, Colorado
potato beetle (CPB), Leptinotarsa decemlineata, and canola flea beetle, Phy-
llotreta striolata, are remarkably sensitive to ingested dsRNAs with LC50
values in the range of 110 ppb (Table 5.1, Baum et al., 2007; Bolognesi
et al., 2012; Zhao et al., 2008). This sensitivity to environmental RNAi
extends to both the larval and adult stages (Rangasamy and Siegfried,
2012; Zhao et al., 2008). To our knowledge, no insect species outside
the Order Coleoptera approaches this level of sensitivity to ingested dsRNA,
with most studies of successful oral delivery in other insect orders reporting
LC50 values >10 ppm. This potent environmental RNAi response is not
necessarily shared among all coleopteran species, however, as studies with
the red flour beetle, Tribolium castaneum, and cotton boll weevil, Anthonomus
grandis, suggest (Baum et al., 2007; Whyard et al., 2009).
Some aspects of the RNAi response in corn rootworm are instructive. Of
the 290 gene targets screened by Baum et al. (2007), approximately 2/5 cau-
sed rootworm mortality or stunting in surface overlay diet bioassays when
applied at the relatively low concentration of
50 ng/cm2, or roughly
equivalent to 0.1 ppm in diet (see Baum et al., 2011 for a listing of efficacious
Table 5.1 Sensitivity of insect species to ingested dsRNAs
LC50 or
Target gene Mortality or (concentration mRNA
Organism product Stage Assay stunting tested) silencing Reference
Coleoptera
Diabrotica virgifera Multiple targets Neonates Artificial diet Yes 110 ppba Yes Baum et al. (2007)
virgifera
V-ATPase A Neonates Transgenic Yes ND Yes Baum et al. (2007)
plant
Snf 7 Neonates Artificial diet Yes 4.3 ppb Yes Bolognesi et al.
(2012)
Snf 7 Neonates Transgenic Yes ND Yes Bolognesi et al.
plant (2012)
Diabrotica Snf 7 Neonates Artificial diet Yes 1.2 ppb Yes Bolognesi et al.
undecimpuctata (2012)
howardii
V- ATPase A Neonates Artificial diet Yes (
0.1 ppm)a Yes Baum et al. (2007)
and E
-Tubulin Neonates Artificial diet Yes (
0.1 ppm)a Yes Baum et al. (2007)
Leptinotarsa V-ATPase A Neonates Artificial diet Yes
10 ppb a
Baum et al. (2007)
decemlineata and E
Multiple targets Neonates Leaf tissue Yes ND Yes Zhu et al. (2011)
Phyllotreta striolata Arginine kinase Adults Leaf tissue Yes 0.8 ppb Yes Zhao et al. (2008)
Tribolium V-ATPase E Neonates Flour Yes 2.5 ppm Yes Whyard et al.
castaneum (2009)
Diptera
Aedes aegypti V-ATPase A Adults Artificial diet (1000 ppm) Yes Coy et al. (2012)
Multiple targets First instars Water Yes (200, 500 ppm) Yes Singh et al. (2013)
ATP-dependent Second Water 30 ppm Yes Figueira-Mansur
efflux pump instars et al. (2013)
Anopholes gambiae Chitin synthase 2 Third Artificial diet Yes Yes Zhang et al. (2010)
instars
Anopheles stephensi 3-HKT First instars Chlamydomonas Yes ND Yes Kumar et al. (2013)
Bactrocera dorsalis Multiple targets Adults Artificial diet Yes/No (2000 ppm) Yes/No Li et al. (2011b)
Glossina morsitans Tsetse EP Male adults Blood meal No (>400 ppm) Yes Walshe et al.
morsitans (2009)
Transferrin Male adults Blood meal No (>400 ppm) No Walshe et al.
(2009)
Hemiptera
Acyrthosiphon Aquaporin Six-day- Artificial diet No (10005000 ppm) Yes Shakesby et al.
pisum old nymphs (2009)
V-ATPase E First instars Artificial diet Yes 3.4 ppm Yes Whyard et al.
(2009)
Continued
Table 5.1 Sensitivity of insect species to ingested dsRNAscont'd
LC50 or
Target gene Mortality or (concentration mRNA
Organism product Stage Assay stunting tested) silencing Reference
V-ATPase E Neonates Artificial diet No ND No Christiaens et al.
(2014)
Hunchback (hb) Neonates Artificial diet Yes (750 ppm) Yes Mao and Zeng
(2012)
Bactericerca cockerelli Multiple targets Adults Artificial diet Yes (5001000 ppm) Yes Wuriyanghan et al.
(2011)
Bemisia tabaci V-ATPase Adults Artificial diet Yes 3,11 ppm Yes Upadhyay et al.
subunit A, rpL19 (2011)
Lygus lineolaris Inhibitor of Neonates Artificial diet No (1000 ppm) No Allen and Walker
apotosis (2012)
Myzus persicae RACK-1, Nymphs Transgenic Yes ND Yes Pitino et al. (2011)
COO2 plant
Hunchback (hb) Neonates Transgenic Yes ND Yes Mao and Zeng
plant (2014)
Nilaparvata lugens Trehalose PO4 Third Artificial diet ? (500 ppm) Yes Chen et al. (2010)
synthase instars
V-ATPase E 2nd instars Artificial diet No (50 ppm) Yes Li et al. (2011a)
Multiple targets Neonates Transgenic No ND Yes Zha et al. (2011)
plant
Peregrinus maidis V-ATPase Third Artificial diet Yes (500 ppm) Yes Yao et al. (2013)
B and D instars
Rhodnius prolixus Nitroporin 2 Second Artificial diet NA (1000 ppm) Yes Araujo et al. (2006)
instars
Sitobion avenae Multiple targets Third Artificial diet Yes (3 7.5 ppm) Yes Zhang et al.
instars (2013b)
Hymenoptera

Apis mellifera Vitellogenin Second Natural diet Nonspecific (5003000 ppm) Yes Nunes and Simoes
instars (2009)
Solenopsis invicta PBAN/pyrokinin Fourth Artificial diet Yes (1000 ppm) ND Vander Meer and
instars Choi (2013)
GNBP Workers Artificial diet Yes (200 ppm) ND Zhao and Chen
(2013)
Isoptera
Reticulitermes Cellulase Workers Paper discs Yes (5.1 g/cm2) Yes Zhou et al. (2008)
flavipes
Hexamerin Workers Paper discs Yes (2.2 g/cm )2
Yes Zhou et al. (2008)
Lepidoptera
Chilo infuscatellus CiHR3 moulting Third Corn kernels Yes (250 ppm) Yes Zhang et al.
factor instars (2012c)
Epiphyas Carboxylesterase Third Droplet No (4000 ppm) Yes Turner et al.
postvittana instars (2006)
Pheromone bp Third Droplet No (4000 ppm) Yes Turner et al.
instars (2006)
Continued
Table 5.1 Sensitivity of insect species to ingested dsRNAscont'd
LC50 or
Target gene Mortality or (concentration mRNA
Organism product Stage Assay stunting tested) silencing Reference
Helicoverpa armigera CYP6AE14 Third Transgenic Yes ND Yes Mao et al. (2007)
instars plant
GST Third Transgenic No ND Yes Mao et al. (2007)
instars plant
CYP6AE14 Third Transgenic Yes ND Yes Mao et al. (2011,
instars plant 2013)
AchE receptor Neonates Artificial diet Yes (
0.35 ppm) Yes Kumar et al. (2009)
AchE receptor Neonates Leaf tissue Yes (
0.35 ppm) Kumar et al. (2009)
Ecdysone Second Transgenic Yes ND Yes Zhu et al. (2012)
receptor EcR instars plant
Ecdysone Third Artificial diet Yes ND Yes Zhu et al. (2012)
receptor EcR instars (Ec)b
HaHR3 Third Transgenic Yes ND Yes Xiong et al. (2013)
moulting factor instars plant
HaHR3 Third Artificial diet Yes ND Yes Xiong et al. (2013)
moulting factor instars (Ec)
 CYP6B6 Third Artificial diet Yes ND Yes Zhang et al.
instars (Ec) (2013c)
Ultraspiracle Third Artificial diet Yes (1000 ppm) Yes Yang and Han
protein, EcR instars (2014)
Manduca sexta V-ATPase E Neonates Artificial diet Yes 11 ppm Yes Whyard et al.
(2009)
Ostrinia nubilalis Chitinase Neonates Artificial diet Yes (2500 ppm) Yes Khajuria et al.
(2010)
Plutella xylostella CYP6BG1 Fourth Droplet (
800 ppm) Yes Bautista et al.
instars (2009)
Rieske protein Second Leaf tissue Yes (6 g/cm2) Yes Gong et al. (2011)
instars
AchE receptor Second Leaf tissue Yes 53.7 ppm - Gong et al. (2013)
instars
Sesamia nonagriodes JH esterase JHER First to Artificial diet No ND Yes Kontogiannatos
sixth instars (Ec) et al. (2013)
Spodoptera exigua Chitin synthase A Neonates Artificial diet Yes ND Yes Tian et al. (2009)
(Ec)
1 integrin Fourth Leaf tissue Yes 100200 ppm Surakasi et al.
subunit instars (2011)
Ecdysone Second Transgenic Yes ND Yes Zhu et al. (2012)
receptor EcR instars plant
Continued
Table 5.1 Sensitivity of insect species to ingested dsRNAscont'd
LC50 or
Target gene Mortality or (concentration mRNA
Organism product Stage Assay stunting tested) silencing Reference
Spodoptera litura Aminopeptidase Neonates Artificial diet No ND No Rajagopal et al.
N (2002)
Spodoptera Allatostatin C Fifth instars Droplet NA (600 ppm) Yes Griebler et al.
frugiperda (2008)
Allototropin 2 Fifth instars Droplet NA (600 ppm) Yes Griebler et al.
(2008)
SfT6 serine Fourth Droplet NA (600 ppm) Yes Rodrguez-
protease instars Cabrera et al.
(2010)
Orthoptera
Gryllus bimaculatus Sulfakinins Adults Droplet NA (100600 ppm) Meyering-Vos and
Muller (2007)
Locusta migratoria Multiple targets Fourth Artificial diet No (
240 ppm) No Luo et al. (2013)
instars
Schistocerca gregaria Tubulin, Adults Artificial diet No ND No Wynant et al.
GAPDH (2012)
Estimated from sample overlay assays in which 20 L samples are infused into 200 L artificial diet.
a

Ec dsRNA expressed in E. coli.

b

ND, not determined or reported; NA, not applicable.

RNAi-Mediated Insect Pest Management 261

targets). While the suppression of certain gene transcripts did not result in a
phenotypic response (Baum et al., 2007, supplemental figure 3), it is clear
that the number of specific gene targets available for successful environmen-
tal RNAi is large. Sectioning of the V-ATPase A coding region into six

300 bp dsRNAs did not reveal dramatic differences in efficacy, suggesting
that a single dsRNA of this size provides a reasonable sampling of target
knockdown efficacy and phenotype. One of the targets identified by
Baum et al. (2007), a Snf7 ortholog, was selected for a more detailed study
of the RNAi response (Bolognesi et al., 2012). Snf7 dsRNAs shorter than
50 bp exhibited dramatically reduced activity in the corn rootworm feeding
assay. This study employed a single Snf7 27-mer sequence embedded in
neutral sequences of varying length, ruling out the possibility that differences
in siRNA composition accounted for differences in activity. An in situ study
of RNA uptake further demonstrated that a Cy3-labelled 240 bp Snf7
dsRNA was taken up by rootworm midgut epithelial cells while a Cy3-
labelled 21 bp Snf7 siRNA was not, corroborating the size dependency of
the RNAi response observed in feeding assays (Bolognesi et al., 2012). Like-
wise, injection studies with corn rootworm larvae have demonstrated the
inability of siRNAs to produce an RNAi response leading to rootworm
mortality (Khajuria et al., 2013). Finally, the RNAi response in corn
rootworm appears to be systemic as judged by qPCR analysis of Snf7
mRNA transcripts in isolated midgut and cadaver tissues (Bolognesi
et al., 2012).
Elements of the rootworm RNAi response can be found in another cole-
opteran species, the red flour beetle T. castaneum, which has become a model
system for studying systemic RNAi in insects (Miller et al., 2012). Injection
studies with a transgenic gfp-Tribolium line permitted visualization of silenc-
ing as suppression of green fluorescent protein (gfp) fluorescence. The
RNAi response was observed to be dose-dependent, systemic, and likewise
dependent on the size of the dsRNA. Injection of
60 ng of a 520-bp gfp
dsRNA/last instar larva resulted in detectable silencing of gfp, demonstrating
that this coleopteran species is very sensitive to systemic RNAi. The effi-
ciency of systemic RNAi appears to drop off with shorter dsRNA molecules
although the precise breakpoint was not determined. Adopting a strategy
similar to that used for characterization of the corn rootworm Snf7 dsRNA
(Bolognesi et al., 2012), a 30-bp gfp dsRNA fused to a 30-bp neutral Ultra-
bithorax (Ubx) dsRNA was shown to be more effective than the 30-bp gfp
dsRNA alone in triggering a systemic RNAi response, presumably due to
less efficient cellular uptake of the smaller dsRNA. Comparing a series of
262 James A. Baum and James K. Roberts

siRNAs and larger dsRNAs targeting the Tc-achaete-scute-homolog (Tc-ASH)

and Ultrabithorax (UBX) genes in Tribolium, Wang et al. (2013) reported that
dsRNAs provided greater silencing for longer periods of time resulting in
developmental phenotypes. Interestingly, the silencing observed with
injected siRNAs (0.40.5 g) was short-lived, lasting from days 2 to 4,
and did not result in a phenotypic response. The dose-dependent systemic
RNAi response observed in Tribolium is consistent with the concentration-
dependent environmental RNAi response observed in Diabrotica that per-
mitted calculation of LC50 values (Baum et al., 2007). The steepness of those
concentrationresponse curves may reflect a threshold effect, as has been
suggested for Tribolium (Miller et al., 2012). Like other insect species, neither
Tribolium nor Diabrotica contains a recognizable RNA-dependent RNA
polymerase that could amplify the production of dsRNAs for a systemic
response (Baum et al., 2007; Tomoyasu et al., 2008). Despite the apparent
absence of this amplification mechanism, corn rootworm larvae are remark-
ably sensitive to ingested dsRNAs and require no more than a 3-h exposure
to dietary dsRNA to observe a lethal phenotype (Bolognesi et al., 2012).
Comparative genomic analysis of the Tribolium and Diabrotica genomes
may shed light on the apparent differences in sensitivity to ingested dsRNAs
observed between these two species.

3.2. Diptera
Belles (2010) provides some historical context for the pioneering RNAi
studies conducted with the fruit fly, Drosophila melanogaster, a model system
for understanding both the mechanism of RNAi and its role in mediating
antiviral immunity in invertebrates (Nayak et al., 2013). Reverse genetic
studies of gene function have been enabled through the use of cultured cells,
employing both genome-wide (Boutros and Ahringer, 2008; Boutros et al.,
2004) and pathway-specific screens (e.g. Clemens et al., 2000). Functional
studies of genes involved in Drosophila development have relied on micro-
injection of dsRNA into embryonic tissue (e.g. Kennerdell and Carthew,
1998; Koizumi et al., 2007; Pilot et al., 2006). Libraries of transgenic Dro-
sophila lines containing conditionally expressed dsRNA hairpins have been
generated for use in whole-animal RNAi screens (e.g. Dietzl et al., 2007).
Abundant tools for both in vivo and in vitro RNAi studies in Drosophila, as
well as relevant literature, can be found at http://www.flyrnai.org/.
Drosophila appears deficient in systemic RNAi when confronted with
endogenously expressed dsRNA hairpins (Roignant et al, 2003), but is
RNAi-Mediated Insect Pest Management 263

capable of mounting a systemic RNAi response to viral infection (Saleh

et al., 2009). The systemic RNAi response to injected dsRNAs is also lim-
ited. Using transgenic Drosophila lines expressing Gal4-regulated enhanced
green fluorescent protein (EGFP) or GFP, Miller et al. (2008) evaluated the
systemic RNAi response in last instar larvae and reported that only hemo-
cytes were responsive to injected (hemolymph) dsRNA, whereas in Tri-
bolium larvae, virtually all cell types were responsive. Co-expression of an
EGFP dsRNA hairpin resulted in down-regulation of EGFP in all tissues
examined, demonstrating that the limited systemic RNAi response observed
in Drosophila larvae is not due to the absence of cell-autonomous RNAi
machinery. Drosophila is likewise recalcitrant to environmental RNA, but
larvae fed with dsRNAs formulated with transfection agents exhibited both
specific target gene knockdown and significant mortality (Whyard et al.,
2009), suggesting that the presence of a robust systemic RNAi response
may not be a prerequisite for RNAi-mediated insect control.
Extensive RNAi studies have likewise been conducted with dipteran
vectors of human diseases to study gene function related to vector biology
and vectorpathogen interactions (reviewed in Barnard et al., 2012; Belles,
2010; Manzano-Roman et al., 2012). Although the majority of studies with
mosquito species have relied on microinjection of dsRNAs into the hemo-
lymph, other methods such as topical delivery of dsRNA to adults (Pridgeon
et al., 2008) and deployment of a recombinant densovirus-mediated RNAi
system (Gu et al., 2011) have been described. Several studies have reported
success in the oral delivery of dsRNAs to dipteran species, including the
mosquitoes, Aedes aegypti and Anopheles gambiae, and the tsetse fly, Glossina
morsitans morsitans (Table 5.1). Ingestion of a 10% sucrose solution con-
taining dsRNA (1000 ppm) targeting the vacuolar ATPase subunit
A gene resulted in significant knockdown of the target transcript in
A. aegypti adults as early as 12 h after the onset of feeding, but no mortality
data were reported (Coy et al., 2012). Silencing of a P-glycoprotein (P-gp;
ATP-dependent efflux pump) gene in A. aegypti larvae following ingestion
of dsRNA resulted in enhanced sensitivity to the insecticide temephos
(Figueira-Mansur et al., 2013), consistent with evidence that P-gp proteins
are involved in resistance to organophosphate insecticides. Singh et al.
(2013) evaluated several dsRNAs in feeding assays with A. aegypti and
observed both silencing and mortality/stunting of larvae treated with the
-tubulin and chitin synthase-1 dsRNAs at dietary concentrations of
200500 ppm. DsRNAs targeting chitin synthase-2 induced silencing but
had no apparent effect on larval survival while silencing of heat shock protein
264 James A. Baum and James K. Roberts

83 manifested itself by increased larval mortality under heat stress. In these

studies with mosquito larvae, it is not clear whether the route of RNA deliv-
ery is oral or via penetration of the larval cuticle. DsRNA complexed with
chitosan and packaged in a gel-based diet was used to silence chitin synthase
genes in A. gambiae larvae resulting in increased larval sensitivity to agents
that either inhibit chitin biosynthesis in insects (diflubenzuron) or act to dis-
rupt the organization or integrity of the peritrophic matrix (calcofluor white,
dithiothreitol) (Zhang et al., 2010). Expression of dsRNAs in transgenic
Chlamydomonas has been proposed as an alternative delivery vehicle for
RNAi-mediated control of mosquito larvae (Kumar et al., 2013).

3.3. Lepidoptera
Terenius et al. (2011) provided a useful overview of the status of RNAi stud-
ies in lepidopteran species and noted that, with respect to oral delivery, gene
suppression only appears to be successful when high concentrations of
dsRNA are provided in the diet. In the first reported example, Turner
et al. (2006) achieved a significant suppression of several target genes in
the brown apple moth, Epiphyas postvittana (Walker), by droplet feeding a
4000 ppm dsRNA solution. Subsequently, silencing via oral delivery of
dsRNA was reported in a wide range of lepidopteran species including
the tobacco hornworm, Manduca sexta, diamondback moth (DBM), Plutella
xylostella, beet armyworm, Spodoptera exigua, fall armyworm, Spodoptera
frugiperda, European corn borer, Ostrinia nubilalis, sugarcane stem borer,
Chilo infuscatellus, and the cotton bollworm, H. armigera (Bautista et al.,
2009; Gong et al., 2011; Khajuria et al., 2010; Mao et al., 2007;
Rodrguez-Cabrera et al., 2010; Surakasi et al., 2011; Tian et al., 2009;
Whyard et al., 2009; Xiong et al., 2013; Zhang et al., 2012c; Zhu
et al., 2012).
For many early RNAi studies, the object was not to kill an insect but to
selectively down-regulate a gene to study its function in a metabolic or
developmental process (Belles, 2010). For example, environmental RNAi
was used to examine the role of two chitinase genes in regulating chitin con-
tent in the peritrophic matrix of the European corn borer (Khajuria et al.,
2010), to demonstrate that the cytochrome P450 gene, CYPBG1, in
DBM is involved in larval resistance to the insecticide permethrin
(Bautista et al., 2009), to study the role of the beet armyworm 1 subunit
integrin (Se1) in development and cellular immunity (Surakasi et al.,
2011), and to demonstrate that a serine protease gene in the fall armyworm
RNAi-Mediated Insect Pest Management 265

plays an important role in the processing of the B. thuringiensis Cry1Ca1

insecticidal protein and the insects pathogen response to Bt toxins
(Rodrguez-Cabrera et al., 2010). In these studies, high concentrations of
dsRNA (502500 ppm) were delivered by droplet feeding, incorporation
into artificial diet, or by soaking leaf tissue prior to feeding. Surakasi et al.
(2011) reported concentration-dependent mortality upon silencing of the
Se1 subunit gene in the beet armyworm from which one can interpolate
an LC50 of 100200 ppm for this topical application. Silencing of the
vacuolar ATPase E subunit gene in M. sexta resulted in concentration-
dependent mortality and an LC50 of only 11 ppm (Whyard et al., 2009).
Even so, this level of activity is still three orders of magnitude lower than
that observed among the sensitive coleopteran species.
Transgenic plants expressing insect-specific dsRNAs have been reported
to impact the growth and survival of certain lepidopteran species, most nota-
bly the cotton bollworm, H. armigera. Larvae of H. armigera fed on tobacco
plants expressing a dsRNA targeting the H. armigera ecdysone receptor
(EcR) gene showed elevated mortality (
40% compared to 10% in the
gfp control group), growth reduction, and significant suppression of the
EcR transcript (Zhu et al., 2012). Larvae of another lepidopteran species,
the beet armyworm S. exigua, also showed elevated mortality and develop-
ment aberrations when fed on the same transgenic plant material, presum-
ably because the EcR target sequences in these two species share regions of
>21 bp sequence identity (Zhu et al., 2012). Likewise, tobacco plants
expressing a dsRNA targeting a moult-regulating transcription factor,
HaHR3, were shown to suppress the HaHR3 transcript in fed H. armigera
larvae. In this study, larval mortality of 2230% and >50% mass reduction
was observed after 5 days of feeding on transgenic leaf discs (Xiong et al.,
2013). Mao et al. (2007) was the first to use RNAi as a means to alter an
insects ability to cope with xenobiotic compounds, in this case gossypol.
Transgenic cotton plants expressing a dsRNA hairpin derived from the
H. armigera gossypol-inducible cytochrome P450 gene CYP6AE14 showed
increased tolerance to the cotton bollworm, H. armigera (Mao et al., 2011)
but were not lethal to the larvae. This response can be enhanced by
co-delivery of a cysteine proteinase to damage the larval peritrophic matrix,
leading to higher gossypol accumulation in the midgut and a modest increase
in larval stunting when fed on cotton (Mao et al., 2013). This strategy of
targeting detoxification mechanisms in the insect midgut to mitigate plant
feeding damage seems promising, particularly since it may not require a sys-
temic RNAi response in the insect. In principle, this strategy is no different
266 James A. Baum and James K. Roberts

than the use of RNAi to restore sensitivity to insecticides among resistant

insect species (Bautista et al., 2009; Figueira-Mansur et al., 2013; Tang
et al., 2012).
Difficulty in delivering sufficient dsRNA to lepidopteran gut epithelial
cells may be inferred from the paper of Gong et al. (2011) which describes
the use of chemically modified siRNAs to target the Rieske iron-sulphur
protein gene in the DBM. Both 20 -O-methoxy nucleotides and
deoxythymidine were incorporated into the sense and antisense strands to
stabilize the siRNAs. When provided to the larvae on cabbage leaves, again
at relatively high concentrations (6 g/cm2), several siRNAs caused sup-
pression of the target gene transcript and mortality. This approach was
extended to include 50 PEG modification of siRNAs designed to target
the acetyl cholinesterase (AchE) receptor in the DBM (Gong et al.,
2013). One such siRNA, Si-ace2_001, exhibited an LC50 of 53.7 ppm
when sprayed on DBM-infested cabbage leaf discs. Kumar et al. (2009) also
employed modified siRNAs targeting the AchE receptor of H. armigera, in
this case substituting a pair of deoxythymidine residues at the 30 end of both
the sense- and antisense strands. Larvae showed reduced growth (stunting)
and increased pupal malformation when fed diet containing siRNA at

0.35 ppm. These results suggest that there might not be a strict dsRNA
size dependency to the environmental RNAi response in lepidopterans,
at least with chemically modified siRNAs. Larger dsRNAs expressed and
encapsulated in Escherichia coli have also been reported to impact the growth
and survival of S. exigua and H. armigera larvae (Tian et al., 2009; Xiong et al.,
2013; Zhang et al., 2013c; Zhu et al., 2012) but neither the concentration of
dsRNA in diet nor the effect of dsRNA alone is reported in these studies.
Yang and Han (2014) reported that E. coli-encapsulated dsRNAs appear to
be more effective than naked dsRNA (1000 ppm in diet) in blocking pupa-
tion and causing mortality of H. armigera larvae. Suppression of a juvenile
hormone esterase-related gene in the corn stalk borer, Sesamia nonagriodes,
via bacterial delivery of dsRNA did not result in a phenotype
(Kontogiannatos et al., 2013). In the case of the sugarcane stem borer,
C. infuscatellus, naked dsRNA (at 250 ppm) and bacterial-expressed dsRNA
applied to corn kernels as a diet appeared equally effective in promoting
silencing and stunting larval growth (Zhang et al., 2012c).
In some cases, the insect gut may be bypassed by the use of topical sprays
that rely on penetration or adsorption through the insect cuticle (Wang
et al., 2011). Topical application of dsRNAs (at 50 ppm) targeting larval
stage-specific transcripts in the Asian corn borer, Ostrinia furnacalis, led to
RNAi-Mediated Insect Pest Management 267

significant larval mortality and gene silencing at 5 days post-sprays. The

route of dsRNA penetration into the larvae is not known but could involve
transit to the hemolymph via the tracheoles. Many of the treatments in this
study caused significant mortality in the absence of significant gene silencing
at day 3 (Wang et al., 2011), suggesting that either a non-RNAi mechanism
is at work or the method used to measure transcript knockdown (qRT-PCR
on whole insects) could not detect localized tissue-specific silencing leading
to mortality.

3.4. Hemiptera
RNAi techniques have been used successfully in a wide variety of hemip-
teran species encompassing phloem feeders such as aphids and piercing
sucking insects such as plant bugs (Li et al., 2013b). Since many hemipteran
species are relatively small and fragile as nymphs, oral delivery of dsRNAs for
gene silencing has been an attractive alternative to microinjection. As was
the case with lepidopterans, early studies were not necessarily focused on
killing insects but on studying gene function (e.g. Belles, 2010; Paim
et al., 2013). Across all studies with hemipterans, the dietary concentrations
of dsRNA required for silencing and/or lethal phenotypes vary widely, even
between studies with the same organism, but tend to be at least three orders
of magnitude higher than effective concentrations used with sensitive cole-
opteran species. Examples of gene silencing following ingestion of dsRNA
include the nitrophorin 2 (Np2) gene in the triatomine bug, Rhodnius prolixus
(Araujo et al., 2006), the aquaporin 1 (ApAQP1) gene in the pea aphid,
Acyrthosiphon pisum (Shakesby et al., 2009), the vacuolar ATPase subunit
E gene in A. pisum (Whyard et al., 2009), the trehalose phosphate synthase
(tps) gene in brown planthopper, Nilaparvata lugens (Chen et al., 2010), the
gap gene hunchback in A. pisum (Mao and Zeng, 2012), and the vacuolar
ATPase subunit E gene in N. lugens (Li et al., 2011a). Drawing from a list
of efficacious gene targets identified for the WCR, Upadhyay et al.
(2011) reported silencing of the vacuolar ATPase subunit Aand ribosomal
protein L9 genes in the whitefly, Bemisia tabaci, as well as mortality with
LC50 values in the 311 ppm range. Focusing on gene targets that are highly
or specifically expressed in the midgut, Wuriyanghan et al. (2011) demon-
strated target gene suppression and lethality in the potatotomato psyllid,
Bactericerca cockerelli, when dsRNAs were presented at high concentrations
(5001000 ppm) in a 15% sucrose diet. Both studies also reported lethality
upon ingestion of siRNAs, an effect that has not been reported in
268 James A. Baum and James K. Roberts

coleopteran species. Ingestion of dsRNA (750 ppm) targeting the hunchback

gene led to target gene suppression and increased mortality of A. pisum
nymphs fed on an artificial diet (Mao and Zeng, 2012). Tobacco plants
expressing a dsRNA targeting the orthologous hunchback gene of the peach
aphid, Myzus persicae, caused
30% target mRNA suppression and
13%
inhibition of aphid reproduction (Mao and Zeng, 2014). Feeding large
dsRNAs for multiple gene targets at dietary concentrations of 7.5 ppm
resulted in target gene suppression and increased mortality in the grain aphid,
Sitobion avenae F. (Zhang et al., 2013b). In this study, feeding 37.5 ppm
dsRNA of C002, a gene encoding an unknown protein required for normal
plant foraging behaviour in A. pisum (Mutti et al., 2008), was also reported to
cause increased mortality, although it is not clear from the literature whether
silencing of this gene should even impact feeding on artificial diet (Mutti
et al., 2008). In contrast, Inhibitor of Apoptosis (IAP) dsRNA presented in diet
at 1000 ppm had no effect on the survival of tarnished plant bug, Lygus lin-
eolaris, nymphs (Allen and Walker, 2012) despite evidence that silencing of
IAP via dsRNA injection results in increased Lygus mortality (Walker and
Allen, 2011). Consistent with many studies, dsRNA injection was far more
effective than dsRNA feeding in silencing target genes and causing a lethal
phenotype in the corn planthopper, Peregrinus maidis (Yao et al., 2013). In
this study, it is worth noting that early (day 2) and prolonged (day 6) sup-
pression of the vacuolar ATPase subunit B transcript by diet feeding
(500 ppm) did not result in the expected lethal phenotype observed via
injection while late silencing (day 6) of the vacuolar ATPase subunit
D transcript resulted in a detectable lethal phenotype, but at a time point
when the majority (
60%) of the control insects had already died. This
illustrates the difficulties encountered with artificial diet feeding assays con-
ducted with many hemipteran species: phenotypes caused by environmental
RNAi can be slow to emerge and keeping insects alive on an artificial diet for
>7 days can be challenging.
A number of research groups apparently eschewed artificial diet feeding
assays altogether and evaluated insect feeding on transgenic plants expressing
dsRNAs designed to target essential insect genes. Pitino et al. (2011) selected
two target genes in the aphid, M. persicae, for knockdown using transgenic
Arabidopsis thaliana; receptor of activated kinase C (Rack-1) and C002. When
fed on transgenic lines expressing the target dsRNAs, M. persicae nymphs
exhibited a >50% knockdown in target gene expression after 16 days,
but only a small reduction in progeny when compared to nymphs fed on
the dsGFP control line or on Rack-1 lines that did not suppress the Rack-1
gene. Injection of C002 dsRNA into A. pisum nymphs results in both rapid
RNAi-Mediated Insect Pest Management 269

gene silencing and 100% mortality after 7 days of feeding on plants (Mutti
et al., 2006), suggesting that the delivery of dsRNA via plant expression was
simply inadequate for control of this species. A similar outcome was reported
using transgenic rice plants expressing dsRNAs targeting three midgut-
expressed genes in N. lugens (Zha et al., 2011). In this study, significant
reductions in target transcript levels (as high as 73% for the Nltry gene) were
detected 24 days after feeding but no lethal phenotype was observed.
Comparisons across related studies reveal marked variation in the hemip-
teran response to environmental RNAi. For example, Chen et al. (2010)
reported significant silencing of the trehalose phosphate synthase (tps) gene
in N. lugens at dsRNA diet concentrations of 500 ppm but no silencing at
concentrations of <100 ppm while Li et al. (2011a) reported significant
silencing of the vacuolar ATPase subunit E gene in N. lugens at dsRNA diet
concentrations of only 50 ppm. Silencing of the same essential gene in two
distantly related hemipteran species can apparently yield different pheno-
types: a dsRNA silencing the vacuolar ATPase subunit E gene in
A. pisum yielded a reported LC50 of 3.4 ppm (Whyard et al., 2009) while
a dsRNA silencing the N. lugens ortholog caused no phenotype (Li et al.,
2011a). Moreover, different groups working with the same insect species
and gene target have reported conflicting outcomes. Whereas Whyard
et al. (2009) reported oral efficacy with a dsRNA targeting the vacuolar
ATPase subunit E gene in A. pisum and Mutti et al. (2006) reported lethality
following injection of C002 dsRNA in A. pisum, Christiaens et al. (2014)
failed to observe a phenotypic response with either dsRNA and delivery
method in this species. In this case, the decision to deploy a long COO2
dsRNA rather than pre-diced siRNAs could have influenced the outcome
of the injection experiment. Other factors that could account for these dis-
parate results include insect colony source and health, viral load, life stage,
dsRNA stability in different diets, and target gene propensity for silencing.

3.5. Other agricultural pests

Environmental RNAi has been studied in a number of other arthropod pests
for which systemic RNAi has already been demonstrated via injections stud-
ies. Feeding studies with the invasive fire ant, Solenopsis invicta, have dem-
onstrated that dsRNAs ingested by worker ants can be brought back to
the colony and shared with the brood (trophallaxis), leading to significant
mortality in the brood (larval) population. Worker ant feeding on a 10%
sucrose diet containing 1000 ppm dsRNA targeting the PBAN/pyrokinin
gene resulted in increased mortality among the nursing fourth instar larvae
270 James A. Baum and James K. Roberts

(Vander Meer and Choi, 2013). In contrast, a dsRNA targeting a guanine

nucleotide-binding protein (GNBP) was reported to be directly toxic to
S. invicta worker ants when presented in diet at 200 ppm (Zhao and
Chen, 2013). Although not an insect, spider mites can be a significant agri-
cultural pest in both vegetable- and row crops. Using a dsRNA permeated
leaf disc assay, Kwon et al. (2013) demonstrated gene silencing and signifi-
cant mortality in the two-spotted spider mite, Tetranychus urticae, by
targeting several genes at applied concentrations of 160 ppm dsRNA. Most
of the efficacious targets surveyed, including genes encoding the COPI
coatomer 0 subunit, V-ATPAse subunits A and B, and ribosomal protein
S4, had been previously identified as efficacious targets in the coleopteran
species D. virgifera virgifera (Baum et al., 2007; Khajuria et al., 2013) and
L. decemlineata (Baum et al., 2007; Zhu et al., 2011). The mite, Varroa
destructor, an ectoparasite of the honey bee, Apis mellifera, and a major suspect
contributing to the phenomenon known as bee colony collapse disorder, is
also sensitive to environmental RNAi but the route of delivery is unusual in
this case. Immersion of Varroa mites in a saline solution containing dsRNA
can lead to gene silencing (Campbell et al., 2010), but so can feeding dsRNA
to the honey bees upon which the mites subsequently feed (Garbian et al.,
2012). This unique interspecies transfer of dsRNA from bee to mite presents
a strategy for control of this debilitating agricultural pest. Finally, it is worth
noting here that locust species (order Orthoptera) display a highly sensitive
systemic RNAi response but are refractory to environmental RNAi. As little
as 15 pg of dsRNA per mg body mass (
10 ng/insect) is required for sig-
nificant knockdown of target gene expression in the desert locust, Schistocerca
gregaria (Wynant et al., 2012). As is the case for T. castaneum, the systemic
response is dose-dependent, continues to increase over time, and can lead
to mortality 7 days post-injection. Likewise, the migratory locust, Locusta
migratoria was observed to be highly responsive to injected dsRNAs,
exhibiting a dose-dependent response leading to target gene suppression
and lethality, but was not responsive to environmental RNAi (Luo et al.,
2013). A survey of the Locusta genome revealed putative orthologs for almost
all of the genes implicated in dsRNA uptake in Drosophila melanogaster (Saleh
et al., 2006), another species unresponsive to environmental RNAi.

4. BARRIERS TO DELIVERY AND EFFICACY

IN RECALCITRANT SPECIES
Many recent studies have focused on the optimization of RNAi effi-
ciency in recalcitrant species via selection of optimal target genes (Li et al.,
RNAi-Mediated Insect Pest Management 271

2013a; Wang et al., 2011; Yao et al., 2013; Zhang et al., 2013b). Contrary to
this view, the results of dsRNA screens with sensitive coleopteran species
yielding high hit rates among housekeeping gene targets using low dietary
concentrations of dsRNA support the argument that biological barriers to
RNAi, not target gene selection, are the problem. Likewise, the need to pro-
vide dsRNAs targeting obviously essential genes (e.g. the V-ATPase subunit
genes) at dietary concentrations more than 1000-fold higher than are
required for an RNAi phenotype in sensitive coleopteran species suggests
that there are great inefficiencies in triggering and sustaining an RNAi
response in recalcitrant species. It seems unlikely that the selection of alter-
native gene targets will close this efficacy gap. The absence of RNAi-induced
lethal phenotypes in recalcitrant species may simply reflect the degree to
which gene silencing actually occurs. While many studies report target gene
silencing upon ingestion of dsRNA, the level of transcript knockdown sel-
dom exceeds 60% and silencing is frequently transient (Huvenne and
Smagghe, 2010; Li et al., 2013b). In the case of sensitive coleopteran species,
target gene knockdown is frequently 90% (Baum et al., 2007; Bolognesi
et al., 2012; Rangasamy and Siegfried, 2012; Zhu et al., 2011). The typical
twofold reduction in mRNA transcript levels observed in many species may
fall well within the range of normal variation observed in biological systems
and, in any case, may not manifest immediately during the course of a feeding
assay since the target protein itself needs to turnover first.
If we set aside the question of target gene selection and dsRNA design,
the barriers to efficient RNAi in recalcitrant insects can be pictured as dis-
creet steps in the mechanism of action of RNAi. DsRNAs, whether applied
topically to an insect diet or expressed in transgenic plants, must be able to
survive long enough to be taken up by insect cells. The insect midgut with its
brush border membrane topography (Chapter 1) represents a large surface
area that facilitates the absorption of nutrients and is the presumed site of
entry for dsRNAs in most studies. It is not clear to what extent the
peritrophic matrix in lepidopteran- and coleopteran midguts (Hegedus
et al., 2009; Lehane, 1997) or the perimicrovillar membrane in hemipteran
midguts (Silva et al., 2004) represents a physical barrier to dsRNA delivery.
Upon safe arrival at the gut membrane surface, dsRNAs must be efficiently
taken up by epithelial cells and delivered to the intracellular RNAi machin-
ery. If the route of cellular uptake involves endocytosis, then release or
escape from the endosome becomes a critical step for delivery to the cyto-
plasm (Varkouhi et al., 2011). The core RNAi machinery, being agnostic
with respect to the source of the dsRNA, processes the dsRNA to generate
siRNARISC complexes that can trigger a silencing event, provided
272 James A. Baum and James K. Roberts

transcripts of the gene target are present in that host cell. It is likely that
expression of the RNAi core machinery, including Dicer and Argonaute
proteins, varies among insect cell types and species, contributing to differ-
ences in the efficiency of cell-autonomous RNAi. Factors impacting
RNA amplification and/or systemic movement of the silencing signal from
the site of cellular entry represent additional barriers to RNAi efficiency and
the generation of lethal phenotypes.
The alkaline nature of the lepidopteran midgut (Dow, 1992) could cer-
tainly represent one barrier to RNA delivery. Across insect orders, the pres-
ence of midgut and salivary nucleases that can rapidly degrade dsRNA
molecules likely constitutes a more significant barrier (Arimatsu et al.,
2007a,b; Christiaens et al., 2014; Furusawa et al., 1993; Liu et al., 2013;
Luo et al., 2013; Rodrguez-Cabrera et al., 2010; Terenius et al., 2011;
Wynant et al., 2014). In the case of hemipteran species such as the tarnished
plant bug, L. lineolaris, which engage in extra-oral digestion of plant material
prior to ingestion, the presence of dsRNAses in salivary secretions represents
a significant barrier to dsRNA survival (Allen and Walker, 2012). Likewise,
Christiaens et al. (2014) provided evidence for dsRNA degradation by
dsRNAses in both pea aphid salivary secretions and haemolymph. No less
than four different midgut-expressed dsRNAses were observed in the desert
locust, S. gregaria (Wynant et al., 2014), a species that is recalcitrant to
ingested dsRNA but exhibits a potent systemic RNAi response to injected
dsRNA (Wynant et al., 2012). Knockdown experiments implicated at least
one of these nucleases in dsRNA degradation in the midgut. Likewise, Luo
et al. (2013) reported the presence of potent dsRNAses in midgut fluid from
the migratory locust, L. migratoria, and concluded that rapid degradation of
dsRNA in the midgut explains the recalcitrant nature of this species to die-
tary dsRNA. Given the sensitivity of both locust species to injected
dsRNAs, it would be worth understanding the extent to which dsRNAses
are also expressed in the haemolymph. Finally, the importance of a gut envi-
ronment hostile to dsRNA is supported by our own observation that nucle-
ases found in gut extracts from S. frugiperda, a lepidopteran species, are far
more active in degrading dsRNAs than are nucleases found in corn
rootworm gut extracts (Fig. 5.1), particularly at an alkaline pH more typical
of the lepidopteran midgut.
Inefficient uptake by midgut epithelial cells represents the next barrier to
efficacious RNAi-mediated insect control. The mechanism of dsRNA
uptake by insect gut epithelial cells has not been defined, even for highly
sensitive species such as the WCR. In the nematode, C. elegans, several genes
RNAi-Mediated Insect Pest Management 273

Figure 5.1 dsRNAs are rapidly degraded when treated with midgut extracts from the
lepidopteran species, Spodoptera frugiperda, but remain intact when treated with mid-
gut extracts from the western corn rootworm, Diabrotica virgifera virgifera. A 400-bp
V-ATPase A dsRNA (10 g, see arrow) was incubated at 23  C with 50 g of total protein
extracted from isolated midgut tissue. Incubations were carried out in 100 L volumes in
either 25 mM Tris HCl (pH 7.4) or 25 mM sodium carbonate (pH 10.5). Aliquots (20 L)
were drawn at the times indicated, the digestions were quenched by ethanol precipi-
tation, and the recovered dsRNAs were resolved by agarose gel electrophoresis.
MW Invitrogen molecular weight standards.

required for environmental RNAi and systemic spread have been identified.
The Sid-1 gene is required for systemic movement of the silencing signal
between cells and is also required for environmental RNAi by gut epithelial
cells. The Sid-1 gene encodes a transmembrane protein that acts as a
dsRNA-selective and dsRNA-gated channel to passively traffic dsRNAs
between cells (Feinberg and Hunter, 2003; Jose et al., 2009; Shih and
Hunter, 2011; Shih et al., 2009; Winston et al., 2002). The Sid-2 gene
encodes a membrane protein exclusively localized to intestinal cells that is
required for dsRNA uptake from the intestinal lumen (McEwan et al.,
2012; Winston et al., 2007). Environmental RNAi therefore requires coor-
dination between the SID-1 and SID-2 proteins. A model has been pro-
posed whereby dsRNAs are imported from the intestinal lumen by
SID-2 via endocytosis and released from the internalized vesicles via passive
274 James A. Baum and James K. Roberts

movement through the SID-1 channel (McEwan et al., 2012; Whangbo and
Hunter, 2008). While putative Sid-1-like genes have been identified in some
insects, there are no reports of Sid-2-like genes in insect species whose
genomes have been completely sequenced (e.g. Tomoyasu et al., 2008;
Xu and Han, 2008; Zha et al., 2011).
In Drosophila, an insect lacking Sid-1-like genes, evidence has been pres-
ented for an energy-dependent endocytic mechanism of dsRNA uptake by
cultured (S2) cells, apparently mediated by pattern-recognition scavenger
receptors (Saleh et al., 2006; Ulvila et al., 2006). A genomic screen identified
23 genes required for RNAi in S2 cells including many components of the
endocytic pathway involved in vesicle trafficking and protein transport or
sorting. Subsequently, orthologs of these genes were selected for RNAi
screens in C. elegans, resulting in the identification of 10 genes whose silenc-
ing disrupted the RNAi response triggered by feeding (Saleh et al., 2006). As
noted by Hinas et al. (2012), this screen could not distinguish among knock-
outs that affected dsRNA uptake by intestinal cells, systemic RNAi, or the
autonomous cellular RNAi machinery. In a subsequent study, mutations in
several of the endocytic pathway genes required for cellular dsRNA uptake
in Drosophila rendered flies that were hypersensitive to viral infection and
unable to mount a systemic antiviral response (Saleh et al., 2009). Further
support for the role of endocytic pathways in systemic RNAi proceeded
from the characterization of sid-3 and sid-5 mutants in C. elegans, mutants
that are defective in systemic RNAi but are otherwise RNAi competent.
Sid-3 encodes a conserved tyrosine kinase, homologs of which are known
to associate with endocytic vesicles, and is required for the import of dsRNA
into cells ( Jose et al., 2012). Sid-5 encodes an endosome-associated protein
required for the export of dsRNAs from cells (Hinas et al., 2012). Three
other mutants identified in RNAi screens, rsd-2, rsd-3, and rsd-6, are insen-
sitive to ingested dsRNA targeting germline-expressed genes but remain
sensitive to injected dsRNA (Tijsterman et al., 2004). Other mutants iden-
tified in this screen, rsd-8 and rsd-4, were subsequently shown to be alleles of
sid-1 and sid-2, respectively, the former already identified as required for sys-
temic RNAi. While the details are not yet understood, we expect that sys-
temic RNAi in insects will prove to be equally complex and that Sid-1-like
genes will not prove to be the sole determinant of systemic RNAi. Indeed,
it is not entirely clear that the Sid-1-like genes identified to date in insects
are even involved in systemic RNAi. A phylogenetic analysis of Sid-1-like
proteins in Tribolium suggests that these proteins may not be orthologous to
Sid-1, but rather to the C. elegans Tag-130 protein that is not involved in
RNAi-Mediated Insect Pest Management 275

systemic RNAi (Tomoyasu et al., 2008). More recently, Xu et al. (2013)

provided evidence through injection studies that the single Sid-1-like gene
found in N. lugens is required for systemic RNAi.
With respect to environmental RNAi, we observe that immortalized
insect cells scarcely resemble the differentiated cell types found in an insect
midgut. The mechanism of dsRNA uptake by Drosophila S2 cells (Saleh
et al., 2006; Ulvila et al., 2006) may or may not inform us regarding the
mechanism of RNA uptake by gut epithelial cells, particularly since Drosoph-
ila is not sensitive to ingested dsRNAs in the absence of a transfection agent
(Whyard et al, 2009). An alternative endocytic uptake mechanism may be
operational in insect species that respond to bacterial-encapsulated dsRNAs
in diet (Tian et al., 2009; Xiong et al., 2013; Zhang et al., 2013c; Zhu et al.,
2012). In the future, the selection of insects resistant to orally toxic dsRNAs
could provide an experimental system to identify genes involved in dsRNA
uptake. While Tribolium may be the best model for understanding systemic
silencing in insects, Diabrotica (corn rootworm) or L. decemlineata (CPB) may
be the emerging model systems for understanding the mechanism of efficient
environmental RNAi.
The presence of dsRNAses in the insect hemolymph, noted in some lep-
idopteran species, could prove to be a formidable barrier to systemic RNAi.
A comparative study of the RNAi-sensitive cockroach, Blattella germanica,
and the RNAi-recalcitrant lepidopteran, M. sexta, revealed that dsRNAs
are rapidly degraded in the haemolymph fluids from M. sexta but not in
the haemolymph fluids of B. germanica (Garbutt, 2011; Garbutt et al.,
2013). Putative orthologs of the Bombyx mori and M. sexta dsRNAses were
also identified in Spodoptera littoralis and S. frugiperda, two other lepidopteran
species insensitive to environmental RNAi. Although not comprising an
extensive dataset, these findings nevertheless prompted the suggestion that
the capacity to rapidly degrade dsRNA in the hemolymph could represent
an evolutionary adaptation to high viral exposure since lepidopteran-specific
viruses are abundant and lepidopterans appear particularly prone to viral dis-
eases among holometabolic species (Garbutt, 2011). More recently, Swevers
et al. (2013) expanded on this hypothesis to discuss the possible role of per-
sistent virus infections on RNAi efficiency, noting that viruses could inter-
fere with RNAi in insects via number of strategies including expression of
RNAi suppressor genes, production of RNA decoys, and manipulation of
host gene expression. One attractive feature of this hypothesis is that it might
explain why different research groups report such disparate results working
with lepidopteran species (Terenius et al., 2011).
276 James A. Baum and James K. Roberts

5. COMMERCIAL DEVELOPMENT OF RNAi ACTIVES

5.1. Next-generation rootworm-resistant corn
The exceptional sensitivity of corn rootworm larvae to environmental
RNAi and the economic importance of this pest complex for corn produc-
tion in the United States provided an excellent opportunity to develop a
biotechnology-based strategy for corn rootworm management. Replicated
field trials with transgenic corn plants expressing a corn rootworm
V-ATPase dsRNA showed significant root protection (Fig. 5.2) similar to
the pot assay results reported by Baum et al (2007).
While these plants showed significant reduction in root damage due to
corn rootworm feeding, the damage observed was still above the commer-
cially acceptable threshold of 0.25 for root damage ratings. Genetic crosses
between the V-ATPase dsRNA-expressing transgenic event and a corn line
expressing the corn rootworm-active Bt protein Cry3Bb1 served to com-
bine these traits. Corn hybrids expressing both traits gave better root protec-
tion than did hybrids expressing either Cry3Bb1 or dsRNA alone (Fig. 5.2).
The benefit of the RNAi-based trait became even more apparent when
adult emergence was assessed. Corn plants expressing the V-ATPase dsRNA

Figure 5.2 Transgenic plants expressing both the Cry3Bb1 protein and corn rootworm
(CRW)-specific dsRNAs show excellent protection from rootworm feeding damage. Root
damage ratings (03 Iowa scale) are averaged across nine locations from four states (IA,
IL, IN, NE). Standard error bars are shown.
RNAi-Mediated Insect Pest Management 277

exhibited superior suppression of adult corn rootworm emergence in

growth chamber assays (Fig. 5.3).
The identification of numerous efficacious targets through insect feeding
assays (Baum et al., 2007) enabled a rational approach to the disruption of
different cellular pathways including, in the case of the Snf7 ortholog
(Bolognesi et al., 2012), pathways involved in protein trafficking.
Ramaseshadri et al. (2013) used histology and immuno-staining to show that
knockdown of Snf7 gene expression in corn rootworm larvae leads to cel-
lular perturbations that are consistent with a gene encoding an essential com-
ponent of the ESCORT (Endosomal Sorting Complex Required for
Transport) pathway essential for protein trafficking within cells. Further
electron microscopy studies (Koci et al., 2014) with WCR midguts have
confirmed that loss of Snf7 function causes a progressive degeneration of
midgut enterocytes resulting in cell sloughing and lysis. These authors con-
clude that the severe cellular defects caused by loss of Snf7 in midgut
enterocytes is the cause of death for rootworm larvae fed Snf7 dsRNA. This
RNAi-mediated disruption of cellular function represents a very different
cause of insect mortality than that attributed to Bt insecticidal proteins, most
of which bind to specific midgut receptors and create ion channels and pores
to disrupt midgut function in susceptible species (Vachon et al., 2012;
Chapter 2). Accordingly, a single transgenic corn line expressing both a corn
rootworm-active Bt protein and an efficacious dsRNA (e.g. the Snf7
dsRNA) can possess two very different modes of action for the control of

40%
% Beetle emergence

30%

20%

10%

0%
Negative Cry3Bb1 V-ATPase V-ATPase
control + Cry3Bb1
Figure 5.3 Transgenic plants expressing the CRW V-ATPase A dsRNA exhibit superior
control of CRW adult beetle emergence in growth chamber assays. Standard error bars
are shown.
278 James A. Baum and James K. Roberts

corn rootworm. Commercial introduction of the Snf7 RNAi trait in corn

hybrids is expected by the end of this decade (Kupferschmidt, 2013).

5.2. Topical application

Given the recalcitrant nature of most lepidopteran and hemipteran pests
towards environmental RNAi, the future of RNAi-based insect pest man-
agement strategies may depend on the development of topical formulations
that facilitate delivery into insect cells. Significant progress has been made in
the development of RNA delivery agents for RNAi human therapeutics
(Zhou et al., 2013), a field that has wrestled with the challenges of RNA
stability, endocytosis, and endosomal escape to deliver siRNA payloads.
Examples of delivery agents being explored in the pharmaceutical arena
are illustrated in Fig. 5.4.
These include cationic lipids, cationic dendrimers, cyclodextrin poly-
mers, various forms of polethyleneimine, mesoporous silica, cell-
internalizing peptides, and nucleic acid aptamers. Transfection agents have
been shown to aid in the delivery of dsRNAs to a recalcitrant insect species,

Figure 5.4 The mechanism and delivery strategies for RNA interference. RNAi therapeu-
tics (e.g. siRNA) can be internalized into cells via different delivery vehicles.
Figure reproduced with permission from Zhou et al. (2013).
RNAi-Mediated Insect Pest Management 279

in this case Drosophila (Whyard et al., 2009). Zhang et al. (2010) demon-
strated that chitosandsRNA complexes were stabilized in gel-based diets
for delivery to mosquito larvae, but the actual impact of chitosan on dsRNA
delivery to or uptake by mosquito gut cells was not evaluated in this study.
More recently, He et al. (2013) reported that fluorescent nanoparticles
(FNPs) comprised of a perylene-3,4,9,10-tetracarboxydiimide chromo-
phore core, a polyphenylene dendrimer inner shell, and a cationic polymer
outer shell can serve as a delivery vehicle for both DNA and dsRNA. FNPs
coated with a dsRNA targeting the chitinase-like CHT10 gene were fed at a
dietary concentration of 250 ppm to the lepidopteran species, O. furnacalis,
and shown to be more effective than dsRNA alone in arresting larval
development.
A formulation of chemically modified AChE2 siRNA complexed with
chitosan was shown to be toxic to DBM larvae in leaf disc assays and in a field
trial (Gong et al., 2013). Larval mortality (%) in the field appeared to peak at
5 days post-treatment with the siRNA formulation (50, 100, and 200 ppm
treatments), perhaps reflecting foliar degradation of the siRNA active or
alternatively an increase in larval infestation leading to a higher recovery
of survivors. The high sensitivity of CPB larvae to ingested dsRNAs pro-
vides an opportunity to explore the development of dsRNA formulations
for use as foliar sprays in insect control. Zhu et al. (2011) evaluated the
use of E. coli-expressed dsRNA to control CPB, demonstrating consistent
target gene knockdown and high mortality of CPB larvae when fed potato
leaves treated with either bacterial-expressed dsRNA or in vitro-
synthesized dsRNA.
The cost of goods weighs heavily on the development of RNAi spray
formulations, including the cost of dsRNA production as well as any req-
uisite delivery agents. The first outdoor trial with an RNAi product was
conducted by Hunter et al. (2010) to assess the efficacy of a dsRNA product
(Remebee-IAPV) in reducing Israeli acute paralytic disease in honey bees,
one of many viral diseases that afflict honey bee colonies worldwide. In this
case, dsRNA was provided to honey bees in a sugar solution with the aim of
blocking virus replication and transmission. Formulations that improve
RNA delivery and insecticidal activity while enhancing environmental sta-
bility could enable a more favourable cost of goods (through lower use rates)
and the development of economically viable topical applications for insect
control. Factors that likely impact the environmental stability of dsRNA are
not so different from those impacting the stability of synthetic insecticides
and include UV damage from exposure to sunlight, microbial degradation
280 James A. Baum and James K. Roberts

(e.g. on foliar surfaces), and exposure to plant exudates. It is uncertain how

formulations that improve the environmental stability of dsRNAs will affect
bioavailability and efficacy. Likewise, it is too early in the development of
formulation technologies to determine whether approaches taken in the
pharmaceutical field can be applied to the development of effective crop
protection products.

6. SAFETY CONSIDERATIONS
Safety assessments for the use of RNAi strategies in insect pest man-
agement include both the evaluation of environmental safety and food and
feed safety. The impact of dsRNAs on non-target organisms, including
beneficial insects, and the persistence of applied dsRNAs in the environ-
ment, are also addressed during the registration of Bt-based bioinsecticides
and insect-protected crops. The current safety assessment approach to
biotechnology-derived plants is appropriate for assessing the safety of plants
produced using RNA-mediated gene regulation (Codex, 2003; EFSA,
2006; FDA, 1994; Parrott et al., 2010; Petrick et al., 2013; Redenbaugh
et al., 1992), and the current environmental risk assessment approach can
be applied to characterize risk for RNA-based technologies (Auer and
Frederick, 2009; ILSI-CERA, 2011).
RNAi technology has the potential for high specificity towards target
pest organisms, even more so than Bt-based technology (Bachman et al.,
2013; Baum et al., 2007; Whyard et al., 2009). Larval feeding assays with
orthologous CPB- and WCR dsRNAs demonstrated that the accumulation
of base pair mismatches between the dsRNA sequence and target gene
sequence results in reduced insecticidal activity (Baum et al., 2007). For
example, a V-ATPase A dsRNA from WCR sharing 83% sequence identity
with the CPB V-ATPase A coding region showed >10-fold lower activity
against CPB larvae than did the orthologous CPB V-ATPase A dsRNA.
Note that this residual activity towards CPB was expected because the
WCR dsRNA sequence still retained stretches of >21 bp sequence identity
with the CPB V-ATPase coding region, a result that is entirely consistent
with the work of Bolognesi et al. (2012) and Bachman et al. (2013). The
case for sequence specificity is further supported by the work of Whyard
et al. (2009), who demonstrated that four Drosophila species could be selec-
tively controlled using dsRNAs that target the divergent 30 untranslated
region of the Tub23C tubulin gene, a region lacking 21 bp matches among
the four species. Other factors, including the barriers to RNAi described in
RNAi-Mediated Insect Pest Management 281

this review, clearly play a role in determining sensitivity to dsRNA and

assessing risk to non-target organisms. The aggregate of these factors pro-
vides outcomes such as those reported by Bachman et al. (2013) showing
that a Snf7 dsRNA from WCR has no effect when tested on a wide range
of insect species. Indirect WCR feeding assays with Snf7 orthologs from
other coleopteran species further demonstrated that the spectrum of activity
for the WCR Snf7 dsRNA is narrow and only evident in a subset of beetles
within the Galerucinae subfamily of Chrysomelidae (>90% identity with
the WCR Snf7 240 bp dsRNA) that contain a shared sequence length of
21 bp within the Snf7 coding sequence (Bachman et al., 2013). In sum-
mary, the careful selection of dsRNA sequences with no 21 bp match to
orthologous genes further reduces the risk of unintended effects on non-
target species that may be sensitive to ingested dsRNAs. With respect to
RNAi-based strategies for corn rootworm control, however, any assessment
of risk towards non-target insects or arachnids should start with the
acknowledgement that non-coleopteran species will likely be at least three
orders of magnitude less sensitive to environmental RNAi than the target
pest itself (Table 5.1).
Regarding food safety, the natural occurrence of long dsRNAs and small
RNAs in plants and other foods provides a very long history of safe use by
humans (Heisel et al., 2008; Ivashuta et al., 2009; Jensen et al., 2013; Parrott
et al., 2010; Petrick et al., 2013), even though sequence complementarity
exists between small- and long dsRNAs in crops and human genes
(Ivashuta et al., 2009; Jensen et al., 2013). The long list of biological barriers
to oral activity of dietary small RNAs and longer dsRNAs in mammals and
other vertebrates has been summarized (Petrick et al., 2013). Vertebrate
digestive systems display common structural and functional features
(Stevens and Hume, 2004); therefore, the same digestive barriers that greatly
limit the potential for oral activity of ingested RNA in mammals are likely to
be present in lower vertebrates. These barriers include salivary RNAses,
harsh acidic conditions in the stomach that lead to depurination and dena-
turation of nucleic acids, nucleases in the lumen of the gastrointestinal tract,
lytic enzymes from pancreatic secretions in the duodenum and nucleases in
the blood (Houck and Berman, 1958; Loretz et al., 2006; ONeill et al.,
2011; Park et al., 2006; Petrick et al., 2013). Cellular membranes of the
gut epithelium provide a physical barrier to uptake of high molecular
weight, hydrophilic compounds like siRNAs (Akhtar, 2009; Jain, 2008).
Recent studies employing small RNA sequencing and/or quantitative
PCR support the view that dietary dsRNAs have extremely low
282 James A. Baum and James K. Roberts

bioavailability in mammals, at a level that is orders of magnitude below that

needed for biological activity (Cottrill and Chan, 2014; Dickinson et al.,
2013; Snow et al., 2013; Witwer and Hirschi, 2014; Witwer et al.,
2013). Likewise, a recently reported survey of numerous animal small
RNA datasets from public sources has not revealed evidence for any major
plant-derived miRNA accumulation in animal samples (Zhang et al.,
2012b), consistent with the lack of success in oral delivery of RNA-based
therapeutics experienced by the pharmaceutical industry (Petrick et al.,
2013). Current pharma approaches employ local and systemic delivery of
RNA therapeutics through methods that bypass the oral route (Zhou
et al., 2013). Based on the history of safe consumption of dsRNA in the diet,
the extensive barriers to ingested RNA, and the lack of any appreciable
dsRNA uptake from the diet, dietary dsRNA is not anticipated to represent
any hazard or risk to humans, mammals, or other vertebrates.

7. INSECT RESISTANCE MANAGEMENT

In order to reduce the intensity of selection for resistance, insecticides
can be applied judiciously when established economic thresholds of pest
infestation have been crossed, tank mixed with insecticides providing a dif-
ferent MOA, or rotated with those insecticides during the growing season to
alter the selective pressure on insect populations. The strategy of tank-
mixing insecticides is fundamentally the same as stacking or pyramiding
insect control traits with different MOAs in plants. RNAi-based insecticides
would fit well with IPM strategies that rely on the use of multiple control
strategies, including synthetic and biological insecticides, and that leverage
host plant resistance traits and natural predators to provide economic control
of insect pests. The selectivity and slow knockdown anticipated for RNAi
insecticidal actives might encourage their use in the context of an IPM
framework, perhaps as early-season applications to suppress target pest
populations while allowing beneficial insects and predators to thrive.
For transgenic delivery of RNAi-based insect control traits, the path
towards resistance management is well worn. Strategies were modelled
and deployed in the United States and elsewhere to delay the emergence
of insect resistance to insect-protected crops expressing Bt insecticidal pro-
teins (Gassmann et al., 2009; Huang et al., 2011; Tabashnik, 2008). The high
dose/refuge strategy for insect-protected crops relies on the out-crossing of
rare resistant individuals with sensitive individuals in the non-transgenic ref-
uge, but is only effective if the resistance phenotype is recessive and the dose
RNAi-Mediated Insect Pest Management 283

sufficiently high to kill heterozygous progeny. In recent years, the stacking

of insect resistance traits deploying different MOAs (Roush, 1998) to delay
resistance development has become the standard for new commercial trans-
genic plant offerings. Examples include BollGard II cotton expressing the
Cry1Ac and Cry2Ab proteins for lepidopteran control, Genuity VT Dou-
ble PRO corn expressing the Cry1A.105 and Cry2Ab proteins for lepi-
dopteran control, and Genuity SmartStax corn expressing the Cry3Bb1
and Cry34AbCry35Ab proteins for corn rootworm control, and the
Cry1A.105, Cry1Fa, and Cry2Ab proteins for broad-spectrum lepidopteran
control (www.monsanto.com).
To ensure their durability, RNAi-based insect resistance traits should
also be stacked with traits employing a different MOA. This is where the
work in C. elegans provides additional value: the isolation of multiple
mutants impaired in environmental and/or systemic RNAi in the nematode
provides a cautionary tale for those of us in the field of insect pest manage-
ment. The risk is not that target genes will be selected for single nucleotide
polymorphism variants that evade RNAi processing (large dsRNAs can pre-
sumably mitigate this risk; Bachman et al., 2013; Bolognesi et al., 2012) but
that, for instance, up-regulation of nucleases or defects in dsRNA uptake,
processing, or systemic spread will compromise the triggering and spread
of the RNAi response in the pest organism. The observation that most spe-
cies of Caenorhabditis are not sensitive to environmental RNAi (Nuez and
Felix, 2012; Whangbo and Hunter, 2008) and that several grassland nema-
tode species are not sensitive to RNAi altogether (Wheeler et al., 2012) fur-
ther suggests that this capability is under rapid evolution in nematodes. In the
case of next-generation rootworm-resistant corn, the stacking of the Snf7
RNAi trait with Bt genes encoding two rootworm insecticidal toxins with
distinct MOAs could provide strong protection for all three traits. Likewise,
one can envision the stacking of RNAi traits for lepidopteran control with
any number of Bt genes encoding highly efficacious lepidopteran-active
proteins. In contrast, the deployment of RNAi traits for hemipteran control
is problematic because there are no viable biotech traits that confer effective
protection from hemipteran feeding damage and consequently no traits to
stack with. Progress has been made in the development of engineered Bt
proteins for control of Lygus species in cotton (Baum et al., 2013) but, in
general, Bt proteins tend to be ineffective against hemipteran pests. Trans-
genic plants expressing lectins have been shown to impact the growth,
development, and fecundity of hemipteran pests, but regulatory approval
of such traits is not assured given their apparent MOA in binding complex
284 James A. Baum and James K. Roberts

glycans that are also found along the intestinal tract of mammalian herbivores
(Vandenborre et al., 2009). Without complementary traits for resistance
management, the commercialization of durable RNAi-based hemipteran
control traits may prove elusive.

8. CONCLUDING REMARKS
The mechanism of gene suppression manifested as RNAi provides an
MOA unique among insecticidal agents. In addition, the potential for highly
tailored insecticidal specificity afforded in part by the mechanism of RNAi
differentiates it from other insect pest management strategies. Successful
development of RNAi actives, either expressed in plants or applied topically
as biological insecticide sprays, would provide growers with an important
tool for sustainable insect pest management.
Despite the exceptional sensitivity of corn rootworm larvae to ingested
V-ATPase- or Snf7 dsRNAs, transgenic corn plants expressing these
dsRNAs do not provide complete protection from rootworm feeding dam-
age in the field, presumably because of the slow speed-to-kill typical of
dsRNA actives. Transgenic corn plants combining the Snf7 RNAi trait with
the Cry3Bb1 gene, however, do provide superior control of corn rootworm
larvae and emerging adults in the field. Accordingly, stacking the Snf7 RNAi
trait with suitable Bt traits in corn offers the best opportunity for efficacious
and durable control of corn rootworm species. Looking towards the future,
it seems likely that the slow speed-to-kill exhibited by insecticidal dsRNAs
will affect how these agents are deployed commercially.
It is now firmly established that insect species differ greatly in their
response to environmental RNA. Most notably, the lepidopteran and
hemipteran species studied to date are far less sensitive to ingested dsRNAs
than are the responsive coleopteran species exemplified by the corn
rootworm. This differential sensitivity to environmental RNAi helps inform
the environmental safety assessment of RNAi for management of coleop-
teran pests. It will also dictate the approach taken to exploit the phenomenon
of RNAi for insect pest management. In order to achieve parity with the
WCR Snf7 dsRNA active, for example, dsRNAs for lepidopteran- or
hemipteran pests will need at least a 1000-fold improvement in oral activity.
Given the recalcitrant nature of most lepidopteran and hemipteran species to
environmental RNAi and the minimal impact target gene selection will
likely have on closing this efficacy gap, a clear path towards development
of RNAi-based plant traits for lepidopteran- and hemipteran pest
RNAi-Mediated Insect Pest Management 285

management is not yet apparent. For example, while transgenic plants

expressing dsRNAs have been shown to impact the growth and survival
of a few lepidopteran or hemipteran species in controlled environment tests,
the effects are largely sub-lethal and have not been confirmed with field effi-
cacy data demonstrating economic control of the pest species. Given this
apparent ineffective dose activity towards recalcitrant species, it would
be difficult to justify trait commercialization in light of the significant devel-
opment and regulatory costs involved (McDougall, 2011; Prado et al.,
2014). In the case of hemipteran species, the general lack of complementary
(e.g. Bt) traits for use in insect resistance management strategies provides an
additional hurdle towards commercial development of transgenic crops
employing an RNAi-based trait.
For these reasons, the development of topically applied formulations that
facilitate dsRNA delivery into insect cells provides a sensible alternative to
the transgenic plant approach and may enable RNAi-based insect pest man-
agement for otherwise recalcitrant species. The success of this strategy
depends on a detailed understanding of the barriers to efficient environmen-
tal RNAi, the development of cost-effective, stable formulations that over-
come those barriers, and efficient robust systems for dsRNA production. In
some insect species, the absence of an efficient systemic RNAi mechanism
may mute any efficiency gained through the use of RNA delivery agents that
enable environmental RNAi. Despite these uncertainties, initial studies with
formulated dsRNAs suggest this is a promising approach.

ACKNOWLEDGEMENTS
The authors wish to thank Greg Heck, Sergey Ivashuta, Steve Levine, Bill Moar, Jay Petrick,
Gerrit Segers, and Greg Watson for their thorough review of the manuscript.

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 CHAPTER SIX

Detection and Mechanisms

of Resistance Evolved in Insects
to Cry Toxins from Bacillus
thuringiensis
Yidong Wu
*College of Plant Protection, Nanjing Agricultural University, Nanjing, China

Contents
1. Introduction 298
2. Detection Methods and Current Status of Insect Resistance to Bt Crops 299
2.1 Definition of resistance 299
2.2 Resistance detection methods 301
2.3 Current status of field-evolved resistance to Bt crops 312
3. Resistance Mechanisms 314
3.1 Mode of action of Bt Cry toxins 314
3.2 Alterations in proteolytic processing of Cry toxins in resistant insects 318
3.3 Modifications of Cry toxin receptors in resistant insects 319
4. Genetic Diversity of Resistance and Implications for Resistance Management 324
4.1 Laboratory-selected and field-evolved resistance 324
4.2 Resistance dominance and the refuge strategy 328
4.3 Cross-resistance and the pyramid strategy 329
5. Conclusions 331
Acknowledgements 332
References 332

Abstract
Transgenic crops producing Bacillus thuringiensis Cry toxins (Bt crops) have been planted
globally to control some key pests. Benefited from implementation of proactive resis-
tance management strategies such as the refuge strategy and the pyramid strategy in
many countries, most of the target pests of Bt crops have been sustainably and effec-
tively controlled for nearly 20 years. However, several cases of field-evolved resistance to
Bt corn and Bt cotton have been documented, causing reduced field efficacy. Evolution
of resistance by target pests is a real threat to the continued success of Bt crops. It is
crucial to employ sensitive detection methods to monitor the evolution of the resis-
tance in the target insects and thereby adapt resistance management strategies

Advances in Insect Physiology, Volume 47 # 2014 Elsevier Ltd 297

ISSN 0065-2806 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800197-4.00006-3
298 Yidong Wu

proactively to delay resistance evolution. Recent progress and considerations were

reviewed on the four resistance detection approaches: concentration-response assay,
F1 screen, F2 screen and DNA screen. Diverse genetic options for target pests to cope
with Bt crops are challenging the efforts to understand mechanisms of resistance and to
design rational resistance management strategies. Understanding the molecular
genetic basis of Bt resistance is essential for developing sensitive resistance detection
methods and intelligent resistance management strategies. Clarifying resistance mech-
anisms have facilitated and advanced our understanding on modes of action of Bt Cry
toxins. The most recent advances on resistance mechanisms to Bt toxins are summa-
rized in both laboratory-selected strains and field-evolved populations. According to
current knowledge of Bt resistance mechanisms, some implications for resistance man-
agement and directions for future research are suggested.

1. INTRODUCTION
Bacillus thuringiensis (Bt) is a gram-positive bacterium that is character-
ized by producing parasporal crystal proteins with insecticidal activity (Cry
toxins) during sporulation (Knowles, 1994; Schnepf et al., 1998). Bt sprays
have been widely used as a bioinsecticide in agriculture, forestry and mos-
quito control for several decades (Sanahuja et al., 2011). Transgenic crops
expressing genes encoding Bt toxins (Bt crops) were commercialized for
the first time in 1996 in the United States; since then, the cumulative total
of more than 570 million ha of Bt crops (mainly corn and cotton) has been
adopted globally ( James, 2013; Tabashnik et al., 2013). Intensive planting of
Bt crops inevitably creates strong selection pressure on the target insect pests,
thereby the resistance to these Bt crops evolved by target insect pests is con-
sidered a major threat to the durability of Bt crops (Carriere et al., 2010;
Gould, 1998; Huang et al., 2011; Tabashnik, 1994).
As a proactive effort to preserve the value and benefits of Bt crops, the
refuge strategy has been widely used to delay development of insect resis-
tance to Bt crops (Tabashnik et al., 2004). In theory, three key conditions
should be met in order to maximize the effectiveness of the refuge strategy:
recessive inheritance of resistance phenotype, low frequency of initial resis-
tance alleles and abundant susceptible insects provided by non-Bt host plants
nearby (Tabashnik et al., 2013). In practice, target insects have variable and
unpredictable inheritance patterns of the resistance trait and the initial fre-
quencies of resistance alleles within geographical populations. So, it is crucial
to employ sensitive detection methods to monitor the dynamic evolution of
the resistance alleles over space and time for the target insects and thereby
Insect Resistance to Bt Cry Toxins 299

adapt resistance management strategies proactively to delay resistance evo-

lution for a given pest speciesBt crop combination. This review summarizes
recent progress and considerations on different resistance detection
approaches.
The genetic capacity of insect populations to evolve resistance to Bt toxins
has been well demonstrated in many species within several insect orders.
Numerous Bt-resistant strains have been developed by laboratory selection
(Bauer, 1995; Ferre and Van Rie, 2002; Ferre et al., 2008). Other insects such
as diamondback moth, Plutella xylostella, and cabbage looper, Trichoplusia ni,
have evolved resistance to Bt sprays in the open field and green houses, respec-
tively ( Janmaat and Myers, 2003; Tabashnik et al., 1990). Recently, field-
evolved resistance to Bt corn in three pests (maize stem borer, Busseola fusca;
western corn rootworm, Diabrotica virgifera virgifera; and fall armyworm,
Spodoptera frugiperda) and to Bt cotton in two pests (American bollworm, Hel-
icoverpa zea; pink bollworm, Pectinophora gossypiella) has been documented and
reported to cause reduced field control efficacy (Tabashnik et al., 2013, 2014).
Therefore, understanding the molecular genetic basis of Bt resistance is essen-
tial for developing sensitive resistance detection methods and intelligent resis-
tance management strategies (Ferre and Van Rie, 2002; Heckel, 1994, 2012;
Heckel et al., 2007). This review will also focus to describe the most recent
advances on resistance mechanisms to Bt toxins in both laboratory-selected
strains and field-evolved populations. Given current knowledge of Bt resis-
tance mechanisms, some implications for resistance management and direc-
tions for future research are suggested.

2. DETECTION METHODS AND CURRENT STATUS

OF INSECT RESISTANCE TO Bt CROPS
2.1. Definition of resistance
The WHO Expert Committee on Insecticides defined insecticide resistance
as the development of an ability in a strain of insects to tolerate doses of
toxicants which would proved to be lethal to the majority of individuals
of the normal population from the same species (WHO, 1957). This def-
inition was the established operational definition for years and has been
adopted widely. However, this definition is considered not complete
because it is not applicable to individual insects, and it is difficult to define
an unselected or susceptible normal population (Sawicki, 1987). Crow
(1960) proposed a more general definition as Insecticide resistance marks
a genetic change in response to selection. Crows definition is applicable
300 Yidong Wu

to both single insects and whole populations, and the severity of resistance
can range from no effect on field control to field failure of an insecticide
application. Brent (1986) defined resistance as any heritable decrease in sen-
sitivity to a chemical within a pest population. Brent (1986) emphasized
that insecticide resistance can cause complete loss of action of an agrochem-
ical or may have little practical significance. Sawicki (1987) modified Crows
definition to Insecticide resistance marks a genetic change in response to
selection by toxicants that may impair control in the field. In the definitions
mentioned above, both a change in susceptibility to the insecticide detect-
able with bioassays in the laboratory and field control failure are considered
resistance. In other words, resistance to an insecticide need not result in loss
of insect pest control.
Tabashnik et al. (2009a, 2014) defined the term field-evolved
resistance as a genetically based decrease in susceptibility of a population
to a pesticide caused by exposure to the pesticide in the field. The term
field-evolved resistance applies to resistance in both pest and beneficial
organisms and does not necessarily imply loss of economic efficacy in the
field (Tabashnik et al., 2009a). This general definition favours proactive
detection and management of resistance.
Another definition of resistance proposed by the Insecticide Resistance
Action Committee (IRAC) is a heritable change in the sensitivity of a pest
population that is reflected in the repeated failure of a product to achieve the
expected level of control when used according to the label recommendation
for that pest species (http://www.irac-online.org/about/resistance). This
definition emphasizes a causal relationship between resistance and field con-
trol failure. Agrochemical industry intends to use this definition to validate
and confirm resistance as the cause of observed losses in field efficacy.
There are debates on the term, field-evolved resistance, in defining
insect resistance to Bt crops. A general definition of field-evolved
resistance considers that the primary goal of monitoring resistance to Bt
crops is to detect evolution of resistance early enough to enable proactive
management of resistant insects, and field control problems associated with
field-evolved resistance vary from none to severe (Tabashnik et al., 2008,
2013). A narrow definition of field-evolved resistance regards decreased
field efficacy and/or survival on Bt plants as the decisive criteria for defining
a case of field-evolved resistance (Moar et al., 2008; Sumerford et al., 2013).
To avoid this confusion, Tabashnik et al. (2014) defined practical
resistance as field-evolved resistance that reduces the efficacy of a pesticide
and has practical consequences for pest control.
Insect Resistance to Bt Cry Toxins 301

2.2. Resistance detection methods

The major objectives of resistance detection and monitoring should include
(1) determining baseline susceptibility and initial resistance allele frequency
of target pests to pesticides; (2) detecting changes in resistance frequency to
gain early warning of a resistance problem; (3) determining the effectiveness
of the implemented resistance management strategies; and (4) documenting
losses of insect control in the field associated with resistance (Brent, 1986;
Caprio and Sumerford, 2007). The primary objective of resistance monitor-
ing to Bt crops is to detect field-evolved resistance early enough to enable
proactive management before field failure occurs (Tabashnik et al.,
2009a, 2013).
Resistance detection means identifying a significant change in the sus-
ceptibility of a pest population to pesticides. Effective resistance detection
techniques are essential for useful early warning systems and in defining
the extent and severity of resistance. This information is also critical for
evaluating resistance management strategies. There are a number of methods
developed for detection of Bt resistance in field insect populations (Caprio
and Sumerford, 2007; Huang, 2006). The four methods reviewed here for
Bt resistance detection are divided into two types: (1) the concentration
response and diagnostic concentration assays used for determining resistance
intensity and the frequency of resistant individuals and (2) F1 screens, F2
screens and DNA screens used for detecting resistance allele frequency.
These two types of detection approaches are complementary and mutually
confirmatory, and the combination of these two types of detection methods
has proved to be especially effective for Bt resistance monitoring in Hel-
icoverpa armigera from China (Zhang et al., 2011, 2012a, 2013).

2.2.1 Concentrationresponse and diagnostic concentration assays

Concentrationresponse assays are commonly used to estimate the toxin
concentration that kills 50% of a pest population (LC50). Resistance ratios
(RRs) are then measured by comparing the LC50 values obtained with insect
strains derived from populations exposed to the toxin against those of sus-
ceptible laboratory strains (Tabashnik et al., 2008, 2009a). An RR greater
than 10 is more likely to indicate heritable decrease in susceptibility and
higher RRs provide stronger evidence of resistance (Tabashnik, 1994,
Tabashnik et al., 2009a).
The frequency of resistant individuals can be measured as the proportion
of a field population surviving at an appropriate diagnostic concentration
302 Yidong Wu

which kills all or nearly all individuals of the susceptible population, but few
or no resistant individuals. Resistance allele frequency can be readily esti-
mated if the genetic basis of resistance is known (Tabashnik et al., 2009a;
Zhang et al., 2012a). Compared with the concentrationresponse assay,
the diagnostic concentration test is more efficient for detecting an incipient
resistance outbreak (Roush and Miller, 1986).
When designing and implementing an efficient detection programme for
Bt resistance based on the concentrationresponse and/or diagnostic con-
centration assays, several key factors should be considered:
(1) Establishment of baseline susceptibility data and the calibration of a diag-
nostic concentration. It is important to survey the baseline susceptibility
of target pest populations sampled from geographical areas prior to wide
commercial adoption of a Bt crop. The baseline data can reveal the range
of geographical variations of unexposed field populations which will then
be necessary for defining susceptibility changes relating to exposure to Bt
crops. Similarly, it is necessary to calibrate the diagnostic concentrations
against a range both of unexposed field populations and of susceptible lab-
oratory strains (Forrester et al., 1993; Khakame et al., 2013; Wang et al.,
2010). Compared with the appropriate baseline comparators considering
natural susceptibility variations, a statistically significant increase in sur-
vival at a diagnostic concentration and/or in LC50 values or LC90 will
provide initial evidence of resistance evolution in the field.
(2) Maintaining a susceptible laboratory strain for comparison across years and
regions. For valid comparison with the susceptibility of field populations, it
is necessary to choose a reference strain to be kept in the laboratory, which
should be neither extremely susceptible nor unusually tolerant to the Bt
toxin concerned. Concurrent comparative susceptibility data for a specific
Bt toxin from the susceptible strain are essential for comparison with field
population susceptibility across years and regions.
(3) Sampling and testing. The scale of sampling depends on the aims and
types of resistance detection as well as on the mobility and population
structure of the target pest. Each collection needs to be representative of
the local field population. It is suggested that hundreds of insects should
be collected from many locations instead of thousands of insects from a
few locations (ffrench-Constant and Roush, 1990). Considering costs
of time and labour, it is often best that an extensive survey be done first,
followed by a more intensive monitoring of selected locations, which
have intensive adoption of Bt crops and possibly a higher risk of resis-
tance evolution.
Insect Resistance to Bt Cry Toxins 303

Comprehensive resistance monitoring requires evaluation of insect

field populations on Bt crops as well as from other sources including
non-Bt plants. Sampling and testing of target pest insects surviving
on or near Bt crops is essential for early detection of field-evolved resis-
tance (Tabashnik et al., 2008, 2013).
Field-collected insects are pooled in large groups for mating in the
laboratory to generate field-derived strains for bioassays which are nor-
mally carried out on the damaging life stages, usually larval or nymphal.
Both diet incorporation and diet-overlay methods are commonly used
to determine responses of the target pest to a series of concentrations
and/or a diagnostic concentration of Bt toxin. Although the diet incor-
poration method is closer to exposure of target pests to Bt plants, diet-
overlay assays are simpler and require less Bt toxin (Sivasupramaniam
et al., 2007). In addition, precautions should be taken to avoid high tem-
peratures that would degrade the Bt toxin and give misleading results.
(4) Bt protein source and forms. Provision and tracking of a reliable Bt pro-
tein source is extremely important in interpreting data across laborato-
ries and making valid comparison over time (Sivasupramaniam et al.,
2007). In the United States and some other countries, Bt proteins
required for resistance monitoring programmes are mainly supplied
by industry. Centralized supplies of Bt proteins can make sure reliable
and consistent quality throughout monitoring programmes. It is always
important to use a tester strain (a susceptible laboratory strain) of a target
pest to check the efficacy of new protein sources, or new batches from
the same source. Mortality in the laboratory susceptible strain should
remain constant at a given toxin dose, and conversion factors should
be used when comparing results using proteins of different toxicity.
The toxin form to be used for a monitoring programme needs to be
as similar as possible to the form ingested by the target pest when feeding
on a Bt crop. Adoption of the most appropriate form of a Bt protein for
resistance selection in the laboratory or resistance monitoring in the
field has been an issue for debate (Anilkumar et al., 2008). A partially
activated form of Cry1Ac toxin (C-terminal removed, but
N-terminal retained) is expressed in transgenic cotton (Perlak et al.,
1990), but a formulation called MVPII has been widely used for mon-
itoring resistance of cotton pests to Cry1Ac (Sivasupramaniam et al.,
2007). MVPII contains a hybrid protoxin that is identical to Cry1Ac
in its active region and is encapsulated in Pseudomonas fluorescens
(Tabashnik et al., 2002). One resistant strain of cotton bollworm,
304 Yidong Wu

H. armigera, and one resistant strain of H. zea, both pests that are targeted
by Bt cotton, were ca. 10 times more resistant to Cry1Ac toxin than to
Cry1Ac protoxin. The SCD-r1 strain of H. armigera had ca. 500-fold
resistance to Cry1Ac toxin, but only 39-fold resistance to Cry1Ac
protoxin (Tabashnik et al., 2011; Xu et al., 2005). Similarly, the AR
strain of H. zea had more than 100-fold resistance to Cry1Ac toxin,
but only 10-fold to Cry1Ac protoxin (Anilkumar et al., 2008). For
Bt resistance monitoring of these two cotton pests, it could be better
to use Cry1Ac toxin for early detection of resistance.

2.2.2 F1 screen
The principles of complementation tests are as follows (Zhang et al., 2012a):
If two resistant strains are crossed, each with recessive alleles for resistance at
separate loci, allelic complementation will restore susceptibility (the wild-
type phenotype) in the progeny. However, if the recessive resistance alleles
occur at the same locus in different strains, the progeny from the cross
between strains will be resistant because they will inherit resistance alleles
at the same locus from both parents. Based on the principle of complemen-
tation tests, Gould et al. (1997) developed an F1 screen method to estimate
the frequency of Cry1Ac-resistance alleles in field populations of the tobacco
budworm, Heliothis virescens. Over 2000 single-pair families were made
among field-collected male moths and virgin female moths from the
laboratory-selected YHD2 strain of H. virescens, which has a high level of
Cry1Ac resistance conferred by a recessive gene (producing a truncated
cadherin). F1 offspring from each of 1025 families were screened with a dis-
criminating concentration of Cry1Ac that could distinguish heterozygous
from homozygous resistant individuals. Four of these families produced
3042% offspring with resistance to Cry1Ac, indicating that in each of
the four field-collected males was heterozygous and carried a recessive resis-
tance allele. By the F1 screen, the initial frequency of alleles for Cry1Ac resis-
tance was able to be estimated as 0.0015 in field populations of H. virescens
(Gould et al., 1997).
The F1 screen method has been used to determine initial Bt resistance
allele frequencies in field populations of several pests other than
H. virescens. A total of 286 single-pair crosses were made between field-
derived adults of the poplar leaf beetle, Chrysomela tremulae, and adults of
a laboratory strain with recessive resistance to Cry3Aa. F1 neonates from
each of the 176 single-pair families with enough F1 offspring were screened
with leaf discs from a Bt poplar line. Three of the 176 families screened
Insect Resistance to Bt Cry Toxins 305

produced resistant F1 larvae, and the estimated frequency of resistance allele

was 0.0113 (Wenes et al., 2006). In the sugarcane borer, Diatraea saccharalis
(F.), a total of 364 single-pair matings were made between field-collected
individuals with those from a laboratory strain with recessive resistance to
Cry1Ab. F1 neonates from each of 256 single-pair families with sufficient
F1 offspring were screened with Bt maize leaf tissue. One of the 256 families
screened produced resistant F1 larvae, and the estimated frequency of resis-
tance allele was 0.0028 for the combined five populations collected from
Louisiana and Texas of the United States (Yue et al., 2008).
The F1 screen method has been confirmed to be effective for detecting
early shifts in the resistance frequency of target pests after exposure to Bt
crops. F1 screens were conducted in Australia to monitor Cry2Ab resistance
allele frequencies in field populations of the Australian bollworm, Helicoverpa
punctigera, sampled from both Bt cotton (Bollgard II, pyramided with
Cry1Ac and Cry2Ab) cropping areas and non-cropping areas in Australia
(Downes et al., 2010a). Cry2Ab resistance allele frequency of populations
of H. punctigera from cropping areas increased from 0.015 in 2007/2008
to 0.048 in 2008/2009 cotton season. The Cry2Ab resistance allele fre-
quency for cropping populations collected in 2008/2009 is eightfold higher
than for non-cropping populations sampled in 2009 (Downes et al., 2010a).
Bt cotton expressing only Cry1Ac protein has been intensively planted in
northern China for more than a decade, but planting of this cotton has been
limited in northwestern China (Zhang et al. 2011). F1 screens conducted by
Zhang et al. (2012a) with 593 H. armigera males caught in the field during
20092010 show that the proportion of males in which resistance to Cry1Ac
was detected three times higher for northern China field populations (0.16)
than for a field population from northwestern China (0.057).
The F1 screen method is designed to detect rare recessive resistance
alleles in field-derived individuals that occur at the same locus as the resis-
tance alleles in the resistant laboratory strain. In theory, the F1 screen can also
detect non-recessive resistance alleles at any locus. Zhang et al. (2012a)
extended the F1 screen method for detection of both recessive and non-
recessive alleles from field populations of H. armigera. Figure 6.1 is a sche-
matic diagram of the F1 screen programme for resistance alleles in
H. armigera. With this approach, resistance alleles detected by F1 screen in
northern China were composed of 84% recessive cadherin alleles and
16% non-recessive alleles at either a cadherin locus or another locus. In con-
trast, for northwestern China, all of the resistance alleles were recessive
cadherin alleles (Zhang et al., 2012a).
306 Yidong Wu

Figure 6.1 F1 screen for resistance alleles in Helicoverpa armigera (Zhang et al., 2012a).
Field-caught male moths were crossed individually to homozygous female moths with a
recessive resistance allele at the cadherin locus (rcrc) from a laboratory strain of
H. armigera. F1 offspring from single-pair families were tested at a diagnostic concen-
tration of Cry1Ac. Expected survival of the F1 progeny in these bioassays depends on the
genotype of the field-caught male: 0% for a susceptible homozygote (ss), 50% for indi-
viduals with one susceptible allele and either any recessive resistance allele at the
cadherin locus (rc) or a dominant resistance allele at any locus (Rx) (genotypes rcs or
Rxs) and 100% for individuals with the genotypes rcrc or RxRx. To determine the domi-
nance of the resistance alleles detected with the F1 screen, survivors of the F1 screen are
crossed with a susceptible strain (ss) and the progeny tested at the diagnostic concen-
tration. Expected survival of the progeny of these F1 survivors
ss crosses is 0% if the
resistance allele is recessive (progeny are rcs) and 50% if the allele is dominant (half of
progeny are Rxs and half are ss). It should be noted that F1 screen does not detect reces-
sive alleles at loci other than the same resistance locus as in the tester laboratory strain.

The prerequisite for the success of an F1 screen is to possess a laboratory

strain which has recessive resistance conferred by a single locus. If the resis-
tance genes are well characterized, such as cadherin mutations in the three
cotton pests (H. virescens, P. gossypiella and H. armigera), the F1 screen can be
used to recover resistance alleles from field populations. With F1 screens and
molecular cloning, 15 cadherin alleles (r1r15) associated with Cry1Ac resis-
tance were discovered from field populations of H. armigera from China
(Yang et al., 2007; Zhang et al., 2012a,b; Zhao et al., 2010).
Insect Resistance to Bt Cry Toxins 307

2.2.3 F2 screen
Andow and Alstad (1998) developed the F2 screen technique to detect reces-
sive alleles in field populations. The principle of an F2 screen is that F1 off-
spring of each mated female collected from the field are sib-mated to
produce F2 iso-female families. Each mated female carries four haploid
genomes, two from her own and two from her mate. If the field-collected
female or her mate carries one recessive resistance allele (r), the F2 offspring
of the iso-female family will have approximately 6.25% survival (rr) when
screened with a diagnostic concentration of Bt toxin or with Bt plants. Com-
pared with the diagnostic concentration assay, the F2 screen extends the sen-
sitivity to detect recessive resistance traits by more than an order of
magnitude. Compared with the F1 screen, the F2 screen can detect resistance
alleles at any locus rather than just the same locus where resistance alleles
occur in the laboratory strains (Andow and Alstad, 1998).
Because of its robustness, the F2 screen has been employed to detect
resistance allele frequency to Bt toxins for at least eight lepidopteran pests:
European corn borer, Ostrinia nubilalis (Andow et al., 1998, 2000;
Bourguet et al., 2003; Siegfried et al., 2014; Stodola et al., 2006), rice yellow
stem borer, Scirpophaga incertulas (Bentur et al., 2000), southwestern corn
borer, Diatraea grandiosella (Huang et al., 2007a), Mediterranean corn borer,
Sesamia nonagrioides (Andreadis et al., 2007), sugarcane borer, D. saccharalis
(Huang et al., 2007b, 2008, 2009), H. armigera (Liu et al., 2010; Mahon
et al., 2007, 2012; Xu et al., 2009; Zhang et al., 2012a), H. punctigera
(Downes et al. 2009, 2010a; Mahon et al. 2012) and H. virescens (Blanco
et al., 2009) and one coleopteran pest: C. tremulae (Genissel et al., 2003).
Because the F2 screen involves intensive input of time and resources, it
has been used only to determine initial resistance allele frequency for most
monitoring plans. However, F2 screen has been practically used as the main-
stay of the routine monitoring of Bt resistance in two cotton pests,
H. armigera and H. punctigera in Australia (Downes and Mahon, 2012a,b).
From 2002/2003 until 2010/2011, F2 screens were used to test 1222 iso-
female lines (4888 alleles) of H. armigera and 1558 (6232 alleles) iso-female
lines of H. punctigera for Cry1Ac resistance. Two H. armigera and three
H. punctigera lines were positive for a resistance allele to Cry1Ac. Based
on data pooled since 2002/2003, the estimated Cry1Ac resistance allele
frequency for H. armigera is 0.0006 (95% CI between 0.0001 and 0.002)
and for H. punctigera is 0.0006 (95% CI between 0.0002 and 0.001).
During the same period, F2 screens were used to test 1303 iso-female
lines (5212 alleles) of H. armigera and 1642 iso-female lines (6566 alleles)
308 Yidong Wu

of H. punctigera for Cry2Ab resistance. Forty-seven H. armigera and

33 H. punctigera lines were positive for a resistance allele to Cry2Ab. Based
on data pooled since 2002/2003, the estimated Cry2Ab resistance allele fre-
quency for H. armigera is 0.009 (95% CI between 0.007 and 0.012) and for
H. punctigera is 0.005 (95% CI between 0.004 and 0.007) (Downes and
Mahon, 2012a,b). The F2 screen data clearly showed that there has been
a statistically significant increase in the frequency of Cry2Ab resistance alleles
in H. punctigera since the widespread adoption of Bollgard II in 2004/2005 in
Australia. The F2 screen therefore fulfilled the intended function of resis-
tance monitoring for providing an early warning of resistance evolution
(Downes et al., 2010a). In China, the F2 screen data of 363 field-derived
iso-female families of H. armigera showed that the frequency of Cry1Ac resis-
tance was three times higher for a field population from northern China
(0.052), where there is intensive Bt cotton planting than for field populations
from northwestern China (0.016), where there is limited Bt cotton planting
(Zhang et al., 2012a).
If the resistance of the isolated strain from an F2 screen is recessive, it can be
used to perform F1 screens. From the positive lines with a major resistance
allele detected by F2 screens, strains with recessive Bt resistance at a single locus
have been successfully isolated from C. tremulae, D. saccharalis, H. armigera and
H. punctigera. The R#60 strain of C. tremulae resistant to Cry3Aa, the BtRR
strain of D. saccharalis resistant to Cry1Ab, the SP15 strain of H. armigera resis-
tant to Cry2Ab and the Hp4-13 strain of H. punctigera resistant to Cry2Ab
were subsequently employed in performing F1 screens (Augustin et al.,
2004; Downes et al. 2010b; Huang et al., 2007b; Mahon et al., 2007).
If resistance is controlled by a single locus in field populations of a target
pest, F1 screens and F2 screens should detect similar frequencies of resistance
alleles. If there are different resistance genes at more than one locus, F2
screens are expected to detect higher resistance frequencies than F1 screens
because F1 screens detect only the type of resistance present in the resistant
tester strain. It is very interesting to note that Bt resistance frequencies are
generally higher when estimated by F1 screen than by F2 screen in several
pests (Table 6.1). For C. tremulae and D. saccharalis, the discrepancy in fre-
quencies identified using the two methodologies might be explained by the
fact that the field populations were sampled from different locations and in
different years. For H. armigera and H. punctigera from Australia, F1 screens
and F2 screens were conducted concurrently on same batches of field
populations, but resistance frequencies to Cry2Ab were 35.5 times higher
estimated by F1 screens than by F2 screens. Mahon et al. (2010) hypothesized
Table 6.1 Comparison of Bt resistance allele frequencies estimated from the F1 screens and F2 screens
Insect Frequency estimated by Frequency estimated
species Bt toxin Location Year F2 screen (95% CI) by F1 screen (95% CI) References
Chrysomela Cry3Aa La Chesnaye, France 19992001 0.0037 (0.000450.008) NA Genissel
tremulae et al. (2003)
Bar-le-Duc, France 2003 NA 0.0113 (0.00310.247) Wenes et al.
(2006)
Diatraea Cry1Ab Winnsboro, LA, USA 2004 0.0012 NA Huang et al.
saccharalis (2007b)
Cry1Ab Franklin, Tensas, East Carroll, 2006 NA 0.0030 (0.00040.0084) Yue et al.
and Rapides, LA, USA (2008)
Ostrinia Cry1F Saunders, NE, USA 20032005 NA 0.0286 Siegfried
nubilalis et al. (2014)
Cry1F Saunders, NE, USA 20062008 NA 0.0253 Siegfried
et al. (2014)
Cry1F Saunders, NE, USA 20082009 0.00930.0142 NA Siegfried
Story, IA, USA et al. (2014)
Helicoverpa Cry2Ab Cotton regions, Estern 20072008 0.006 (0.00020.013) 0.033 (0.0210.047) Mahon et al.
armigera Australia (2010)
Cry1Ac Anyang, Henan, China 2009 0.052 (0.0340.076) 0.091 (0.0660.123) Zhang et al.
(2012a)
Cry1Ac Shawan, Xinjiang, China 2010 0.019 (0.00840.041) 0.029 (0.0120.064) Zhang et al.
(2012a)
Helicoverpa Cry2Ab Cotton regions, Estern 20072008 0.005 (0.0020.010) 0.015 (0.0030.036) Downes
punctigera Australia et al. (2010a)
Cry2Ab Cotton regions, Estern 20082009 0.012 (0.0060.020) 0.048 (0.0330.065) Downes
Australia et al. (2010a)
310 Yidong Wu

that some resistance alleles are homozygous lethal if autozygous (as gen-
erated in F2 tests) but not as allozygous homozygotes (as generated in F1
tests). The hypothesis was extended to accommodate the possibility that
alleles at linked loci may be homozygous lethal. However, neither of two
tests of the hypothesis carried out to date provided evidence that any alleles
that confer resistance are associated with severe fitness costs (Mahon et al.,
2010), which does not support the hypothesis.
In China, diverse cadherin mutants associated with Cry1Ac resistance in
H. armigera were detected from F1 screens and F2 screens (Yang et al., 2007;
Zhang et al., 2012a; Zhao et al., 2010). A number of resistant strains with
autozygous genotypes of cadherin showed various resistance intensities to
Cry1Ac, ranging from 31- to 530-fold (Zhang et al., 2012a). Under a diag-
nostic concentration of Cry1Ac (1 g/cm2 diet surface), the AY148 strain
(r9r9) had 46% survival, the SCD-r1 strain (r1r1) had 92% survival and F1 off-
spring (r1r9) from AY148
SCD-r1 had 81% survival (Zhang et al., 2012a).
If one of the field parents of an iso-female line in an F2 screen carries a copy
of the r9 allele, the F2 offspring will have only 2.9% survival (6.25%
0.46)
under the diagnostic concentration, which will be scored negative. In con-
trast, if one of the field males carrying a copy of r9 allele (r9s) is crossed with a
female from SCD-r1 (r1r1) in an F1 screen, the F1 offspring (r1r9) will have
41% survival (81%
0.5) under the diagnostic concentration, which will be
scored positive. In F2 screens of Cry1Ac resistance alleles in two populations
of H. armigera from China, the parents of iso-female lines were scored as hav-
ing at least one resistance allele if F2 progeny survival was >3% instead of
>6.25% (Zhang et al., 2012a). Thus, Cry1Ac resistance allele frequencies
estimated by F1 screens were similar to that by F2 screens (Zhang et al.,
2012a). So, variability in resistance intensities among different autozygotes
(such as rara and rbrb) and/or allozygotes (rarb) might cause discrepancy in
results of F1 screens and F2 screens.

2.2.4 DNA screen

Bioassay-based screens (including concentration-response assays, F1 screens
and F2 screens) have been used as primary tools to detect and monitor Bt
resistance, but these approaches need huge manpower, material resources
and time for sampling insects from the field and rearing their progeny in lab-
oratory conditions. Compared with bioassay-based screens, DNA screens
can save labour and time, for it can detect resistance genotypes on individual
insects at any life stages. Further, DNA screens are efficient for detecting
recessive resistance alleles at low frequencies (Zhang et al., 2013). In other
Insect Resistance to Bt Cry Toxins 311

words, DNA screen can only pick up resistance data for which genetic basis
is already well characterized.
DNA-based screening has become applicable to Bt resistance since
cadherin mutations were confirmed to cause Cry1Ac resistance in three lep-
idopteran pests of cotton (Gahan et al., 2001; Morin et al., 2003; Xu et al.,
2005). In P. gossypiella, >8000 insects collected from cotton fields in
Arizona, California and Texas during 20012009 were screened with a
DNA-based diagnostic PCR for three resistance alleles from laboratory-
selected strains, and none of these three resistance alleles were detected
(Tabashnik et al., 2006, 2010). Similarly, the cadherin resistance allele from
a laboratory-selected strain of H. virescens was not detected in >7000 field-
collected individuals (Gahan et al., 2007). These results indicate extremely
low frequencies of the cadherin resistance alleles that could be detected, but
they do not exclude the presence of resistance alleles at either the cadherin
locus or other loci that could not be detected by the PCR methods used. In
both cases, however, bioassay data show that resistance remained rare in field
populations (Blanco et al., 2009; Tabashnik et al., 2010).
In H. armigera, an allele (r15) with a 55aa deletion in the intracellular
domain of cadherin (HaCad) was identified to confer non-recessive resis-
tance to Cry1Ac (Zhang et al., 2012b). Subsequently, a DNA-based
PCR method was developed to screen for the r15 allele in field populations
collected from the main cotton planting areas of China in 2011 and 2012
(Zhang et al., 2013). Three heterozygous r15 alleles were detected from
562 moths collected from northern China (with intensive Bt cotton plant-
ing), and the frequency of r15 allele was estimated to be 0.0027. However, no
r15 allele was detected from 314 moths collected from Xinjiang (with limited
Bt cotton use). Although all the r15 alleles have the same deletion in the
cDNA sequence, at least four different indels causing loss of exon 32 have
been detected in the genomic DNA sequences flanking exon 32 of HaCad.
Thus, designing a new method to detect the resistance alleles such as r15 at
cDNA level can avoid underestimating their frequency.
DNA screening is still in its infancy for Bt resistance monitoring. There
are two major limiting factors to be resolved yet. The first is the fact that
diverse mechanisms of resistance may exist in field populations (Zhang
et al., 2012a), challenging the accurate prediction of different or novel
resistance mechanisms evolved in the field. Selection for resistance in the
laboratory is a routine approach to predict likely mechanisms, but the resis-
tance may not be representative of resistance found in the field. F2 screens
might be applicable for isolating a collection of resistant strains representing
312 Yidong Wu

the important resistance types in the field populations, molecular mecha-

nisms for which can be further characterized in the laboratory. The second
limiting factor is multiple mutations occurring at a single resistance locus
complicating DNA screens. At least 15 cadherin alleles have been identified
associated with Cry1Ac resistance in H. armigera (Xu et al., 2005; Yang et al.,
2006, 2007; Zhang et al., 2012a,b; Zhao et al., 2010). Even the single r15
cadherin allele has been shown to have at least four origins, each of which
has a different indel in the genomic DNA sequence (Zhang et al., 2013). So,
the use of DNA-based diagnostics to screen for specific resistance-associated
alleles is prone to underestimating resistance gene frequencies as new muta-
tions within the same gene may be overlooked.

2.3. Current status of field-evolved resistance to Bt crops

The area planted with Bt crops worldwide increased from 1.1 million ha in
1996 to 75 million ha in 2013 ( James, 2013). Bt corn accounted for 67% of
corn planted in the United States during 2012 and Bt cotton accounted for
7995% of cotton planted in Australia, China, India and the United States
during 20102012 (Tabashnik et al., 2013). Considering the strong adapta-
tion capacity of insects to insecticides, large scale and intensive planting of Bt
crops will inevitably result in evolution of resistance in the field by the target
pests to Bt crops.
Monitoring data on insect resistance to Bt crops were recently reviewed
by Tabashnik et al. (2013) from 77 studies conducted in eight countries.
Whereas most previous assessments characterized pest populations only as
resistant or not, the new analysis by Tabashnik et al. (2013, 2014) introduced
a series of four levels of field-evolved resistance to Bt crops, ranging from
incipient resistance, to the most serious cases of resistance practical resis-
tance. Among the 13 cases examined with field-evolved resistance
(Table 6.2), 3 cases were categorized as incipient resistance (a statistically
significant increase in resistance occurred, but <1% of the individuals were
resistant), 4 cases as early warning of resistance (with 16% of individuals
with resistant phenotypes), 1 case as >50% resistant individuals and reduc-
tion of the expected efficacy, and 5 cases as practical resistance (>50%
resistant individuals and reduced efficacy reported).
Analysis of the available data from resistance monitoring suggests that the
high-dose/refuge strategy has helped to delay pest resistance to Bt crops
(Huang et al., 2011; Tabashnik et al., 2013). For the high-dose/refuge strat-
egy to be effective, there are three conditions to be met: (1) high dose. The
Insect Resistance to Bt Cry Toxins 313

Table 6.2 Status of field-evolved resistance to Bt crops in nine species of major insect
pests (Tabashnik et al., 2013, 2014)
Pest Crop Toxin Country References
Incipient resistance (<1% resistant individuals)
H. armigera Cotton Cry1Ac Australia Downes and Mahon (2012b)
H. armigera Cotton Cry2Ab Australia Downes and Mahon (2012b)
H. punctigera Cotton Cry2Ab Australia Downes et al. (2010a)
Early warning (16% resistant individuals)

D. saccharalis Corn Cry1Ab United Huang et al. (2012)

States
H. armigera Cotton Cry1Ac China Zhang et al. (2011, 2012a) and Jin
et al. (2013)
O. furnacalis Corn Cry1Ab Philippines Alcantara et al. (2011)
P. gossypiella Cotton Cry1Ac China Wan et al. (2012)
>50% resistant individuals and reduced efficacy expected

H. zea Cotton Cry2Ab United Ali and Luttrell (2007) and Tabashnik
States et al. (2009a, 2013)
Practical resistance (>50% resistant individuals and reduced efficacy reported)
B. fusca Corn Cry1Ab South Van Rensburg (2007) and Kruger
Africa et al. (2011)
D.v. virgifera Corn Cry3Bb United Gassmann et al. (2011, 2012)
States
H. zea Cotton Cry1Ac United Luttrell et al. (2004), Ali et al. (2006)
States and Tabashnik et al. (2008)
P. gossypiella Cotton Cry1Ac India Dhurua and Gujar (2011)
S. frugiperda Corn Cry1F United Storer et al. (2010, 2012)
States

concentration of toxins produced by Bt plants should be high enough to kill

all or nearly all heterozygotes, rendering resistance as a recessive trait; (2)
abundant non-Bt refuges to provide susceptible insects; and (3) low initial
resistance allele frequency. Among the five cases of practical resistance
(Table 6.2; Tabashnik et al., 2013), at least one of the key requirements
for the high-dose/refuge strategy was violated. For B. fusca, D.v. virgifera,
H. zea and S. frugiperda, the high-dose standard is not met. For
314 Yidong Wu

P. gossepiella, the first-generation Bt cotton (producing Cry1Ac) meets the

high-dose standard, but P. gossepiella resistance to Bt cotton evolved quickly
in western India, though not in the southwestern United States. Although
both countries had regulations requiring refuges of non-Bt cotton to be
planted near Bt cotton, poor compliance with the refuge regulations by
Indian cotton farmers may play a key role in the rapid evolution of resistance
and reduced field control in India (Tabashnik et al., 2013).

3. RESISTANCE MECHANISMS
Mode of action of Bt Cry toxins has been intensively studied and fre-
quently reviewed (Bravo et al., 2011; Ferre and Van Rie, 2002; Griffitts and
Aroian, 2005; Ibrahim et al., 2010; Knowles, 1994; Pardo-Lopez et al.,
2013; Pigott and Ellar, 2007; Soberon et al., 2009; Vachon et al., 2012;
Chapter 2). Although many details of mode of action of Bt Cry toxins
are far from understood, the major steps (crystal solubilization, proteolytic
activation, receptor binding, membrane insertion and pore formation) are
generally agreed on (Knowles, 1994; Schnepf et al., 1998). Insects could
develop resistance to Cry toxins due to alteration at any step of the sequential
procession of intoxication, and as many as 10 potential Bt resistance mech-
anisms were proposed (Heckel, 1994). For brevity, major steps of mode of
action, potential and observed resistance mechanisms are represented in
Fig. 6.2. This review focuses on resistance mechanism involving alterations
in receptor binding and proteolytic activation of Cry toxins in lepidopteran
insects. Other mechanisms such as more efficient repair of damaged midgut
cells and elevated immune responses in Bt-resistant strains are not included.

3.1. Mode of action of Bt Cry toxins

3.1.1 Structure of Bt Cry toxins
B. thuringiensis bacteria can produce various kinds of proteins with insecti-
cidal activity. The major insecticidal toxins include the -endotoxins pro-
duced as parasporal crystals during sporulation growth phase of Bt, and
the vegetative insecticidal proteins (VIP proteins) synthesized during the
vegetative phase of growth and secreted into the medium. The -endotoxins
are mainly composed of two families, Cry and Cyt, which are significantly
different in primary sequence. Cry toxins are insecticidal to the insects from
Lepidoptera, Coleoptera, Hymenoptera and Diptera orders, and also to
nematodes. Cyt toxins, characterized for possessing cytolytic activity, are
specifically toxic to the Dipteran insects.
Insect Resistance to Bt Cry Toxins 315

Figure 6.2 Schematic diagram of the steps of Cry toxin action and possible resistance
mechanisms. Receptor mutations and/or proteolytic alterations have been identified in
a number of Bt-resistant lepidopterans, whereas resistance mechanisms involving sol-
ubilization failure or abnormal membrane insertion/pore formation have not been
reported yet.

Since identification of the first cry genes (Schnepf and Whiteley, 1981),
more than 700 cry genes divided into 72 groups have been reported
(Crickmore et al., 2014). Cry toxins are grouped in three families that are
not related in structure: Bin-like, Mtx-like and 3d-Cry (Pardo-Lopez
et al., 2013). Although the sequence identity is low between different groups
of 3d-Cry proteins, the three-dimensional structure is highly conserved.
The structure of 3d-Cry toxins is composed of three structural domains
(Li et al., 1991). Domain I consists of a bundle of seven -helices with a cen-
tral helix -5 surrounded by six amphipathic helices. Domain I is the most
conserved domain among Cry toxins and is responsible for pore formation in
the cell membrane. Domain II is composed of three antiparallel -sheets
316 Yidong Wu

packed around a hydrophobic core to form a -prism. Domain II is the

most divergent domain among Cry toxins and is involved in defining recep-
tor binding specificity. Domain III is a -sandwich of two antiparallel
-sheets that form a jelly-roll topology. Domain III is implicated in both
receptor binding and pore formation in the cell membrane (de Maagd et al.,
2001; Schwartz et al., 1997). An extensive discussion of these toxins, its sol-
ubilization and activation is presented in Chapter 2.

3.1.2 Receptors for Bt Cry toxins

A variety of proteins have been identified and characterized as receptors or
putative receptors for Cry toxins. These include cadherins (Chen et al.,
2009a; Contreras et al., 2013; Fabrick et al., 2009; Flannagan et al., 2005;
Gahan et al., 2001; Hua et al., 2008, 2013; Morin et al., 2003; Nagamatsu
et al., 1998; Ren et al., 2013; Sayed et al., 2007; Vadlamudi et al., 1995;
Xu et al., 2005), aminopeptidases (APNs; Chen et al., 2009b; Crava et al.,
2013; Gill et al., 1995; Gill and Ellar, 2002; Knight et al., 1994, 1995; Luo
et al., 1997; Oltean et al., 1999; Rajagopal et al., 2002; Sivakumar et al.,
2007), alkaline phosphatases (ALPs; Hua et al., 2009; Jimenez et al., 2012;
Martins et al., 2010; McNall and Adang, 2003; Ning et al., 2010; Perera
et al., 2009; Zuniga-Navarrete et al., 2013) and ATP-binding cassette
(ABC) transporters (Atsumi et al., 2012; Gahan et al., 2010; Tanaka et al.,
2013). Please refer to Chapter 2 for additional details on the receptor proteins.

3.1.3 Models for mode of action

Crystal proteins ingested by the larvae are solubilized in the insect midgut to
release protoxins, and protoxins are then proteolytically cleaved to active
toxins. These activated toxins bind to specific receptors at the surface of mid-
gut epithelial cells and insert into the membrane to form non-selective pores
which are permeable to ions and small molecules. The presence of such
pores in the plasma membrane leads to osmotic lysis and death of the cells
(Carroll and Ellar, 1993; Knowles, 1994; Knowles and Ellar, 1987; also refer
to Chapter 2 for more details). Although the mode of action of Cry toxins
described above is widely accepted, many details in the process of intoxica-
tion are still lacking or controversial. Two models have been proposed to
depict details of the mode of action of Cry toxins: the sequential binding
model (Bravo et al., 2004) and the signalling pathway model (Zhang
et al., 2005, 2006). These two models were critically reviewed in detail
by Vachon et al. (2012).
Insect Resistance to Bt Cry Toxins 317

The sequential binding model comprises several steps: the monomeric

toxin binds to ALP and APN receptors in a low-affinity interaction to
locate the toxin in close proximity to the membrane surface; the mono-
meric toxin binds to the cadherin receptor in a high-affinity interaction
to induce proteolytic cleavage of the N-terminal end containing helix
-1 of domain I, and removal of helix -1 enables the rest of the toxin
to form a toxin prepore oligomer; the oligomeric prepore structure binds
to ALP and APN receptors with high affinity; and this binding is conducive
to the insertion of the prepore oligomer into the membrane, creating per-
meable pores in the apical membrane of midgut cells (Pardo-Lopez et al.,
2013). Recently, additional evidence supporting the sequential binding
model indicate that pore formation is an important step in the mechanism
of action of Cry proteins and that protoxin binding to cadherin receptor
may have a functional role inducing an alternative oligomer formation dif-
ferent from the oligomer induced by the activated toxin (Gomez et al.,
2014). Upon identification of ABCC2 as a novel receptor for Cry toxins,
Heckel (2012) proposed that ABCC2 may be involved in facilitating the
insertion of the prepore structure into the membrane as an extension of
the sequential binding model.
The signalling pathway model proposed that the toxicity of Cry pro-
teins is due to the activation of a Mg2+-dependent signal cascade pathway
that is triggered by the interaction of the monomeric toxin with the
cadherin receptor. Binding of Cry1Ab monomer to cadherin activates a
guanine nucleotide-binding protein (G protein), which in turn activates
an adenylcyclase promoting the production of intracellular cAMP. The
increased cAMP levels activate protein kinase A which activates an intra-
cellular pathway resulting in cell death (Zhang et al., 2005, 2006). A recent
study by Zhang et al. (2012b) demonstrated that a deletion in the intracel-
lular domain of cadherin is genetically linked with non-recessive resistance
to Cry1Ac in H. armigera, providing evidence that the cytoplasmic domain
of cadherin is involved in mediating toxicity. This evidence is consistent
with the vital role of the intracellular region of cadherin proposed by
the cell signalling model.
Based on work dealing with Cry1A receptors and resistance mechanisms
in H. virescens, a combination of the above two models has also been pro-
posed. In the combined model, osmotic cell lysis due to pore formation
and programmed cell death are not mutually exclusive, and both events
are possibly involved and have a direct effect in toxicity ( Jurat-Fuentes
and Adang, 2006).
318 Yidong Wu

3.2. Alterations in proteolytic processing of Cry toxins

in resistant insects
Proper activation of Bt Cry protoxins is critical for mediating toxicity
to target insects, and improper processing of a protoxin (insufficient acti-
vation or increased degradation) can result in insect resistance to the
protoxin (Oppert, 1999). The 198r strain of Indian meal moth, Plodia
interpunctella was selected with B. thuringiensis subsp. entomocidus HD198
resulting in high levels of resistance to Cry1Ab protoxin (Herrero et al.,
2001). A major gut protease (T1) that activates Cry1A protoxins was
found to be absent in the gut extracts from the resistant 198r strain. Sub-
sequently, bulked segregant analyses revealed that the absence of T1 was
genetically linked to resistance to HD198 Bt-strain (Oppert et al., 1997).
A laboratory-selected strain of H. armigera (designated Akola-R) exhibited
72-fold resistance to Cry1Ac protoxin. The larval midgut juice from
Akola-R strain did not activate Cry1Ac protoxin properly, producing a
mixture of 95 and 68 kDa Cry1Ac polypeptides instead of the biologically
active 65 kDa toxin core. N-terminal sequencing of these 95 and 68 kDa
polypeptides produced by gut juices of resistant insects revealed an intact
N-terminus (Rajagopal et al., 2009). Considering removal of N-terminal
of Cry1Ac is an essential early step in the mode of action (Bravo et al.,
2002), retention of the N-terminal of Cry1Ac protoxin is expected to
impair Cry1Ac toxicity. Further studies by Rajagopal et al. (2009) identi-
fied a proteinase (HaSP2) responsible for Cry1Ac activation, and down-
regulation of HaSP2 was claimed to cause improper processing of
the protoxin. But the deduced amino acid sequence of HaSP2 lacked
two (Asp102 and Ser195) of the three conserved residues (His57,
Asp102 and Ser195) important for the catalytic triad, suggesting that HaSP2
may be a serine protease homologue without proteinase activity. The func-
tion of HaSP2 on Cry1Ac protoxin activation needs to be further
investigated.
The 198r strain of P. interpunctella showed 264-fold resistance to Cry1Ab
protoxin but only 25-fold to Cry1Ab toxin (Herrero et al., 2001). The
Akola-R strain of H. armigera showed 72-fold resistance to Cry1Ac
protoxin, but no resistance to Cry1Ac toxin (Rajagopal et al., 2009). Much
higher levels of resistance to a protoxin than to its toxin form in a Bt-resistant
strain may be a good indication of the involvement of proteinases in Bt
resistance.
Insect Resistance to Bt Cry Toxins 319

3.3. Modifications of Cry toxin receptors in resistant insects

3.3.1 Cadherin
The YHD2 strain of H. virescens is a laboratory-selected strain that is about
10,000-fold resistant to Cry1Ac toxin (Gould et al., 1995). A single major
gene (BtR-4) is responsible for 4080% of Cry1Ac resistance levels in
YHD2 and was mapped to linkage group 9 (LG9; Heckel et al., 1997).
At that time, two types of Bt receptors were identified: APNs and cadherins
(Knight et al., 1994; Vadlamudi et al., 1995). While two APNs were mapped
on other linkage groups, a cadherin gene (HvCad) was mapped on the same
linkage group (LG9) as BtR-4. Further genetic and molecular analysis rev-
ealed that the HvCad in the YHD2 strain was inactivated by a ret-
rotransposon, introducing a premature stop codon and preventing the
translation of the full-length protein (Gahan et al., 2001). After the first iden-
tification of cadherin as a Bt resistance gene in H. virescens, linkage between
Cry1Ac resistance and the cadherin gene were tested in another two cotton
pests: P. gossypiella and H. armigera (Morin et al., 2003; Xu et al., 2005).
In P. gossypiella, three mutated cadherin gene alleles (r1, r2 and r3) were
genetically linked to resistance to Cry1Ac in the AZP-R strain (Morin et al.,
2003), and a fourth resistance allele (r4) was identified from the Bt4R strain
(Fabrick and Tabashnik, 2012). The r1 allele has a deletion mutation of 24 bp
that results in two amino acid substitutions and the omission of eight amino
acids. The r2 allele has a 202-bp deletion introducing a premature stop
codon, the r3 allele has a 126-bp deletion that eliminates 42 amino acids
and the r4 allele has a 15-bp deletion that causes loss of 5 amino acids. All
four deletions were located upstream of the Cry1Ac-binding region of
the cadherin protein (Fabrick and Tabashnik, 2012; Morin et al., 2003).
Recently, eight novel alleles (r5r12) associated with Cry1Ac resistance were
revealed from two field populations of P. gossypiella from western India. For
seven of the eight alleles, each produced two or more different transcript
isoforms by alternative splicing (Fabrick et al., 2014).
In H. armigera, a deletion between exons 8 and 25 of HaCad (r1 allele) cre-
ated a premature stop codon that was genetically linked to Cry1Ac resistance
in the laboratory-selected GYBT strain (Xu et al. 2005; Yang et al., 2006).
Through F1 screens and F2 screens of field populations of H. armigera from
northern China, where Bt cotton expressing Cry1Ac has been intensively
adopted, a total of 15 resistance alleles of HaCad (r1r15) were identified
(Yang et al., 2007; Zhang et al., 2012a; Zhao et al., 2010). Unlike most
320 Yidong Wu

cadherin mutants previously identified, which have mutations at the

ectodomain and confer recessive resistance, the r15 allele of HaCad showed
a 55 amino acids deletion in the intracellular domain of cadherin and it
was reported to cause non-recessive resistance to Cry1Ac in H. armigera
(Zhang et al., 2012b). Although all the r15 alleles have the same deletion in
the cDNA sequence, at least four different indels causing loss of exon 32 have
been detected in the genomic DNA sequences flanking exon 32 of HaCad.
Multiple origins of the r15 alleles illustrate parallel genotypic adaptation of
H. armigera to the selection pressure of Bt cotton (Zhang et al., 2013).
Unlike mutations that occurred directly in the cadherin gene, reduced
expression levels of cadherin (DsCad) were reported to be associated with
Cry1Ab resistance in the Cry1Ab-RR strain of D. saccharalis (Yang et al.,
2011). Whereas cDNA sequences of DsCad were identical between the
resistant and susceptible strains, the transcript levels of DsCad was signifi-
cantly lower in Cry1Ab-RR. The role of DsCad protein on Cry1Ab tox-
icity was further confirmed by RNAi analysis, which demonstrated that
knockdown of DsCad expression correlated with increased tolerance to
Cry1Ab in the susceptible strain (Yang et al., 2011).
So far, a total of 28 cadherin alleles genetically linked to Cry1Ac resis-
tance were identified from three lepidopteran pests: 1 from H. virescens,
12 from P. gossypiella and 15 from H. armigera (Fabrick and Tabashnik,
2012; Fabrick et al., 2014; Gahan et al., 2001; Morin et al., 2003; Xu
et al., 2005; Yang et al., 2006, 2007; Zhang et al., 2012a,b; Zhao et al.,
2010). These 28 resistance alleles can be grouped into four types (illustrated
in Fig. 6.3): (1) Truncation caused by premature stop codons at the extra-
cellular domain are expected to create truncated proteins that lose the trans-
membrane anchor region and cannot interact with Cry1Ac. (2) Deletions
from the Cry1Ac-binding region or somewhere else at the extracellular
domain are expected to damage normal interaction between cadherin and
Cry1Ac. (3) Deletion at the intracellular domain (such as r15 of HaCad) does
not affect toxin binding, and this mutation is suggested to affect post-binding
events (Zhang et al., 2012b). Evidence of involvement of the intracellular
cadherin-domain in the Cry1Ac toxicity suggest that both pore formation
and cell signalling pathways contribute to the efficacy of Bt toxins (Zhang
et al., 2012b). (4) Single amino acid mutations in the Cry1Ac-binding
region of the cadherin receptor from H. virescens were demonstrated to affect
its toxin-binding ability to Cry1Ac (Xie et al., 2005). Resistance alleles
r10r14 of HaCad were suggested to confer resistance through amino acid
substitutions, although exact amino acid substitutions have not been func-
tionally confirmed yet (Zhang et al., 2012a).
Insect Resistance to Bt Cry Toxins 321

Figure 6.3 Types of cadherin mutations associated with Cry1Ac resistance in three
cotton pests (Heliothis virescens, Pectinophora gossypiella and Helicoverpa armigera).
(1) Truncation: a premature stop codon is present at any sites on the extracellular
domain of cadherin. (2) Deletion in the extracellular domain: a stretch of amino acid res-
idues on the extracellular domain, present within or ahead of the toxin-binding region.
(3) Deletion in the intracellular domain: lack of 55 amino acid residues in the r15 allele of
HaCad, which confers non-recessive resistance to Cry1Ac (Zhang et al., 2012b). (4) Amino
acid substitution: the L1425R mutant of HvCad of H. virescens can decrease binding to
Cry1Ac and has the potential to confer resistance (Xie et al., 2005). The r10r14 alleles of
HaCad in H. armigera were suggested to confer Cry1Ac resistance by amino acid sub-
stitutions (Zhang et al., 2012a).

3.3.2 Aminopeptidase
Mutations or altered expression of APN has been suggested or confirmed to
confer high levels of resistance to Cry1 toxins in three lepidopterans:
S. exigua, T. ni and H. armigera (Herrero et al., 2005; Tiewsiri and Wang,
2011; Zhang et al., 2009).
Lack of APN1 expression was detected in a Cry1Ca-resistant strain of
S. exigua by using suppression subtractive hybridization. Northern blot anal-
ysis confirmed APN1 was not expressed in the resistant strain, whereas other
three APNs (APN2, APN3 and APN4) had no difference in expression
between the resistant and susceptible strains (Herrero et al., 2005). These
data suggest that the lack of APN1 expression plays a role in Cry1Ca resis-
tance in S. exigua, although a linkage between Cry1Ca resistance and the
lack of APN1 expression needs to be determined. Cry1Ac resistance in
the GLEN-Cry1Ac-BCS of T. ni was introgressed into a susceptible strain
322 Yidong Wu

(Benzon) by eight generations of backcrossing. A proteomic screen of mid-

gut BBMV proteins from the two isogenic strains revealed that Cry1Ac
resistance is associated with differential alteration of two midgut APNs,
down-regulation of APN1 and up-regulation of APN6, conferred by a
trans-regulatory mechanism. Genetic analysis showed that only down-
regulation of APN1 was linked to Cry1Ac resistance (Tiewsiri and
Wang, 2011), supporting the importance of APN1 on Cry1Ac resistance
and on the mode of action of Cry1Ac toxin in T. ni.
Interestingly, a deletion mutation of HaAPN1 (at amino acids 9381004)
was suggested to confer Cry1Ac resistance in the BtR strain of H. armigera
(Zhang et al., 2009). A peptide fragment of HaAPN1 with the deletion
expressed in Escherichia coli cells lost binding with Cry1Ac, whereas the pep-
tide fragment without the deletion bound to Cry1Ac (Zhang et al., 2009).
Both the wild type and the short type of HaAPN1 (i.e. heterozygote) were
present in many individuals from the resistant BtR strain, suggesting that
other resistance mechanism(s) may exist in this resistant strain. Genetic anal-
ysis showed Cry1Ac resistance in BtR is incompletely recessive and poly-
genic (Liang et al., 2008), which confirms that there are other resistance
mechanisms in this resistant strain besides the HaAPN1 mutation.

3.3.3 Alkaline phosphatase

Reduced expression of ALP protein and mRNA has been associated with
Cry1 resistance in some strains of three Noctuid species (H. virescens,
H. armigera and S. frugiperda) with different resistance phenotypes to Cry1
toxins ( Jurat-Fuentes and Adang, 2004; Jurat-Fuentes et al., 2011). In the
YHD3 strain of H. virescens, two genes at different loci (HvCad and
HvABCC2) have been identified to confer extremely high levels of resis-
tance to Cry1Ac (Gahan et al., 2001, 2010). Although reduced binding with
Cry1Ac mediated by HvALP was observed in YHD3 ( Jurat-Fuentes and
Adang, 2004), a direct correlation between HvALP levels and resistance
is still lacking ( Jurat-Fuentes et al., 2011). Two field-derived strains of
S. frugiperda (456 and 512) with extremely high levels of resistance to Cry1Fa
showed three- to fourfold reduction in ALP activity and protein levels, but
no differences in APN activity in midgut BBMVs when compared with two
susceptible strains ( Jurat-Fuentes et al., 2011). In the Cry1Ac-selected AR1
strain of H. zea, a 10-fold increase in specific ALP activity in the midgut
lumen was suggested as a resistance mechanism. The soluble form of ALP
in midgut lumen may sequester some of Cry1Ac toxins and, therefore, affect
toxin binding with other receptors (Caccia et al., 2012). Further studies are
Insect Resistance to Bt Cry Toxins 323

needed to verify if alteration of ALP expression in resistant strains is just an

adaptive consequence or directly impact toxin binding resulting in insect
resistance.

3.3.4 ABCC2
In the YHD2 strain of H. virescens, a mutation in the cadherin gene HvCad
was identified as a major mechanism conferring high levels of resistance to
Cry1Ac toxin (Gahan et al., 2001). YHD2 lost binding with Cry1Aa, but
not with Cry1Ab and Cry1Ac (Lee et al., 1995). The YHD2 strain was fur-
ther selected with Cry1Ac to produce the YHD3 strain, which had much
higher resistance levels to Cry1Ac than YHD2 and loss binding with all three
Cry1A toxins ( Jurat-Fuentes and Adang, 2004). A second resistance gene
(HvABCC2) contributing to the additional resistance and lost of binding
with Cry1Ab and Cry1Ac in YHD3 was then identified by genetic mapping
and positional cloning (Gahan et al., 2010). A 22-bp deletion in exon 2 of
HvABCC2 in YHD3 was predicted to result in a truncated 99-residue pro-
tein instead of the full-length protein composed of 1339 amino acids. The
inactivation mutation of HvABCC2 was correlated with both higher
resistance levels and the loss of Cry1Ab and Cry1Ac binding to midgut
membranes (Gahan et al., 2010).
Resistance to Cry1Ac spray formulations has also evolved in field
populations of P. xylostella and T. ni ( Janmaat and Myers, 2003;
Tabashnik et al., 1990). Genetic mapping demonstrated that field-evolved
resistance to Cry1Ac in the NO-QA strain of P. xylostella and the
GLEN-Cry1Ac-BCS strain of T. ni was also genetically linked with ABCC2
gene, named PxABCC2 and TnABCC2, respectively (Baxter et al., 2011).
In the NO-QA strain of P. xylostella, a 10-residue deletion in the middle of
transmembrane helix 12 of PxABCC2 was predicted to be located in the
extracellular region of the second NBD domain (Baxter et al., 2011). How-
ever, the mutation of TnABCC2 has not yet been identified in the GLEN-
Cry1Ac-BCS strain of T. ni.
In the C2 strain of B. mori, a candidate locus for recessive resistance to
Cry1Ab was mapped to a region of 82 kb (containing BmABCC2) on chro-
mosome 15. Comparisons of BmABCC2 sequences among 10 susceptible
and 7 resistant strains revealed a common tyrosine insertion in resistant
alleles which is located in an outer loop of the predicted transmembrane
structure. Introduction of a wild-type allele of BmABCC2 into a resistant
strain by using germline transformation restored susceptibility to Cry1Ab
in the resistant strain, which provides very strong evidence for the role of
324 Yidong Wu

this single tyrosine insertion of BmABCC2 in conferring Cry1Ab resistance

(Atsumi et al., 2012). As mentioned above, the additional tyrosine at posi-
tion 234 is located in one of the extracellular loops of a transmembrane
domain of BmABCC2, and it was proposed that this loop is involved in
binding to the toxin in the midgut lumen. Expression of BmABCC2 with
and without Tyr234 in Sf9 cells showed that insertion of Tyr234 caused
reduced Cry1Ab cytotoxicity (Tanaka et al., 2013). Reduced binding of
Cry1Ab was associated with this insertion in Sf9 cells (Tanaka et al.,
2013), but not in brush border membrane vesicles from larval midguts
(Atsumi et al., 2012).
Diversified resistance mutations in the same ABCC2 gene of at least four
lepidopteran species, from single amino acid insertions to deletion of the
entire protein, strongly suggest that this ABC transporter plays a crucial role
in the toxin mode of action as a functional receptor (Heckel, 2012; Tanaka
et al., 2013). According to the existing evidence, it is concluded that the
ABCC2 protein is a functional receptor for Cry1Ab and Cry1Ac but not
for Cry1Aa, and mutations in this gene confer high levels of resistance to
Cry1Ab and Cry1Ac with recessiveness.

4. GENETIC DIVERSITY OF RESISTANCE AND

IMPLICATIONS FOR RESISTANCE MANAGEMENT
4.1. Laboratory-selected and field-evolved resistance
A prominent hypothesis about insecticide resistance is that selection in the
laboratory favours a polygenic response, whereas selection in the field
favours a monogenic response (McKenzie and Batterham, 1994; Roush
and McKenzie, 1987). The idea is that the starting laboratory populations
for selection are usually small and not likely to contain rare, major resistance
mutations; thus, laboratory selection is prone to pick up pre-existing genes
of minor effect (ffrench-Constant, 2013). However, for conventional insec-
ticides, a review of empirical data and simulation results did not support this
hypothesis (Groeters and Tabashnik, 2000). Likewise, this hypothesis is not
supported by the available data on Bt resistance. First, several strains with
high levels of resistance to Cry1Ac conferred by major genes (cadherin or
ABCC2) were selected for each of the three lepidopterans: H. virescens,
P. gossypiella and H. armgiera (Gahan et al., 2001, 2010; Morin et al.,
2003; Xu et al., 2005; Zhang et al., 2012a,b). Second, the correspondence
between the genetic basis of laboratory-selected resistance and field-selected
resistance to Cry1Ac has been established in two cases. In northern China,
Insect Resistance to Bt Cry Toxins 325

the r1 resistance allele of cadherin of H. armigera was first identified in a

laboratory-selected strain collected from Hebei Province in 2001
(Xu et al., 2005) and the r1 allele was also detected in three resistant strains
isolated in 2009 from the field-selected Anyang population in Henan Prov-
ince of China (Zhang et al., 2012a). Mutations in the same cadherin gene are
associated with P. gossypiella resistance to Cry1Ac in laboratory-selected
strains from Arizona of the United States and field-selected populations from
India (Fabrick et al., 2014). It shows that laboratory-selected strains can be a
valuable tool for finding loci that are important in field-evolved resistance to
Bt crops.
In view of diverse and complex genetic basis of insect resistance to Bt
proteins that have been identified (as summarized in Table 6.3), we can
say that the use of laboratory selection to predict the exact types of field-
selected resistance is facing challenges and should be taken with caution.
Screen of F1 and F2 progeny from several geographic field-derived
populations for Bt resistance in the laboratory will provide better prediction
of resistance mechanism than selection from a single field population or from
a susceptible strain kept in the laboratory for a long period.
Since resistance to Cry toxin may result from multiple mutational events
at a single resistance locus (cadherin) such as have been revealed in field-
selected populations of two cotton pests H. armigera from China and
P. gossypiella from India (Fabrick et al., 2014; Yang et al., 2007; Zhang
et al., 2012a; Zhao et al., 2010), the F1 screen will be a practical and reliable
method to recover resistance alleles at a known resistance locus from field
populations.
The F2 screen is an efficient approach to isolate a collection of strains rep-
resenting the important types of resistance mechanisms that are present in
field populations of the target pests. Large-scale F2 screens have been suc-
cessfully employed to monitor resistance frequency and also to isolate differ-
ent Bt-resistant strains from geographical field populations of H. armigera in
China (Zhang et al., 2012a) and H. armigera and H. punctigera in Australia
(Downes et al., 2009, 2010a; Mahon et al., 2007, 2008, 2012). Eight strains
of H. armigera resistant to Cry1Ac were isolated from an F2 screen of field
populations in China, and resistance types of these eight strains were deter-
mined through an approach involving crossing with a susceptible or resistant
strain, bioassay and sequencing of the cadherin gene (Fig. 6.4). Among these
eight resistant strains, six strains had recessive resistance alleles at a cadherin
locus, one strain had a non-recessive resistance allele at the cadherin locus
and the other strain had non-recessive resistance but not linked with the
Table 6.3 Mechanisms of resistance to Cry1 toxins in lepidopterans
Binding site modification
Altered proteolytic
Strain Toxin RRa processing Cadherin ABCC2 APN ALP References
H. virescens
YFO Cry1Ac >78 4b Gahan et al. (2010)
YEE Cry1Ac >780 4 Gahan et al. (2010)
YHD3/ Cry1Ac 73,700 4 4 4 Gahan et al. (2010) and Jurat-
YHD2-B Fuentes et al. (2011)
H. armigera
GYBT Cry1Ac 564 4 Xu et al. (2005)
SCD-r1 Cry1Ac 438 4 Yang et al. (2009)
Xj-r15 Cry1Ac 140 4 Zhang et al. (2012b)
BtR c
MVP/Cry1Ac 2971 4 4 Zhang et al. (2009) and Jurat-
Fuentes et al. (2011)
Akola-R Cry1Acc 72 4 Rajagopal et al. (2009)
H. zea
AR1 Cry1Ac 100 4 Caccia et al. (2012)
P. gossypiella
AZP-R Cry1Ac 3100 4 Morin et al. (2003)
AGJ, KMP Cry1Ac NA d
4 Fabrick et al. (2014)
P. xylostella
NO-QA/ MVP/Cry1Acc >6800 4 Baxter et al. (2011) and Tabashnik
NO-QAGE et al. (1993)
T. ni
GLEN- Cry1Ac 2344 4 4 Tiewsiri and Wang (2011) and
Cry1Ac-BCS Baxter et al. (2011)
B. mori
C2 Cry1Ab >2860 4 Atsumi et al. (2012)
S. frugiperda
456, 512 Cry1Fa 772010,000 4 Jurat-Fuentes et al. (2011) and
Blanco et al. (2010)
P. interpunctella
198-r Cry1Abc 264 4 Oppert et al. (1997) and Herrero
et al. (2001)
S. exigua

Resistant Cry1Ca 850 4 Herrero et al. (2005) and Moar

et al. (1995)
a
RR at LC50.
b
Presence of a specific resistance mechanism.
c
The proteins used for bioassays are protoxins.
d
Survivors collected from Bt cotton in western India.
328 Yidong Wu

Figure 6.4 Diverse genetic basis of Cry1Ac resistance in field populations of Helicoverpa
armigera from China (Zhang et al., 2012a). Survival at the diagnostic concentration of
Cry1Ac of nine resistant (R) strains (red bars; eight strains isolated from the F2 screen
and the laboratory-selected strain SCD-r1 with the r1 allele of HaCad at the cadherin locus),
and progeny from crosses between each resistant strain and either the susceptible SCD
strain (green bars) or the resistant SCD-r1 strain (blue bars). Asterisks indicate 0% survival
for progeny from crosses between the SCD strains. Rec, recessive; Dom, dominant.

cadherin locus (Zhang et al., 2012a). In contrast with diverse genetic basis of
Cry1Ac resistance in H. armigera from China, five Cry2Ab-resistant strains of
H. armigera, which were isolated from an F2 screen of field populations in
Australia, were all recessive and shared a common resistance locus
(Mahon et al., 2008).

4.2. Resistance dominance and the refuge strategy

The refuge strategy has been the primary approach to delay evolution of pest
resistance to Bt crops and has been mandatory in the United States, Australia
and elsewhere. The refuge is expected to produce relative abundant suscep-
tible target insects that will be able to mate with rare survivors from Bt crops.
If resistance is recessive, the progeny from such matings will be killed by Bt
crops and the evolution of resistance will be substantially delayed (Tabashnik
et al., 2009a, 2013). The refuge strategy will be most effective if Bt crops
express consistently a high dose of Bt proteins to render resistance a func-
tionally recessive trait and thus kill almost all of the heterozygous progeny.
Insect Resistance to Bt Cry Toxins 329

In contrast, effectiveness of the refuge strategy will be largely compromised if

resistance is non-recessive or partial recessive and Bt crops do not meet the
high-dose standard for the target pest.
Selection for resistance to Bt Cry toxins in the laboratory has shown that
the levels of insect resistance to Cry proteins can vary from complete reces-
siveness to complete dominance (Ferre et al., 2008). Even for a single species
H. armigera, resistance to Cry1Ac is completely recessive in SCD-r1 strain (r1
allele, with a truncated cadherin) and completely dominant in AY2 and
QX7 strains at the diagnostic concentration of Cry1Ac ( Jin et al., 2013).
Even further, different alleles at the same locus of cadherin in H. armigera
can confer different levels of dominance, r1 allele is recessive and r15 allele
(with a 55aa deletion in the cytoplasmic domain of cadherin) is non-
recessive (Zhang et al., 2012b).
Similar to results from laboratory selection, dominance of field-evolved
resistance also ranges from being recessive to dominant. Resistance to Cry1F
corn is recessive in S. frugiperda from Puerto Rico (Storer et al., 2010). Based
on results from laboratory-selected resistant strains, resistance to Cry1Ac
cotton is expected to be recessive in P. gossypiella from India (Fabrick
et al., 2014). In contrast, resistance to Bt corn (Cry1Ab) is dominant in
B. fusca from South Africa (Campagne et al., 2013), and resistance to Cry3Bb
corn is expected to be non-recessive in D.v. virgifera from the United States
(Devos et al., 2013; Petzold-Maxwell et al., 2012).
Results from evidence of both the laboratory-selected and field-evolved
resistance to Cry toxins, the diversity of evolved resistance dominance to Bt
toxins is high. It is implying that target pests could evolve dominant resis-
tance alleles that could cope with the refuge strategy. Compared with the
progress on understanding of the molecular mechanisms mediating recessive
resistance to Bt toxins, our understanding on mechanisms of dominant resis-
tance is limited. It deserves more attention because it is tougher for us to deal
with such mechanism of resistance to Cry proteins.

4.3. Cross-resistance and the pyramid strategy

Cross-resistance means resistance to a particular pesticide that results in resis-
tance to other pesticides through a common resistance mechanism. Cross-
resistance is usually present among pesticides sharing similar binding target
sites or similar detoxifying pathways. For example, selection with Cry1Ac in
H. armigera caused cross-resistance to Cry1Aa and Cry1Ab, which is con-
ferred by cadherin mutations (Xu et al., 2005). Multiple resistance is defined
330 Yidong Wu

as resistance to more than one class of pesticides in a single pest through dif-
ferent resistance mechanisms. Multiple resistance is generally caused by
sequential or mosaic uses of different classes of pesticides. For example, a
strain of P. xylostella (NO-95C), selected in the field with Bt formulations
(containing Cry1A and Cry1C toxins) and further selected with Cry1C
in the laboratory, developed high levels of resistance to Cry1A and moderate
levels of resistance to Cry1C (Liu and Tabashnik, 1997; Liu et al., 1996).
Bioassays of progeny from split broods of single-pair families demonstrated
that resistance to Cry1C segregates independently of resistance to Cry1Ab in
NO-95C (Liu and Tabashnik, 1997). Resistance to Cry1A and Cry1C in
the NO-95C strain of P. xylostella is a typical phenomenon of multiple
resistance.
The pyramid strategy for Bt resistance management uses crops that
produces two or more dissimilar toxins that kill the same target pest
(Roush, 1998). Toxins to be used for pyramiding should have no cross-
resistance, so that the risk to evolve a single mechanism of resistance against
both toxins could be dramatically reduced. Mathematical models and empir-
ical studies have suggested resistance to pyramided two-gene plants that
show no cross-resistance can be significantly delayed as compared with resis-
tance to single-gene plants adopted sequentially or in mosaics (Roush, 1998;
Zhao et al., 2003).
However, target pests can possibly evolve a single gene that can over-
come both Bt genes to be used in the pyramid, even if they have different
binding sites. The most widely used pyramid is the second-generation Bt
cotton that produces Cry1Ac and Cry2Ab. No cross-resistance between
these two toxins is presumed because they bind to different larval midgut
target sites. However, several strains of H. virescens selected with Cry1Ac
in the laboratory caused significant cross-resistance to Cry2Aa (Gould
et al., 1992, 1995; Jurat-Fuentes et al., 2003). In addition, laboratory selec-
tion of a strain of P. gossypiella with Cry2Ab resulted in 420-fold cross-
resistance to Cry1Ac as well as 240-fold resistance to Cry2Ab (Tabashnik
et al., 2009b). Also, two Cry1Ac-resistant strains of H. armigera isolated from
field-selected populations from northern China had low, but significant
cross-resistance to Cry2Ab (46-fold; Jin et al., 2013), and a strain of
H. zea selected for resistance to Cry1Ac increased survival on Bt cotton
expressing Cry1Ac and Cry2Ab (Brevaulta et al., 2013). All these results
show that cross-resistance occurs between Cry1Ac and Cry2Ab in at least
three key cotton pests. Even though there are no shared binding sites
between two Cry proteins, there are several potential resistance mechanisms
Insect Resistance to Bt Cry Toxins 331

that could result in cross-resistance in the target insects. For example, if dif-
ferent classes of Bt toxins are activated by the same proteases, alterations of
the proteases could cause cross-resistance. If a novel proteolytic mechanism
is evolved to degrade the activated toxin core, it could cause cross-resistance.
Pore formation is believed to be general and an important step in the mech-
anism of action of many Cry proteins, thus any change affecting pore for-
mation or pore function may result in broad cross-resistance (Heckel,
1994). With increasing deployment of the pyramid Bt cotton and corn
crops, more work is needed to identify potential molecular basis of cross-
resistance between two distinct toxins such as Cry1Ac and Cry2Ab. In
the future, the toxins for stacking varieties should be as disparate as possible,
showing highly different mechanism of actions such as a Bt toxin and a non-
Bt factor, to minimize the chance that mutations in a single gene could con-
fer resistance to both factors.

5. CONCLUSIONS
Large-scale adoption of Bt crops have brought both ecological benefits
for environment and economic benefits for farmers. In the foreseeable
future, adoption of Bt crops will keep increasing globally. Benefited from
implementation of proactive resistance management strategies such as the
refuge strategy and the pyramid strategy in many countries, most of the tar-
get pests of Bt crops have been sustainably and effectively controlled for
nearly 20 years. However, several cases of field-evolved resistance to Bt corn
and Bt cotton have been documented causing reduced field efficacy. Evo-
lution of resistance by target pests is a real threat to the continued success of
Bt crops.
The genetic capacity of insect populations to evolve resistance to Cry
toxins has been well demonstrated by both laboratory-selected and field-
evolved resistance in many species within several insect orders. Diverse
genetic options for target pests to cope with Bt crops are challenging the
efforts to understand mechanisms of resistance and to design rational resis-
tance management strategies. Genetic mapping approach has been proved to
be a powerful tool and it is anticipated that it will continue to be essential in
dissecting the complex genetic basis of Bt resistance. Clarifying resistance
mechanisms can facilitate and advance our understanding on modes of action
of Cry toxins.
Proactive evaluation of the inheritance and initial frequency of resistance
are useful for assessing the risk of resistance problems in the close future.
332 Yidong Wu

Although laboratory selection can provide useful information on characters

of Bt resistance, employing the F1 screens and F2 screens to isolate various
resistance alleles from geographical field populations and to track evolution
trajectory of resistance are greatly encouraged. Detection of early resistance
is critical for the establishment of an adaptive and intelligent resistance man-
agement strategy.
Intensive research on Bt resistance focuses primarily on resistance to
Cry1A toxins in lepidopteran insects. More work is needed to cover resis-
tance in other insect orders and resistance to Bt toxins from other Cry pro-
teins and families. Resistance mechanisms and resistance evolution for
several cases of field-evolved resistance deserve further studies. Retrospec-
tive analysis of these cases can provide better understanding of resistance
mechanisms to Cry proteins to help improve Bt resistance management
practices.

ACKNOWLEDGEMENTS
I am grateful to Yihua Yang, Derek Russell, Alejandra Bravo, Mario Soberon, Bruce
Tabashnik and the editors of this volume for reading the text and making helpful
comments. This work was supported in part by grants from the Ministry of Agriculture of
China (Grant no. 2014ZX08012-004) and the NSFC of China (Grant no. 31272382).

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 CHAPTER SEVEN

Photorhabdus Toxins
Richard H. ffrench-Constant, Andrea J. Dowling
Biosciences, University of Exeter, Cornwall, United Kingdom

Contents
1. Photorhabdus Lifestyles, Relatives and Genomes 344
1.1 The life cycle of Photorhabdus temperata and Photorhabdus luminescens 344
1.2 The unusual life cycle of Photorhabdus asymbiotica 345
1.3 Xenorhabdus and comparative genomics 346
2. The Toxin Complexes 347
2.1 Tc discovery, gene cloning and ABC nomenclature 348
2.2 Diversity of tc-like genes from other bacteria 349
2.3 Structure, function and biophysics of the Tc ABC complexes 350
2.4 The Tcs as polymorphic toxins 361
2.5 The role of the Tcs in the biology of infection 361
2.6 The potential role of Tcs in crop protection 363
3. Photorhabdus Virulence Cassettes 364
3.1 Discovery and organization of PVCs 365
3.2 The role of PVC-like structures in other bacteria 367
3.3 Implications for PVC biology 369
4. The Mcf Toxins 370
4.1 Discovery and mode of action of Mcf1 370
4.2 Diversity of Mcf-like toxins 372
4.3 Studying Mcf-like toxins in vivo 374
5. Patox and Photox 376
5.1 PaTox structure and function 376
5.2 Photox as a novel mART toxin 378
6. Binary Toxins 379
6.1 The PirAB binary toxins 379
6.2 The XaxAB and YaxAB cytotoxins 379
7. Classical Secretions Systems and Novel Screens 380
7.1 Type III and other classical secretion systems 380
7.2 RVA-like screens for novel effectors 381
7.3 Clustering methods to look for novel effector phenotypes 382
8. Perspectives for the Future of Photorhabdus Toxins 383
Acknowledgements 383
References 383

Advances in Insect Physiology, Volume 47 #2014 Elsevier Ltd 343

ISSN 0065-2806 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800197-4.00007-5
344 Richard H. ffrench-Constant and Andrea J. Dowling

Abstract
The last 10 years has seen an explosion in our knowledge of the diversity of toxins pro-
duced by the insect pathogenic bacteria Photorhabdus and Xenorhabdus. Here we
review new data on the structure, mode of action and biophysics of Photorhabdus
toxins and place them into context with similar toxins made in other bacteria such
as those of the genus Yersinia and Serratia. We look in detail at structurefunction stud-
ies of the Toxin complexes or Tcs and suggest that these might constitute a new mode
of bacterial secretion, here termed the Toxin complex secretion system or TC-SS. We
examine current data on the Makes Caterpillars Floppy or Mcf toxins both from insect
pathogenic bacteria and also the related Mcf-like Fit toxin from Pseudomonas. We also
review data on the Photorhabdus Virulence Cassettes or PVCs and their equivalent
phage-like secretion systems in other bacteria. Again these appear to be a totally novel
secretion system, termed PVC-SS, derived from tailed myophages. Finally, we review the
sheer diversity of other candidate toxins identified from Photorhabdus and Xenorhabdus
genomes, such as PaTox and Photox, and from simple gain-of-function screens devised
to identify novel effectors when expressed in recombinant Escherichia coli, termed
Rapid Virulence Annotation or RVA. Taken together, available data support the early
contention that Photorhabdus genomes do indeed encode more candidate toxins than
those of other bacteria and suggest that this unique genus demands more time from
serious toxinologists than has been devoted to date.

1. PHOTORHABDUS LIFESTYLES, RELATIVES

AND GENOMES
Before we can fully appreciate the stunning diversity of toxins made by
Photorhabdus bacteria, and indeed their potential utility, we first need to under-
stand their lifestyles and how they are related to other entomopathogenic bac-
teria such as those from the related genus Xenorhabdus.

1.1. The life cycle of Photorhabdus temperata

and Photorhabdus luminescens
Photorhabdus are -proteobacteria that live in a bizarre symbiosis of patho-
gens with nematodes of the genus Heterhorhabditis and their life cycle has
been extensively reviewed previously (Forst and Nealson, 1996; Forst
et al., 1997; Waterfield et al., 2009). While the bacteria appear to act as
benign symbionts of the nematode gut, once regurgitated into the open
blood system (hemocoel) of their insect hosts they switch to becoming
potent pathogens that rapidly overcome the insect immune system and then
kill the insect (Forst et al., 1997). The insect cadaver is then bioconverted
into a realm of future bacteria and their nematode partners in a life cycle
Photorhabdus Toxins 345

involving exquisitely precise interactions with nematodes of different stages

(Somvanshi et al., 2012). The transmission of Photorhabdus to the infective
juvenile nematode progeny requires bacterial colonization of maternal rectal
glands and hatching of the progeny within the mother, in a process termed
endotokia matricida (Somvanshi et al., 2012). This is a stunning piece of natural
history that effectively reclones the bacterium prior to its repackaging in the
infective juvenile. However for the purposes of this review, which focuses
on insecticidal toxins, it is really only the biology of insect infection that
demands our immediate attention.
The infective juvenile nematodes are the vectors that actively seek out and
penetrate their insect hosts. They then individually regurgitate 50250 bacte-
rial cells from their guts directly into the insect hemocoel (Ciche and Ensign,
2003). Unfortunately from then on, the precise path of the insect infection and
the stages at which different toxins are expressed is still largely a black box
(ffrench-Constant et al., 2003). Part of this problem results from the difficulty
of relating bacteria grown in aerobic, nutrient-rich broth in the laboratory to
the different stages of insect infection and eventual death. In fact, only a very
few studies have attempted to correlate the production of putative anti-insect
virulence factors with their production in infected insects themselves (Daborn
et al., 2001). In reality, therefore, we remain with a series of isolated obser-
vations about different classes of toxins and a central failure to relate even
the most detailed biochemistry to any aspect of infection itself (ffrench-
Constant et al., 2003). While this continues to be a depressing concept for
the biologist, we have been lucky enough to be able to identify and isolate
a plethora of insecticidal toxins from Photorhabdus and these will, therefore,
be the focus of this review.

1.2. The unusual life cycle of Photorhabdus asymbiotica

Most attempts to develop a family tree for Photorhabdus divide the genus
into three species groups P. temperata, P. luminescens and P. asymbiotica
(Peat et al., 2010). The strictly insect-associated life cycle briefly described
above is undertaken by bacteria from the two groups termed P. temperata and
P. luminescens. These two groups seem to lack plasmids and have been widely
used with their nematode vectors in biological control. However, the third
group P. asymbiotica, which carry plasmids (Wilkinson et al., 2010), seems to
have evolved a recent and more sinister side to its life cycle. As its specific
name suggests, P. asymbiotica was originally named as such as it did not appear
to have a nematode vector, it was therefore literally termed a-symbiotic.
346 Richard H. ffrench-Constant and Andrea J. Dowling

However over time, a number of cases of human infection by P. asymbiotica

have been reported both from North America and from Australia (Gerrard
et al., 2004), and a vector nematode was eventually discovered after one
particular Australian patient had been digging in the soil with his bare hands
(Gerrard et al., 2006). While the number of cases of P. asymbitioca infection
can hardly be described as an epidemic, there are several factors we should
probably take into account in relation to both the evolution of human
virulence and also the continued use of these bacteria in biocontrol. (1)
At this stage, we cannot determine if the infection of humans is due solely
to inappropriate infection of patients by infective juvenile nematodes or if
there are accompanying changes in virulence factor expression, for example,
gains or losses of specific genes and/or changes in their genetic regulation.
(2) Because of the relative awareness/watchfulness of clinicians in America
and Australia, we cannot be sure that P. asymbiotica infections are also occur-
ring at low frequency in other countries and that they are simply not being
diagnosed as such. (3) What is clear is that all bacteria associated with human
infection seem to carry one or more plasmids and, so far, those used in crop
protection do not. So it would seem prudent to avoid the use of plasmid-
carrying bacteria in crop protection.
Why is all this relevant to a comprehensive review on insecticidal toxins?
First, the relationship between bacteriainvertebrate and bacteriavertebrate
interactions is clearly important from the point of view of disease emer-
gence, specifically, when the bacterial vector starts to change its host spec-
ificity. Second, if any of the insecticidal toxins discovered from the system
are seen to have any potential utility in crop protection, it may be useful to
have some understanding of their role in virulence in order to make
informed decisions about their likely mode of action and safety.

1.3. Xenorhabdus and comparative genomics

In a remarkable example of parallel evolution, Xenorhabdus bacteria have a very
similar lifestyle to Photorhabdus. Xenorhabdus are also vectored by symbiotic
entomopathogenic nematodes, this time from the family Steinernematidae.
The closer relationship between Xenorhabdus and Photorhabdus than between
either of their nematode hosts may suggest that their recent common ancestor
may have been able to colonize both Steinernema and Heterorhabditis nematodes
(Chaston et al., 2011). Xenorhabdus bacteria are also subdivided into several
species groups, specifically Xenorhabdus nematophila and Xenorhabdus bovienii.
Given the relatively small size of both Photorhabdus and Xenorhabdus genomes
Photorhabdus Toxins 347

(45 Mb) and recent rapid advances in next-generation sequencing, we are

beginning to see a steady stream of finished and draft genome sequences appe-
aring in the genome databases. As early as the year 2000, a simple sample
sequence (2000 random reads) from the genome of P. luminescens strain
W14 began to demonstrate that these bacteria were a potential gold mine
for novel bacterial effectors (ffrench-Constant et al., 2000). The first full
genome to be sequenced was that of P. luminescens strain TT01 which was
5,688,987 bp in length and contained 4839 predicted protein-coding genes
(Duchaud et al., 2003). At the time in 2003, the authors claimed that the
TT01 genome encoded more predicted toxins than any other bacterial
genomes sequenced. Some 10 years later in 2014, this claim may still hold true
as Photorhabdus genomes are literally stuffed with genes encoding proven or
putative toxins. Now we also have a finished genome for the North American
P. asymbiotica ATCC43949 (Wilkinson et al., 2009) and a draft genome of the
Australian P. asymbiotica strain Kingscliff (Wilkinson et al., 2010), both of
which can infect both insects and man. The genome of P. asymbiotica will
hopefully, therefore, not only be a source of novel insecticidal toxins but will
help us understand why the vector nematode sometimes infects man rather
than insects and indeed, having done so, how the P. asymbiotica bacteria them-
selves persist in the face of the human immune system. Several genomes for
the closely related Xenorhabdus bacteria are also now available and the com-
parative genomics of different Photorhabdus and Xenorhabdus stains has proved
to be a powerful tool in the identification of novel insecticidal toxins. The type
strain of X. nematophila ATCC 19061, a North American strain isolated from
the nematode symbiont S. carpocapsae, has been fully sequenced (Chaston
et al., 2011), and more recently, a draft genome of a French strain
X. nematophila strain F1 has also been completed (Lanois et al., 2013). For
X. bovienii, we also now have the complete sequence of strain SS-2004
(Chaston et al., 2011). The utility of all these genome sequences to the hunt
for Photorhabdus toxins will become clear when we examine how they can be
mined both in silico and biologically for novel effectors.

2. THE TOXIN COMPLEXES

Much of the original interest in Photorhabdus as a source of novel insec-
ticidal toxins was centred on the discovery that the cell-free supernatant of
certain strains was orally active to the model insect, Tobacco (tomato) horn-
worm (hawkmoth), Manduca sexta (Bowen and Ensign, 1998). It is worth
noting at the outset that the delivery of the Photorhabdus bacteria directly into
348 Richard H. ffrench-Constant and Andrea J. Dowling

the insects hemocoel does not make it obvious as to why any Photorhabdus
toxins are indeed active on the insect midgut, unless they are somehow able
to exert their effects from either side of the gut (gut lumen or insect hemo-
coel). This section, therefore, describes the discovery, purification, cloning
and structure of the toxins conferring this oral activity, namely the Toxin
complexes or Tcs, and finally attempts reconcile the mode of action of these
important toxins with their potential role in infection.

2.1. Tc discovery, gene cloning and ABC nomenclature

The oral activity of the cell-free bacterial supernatant of Photorhabdus was
first demonstrated in P. luminescens strain W14 (Bowen and Ensign,
1998). The toxicity was associated with large protein complexes with an
estimated molecular weight of >1,000,000 which migrate as single com-
plexes on a native gel but could be resolved into a range of different poly-
peptides (30200 kDa) under denaturing conditions (Bowen and Ensign,
1998). Four such million molecular weight complexes were isolated from
strain W14 and were termed Toxin complex (Tc) A, B, C and D or
TcA, TcB, TcC and TcD (Bowen et al., 1998). By raising a polyclonal anti-
body against a mixture of all four complexes, the loci encoding each com-
plex were identified from an expression library and the corresponding loci
were termed the toxin complex (tc) encoding loci tca, tcb, tcc and tcd, encoding
complexes A, B, C and D, respectively (Bowen et al., 1998). At the time, this
seemed like a remarkably convenient and appropriate nomenclature, with
four genomic tc loci encoding four final Tc complexes. However, this
nomenclature was to be undone by two further findings. First, each tc locus
was usually split into two or three separate open reading frames (Bowen
et al., 1998). Thus, individual open reading frames in the tca locus had to
be termed tcaA, tcaB and tcaC. Second, it rapidly became clear from the
growing number of genome sequences that there was no limit to the number
of potential tc loci in any one genome and that we were simply going to run
out of enough letters and/or numbers to name each locus sensibly.
The growing confusion around the rapidly growing tc nomenclature
came hand in hand with a more detailed understanding of what minimum
set of genes was required to reconstitute recombinant oral toxicity in
Escherichia coli. Specifically, expression of all three A, B and C subunit-
encoding genes (e.g. tcaA, tcaB and tcaC) was necessary to reconstitute
recombinant oral toxicity (Waterfield et al., 2001a; Yang and Waterfield,
2013). Moreover, for reasons we will return to later, expression of tcaB
Photorhabdus Toxins 349

and tcaC (encoding B and C) in the same bacterial cytoplasm was also essen-
tial (Waterfield et al., 2001a), whereas the tcaA open reading frame
(encoding A) could be expressed alone and then mixed with the products
of tcaB/tcaC (B and C) to reconstitute full oral toxicity (Waterfield et al.,
2001a). To put it most simply, a mixture of A, B and C toxin subunits
was required and, for some mysterious reason, B and C needed to be
made together in the same bacterial cytoplasm. This mechanistic explana-
tion of the A, B and C subunits also provided an instant cure to the growing
nomenclature problem as the massive list of gene names derived for different
tc loci within the same genome or the genomes of different bacteria, such
as Xenorhabdus, Serratia entomophila, Yersinia entomophaga and Yersinia pestis,
could all be resolved down to whether they encoded an A, B or
C subunit of these ABC toxins.

2.2. Diversity of tc-like genes from other bacteria

As discussed above, following the cloning of the tc genes from P. luminescens,
a number of tc-like genes were cloned from a range of other bacteria. These
were all given a range of very different names resulting in considerable con-
fusion in the resulting rather complex literature. For example, the tc-like
genes from S. entomophila are found on the amber disease-associated plas-
mid or pADAP which is responsible for amber disease in the New Zealand
grass grub, Costelytra zealandica. These were termed Serratia entomophila path-
ogenicity or sep genes A, B and C, specifically sepA, sepB and sepC (Hurst
et al., 2000). Insertion of a transposon into either of the three genes abolished
the gut clearance and cessation of feeding associated with amber disease
(Hurst et al., 2000) and of course, knowing what we now know from the
Photorhabdus tc genes above, it is now clear that they simply encode the three
different A, B and C subunits of the S. entomophila Tc-like toxin. Similarly
another bacterium, Y. entomophaga strain MH96, that kills the New Zealand
grass grub (Hurst et al., 2011) also carries tc-like genes. Here the three A,
B and C subunit-encoding genes are found in a 32-kb pathogenicity island
together with two chitinase genes (Hurst et al., 2011), whose functions will
be discussed below. In a more biochemical approach, Tc-like toxins were
also purified from X. nematophila strain PMF1296 (Morgan et al., 2001;
Sergeant et al., 2003). Here they were termed Xenorhabdus particulate toxins
or xpt and again genes encoding each of the A, B and C subunits are found,
namely xptA1, xptB1 and xptC1 (Morgan et al., 2001), although to add fur-
ther to the growing confusion the B and C subunit-encoding genes are in
350 Richard H. ffrench-Constant and Andrea J. Dowling

fact in a different order due to the split nature of the first gene in the
operon. Interestingly, and importantly, in some species of bacteria genes
encoding B and C subunits are found as gene fusions. In other words,
both the B and C subunits are encoded together in the same polypeptide.
In fact, Dow AgroSciences still holds patents that protect the fusion of
B and C encoding genes for their expression in transgenic plants (see
Section 2.6 on the use of Tc genes in crop protection below). This clearly
suggests that the B and C subunits must have some very close association in
formation of the final ABC complex, a point to which we will return below.

2.3. Structure, function and biophysics of the Tc ABC

complexes
2.3.1 A subunit assembly
While the list of genes encoding different tc-like homologs from different
bacteria continued to grow, a few laboratories began to focus on resolving
the structure of the Toxin complexes in order to begin to understand how
these huge structures actually conferred their toxicity. The first stoichiomet-
ric and biophysical studies of the Tcs were from Xenorhabdus. Here, using a
combination of biophysical techniques including electron microscopy, Lee
and co-workers suggested that the A subunits formed a large (1.15 MDa)
bottle-shaped tetramer with an open central channel (Lee et al., 2007). This
A subunit tetramer was inferred to bind to the insect brush border mem-
brane and then co-associate with B and C subunits. Four years later, this
A subunit tetramer was again shown to bind to insect brush border mem-
branes and to cause pore formation in black lipid membranes (Sheets
et al., 2011). This confirmed that it was an assembly of the A subunits that
were responsible for recognition and attachment to the insect gut but left the
role of the B and C subunits unclear. Further work on the Xenorhabdus Tc
subunits also showed that the B and C subunits bound together in a 1:1 stoi-
chiometry and that B/C then binds irreversibly to the A subunit assembly,
giving a final A, B, C stoichiometry of 4:1:1 (Sheets et al., 2011). Excitingly,
these studies also revealed that the A and B/C subunits from Photorhabdus
and Xenorhabdus were functionally interchangeable, suggesting that the
A attachment vehicle (four A subunits as a tetramer) could be combined with
B/C components from other bacterial species. In other words, the Tc toxins
could be mixed and matched (ffrench-Constant et al., 2007) and it did not
matter where individual component subunits came from as long as they were
combined in the correct manner (B and C subunits made in the same bac-
terial cytoplasm and then combined with A subunits). Similar studies of the
Photorhabdus Toxins 351

ABC complex from Y. entomophaga appeared to show, in contrast, that the

A subunits form a pentamer, rather than a tetramer (Landsberg et al., 2011).
Again in an apparent departure from the Xenorhabdus toxin, the Yersinia ABC
complex is also decorated with chitinase enzymes that confer chitinase activ-
ity on the purified native complex. This chitinase activity may be responsible
for the disappearance of the peritrophic membrane in the guts of toxin-
treated Diamondback moth, Plutella xylostella, caterpillars (Landsberg
et al., 2011) which could represent the first step in allowing the mature toxin
access to the brush border membrane.

2.3.2 Low-resolution structure of the Tc ABC holotoxin

At this point, we are left with a picture whereby the A subunits combine to
form either a pentamer (as suggested by the Yersinia data) or a tetramer (as
suggested by the Xenorhabdus data) with an open central channel. This cage-
like structure then attaches to the brush border membrane where it exerts its
toxic effect. However, the precise role of the integral channel and its inter-
action with the B and C subunits of the toxin had to await further detailed
analysis of the Photorhabdus toxin at 6.3 A resolution by cryo-electron
microscopy and single particle analysis (Gatsogiannis et al., 2013). This very
elegant work showed that the A subunits (formed from TcdA1-encoded
A subunits) formed a syringe-like pentamer with a molecular mass of
1.14 MDa and a long funnel-like central pore that is capable of conducting
ions. These channels are cation selective due to a ring of negative charges
displayed at the mouth of the channel. The structure of the Tc channel is
also very different from that of other AB-type toxins as the binding compo-
nents of anthrax, C2 and iota toxins all form -hairpins whereas the cryo-
EM structure of the Photorhabdus A subunit suggests it is composed entirely
of -helices with a minimal channel diameter of 1.5 nm (Gatsogiannis
et al., 2013). Further, the A subunit-mediated channel is reconstituted in
a fully oriented manner when added to only one side of the lipid bilayer
and its conductance can be blocked when either the B or the C subunits
are added to the same side of the membrane (Lang et al., 2013). This suggests
that B and C co-assemble with the A assembly in a very specific manner and
that their interaction blocks the pore formed by the A subunits themselves.
But what do the B and C subunits actually do and how are they involved in
toxicity?
The precise role of the C subunits of the Tcs was an intriguing mystery
due to the unusual structure of the tccC-like genes that encode them. These
genes clearly encoded proteins with three separate domains (Waterfield
352 Richard H. ffrench-Constant and Andrea J. Dowling

et al., 2001b). The central core of a tccC gene encodes a highly conserved
motif with similarity to sequences previously termed rearrangement hotspots or
rhs elements in E. coli. The N-terminal sequence is also well conserved but
the C-terminal tail is extremely variable. This variability of the C-termini of
tccC-like genes is found across all tccC genes in one genome or indeed across
all tccC-like genes in any bacterial genome. Critically, the laboratory of Klaus
Aktories has recently shown that two of these C subunits (termed TccC3
and TccC5) are ADP-ribosyltransferases that selectively modify unusual
amino acids with actin and Rho proteins, respectively (Gatsogiannis
et al., 2013; Lang et al., 2010, 2011). Specifically, TccC3 ADP-ribosylates
threonine-148 in actin, resulting in actin polymerization, and TccC5 ADP-
ribosylates Rho at glutamine-61/63, inducing Rho activation (Gatsogiannis
et al., 2013), and the net effect of these two enzymatic activities is to cause
extensive polymerization and clustering of actin in target cells. Intriguingly,
given that the active site of the ribosyltransferase is encoded within the
C-termini of the C subunits confirms the hypothesis that the C-termini
of the C subunits encode a range of different potential effectors
(Waterfield et al., 2001b). The tccC genes that encode the C subunits are,
therefore, a potential gold mine for the identification of further enzymatic
activities and translocated effectors. For example, the C-terminal end of one
C subunit from Y. pestis (termed YipA) carries a consensus sequence for pro-
tein tyrosine phosphatase (Spinner et al., 2012), suggesting one possible fur-
ther mode of enzymatic action. All of these data suggest a model whereby
the A subunits of the Tc toxins come together to form a syringe-like injec-
tion mechanism that somehow translocates the C-terminus of the C subunit
into the target cell where it exerts its toxic effect. This hypothesis is consis-
tent with data showing rearrangement of the A subunits upon membrane
insertion, suggesting that they do indeed act like a syringe for insertion
of the toxic moiety into the host cell. However, the precise interaction
of the B and C subunits and how it leads to the release of the C-terminus
of the C subunit awaited further detailed structural studies, this time again
from the Yersinia toxin.

2.3.3 Encapsulation and auto-proteolysis of the C subunit

The precise interaction of the B and C subunits had to await the crystalli-
zation of the Y. entomophaga B/C in complex and structural determination
to a resolution of 2.5 A. This crystal structure showed that the B/C complex
forms a large hollow structure that encapsulates the cytotoxic C-terminus of
the C subunit like the shell of an egg (Busby et al., 2013), thus presumably
Photorhabdus Toxins 353

protecting that host bacterium from any of the toxic effects that the
C subunit might exert on the host itself. The shell of the egg has a
-propeller domain at one end which it uses to attach to the A subunit in
the mature native ABC complex. Moreover, when the C subunit is folded
in complex with B, it auto-proteolyses to release its toxic C-terminus for
translocation into the target cell (Busby et al., 2013). This correlates
with the earlier observation that the Photorhabdus C subunit, specifically
TccC3, is cleaved at its hyper-variable sequence but only in the presence
of the B subunit, in this case TcdB2. This superb piece of work in
Y. entomophaga finally explains why the central core of the tccC-like genes
is so conserved, as it encodes the precise site at which auto-proteolysis occurs
and it must therefore always remain invariant for the C subunit cleavage to
work. The structure of the C subunit is also important as it is the first struc-
ture determined for proteins containing Rearrangement hotspot (Rhs)
repeats, similar structures to which are found in the eukaryotic tyrosine
aspartate YD-repeat-containing family, including the teneurins which are
type II integral membrane proteins that help establish neuronal cell connec-
tions during development (Hong et al., 2012). This may suggest that the Tc
B/C-mediated encapsulation device is used in other systems for protein
encapsulation and delivery. Indeed, recent work has suggested that
Gram-negative Rhs proteins and the distantly related wall-associated protein
A (WapA) from Gram-positive bacteria mediate intercellular competition in
bacteria. Specifically, Rhs and WapA carry polymorphic C-terminal toxin
domains that are deployed to inhibit the growth of neighbouring bacterial
cells (Kung et al., 2012). Similarly, RhsT from Pseudomonas aeruginosa PSE9
is translocated into mouse phagocytic cells and mice survive infection with
rhsT mutants, showing that RhsT is a real virulence factor (Kung et al.,
2012). These striking findings show that YD-repeat proteins in bacteria
are involved in contact-dependent growth inhibition and/or virulence,
and suggests that B/C-like YD-repeat containing egg shell structures could
play a more general role in protecting host bacterial cells from their delivered
bacterial toxins.

2.3.4 Insights from the high-resolution structure of the ABC holotoxin

During the final drafting of this chapter, Meusch et al. published a high-
resolution structure of the complete P. luminescens ABC Tc holotoxin at a
resolution of 4.0 A (Meusch et al., 2014), which now allows us to begin
to put all of the pieces of this complex puzzle together into a novel picture.
To add to the already confusing nomenclature, they call the Tc holotoxin
354 Richard H. ffrench-Constant and Andrea J. Dowling

PCT3 as it is composed of TcdA1, TcdB2 and TccC3 polypeptides,

whereby presumably PCT5 would contain TccC5. However, here
again we will try and ignore the over-complicated nomenclature and simply
refer to the A, B and C subunits of the Tc holotoxin. This exquisite
holotoxin structure reveals how the A subunits come together to form four
receptor-binding sites and a neuraminidase-like region. The neuraminidase-
like region is structurally homologous to neuraminidases and closes the bot-
tom of the pentameric Tc shell. Neuraminidases are exosialidase enzymes
that cleaves -ketosidic linkage between the sialic (N-acetylneuraminic)
acid and an adjacent sugar residue. The precise role of this domain in the
Tc toxins is unclear. However, the influenza virus neuraminidase may take
part in fusion of the viral and host cell membranes (Wagner et al., 2000),
suggesting that it may somehow be involved in docking of the Tc
A subunits onto the host membrane. The four receptor-binding domains
have an immunoglobulin-like -sandwich fold structurally similar to the
receptor-binding domains of the well-studied diphtheria and anthrax toxins.
However, it is notable that the Tc toxins have four such receptor-like
domains compared to other toxins that have only one or two (Choe
et al., 1992; Petosa et al., 1997). Meusch et al. (2014) argue that the very
closely spaced (only 8 A apart) amino- and carboxy-terminal regions of
the two insertions that form the receptor-binding domains suggest that
the inserted domains were added to an originally much simpler shell
(Meusch et al., 2014). The authors also argue that the variability of this
region between different Tc toxins might begin to explain their putative
host specificity. It is, however, worth reminding ourselves that in Photo-
rhabdus at least, host specificity (choice of insect host) is determined by
the nematode vector and not the bacterium. An alternative role for the
Tc receptor variability found in different toxins in the same bacterium is that
the different Tcs formed might target different tissues within the same insect.
To understand how the Tc toxin changes from its pre-pore to pore state,
Meusch et al. (2014) built a molecular model by fitting the crystal structure
of the A pre-pore into the cryo-EM structure. This analysis shows in incred-
ible detail how formation of the pore is associated with dramatic conforma-
tional changes that open the shell and shift the central channel by 12 nm.
This dramatic change in structure of the of the A pore complex is facilitated
by three defined hinge regions and results in a large-scale re-organization of
the four receptor domains (labelled Receptor-binding domain A, B, C and
D in Fig. 7.1), suggesting a potential alteration in their binding geometry
and/or affinity.
Photorhabdus Toxins 355

Figure 7.1 Ribbon diagram showing the TcA protomer in (A) pre-pore state and (B) in
pore-forming state. Receptor regions AD and neuramidase-like region are labelled,
along with the Tcb-binding domain, linker, pore-forming domain and -helical domains
(the small and large lobes). Note the dramatic changes that occur as the TcA protomer
switches from pre-pore to pore-forming states, specifically, the collapse of the elastic
linker, the extension of the pore-forming domain and the re-organization of the recep-
tor domains AD. Reprinted from Meusch et al. (2014) with permission from MacMillan.

How is the energy provided for such a dramatic change of conformation?

The answer comes from the linker (labelled Entropic spring in Fig. 7.2)
which connects that A subunit shell and the channel in the pre-pore state.
This linker has an unusually elongated structure with a length of 113 A cov-
ered by only 48 residues. The authors therefore suggest that this linker acts
like a stretched elastic band or entropic spring that is stretched in the pre-
pore state and can only achieve its preferred relaxed state when the shell is
opened and the channel extended. This begins to explain how the channel is
driven through the complex for entry into the host target membrane. The
concept behind this entropic spring mechanism can be seen very clearly in
Fig. 7.2 as the spring contracts to send the pore shooting down into the
356 Richard H. ffrench-Constant and Andrea J. Dowling

Figure 7.2 Another view of the structure of (A) Tca pre-pore and (B) pore-forming com-
plex. Note the entropic spring or linker (black), release of which provides the elastic
energy to drive the pore-forming channel down towards the membrane. The
neuramidase-like domain is shown in blue (see text for discussion). Reprinted from
Meusch et al. (2014) with permission from MacMillan.

membrane following the release of the electrostatic lock that otherwise

keeps the delivery vehicle in its pre-pore state.
A question remains as to how the C subunit toxin is delivered down this
dramatically extended pore? To answer this question, Meusch et al. also
solved the crystal structure of the B and C subunits in complex (Meusch
et al., 2014). To get around the fact that B and C subunits need to be made
together in the same bacterial cytoplasm, as discussed above, they made a
B/C fusion protein (by fusing the genes encoding TcdB2 and TccC3).
Although the P. luminescens B/C complex only shares 56% amino acid iden-
tity to the equivalent structure in the Y. entomophaga B/C complex (termed
YenB-YenC2), the eggshell-like structure formed by the B subunit and the
manner in which it encases the toxic C-terminus of the C subunit are almost
identical. Similarly, as suggested by the work in the Yersinia Tc toxin, the
C subunit acts as an aspartyl autoprotease with aspartates 651 and 674 for-
ming the catalytic dyad. This means that the toxic C-terminal of the
C subunit is again cleaved with the shell provided by the B subunit. This
does not explain how the C subunit is translocated down the very narrow
channel so dramatically extended by the A subunits and how it gets into the
channel in the first place?
The inner surface of the B/C-formed shell is positively charged and
contains large hydrophobic patches. Meusch et al. (2014) suggest that the
inside of the B/C shell creates a hostile milieu for protein folding
Photorhabdus Toxins 357

(Meusch et al., 2014). They propose that this hostile environment might be
able to directly unfold native proteins and suggest that the lack of resolution
of the cleaved ADP-ribosyltransferase domain residing in the shell is consis-
tent with it being either unfolded or in static disorder. This leaves us with a
picture of the unfolded ADP-ribosyltransferase domain sitting within the
B/C shell and awaiting translocation into the delivery channel formed by
the A subunits. So how does it get into the channel? The -propeller of
the B subunit interacts strongly with the highly conserved central funnel
formed by the A subunits, as shown in Fig. 7.3AC. The narrow gate
formed by this -propeller is open in the holotoxin, suggesting that it forms
the entrance to the delivery channel, as depicted in Fig. 7.3B. The open
-propeller pore is also hydrophobic and the authors suggest it therefore
plays a similar role to the -clamp in anthrax toxin which protects hydro-
phobic patches in the translocated protein.
The narrow passage of the -propeller suggests that the ADP-
ribosyltransferase must be unfolded before it passes this gate and critically
the authors found extra density (labelled in red in Fig. 7.4B) within the trans-
location channel after holotoxin formation, confirming the idea that the
unfolded ADP-ribosyltransferase passes through the -propeller gate and
enters the translocation channel before membrane permeation. Finally,
before we leave this description of a stunning new delivery mechanism, it
is worth returning to the unexplained diversity of potential toxins encoded
by the C-termini of different TccC-like proteins. Meusch et al. (2014) point
out that the C-terminal (hyper-variable) regions of TccC3 and TccC5 are
both cationic with isoelectric points of 9.68 and 8.65, respectively
(Meusch et al., 2014). This suggests that translocation of the ADP-
ribosyltransferase domains of TccC3 and TccC5, and indeed equivalent
domains from other unstudied TccC-like proteins are pH-independent. This
is in stark contrast to the pH gradient-dependent, unidirectional, transloca-
tion of anthrax lethal factor across the cation-selective anthrax translocation
pore (Brown et al., 2011). This also suggests that the other cleaved toxic
C-termini of other C subunits should also be cationic (have similar isoelectric
points) to take advantage of the same delivery mechanism.

2.3.5 Potential Tc chaperones, release factors and delivery co-factors

Finally, before we leave the detailed study of Tc structure and its mode of
action, it is worth noting that there are several unresolved questions relating
to both toxin complex assembly and toxin delivery itself. Specifically, what
are the roles of molecular chaperones in assembling the ABC complex itself?
358 Richard H. ffrench-Constant and Andrea J. Dowling

Figure 7.3 Interaction of the B/C (TcB/TcC) subunits with the A (TcA) subunit assembly.
(A) The B subunit (labelled TcB in blue) and the C subunit (TcC in yellow) interact to form
a large cocoon- or egg-shell-shaped structure. The B subunit also comprises the
A subunit-binding domain (labelled Tca-binding domain in light blue) which folds into
an asymmetric six-bladed -propeller. (B) A vertical section through the egg-shell-like
structure formed by B/C to show the upper and lower chambers and the pre-chamber
formed by the -propeller. Note that the gate out of the B/C chamber is closed. Subunit
domains are coloured: Tca-binding domain (light blue), TcB (dark blue), TcC (yellow) and
Tcb N-terminal region (red). (C) Cryo-EM structure of the ABC holotoxin. Subunits and
domains are labelled: TcA (grey) and TcB/TcC (yellow). The inset labelled TcBTcC shows
that the -propeller gate is open here. (D) Final molecular model of the ABC holotoxin
formed by fitting the crystal structure of the A subunits and the crystal structure of B/C
into the overall cryo-EM structure. Reprinted from Meusch et al. (2014) with permission
from MacMillan.
Photorhabdus Toxins 359

Figure 7.4 Vertical sections through the density of the ABC holotoxin channel.
(A) Section showing the continuous channel between the egg-shell-like structure of
B/C in yellow and the channel of the A subunits in grey. (B) Difference map between
the cryo-EM structure of the ABC holotoxin and the cryo-EM structure of the
A subunits alone. The difference map is overlaid in red. Note the extra density present
in the channel which may correspond to the delivered ADP-ribosyltransferase domain
of the C subunit (see text above for discussion). Reprinted from Meusch et al. (2014) with
permission from MacMillan.

How are such large proteins extruded through such a small pore and exactly
how are they unfolded and re-folded? How are the mature holotoxins
released from the bacterial outer membrane to be found free in the bacterial
supernatant? In relation to the first question, it is interesting that the ABC
complexes of Yersinia appear to be decorated with chitinases, whereas no
similar companions have been noted for Tc holotoxins from Photorhabdus
or Xenorhabdus. There are clearly several potential and simple explanations
for the lack of these co-inhabitants in Photorhabdus and Xenorhabdus. (1) It
may simply be that the operons studied in Photorhabdus and Xenorhabdus lack
nearby genes encoding chitinases. There is, therefore, the formal possibility
that chitinases will form partners in ABC complexes if encoded within, or
nearby, the tc-encoding locus itself. (2) The potential work of the chitinases
as chaperones (i.e. the presence of chitinases is required for correct folding
and assembly of the mature ABC toxin) might be performed by other part-
ners in other bacteria. To this end, it is noteworthy that we recovered
GroEL-related amino acid sequences from our original purification of
native P. luminescens W14 Toxin complex A (D. Bowen, M. Blackburn
360 Richard H. ffrench-Constant and Andrea J. Dowling

and R.H. ffrench-Constant, unpublished data). It is, therefore, possible that

this well-known molecular chaperone does indeed help fold and assemble
the massive ABC toxin itself.
Little work has been done to look at the localization of the mature Tc
holotoxins on the bacteria themselves. Using a polyclonal antibody raised
against the complete Tca holotoxin in P. luminescens W14, we showed that
the mature Tc particles are displayed on the outer membrane of the host
bacterium (ffrench-Constant et al., 2003). In some members of the
P. luminescens clade, the Tc complexes are released into the bacterial super-
natant when grown in culture, resulting in an oral toxicity to caterpillars of
the cell-free supernatant. In other members of this clade, the Tc toxins are
not released from the outer membrane when grown in culture in the
laboratory (Waterfield et al., 2001a). The release of the mature holotoxin
from the bacterial outer membrane is mediated by the pdl1-encoded
lipase, a gene that lies within the tcd locus of P. luminescens W14 but is lacking
from the equivalent region of the P. luminescens TT01 genome (Yang and
Waterfield, 2013). These data are therefore consistent with the hypothesis
that the PDL1 lipase releases Tc complexes from the outer membrane of
strain W14 but not from strain TT01. The precise role of such release in
the biology of insect infection is not shown and presumably for most strains
of Photorhabdus the Tc holotoxins must remain displayed on the bacterial
outer membrane, presumably for use only in a contact-dependent manner.
To determine if factors within the host cell itself can also influence Tc
delivery, perhaps by refolding the extruded C subunit toxin, Lang et al.
(2014) looked at the effects of different drugs which block different
co-factors in the target cell. They discovered that both the cellular uptake
and resultant toxicity of the two C subunits TccC3 and TccC5 could be
blocked by radiciol, cyclosporine A and FK506 or tacrolimus (Lang et al.,
2014). The blocking effect of these drugs implicates the molecular chaper-
one heat shock protein (Hsp) 90 and the peptidyl prolyl cis/trans isomerases
(PPIases) of the cyclophilin and FKBP families in the uptake or refolding of
the ADP-ribosylating C subunits. Specifically, given the tiny size of the pore
formed by the A subunits (1.5 nm minimal diameter), the authors specu-
late that these putative chaperones (such as Hsp90 and FKBP51) might play a
role in refolding translocated proteins after passage through the pore in an
unfolded state (Lang et al., 2014). In summary, there is clearly still much
we need to learn both about how these enormous ABC complexes are made
and secreted by bacteria and also how they exert their toxic action on the
host target cell.
Photorhabdus Toxins 361

2.4. The Tcs as polymorphic toxins

Before we leave the structure of this fascinating group of toxins, it is worth
looking at the Tcs in their broadest context; in other words, recognizing that
they belong to a large and diverse group of toxins recently defined as poly-
morphic toxin systems (Zhang et al., 2012). Polymorphic toxin systems are
characterized by an amazing diversity of C-terminal toxin domains which
appear to have been generated by recombination with standalone toxin-
coding cassettes. They are found across all bacterial lineages and are delivered
by seven distinct secretory systems including type II, V, VI, VII and the
PVCs (see Section 3), PrsW (a putative site 1 protease)-dependent and
MuF (a component of the phage prohead)-phage-capsid-like systems
(Zhang et al., 2012). These have a general domain template of (a) a traffick-
ing domain, (b) a series of repeats, (c) a pre-toxin domain, (d) a releasing
auto-peptidase domain and (e) a toxin domain. Note that the TcB and
TcC proteins share some of the characteristics of this system. Specifically,
TcB proteins have an N-terminal Salmonella plasmid-borne virulence factor
B-like trafficking domain, followed by an integrin-like -propeller, whereas
the TcC proteins carry multiple Rearrangement Hot Spots repeats followed
by a variable C-terminal toxin domain (as discussed above).

2.5. The role of the Tcs in the biology of infection

Despite our detailed knowledge about the structure, mode of action and even
biophysics of the Tcs, it is worth considering if any of this new knowledge
really tells us anything about their potential role in infection. For example,
it is notable that the bacteria that carry tc gene homologs have very different
lifestyles and only some of them currently have known associations with
insects, or other invertebrates, at any stage within their lifecycles. The most
notable bacterium outside of Photorhabdus and Xenorhabdus that carries copies
of tc-like genes is none other than Bacillus thuringiensis itself (Blackburn et al.,
2011). These tc-like genes in B. thuringiensis may be important in increasing
the potency of this well-known bacterium against insects particularly when
it comes to oral routes of infection. In contrast, other bacteria which carry
tc-like genes, such as Pseudomonas syringae pv. tomato (a plant pathogen),
Fibrobacter succinogenes (a cellulolytic bacterium found in ruminants) and Trep-
onema denticola (associated with periodontal disease), currently have no known
associations with insects. Of course, the simplest hypothesis is indeed that the
presence of tc-like genes within a bacterial genome somehow predicts an inter-
action with insects at some stage in the bacterias lifecycle. However, the
362 Richard H. ffrench-Constant and Andrea J. Dowling

natural history of most bacteria is far from complete and we are therefore only
left to speculate on the role of tc-like genes based on the bacteria for which
some insect-associated natural history is available. For example, in the well-
studied insect pathogen S. entomophila, the pADAP plasmid carrying A,
B and C subunit-encoding tc-like genes (sepA, sepB and sepC) clearly does
indeed cause amber disease in grass grubs (Tan et al., 2006). Similarly, three
tc-like genes are also found in Y. entomophaga, suggesting that the resultant
ABC toxin could have effects on the C. zealandica midgut (Hurst et al.,
2011). In fact, examination of the midgut histopathology of C. zealandica
treated with purified Yersinia Tc toxins (Marshall et al., 2012) is strikingly sim-
ilar to the effects seen on feeding purified native Photorhabdus holotoxin A to
M. sexta caterpillars (Blackburn et al., 1998). Specifically, first the peritrophic
membrane that surrounds the food in the gut is destroyed and then the micro-
villi of the midgut itself are disrupted. While such studies make for elegant
cross sections of the insect midgut, they may be failing to tell us much about
how the Tc toxins are really attaching to and destroying the midgut cells.
Thus, it is also noteworthy that the midgut histopathology of both Bt toxins
and indeed cholesterol oxidase (Purcell et al., 1993) are little different from
that of the Tc toxins. This histopathology may therefore simply reflect a com-
mon pattern of midgut self-destruction, whatever the proximal toxin or toxic
insult. However, taken together, the available data suggest that different insect
pathogens do indeed use the Tcs to disrupt the insect midgut, presumably
either from the gut lumen itself (Y. entomophaga or B. thuringiensis) or from
the hemocoel side (Photorhabdus and Xenorhabdus). Indeed, some pathogens
that carry tc-like genes, such as Y. entomophaga, have been shown to be able
to pass from the gut lumen into the hemocoel itself (Marshall et al., 2012),
suggesting the tc-like genes may somehow facilitate this passage across the gut.
Insects can also act as reservoirs or vectors of human diseases. To this end,
the role of tc-like genes in the plague bacillus Y. pestis has, therefore, attracted
considerable recent attention. In this case, Y. pestis is normally maintained by
flearodent enzootic cycles but is occasionally transferred to man through
the bite of an infected flea. Originally when the first genomes of Y. pestis
were sequenced, these tc-like genes were found disrupted in some strains
leading to the original hypothesis that their loss may somehow be involved
in increased pathogenicity to man. More recently, the genes encoding the
A and B subunits (termed yitAB and yitC) and two C subunits (the tccC
homologues yipA and yipB) have been shown to be largely intact in the
genomes of most other sequenced Y. pestis strains, suggesting that they
may in fact play an undescribed role in interacting with either the flea vector
Photorhabdus Toxins 363

or incidental human host (Spinner et al., 2012). The currently available data
on exactly what role the Tc toxins might perform in the unusual lifecycle of
the plague bacillus are however potentially conflicting. If we ignore the sug-
gestion that Y. pestis Tcs are type III secreted (Gendlina et al., 2007), which
now seems hard to believe given that they effectively form their own secre-
tion machinery, and concentrate on data available for tc gene expression, we
can see several interesting points. First, Y. pestis tc genes (yitAB and yitC) are
up-regulated in J774A.1 macrophages. Second, repression of tc genes via
knock-out of their yitR regulator allowed for increased phagocytosis of
Y. pestis isolated directly from fleas (Vadyvaloo et al., 2010). Suggesting that
the tc genes may be playing some role in inhibiting phagocytosis of the flea-
borne bacteria by the macrophages. Third expression of yitR, the regulator
of Y. pestis tc expression, is up-regulated in the flea itself (Vadyvaloo et al.,
2010) but the Tc proteins themselves do not play a detectable role in flea
infection or the ability to produce a transmissible infection (Spinner et al.,
2012). Fourth, the Y. pestis Tc proteins (A subunit protein, YitA and
C subunit protein, YipA) themselves are highly expressed in the flea but
not at the same temperature (22  C) in the laboratory (Spinner et al.,
2012). Fifth, Tc (YitA and YipA) production is higher at lower temperature
(<22  C) and is cut-off at 37  C, the temperature the bacterium would
reach after being released into a human, but the proteins persist for over
9 h (Spinner et al., 2012). Finally, Y. pestis Tc proteins are localized to
the outer membrane of the bacterium (Spinner et al., 2012), like the Tcs
of Photorhabdus. Taking all of this available evidence together, this suggests
that Tc proteins are made within infected fleas and then displayed on the
outer membrane of Y. pestis as it is introduced into mammalian host upon
flea feeding. This role would be consistent with them resisting phagocytosis
via rodent or human phagocytes after their release from the flea. Such a role
is consistent with them playing a role in pathogenicity against mammalian
hosts but potentially via the nature of the delivered C subunit toxin (i.e.
in preventing phagocytosis) rather than via the loss of insect-specific tc loci,
as originally suggested by the sequencing of the first Y. pestis genome.

2.6. The potential role of Tcs in crop protection

Aside from these major gaps in our understanding of how tc-like genes con-
tribute to lifestyle changes in the bacteria in which they are found, much of the
original interest around the tc genes was for their potential inclusion in trans-
genic crops, specifically as alternatives to the widely deployed toxins from Bt.
364 Richard H. ffrench-Constant and Andrea J. Dowling

Unfortunately our detailed knowledge of the structure and function of the Tcs
has not come hand in hand with their successful expression in transgenic crops,
despite their obvious potential utility as insecticidal proteins. Here we will not
provide a detailed breakdown of the patent literature on this area, which is
considerable, but it is worth considering why the expression of these genes
in plants is so technically demanding. First, before it was understood that
all three A, B and C subunits needed to be expressed in plants, considerable
effort was placed into making plants that only expressed A subunits, with the
underlying assumption that they would kill insects. For example, as a model
system the A subunit from P. luminescens W14 was expressed in transgenic Ara-
bidopsis and its toxicity to neonate M. sexta was examined (Liu et al., 2003).
We now clearly know that there are a number of reasons as to why we would
not expect this experiment to work. First, M. sexta (Tobacco or Tomato
hornworm) does not feed on this plant by choice, so while Arabidopsis is a use-
ful model for testing the success of expressing a large gene (the intact tcdA
mRNA expressed was 8 kb) and resulting protein (TcdA at 283 kDa) in
plants, we would not expect the feeding results to be very meaningful. Sec-
ond, and most importantly, apart from the potential for pore formation, it is
hard to see why expression of the A subunits alone would be toxic in the
absence of B and C subunits, specifically the delivered C toxin, unless binding
and pore formation by A subunits alone are indeed enough to trigger oral tox-
icity. However, despite all this, bioassays of 234 T0 lines transformed with
construct pDAB7026 (osm-tcdA) identified nine transformed lines that caused
100% mortality in neonate M. sexta infested on transgenic Arabidopsis (Liu
et al., 2003). These data are therefore consistent with the hypothesis that bind-
ing and potential pore formation by A subunits alone are enough to trigger
insect mortality, at least in this model insect. However, full recombinant tox-
icity clearly requires full assembly of the ABC holotoxin which to our knowl-
edge has not been successfully achieved in a transgenic plant. The simplest
reason for this is that expression of all three relatively large genes encoding
A, B and C subunits (or expression of A with a B/C fusion) is still beyond
the limits of current plant transformation technology or that the plant is simply
not able to perform all of assembly/chaperone functions required to assemble a
functional Tc holotoxin.

3. PHOTORHABDUS VIRULENCE CASSETTES

The recognition that the Tc toxins form part of the super-family of
polymorphic toxin systems also helps us understand the next toxin delivery
Photorhabdus Toxins 365

vehicle from Photorhabdus that we will discuss, termed the phage-like PVCs.
Belonging to a group of phage-tail-like particles termed tailocins, PVCs are
a fascinating structure with a potentially very interesting biology.

3.1. Discovery and organization of PVCs

Following the sequencing of the genomes of both P. luminescens TT01 and
P. asymbiotica ATCC43949, a large number of pro-phage-like loci were
noted in each genome and termed the PVCs. The number and location
of these phage-like loci differed between the different sequenced genomes.
For example, in P. luminescens four PVC encoding loci were found in
a tandem array between a type IV DNA conjugation pilus operon and a
mukB replicon partitioning gene. However at the equivalent position in
P. asymbiotica genome, only one PVC locus was found and in P. temperata
K122, the PVC loci have been replaced by a Mu-like phage (Yang et al.,
2006). This suggests that the PVC loci are not only phage-like in sequence
but also that they can readily move around and between bacterial genomes,
as also supported by the presence of the PVC-like anti-feeding pro-phage
(Afp) locus on the virulence plasmid pADAP in S. entomophila (as discussed
in Section 3.2). The PVC loci themselves have a specific overall structure
with 1215 open reading frames encoding a conserved PVC unit followed
by 24 loci encoding putative effector proteins. The conserved PVC unit
encodes caudate phage-derived proteins, such as the tail sheath protein
and gp19, which both form the tail tubule, gp25 which forms the baseplate
and a distinct clade of AAA + ATPases (AAA representing ATPases associ-
ated with diverse cellular activities) that are related to CDC48 (Yang et al.,
2006). This overall picture suggests that the PVC-Secretion System (PVC-
SS) is similar to the type VI secretion system in being derived from the tails of
pro-phage but differs in its associated AAA + ATPase, which in the type VI
system is a member of the ClpB clade (ClpV). We are therefore left with a
picture that both the type VI-SS and PVC-SS have both independently
evolved from pro-phage tails using different AAA + ATPases to recycle
the injection apparatus after deployment (Zhang et al., 2012).
The putative PVC encoded effectors appeared to correspond to proteins
with similarity to a range of different toxins including TccC, from the Toxin
complexes (discussed above); part of Makes Caterpillars Floppy 1 (Mcf1)
toxin (discussed in Section 4); LopT (a homologue of the Yersinia cysteine
protease YopT) an effector delivered by the Type III secretion system in
other bacteria and the Photorhabdus necrosis factor (Pnf; a homolog of
366 Richard H. ffrench-Constant and Andrea J. Dowling

Cnf ). The open reading frames encoding these putative effectors are also
regularly flanked by transposons, suggesting that the different effector genes
may be swapped between different PVC loci. This array of putative toxins
encoded at the end of each PVC locus, and the potential to swap these toxin-
encoding genes around, suggests that the PVCs also belong to the polymor-
phic toxin system super-group (Zhang et al., 2012), as discussed above for
the Tc-encoding genes. Zhang and co-workers have noted that several toxin
domains are secreted by the PVC-SS across most major bacterial lineages and
even the euryarchaea. Moreover, proteins secreted via the PVC-SS carry a
highly conserved metallopeptidase domain (identified by the motif
HExxHxxQ-E) N-terminal to the toxin domain itself. So again we see a
system, like the TccCs, whereby a constant N-terminal peptidase domain
can recombine to gather different variable C-terminal toxin domains (Zhang
et al., 2012) and again this suggests that these peptidase domains are likely to
act as auto-proteolytic domains that release the toxin during or after its secre-
tion by the PVC-SS. The C-terminal toxins found to be associated with
these PVC metalloproteases span an incredible diversity of nucleases, nucleic
acid deaminases, peptidases, pore-forming domains and several other enzy-
matic domains (Zhang et al., 2012).
PVCs can be readily expressed in recombinant E. coli and expression of
the proteins corresponding to the first three open reading frames of the
P. asymbiotica PVC carrying Photorhabdus necrosis factor (PaPVCpnf ) was
confirmed via 2D gel analysis and protein sequencing. Transmission electron
microscopy (TEM) of the recombinant PVCs revealed a phage-tail-like par-
ticle very similar to R-type pyocins (Yang et al., 2006). The PVCs are
30 nm wide but show a huge variety in length (up to 800 nm long)
depending on whether they are found in a contracted (needle within sheath)
or extended state (needle protruding from sheath). Interestingly, polyclonal
antibodies raised against the putative effector proteins do seem to label pro-
teins extruded from the PVC when anti-effector antibody-conjugated gold
particles are viewed under TEM (N.R. Waterfield and R.H. ffrench-
Constant, unpublished data). The candidate effector proteins also cause
extensive rearrangement of the actin cytoskeleton when expressed directly
inside transfected NIH-3T3 cells, consistent with them being bona fide effec-
tors. Finally, injection of larvae of the wax moth, Galleria mellonella, with
recombinant PaPVCpnf killed the larvae and caused dramatic rearrangement
of the actin cytoskeleton of hemocytes recovered from injected larvae (Yang
et al., 2006). Such effects disappeared when transposons were inserted into
the open reading frames encoding the putative effector proteins (Yang et al.,
Photorhabdus Toxins 367

2006). Taken together, these rather preliminary data support a hypothesis

whereby the PVCs are phage-tail-like delivery vehicles that export candi-
date effectors (encoded at the end of each of the individual PVC encoding
loci) into their target cells. However at this point, we have no idea as to what
cells are targeted and indeed what the full range of potential effectors might
be, as their predicted amino acid sequences yield few clues as to their likely
modes of action.

3.2. The role of PVC-like structures in other bacteria

To get some idea of how a phage-tail-like PVC structure might be playing a
role in the delivery of bacterial effectors to eukaryotic cells, we need to look
at the overall diversity of phage-like structures made by bacteria. Phages
with contractile tails, or myophages, infect a wide range of bacteria and
are important for our discussions here as they are related to several other
structures made by bacteria, including Photorhabdus. First, the important type
VI secretion system of bacteria is evolutionarily related to myophage tail
structures and shares several common components (Leiman et al., 2009).
Second, the bacteria killing bacteriocins, such as the R-type pyocins of
P. aeruginosa, are also closely related to phage (Nakayama et al., 2000). These
phage-tail-like bacteriocins have a contractile sheath, inner tube, baseplate
components and tail fibres, but lack a DNA-filled head and therefore cannot
replicate on their own. Third, the bacteriocin-like structures made by the
insect killing S. entomophila, where they are termed the Afp, and the meta-
morphosis inducing Pseudoalteromonas luteoviolacea, where they are termed
Metamorphosis-associated contractile structures or Macs (Shikuma
et al., 2014), are also very similar in their overall morphology to the PVCs
of Photorhabdus. But what does this tell us about the potential function of the
PVCs themselves? To address this question, we need to look in more detail
at the structure and biology of the Afp and the Macs and ask how they medi-
ate their specific bacteriaanimal interactions.
In S. entomophila, the genes encoding the Afp are found on the same
pADAP plasmid as those (sepABC) encoding the Tc-like ABC toxin.
The Afp toxin delivery system causes the initial cessation in feeding of
the New Zealand grass grub (C. zealandica) larva, and the Tc-like toxin then
causes clearance of the gut leading to amber colouration in the dying grub
(Hurst et al., 2000). Eighteen open reading frames in the pADAP plasmid are
associated with the anti-feeding activity and these appear to encode struc-
tures similar to different components of the sheathed tail of bacteriophage
368 Richard H. ffrench-Constant and Andrea J. Dowling

T4 and also some components of the type VI secretion system. Using cryo-
EM and electron tomography of negatively stained Afp particles, Heymann
et al. (2013) have recently shown that the Afp sheath is similar to that of the
T4 phage tail but with a different sub-domain arrangement of the polymer-
izing sheath proteins. The central channel, which presumably delivers the
putative effectors encoded at the end of the PVC-like locus, is similar in
diameter and axial width to the Hcp1 hexamer of the P. aeruginosa type
VI secretion system. This tube extends through the baseplate into
a needle, resembling the puncture device of the T4 phage tail. Interest-
ingly, the tube itself appears to contain either the delivered toxin or poten-
tially a ruler protein which may measure the length of the tube while
under assembly. Taken together, the available Afp data support a hypothesis
whereby one of two different candidate effectors, also encoded on pADAP,
is delivered by the modified phage tail into target cells in the insect midgut.
At the time of writing, however, the exact receptors for the Afp particle,
how it binds to the midgut cell and also exactly how the toxin is delivered
all remain a mystery.
More recently, another stunning piece of bacterial natural history has
been described around a second PVC-like structure, the Mac, of the marine
bacterium P. luteoviolacea. Larvae of the serpulid polychaete tubeworm,
Hydroides elegans, require contact with surface-bound P. luteoviolacea bacteria
in order to undergo metamorphosis. This has recently been shown to be
determined by an array of PVC-like phage-tail-like structures that the bac-
terium makes. These Mac arrays are formed from 100 contractile struc-
tures with outward-facing baseplates, linked by tail fibres and a dynamic
hexagonal net (Shikuma et al., 2014). These arrays are synthesized intracel-
lularly, are released by cell lysis and then expand extracellularly into an
ordered multi-Mac array. Previously, Huang and co-authors had specu-
lated that the Macs might be involved in transporting specific molecules
either into the biofilm matrix or into larval cells directly that then leads
to larval metamorphosis (Huang et al., 2012). The presentation of the Macs
in an ordered array may therefore suggest that their firing or delivery is syn-
chronized or that the array somehow guarantees the correct dosage of deliv-
ered effector to trigger metamorphosis when contacted by a settling worm
larva. Either way, this incredible piece of biology supports the idea that a
PVC-like structure can deliver an unknown bacterial effector into a marine
worm larva that is then triggered to undergo metamorphosis. This piece of
biology reminds us that the relationship between bacteria, phage and
eukaryotes is very ancient and that bacterial effectors that we view simply
Photorhabdus Toxins 369

as toxins due to their effects on isolated cells may in fact be effectors that
modulate exquisite aspects of bacteriumanimal interactions. Thus in the
case of the PVCs expressed and released by Photorhabdus in the infected
insect, these phage-like delivery vehicles might even be interacting with
the infective (dauer) juvenile nematode hosts and triggering their develop-
ment (metamorphosis) into reproductive hermaphrodites.
How do PVC-like secretion systems recognize their target cells? To
begin to come up with some hypotheses to answer this question, its notable
that some tailed phage also carry virion proteins that show peptidoglycan
hydrolytic activity (Moak and Molineux, 2004), suggesting that these are
used by the phage to penetrate the peptidoglycan cell wall during the deliv-
ery of DNA. Phage P2 gpX also carries LysM domains which have been
implicated in binding to both peptidoglycan and chitin, the latter of which
would clearly be important in the recognition of insect tissue. In conclusion,
and at this point in time, we are clearly not certain as to how the released
Photorhabdus PVCs bind to or indeed penetrate host cell walls but, given
the above, it is interesting to speculate that they may be redeploying mech-
anisms evolved to get into prokaryotic cells (Gram-positive and Gram-
negative bacteria) in their interactions with eukaryotic cells.

3.3. Implications for PVC biology

To conclude this section, the PVC-like tailocins are modified phage tails
that appear to be capable of delivering candidate effectors into target eukary-
otic cells. While the nature of the delivered effectors, and exactly how they
enter the target cell, remains unclear, what is clear is that PVC-like particles
seem to have clearly defined phenotypes (either death, cessation of feeding
or the triggering of metamorphosis). This seems to suggest that the PVCs are
binding to defined receptors in their target cells or tissues and that they can
recognize specific structures (e.g. to destroy midgut cells) or indeed specific
species (e.g. the settling Hydroides tubeworm larva). We can, therefore, spec-
ulate that the PVCs have defined receptors capable of recognizing displayed
sugars or proteins (like most phage) and that the effectors they deliver are
likely to interfere with deep physiological processes that alter the develop-
mental trajectory of the target tissue. In this context, it is fascinating that Pho-
torhabdus genomes carry so many PVC loci and it raises the interesting
possibility that the effectors they deliver may be involved both in destruction
of the insect midgut (like the Afp particle from Serratia) or indeed in trigger-
ing developmental changes in their nematode hosts (by analogy with the role
370 Richard H. ffrench-Constant and Andrea J. Dowling

of Macs in triggering tubeworm metamorphosis). This latter possibility is

particularly interesting given that Photorhabdus must somehow trigger repro-
ductive development in its nematode host following its delivery from the
infective juvenile, which is equivalent to the dauer juvenile of
Caenorhabditis elegans and is therefore effectively a resting stage.

4. THE MCF TOXINS

The Mcf toxins are another group of large insecticidal toxins that have
close relatives in other bacteria. Here we will look at their discovery in Pho-
torhabdus, their close relatives in Pseudomonas and what little we know about
their potential mode(s) of action. We will also begin to examine how the
discovery of the Mcf toxin led to a series of very simple but very effective
gain-of-function screens carried out in recombinant E. coli.

4.1. Discovery and mode of action of Mcf1

Due to the relatively small genome size (45 Mb) of Photorhabdus, it is easy to
envisage screening a relatively small number of recombinant E. coli clones
with large insert sizes 3050 kb. Although standard laboratory cloning
strains of E. coli lack specific secretion machineries, such as the well-studied
type III secretion syringe, if we are simply interested in looking for novel
toxins capable of their own export then a simple gain-of-function screen
becomes easy. To this end, we arrayed 50 kb cosmids from P. luminescens
W14 into 96 well plates and then simply grew up individual clones and
injected them into individual M. sexta caterpillars (Daborn et al., 2002).
On the face of it, the caterpillar immune system, which has both cellular
(hemocytes) and microbial (anti-microbial peptides) components, is another
good reason why such a screen should not work. For example, without any
mechanism of evading their own phagocytosis, all the recombinant clones
might be successfully engulfed by the Manduca hemocytes. However to
our surprise, we discovered that 1 in every 300 or so clones killed the
injected caterpillars within 24 h. These caterpillars not only died but within
12 hours they exhibited a significant loss of body turgor, in other words they
became floppy (Daborn et al., 2002). We bombarded this positive clone
with 126 transposons and then sequenced the complete 33 kb insert using
the transposons to sequence from. On retesting all these mutants, we found
that 39 insertions negated both the lethal and the floppy phenotypes and that
all 39 insertions were in a single long open reading frame (8.8 kb) termed the
makes caterpillars floppy gene or mcf. When sub-cloned alone, this gene still
Photorhabdus Toxins 371

had the ability to kill caterpillars and also caused apoptotic cell death in both
isolated insect hemocytes and also the midgut of injected caterpillars,
suggesting that Mcf may be promoting apoptosis (Daborn et al., 2002).
The mcf gene predicts a 2929-amino acid protein with a molecular
weight of 324 kDa. Homology searches suggested that this large protein
was divided into three overall domains (Daborn et al., 2002). First, the
N-terminal domain carries a domain similar to a Bcl2-homology 3-like
(BH3-like) domain (labelled as BH3 in Fig. 7.5), whose putative function
we will return to. Second, the central domain is similar to Clostridium difficile
toxin B over a region important for toxin translocation (labelled as Cyto-
toxin B-like in Fig. 7.5). Third, the C-terminus carries a region similar to
repeats in a Repeats in toxins (RTX)-like toxin from Actinobacillus
pleuropneumoniae (labelled as RTX in Fig. 7.5).
While none of these homologies are particularly striking, we wanted to
test the possibility that Mcf was promoting apoptosis via its similarity to a
BH3-domain-only protein. BH3-domain-only proteins are important in
the homeostatic balance of Bcl2 proteins in the outer membrane of the mito-
chondrion, and overexpression of BH3-domain-only proteins in mammals
causes mitochondrially mediated apoptosis (Budd, 2001). To test this
hypothesis, we looked at the effects of recombinant Mcf toxin on mamma-
lian cell lines. We found that Mcf-treated cells showed apoptotic nuclear

Figure 7.5 Diagram showing the inter-relationship between the different domains in
Mcf-like toxins. The Photorhabdus toxin Mcf1 carries similarities to the predicted
MCF1/SHE proteolysis domain (domain labelled SHE), a BH3-like domain (BH3) and
two regions that carry similarity to Repeats in toxins (RTX) toxins. The central domain
is similar to Clostridium difficile toxin B over a region important for toxin translocation
(labelled as Cytotoxin B-like). The Photorhabdus toxin Mcf2 has a different N-terminal
domain which carries similarity to the type III delivered plant effector HrmA (domain
labelled HrmA-like). Finally, the Mcf-like FitD toxin carries the SHE, BH3 and one of
the RTX-like domains but lacks similarity to RTX-like toxins at its C-terminus (see text
for further discussion).
372 Richard H. ffrench-Constant and Andrea J. Dowling

morphology, activation of the effector caspase-3 and DNA laddering after

6 h and the presence of cleaved PARP after 16 h (Dowling et al., 2004).
All of these cellular phenotypes could be prevented by incubation of the
treated cells with the apoptosis inhibitor zVAD-fmk. Further, transfection
of cells with only the N-terminal 1280 amino acids, which contains the
BH3-like domain, also triggered cell destruction (Dowling et al., 2004).
Taken together, these data are consistent with the hypothesis that Mcf causes
apoptosis and that the domain responsible is within the N-terminal 1280
amino acids of the protein.
To test the hypothesis that the BH3-like domain was indeed responsible
for the apoptotic activity of Mcf, we target the domain using site-directed
mutagenesis. A double mutant within the BH3 domain dramatically decreases
apoptosis in treated cells, again consistent with the hypothesis that Mcf is
acting via its BH3-like domain on the mitochondrion (Dowling et al.,
2004). Topical treatment of cells with the Mcf toxin also directly alters
mitochondrial membrane potential, causes the translocation of the Bcl2-
associated X protein to the mitochondrion and triggers the release of cyto-
chrome c. Further, and perhaps most convincingly, cells over-expressing
Bcl-xL, a member of the Bcl-2 family that is anti-apoptotic, are resistant to
Mcf-mediated apoptosis (Dowling et al., 2004). All of these data are consistent
with the idea that Mcf mimics a BH3-domain-only protein and thereby inter-
feres with the balance of Bcl-2-containing proteins in the outer membrane of
the mitochondrion, thus promoting mitochondrially mediated apoptosis.

4.2. Diversity of Mcf-like toxins

4.2.1 The Mcf toxin 2
Following the sample sequencing of P. luminescens strain W14, we also discov-
ered a second mcf-like gene in the W14 genome which we termed mcf2
(Waterfield et al., 2003). The mcf2 gene predicts another large toxin with a
similar three domain structure. However this time, the N-terminus of
the predicted Mcf2 shows similarity to the type III secreted plant avirulence
gene (HrmA) from the plant pathogenic bacterium P. syringae (see Fig. 7.5 for
location of HrmA-like region), the transient expression of which in tobacco
cells results in cell death. This suggested to us that the N-terminus of Mcf-like
toxins encodes the toxic activity and that this activity differs between Mcf-like
homologs. To test the role of the Mcf2 N-terminus, we tagged it with a
c-Myc antibody tag and showed that it was translocated to the nucleus of
transfected mammalian cells which were subsequently destroyed by an
uncharacterized mechanism (Waterfield et al., 2003).
Photorhabdus Toxins 373

4.2.2 The Mcf-like Fit toxin from Pseudomonas

In parallel work, an Mcf-like toxin was also found in Pseudomonas fluorescens
CHA0 (recently renamed as Pseudomonas protegens), a bacterium found in
the plant rhizosphere that can protect plants against fungi. This insecticidal
toxin, termed P. fluorescens insecticidal toxin or Fit, is also encoded by a
single long open reading frame ( fitD) which lies within the fitABCDEFGH
operon (Pechy-Tarr et al., 2008). The sub-cloned fit gene produced the
same floppy phenotype in injected Manduca larvae as the mcf1 gene.
Knock-out mutants of the fit gene also showed reduced toxicity to insects,
although they still killed injected insects presumably due to a similar level of
functional redundancy to that seen in single gene knock-outs in Photo-
rhabdus. In other words, both Photorhabdus and Pseudomonas genomes encode
so many genes with insecticidal activity that knock-out of one alone is not
sufficient to eliminate toxicity in the resulting mutant. This is a point that has
played a large part in our development of gain-of-function screens, and one
which we will return to below. Similarly, other rhizosphere-inhabiting
pseudomonads that naturally lack the fit-encoding locus also showed little
or no insecticidal activity, suggesting that Fit is indeed like Mcf a dominant
toxin. In conclusion, while the Fit toxin has a similar three-part domain
structure to Mcf1 and Mcf2, and even similarity to the consensus BH3
domain within its N-terminus, its precise mode of action is currently
unclear.

4.2.3 The MCF1-SHE domain as a novel serine peptidase

Before we leave our consideration of the primary structure of Mcf-like
toxins, it is worth returning again to the work of Zhang and co-workers
who have identified the MCF1-SHE domain as a possible serine peptidase
shared both by members of the polymorphic toxin super-family and also by
secreted effectors (Zhang et al., 2012). They initially identified this con-
served domain as a region shared by certain polymorphic toxins and the
PVC-SS toxins. However, iterative PSI-BLAST searches also revealed that
a similar domain is present in Mcf1, FitD and the phytotoxin HopT1-1
which is secreted by the type III-SS in P. syringae. The domain highlights
a putative catalytic triad and suggests that the C-terminus of Mcf1 and FitD
might be released by auto-proteolysis, in an analogous fashion to the release
of the C-terminal bullet from TccC. However, exactly how Mcf-like
toxins enter cells (presumably via endocytosis) and how they are cleaved
within their target cells (presumably within endosomes) remains unclear.
374 Richard H. ffrench-Constant and Andrea J. Dowling

4.3. Studying Mcf-like toxins in vivo

Both Mcf1 and the Mcf-like Fit toxin are dominant toxins that appear to act
quickly on their insect hosts. While it is possible to isolate hemocytes from
toxin-injected insects and to study the histopathology of other tissues such as
the midgut, these rather static end-point-mediated assays do not really tell us
much about how the toxins are really acting in vivo. Moreover, they do not
really tell us why such active toxins are not active on the host bacterium, or
in the case of Photorhabdus why they do not kill the nematode vector as well.
To this end, recent studies on Mcf-like toxins have focused on observations
of both toxin activities in vitro and in real time and also on their precise
genetic regulation in insects.

4.3.1 The Drosophila embryo as a microcosm for toxin mode-of-action

studies
To look at how quickly Mcf1 acts on insect hemocytes, Vlisidou and
co-workers have co-opted the Drosophila embryo system to look at toxin
mode of action in real time (Vlisidou et al., 2009). The Drosophila embryo
system was pioneered to look at the role of insect hemocytes in development
and wound repair but the system has also recently been used to look at the
effects of Mcf1 toxin on the hemocytes in real time. The advantage of this
system is that labelled hemocytes can be visualized in real time and the
response of these hemocytes to toxin action can be tested in Drosophila strains
of differing genetic background. In other words, the role of potential toxin
targets can be tested directly via genetic manipulation of the insect host. For
this study, a strain of the insect and human pathogen P. asymbiotica was
injected into Drosophila embryos and the response of the patrolling hemo-
cytes recorded using confocal microscopy of the whole embryo. Vlisidou
et al. (2009) found that injection of P. asymbiotica caused a dramatic freezing
of the hemocytes shortly following introduction of the bacteria into the
hemocoel. This was in stark contrast to wild-type E. coli which was phago-
cytosed by the embryonic hemocytes in a Dscam-independent manner
(Vlisidou et al., 2009). This freezing phenotype was seen both with injection
of purified Mcf1 and also with recombinant E. coli expressing the mcf1 gene,
suggesting that the recombinant bacteria make enough Mcf1 toxin and that
it is displayed/released in a biologically meaningful manner. This freezing
phenotype is accompanied by a dramatic rearrangement of the actin cyto-
skeleton of affected hemocytes (Vlisidou et al., 2009). Importantly, genetic
modulation of the different Drosophila strains injected with Mcf1 showed
Photorhabdus Toxins 375

that the freezing phenotype is shibire dependent (shibire is a temperature-

sensitive Drosophila dynamin mutant that results in the inability of endocytic
vesicles to be separated from the parent membrane), suggesting that endo-
cytosis of the Mcf1 toxin is required for toxicity, and that effects on the actin
cytoskeleton could be modulated by dominant negative or constitutively
active Rac expression. Taken together, these results not only elegantly illus-
trate how the Drosophila embryo can be used to test bacterial toxins in real
time and in the presence of different host cell backgrounds but also show
unexpected and very early effects of Mcf1 on the actin cytoskeleton. The
interaction between the BH3-domain-related apoptosis triggered by
Mcf1 and these early effects on the actin cytoskeleton should therefore form
a productive area for future research on this fascinating toxin.

4.3.2 Regulation of Fit toxin expression in the insect host

Finally, the broad spectrum of activity of Mcf-like toxins (the effect of Mcf1
on a range of insect and mammalian cells) suggests that they must be very
tightly regulated during insect infection; otherwise, toxin production might
kill other unintended partners, such as the host nematodes in the case of Pho-
torhabdus. To this end, recent studies on the regulation of the Mcf-like Fit
toxin in Pseudomonas are extremely interesting. As described above, the
fitABCDEFGH operon contains the fitD gene which encodes the Fit
Mcf-like toxin. However, fitF, fitG and fitH appear to code for regulatory
proteins. Until recently, the precise role of these candidate regulators was
obscure; however, recent work by Pechy-Tarr et al. has shown that expres-
sion of Fit in culture can be activated by over-transcription of fitG or dele-
tion of fitH, thus suggesting that the encoded proteins are Fit activators and
repressors, respectively (Pechy-Tarr et al., 2013). More recently, the same
group has shown that the histidine kinase encoded by fitF is the primary sen-
sor that signals the presence of the appropriate conditions for making the
toxin (Kupferschmied et al., 2014), which presumably activates a pho-
sphorelay from the histidine kinase to the receiver and phosphotransfer
domain of FitF. FitF is then thought to inactivate FitH via phosphorylation
of its conserved aspartate, as replacement of this amino acid locks the protein
in it repressing state. The periplasmic sensory domain of FitF is proposed to
have evolved via domain shuffling from a common ancestor with DctB (a
histidine kinase that regulates the uptake of C4-dicarboxylates in Prote-
obacteria), and the authors speculate that FitF is inhibited by plant-associated
small molecules which then block expression of the insecticidal toxin when
the bacterium is in the rhizosphere (Kupferschmied et al., 2014). Similarly,
376 Richard H. ffrench-Constant and Andrea J. Dowling

toxin expression may then be switched on in the insect by a combination of

the absence of plant-associated molecules and/or the presence of signals
within the insect. Either way, this begins to demonstrate how such a toxic
toxin can be tightly shut-off in situations where its production could be det-
rimental but quickly turned on as the bacterium switches to an insect host.
The equivalent genes for the control of Mcf1 in Photorhabdus are not known,
but a number of such sensor kinases such as PhoQ are present in the genome
and could play a similar role to limit toxin expression in the nematode host.

5. PATOX AND PHOTOX

The toxin classes we have discussed so far illustrate three very different
approaches to toxin gene isolation from Photorhabdus. First, in the case of the
tc genes, Tc proteins were purified, anti-Tc antibodies raised and then the
Tc-encoding genes were recovered from expression libraries. Second, in
the case of the PVCs, phage-like loci were expressed in recombinant
E. coli and found to produce visible phage-tail-like structures. Third, in
the case of Mcf, we have seen how gain-of-function screens can be used
to identify novel toxins with killing caterpillars being the simple gain-of-
function phenotype. Clearly, however, given the large amount of genomic
sequence now available for different strains and species of Photorhabdus, one
can also use bioinformatic tools to simply search for motifs found in other
toxins. This seems to have been the approach used in the discovery of
the next group of toxins we are going to talk about and it is the one that
has probably been underutilized in the analysis of all the candidate toxins
identified from Photorhabdus and Xenorhabdus genomics.

5.1. PaTox structure and function

Jank and co-workers ( Jank et al., 2013) have recently used BLAST
sequence alignment tool to look for sequence motifs in the P. asymbiotica
genome that are similar to those found in the glycosylating toxins of Clos-
tridium and Legionella. These toxins are characterized by an aspartic acid-
X-aspartic acid (or DxD) motif which is thought to be important for the
coordination of a divalent cation and for nucleotidesugar binding
(Wiggins and Munro, 1998). Jank et al. (2013) discovered a gene in the
genome encoding a putative glycosylating toxin which they termed
P. asymbiotica Toxin or PaTox. They then cloned the associated gene, puri-
fied the PaTox protein and tested its activity by injecting it into larvae of the
Greater wax moth, G. mellonella. They found that the full-length toxin killed
Photorhabdus Toxins 377

all of the injected larvae in 88 h. They also found that when they changed the
DxD motif in PaTox to asparagine-X-asparagine (NxN), 61% of the larvae
survived ( Jank et al., 2013). The authors also tested the role of the putative
glycosyltransferase domain (termed PaToxG) in inhibiting phagocytosis by
macrophages using protective antigen (the component of anthrax toxin
responsible for binding and translocation) as a delivery system. Strikingly,
PaToxG strongly inhibited phagocytosis of labelled E. coli, an effect that
was eliminated when the DxD motif was again changed to NxN. Similarly,
the PaToxG domain also caused disassembly of actin filaments in HeLa cells,
suggesting that phagocytosis is blocked via interference with the actin cyto-
skeleton. Jank and others went on to solve the structure of PaTox using
X-ray crystallography to a resolution of 1.8 A ( Jank et al., 2013). PaTox
is extremely interesting as it effectively performs two functions and the
crystal structure helps us understand how it performs both functions. First,
it targets Rho proteins (like TccC5 which is delivered by the Toxin Com-
plex Secretion System discussed above) by glycosylation of tyrosine
(GlcNAcylates) at Y32. Second, it also attacks heterotrimeric G proteins
via deamidation. Interestingly, P. asymbiotica does not carry a copy of TccC5,
and therefore, the authors only detected tyrosine GlcNAcylation during
P. asymbiotica infection of insect cells but not following infection with
P. luminescens. This might suggest that PaTox is somehow unique to, or
required by, Photorhabdus strains that can infect man but this hypothesis
remains to be tested.
The overall structure of the toxin is a single-chain AB toxin with two
catalytic domains within the C-terminus and a receptor-translocation
domain at the N-terminus ( Jank et al., 2013). Much like Mcf, its precise
mechanism of entry into the cell is uncertain, but it has been speculated that
it enters host cells from early endosomes ( Jank et al., 2013). The crystal
structure of PaToxG shows a unique GT-A fold, which is the smallest found
in currently known glycosyltransferase toxins, and lacks extending sub-
domains. As these sub-domains are critical in distinguishing Rho from
Ras as targets for enzymic attack, PaToxG is proposed to target Rho in a
highly specific manner. Downstream of the glycosyltransferase domain in
the crystal structure is a deamidase with amino acid similarity to Salmonella
SseI, and this PaTox domain is termed PaToxD ( Jank et al., 2013). Salmonella
SseI is secreted into host cells by the SPI-2 type III secretion system and is
thought to control the migration of host immune cells (McLaughlin et al.,
2009). The SseI-like domain of PaTox activates Gi and Gq/11 via
deamidation of a critical glutamine residue involved in the GTP hydrolysis
378 Richard H. ffrench-Constant and Andrea J. Dowling

of the G proteins. However, because PaTox appears to activate several dif-

ferent heterotrimeric G proteins, the downstream consequences of this
activation are currently unclear. What is clear, however, is that the preferred
substrate of Rho inhibition by glucosylation is the GTP-loaded form of
Rho, suggesting that the two functions are closely linked. In conclusion, this
elegant structurefunction analysis of PaTox has shown that the same toxin
(PaTox) causes both activation (via PaToxD) and inactivation (via PaToxG)
of the same Rho protein target. Although glycosylation-induced inactiva-
tion appears to be the dominant activity, this suggests that PaTox is designed
to precisely modulate the activity of Rho in a delicate balance between acti-
vation and inactivation. Perhaps more importantly, this begins to suggest
that the other large toxins made by Photorhabdus, such as Mcf, may also carry
more than one enzymatic activity. Indeed, this may thus begin to explain the
complex phenotypes observed following the treatment of different cell types
with Mcf.

5.2. Photox as a novel mART toxin

Another Photorhabdus toxin was identified using bioinformatic approaches in
the mono-ADP-ribosyltransferase (mART) toxin termed Photorhabdus
toxin or Photox. The plu0822 gene was identified as the putative gene
encoding mART toxin protein using an array of parallel bioinformatic
approaches that basically looked for the characteristic primary amino acid
sequence and predicted folding pattern of the mART toxins (Visschedyk
et al., 2010). Amino acid alignments over the three key regions (termed
regions 13) of similarity provided strong support for the hypothesis that
plu0822 encodes a mART toxin. Specifically, in region 1, the catalytic
Arg288 is preceded by a Tyr. In region 2, the Ser-Thr-Ser motif is preceded
by aromatic and hydrophobic residues. Finally in region 3, both primary and
secondary glutamate residues are found, namely Glu355 and Glu353. The spe-
cific site of Photox modification of actin was analyzed in vivo using a yeast-
based assay system and showed that Photox specifically ADP-ribosylates
actin at Arg177. Photox has an unusual domain structure with the
N-terminal domain (residues 1185) lacking any similarity to other toxin
domains and being predicted to be disordered in structure. The authors
speculate that this N-terminal domain may be involved in entry of Photox
into the target cell, potentially using VgrG as a translocator during type VI
secretion (Visschedyk et al., 2010). In conclusion, Photox is clearly a novel
mART that ADP-ribosylates actin, but the exact point at which it is
Photorhabdus Toxins 379

expressed during Photorhabdus infection and therefore its potential interac-

tion with other toxins that alter actin assembly remains unclear.

6. BINARY TOXINS
Finally, we will briefly review the binary toxins that have been found
in either Photorhabdus or Xenorhabdus. These toxins are clearly smaller and
potentially easier to get to grips with than the large Photorhabdus toxins
and toxin delivery systems discussed so far. They may, therefore, also be eas-
ier to clone and utilize in insect control.

6.1. The PirAB binary toxins

The products of genes (plu4093-plu4092 and plu4437-plu4436) in
P. luminescens TT01 show oral activity against mosquito larvae (Waterfield
et al., 2005). Because the encoded proteins showed similarity to both
delta-endotoxins from B. thurigiensis and also a developmentally regulated pro-
tein from the Colorado potato beetle, Leptinotarsa decemlineata, they were ter-
med the Photorhabdus insect related or Pir toxins. The developmentally
regulated beetle protein has been inferred to possess juvenile hormone esterase
activity but the PirAB toxins themselves show no such activity (Waterfield
et al., 2005). Instead, they show injectable activity against the wax moth,
G. mellonella, but only when injected together and not when injected individ-
ually, suggesting that both pirA and pirB are necessary as a binary toxin to
cause insecticidal activity. The mode of action of these interesting proteins
is still unclear but they are clearly potentially useful as mosquito larvicides.
Specifically, PirAB shows good activity against the larvae of both Aedes aegypti
(the vector of Dengue fever) and Aedes albopictus, while not affecting the Mes-
ocyclops thermocyclopoides predator (Ahantarig et al., 2009). Photorhabdus PirAB
may, therefore, form a useful alternative to toxins from B. thuringiensis and
Bacillus sphaericus in the control of mosquito larvae.

6.2. The XaxAB and YaxAB cytotoxins

The XaxAB cytotoxin was first purified from the supernatant of
X. nematophila grown in laboratory culture and was shown to have activity
both against insect hemocytes and mammalian red blood cells (Vigneux
et al., 2007). Pairs of genes encoding similar proteins are found in the insect
pathogens P. luminescens (where they would presumably be termed paxAB)
and Pseudomonas entomophila, the plant pathogen P. syringae and the human
380 Richard H. ffrench-Constant and Andrea J. Dowling

pathogens Yersinia enterocolitica (see following discussion) and Proteus mirabilis.

The XaxAB toxin appears to trigger apoptosis in treated HeLa cells, and pre-
treatment of the cells with the pan-caspase inhibitor z-VAD-fmk inhibits
XaxAB cytotoxicity. Interestingly, maximal haemolytic activity is observed
with exactly equal concentrations of both XaxA and XaxB. Moreover, addi-
tion of XaxA then XaxB in that specific order is required for toxicity. This is
interesting and hard to understand that in some AB toxins, the A subunit
carries the enzymatic activity and the B subunit is required for toxin binding,
in which case one might expect the binding subunit (B) to have to be added
before the toxic A subunit (Barth et al., 2004). Most recently, the activity of
the XaxAB-like toxin in P. luminescens TT01 (here termed PaxAB), encoded
by plu1961 and plu1962, has also been examined (Zhang et al., 2014). Again
both PaxA and PaxB are required for insecticidal activity and in combination
showed effects on a Spruce budworm, Choristoneura fumiferana, a midgut-
derived cell line FPMI-CF-203/2.5 or CF-203. Finally, homologous pro-
teins to Xenorhabdus XaxAB are also found in Y. enterocolitica and have been
named YaxAB (Wagner et al., 2013). Mutant Y. enterocolitica lacking yaxAB
genes shows similar final levels of infection as wild-type bacteria, but the
time course of infection is delayed and a different pathology results in the
mouse spleen. Again, the two proteins YaxA and YaxB are required for
cytotoxicity which appears to be related to osmotic lysis triggered by pore
formation. Indeed, protein modelling work shows similarities between
YaxA and pore-forming toxins from Bacillus cereus (HBL-B) and E. coli
(HlyE). Taken together, these data are consistent with the hypothesis that
YaxAB is a pore-forming toxin that plays an important role in the progres-
sion of mouse infection. Overall, this suggests that the XaxAB-like binary
toxins demand further work on their specific mode of action.

7. CLASSICAL SECRETIONS SYSTEMS AND NOVEL

SCREENS
7.1. Type III and other classical secretion systems
It is not the intention of this review to produce another long list of the
incredible diversity of secretion machinery found in the genomes of Photo-
rhabdus and Xenorhabdus. If the reader is specifically interested in toxin secre-
tion, then the reader is referred to previous reviews which list all known
secretion systems as having representatives in P. luminescens except for those
of the type IV secretion system (Rodou et al., 2010). Many of these classical
secretion systems, such as the molecular syringe encoded by the type III
Photorhabdus Toxins 381

secretion system, are clearly important in Photorhabdus infection but gener-

ally they are more highly described in other more human pathogenic bac-
teria such as Salmonella. That is not to say that study of the P. luminescens type
III secretion device does not demand attention. Thus, elegant early studies
by Brugirard-Ricaud et al. showed that Photorhabdus LopT, which is homol-
ogous to the Yersinia cysteine-protease YopT, could be translocated into
mammalian cells in an active form when heterologously expressed in Yersinia
(Brugirard-Ricaud et al., 2005). Moreover, the Photorhabdus lopT gene itself
only appears to be transcribed in interactions with insect hemocytes, and
knock-out of the type III secretion system increased phagocytosis and
reduced nodulation (the formation of a melanin case around invading bac-
teria by a body of hemocytes). It, therefore, appears that the delivered LopT
effector is important in reducing the phagocytosis of Photorhabdus and also in
reducing the capacity of the insect hemocytes to envelop the invading bac-
teria in a melanin nodule. Similarly, Photorhabdus type III secretion system
also delivers the cycle-inhibiting factor, named Cif (Chavez et al., 2010).
Cycle-inhibiting factors induce the arrest of the cell cycle at the G2/M or
G1/S transition by catalyzing the deamidation of a specific glutamine residue
(Gln40) in the Neural-precursor-cell-Expressed Developmentally Down-
regulated 8 (NEDD8) protein and the related protein ubiquitin (Crow
et al., 2012). This is interesting from an evolutionary perspective as type
III effectors often act as proteases or phosphatases but here a cysteine-
protease-like fold in Cif has been adapted to perform a deamidation reaction.
The P. luminescens Cif blocks the cell cycle at the G2/M transition and trig-
gers apoptosis in treated cells. Cif is expressed in the hemolymph of infected
insects but is not essential for anti-insect virulence. Both of these elegant
studies therefore show that the type III secretion system is deployed during
infection of the insect by Photorhabdus and that an array of other undocu-
mented effectors probably also have subtle but critical effects on the resulting
infection.

7.2. RVA-like screens for novel effectors

Given the brief discussion of the array of classical secretion systems present in
Photorhabdus, it does not seem likely that expressing random fragments of
Photorhabdus DNA in laboratory strains of E. coli which are not secretion
proficient or indeed armed with any specific secretion machinery (such as
the type III-SS) would be useful in producing biologically active toxins.
However, we reasoned that if a toxin was sufficiently toxic (like Mcf1) or
382 Richard H. ffrench-Constant and Andrea J. Dowling

if a toxin effectively encoded its own secretion system (like the Tc secretion
system or TC-SS), then it should be possible to perform simple gain-of-
function screens to look for novel toxins active in recombinant E. coli.
We, therefore, designed a suite of simple gain-of-function screens that
we termed Rapid Virulence Annotation or RVA (Waterfield et al.,
2008). The first RVA-like screen obviously led to the discovery of Mcf1
and simply involved injecting individual recombinant E. coli clones carrying
large (3050 kb) insert sizes. As discussed above, this rapidly led to the dis-
covery of the dominant Mcf1 toxin which has then also served as a useful
positive control to test for the quality and insert size of any screened library.
Since Photorhabdus has to interact with, and suppress, a range of different
organisms, including its host nematode and a range of saprophytes such as
bacteria, nematodes and fungi that invade the infected insect corpse, we
expanded these simple screens to look at different insects, the model nem-
atode, C. elegans, amoebae (as models for phagocytosis), insect phagocytes
and macrophages (mammalian phagocytes). These screens are not only
highly effective in recovering loci that encode toxic proteins but also recov-
ered polyketide synthase and non-ribosomal peptide synthase encoding loci
that make a range of different small molecules and peptides (Waterfield et al.,
2008). Again, it is not the point of this review to simply provide a shopping
list of all the hits from these screens, which are reported elsewhere
(Waterfield et al., 2008), but to simply point out that the screens have rev-
ealed a further wealth of potentially bioactive proteins and small molecules
from Photorhabdus that demand further detailed investigation.

7.3. Clustering methods to look for novel effector phenotypes

Finally, one might question the validity of isolating hundreds of new recom-
binant clones without the ability to rapidly characterize their potential
modes of action. To this end, we have used high content microscopy and
cluster analysis of the resulting phenotypic data to assign candidate clones
into their likely phenotypic clusters (Dowling and Hodgson, 2014). In other
words, we have taken detailed measurements of everything from treated cell
shape, cell density, nuclear morphology, actin clustering and mitochondrial
permeability to provide a snapshot of the treated cell. By then clustering
unknown effectors with effectors for which we have a known mode of
action, we can determine whether any new candidate effectors fall into clus-
ters with known toxins or if their phenotype(s) suggest a novel mode of
action (Dowling and Hodgson, 2014).
Photorhabdus Toxins 383

8. PERSPECTIVES FOR THE FUTURE OF PHOTORHABDUS

TOXINS
Since 1998 and the first descriptions of the orally active Toxin com-
plexes, we have come a long way in our understanding of Photorhabdus
toxins. The study of Photorhabdus has been important not only because of
the promise of producing an orally active agent for insect-resistant transgenic
crops, but perhaps more importantly because Photorhabdus has now led the
way in highlighting new secretion systems such as the TC-SS and the PVC-
SS. Where do we go from here? Clearly, one central rate-limiting step in the
discovery of insecticidal toxins is the assumption that they must be orally
active against insects in their native recovered state. In this context, since
we now understand something about how Photorhabdus toxins are actually
delivered to their target cells, this demands a more thorough investigation
of the receptors on those target cells and, therefore, of the likely tissue spec-
ificity (insect vs. mammalian or insect gut vs. insect hemocyte for example)
of any delivery device. In fact, Raunser and co-workers specifically imply at
the end of their beautiful paper on the mode of action of the Tcs that the
A subunit receptors could be specifically modified or adapted to act as a drug
delivery syringe in medicine. This makes the critical point that any given
toxin is not specifically insecticidal because of the nature of the toxin that
it delivers, which may be equally active on the cellular components of both
insects and man, but that it is the nature and specificity of the cell surface
receptor that must dictate a specific interaction with the insect midgut.
Clearly, any successful use of Photorhabdus toxins in agriculture must there-
fore demand a further detailed investigation of their receptors in the insect
midgut.

ACKNOWLEDGEMENTS
We would like to thank all members of the Richard ffrench-Constant laboratory, past and
present, for their work on Photorhabdus. We would also like to thank the BBSRC,
Leverhulme Trust and Royal Society of London for research funding.

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 CHAPTER EIGHT

Methods for Deployment

of Spider Venom Peptides
as Bioinsecticides
Volker Herzig*, Niraj S. Bende*, Md. Shohidul Alam*,
H. William Tedford, Robert M. Kennedy, Glenn F. King*
*Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University
of Queensland, St. Lucia, Queensland, Australia

Vestaron Corporation, Kalamazoo, Michigan, USA

Contents
1. Introduction 390
2. Spider Venom Peptides as Bioinsecticides 391
3. Transcytosis of Spider Venom Peptides Across the Insect Gut Epithelium
via Fusion to Molecular Transport Vehicles 394
4. Use of Entomopathogens for ISVP Delivery 397
4.1 Entomopathogens as bioinsecticides 397
4.2 Entomopathogens as a delivery vehicle for ISVPs 398
5. In Planta Expression of Spider Venom Peptides 399
6. ISVP Mimetics 401
6.1 Synthetic mimics of venom peptides 401
6.2 Hv1a pharmacophore analysis and modelling 402
6.3 Optimisation and mechanism of action of the Type II mimic VNX-440 405
6.4 Conclusions from development of Type-II mimetics of Hv1a 407
7. Outlook 407
Acknowledgements 408
References 408

Abstract
Despite intensive control measures, insect pests cause enormous damage to crops and
stored grain. In addition, insects vector some of the world's most devastating human
diseases, including malaria, dengue and Chagas disease. Chemical insecticides remain
the dominant method of controlling insect pests but the arsenal of extant insecticides is
rapidly diminishing due to the evolution of resistance in pest species and a more dif-
ficult regulatory environment due to heightened concern about the potential adverse
impacts of chemical insecticides on human health and the environment. Along with
predatory beetles, spiders are the most successful insect predators on the planet and

Advances in Insect Physiology, Volume 47 # 2014 Elsevier Ltd 389

ISSN 0065-2806 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800197-4.00008-7
390 Volker Herzig et al.

their venoms contain a diverse array of small, disulfide-rich insecticidal peptides. In addi-
tion to being potent and highly selective for insects, they collectively offer very diverse
pharmacology and should degrade to innocuous breakdown products in the field.
However, their major disadvantage is a low level of intrinsic oral activity. Consequently,
much research has been directed towards improving the oral activity of these peptide
toxins or, alternatively, obviating the oral activity problem altogether by incorporating
transgenes encoding the toxins into suitable biological delivery vehicles. Here, we dis-
cuss recent advances in both of these areas. We also discuss an approach that merges
the advantages of small-molecule and peptide-based insecticides, namely using the
pharmacophore of an insecticidal spider-venom peptide to rationally develop a
small-molecule mimetic that has improved oral activity, but retains activity for a novel
insecticide target.

1. INTRODUCTION
The human population is projected to reach to 9.3 billion by 2050
(Bloom, 2011). This expansion of the population will create enormous pres-
sure on food production since there is limited scope for increasing the
amount of cultivated land (Oerke, 2006). Despite intensive control mea-
sures, herbivorous insects reduce world crop yields by 1014% (Oerke,
2006) and damage as much as 30% of stored grain (Boyer et al., 2012),
and hence their control is a key factor in determining food productivity.
Increases in human population density combined with global warming
and a dramatic escalation in international travel has also placed considerable
pressure on the public health sector to control the spread of insects that vec-
tor human diseases such as malaria, dengue and Chagas disease.
Unfortunately, the arsenal of chemical insecticides available to combat
insect pests is in rapid decline. This is largely due to the fact that most chem-
ical insecticides target a handful of enzymes, receptors and ion channels in
the insect nervous system, and consequently their long-term use has led to
widespread resistance in pest species (King and Hardy, 2013; Smith et al.,
2013). Concerns about the adverse impact of certain classes of chemical
insecticides on the environment and human health have also led to wide-
spread de-registrations and use cancellations; for example, over the period
20052009, the U.S. Environmental Protection Agency (EPA)
de-registered or limited the use of 169 insecticides, while only nine new
insecticides were registered during the same time period (Windley et al.,
2012). In simple terms, we have to combat an increasing insect pest problem
with a rapidly diminishing chemical arsenal.
Spider Venom Peptides as Bioinsecticides 391

2. SPIDER VENOM PEPTIDES AS BIOINSECTICIDES

Spiders are among the most successful and abundant insect predators
on the planet. They play a key role in keeping insect populations at bay in a
wide range of ecosystems (Nyffeler and Sunderland, 2003). During more
than 300 million years of co-evolution with insects, spiders not only evolved
sophisticated ways of using silk for prey capture (Blackledge et al., 2009;
Vollrath and Selden, 2007), but also acquired powerful venoms that rapidly
paralyse insect prey. Spider venoms are complex chemical cocktails of salts,
small organic molecules, peptides and proteins, but the dominant compo-
nents in most spider venoms are small, disulfide-rich peptides (King and
Hardy, 2013; Kuhn-Nentwig et al., 2011). Individual spider venoms can
contain more than 1000 small peptides (Escoubas et al., 2006). These pep-
tides simultaneously attack virtually every component of the synaptic
machinery in the central or peripheral nervous system of envenomated prey,
which is tantamount to shock-and-awe at the molecular level
(Ikonomopoulou and King, 2013; King and Hardy, 2013).
Given that more than 44,500 extant species of spiders have been
described to date (Platnick, 2013), then a conservative estimate of 250 pep-
tides per venom indicates that as a group spider venoms are likely to contain
>10 million bioactive peptides (King, 2011), the vast majority of which are
expected to be insecticidal. Hence, spider venoms provide an almost inex-
haustible source of insecticidal molecules. To date, more than 200 disulfide-
rich insecticidal spider-venom peptides (ISVPs) have been sequenced,
ranging in size from 3.3 to 9.0 kDa and containing three to six disulfide bonds
(King and Hardy, 2013). Several of these peptides are devoid of vertebrate
activity and sufficiently potent against pest insects (i.e. LD50 < 1500 pmol/g)
and to warrant serious consideration as bioinsecticides (Table 8.1).
Almost all ISVPs isolated to date contain a structural motif known as an
inhibitor cystine knot (King et al., 2002; Pallaghy et al., 1994) that provides
these peptides with remarkable chemical and thermal stability as well as resis-
tance to proteases (King and Hardy, 2013; Saez et al., 2010). Thus, in con-
trast with most other peptides and proteins, biological stability is not a
concern with respect to use of ISVPs as bioinsecticides. ISVPs should be sta-
ble during long periods of storage as well as in the field, and they should
degrade to innocuous breakdown products. Spider-venom peptides can also
be produced using a range of heterologous expression systems that enable
cost-effective large-scale production (Klint et al., 2013).
Table 8.1 Properties of ISVPs with potential for development as bioinsecticidesa
Mass Disulfide Molecular Phyletic LD50 Oral Vertebrate
Rational name Synonyms Species ArachnoServer IDb (kDa) bonds target specificity (pmol/g)c activity activity
-AGTX- -Agatoxin Agelenopsis 381 4.20 4 NaV D, L, Od 30 (D) N.D.e No
Aa1d IV aperta channel
-CUTX- Aptotoxin Apomastus 403 3.76 4 NaV D,d L 133 (L) N.D. N.D.
As1a 3; Aps III schlingeri channel
U1- Aptotoxin Apomastus 407 8.31 4 N.D. L 2 (L) N.D. N.D.
CUTX-As1c 6; Aps VI schlingeri
U1- Peptide A Calisoga sp. 28 4.30 4 N.D. L 551 (L) N.D. No
NETX-Csp1a
-DGTX- DTX9.2 Diguetia 352 6.39 4 NaV B,d D,d L 380 (L) N.D. No
Dc1a canities channel
-HXTX- J-ACTX- Hadronyche 174 3.76 4 KCa B, C,d D, 91 (D) Lowd No
Hv1c Hv1c versuta channel L, O
-HXTX- V-1 Hadronyche 193 4.05 3 CaV B, C, D, 77 (D) Lowd No
Hv1a versuta channel H, L, O
-HXTX- Hadronyche 204 4.48 3 CaV B,d C,d 160 (C) N.D. No
Hv2a versuta channel D, O
-CNTX- Tx4(5-5); Phoneutria 279 5.17 5 Glu B, D, O <100 N.D. No
Pn1a Pn4A nigriventer receptor (D)
(NMDA
subtype)
-CNTX- Tx4(6-1); Phoneutria 280 5.24 5 NaV B, D 36 (D) N.D. No
Pn1a PnTx4(6-1) nigriventer channel
-AMATX- -palutoxin Pireneitega 301 4.03 4 NaV D, H, L 2347 (L) No N.D.
Pl1a IT1 luctuosa channel
U1- Plectoxin- Plectreurys 405 5.07 5 N.D. D,d L 14 (L) N.D. N.D.
PLTX-Pt1a V/VI tristis
U2- Toxin SF12; Segestria 117 5.12 4 N.D. L 1365 (L) N.D. No
SGTX-Sf1b Toxin F5.7 florentina
U1- OAIP-1 Selenotypus 1495 3.81 3 N.D. B, C, L 2 (C) Moderate N.D.
TRTX-Sp1a plumipes
U1- TaITX-1 Tegenaria 349 5.68 3 N.D. C, D,d L 780 (L) N.D. No
AGTX-Ta1a agrestis
a
Table is modified and updated from King and Hardy (2013) and includes only ISVPs reported to have no toxicity on vertebrates. Phyletic specificity refers to the insect orders
in which activity has been reported: Blattaria (B), Coleoptera (C), Diptera (D), Hemiptera (H), Lepidoptera (L) and Orthoptera (O). NaV, voltage-gated sodium channel;
CaV, voltage-activated calcium channel; KCa, calcium-activated potassium channel.
b
Refers to the toxin ID number in the ArachnoServer database. To navigate to the toxin card, use http://www.arachnoserver.org/toxincard.html?idID-number.
c
LD50 value is given for the insect order (in parentheses) in which the peptide was found to be most potent.
d
V. Herzig, N. Bende, and G.F. King (unpublished data).
e
N.D., not determined.
394 Volker Herzig et al.

The biggest potential disadvantage of ISVPs is that they are not orally or
topically active. Spiders have solved the delivery problem by evolving fangs
that act like hypodermic syringes to deliver their venoms into the
haemolymph of prey, thus enabling ISVPs to access their targets in the insect
nervous system. We have found that the LD50 values for ISVPs fed to insects
are about 100-fold higher than when they are injected (Hardy et al., 2013;
King and Hardy, 2013), presumably due to a slow rate of absorption in
the insect gut, as observed previously for insect neuropeptides (Audsley
et al., 2008) and disulfide-rich peptides from scorpion and snake venoms
(Casartelli et al., 2005). Nevertheless, the low level of intrinsic oral activity
of ISVPs is not restricted to insects, as other arthropod pests such as ticks
were found to be sensitive to oral administration of -hexatoxin-Hv1a
(hereafter Hv1a) (Mukherjee et al., 2006).
There is growing evidence that some peptides and proteins can access
paracellular and transcellular transport routes in the insect midgut. Septate
junctions in insects are leaky for molecules smaller than 5 kDa (Casartelli
et al., 2005; Skaer et al., 1987), and this paracellular route might explain
the observed uptake of insect neuropeptides and ISVPs (Audsley et al.,
2007; Hardy et al., 2013). However, size alone is not the sole determinant
of uptake rate, as the 0.9-kDa insect neuropeptide cydiastatin-4 is translocated
across the midgut of the lepidopteran Manduca sexta at a rate 42 times lower
than for Hv1a, a 4-kDa ISVP (V. Herzig, N. Audsley, and G.F. King,
unpublished data). In addition to the paracellular route, the translocation of
large proteins such as albumin across the insect midgut epithelium involves
active energy-dependent processes such as transcytosis (Casartelli et al., 2005).
Even though our understanding of transport routes across the insect mid-
gut is still in its infancy, it is clear that some peptides can pass undegraded
through the midgut, consistent with the observed insecticidal effects of ISVPs
after oral administration. Further research in this area might facilitate the
rational introduction of chemical modifications and/or the use of formula-
tions that will enhance the oral activity of ISVPs and facilitate their direct
deployment as bioinsecticides. However, the remainder of this chapter will
focus on alternative methods of delivering ISVPs to target insect species as
well as the option of developing orally active small-molecule ISVP mimetics.

3. TRANSCYTOSIS OF SPIDER VENOM PEPTIDES ACROSS

THE INSECT GUT EPITHELIUM VIA FUSION TO
MOLECULAR TRANSPORT VEHICLES
ISVPs appear to be able to access the insect haemolymph via para-
cellular transport. Although this route of uptake is extremely inefficient,
Spider Venom Peptides as Bioinsecticides 395

it could be enhanced by formulation with appropriate solvents and adju-

vants. However, a more generic approach for improving the oral activity
of these peptides might be to fuse them to a protein that is capable of being
transcytosed across the insect gut epithelium. These fusion proteins would
thus serve as molecular transporters that could ferry ISVPs across the
insect gut.
This approach was first demonstrated via fusion of ISVPs to a plant lectin.
Lectins are protease-resistant, carbohydrate-binding proteins that are widely
distributed in animals, plants and micro-organisms. Insecticidal plant lectins
play an important role in defence against herbivorous insects (Michiels et al.,
2010). Galanthus nivalis agglutinin (GNA), a small 14-kDa mannose-specific
lectin from the snowdrop plant, is taken up by receptor-mediated endocy-
tosis in the insect midgut after binding to aminopeptidase N, a membrane-
bound glycoprotein. Subsequent transcytosis leads to its accumulation in
haemolymph, Malpighian tubules, fat bodies, ovarioles and the central nerve
cord (Fitches et al., 2001, 2012) (Fig. 8.1). Thus, the transcellular uptake of
GNA can be exploited as a mechanism for delivering ISVPs to the insect
haemolymph and nervous system (reviewed in Bonning and Chougule,
2014). For example, the oral activity of both Hv1a and the ISVP U2-
SGTX-Sf1a (SFI1) was improved dramatically by fusion to the
N-terminus of GNA. Second instar cabbage moths, Mamestra brassicae, fed
on cabbage leaves coated with 0.2% Hv1aGNA fusion protein suffered
85% mortality after 10 days, whereas control mortality was <20%
(Fitches et al., 2012). Similarly, although neither GNA nor SFI1 was toxic
when fed to fourth instar tomato moths, Lacanobia oleracea, the inclusion of a
GNASFI1 fusion protein as 2.5% of dietary protein was insecticidal to first
instar larvae, causing 100% mortality after 6 days, and to fifth instar larvae,
causing 20% mortality and threefold reduced weight gain compared to con-
trols (Fitches et al., 2004).
An alternative fusion-protein approach recently introduced for specific
delivery of ISVPs to aphids (Bonning et al., 2014) relies on the fact that
aphids and other sap-sucking hemipterans vector a large number of plant
viruses. Luteoviruses are specifically vectored by aphids; the virus is ingested
during feeding on an infected plant and then it persists in the aphid without
replicating. Remarkably, the virus is able to move from the aphid gut into
the haemocoel via transcytosis, which relies on specific recognition of the
viral coat protein (CP) via gut receptors followed by receptor-mediated
endocytosis (Bonning and Chougule, 2014). Thus, as for GNA, the virus
CP can be used as a delivery vehicle for ISVPs. Hv1a has very little intrinsic
oral activity against aphids (Pal et al., 2013), but it was recently demonstrated
to be highly orally active against a wide range of aphids when fused to the
396 Volker Herzig et al.

Figure 8.1 Summary of methods for delivering ISVPs to the insect haemocoel: (1) Some
ISVPs appear to be able to access the haemocoel via paracellular uptake through leaky
septate junctions. (2) ISVPs can be fused to carrier proteins such as snowdrop lectin
(GNA) and the coat protein of pea enation mosaic virus, which are recognised by specific
receptors on gut epithelial cells. These fusion proteins can be taken up via receptor-
mediated endocytosis and delivered to the haemocoel by transcytosis. (3) Baculovirus
can be engineered to encode an ISVP transgene. Upon recognition by specific gut
receptors, budded virions infect gut epithelial cells and other body tissues and release
ISVPs. (4) Entomopathogenic fungi such as Metarhizium and Beauveria can also be
engineered to encode an ISVP transgene. Fungal spores (conidia) adhere to the insect
cuticle, germinate and then invade the integument. The fungus then proliferates in the
haemocoel in the form of wall-less blastopores, which produce and release ISVPs.
Adapted from Bonning and Chougule (2014).

C-terminus of the CP of pea enation mosaic virus (PEMV); the CPHv1a

fusion protein was even orally active against the soybean aphid, Aphis gly-
cines, which is not a vector for PEMV (Bonning et al., 2014). The CPs from
two other luteoviruses, barley yellow dwarf virus and soybean dwarf virus,
are also effective vehicles for ISVP delivery (Miller and Bonning, 2007).
A significant advantage of the GNA and CP delivery methods is that the
fusion proteins can be produced cheaply by recombinant techniques, and
transgenes encoding the fusion proteins can be engineered into
entomopathogens and plants (see Sections 4 and 5). GNA is a highly generic
Spider Venom Peptides as Bioinsecticides 397

delivery vehicle as it has been used to deliver insecticidal peptides to lepi-

dopterans, hemipterans, coleopterans and dipterans. However, GNA does
have deleterious effects on some parasitoids, although only at very high doses
(Gatehouse et al., 2011). Thus, it will be important, especially in the context
of integrated pest management programs, to establish whether attachment to
GNA alters ISVP selectivity. In contrast with GNA, CP is a highly specific
delivery vehicle that is currently limited to aphids and possibly other sap-
sucking hemipterans. However, development of a deeper molecular under-
standing of the mechanism of CP recognition and uptake in the insect gut
might allow this technology to be adapted to the control of other insect pests
(Bonning and Chougule, 2014).

4. USE OF ENTOMOPATHOGENS FOR ISVP DELIVERY

4.1. Entomopathogens as bioinsecticides
Entomopathogens, which can be bacterial, viral or fungal, are pathogens that
kill or seriously disable insects. They play a vital role in natural regulation of
insect pests (Sandhu et al., 2012). A wide range of entomopathogens have
been studied and used as bioinsecticides, including the soil bacterium Bacillus
thuringiensis (Bt), baculoviruses and entomopathogenic fungi such as
Metarhizium and Beauveria species.
Bt is a naturally occurring spore-forming bacterium from the family
Bacillaceae. It is widespread in soil and lethal to a range of insects due to
the production of pore-forming Cry toxins (also known as -endotoxins)
(Gatehouse et al., 2011; Pardo-Lopez et al., 2013; also refer to Chapters
2, 3 and 6). Cry toxins are toxic to specific species of insects, especially larval
stages, and they have proved to be safe to humans and other vertebrates.
Heterologously expressed Cry toxins supply the insect-resistance trait in
Bt transgenic crops (see Section 5; Chapter 6). Most Bt species are only active
against Lepidoptera, Diptera and Coleoptera, although the subspecies Bt
israelensis is effective against the larval stage of mosquitoes (Nartey et al.,
2013; Chapter 3).
Baculoviruses are pathogenic to the larval stage of a limited range of
insects, mostly within the orders Lepidoptera, Hymenoptera and Coleoptera
(Bonning and Hammock, 1996; Inceoglu et al., 2006). As for Bt,
baculoviruses have to be ingested by the insect to cause mortality. Although
wild-type baculoviruses have been used successfully for control of soybean
and forest pests, they have not competed successfully with chemical insec-
ticides. This is because baculoviruses take 514 days to kill infected insects,
398 Volker Herzig et al.

during which time the insect continues to feed and cause crop damage
(Bonning and Hammock, 1996; Inceoglu et al., 2006).
There are 400500 species of entomopathogenic fungi and some have
been used, without modification, for biocontrol of pest insects for 150 years
(Bailey and Boyetchko, 2010; Shah and Pell, 2003). Compared to other
micro-organisms, fungi infect a broader range of insects including lepidop-
terans, homopterans, hymenopterans, coleopterans and dipterans (Vilcinskas
et al., 1997). The EPA have registered more than 100 fungus-based insect
control products since 1995 (De Faria and Wraight, 2007; Shah and Pell,
2003). Mycotrol, for example, is a Beauveria-based mycoinsecticide used
for control of whiteflies, thrips, mealybugs and aphids (De Faria and
Wraight, 2007), while Green Muscle contains spores of Metarhizium
acridum, which are selectively pathogenic to locusts. Green Muscle showed
no detrimental effects on non-target organisms and it is now registered and
recommended for locust control by the United Nations (Lomer et al., 2001).
However, as for baculoviruses, a major disadvantage of entomopathogenic
fungi compared with chemical insecticides is their slow kill time (typically
>7 days).

4.2. Entomopathogens as a delivery vehicle for ISVPs

Since ISVPs are gene-encoded mini-proteins, transgenes encoding these
peptides could be engineered into a variety of entomopathogens, which
would mitigate two of their potential disadvantages. First, in this scenario,
ISVPs would be produced systemically in the insect host after
entomopathogen infection, and consequently their low level of oral activity
would not be an impediment to toxin deployment. Second, the phyletic
selectivity of the ISVP would become less important as the range of affected
insects would be determined primarily by the host range of the selected
entomopathogen. Off-target effects on predators and parasitoids could be
limited by choosing entomopathogens with restricted host selectivity; for
example, M. acridum exclusively infects locusts in the suborder Caelifera,
making it valuable as a locust-specific bioinsecticide.
Transgenic baculovirus have been engineered that express insecticidal
venom peptides from sea anemones, scorpions or spiders; in all cases, the
toxin transgene reduced the time between virus application and cessation
of feeding or death. The most dramatic improvement in insecticidal activity
resulted from incorporation of a transgene encoding an ISVP (Maggio et al.,
2010). The potency and speed of kill of entomopathogenic fungi can also be
Spider Venom Peptides as Bioinsecticides 399

enhanced by engineering them to express insecticidal venom peptides. For

example, the time as well as the dose required for Metarhizium anisopliae to
kill the tobacco hornworm, M. sexta, and the dengue vector, Aedes aegypti,
was reduced when the fungus was engineered to express the scorpion-
venom peptide AaIT (Wang and St Leger, 2007). Entomopathogenic fungi
are desirable insect control agents as they directly parasitise arthropods via
penetration of the host integument, which sets them apart from bacteria
and viruses, which must be ingested to gain host entry (Bailey and
Boyetchko, 2010; Roberts, 1973). Engineering entomopathogenic fungi
to express ISVPs removes two of the major impediments to their commer-
cial developmentthe kill time is dramatically decreased and the spore dose
required for effective insect control is lowered, thereby reducing
application costs.

5. IN PLANTA EXPRESSION OF SPIDER VENOM PEPTIDES

The introduction of genetically modified (GM) crops in the mid-
1990s revolutionised global crop production. As of 2012, 170 million hect-
ares of GM crops were grown by 17.3 million farmers in 30 countries
( James, 2012). More than 90% of growers are small resource-poor farmers
in developing countries ( James, 2012). Four GM crops currently dominate
global agriculture: cotton (81% of all cotton planted globally), soybeans
(81%), maize (35%) and canola (30%) ( James, 2012).
The introduction of insect-resistant corn and cotton expressing insecti-
cidal Cry toxins from Bt dramatically reduced insecticide use, improved crop
yields and helped conserve beneficial natural enemies (Que et al., 2010;
Tabashnik et al., 2013; also refer to Chapter 6). However, constitutive
expression of the toxin in transgenic plants has expedited resistance devel-
opment in a number of pest species; field-evolved resistance to Bt corn has
been reported for the maize stalk borer, Busseola fusca; the Western corn
rootworm, Diabrotica virgifera virgifera; and the fall armyworm, Spodoptera
frugiperda; while resistance to Bt cotton has been reported for the corn
earworm, Helicoverpa zea, and the pink bollworm, Pectinophora gossypiella
(Gassmann et al., 2014; Tabashnik et al., 2013; this chapter). In China,
2.6% of the population of the cotton bollworm, Helicoverpa armigera, are
now resistant to Cry1Ac cotton, which might be a prognostic indicator
of future widespread resistance (Tabashnik et al., 2013). Moreover, since
Cry toxins primarily target lepidopterans and coleopterans, other insects
(especially sap-sucking hemipterans) that were only secondary pests prior
400 Volker Herzig et al.

to the introduction of Bt crops have now become a major pest on some crops
(Bonning and Chougule, 2014).
Resistance to Bt crops can be delayed through the use of non-GM ref-
uges and/or by engineering Bt plants to express additional insecticidal genes
that act via different mechanisms, an approach known as pyramiding or trait
stacking (Moar and Anilkumar, 2007; Que et al., 2010; Chapter 6). ISVP
transgenes are good candidates for trait stacking with Bt since (i) they have
completely different mechanisms of action; (ii) they are likely to be
synergised by Cry toxins which lyse midgut epithelial cells (Soberon
et al., 2007), a property that should facilitate translocation of ISVPs into
the insect haemocoel; and (iii) whereas Bt toxins are largely specific for
the insect orders Lepidoptera and Coleoptera, ISVPs with complementary
selectivity, particularly against sap-sucking hemipterans, can be selected
for trait stacking.
Attempts to engineer plants expressing ISVPs began almost two decades
ago with the demonstration that transgenic tobacco expressing -HXTX-
Ar1a, a 37-residue insect-specific calcium channel blocker from the
Australian funnel-web spider, Atrax robustus, had enhanced resistance to
H. armigera ( Jiang et al., 1996). Transgenes encoding this ISVP (or its
orthologue Hv1a) have subsequently been engineered into cotton (Omar
and Chatha, 2012), tobacco (Khan et al., 2006) and poplar (Cao et al.,
2010). All of these ISVP-expressing transgenic plants were shown to have
significantly increased resistance to insect pests. For example, the mortality
of second instar H. armigera fed on transgenic tobacco expressing Hv1a was
75100% after 72 h compared to 0% for larvae fed on untransformed plants,
regardless of whether the ISVP was expressed under the strong 35S pro-
moter (Khan et al., 2006) or weaker phloem-specific promoters (Shah
et al., 2011). It has even been claimed that transgenic cotton expressing
Hv1a is as effective as Bollgard II cotton in controlling major cotton pests
(Omar and Chatha, 2012).
Tobacco plants engineered to express U7-HXTX-Mg1a (Magi-6), a
36-residue ISVP from the venom of the hexathelid spider Macrothele gigas
were found to be significantly more resistant than wild-type plants
against S. frugiperda (Hernandez-Campuzano et al., 2009). More recently,
transgenic Arabidopsis expressing Hv1a fused to the CP of PEMV were
shown to be resistant to a diverse range of aphid species (Bonning et al.,
2014). Thus, ISVP transgenes appear to have significant potential
either as a standalone insect-resistant plant trait or for trait stacking with
Cry toxins.
Spider Venom Peptides as Bioinsecticides 401

6. ISVP MIMETICS
6.1. Synthetic mimics of venom peptides
Venom peptides present structural characteristics that are far from the norm
for synthetic agrochemicals and drugs (Tice, 2001), and their size and polar-
ity impede passive diffusion through cell membranes. Instead, their oral bio-
availability to targets beyond the gut lumen relies on paracellular or active
transport (see Section 2). Given the diversity and potential complexity of
mechanisms that can mediate such paracellular/active transit through the
physical barriers of the gut wall, it seems unlikely that a simple set of general
rules could be derived that describe the oral bioavailability of venom pep-
tides. While the oral activity of ISVPs might be improved by chemical mod-
ifications (e.g. PEGylation and acylation) or by packaging them in
liposomes, microparticles and/or nanoparticles, these are complex and
expensive modifications that would significantly increase production costs.
An alternative approach to improving oral activity is to create
peptidomimetics wherein the functionally critical chemical features of the
peptide are displayed on a smaller structural scaffold with better pharmaco-
kinetic properties.
This alternative has been most extensively pursued for the conotoxin
family of venom peptides. The physicochemical properties of Ziconotide/
Prialt (-conotoxin MVIIA), a cone snail venom peptide used for manage-
ment of intractable pain, result in insignificant oral bioavailability and
consequently this drug requires intrathecal administration (Bingham et al.,
2010). In order to avoid the complexity of this drug delivery method, efforts
have been undertaken to design small-molecule mimetics that might show
oral bioavailability and bloodbrain barrier permeability (Brady et al., 2013).
Peptidomimetics that could surmount these challenges are classifiable
into three types (Ripka and Rich, 1998): (Type I) molecules in which pep-
tide bonds are replaced with more tractable bioisosteres; (Type II) molecules
which use moieties that are not structurally equivalent to the native peptide
to interact with the receptor; and (Type III) molecules which utilise a scaf-
fold that can spatially display the critical amino acid residues in the same way
as the native peptide. Several papers have been published on the design of
Type-III mimetics of -conotoxins due to the medical and commercial
need (Baell et al., 2001, 2004; Menzler et al., 2000). Despite early progress,
the resultant conotoxin mimetics were much less potent than the native pep-
tide (Brady et al., 2013).
402 Volker Herzig et al.

Recently, we took a more aggressive approach in the design of Type-II

mimetics of Hv1a (Tedford et al., 2013). Hv1a is a 37-residue ISVP that blocks
insect but not mammalian voltage-gated calcium (CaV) channels (Fletcher
et al., 1997; Tedford et al., 2004b). It is harmless when injected into newborn
mice (Atkinson et al., 1998) and a closely related homologue (Ar1a) is inactive
against nervemuscle preparations from chicken and rat (Chong et al., 2007).
We developed 3D models of the Hv1a pharmacophore and deployed them as
search terms for an in silico screen of a database of millions of commercially
available compounds. Hits from this screen were sorted and filtered as part
of a multiple-round selection process, ultimately yielding an Hv1a mimetic
collection that was tested for activity against A. aegypti and Spodoptera exigua.
This testing identified several families of viable prototype molecules, including
the VNX-000440 (VNX-440) family of triazine molecules that were further
optimised by iterative synthesis and analogue testing.

6.2. Hv1a pharmacophore analysis and modelling

The 3D solution structure of Hv1a comprises a core region rich in turns
and disulfide bonds (residues 421) and a hairpin (residues 2237) that pro-
jects from the disulfide-rich core (Fletcher et al., 1997). Site-directed muta-
genesis and synthetic truncates were used to elucidate the Hv1a
pharmacophore (i.e. the set of key structural features contributing to insec-
ticidal activity, presumably by directing interactions with one or more classes
of insect CaV channels) (Tedford et al., 2001, 2004a). The primary
pharmacophore residues (Pro10, Asn27 and Arg35) form a small, contiguous
patch of 200 A2 on one face of the toxin (Tedford et al., 2001, 2004a). To
build a viable peptide pharmacophore model, key functionalities of the crit-
ical amino acids were converted into pharmacophoric features and mapped
out in three dimensions (Fig. 8.2). Analyses of the structure of Hv1a and
several related peptides suggested that certain chemical features not identi-
fied previously by scanning mutagenesis, such as the Gly8 carbonyl oxygen
and the hydrophobic Val33 isopropyl functionality, are involved in Hv1a
activity. This conclusion was taken into account while building different
versions of the pharmacophore model: the Val33 side chain was represented
in two versions of the pharmacophore model as a hydrophobic feature,
while the backbone carbonyl oxygen of Gly8 was included as a
hydrogen-bond acceptor (HBA) in one of the models (Table 8.2 lists fea-
tures built into each version of the pharmacophore models that were used
for database searches).
Spider Venom Peptides as Bioinsecticides 403

Figure 8.2 Development of small-molecule mimetics of Hv1a. (A) Richardson schematic

of Hv1a (first molecule in the PDB file 1AXH) showing the key chemical features that
were used for pharmacophore modelling (i.e. the side chains of Pro10, Asn27, Val33,
and Arg35 and the backbone carbonyl of Gly8). Hydrogen, carbon, oxygen and nitrogen
atoms are shown in white, orange, red and blue, respectively. The N- and C-termini are
labelled. (B) Bioassay screening hit VNX-440. (C) Three-dimensional stick representation
of VNX-440; atoms are coloured as in (A) except that carbons are grey and chlorine
atoms are green. Superimposed on VNX-440 are representations of the eight chemical
features of the residues that were included in pharmacophore models of the peptide.
Cyan spheres indicate hydrophobic features matching the hydrophobic side chains of
Val33 and Pro10; magenta spheres and sticks indicate hydrogen-bond donor (HBD)
features projecting from Asn27 and Arg35; green spheres and sticks indicate
hydrogen-bond acceptor (HBA) features projecting from Asn27 and Gly8 (leftmost
and rightmost residues, respectively). (D) Optimised lead compound VNX-443, an ana-
logue of VNX-440.

Available SAR data suggested that the most functionally important res-
idue of Hv1a is Arg35 (Tedford et al., 2004a). This residue contributed three
features to the pharmacophore models: two hydrogen-bond donors (HBDs)
and one positively ionisable group. Asn27 contributed two features: one
HBD and one HBA. Pro10 and Val33 presumably mediate hydrophobic
interactions with CaV channels targeted by the peptide; hence, hydrophobic
features represented their side chains in the pharmacophore. Gly8, which
adopts an unusual backbone configuration (backbone dihedral angles:
90 and 20 ), is conformationally restricted by the cystine knot
in a way that allows the Gly8 carbonyl oxygen to contribute to the toxin
pharmacophore.
404 Volker Herzig et al.

The five toxin residuesGly8, Pro10, Asn27, Val33 and Arg35whose

substituents were used to derive features of the pharmacophore models
are illustrated in the context of the overall structure of Hv1a in
Fig. 8.2A. The minimal set of pharmacophore features is formed by Arg35,
Pro10 and Asn27, yielding a 6-feature pharmacophore (see 6 point 1,
Table 8.2).
The pharmacophore model was deployed to search the Accelrys
Chemicals Available for Purchasing (CAP) database. Low-energy con-
formers of the compounds were measured and scored for their fit to chemical
features of the pharmacophore. The 8-point model proved too stringent
as a search term, so two 6-feature models were constructed and used to
query the CAP database. After searches using all three versions of the
pharmacophore (Table 8.2), 8773 compounds were identified as hits that
showed a substantial structural match to the Hv1a pharmacophore model.
A set of physical properties considered relevant for this exercise were then
used to filter, sort and annotate the hit compound set. This resulted in the
purchase of 1370 unique compounds that were each tested for insecticidal
activity against A. aegypti larvae. This led to the identification of several fam-
ilies of compounds, including one containing the screening hit VNX-440
(Fig. 8.2B). VNX-440 is a commercially available compound that was pre-
viously shown to have modest antimicrobial activity (McKay et al., 2006).
The remarkable fit of VNX-440 to the pharmacophore model used for
in silico screening, presented in Fig. 8.2C, can be described as follows: a nitro-
gen of the triazine core ring and the oxygen of the morpholine functionality
of the compound overlap with the HBA features of the pharmacophore; two
amine substituents on the triazine core of the compound overlap with the
models HBD features; the basic nitrogen of the morpholine overlaps with

Table 8.2 Chemical features of key Hv1a residues used to build 3D pharmacophore
models
Arg35 Asn27 Val33 Pro10 Gly8
Model HBD HBD PI HBA HBD HPH (C, HPH (C, HBA
version (N1) (N2) (N) (O1) (N2) C1, C2) C, C) (O)
8-point Y Y Y Y Y Y Y Y
6-point 1 Y Y Y Y Y N Y N
6-point 2 N Y Y Y Y Y Y N
HBD, hydrogen-bond donor; PI, positively ionisable group; HBA, hydrogen-bond acceptor; HPH,
hydrophobe.
Spider Venom Peptides as Bioinsecticides 405

the cationic feature; and, finally, the remaining anilide has modest overlap
with the two hydrophobic features.

6.3. Optimisation and mechanism of action of the Type II mimic

VNX-440
Iterative analoguing, guided by the results of bioassays performed against
A. aegypti and S. exigua, allowed us to further characterise the VNX-440
family of hits and to develop a lead molecule (VNX-443; Fig. 8.2D) with
significantly improved activity compared with the initial screening hit.
The structural changes between VNX-440 and the latter analogue are
summarised as follows (cf. Fig. 8.2B and D): first, the amine of the anilide
that roughly matched the hydrophobic features in the model was excised to
provide a direct connection of a phenyl group to the triazine core. Second,
the anilide that only matched a single HBD feature was replaced with het-
erocycles to improve water solubility. Third, the hydrophilic tail was altered
to change the positioning and basicity of the amine. These alterations
retained a match with HBD and cationic features of the model for this pen-
dant group and increased insecticidal activity. In aggregate, these structural
changes increased insecticidal activity as much as 2200-fold (against
S. exigua) and resulted in a class of novel compounds (Kennedy and
Steinbaugh, 2013).
To confirm that VNX-440 acted against insects via a mechanism similar
to Hv1a, its effect on insect ion channels was examined using whole-cell
voltage-clamp recordings from dorsal unpaired median (DUM) neurons
harvested from the ventral nerve cord of the American cockroach, Peri-
planeta americana (Tedford et al., 2013). Figures 8.3A and B show examples
of the effects of 100 M VNX-440 on IBa (a measure of CaV channel activ-
ity) recorded from clamped DUM neurons. Control superfusion with bath
solution had no effect on IBa, but superfusion with 100 M VNX-440
reduced IBa dramatically, with maximum inhibition reached within
10 min of exposure to VNX-440.
The IC50 for inhibition of peak IBa by VNX-440 was estimated from the
doseresponse curve to be 23.1 M (Fig. 8.3C), which is only 20 times
higher than the IC50 (1.08 M) reported for inhibition of high voltage-
activated CaV channels by Hv1a (Chong et al., 2007). Unfortunately, this
activity was not replicated when VNX-443 was tested in this system.
Regardless, VNX-443 and various other analogues in this series caused
no adverse effects in an acute oral toxicity study in which rats were dosed
at 100 mg/kg (R.M. Kennedy, unpublished results).
Figure 8.3 Inhibition of Ba2+ currents (IBa) in American cockroach DUM neurons by VNX-440. (A) IBa recorded at 10 mV: black trace shows
control IBa, green trace shows IBa during superfusion with bath solution and the red trace shows IBa following with superfusion with 100 M
VNX-440. (B) Time course of peak IBa values recorded at a test potential of 10 mV after addition of 100 M VNX-440. (C) Doseresponse curve
for VNX-440 inhibition of peak IBa at a test potential of 10 mV. (D) IV curves for IBa obtained before, and 10 min after, application of 100 M
VNX-440.
Spider Venom Peptides as Bioinsecticides 407

6.4. Conclusions from development of Type-II mimetics

of Hv1a
Our in silico modelling and screening using the pharmacophore of Hv1a led
to a small-molecule hit (VNX-440) that showed properties consistent with
mimicry of the native peptides biological activity. To our knowledge, this
remains the only published example of the discovery of an insecticidal lead
compound through testing of a small set of candidate compounds chosen to
match the pharmacophore of a peptide. However, since the screening lead
VNX-440 was optimised solely via an in vivo assay (with insect mortality as
the read-out), it is not entirely clear if fidelity was maintained with respect to
the peptides mechanism of action, or if another property was optimised. An
additional consideration is that while VNX-443 has good per os activity, it
has only limited contact activity in adult mosquitocidal tests. The physico-
chemical properties enabling topical bioavailability are considerably stricter
than for oral bioavailability and may not allow full coverage of the 200 A2
pharmacophoric hot spot on peptides such as Hv1a.

7. OUTLOOK
An unmodified ISVP developed by Vestaron Corporation was
recently approved by the U.S. EPA for use against cabbage looper, Tricho-
plusia ni, marking the start of new era of peptide-based insecticides. ISVPs
have many properties that make them attractive as insect control agents: they
are potently insecticidal, highly selective for insects, stable to extremes of
temperature and should degrade in the field into innocuous breakdown
products. Although they typically have low levels of intrinsic oral activity,
simple formulation can improve their oral activity sufficiently to make them
competitive with chemical insecticides. The oral activity of ISVPs can also
be markedly improved by fusing them to proteins that ferry them via
transcytosis across the insect gut, including plant lectins and the CP of
insect-vectored viruses. Transgenes encoding these fusion proteins could
be incorporated into crop plants either as a standalone insect-resistance trait
or they could be pyramided with Bt transgenes in order to reduce the like-
lihood of resistance development and expand the range of susceptible
insects. Entomopathogens can also be engineered to express transgenes
encoding ISVPs or ISVP fusion proteins with enhanced oral activity. This
approach has the advantage that the selectivity of the ISVP can be tailored
by taking advantage of the limited host range of many entomopathogens.
408 Volker Herzig et al.

Thus, in summary, there are a wide variety of methods by which ISVPs can
be deployed as natural insecticides. We also recently demonstrated for the
first time that the pharmacophore of ISVPs can be used to rationally develop
small-molecule mimetics with improved oral activity, thereby providing
another approach by which these peptides can be exploited for the control
of insect pests.

ACKNOWLEDGEMENTS
The authors would like to thank the Australian Research Council for financial support. R.M.
Kennedy was funded in part by a grant from the Foundation for the National Institutes of
Health through the Vector-based Transmission of Control: Discovery Research (VCTR)
program of the Grand Challenges in Global Health initiative.

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INDEX

Note: Page numbers followed by f indicate figures and t indicate tables.

A Anti-feeding pro-phage (Afp) locus, 365,

ABCC2 proteins 367368
chromosome 15, 323 APN. See Aminopeptidase-N (APN)
Cry1Ab resistance, 323 Arabidopsis thaliana, 251, 307308
HvCad, 323 Aspartyl autoprotease, 356
YHD2 strain, 323 ATP-binding cassette (ABC) transporter,
A disintegrin and metalloprotease (ADAM), 6162, 121, 316
6263, 191
ADP-ribosyltransferases, 351352, 356357 B
Aedes Bacillus thuringiensis (Bt)
A. aegypti biopesticides, 179184
glucosidases, 118t and BTI, 46
larvae, 46 BT-R1, 5960
SEM image, 1012 characteristics, 4142
ALPs, 6061 cotton, 399400
Bin toxin, 9697 crops (see Bt crops)
cadherin, 15 Cry35 and Cry36, 102
Cry11Aa and Cry11Ba, 59 Cry11Ba toxin, 17
and Culex, 127129 Cry toxins (see Crystal toxins (Cry toxins))
GC cells, 1720, 21f Cyt1Aa protein, 99
SEM image, 46, 5f, 1012, 11f description, 179
Agrobacterium, 196197 discovery and development, 4041
Alkaline phosphatase, 6061 -endotoxins, 178179
Amber disease-associated plasmid (pADAP), GE crops (see Genetically engineered
349350 (GE) crops)
AMG. See Anterior midgut (AMG) GM crops, 3132
Aminopeptidase-N (APN), 5859 insecticidal proteins (see Bt insecticidal
Anopheles proteins)
A. gambiae and Ls strains, 128t
alimentary canal, 22f mosquitocidal toxins, 145148
dsRNAs, 263264 phenotypic trait, 41
human HeLa cells, 106107 S-layer protein, 109
maltase 3, 114 Baculoviruses, 397398
mosquito control, 128t Barriers to RNAi
A. merus, 1314 B. germanica and M. sexta, 275
and Culex, 93 cellular uptake, 271272
larvae, 125126 Dicer and Argonaute proteins, 271272
Anterior midgut (AMG) Drosophila, 274275
caecal neck adjoins, 10 dsRNA degradation, 272, 273f
GC and PMG, 10 endocytic pathways, C. elegans, 274275
lumen pH, 46 housekeeping gene, 270271
proton pump, 1720 mechanism, dsRNA uptake, 275

413
414 Index

Barriers to RNAi (Continued ) microvillar array, 2830

mRNA transcript levels, 270271 midgut epithelium, 55f
nutrients, absorption, 271272 physiological targeting/binding, 2223
Sid-1 and Sid-2 genes, 272274 and PMG, 18f
virus infections, 275 topography, 271272
BBM. See Brush border membrane (BBM) Brush-border membrane fractions (BBMF),
BBMF. See Brush-border membrane 113114, 115f, 135
fractions (BBMF) Bt biopesticides
Binary (Bin) toxins DipelT and ThuricideT, 180
analysis, 99 endophytes, 184
BinA and BinB proteins, 9799 israelensis (H 14), 180181
binding, receptor, 101 lepidopteran pests, 180181
block mutation, BinB, 102104 plasmid-based DNA cloning, 181182
circular dichroism analysis, 99100 products, 180, 181t
cross-resistance, 102104 properties, 182183
Cry49, 102 Pseudomonas fluorescens, 183184
Cx. pipiens, 101 recombinant strains, 182183, 183t
gastric caecum and posterior midgut, 100 sporeine, 179180
ingestion, mosquito larvae, 100 Bt corn
mosquitocidal, 4346 Cry1A.105 and Cry2Ab, 217
N-and C-termini, 102104 Cry1Ab, Cry1Ac, Cry9C, Cry1Fa and
patch-clamp experiments, 101102 Vip3Aa, 215
PirAB, 379 cultivation and commercialization, 215,
pore formation, 102 216t
P20 protein, 99 high-rootworm pressure, 217224, 224f
receptors (see Receptors, Bin toxin) MON810 and Bt11, 215217
tryptophan residues, 104 mycotoxin contamination, 214
Vip1Aa1 and Vip2Aa1, 186187 products, 217224, 218t
XaxAB cytotoxin, 379380 Bt cotton
YaxAB cytotoxin, 379380 BollGard, 225
Bioinsecticides cultivation and commercialization, 225,
entomopathogens, 397399 227t
ISVPs (see Insecticidal spider-venom development, 224225, 226f
peptides (ISVPs)) H. zea, 208
Biolarvicides products, United States, 225, 228t
accumulation of spores, 111112 reductions, 214
in China, 131134 Bt crops
Lysinibacillus sphaericus, 93 adoption, 214
vector-control programmes, 127129 Cry GE plants, 196197
Blattella germanica, 275 Cry toxins, 314317
Bollgard II cotton, 225, 400 discovery and development process,
Bombyx mori 193194
ABCC2 genes, 6162 DNA shuffling, 231
C2 strain, 323324 domain 3 exchange, 231232
polycalin family, 63 field-evolved resistance, 300, 312314
Brush border membrane (BBM) gene discovery, 194196
black lipid membranes, 350351 germplasm, 198
B. thuringiensis spore, 5556 insect resistance management, 207210
GC cells, 18f IRAC, 300
Index 415

lepidopterans, 314, 315f Dibrotica virgifera virgifera, 3031

protease activation, 230231 extracellular matrix, 30
refuge strategy, 298299 housekeeping gene, 270271
resistance detection methods midguts, 271272
(see Resistance detection, Bt) red flour beetle, 261262
resistance management, 324331 sensitivity level, ingested dsRNA, 253
resistance mechanisms, 314324 Snf7 dsRNAs, 253261, 280281
site directed mutagenesis, 231 Tc-ASH and UBX genes, 261262
transformation technologies, 197198 Tribolium and Diabrotica, 261262
transgenic crops, 298 type I peritrophic matices, 3031
Bt insecticidal proteins Concentrationresponse and diagnostic
ABCC2, 193 concentration assays
Bt Toxin Specificity Database, Bt protein source and forms, 303
185186 diagnostic concentration test, 301302
cross-order activity, 185186 laboratory strains, 301, 302
Cry proteins, 187190 MVPII, 303
Cyt proteins, 190191 protein source, 303
diversity, 184185 target pest populations, 302
mechanisms of resistance, 191193 Corn events with Bt genes, 216t
receptors, 191 Costelytra zealandica, 349350, 361362
Vip3 proteins, 186187 Cotton events with Bt genes, 227t
Bt potato, 226230 CP. See Coat protein (CP)
Bt soybean, 225226 Cqm1 vs. Aam1 glucosidases, 119121
Crystalline (Cry) proteins
C ALPs, 6061
Cabbage moths, 395 Cry1A.105, 211
Cadherins (CADs) Cry1Ab, 210
BBM, 2223 Cry1Ac, 210211
BtR1, 8 Cry1Fa2, 211
Bt4R strain, 319 Cry2Ab, 211
cellcell interactions, 5960 Cry2Ae, 212
Cry3Aa and Cry3Bb toxins, 5960 Cry3Aa1 and Cry1Aa1, 187188, 188f
Cry1Ac resistance, 320 Cry34Ab1/Cry35Ab1, 213
Cry toxin receptors, 5960 Cry3Bb1, 213
DsCad, 320 eCry3.1Ab, 212213
extracellular and intracellular domain, -endotoxins, 187
320, 321f GPI-anchored APN, 192193
HvCad, 319 mCry3Aa, 212
Lepidoptera, 192 mechanism of action, 188190, 190f
locus, 306f pore formation, 317
mutants, 310 structures, PDB accession, 188, 189t
Caenorhabditis elegans, 191, 250251, Vip3Aa, 212
283284 Crystal toxins (Cry toxins)
Caudate phage-derived proteins, 365 ABCC2, 323324
Coat protein (CP), 395396, 396f alkaline phosphatase, 322323
Coleopterans aminopeptidase, 321322
agricultural pests, 3031 bacterial pore-forming, 67
corn roots, 186187, 253261 cadherin, 319320
Cry3Bb toxins, 5960 cell death, 317
416 Index

Crystal toxins (Cry toxins) (Continued ) Aedes aegypti and Anopheles gambiae,
Cry1Aa, 4950, 50f 263264
Cry1Ac protoxin, 318 Drosophila, 262263
Cry34 protein, 6768 DNA screening
definition and classification, 4243 bioassay, 310311
diversity, 4346 cadherin mutations, 311
domain I, 5051 HaCad, 311
domain II, 5153 PCR method, 311
domain III, 5355 r15 cadherin allele, 311312
-endotoxins, 314 DNA shuffling, 231
enterocyte death, 6667 Dolichus biflorus agglutinin (DBA),
genomic sequencing, 49 1517, 16f
4 helix lining, 66 Dorsal anterior rectum (DAR) cells,
insect-resistance trait, 397 1314, 26f
intoxication process, 5556, 55f Drosophila melanogaster
midgut Cry-binding proteins, 5763 Cry1Ac, 6465
models, 6368 dsRNA, 262
oligomer formation, 317 embryo system, 374375
parasporin, 4748 S2 cells, 275
PCR and next-generation sequencing, 49
proteins and molecules, 6263 E
protoxin, 4950 Egg shell structures, 352353
receptors, 316 -Endotoxins, 178179, 187, 314, 379,
ricin domain, 48 397
sequential binding model, 316 Entomopathogens
signalling pathway, 316 Bacillus thuringiensis (Bt), 397
solubilization and proteolytic processing, baculoviruses, 397398
5657 bioinsecticides, 397398
structural domains, 315316 Cry toxins, 397
Culex -endotoxins, 397
and Aedes species, 127129 fungi and parasites (nematodes), 149150
and Anopheles, 93 Green Muscle, 398
C. pipiens, 97t, 102104, 113114, 132t ISVPs, 398399
C. quinquefasciatus Mycotrol, 398
Cry48Aa/Cry49Aa toxin, 105f Entropic spring mechanism, 355356, 356f
growth and mortality, 102104 Environmental effects, GE crops
maltase 1, 114, 132t agronomic properties, 205
Synergism, 6970 cultivation, 203
larvae, 99 gene flow, 205
mosquitoes, 104 grain importation, 205
Cytolytic (Cyt) proteins hazard testing, 203204
description, 184185 monarch butterflies, larvae, 204
mosquitoes and blackflies, toxicity, 187 non-GE isoline, 205
structure and function, 190191, 190f Environmental RNAi
arthropod pests, 269270
D Coleoptera, 253262
Dibrotica virgifera virgifera, 3031, 253 components, 252253
Diptera definitions, 252
Index 417

Diptera, 262264 product identification and

Hemiptera, 267269 characterization, 201
ingested dsRNAs, 252253, 254t recombinant DNA techniques, 198199
Lepidoptera, 264267 regulatory systems, 199
Escherichia coli safety assessments, 199, 200t
Bin toxin, 148 stacks, 206
clones, 370371 GNA. See Galanthus nivalis agglutinin (GNA)
dsRNAs, 266 Gram-negative Rhs proteins, 352353
host cells, 106107 Green Muscle, 398
Guanine nucleotide-binding protein
F (GNBP), 269270
Fibrobacter succinogenes, 361362
Field-evolved resistance, Bt crops H
Cry1Ac resistance, 325328, 328f HBA. See Hydrogen-bond acceptor (HBA)
F1 and F2 screens, 325 HBDs. See Hydrogen-bond donors (HBDs)
high-dose/refuge strategy, 312314 Heat shock protein (Hsp) 90, 360
hypothesis, 324325 Helicoverpa armigera
incipient and practical resistance, 312 Bt cotton, 224225
lepidopterans, 325, 326t Cry1Ac binding, 58
target pests, 312, 313t, 325328 EcR gene, 265266
Flea-rodent enzootic cycles, 362363 and 1642 iso-female lines, 307308
Fluorescent nanoparticles (FNPs), 278279 RNA interference, 59
Food safety, 202204, 281282 Helicoverpa punctigera
Fruit fly, 262 in Australia, 325328
F1 screen, resistance detection cropping areas, 305
Cry1Ab, 304305 Cry2Ab resistance, 307308
Cry1Ac, 304 Cry1Ac resistance, 307308
laboratory strain, 305, 306f Helicoverpa zea
locus, 306 Bt cotton, 208
resistance allele frequencies, 304305 Cry1Ac-selected AR1 strain, 322323
F2 screen, resistance detection GE plants resistance, 196197
cadherin mutants, 310 Heliothis virescens
Cry1Ac resistance, 307308 and cotton bollworm, 5355
diagnostic concentration assay, 307 Cry1Ac-expression, 225
lepidopteran pests, 307 and M. sexta, 6061
resistance alleles, 308310, 309t YHD3, 323
target pests, 308310 Hemimetabolous
macroscopic features, 2728
G nymph to adults, 4
Galanthus nivalis agglutinin (GNA), 395397 Hemipterans
Gene duplication, 107108 aphids, 3132
Genetically engineered (GE) crops dietary concentrations, 267268
environmental effects, 203205 dsRNA feeding, 267268
environmental, food and feed safety hunchback gene, 267268
profile, 199 nitrophorin 2 (Np2) gene, 267268
EPA, 206207 N. lugens, 269
human health assessment, 201203 Rack-1 and C002, 268269
PMM, 207 Herbivorous insects, 23, 390
418 Index

Hexatoxin (Hv1a) human development, 23

Asn27, 403 human disease, 2
development, small-molecule mimetics, mosquito, 327
402, 403f structure and function, 2325
3D solution structure, 402 Insecticidal spider-venom peptides (ISVPs)
Gly8, 403 chemical cocktails, 391
pharmacophore model, 404, 404t description, 391
physical properties, 404405 disadvantages, 394
primary pharmacophore residues, 402 entomopathogens, 397399
residues, 3D pharmacophore, 404, 404t heterologous expression systems, 391
SAR data, 403 in planta expression, 399400
site-directed mutagenesis and synthetic inhibitor cystine knot, 391
truncates, 402 insect control agents, 407408
type-II mimetics, development, 407 insecticidal effects, 394
Val33 side chain, 402 mimetics, 401407
VNX-000440, 404405 oral activity, 394, 407408
Holometabolous paracellular and transcellular transport
adult stages, 4 routes, 394
dipterans, 34 properties, 391, 392t
Human health assessment septate junctions, 394
allergenic potential, 201202 transcytosis, 394397
breeding and crop improvement Insecticide Resistance Action Committee
techniques, 202203 (IRAC), 300
Bt proteins, 201 Insect pest management. See also RNA
nutritional profile, 202 interference (RNAi)
protein levels, 202 Arabidopsis, 251
soil and phylloplane description, 252
microorganism, 201 development, insecticides, 251
toxicity, 202 environmental RNAi (see Environmental
Hv1a. See Hexatoxin (Hv1a) RNAi)
Hydrogen-bond acceptor (HBA), 402 eukaryotic cells, 250
Hydrogen-bond donors (HBDs), 403 RISC, 250251
Insect resistant crops, Bt. See Bacillus
I thuringiensis (Bt)
Influenza virus, 353354 IRAC. See Insecticide Resistance Action
Inhibitor cystine knot, 391 Committee (IRAC)
In planta expression, ISVPs ISVP mimetics
Bt corn, 399400 Hv1a pharmacophore, 402405, 407
Bt crops, 400 type II mimic VNX-000440, 405406
Cry toxins, 399400 venom peptides, 401402
engineer plants, 400
J
genetically modified (GM) crops, 399
Juvenile hormone (JH), 266, 379
Lepidoptera and Coleoptera, 400
Juvenile nematodes, 345
transgenic tobacco, -HXTX-Ar1a, 400
U7-HXTX-Mg1a (Magi-6), 400 L
Insect alimentary canal Larval feeding assays, 280281
disease control strategies, 2 Lepidopteran larvae (caterpillars), 2830,
gut function, 12 29f, 5960
Index 419

Lepidopteran pests Malaria

beet armyworm 1 subunit integrin in Africa, 121122
(Se1), 264265 Plasmodium falciparum, 17
brown apple moth, 264 Manduca sexta
Bt genes, 283284 BtR1, 5253
chitinase genes, 264265 caterpillars, 370371
Cry1Ac and Cry2Ab proteins, 282283 and coleopterans, 34
Escherichia coli, 266 GalNAc, 58
H. armigera, 265266 tobacco hornworm, 264
20 -O-methoxy nucleotides and Marine worm larva, 368369
deoxythymidin, siRNAs, 266 Mcf-like fit toxin in vivo
Ostrinia furnacalis, 266267 Drosophila embryo, 374375
Spodoptera frugiperda, 272, 273f regulation, Fit toxin expression, 375376
tobacco plants, 265266 Midgut Cry-binding (receptor) proteins
WCR Snf7 dsRNA, 284285 ABC transporter, 6162
Luteoviruses, 395396 alkaline phosphatase, 6061
Lymphatic filariasis transmission, 126127 APN, 5859
Lysinibacillus sphaericus (Ls) cadherin-like proteins, 5960
Bin toxin, 97104 Molecular transport vehicles, 394397
biolarvicides, 9596 Mono-ADP-ribosyltransferase (mART)
biological control agents, 149150 toxin, 378379
classification, 9192 Mosquitocidal toxin 1 (Mtx1)
Culex spp., 9697 ADP-ribosyl transferase, 106107
in DNA group IIA, 9596 catalytic subunit, 107
insecticidal factors, 91 double mutants, 107108
micrography, 91, 92f gene encoding, 106
mosquitocidal toxins, 148149 gut enzymes, 106107
as mosquito-control agent, 9395 low-toxicity strain SSII-1, 107108
pathogen, mosquito, 9091 LP1-G, 104106
safety studies, 109111 Mosquito control
saprophytic organism, 91 anophelines, 122125
SSII-1 strain, 9091 aquatic habitats, 121122
strains and larvicidal properties, 9293, biotic and abiotic factors, 125126
94t large-scale field tests, 127129, 128t
LS-based products, 122125, 123t
M lymphatic filariasis, 126127
Makes caterpillars floppy (Mcf ) toxins operational use, 129130
2929-amino acid protein, 371 small-and medium-scale, 122125
Bcl2-homology 3-like (BH3-like) transgenic corn plants, 276
domain, 371372, 371f Mosquito larval alimentary canal
caterpillar immune system, 370371 Aedes aegypti larvae, SEM, 46, 5f
cytotoxin B-like, 371, 371f AgAPN1, 17, 18f
description, 370 Anopheles gambiae, 1720, 19f
in vivo, 374376 anterior intestine/ileum, 13
Mcf2, 372 apical-to-basal movement, 14
MCF1-SHE domain, 373 caecal diverticuli, 10
Pseudomonas, Fit toxin, 373 CAP cells, 810
repeats in toxins (RTX), 371, 371f cell biology and polarity, 1415
420 Index

Mosquito larval alimentary canal (Continued ) Hv1a and SFI1, 395

Cry-IVB toxin damage, 2022 ISVPs, 394, 401
culicenes, 2526, 26f Ostrinia furnacalis, 266267, 278279, 313t
disease vector species, 4 Ostrinia nubilalis
embryonic development, 3, 4f Cry1Ab, 215217
endodermal epithelial cells, 810 resistant strains, 192193
gastric caeca cells, 810, 9f stalk boring Lepidoptera, 215217
GC and CM, 8
glycocalyx-type extracellular matrix, P
1517 Paracellular transport, 394
hemimetabolous and holometabolous, Parasporin
34 ETX/MTX family, 4748
ileum/posterior intestine, 2526 graphical representation, 44f, 4748
lepidopterans, 34 P. asymbiotica toxin (PaTox)
midgut, Aedes aegypti, 1012, 11f description, 376377
MT cell biology, 2223, 24f DxD motif, 376377
Na+/K+-ATPase and CA9, 2022, 22f heterotrimeric G proteins, 376377
pH gradient, 1720 PaToxG domain, 376378
physiological and immunohistochemical Rho proteins, 376378
analyses, 1213 SseI-like domain, 377378
PM, 78 structure-function analysis, 377378
pyloris and MTs, 1213 Pea enation mosaic virus (PEMV), 395396
rectum, 1314 Pectinophora gossypiella
salivary glands, 67, 6f Bt crops, 313t
SG function, 15 field control efficacy, 299
V-ATPase, 1720, 21f Peptidomimetics
Mtx1. See Mosquitocidal toxin 1 (Mtx1) classification, 401
Mu-like phage, 365 pharmaco-kinetic properties, 401
Mycotrol, 398 Peptidyl prolyl cis/trans isomerases (PPIases),
360
N Photorhabdus temperata, 344345
Neuraminidases, 353354 Photorhabdus toxin (Photox)
Next-generation DNA sequencing binary toxins, 379380
technology, 195196 Mcf Toxins, 370376
Next-generation rootworm-resistant corn P. asymbiotica, 345346
Bt insecticidal proteins, 277278 and Patox, 376379
Cry3Bb1 protein, 276277, 276f P. temperata and P. luminescens, 344345
Snf7 gene expression, 277278 PVCs (see Photorhabdus virulence cassettes
V-ATPase dsRNA, 276277, 277f, 284 (PVCs))
Nilaparvata lugens, 267268, 269 toxin complexes (Tcs), 347364
N-terminal peptidase domain, 365366 Photorhabdus virulence cassettes (PVCs)
Afp toxin delivery system, 367368
O bacteriocins, 367
Oral activity candidate effector proteins, 366367
cell-free bacterial supernatant of caudate phage-derived proteins, 365
Photorhabdus, 348 description, 364365
dietary small RNAs and longer dsRNAs, discovery and organization, 365367
281282 E. coli, 366367
Index 421

infective juvenile, 369370 -Propeller, 352353, 356357

injection of larvae, 366367 Pseudomonas fluorescens, 183184, 373
Macs, 367, 368369 Pseudomonas syringae, 361362, 373
marine worm larva, 368369 PVCs. See Photorhabdus virulence cassettes
open reading frames, 365366 (PVCs)
pADAP plasmid, 367368 Pyramid, Bt crops
PaPVCpnf, 366367 Cry2Ab, 211
peptidoglycan hydrolytic activity, 369 Cry proteins, 330331
phage-like structures, 367 insect resistance, 282
Phage P2 gpX, 369 pesticides, 329330
P. luminescens TT01 and P. asymbiotica target pests, 330331
ATCC43949, 365 Pyrethroid, 127129, 139140
putative PVC encoded effectors, 365366
PVC-SS, 365366, 369 R
receptors, 369370 Rapid virulence annotation (RVA)-like
tailocins, 369370 screens, 381382
TEM, 366367 Rearrangement hotspot (Rhs), 351353
type VI secretion system, 367 Receptors, Bin toxin
PirAB binary toxins, 379 Cpm1 -glucosidases, 117, 118t
P. luminescens ABC Tc holotoxin Cqm1/Cpm1 proteins, 119
ADP-ribosyltransferase domain, 356357 Cry toxins, 119
B/C-formed shell, 356357 fluorescent-labelled Bin toxin, 112113,
cryo-EM structure, 357, 359f 113f
crystal structure B and C subunits, 356 -glucosidases, 117
egg-shell-like structure, 357, 359f in vitro saturation, 113114, 115f
entropic spring/linker, 355356, 356f larvae midgut, 112114
interaction of B/C (TcB/TcC) subunits, midgut-bound proteins, 114
356357, 358f mosquito susceptibility to Ls, 113114,
neuraminidase-like region, 116t
353354 quantitative assays, 113114
PCT3 and PCT5, 353354 Spodoptera frugiperda, 117119
pre-pore to pore state, 354 Refuge, Bt crops
-propeller, 356357 Cry toxins, 329
receptor domains, 353354, 355f field-evolved resistance, 329
PMM. See Post market monitoring (PMM) heterozygous progeny, 328329
Polymerase chain reaction (PCR) high dose, 209
Bt cry gene, 195 insect resistance, 208
RNAi, 253261 pest resistance, 328329
toxin genes, 49 Resistance detection, Bt
Polymorphic toxin systems, 361 allele frequency, 301
Posterior midgut (PMG) concentration-response and diagnostic
and BBM, 18f concentration assays,
Culex quinquefasciatus, 11f 301304
and GC cells, 10 DNA screen, 310312
granular surface, 1012 field-evolved resistance, 301
larval mosquitoes, 2223 F1 screen, 304306
Post market monitoring (PMM), F2 screen, 307310
207 pesticides, 301
422 Index

Resistance management Serine peptidase, 373

Bin-toxin, 145150 Site directed mutagenesis, 231
biological cost, 141143 S-layer proteins, 109
biological risks, 209210 Soybean and potato events with Bt genes,
Bt proteins, 283284 229t
Caenorhabditis, 283284 Sphaericolysin, 108109, 110f, 112f
cqm1 gene, 135139, 137f Spodoptera frugiperda, 117119, 272, 273f
development, 207208 Systemic RNAi
diagnosis and field survey, 139140 arthropod pests, 269270
factors, 129130 Drosophila, 262263
field-evolved resistance, 324328 dsRNA degradation, 272
heterozygous/homozygous, alleles, 209 nematode, 283284
high dose/refuge strategy, 282283 sid-3 and sid-5 mutants, C. elegans, 274275
host plants, 208 Snf7 dsRNA, 261262
inheritance, 135
integrated mosquito-control T
programmes, 143144 Tail sheath protein, 365
laboratory and field reports, 131134, Tc ABC complexes
132t chaperones, 357360
natural refuges, 208 encapsulation and auto-proteolysis,
O. nubilalis, 208 C subunit, 352353
population genetics theory and simulation holotoxin
models, 209 high-resolution structure, 353357
prevention factors, 144 low-resolution structure, 351352
pyramid strategy, 329331 A subunit assembly, 350351
refuge strategy, 328329 Tomato moths, 395
stacking/pyramiding, 282 Toxin complexes (Tcs)
Ricin domain, 48 ABC complexes, 350360
RNAi. See RNA interference (RNAi) crop protection, 363364
RNA-induced silencing complex (RISC), description, 347348
250251, 271272 discovery, gene cloning and ABC
RNA interference (RNAi) nomenclature, 348349
barriers to delivery, 270275 infection
cost of goods, 279280 Bacillus thuringiensis, 361362
environmental, 252270 C. zealandica midgut, 361362
FNPs, 278279 Fibrobacter succinogenes, 361362
insect resistance management, 282284 plague bacillus, 362363
mechanism and delivery strategies, 278, Pseudomonas syringae pv. tomato, 361362
278f Treponema denticola, 361362
next-generation rootworm-resistant corn, Y. pestis, 362363
276278 polymorphic toxins, 361
safety assessments, 280282 tc-like genes, diversity, 349350
siRNA formulation, 279 Toxin complex secretion system (TC-SS),
transfection agents, 278279 381382, 383
Transcellular transport route, 394
S Transcytosis, ISVPs
Septate junctions, 394 aphids, 395396
Sequential binding model, Cry1A, 6465 CP, 395397
Index 423

GNA, 395397 Whiskers, 197198

insect haemocoel, 395, 396f
lectins, 395 X
luteoviruses, 395396 XaxAB cytotoxin, 379380
PEMV, 395396 Xenorhabdus
Transgenic baculovirus, 398399 and Photorhabdus, 357360, 361362,
Treponema denticola, 361362 380381
Tribolium castaneum, 6263, 191 Tc subunits, 350351
Type II integral membrane proteins, X. bovienii, 346347
352353 X. nematophila, 346347, 349350
Type III secretion systems, 380381 X. particulate toxins (xpt), 349350
Type II mimic VNX-000440, 405406,
406f Y
YaxAB cytotoxin, 379380
V YD-repeat proteins, 352353
Vacuolar-ATPase (V-ATPase), 1720, Y. entomophaga strain MH96, 349350
270271, 280281 Yersinia pestis, 351352, 362363

W Z
Wall-associated protein A (WapA), 352353 Ziconotide/Prialt, 401