Chapter 12

From Silkworms to Bees: Diseases

of Beneficial Insects
Rosalind R. James* and Zengzhi Liy
*
United States Department of Agriculture, Agricultural Research Service, Logan, Utah, USA, y Anhui Agricultural University, Hefei, Anjui, China

Chapter Outline
12.1. Introduction 425 12.3.2.Bacteria 439
12.2. Diseases of Silkworms 426 12.3.3.Fungi: Filamentous 444
12.2.1. Viruses 427 12.3.4.Fungi: Microsporidia 446
12.2.2. Bacteria 431 12.3.5.Protists 446
12.2.3. Fungi: Filamentous 432 12.3.6.Non-infectious Biotic Agents 447
12.2.4. Fungi: Microsporidia 433 12.3.7.Abiotic Agents 448
12.2.5. Protists 435 12.3.8.Colony Collapse Disorder 448
12.2.6. Non-infectious Biotic Agents 435 12.3.9.Disease Control Methods for Managed
12.2.7. Abiotic Agents 436 Bees 449
12.2.8. Disease Control Methods for Sericulture 437 12.4. Future Research Directions 453
12.3. Diseases of Bees 438 References 454
12.3.1. Viruses 439

12.1. INTRODUCTION
Summary
Diseases of the silkworm (Bombyx mori) and managed bees, Despite the predominance of attention to insects as pests,
including the honey bee (Apis mellifera), bumble bee (Bombus many insects are directly beneficial to humans, and as
spp.), alfalfa leafcutting bee (Megachile rotundata), and mason a result, mass-production methods have been developed.
bee (Osmia spp.), are reviewed, with diagnostic descriptions and The most notable examples are silkworms and honey bees,
a summary of control methods for production systems. Silkworms although many other insects are mass produced for various
and honey bees have been managed by humans for a few thousand purposes. For example, screw worms and fruit flies are
years. This close association has provided insights into the nature mass produced for the sole purpose of sterilizing the males
of infectious diseases, influencing microbiologists such as Agos-
and releasing them to mate with wild females in what is
tino Bassi and Louis Pasteur. Silkworms and bees are susceptible
to a wide range of pathogens, including viruses, bacteria, fungi,
known as the sterile insect technique. Lacewings, ladybird
and protists. However, pathogenic nematodes are conspicuously beetles, and some parasitoids are mass produced and sold as
absent. These insects are also susceptible to parasitoids, mite biological control agents against various pests. Bumble
parasites, pesticides, and environmental toxins. Control measures bees and alfalfa leafcutting bees are mass produced as
for silkworm diseases center around sanitation, as chemical pollinators for greenhouse crops and alfalfa seed produc-
treatment options are extremely limited. During the past century, tion, respectively. A major concern in any kind of mass
honey beekeepers have developed a reliance on chemical treat- rearing facility is the risk for disease outbreaks. If diseases
ment options, but this approach has produced resistant pathogens are not controlled, a sudden collapse of the insect colony
and parasites, and beekeepers are vulnerable to the introduction of can occur and the quality and quantity of their product may
new diseases that do not yet have control methods. An integrated be reduced.
approach, with an emphasis on prevention and sanitation
In contrast to other chapters, where the main focus is the
measures, would help to reduce these problems.
use of pathogens to kill insects, what is special about

Insect Pathology. DOI: 10.1016/B978-0-12-384984-7.00012-9 425

Copyright Ó 2012 Elsevier Inc. All rights reserved.
426 Insect Pathology

beneficial insects is our desire to protect them from path- The history of sericulture is also a history of the war
ogens. Diseases occur in beneficial insects in natural and against silkworm diseases (see Chapter 2). The earliest
agricultural settings, and undoubtedly have an impact on record of a silkworm disease was the announcement by
the population dynamics of these insects, just as they do on Guan Zhong (725e645 BCE) that any man who could
herbivorous insects. However, insect production systems produce silkworms free of diseases in the spring and
lead to very large, crowded populations, conditions that autumn (fall) would be awarded 0.5 kg of gold. In the
provide ideal opportunities for disease development and earliest Chinese pharmaceutical work, “Classic of Herbal
spread. In addition, insect production systems provide Medicine” (221e220 BCE), Beauveria bassiana-infected
a unique opportunity to observe pathogens that may be silkworms were used as a folk medicine for childhood
more difficult to find in nature because the insects are convulsions. Chen Fu’s “Book on Agriculture”, published
confined and reared in high numbers, and are under careful in 1149, describes black, white, and red muscardines
observation. (diseases caused by fungi) and some typical symptoms of
This chapter presents a discussion of diseases that occur polyhedrosis-like infections in silkworms. The relationship
in two insect groups, silkworms and bees, and methods for between the occurrence of the muscardines and weather
controlling these in productions systems. Many of the most conditions was analyzed in the “Handbook of Agriculture
well-studied insect pathogens were first found in silkworms and Sericulture”, published in 1273. This book also
or honey bees, as these insects have been managed by describes the symptoms of silkworm adults suffering from
humans for millennia. Silkworms have provided scientists pébrine (a disease caused by a microsporidium). In the
with key insights into the very existence of pathogens (see famous ancient Chinese encyclopedia written by Yingxing
Chapter 2). Likewise, important discoveries are certain to Song in 1637 and entitled “Tiangong Kaiwu” (“The
be made as researchers seek to identify the causes of Exploration of the Works of Nature”), typical symptoms of
current problems in honey bee production. grasserie (viral disease) and flacherie (either viral or
bacterial disease) are described, including methods of
control that involved segregating and eliminating diseased
12.2. DISEASES OF SILKWORMS
larvae, suggesting that the contagious nature of these
Several silkworm species that are indigenous to Asia and diseases had been recognized.
Africa have been domesticated and are raised for silk The first scientific study on silkworm diseases was
production throughout most of the temperate zone. These conducted by Agostino Bassi in 1807 on the white mus-
species vary in the quality of the silk they produce, the host cardine (known as “mal de segno” and “calcinaccio” in
plant on which they feed, and the number of generations Italian) (see Chapter 2). This disease initially appeared in
produced per year. The most widely raised species and the Italy around 1805, and in France by 1841. Bassi (1835)
producer of the finest silk is Bombyx mori, which is of determined that muscardine was caused by a living entity (a
Chinese origin and feeds on mulberry (Morus spp.) leaves. fungus) that formed the powdery appearance on the dead,
Silkworm rearing and silk weaving can be traced back to diseased silkworms, and that this condition was contagious.
the ancient Chinese, as early as at least 6000 years ago. He is credited with rescuing the economically important
Some classical Chinese books such as “The Book of silk industry with his recommendations for the use of
Songs” (sixth to eleventh century before the common era, disinfectants, separating the rows of feeding caterpillars,
BCE) and “The Chronicle of Zuo” (453 BCE) described isolating and destroying infected caterpillars, and keeping
thriving sericulture and silk textile industries in northern the farms clean. His paper was translated into French and
and southern China 2500e3000 years ago. Silk became one distributed throughout Europe and greatly influenced Louis
of the main cloth materials of the ancient Chinese, and as Pasteur (Porter, 1973).
a result, sericulture became an important component of In 1865, epizootics were devastating the silk industry in
Chinese agriculture. The westward exportation of silk France. Louis Pasteur was asked by the French Government
started at least 3000 years ago, based on Chinese silk found to investigate, and after several years, he discovered that the
on Egyptian mummies. Starting with the Han Dynasty (202 silkworms had been suffering from two diseases, called
BCEe220 common era, CE), large quantities of silk and pébrine and flacherie, and proved that the spread of these
silk products were exported along the famous Silk Road, diseases could be prevented by carefully segregating
westwards to Central and Western Asia, the Mediterranean, healthy and diseased larvae from each other (Debré, 1998).
and Europe, and eastwards to Korea and Japan. Silkworm He published a two-volume treatise on silkworm diseases
eggs and sericultural techniques were introduced into Japan to describe his findings (Pasteur, 1870). From the 1870s
via Korea in the second century, and then into Turkey, onwards, advanced sericultural techniques were further
Egypt, Arabia, the Mediterranean, and Rome in the sixth developed in Japan. Japanese studies on silkworm viruses
century. Since then, sericulture has spread throughout much also improved the understanding of other silkworm
of the world. diseases. In the second half of the twentieth century,
Chapter | 12 Silkworms and Bees 427

silkworm pathology entered the age of biochemical and incubation period for viral diseases (the time from infection
molecular biology. Currently used mainly for disease until symptoms appear) is usually six to 10 days. Most
diagnosis, these techniques could lead to molecular silkworms become infected during the fourth to early fifth
marker-assisted selection, transgenic breeding of resistant instars, whereas the disease outbreaks usually occur in late
varieties, and gene therapy. fifth instars.
To understand the biology of silkworm diseases today, it Bombyx mori nucleopolyhedrovirus (BmNPV) causes
is important to become familiar with some aspects of a disease referred to as nuclear polyhedrosis, jaundice, or
modern silkworm production. Silkworm rearing can be blood-type grasserie (from the French word “grasse”,
“batched” or “multibatched”, depending on the duration of which means thick and sticky and describes the exudates
the frost-free period and availability of mulberry trees in seen in dead silkworms). BmNPV is a double-stranded
the region. In batched rearing, larvae are reared five or circular DNA (dsDNA) virus that has a genome consisting
fewer times in a year, while 12 or more cycles are produced of 128,413 nucleotides, a GC content of 40% (GenBank
per year in multibatched rearing. The batches overlap in Accession No. L33180), and 143 genes (Kamita and
time with multibatched rearing but are consecutive in Maeda, 1997; Gomi et al., 1999) (see Chapter 4). The
batched rearing. Accordingly, there is time to disinfect virions are occluded in an occlusion body. Healthy (i.e.,
rearing facilities between batches for batched rearing but uninfected) larvae are slightly green and appear somewhat
not for multibatched rearing. Thus, multibatched rearing transparent white, whereas infected larvae become ivory
systems are more prone to disease outbreaks. with swollen intersegmental membranes. Diseased larvae
In a typical production system, silkworm eggs and behave differently, becoming easily agitated when
young instars (first to third) are produced initially in large disturbed, and are frequently found lying at the edge of the
professional rearing facilities or by experienced and well- rearing beds before death. In addition, the integument
equipped farmers. The fourth instars are then distributed to becomes very fragile, and the hemolymph turns milky and
smaller facilities at farmers’ homes. In China, the facilities turbid.
used to produce the eggs and early instars are mostly state Bombyx mori cypovirus-1 (BmCPV-1) (also called
owned. These facilities have specially designed rearing cytoplasmic polyhedrosis or cypovirus) is a circular, double-
rooms that are easy to clean and disinfect (commonly, stranded RNA (dsRNA) virus. Its genome consists of 10
rooms with steel walls and ceilings and cement floors). The dsRNA segments that range in size from 0.3e0.8 to 2.2e2.6
rearing rooms are also well ventilated and somewhat heat kilobases (kb), each possessing a single, complete open
insulated, often with heating and/or cooling systems. reading frame. Segments IeIV, VI, and VII encode struc-
Young silkworms are usually reared on rearing trays made tural capsid and occlusion body proteins, segments VIIIeX
of either bamboo or wood and fed the tender new leaves of encode non-structural proteins, and the function of segment
mulberry. The trays are stacked in racks, seven to 10 tiers V is still uncertain (Ikeda et al., 2001; Hagiwara et al., 2002;
high, and the racks are usually either wood or metal. The Lin et al., 2002) (see Chapter 4). This virus forms occlusion
facilities for the late instars are simpler, with the silkworms bodies that are mostly hexagonal, icosahedral, tetragonal, or
reared directly on the floor of a small room (often on occasionally triangular, ranging in size from 0.5 to 10 mm
a wooden or dirt floor), and heating and cooling systems and averaging 2.6 mm (Jin, 2001). Larval development in
may be lacking. These are called “rearing beds”, The late infected insects becomes asynchronous, with prolonged
instars can be fed mature, fresh mulberry leaves. Thus, the instar duration. Infected individuals are thin and small, show
production system is not entirely self-contained, as insects a loss of appetite, and respond slowly to stimuli. The thorax
are shipped from one facility to another and field-collected becomes translucent, the abdomen appears shrunken, and
fresh mulberry leaves are brought in daily. grayish white feces are produced when the larvae are
Disease control is an important part of sericulture, even squeezed. The midgut and hindgut become milky, striated in
in the best facilities. The infectious diseases of silkworms appearance, and shrunken.
are caused by viruses, bacteria, fungi, and protists (Table In sericultural practice, the French term “flacherie” has
12.1). The most serious diseases are the blood-type grass- been used for two forms of dysentery, non-infectious and
erie, white muscardine, and pébrine. Most of the diseases infectious (viral), where the silkworm larvae become
can be readily diagnosed based on signs and symptoms in flaccid. Non-infectious flacherie, also known as “touffee
the larvae or cadavers and by microscopic examination flacherie”, is caused by exposure of the larvae to exces-
(Table 12.1). sively high temperatures. Viral flacherie can be caused by
B. mori infectious flacherie virus (BmIFV), B. mori den-
sovirus (BmDNV) (¼ B. mori densonucleosis virus), or B.
12.2.1. Viruses
mori cypovirus-1. These viral infections, either alone or in
In silkworms, nucleopolyhedrosis virus and viral flacherie combination with bacterial infections, destroy the gut
are common and can cause very serious larval losses. The tissue, resulting in dysentery and larval flaccidity.
 428
TABLE 12.1 Some Useful Characteristics for Preliminary Diagnosis of Infectious Silkworm Diseases
Some Distinct Characteristics

Pathogen Disease General Signs and Symptoms Hemolymph Midgut Integument

a
Viruses

Bombyx mori Nuclear polyhedrosis, Larvae display defensive postures readily; Turbid and white, Not distinct Shiny just before
nucleopolyhedrovirus jaundice, or blood-type molting is delayed or lacking. posterior occlusion molting. Fragile,
(BmNPV) grasserie abdominal segments swollen; cadavers bodies are present exuding fluids. In late
grayish black and flaccid instars, spiracles black,
and cuticle with black
spots

B. mori cypovirus-1 Cytoplasmic Larvae not easily agitated, pale, thin and Clear, no occlusion Posterior midgut Not distinct
(BmCPV-1) polyhedrosis or small; growth slow and asynchronous; bodies present ivory in color,
cypovirus feces remains attached to the anus; shrunken, and striated.
cadavers flaccid Occlusion bodies
are present

B. mori infectious Viral flacherie Larvae vomit; head raised; growth Clear, without Contains yellowebrown Not fragile
flacherie virus (BmIFV) and molting asynchronous; head and occlusion bodies fluids. Pink viral
thorax translucent; larvae discolored, spherical bodies
flaccid; feces watery; cadavers have a present. Stains with
flattened shape pyronin-methyl green

B. mori densovirus Densoviral flacherie Like flacherie or bacterial flacherie; Clear, without Contains yellowish Not distinct
(BmDNV) (¼ B. mori anorexia; late molting; bodies shrunk; occlusion bodies green fluids. Cocci and
densonucleosis virus) integument not fragile; thorax mostly diplococci very
translucent abundant but no
occlusion bodies
Bacteria

Bacillus spp. Fuliginosa septicemia Large thorax and small abdomen; anterior Brownish black. Not distinct Not distinct
segments of abdomen with greenish Bacteria abundant,
black spots; vomiting and diarrhea; large bacilliform rods
cadavers turn black, decayed and fetid

Serratia marcescens Serratia-type Larvae with light specks; Reddish brown. Not distinct Not distinct
septicemia cadavers appear shortened, become Bacteria abundant,
flaccid, and turn pink or dark pink short bacilliform rods

Aeromonas sp. Green thorax Head and thorax of cadavers Turbid. Bacteria Not distinct Not distinct

Insect Pathology
septicemia translucent green, and turned down ventrally; present, small
cadavers are flaccid and become putrid bacilliform rods

Bacillus thuringiensis Sotto disease or Larvae stop feeding, turn dark with Not distinct Midgut cells disassociating. Not distinct
subsp. sotto (Bt sotto) bacillary paralysis internal nodules; posterior is translucent; Bacteria present, long
cadavers turn flaccid bacilliform rods.
 Chapter | 12
Enterococcus spp. Bacterial flacherie; Larvae not uniform in development, Not distinct Empty except for Translucent on the head
and other intestinal thoracic or wrinkling become thin and small. Diarrhea yellowegreen mucus. and thorax.
bacterial species disease Bacteria present, large,
bacilliform rods

Fungi: Filamentous
Beauveria bassiana White muscardine Larvae sluggish, with oil-colored Turbid. Becomes Not distinct Larvae with powdery

Silkworms and Bees

specks; cadavers soft at first and then full of cylindrical white appearance due
stiffen, and finally covered with hyphal bodies or to presence of large
white conidia hyphae masses of globose
conidia

Nomuraea rileyi Green muscardine Larvae sluggish, with oil-colored specks; Turbid. Gradually Not distinct Presence of large
cadavers soft at first and then stiffen, full of beaded masses of ovoid conidia
and finally covered with bright hyphal bodies or results in bright green
green conidia hyphae appearance

Aspergillus spp., Aspergillosis Newly hatched larvae stop feeding, stop Turbid. Hyphal Not distinct Sporulation occurs in
e.g., A. flavus, A. moving, frequent vomiting, and die in bodies not present localized spots, color
ochraceus, A. oryzae, 2e3 days; late instars develop a lesion, varies, often brown,
A. parasiticus, A. tamari thorax protrudes, frequent vomiting, die usually dark. Hyphae
in 4e5 days; cadavers stiff with hyphae visible around spots
penetrating

Fungi: Microsporidia
Nosema bombycis Pébrine Growth slow and typically asynchronous, Turbid, spores Swollen in appearance, Sometimes peppered
molting may be incomplete; no cocoon present ivory color with black with black specks and
formation; spores present in the hemolymph, specks thin, irregular setae
midgut, silk gland, feces and eggs; ovoid
2.9e4.1  1.5e2.1 mm
Protists
Entamoeba spp. Amoebiasis Late instar larvae, growth slowed, the caudal Not distinct Cells swell due to Not distinct
end of the larvae collapses, posterior end presence of amoebic
shrunken; feces green; cadavers black, cysts, pathogen released
mummified into the gut and feces.
Cysts tetranucleate

Herpetomonas bombycis Trypanosome infection Infected larvae are slightly grayish white, Not distinct Not distinct Not distinct
with decreased appetite, turbid hemolymph,
pulsating dorsal vessel, and die gradually;
cadavers turn blackish brown

Undescribed coccid Coccid disease Larvae develop poorly, shrunken, diarrhea, Not distinct Infection occurs though Not distinct
brown mucus forms around the anus; cysts midgut epithelial cells
detected in the feces
a
In viral nomenclature, only the type species is in italics.

429
430 Insect Pathology

BmIFV is the most common cause of viral flacherie in densoviruses are single-stranded DNA (ssDNA) with no
silkworms (see Chapter 5). It is a single-stranded, posi- occlusion body. The virions are globose, with a diameter
tive-strand RNA virus with no occlusion bodies. The of 22 nm for BmDNV-I and 24 nm for BmDNV-II.
virions are equilateral icosahedrons with a diameter of BmDNV-II has two sets of non-homogeneous linear
26  2 nm. The viral RNA sequence has been completed ssDNAs (VD1 and VD2) that are separately encapsulated.
for a Japanese and two Chinese BmIFV strains (GenBank The genome of two Chinese strains, VD1 (GenBank
Accession No. EF422865, HM245295, and AB000906, DQ017268) and VD2 (GenBank DQ017269), consists of
respectively), and range from 9650 to 9675 base pairs 6543 and 6022 bp, respectively. Symptoms of diarrhea and
(bp), with 99% homology among these three strains vomiting are very similar to those caused by BmIFV, but
(Wang et al. 2006a, b). BmIFV-infected larvae are stunted, the occurrence of a translucent thorax in a BmDNV-
uneven in growth, show a gradual loss of appetite, and infected larva is more prominent. In addition, the midgut is
molt asynchronously. As the disease develops, the larvae full of yellowish brown fluid, but without any remains of
become discolored, shrunken, and flaccid. The prolegs mulberry leaves because the larvae usually have ceased
lose their ability to clasp the surface, the larvae become feeding for an entire week. Large numbers of cocci and
translucent, and vomiting and diarrhea commonly occur in diplococci bacteria are typically present in the cadaver and
the fifth instar. The symptoms are similar to BmCPV-1, can be detected microscopically (Jin, 2001), but these are
but the feces are thin, form in a chain, and appear not the cause of the disease.
yellowish brown. The cadavers have a foul odor and, upon The silkworm viruses can be transmitted both vertically
dissection, the midgut is not milky. and horizontally, making it difficult to control these path-
Six strains of BmDNV have been described and cate- ogens (see for example, BmNPV, Fig. 12.1). Horizontal
gorized into two types: BmDNV-I and BmDNV-II. These transmission occurs when the viruses contaminate

FIGURE 12.1 Transmission routes for the Bombyx mori nucleopolyhedrovirus (BmNPV). Cadavers of virus-infected silkworm larvae become a source
of inoculum for per os infections in other larvae by contaminating the mulberry leaves with occlusion bodies. Occlusion bodies can also be released though
the feces of infected larvae. Infected larvae may acquire the virus and then die (repeating the cycle), or they may survive infections and serve as a source of
infection through vertical transmission to their progeny. The dispersal of adults to new sites occurs primarily via humans when silkworm cocoons are
harvested and the new adults are transferred to egg production facilities. (From Khurad et al., 2004, with permission from the publisher.)
Chapter | 12 Silkworms and Bees 431

mulberry leaves and rearing trays, and infection of the signs are typically a stiffening of the thorax and the first
larvae occurs per os. The viruses can persist in the three abdominal segments. The stiffening then extends
cadavers, feces, and exuviae, leading to contamination of anteriorly and posteriorly, and the segments darken rapidly
the rearing equipment, rearing beds, and leaves (Fig. 12.1). such that the entire cadaver turns black and exudes a dark
Mulberry leaves can also become contaminated with virus brown fluid. With Serratia-type septicemia, cadavers
particles in the field from other lepidopteran insects that become slightly shortened and develop small brown specks.
serve as carriers for the pathogen. Such carriers include the Cadavers of larvae become flaccid and pink and those of
wild silkmoth, Bombyx mandarina, the mulberry pyralid, pupae become black or pink, whereas cadavers of the adult
Glyphodes pyloalis, the mulberry geometrid, Phthonan- abdomen become partially black or pink and exude a pink
dria atrilineata, the mulberry white caterpillar, Rondotia fluid. Septicemia caused by Aeromonas is also called green
menciana, and the mulberry yellow-tail moth, Porthesia thorax septicemia, with mortality occurring in late instar
xanthocampa (Watanabe et al., 1988). However, most viral larvae. Translucent green or greenish specks appear on the
transmission occurs within rearing beds. The cadavers dorsal part of the thorax, and greenish or brownish bubbles
serve as the main reservoir for infection, but infected form under the integument. The hemolymph from cadavers
larvae can also contaminate the mulberry leaves and is turbid and the cadavers emit a putrid odor (Jin, 2001).
rearing beds. Workers can then further spread the virus Bacteria that induce septicemia are transmitted mainly
particles with their hands, and flies in the rearing rooms through wounds; therefore, occurrence of bacterial infec-
can act as carriers. Vertical transmission occurs with tions is most closely related to silkworm age, handling
BmNPV and BmCPV-1, which can persist in a batch of technique, and rearing conditions. Late instar larvae have
larvae as sublethal infections. The moths derived from sharp, well-developed tarsal claws and often injure each
such larvae can successfully spread the viruses venereally other under crowded conditions. In fact, up to 80% of the
to each other during mating and to their offspring by larvae can become injured (Jin, 2001). The large late instar
transovarial transmission (Fig. 12.1) (Aruga, 1971; Khurad larvae also have an enormous feeding rate, resulting in
et al., 2004). a copious production of fecal material. When large amounts
of feces are combined with high humidity, excessive
bacterial growth can result in the rearing beds. Densely
12.2.2. Bacteria
populated rearing beds and excessive handling can exac-
Facultative bacterial pathogens occur widely in nature, erbate the problem, resulting in higher frequency of larval
have broad host ranges, and cause mostly acute diseases in wounding and bacterial infections due to fecal contami-
silkworms. The signs and symptoms of diseased silkworms nation. Accordingly, septicemia rarely occurs in neonates
are often generic and it may be difficult to identify and is most commonly a problem in mature larvae. Newly
a specific bacterium as the causal agent without performing hatched larvae have undeveloped tarsal claws and their
Koch’s postulates. However, diagnosis according to the bodies are protected to some extent by setae.
characteristics of moribund larvae and cadavers, including Another common bacterial disease is Sotto disease, also
tissue discoloration, may provide sufficient information as known as bacillary paralysis, Sottokin, or “sudden death
to the probable causal agent (Table 12.1). Bacterial diseases bacillus” (see Chapters 2 and 8). This disease is caused by
typically occur when larvae are reared in overcrowded Bacillus thuringiensis subsp. sotto (Bt sotto). During
conditions. sporulation, cells of Bt sotto produce endospores with
Bacteria that cause septicemia (a morbid condition parasporal bodies containing the d-endotoxins, and secrete
caused by the multiplication of microorganisms in the the a-, b-, and g-exotoxins into the medium (i.e., hemo-
blood) (Steinhaus and Martignoni, 1970) in silkworms lymph or nutrient-based agar). Silkworms are very
belong to many taxa. The most common bacteria associated susceptible to these toxins. The disease occurs primarily in
with septicemia are Bacillus bombyseptieus (Huang et al., late instar larvae. Both acute and chronic signs and symp-
2009), Serratia marcescens, and Aeromonas mundii toms can occur. Acute symptoms include a sudden cessa-
(Cappelloza et al., 2011). Streptococcus spp. and Staphy- tion of larval feeding; the larvae raise their body anteriorly
lococcus spp. also cause septicemia in larvae. The common and have convulsive tremors and vomiting, and their thorax
symptoms of septicemia are larvae becoming dull and becomes slightly swollen, followed by sudden death
motionless with reduced feeding rates. Moribund larvae with their whole body paralyzed. Infected larvae may die
can be found vomiting and exhibiting spasms while lying 10 min to several hours after consuming the Bt sotto toxins.
on their sides. Signs of a bacterial infection include The chronic signs and symptoms associated with Sotto
a swollen thorax and shrunk abdominal segments. Cadavers disease include reduced larval feeding, darkening of the
are often discolored and are characteristically flaccid. body, and the posterior midgut being empty; the feces may
Fuliginosa septicemia is caused by Bacillus spp., most be reddish brown in color or be a foul-smelling liquid
commonly B. bombyseptieus (Li et al., 2011). The first discharge. Infected larvae become flaccid on the mulberry
432 Insect Pathology

leaves, and after two to three days, die hanging upside named based on these colors. The most common muscar-
down or lying on their sides. Brownish specks appear on the dines are white and green.
cadavers. The cadavers become discolored and then flaccid, White muscardine is caused by B. bassiana. Infected
starting at the dorsal anterior part of the abdomen (Jin, larvae lose their appetite, become sluggish, and eventually
2001). cease to move. Small irregular specks appear on the body,
A third bacterial disease, bacterial flacherie, also known or two or three large, oil-colored specks appear. Immedi-
as thoracic or wrinkling disease, has symptoms that are less ately after death, the cadavers are soft, but they soon harden
distinct than the first two bacterial diseases. Bacterial and appear mummified, and then white mycelia emerge
flacherie occurs sporadically and does not cause heavy from the intersegmental membranes and gradually cover
losses. It is caused by bacteria that normally occur in the the whole body (if conditions are humid enough) and then
silkworm’s digestive tract, including Enterococcus spp. form a white layer of conidia, making the cadavers look
(mainly E. faecalis and E. faecium) and some species of chalky white.
Staphylococcus, Bacillus, Klebsiella, Alcaligenes, and To insect mycologists and microbial control specialists,
Pseudomonas (Nagae, 1974, 1982). Normally, silkworms the term “green muscardine” indicates a mycosis caused by
are not susceptible to these bacterial species unless the Metarhizium spp., mainly M. anisopliae. However, in
larvae are under stress owing to starvation, low-quality sericulture, this term is used for a common mycosis caused
mulberry leaves, or unsuitable rearing conditions (e.g., high by Nomuraea rileyi. Mycoses caused by M. anisopliae are
temperature and humidity). Common signs and symptoms rare and are called black muscardine. This nomenclature
of infected larvae include sluggishness, cessation of arose because the silkworm cadavers infected with N. rileyi
feeding, vomiting, diarrhea, reduced growth, asynchronous become bright or grass green, while those killed by
development, shrunken bodies, and translucent thoraxes. M. anisopliae infections turn dark green. Both fungi infect
The larvae usually die during the molting process. When larvae and pupae. Early stage signs and symptoms are not
death occurs between molts, both ends of the larvae become obvious. In late stages of fungal infection, the larvae lose
swollen and the cadavers become flaccid (Jin, 2001). their appetite, become sluggish, and develop large, dark
Rearing conditions can increase the prevalence of brown specks on the lateral or dorsal sides of the abdomen.
septicemia, Sotto disease, and bacterial flacherie. As Immediately after death, the soft cadavers appear ivory,
described above, the bacteria that cause septicemia can with the thorax overextended. These soft cadavers harden
persist in cadavers and feces of silkworms and contami- gradually, white mycelia emerge from the cuticle, and as
nate mulberry leaves, rearing beds and equipment, and the sporulation occurs, a green, powdery layer of conidia
floors in the rearing facility. Both Bt sotto and Entero- covers the cadavers. The early signs and symptoms of green
coccus spp. commonly occur in the environment at non- muscardine can overlap with those of other diseases. For
epizootic levels, and usually the prevalence of these example, larvae that die in the third and fourth instars from
diseases in the silkworm colonies is low or not detectable NPV infections also look ivory and shiny. Moribund larvae
(Ohba, 1996). However, unfavorable rearing conditions are easily agitated and their integument ruptures easily,
(e.g., high temperatures and humidity) can cause a bacte- releasing the hemolymph contents, differentiating such
rial disease outbreak. High humidity and a continual infections from the muscardines, where this condition does
feeding of wet mulberry leaves will result in the rearing not occur.
beds becoming humid and hot, and this situation Diseases caused by Aspergillus spp. are called asper-
commonly occurs during the rainy season. Such condi- gillosis. As with the muscardine infections, silkworm
tions not only lead to the proliferation of the bacteria on cadavers with aspergillosis become stiff and mycelia
the leaves but are also harmful to silkworm development, emerge from the integument. However, with aspergillosis,
probably increasing their susceptibility to bacterial toxins sporulation occurs only in localized spots, and the color of
and infections. the conidial layer greatly varies but is most commonly
brown. That is why this disease was once called brown
muscardine. The fungi causing this disease in silkworms
12.2.3. Fungi: Filamentous
include more than 10 Aspergillus species, mainly A. flavus,
The muscardines are fungal diseases and are the second A. ochraceus, A. oryzae, A. parasiticus, and A. tamarii. The
most important silkworm diseases in China and Japan fungus kills the early instars in two to three days and the
(following blood-type grasserie in China and viral flacherie later instars in four to six days. The first three instars are
by BmIFV and BmDNV in Japan). The muscardine fungi readily infected, but the infection is infrequent in mature
produce asexual spores (conidia) on the cuticle of cadavers, larvae. When neonates become infected, they stop feeding,
and these conidia are the infective propagules. Depending vomit, become sluggish, and do not move around in the
on the pathogen species, these conidia are white, green, rearing beds. Mycelia emerge throughout the body and
yellow, black, gray, or red, and the muscardine diseases are produce flocculent-like (i.e., woolly) conidiogenous
Chapter | 12 Silkworms and Bees 433

structures after one to two days. The cadavers do not decay. windows, creating conditions favorable for fungal
When grown larvae are infected, brown specks appear infections.
which increase in size to become lesions, usually on an Silkworm resistance to fungi can be high in late larval
intersegmental membrane or the anal area. The thorax instars. For example, Mu et al. (1999) reported that
protrudes, and the larvae vomit. After death, the cadavers compared to newly hatched larvae, newly molted fifth
harden around the lesion, and the rest of the body becomes instars were 86 times more resistant to white muscardine,
black. Mycelia form and conidia are produced one to two 79 times more resistant to green muscardine, and 95 times
days postmortem. Infected pupae become dark brown. Eggs more resistant to aspergillosis. Larvae are also more
can also become infected. The surface of the eggs becomes susceptible to fungal infection during molting than just
moldy and depressed, and soon shrivels. before or after molting (Mu et al., 1999; Chandrasekharan
Other mycoses from filamentous fungi are rare, but and Nataraju, 2011). Thus, both environmental conditions
include gray muscardine caused by Isaria javanica and the age of the larvae can play an important role in
(formerly Paecilomyces javanicus) (Huang et al., 2008), fungal outbreaks.
yellow muscardine caused by I. farinosa (formerly P. far- Outbreaks of white muscardine on silkworms are
inosus), and red muscardine caused by I. fumosoroseus sometimes attributed to the application of B. bassiana to
(formerly P. fumosoroseus). Grassy muscardine is caused control the Masson’s pine caterpillar, Dendrolimus punc-
by Hirsutella necatrix, and fusariosis is caused by Fusa- tatus (Zhu, 2008). Although B. bassiana has a very broad
rium species (Jin, 2001). These fungal pathogens usually do host range, the pathogenicity and virulence of each strain
not pose a threat to sericulture. The cadavers exhibiting tend to be greatest for the hosts from which it was isolated
gray, yellow, or red muscardine infection become grayish (Li, 1988). The LC25, LD25, and LT25 for silkworms caused
white, yellowish white, dark green to black, or pink, by the strain used for control of pine caterpillars were 1327,
respectively. Those that succumb to grassy muscardine 1378, and 1.5 times that of the LC75, LD75, and LT75 caused
look reddish brown or have synnemata emerging from the by a typical pathogenic strain isolated from silkworms.
cadaver. After infection is initiated, the larvae develop Silkworm white muscardine is characterized by an enzootic
blackish brown specks of various sizes. These appear on nature, being commonly present at a low level (usually
almost every segment before death. Fusariosis is charac- below 5%) (Li et al., 2010, 2011), and the application of
terized by a fecal mass on the anus premortem and B. bassiana against pine caterpillars probably has little
postmortem. impact on silkworm white muscardine levels.
For all of the fungi discussed so far, the infective
propagules are conidia, which infect through the insect
12.2.4. Fungi: Microsporidia
cuticle (see Chapter 6), although per os infections can occur
(Yanagita and Iwashita, 1987). Conidia can be transported Pébrine is caused by various microsporidia, predominantly
very easily in air and water, thus having the potential to Nosema bombycis and, to a lesser extent, Vairimorpha,
contaminate rearing rooms and all the rearing equipment. Pleistophora, and Thelohania species. Although micro-
As long as the conidia are not exposed to solar radiation or sporidia belong to the kingdom Fungi (see Chapter 7), the
disinfectants, they can persist for long periods, especially in diseases they cause have symptoms (Table 12.1) that are
soil, although persistence is strongly affected by several quite different from other mycoses, mainly in that the
biotic and abiotic factors (see Chapter 6) (Keller and cadavers are soft and the microsporidia do not produce
Zimmermann, 1989; Zimmermann 2007a, b). In rearing fruiting bodies. For a long time, microsporidia were
rooms conidia can persist for six to 12 months on cadavers thought to be protists and are often listed among this taxon
(Jin, 2001). in silkworm pathology texts and other older literature.
The effect of environmental factors on fungal ento- Pébrine was first recorded in France in 1845 and then
mopathogens has been widely studied, especially for spread to Italy, Spain, Syria, and Romania. It was respon-
B. bassiana and M. anisopliae (Zimmermann, 2007a, b). sible for the collapse of the French and Italian silkworm
The most important abiotic environmental constraints for industries in 1865 (see Chapter 2). Pasteur discovered that
fungi are temperature, humidity, and solar radiation (see the microsporidium was transmitted both on contaminated
Chapter 6). Silkworm rearing rooms, however, are mulberry leaves and transovarially. This discovery led to
a special artificial ecosystem, where temperature extremes disease control methods that involved sampling and
do not commonly occur, moisture levels can be controlled, examining the egg-laying moths, culling the infected eggs,
and solar radiation is limited. Yet, high humidity can occur and supplying disease-free superior stock. Despite the
on rainy days or when mulberry leaves are brought in wet. success of this method, N. bombycis is still the most serious
Under these conditions, microhabitats with high humidity pathogen of silkworms throughout the world.
and temperature can easily form on rearing beds, espe- Nosema bombycis attacks all insect tissues and devel-
cially in poorly ventilated rooms without sufficient opmental stages, and the signs and symptoms are observed
434 Insect Pathology

in all life stages from egg to adult. Infected eggs are Pieris rapae, Porthesia xanthocampa, Spilarctia sub-
irregularly shaped, hatch asynchronously, and do not carnea, and Spilosoma menthastri (Hirose, 1979; Wan
adhere to the substrate. Some may be dead or infertile. et al., 1991; Shen et al., 1996). Some of these moths are
Larvae infected by pébrine exhibit a loss of appetite, have also susceptible to N. bombycis. However, the hoste
reduced growth rate, vary in size, and display incomplete pathogen relationship between B. mori and N. bombycis
molts. The majority of mortality occurs right after the still appears to be fairly specialized. For instance,
second molt for infections in larvae that acquired the N. bombycis invades B. mori both per os and transovarially,
infection transovarially from their mother (i.e., infected but transovarial transmission does not occur with any other
eggs). Those that survive past the third instar are pale and microsporidia that affect silkworms.
flaccid, small, and vomit frequently. Eventually, these Other silkworm pathogenic microsporidia include
larvae become covered with dark brown spots and either are Endoreticulatus, Vairimorpha, Pleistophora, and Theloha-
unable to spin silk or spin the cocoons loosely. However, nia. The taxonomic position of these silkworm pathogenic
the spots do not always occur, and are not common on most genera remains uncertain (see Chapter 7). One species of
of the Chinese silkworm varieties. Larval cadavers remain Endoreticulatus was reported in China (Wan et al., 1995)
rubbery and do not become flaccid after death. Infected and has oval spores with polar filaments arranged in seven
adult moths have deformed wings and distorted antennae, to nine rings, and develops inside a parasitic sack formed
and the scales on the wings and abdomen fall off easily. by the membrane of the endoplasmic reticulum of host
Adults with pébrine mate poorly and have poor egg cells. This pathogen has high oral infectivity. Several
production. isolates of Vairimorpha spp. have been reported from
The spores of N. bombycis are ovoid. A micropyle is silkworm cultures in Japan (Hatakeyama et al., 1997),
located at the anterior end, and in the center is a belt-like China (Zheng et al., 1999; Yang et al., 2001) and India (Rao
sporoplasm, containing four nuclei. A vacuole occurs at et al., 2004). Spores are ovoid and variable in size. Vair-
each end. A polar capsule, polar capsule nucleus, and polar imorpha spp. have a moderate level of oral infectivity. An
filament are present. The polar filament is tubular and unnamed species of Pleistophora (Fujiwara, 1984a) and
filamentous in shape, and wound into a coil, as is typical for Thelohania (Fujiwara, 1984b) has been reported in Japan.
Nosema (see Chapter 7). The spores are highly retractile, Epizootiological studies of microsporidia in silkworms
appearing light green under the microscope, with a smooth have focused on pébrine caused by N. bombycis, and very
outline. Spore dimorphism was detected during spore little is known regarding the epizootiology of the other
development in the epithelium of silkworm larvae (Iwano, microsporidia. The extent of horizontal transmission on
1991). a rearing tray can be extremely high. If only a few diseased
Nosema bombycis cannot be differentiated from other larvae are mixed with healthy ones at an early date, trans-
Nosema species using morphological methods; therefore, mission will increase infection levels quickly. For example,
various serological, protein, and molecular approaches it has been demonstrated that if 3% of the first instar larvae
have been developed. For example, Qiu et al. (2002) are infected, 50e60% of the silkworms will die from
developed a nucleic acid hybridization method that can pébrine by the time they reach the adult stage, and all the
precisely distinguish 10 Nosema species. Research con- emerging moths will be infected (Jin, 2001).
ducted to characterize the genome of N. bombycis should Spores of N. bombycis in the dormant stage are also
improve molecular detection methods (Sironmani, 2000; very persistent, remaining infective for at least three years
Xiang, 2010). in the dry body of female moths and after being submerged
The prevalence of pébrine varies with the variety of in water for five months. Nosema bombycis spores are
silkworm, the developmental stage, and the rearing envi- inactivated by the following treatments: (1) direct sunlight
ronment (Lu, 1991). The Chinese silkworm varieties tend (39e40 C) for 6e7 h; (2) boiling at 100 C for 5 min; (3)
to be the most pébrine resistant, the Japanese varieties less moist heat at 100 C for 10 min; (4) 2% formalin for
so, and the European varieties are the most susceptible. 40 min; (5) 4% formalin for 5 min; (6) 1% chlorine for
This resistance may be related to voltinism. The multivol- 30 min; or (7) 3% chlorine for 10 min (Lu et al., 2000). The
tine varieties are relatively pébrine resistant, followed by spores are very resistant in the environment, surviving not
the bivoltine, and then univoltine varieties. Also, young only in various infected stages of the silkworms and their
larvae, newly molted larvae, and starving larvae are highly cadavers, but also in silkworm frass, the egg chorion of
susceptible and show high mortality rates. hatched eggs, exuviae, cocoons, moth scales, and on
Several species of Lepidoptera are susceptible to other mulberry leaves, rearing room floors and walls, and rearing
Nosema species that are also pathogenic to silkworms. instruments. All these survival places should be considered
These other lepidopteran hosts include Antheraea yama- when control measures are undertaken.
mai, Chilo suppressalis, Diaphania pyralis, Hyphantria Temperature plays a critical role in microsporidian
cunea, Philosamia cynthia, Phthonandria atrilineata, infection (Maddox, 1973). Pébrine mortality can be
Chapter | 12 Silkworms and Bees 435

reduced by keeping the incubation temperatures for the discovered in Japan (Aratake and Kayamura, 1977). Cells
larvae and pupae high and by soaking eggs in hot water are 15 mm long, with a flagellum as long as the cell. Little is
(53e55 C) (Lu et al., 2000). A 40-year study on Nosema known about this Leptomonas in silkworms.
prevalence in stock female moths at the Guangdong Insti- A coccid disease of silkworms has been reported in
tute of Silkworm Egg Production (China) was conducted Japan (Jin, 2001) (Table 12.1), but the taxonomic position of
between 1957 and 1997, and found that ambient tempera- the etiological agent has not been determined. The pathogen
tures in one year affected disease prevalence the next year, cells are globose with a large nucleus. This pathogen can
with high temperatures leading to disease outbreaks and develop to form large, globose cysts. Cells inside the cyst
low temperatures leading to low disease prevalence (Liu can grow to about 100 mm (visible to the naked eye), and
et al., 2001). Humidity also affects Nosema prevalence. then several spores form as the nucleus divides and the
High humidity increases the attachment of spores to protoplasm splits. After the thick membrane of the cysts
mulberry leaves; thus, rearing on moist trays increases the breaks, the spores are released and can invade other tissues.
chance of silkworms ingesting contaminated mulberry The coccids infect silkworms through the epithelial cells of
leaves (Lu et al., 2000). the midgut. Diseased larvae develop poorly, become
shrunken, have diarrhea, and a brown mucus forms around
the anus. Cysts can be detected in the feces.
12.2.5. Protists
Various protists (see Chapter 10) can infect silkworms,
12.2.6. Non-infectious Biotic Agents
including amoebae, trypanosomes and coccids, and infec-
tions have been reported mostly in Japan and Korea. Silkworms can suffer from injuries caused by non-infec-
Amoebae and trypanosomes are transmitted per os or via tious biotic agents such as parasitoids, parasitic mites, and
wounds. No transovarial transmission occurs, but little is urticating hairs of other Lepidoptera.
known about their epizootiology in silkworms. Silkworm larvae are attacked by tachinid parasitoids
Infections ensue after amoebic cysts are ingested by (called uji flies), mainly in the genus Exorista spp.
silkworms and become active in the digestive tract. The (E. sorbillans and E. bombycis), but also including Cross-
amoebae emerge from the cysts and infect silkworm larvae ococmia sericariae, Ctenophorocera pavida, and Ble-
along the epithelial cells of the midgut, causing them to pharipa zebina. These uji flies can threaten the sericultural
swell and collapse, releasing more pathogenic cells into the industry and are most serious during the summer. Losses
gut. When cysts are produced, they are released from the due to tachinids can reach 10e15% in China, and exceed
host into the feces. The species identification of the etio- 30% in areas of southern China that are hot and humid (Jin,
logical agent is still uncertain, although it has been referred 2001). Exorista sorbillans typically produces four to five
to Entamoeba spp. (Jin, 2001). The cells are globose or generations in northern and north-eastern China, six to
subglobose, with a strongly refractive central nucleus and seven generations in eastern China, and 10e14 generations
one to three vacuoles in the cytoplasm, and reproduce by in southern China. Both males and females mate multiple
binary fission. The cells can form cysts, which are times before females start laying eggs, usually on the
predominantly tetranucleate. The disease occurs most second day after sufficient mating events have occurred.
frequently in third instars or later, and larval development Exorista sorbillans uses odor to locate hosts, laying eggs on
becomes retarded. When heavily infected, the caudal end of fifth instar silkworm larvae. Larvae require only four to five
the larvae collapses and the posterior end shrinks. The feces days to mature, with the pupal stage lasting 10e12 days or
are green, and the cadavers turn black and mummified. for several months if the pupae go into winter diapause.
Trypanosomes were discovered in Italy in the early part Exorista sorbillans is a generalist parasitoid with a host
of the twentieth century. The etiological agent in silkworms range that includes more than 10 other species of
is Herpetomonas bombycis, which has 6e12 mm long Lepidoptera (Jin, 2001).
conical cells with a 10e20 mm long anterior flagellum The first sign of attack is a small brown lesion, where the
(Levaditi, 1905). Within the cell, a kinetoplast occurs parasitoid larva penetrated through the cuticle into the
between the centralized nucleus and the flagellum. The hemocoel. The eggshell of the parasitoid can sometimes be
cells can shrink to form a cyst. Reproduction is by binary seen on the lesion, but normally it drops off the host’s cuticle.
fission. Infected larvae are slightly grayish white, with The lesion enlarges over time, becoming a blackebrown,
decreased appetite, turbid hemolymph, and pulsating dorsal horn-shaped sheath as the silkworm larva grows. The
vessel, and die gradually. Cadavers turn blackish brown yellowish parasitoid larva can be found if the lesion is
(Table 12.1). When healthy larvae are injected with body dissected. The silkworms sometimes take on a purplish color,
fluid from an infected larva, H. bombycis can be detected in due to oxidized hemolymph, and larvae often cannot
24 h, and the injected larvae die in two to three days. complete development to the adult stage and often do not
Leptomonas, another genus of Trypanosomatidae, was even pupate. Parasitoids pupate outside the host.
436 Insect Pathology

More than 10 species of mite in various families are necrosis from the poisonous setae. Brown or blackebrown
known to parasitize silkworm larvae, pupae, and adults. spots appear first on the abdomen, extend gradually to the
Among them, the straw itch mite Pyemotes ventricosus is prolegs, and finally aggregate in large numbers along the
the most common. It reproduces ovovivipariously (i.e., intersegmental membranes, resulting in an irregular belt of
eggs, larvae, and nymphs develop inside the maternal black spots or black feet if the spots aggregate on the
body), leading to the emergence of adult mites. The mites prolegs. The toxins can also enter the hemocoel, affecting
are yellow with a large, globose abdomen, and visible other tissues and organs. Physiological effects include
without magnification. Newly emerged adult females mate retarded development and delayed molts, and in some
immediately and actively search for hosts, which they cases, death of small larvae. Some poorly developed larvae
pierce with their needle-shaped chelicerae, injecting a toxin can spin cocoons and pupate, but the quality of the silk is
that paralyzes the silkworm. The mites continue to feed on low.
the hemolymph until the host dies. Pyemotes ventricosus Consumption of mulberry leaves contaminated with
produces several generations per year, and as many as these poisonous setae results in damage to the epithelial
17e18 generations can occur in eastern China. Winter cells of the foregut, extending to the posterior part of the
diapause occurs in the mated females. Another host of midgut. As a result, the silkworm larvae reduce their
P. ventricosus is the pink bollworm, Pectinophora gossy- feeding rate and develop slowly into small-sized larvae.
piella, and therefore, silkworm infestations can be more Larvae that have consumed a large number of setae stop
serious in cotton-producing areas where the rearing rooms feeding entirely, then swing their head from side to side,
are also used for storing cotton. vomiting, and eventually die. If first instars feed on
When early instar silkworms are parasitized, the larvae mulberry leaves contaminated with setae, they die very
stop feeding and often experience spasms and vomiting. quickly.
When the larvae die, the body is typically curved, with the Various slug caterpillars (Limacodidae), such as the
head protruded and the thorax swollen. First and second mulberry slug caterpillar, Thosea postornata, are pests of
instar silkworms are usually killed in less than 10 h. Third mulberries. The slug caterpillar has a broad food preference
instar silkworms develop asynchronously and become and usually produces two generations per year, with mature
grayish yellow and shrunken. The larvae then become larvae overwintering inside their cocoons. The larvae
slightly reddish and swollen, especially the posterior pupate the following spring. The moths emerge and lay
segments, and a reddish brown effluent is defecated. The eggs that hatch in early July. The larvae have tufted stinging
larvae die hanging upside-down. hairs that are long and stiff and secrete acidic toxins. The
The mature larvae are rarely infested, but when stinging hairs damage the integument of silkworm larvae,
attacked, they shrink and exhibit a prolapsed anus. The but the toxins can also enter the hemolymph. Damaged
posterior segments produce a blackebrown or redebrown silkworm integument will bleed small amounts of hemo-
effluent, and spots appear on the thorax and abdomen. lymph, which coagulates into small blackebrown round
These infested larvae usually die just before molting, or spots. These spots are larger than those caused by mulberry
during molting, with symptoms similar to those in the tussock moths. When exposure to the urticating hairs is
younger larvae. Pupae are usually parasitized in the inter- light, the silkworm larvae stop feeding for 30e60 min
segmental membranes on the dorsal side of the abdomen. before gradually resuming their feeding, but larval devel-
Infested pupae also develop a large number of black spots opment will be retarded. Heavy exposure will cause the
and die before emergence. The cadavers are blackebrown silkworms to die within a few hours to a couple of days. The
and show no signs of decay. Adult moths do not show any cadavers become flaccid and black (Jin, 2001).
obvious symptoms when infested. The female mite usually
parasitizes adult silkworms on the intersegmental
12.2.7. Abiotic Agents
membranes of the abdomen, and the moths become less
flexible. Infested males do not behave normally, and the Nosotoxicosis is a disease caused by or associated with
infested females lay fewer eggs, many of which are not a toxin. In sericulture, nosotoxicosis occurs when silk-
viable (Jin, 2001). worms consume or are exposed to insecticide-contaminated
The mulberry tussock moth, Euproctis similis, has mulberry leaves, a common problem during the summer-to-
urticating hairs that can poison silkworm larvae. The moth autumn rearing period. This period is when many pests
is a common pest on mulberry plantations, producing up to occur in the mulberry fields, and as a result, pesticides are
three generations, and overwinters as larvae. Larvae have intensively used. Owing to the wide variety of pesticides
needle-shaped, aggregate setae that secrete formic acid and used and their timing and dosage, the effect on silkworms
toxic proteins. Stings from these setae can occur on silk- varies greatly (Zhang, 2010). Poisonings may be acute or
worms throughout the year. Young silkworm larvae and sublethal. Sublethal poisonings may cause a reduction in
newly molted larvae are affected the most, exhibiting tissue silkworm resistance to viral and other diseases, negatively
Chapter | 12 Silkworms and Bees 437

affecting the cocooning process, as well as negatively l Maintain strict sanitation and hygienic conditions
affecting the number and viability of eggs. The most during rearing.
common effects are immediate cessation of feeding, loss of l Rear resistant varieties and use disease-free eggs. Moths
mobility, convulsions, and ultimately death within a matter used for breeding should be sampled for the presence of
of minutes or hours after exposure to a lethal concentration infections, and if infections are found, the entire batch
of the insecticide. of adults and any eggs produced from it should be
Besides insecticides, industrial wastes can adversely destroyed.
affect silkworm larvae. For example, fluorides may occur as l Practice intensive rearing management to enhance the
an industrial waste gas (from aluminum smelting or phos- vigor of silkworms by maintaining appropriate larval
phate processing plants) that can contaminate mulberry densities and proper humidity. Also, provide suitable
leaves. Fluorides can damage the leaves, causing them to food, adding fresh uncontaminated mulberry leaves as
become necrotic or show signs of chlorosis. Fluorides once needed during rearing.
affected sericultural production heavily in Japan, and they l Dispose of silkworm litter and other waste materials
still can cause losses in various industrial areas in China properly by burying, burning, or disinfecting (such as
(Miao et al., 2005). Poisoned larvae develop asynchro- a steam heat treatment).
nously, molt late, or have supernumerary instars, often two l Closely monitor, collect, and destroy weak, diseased,
or three extra instars. Lesions form on intersegmental and undernourished larvae.
membranes, causing them to swell and look like bamboo l Properly control pests in the mulberry field so that high-
segments. The lesions are easily ruptured, releasing quality leaves can be supplied to the silkworms that are
a yellow fluid. The larvae have difficultly defecating or both pest free and pesticide free.
defecate bead-like feces. The caudal segments (anything
posterior to the fifth abdominal segment) become trans- Silkworm cadavers, feces, and all contaminated materials
lucent, and the larvae usually die from losing food and should be properly disposed of, and all ceilings, walls,
liquids through vomiting. grounds, rearing beds, trays, racks, and other rearing
Gases produced by coal-burning stoves (sometimes appliances in and around rearing rooms properly cleaned
used to heat rearing rooms) can be toxic to eggs, especially and disinfected. Balavenkatasubbaiah et al. (1989) found
when they are incubated at high temperatures to promote that rearing rooms, trays, stands, and the paper linings used
early hatching. Eggs exposed to the stove gases do not in the trays serve as important sources of contamination,
hatch or only a portion of them hatches, whereas larvae and when these were disinfected between uses, disease
have a reduced feeding rate, become sluggish, or lie still on prevalence was significantly reduced. Disinfection is
rearing trays. Larvae exposed to high concentration of these carried out using approved chemical disinfectants, such as
gases stop feeding, vomit, and die with a swollen thorax chloride or quaternary ammonium compounds (product
and shriveled caudal segments. Larvae may die halfway registration for disinfectants varies greatly among coun-
through the molting process or without molting. The tries). Sulfur is used as a disinfectant fumigation against
cadavers sometimes assume a W-shaped position, develop muscardine fungi and mites.
massive black spots, turning completely black, and are rigid The silkworms have a very poor ability for flight, which
and extremely fragile (Jin, 2001). aids in the control of disease spread. Methods used to
ensure that eggs are disease free are an important aspect of
pébrine control. Such efforts in France, Japan, India, and
12.2.8. Disease Control Methods for China have brought the infection rates down from 20% in
Sericulture the nineteenth century to approximately 1% at present (Jin,
2001). To control egg infection levels in China, female
Two key components are central to disease control in any
moth inspections are conducted by provincial authorities.
insect rearing facility: prevention and avoidance. Once
Moths used as breeding or foundation stock are routinely
a pathogen or parasite establishes within an insect colony, it
sampled for the presence of the microsporidium. The
can spread rapidly and be difficult to control. Chemical
inspected female moths are ground, filtered, centrifuged,
treatments can reduce parasites and pathogens, but they are
and then inspected microscopically at 400 for the pres-
rarely completely effective at eliminating them and they
ence of microsporidian spores. For foundation stock, all
may not stop pathogen transmission. In addition, no treat-
females are inspected after they produce eggs, whereas
ments exist for many of the silkworm diseases, such as
occasional samples are taken for breeding stock. If a female
those caused by viruses. The following preventive
is found to be infected, all her egg batches are destroyed.
measures should always be taken:
To further evaluate the health of foundation females,
l Carry out strict disinfection methods for the rearing their eggs are also inspected for viability. Eggs are placed at
houses, rearing equipment, and surrounding areas. 28e30 C and 80% RH to accelerate hatching and
438 Insect Pathology

subsequently checked under a microscope to determine the densovirus is controlled by a few major genes (Qian
whether a high percentage of hatch has occurred. et al., 2006). Resistance to BmCPV-1, BmNPV, and
The health of mature larvae can be assessed by placing BmIFV is controlled by several minor genes that may be
them at 27e29 C and 80e85% RH. The larvae will unrelated to each other (Qian et al., 2006). Host resistance
complete development early, and moths will emerge two to to BmCPV-1 appears to be correlated with resistance to
four days earlier than usual and can then be examined for BmNPV, as well, but not to BmIFV. Thus, resistances to
presence of the pathogen. Larval health should also be BmCPV-1 and BmNPV are probably controlled by some
regularly inspected in the following manner. Several larvae common genes, but those to BmCPV-1 and BmIFV are not.
are selected from those that appear to molt normally, those Resistance to muscardine diseases is also associated with
that molt late, and those that are half molted, and then are silkworm variety. Silkworm strains from Chinese lineages
placed at 29 C and 90e95% RH. High heat and humidity are more resistant to B. bassiana than varieties from
increase disease levels in infected larvae, where infection a European lineage, and hybrids of Chinese lineages with
levels may otherwise go undetected until an outbreak Japanese lineages were even more resistant (Mu et al.,
occurs. Dead larvae are then examined microscopically to 1999).
determine whether any infectious disease has occurred. Uzigawa and Aruga (1966) developed a silkworm
Any dead pupae in cocoons are also inspected. variety resistant to BmIFV after selecting survivors from
In addition to sanitation, resistance can be an important virus-fed larvae for five consecutive generations. Similarly,
tool for reducing diseases. Resistance can be affected by Watanabe (1967) developed a silkworm variety resistant to
various environmental factors. For example, silkworm BmIFV by selecting survivors from virus-fed larvae for
resistance to viruses is reduced when the larvae are starved eight generations. Dingle et al. (2005) successfully trans-
or fed on low-quality mulberry leaves, with some seasonal ferred Bt resistance through a conventional hybridization
weather changes, or by exposure to certain chemicals, such breeding system. However, more efforts are needed to
as those used for disinfection (Jin, 2001). High and low develop silkworm varieties that are resistant to BmNPV, the
temperatures tend to affect the larvae and not the pupae. most serious viral disease of silkworms. Molecular
Changes in susceptibility to viral infections vary with the methods may provide some new assistance to breeding
rearing season: blood-type grasserie occurs most often in efforts. For example, Yao et al. (2005) constructed an
the spring, while BmCPV-1 and densovirus occur most isogenic silkworm line using molecular marker-assisted
often in the summer and fall. Thus, disease levels can rise breeding, which resulted in a new silkworm variety resis-
when the proper conditions are not maintained. tant to BmNPV. In spite of this progress, however, no
Resistance also varies among silkworm strains (Sen varieties with any substantial resistance have been adopted
et al., 1997) and for different ages and genders. For for silk production.
example, resistance to BmNPV increases as larvae mature, The use of genetic resistance to pathogens is still a long
such that resistance increases 10-fold with each instar (Wu way from commercial use; therefore, sanitation and main-
et al., 1983a, b). Resistance to BmIFV increases 1.5-fold taining larvae in healthy conditions are the primary means
with the first molt, 3-fold with the second molt, 134-fold for disease control. For many of the diseases, silkworms
with the third molt, and 10,000 to 12,000-fold with the last generally cannot be cured with medications. The fungicides
molt (Wu et al., 1983a, b). Conversely, resistance to benomyl, thiophanate, and carbendazim are reported to be
BmCPV-1 and BmDNV does not change much as the therapeutically effective against microsporidia. However,
larvae age (Wu et al., 1983a, b). As for gender differences treatment methods are generally lacking and, therefore,
in susceptibility, the male larvae and pupae are twice as disease prevention is key to maintaining productive silk-
resistant as females (Wu et al., 1983a, b). worm cultures.
Theoretically, resistance could also be achieved with
breeding programs, especially since resistance appears to
12.3. DISEASES OF BEES
vary between strains, suggesting a genetic component. The
use of disease-resistant silkworm varieties would greatly Diseases of bees have been summarized by Morse and
assist in disease management (Eguchi et al., 1996, 1998). Nowogrodzki (1990) and Bailey and Ball (1991). However,
Resistance to per os infections of BmNPV is usually greater these books deal only with honey bees, and primarily Apis
in those silkworm strains that have a multivoltine lineage, mellifera. Furthermore, critical new diseases have devel-
and this resistance is controlled polygenically, with a major oped in A. mellifera production systems since these books
dominant gene on a euchromosome and some minor genes were published, and so a new summary of bee diseases is
on the sex chromosome (Qian et al., 2006). The mecha- needed.
nisms for resistance to viral infections are antiviral activity Beekeeping is an ancient industry, and as with silk-
of the gastric secretions of the midgut and resistance in the worms, disease control is critical to its success. The earliest
peritrophic membrane to viral penetration. Resistance to records of hive beekeeping date back to approximately
Chapter | 12 Silkworms and Bees 439

3500 years ago in Mesopotamia (Crane, 1999). Early are very important for agriculture, and their diseases are
beekeepers kept A. mellifera and A. cerana in hollow logs discussed below.
or tree boles, and later in baskets, jars, and pots of various
sorts. Modern honey beekeeping is based on wooden hive 12.3.1. Viruses
designs with removable frames for the honey comb, as
developed by Langestroth and Dadant in the 1850s (Crane, A fairly large number of viruses is known to infect honey
1999). The basic structure of a movable frame hive is bees and they are generally named after some of the signs
a wooden box called a “super” that is filled with wooden or symptoms they cause in the host, although these signs or
frames that hang downward (a typical standard is 10 frames symptoms are not always a reliable method for identifica-
per super). A sheet of wax with a hexagonal grid is mounted tion (Table 12.2 and Fig. 12.2A). These viruses have
across each frame, and this special sheet of wax is called generally been thought of as being highly specific to honey
a “foundation” because it serves as the foundation for the bees because they cannot be grown on other insects or in
bees to build their comb. The purpose of the frames and cell culture. However, with the advent of reliable molecular
foundation is to entice the bees to build their comb where techniques for identifying viruses, some of the honey bee
the beekeeper wants it, and when the bees build their comb viruses are now being found infecting a fairly broad host
in the frames, the beekeeper can easily remove the frame to range within the Apoidea (bees) or even the Aculeate (bees
access the comb. The bees use the comb for raising their and wasps) (Singh et al., 2010) (Table 12.2). Most viruses
brood, storing pollen, and making and storing honey. The that infect bees are non-occluded, picorna-like RNA viruses
physical structure that houses the bees is referred to as the that are isometrically symmetrical, ovoid or spherical, and
“hive” and the bees that occupy the hive are called range in size from 20 to 30 nm (see Chapter 5). Most
a “colony”. Apis cerana is sometimes kept in Asia, but identifications rely on serological (Ball, 1999) or nucleic
A. mellifera, a bee originally from Africa and Eurasia, has acid techniques (Meeus et al., 2010), the latter being the
behavioral characteristics that make it especially amenable most commonly used today.
to human manipulation to improve honey and colony It has been known for a long time that bees can transmit
production, and so it is the most commonly kept bee plant viruses through pollen; that is, pollinators can serve as
throughout the world. vectors for some plant pathogens, especially those that
Honey bees are only a small portion of all the bees in the infect fruit via blossoms. Therefore, it is not surprising that
world (Michener, 2000). The vast majority of bees are flowers and pollen can serve as a reservoir for bee-patho-
actually solitary, and their life cycles differ greatly. In the genic viruses (Bailey, 1975), but the fact that bees can
last half-century or so, people began to recognize the actually become infected orally from this pollen was
importance of some of these bees for pollination of both demonstrated only recently (Singh et al., 2010). Other
crops and native plant communities. Although A. mellifera routes of transmission within a colony have been better
is a broad generalist in its host plant preferences, this studied and include both horizontal and vertical trans-
species can be a poor pollinator in some situations. The mission (Chen et al., 2006). In addition, varroa mites vector
need for better pollinators in certain situations has led to the some viruses, especially Deformed wing virus (Table 12.2)
development of rearing methods for other kinds of bees. and the Kashmir bee virus (Table 12.2) (Chen et al., 2006;
Bumble bees (especially Bombus terrestris in Europe, and de Miranda and Genersch, 2009), and this is one likely
B. impatiens in eastern North America) are produced in reason why varroa mite infestations are detrimental to
insectaries and sold as pollinators for greenhouse crops, a colony. Other hive pests may also contribute to the spread
especially greenhouse tomatoes. The alkali bee (Nomia of viruses. For example, small hive beetles have been
melanderi) is a solitary ground-nesting bee, and alfalfa seed shown to be susceptible to Sacbrood virus (Table 12.2 and
producers have developed methods to create and maintain Fig. 12.2A) (Eyer et al., 2009) and may serve to transmit
specialized nesting beds that are attractive for this bee viruses in honey bees (Eyer et al., 2008).
because it is a better pollinator for alfalfa than are honey
bees (Pitts-Singer, 2008). The alfalfa leafcutting bee
12.3.2. Bacteria
(Megachile rotundata) is a solitary cavity-nesting bee that
is used extensively for pollination in alfalfa seed crops in Bacterial pathogens are not very diverse among bees. None
North America (Pitts-Singer and Cane, 2011). Production are yet known to be pathogenic to bumble bees and solitary
systems have been developed where M. rotundata is enticed bees, although Skou et al. (1963) describes the presence of
to nest in polystyrene blocks that contain rows of holes the unidentified, Gram-negative bacilliform bacteria isolated
size this bee prefers for nesting. Various species of mason from diseased organs in queens of B. terrestris in Denmark.
bees (Osmia spp.) are used for pollinating fruit trees and In honey bees, three bacterial pathogens are well described:
almonds, and they are often provided with either wooden (1) American foulbrood (AFB) caused by Paenibacillus
nesting boards or hollow reeds to nest in. All of these bees larvae; (2) European foulbrood, caused by Melissococcus
 440
TABLE 12.2 Examples of Infectious Diseases and Mite Parasites of Bees

Affected Host
Pathogen/mite Disease Known Hosts Stages Signs and Symptoms
a
Viruses
Acute bee paralysis Acute bee paralysis Apis mellifera, Bombus terrestris Adults Adults may be paralyzed or asymptomatic. Not thought to be
virus (ABPV) transmitted orally, but found in pollen loads from foragers and thoracic
salivary glands. May be vectored by Varroa destructor

Black queen cell Black queen cells Adrena sp., Apis mellifera, Bemix Larval queens, In honey bees, the queen cells develop dark brown to black cell walls.
virus (BQCV) sp., Bombus spp., Megachile adults Immature queens die as prepupae or pupae, turn flaccid. Larvae do not
rotundata, Nomia melanderi become diseased when fed virus particles, but infections are common
and asymptomatic in adult bees. Infection is enhanced by the presence
of Nosema apis infections. Signs and symptoms of other bees are
undescribed

Apis iridescent Clustering disease Apis cerana Adults During summer, flightless bees detach from the colony and cluster or
virus (IV-24) occur crawling on the ground in large numbers. Infected tissues appear
bright blue. Occurs in the fat body, alimentary tract, hypopharyngeal
glands and ovaries

Chronic paralysis Paralysis, chronic Apis mellifera Adults Sections of the hindgut epithelium have basophilic cytoplasmic bodies.
virus (CPV) paralysis, or hairless Type I: abnormal trembling, failure to fly, adults crawling on the ground
black syndrome in large groups, adults cluster at the top of the hive, bloated abdomens
and dislocated wings. Type II: adults become black and hairless,
abdomens distended and shiny, get slightly attacked by older bees in
the colony, often excluded by guard bees; appear like black robber bees

Deformed wing Deformed wing Adrena sp., Augochlora pura, Apis Adults In honey bees and bumble bees, newly emerging adults have small,
virus (DWV) disease mellifera, Apis cerana, Apis florea, deformed wings. Honey bees may also have bloated, shortened and
Bembix sp., Bombus spp., Ceratina discolored abdomens. Varroa destructor is a vector in honey bees and
dupla, Megachile rotundata, Nomia increases disease symptoms
melanderi, Xylocopa sp.

Kashmir bee Apis cerana, Apis mellifera, Larvae, adults KBV and IAPV are closely related, and best described from honey bees,
virus (KBV) Bombus terrestris where all life stages can become infected. Transmission: horizontal

Insect Pathology
from infected adults via glandular secretions applied to food sources;
transovarial; and vector-borne via Varroa destructor. Infected brood
either dies before the cell is capped or recovers from infections.
Infections can persist in adults without symptoms, or cause rapid
mortality within a few days. Virions are non-enveloped, icosahedral
capsids ~ 30 nm wide
 Chapter | 12
Israeli acute paralysis Found in many species Adults Similar to KBV. Presence of this virus in colonies has been correlated
virus (IAPV) of bees with colony collapse disorder in the USA

Sacbrood virus (SBV) Sacbrood Adrena sp., Apis cerana, Apis Larvae, adults In honey bees, mature larvae fail to molt to pupal stage then become
mellifera, Bombus spp., Nomia flaccid, turn from white, to yellow, to brown in a few days. The dry scale
melanderi does not stick firmly to the cell as it does for foulbrood. Larvae
eventually become dry and flat in capped cells. Signs and symptoms

Silkworms and Bees

unknown for other bees
Bacteria
Melissococcus Foulbrood, European Apis mellifera, Apis cerana Larvae Larvae typically die when 4e5 days old, and outbreaks usually occur in
pluton early summer. Dead larvae turn brown and flaccid. Midguts filled with
bacteria in opaque white clumps. Dead larvae may dry to a scale that is
rubbery and does not stick to the wax. Colonies typically recover.
Melissococcus pluton cells ovoid to lanceolate (0.8 mm wide  1.0 mm
long), forms chains. Facultative anaerobe requires CO2

Paenibacillus larvae Foulbrood, American Apis mellifera, Apis cerana Prepupae, pupae Larvae die after spinning the cocoon, then turn putrid and dark brown,
with a distinct fishy odor. If a toothpick is stuck into the larva and pulled
out slowly, the remains draw out as brown, ropy thread. Discoloration
is first seen when larvae reach 10e15 days old. Cell cappings sink in
slightly, often with small hole in the center. After one month, the dead
larvae dry to a small, flat scale that sticks to the wax. The bacterium is
a Gram-positive bacillus, motile, with oval endospores
(~ 2.5  0.5 mm, but highly variable in size)

Spiroplasma apis May disease Apis mellifera Adults Large numbers of adults found quivering and unable to fly, moribund,
or dead. Abdomens swollen and hard. Midgut full of undigested pollen.
Other adults from the colony cluster in small groups away from the
hive. Bees not dark and shiny, no obvious hair loss. Large numbers of
adults may die in 4e5 weeks, but colonies usually recover. Wall-less,
helical bacteria found in hemolymph and digestive tract
Fungi: filamentous
Ascosphaera apis Chalkbrood Apis mellifera Larvae Infected larvae die at a late stage; sometimes after the cell is capped.
The dead larvae are hard, chalk-white, but often mottled with black
spots (the fungal spores). Typically, adult bees remove infected larvae
from the hive, and these dead larvae can be seen in large numbers near
the hive entrance. Microscopic examination will reveal spores formed
in sacs called ascomata, and within the ascomata are spore balls
(ascospores). The ascospores are ovoid, but only slightly so, nearly
spherical

(Continued)

441
 442
TABLE 12.2 Examples of Infectious Diseases and Mite Parasites of Beesdcont’d
Affected Host
Pathogen/mite Disease Known Hosts Stages Signs and Symptoms

Ascosphaera Chalkbrood Megachile rotundata Larvae Infected larvae most often die during the last instar, after they have
aggregata consumed their entire pollen provision and reached full size, but before
cocooning. Dead larvae are hard and black. The surface may easily
disintegrate to a black powder (the fungal spores). Larvae may be
mottled black and white, or be mostly white, but less commonly so than
with chalkbrood in honey bee larvae. Ascospores are long ovoid,
typically 2 mm wide and 4e5 mm long

Ascosphaera torchioi Chalkbrood Osmia lignaria Larvae Similar to A. aggregata above. The ascospores are typically 2 mm wide
and 4e5 mm long

Aspergillus flavus Stone brood Apis mellifera Larvae Infected larvae die soon after cells are capped, before pupation, then
and Aspergillus turn very hard and pale brownish, gray, or yellowegreen. The color
fumigatus comes from the spores. Spores are spherical and highly sculptured
Fungi: Microsporidia
Nosema apis Dysentery Apis mellifera Adults Disease characterized by the presence of spores in the midgut
epithelium (~ 6  3 mm), and milky white appearance of the
ventriculus. Usually < 30 coils to the polar filaments in the spores. No
outward signs of infection, infected adults live about half as long as
uninfected bees. Associated with dysentery in winter and early spring,
but probably not the cause. Spread via fecal contamination

Nosema ceranae Dysentery Apis mellifera, Apis cerana Adults Disease characterized by the presence of spores in the midgut
epithelium (~ 5  2 mm), with few distinct marks. Approx. 20 coils to
the polar filaments in the spores. No outward signs of infection,
infected adults live about half as long as uninfected bees. Associated
with dysentery in winter and early spring, but probably not the cause.
Spread via fecal contamination

Nosema bombi Nosema disease Bombus terrestris, Bombus Adults Disease characterized by the presence of spores throughout the body
occidentalis, Bombus impatiens cavity (~ 4.2e5.4  2.7e3.5 mm), with few distinct marks. In B.
terrestris and B. impatiens, colony growth and the production of
reproductives are greatly reduced, but not with B. occidentalis.
Transmission is transovarial and oral
Protista
Apicysti bombi No name Bombus affinis, Bombus bimaculatus, Adults Poor colony growth, fat body destroyed in infected workers. Large

Insect Pathology
Bombus fervidus, Bombus griseocollis, spores detected in feces
Bombus impatiens, Bombus perplexus,
Bombus terricola, Bombus vagans
 Chapter | 12
Crithidia bombi and No name Bombus terrestris Adults Trypanosome that occurs in hindgut of bumble bees. Transmission
C. expoeki occurs horizontally within a nest, between nests via flower visitations,
and transovarially. Infections affect founding ability of new queens,
survival of workers, and colony reproduction

Crithidia mellificae Flagellate disease Apis mellifera Adults Trypanosome that occurs in gut lumen and epithelium of honey bees.
Does not cause overt disease

Silkworms and Bees

Gregarines Gregarine disease Apis mellifera Adults Flagellates occurring in midgut of adult bees. Cephalont stage oval,
(e.g., Monoica apis, ~ 16  44 mm, in two segments. Sporont stage ~ 35  86 mm.
Apigregarina Infections do not cause overt disease
stammeri, Acuta
rousseaui, Leidyana
apis)

Malpighamoeba Amoeba disease Apis mellifera Adults Cysts occur in the hindgut and rectum. Infections occur in the
mellificae Malpighian tubules. Infected colonies occur most commonly with
other diseases
Invertebrates: Acari
Acarapis woodi Tracheal mites Apis spp., Bombus spp. Adults Affected bees may have disjointed wings and difficulty flying, distended
abdomens, or may be asymptomatic. Mites occur in the prothoracic
tracheae. Females are 143e174 mm long, males 125e136 mm, white,
somewhat transparent, smooth cuticle with a few long hairs on the
body and legs. The impact on the colony size and productivity is
uncertain

Tropilaelaps clareae Tropilaelaps Apis mellifera, Apis dorsata Adults, pupae, External parasitic mite found in Southeast Asia. Parasitize bee adults
larvae and brood, mites ~ 1 mm long  0.6 mm wide. To sample, hit a frame
of comb onto a light-colored surface to dislodge mites, observe under
magnification

Varroa destructor Varroa mite Apis cerana, Apis mellifera Adults, pupae, External parasitic mite. Extremely serious pest of A. mellifera,
larvae eventually causing colony death. Mites parasitize both adults and
brood, but reproduce only on capped brood. Able to vector some
viruses. Female adults oval, flat, redebrown, ~ 1  1.5 mm. Males
smaller, white, less frequent. Mites can be sampled by dislodging from
adult bees using ethyl alcohol, isopropyl alcohol, ether, or powdered
sugar placed in a jar with the bees, then shaking the jar
a
In viral nomenclature, the type species is in italics.

443
444 Insect Pathology

pluton; and (3) Spiroplasma spp., which are quite rare caused by fungi in the genus Ascosphaera, and it
(Table 12.2). AFB is perhaps the most well-known bacterial affects many different taxa of bees, although it has
disease of honey bees. Paenibacillus larvae infects the never been reported in Bombus. Currently, 22 species of
brood and is highly contagious, and thus, it is also strictly Ascosphaera have been described and all are associated
regulated in many countries. Apiary inspection services in with bees either as saprophytes on the pollen and feces
many parts of the world regularly inspect beekeepers’ found in nests, or as pathogens, or as both (i.e., as
colonies to ascertain the status of this disease and prevent facultative and opportunistic pathogens) (Anderson and
its spread. The bacterium forms spores that are extremely Gibson, 1998; Youssef and McManus, 2001). Ascos-
hardy, capable of persisting for many years on hive mate- phaera spp. are found associated with bees as diverse
rials (Lindström, 2006) and able to withstand high as A. mellifera, Megachile rotundata, M. centuncularis,
temperatures (Forsgren et al., 2008; Genersch, 2008). In Osmia lignaria, O. cornifrons, Trigona carbonaria, and
addition, P. larvae is resistant to many antibiotics Chalicodoma spp. The most common of these patho-
(Kochansky et al., 2001). The infection process begins gens are described in Table 12.2. In honey bees, the
when honey bee larvae are inadvertently fed spores that disease is caused by A. apis, but generally, chalkbrood
contaminate food material in the hive (Fig. 12.3). Trans- is not considered a serious disease in honey bees, even
mission is per os, and infected larvae usually die after they though the pathogen is probably present most of the
spin a cocoon. The cadavers then turn dark brown and have time and in most colonies (Fig. 12.2C, D). Outbreaks
a distinct, fishy, putrid odor. If a toothpick is stuck into such are most common during cold, wet weather, especially
a cadaver and pulled out slowly, the material will come out in the spring, or if hives are kept in standing water or
as a thick, ropey thread (Fig. 12.2B). Eventually, the dead in humid regions when the hives do not have adequate
larva dries down to a hard scale that sticks to the wax along ventilation. In addition, varroa mite infestations can
the side of the brood cell in which it died. This scale will be increase chalkbrood prevalence in honey bees (Getchev
full of P. larvae spores, up to 7.5  108/cadaver (James, and Kantchev, 1998).
2011a), which then serve as a source of contamination and Conversely, Ascosphaera aggregata can be a very
spread the disease. serious disease in M. rotundata, and easily the most
Melissococcus pluton also infects the larvae of honey serious disease of this bee when it is managed for alfalfa
bees per os. Larvae typically die when four to five days old, seed pollination (Fig. 12.2E). This fungal species kills, on
that is, before a cocoon has been spun. Cadavers can be average, about 15% of the larvae in commercial systems,
found in the brood cells that are brown and flaccid, similar but in some alfalfa seed fields, as many as 45% of the
to AFB, but generally European foulbrood cadavers do not larvae may be killed (R. James, unpubl.). Ascosphaera
have the same characteristics as described for AFB. Simi- aggregata probably originated in Europe, where this bee is
larly, when the cadavers dry, they become rubbery and do native, and migrated into North America with its host. In
not stick to the wax. Colonies typically can recover from Europe, this pathogen has been found to infect M. cen-
infections on their own. tuncularis and Osmia rufa (Skou, 1975), but it has never
Two species of Spiroplasma have been reported as been isolated from any bee species other than M. rotun-
pathogens of honey bees: S. apis (Mouches et al., 1982, data in North America. Likewise, A. apis is not found
1983, 1984) and S. mellifera (Clark et al., 1985). Even associated with M. rotundata, either. However, not all
though these are typically rare, a recent molecular survey species of Ascosphaera are host specific, and many have
of parasites in honey bee colonies was positive for the broad host ranges.
presence of Spiroplasma (Runckel et al., 2011), indicating Chalkbrood in M. rotundata spreads when spores
that they may be present in honey bees as non-disease- contaminate the body of nesting females, which in turn
causing infections, and thus go undetected much of the deposit the spores in the pollen they collect to feed their
time. Spiroplasma are believed to cause a disease called young (James, 2008). When adult bees emerge from
May disease. This disease is prevalent in the spring, managed nesting systems, they become contaminated with
causing large numbers of moribund adults to cluster spores from the larvae that died in the previous year (James,
around the hive, often quivering and with swollen and 2011b). Chalkbrood occurs at much lower levels in Canada
distended abdomens. Other adults may cluster together than in the USA. It is unusual for the disease to reach 5% in
away from the hive. Colonies usually recover from these any Canadian population, and it generally affects less than
infections. 1% of the larvae. As a result, Canada supplies a significant
portion of the M. rotundata used in the USA (approxi-
mately 3  108 bees/year, which is about half of the total
12.3.3. Fungi: Filamentous
used in the USA). Several other Ascosphaera infect
The most common fungal disease of bees is chalk- M. rotundata (Table 12.2 and Fig. 12.2E), but none is as
brood, which occurs in the larvae. Chalkbrood is common as A. aggregata.
Chapter | 12 Silkworms and Bees 445

(A) (B)

(C) (D) (E)

(F) (G) (H)

FIGURE 12.2 Diseases of bees. (A) Typical cadaver of Apis mellifera larva infected with SBV. (B) Diagnostic characteristic of American foulbrood.
Apis mellifera larva infected with Paenibacillus larvae has decomposed to a soft mass that draws out a “ropey” thread. (C) Brood comb from an Apis
mellifera colony showing chalkbrood. Larvae infected with Ascosphaera apis turn white and chalky (arrow). (D) Apis mellifera larva that has died
from an Ascosphaera apis infection. The black areas are fungal spores. (E) Megachile rotundata larvae that have died from chalkbrood, due to
infections with Ascosphaera aggregata (left) and Ascosphaera proliperda (right). (F) An adult female of the parasitic mite Varroa destructor on the
posterior end of a pupa of Apis mellifera (arrow). (G) Varroa destructor females invading the cells of mature larvae of Apis mellifera just before the
cells will be capped by nurse bees. (H) Tracheal mites, Acarapis woodi, inside the trachea of an Apis mellifera adult worker. [Photo credits: (AeD)
B. Smith; (E) R. James; (F) S. Bauer; (G) Vita-Bee Ltd; (H) L. I. de Guzman.]
446 Insect Pathology

FIGURE 12.3 Life cycle of American foulbrood (Paenibacillus larvae) in Apis mellifera, showing side and top view of a brood cell.

12.3.4. Fungi: Microsporidia vigor. Nosema ceranae appears to be more sensitive to cold
and is thought to be more common in warmer climates
Nosema disease is caused by microsporidia in the genus (Gisder et al., 2010).
Nosema. Two species that infect A. mellifera are wide- Nosema bombi is a pathogen of bumble bees and occurs
spread throughout the world: N. apis and N. ceranae (Klee widely within the genus Bombus (Cameron et al., 2011).
et al., 2006). These pathogens infect per os, and trans- Unlike Nosema in honey bees, N. bombi can be found
mission occurs when bees ingest the spores, probably in infecting all tissues throughout the body except the ovaries
contaminated water, pollen, or honey. Nosema apis infects and proctodeum (Fries et al., 2001). The spores are slightly
the gut and muscle tissue of honey bees and causes both smaller than N. apis and N. ceranae (Table 12.2). Although
dysentery and crawling bees. Another typical symptom is N. bombi has been found in many parts of the world, no
a milky-white coloration of the gut (Table 12.2). other Nosema has been described from Bombus hosts. This
Nosema ceranae was first identified as a pathogen in the pathogen has been investigated as a possible cause for the
Asian honey bee, A. cerana (Fries et al., 1996). It is now current decline of some bumble bees, perhaps after being
widely found in A. mellifera in Asia, Europe, and North distributed outside Europe through cultivation and release
America (Klee et al., 2007; Williams et al., 2008; Chen for greenhouse pollination (Colla et al., 2006).
et al., 2010). It is not clear whether it has spread rapidly as
a new pathogen of A. mellifera, or whether it was always
present but mistaken for N. apis in the past. However,
12.3.5. Protists
N. ceranae does not cause the same symptoms as N. apis.
Nosema ceranae is typically associated with neither Flagellate trypanosomes in the genus Crithidia are known
dysentery nor crawling bees, but it does affect the feeding to infect both honey bees and bumble bees (Table 12.2).
behavior of honey bees, making them less inclined to Recently, it has been proposed that Crithidia bombi is
participate in trophallaxis. The main effects of this path- actually composed of two different species, C. bombi and
ogen are increased bee mortality and decreased colony C. expoeki (Schmid-Hempel and Tognazzo, 2010). Both
Chapter | 12 Silkworms and Bees 447

are gut parasites of bumble bees. Crithidia mellificae is exit at that time. The mother mite can live long enough to
found in A. mellifera. These parasites infect per os, and infest more than one brood cell.
may be transmitted from one colony to the next through Varroa destructor originated as a parasite on A. cerana
contaminated flowers (Durrer and Schmid-Hempel, and adapted to A. mellifera, where it became a serious pest
1994). worldwide. Most populations of the mite in North America
The gregarines are also gut parasites of A. mellifera, but probably originated from Korea and are likely to be the
they do not cause overt symptoms in infected colonies result of several introductions by way of Europe (de
(Table 12.2). A neogregarine, Apicystis bombi (formerly Guzman et al., 1997; Anderson and Trueman, 2000). The
Mattesia bombi), has also been found in several bumble bee varroa mite is important not only as a parasite, but also
species (Liu et al., 1974). This parasite occurs mainly in the because it serves as a vector for honey bee viruses (see
fat body of the host. Mobile sporozoites release sporocysts Section 12.3.1). In addition, the feeding activity of these
into the midgut, and these invade new fat body cells. mites compromises the honey bee immune system, leaving
Schizonts are produced intracellularly and extracellularly parasitized bees more vulnerable to infections (Kanbar and
and are multinucleated. Engles, 2003).
Another protist disease is caused by the amoebozoan The tracheal mite, Acarapis woodi, is a parasite of bees
Malpighamoeba mellificae, and it occurs in managed that lives within the trachea of the thorax and head
colonies of A. mellifera. Infections with this pathogen are (Fig. 12.2H), where it feeds on the host hemolymph. Adult
called amoeba disease, and it has been reported in various females invade the host through the spiracles, and then lay
parts of the world, mainly Europe, but generally it is quite nearly one egg a day, and as many as 21 eggs in a lifetime.
rare. It is not clear whether M. mellificae infections actually The only time that the mites live outside the host is when
cause any disease symptoms or mortality in the bees, young, new females transfer to new hosts (Pettis and
but infections probably affect colony vigor (Morse and Wilson, 1995). In warm climates, honey bees appear to be
Nowogrodzki, 1990). The pathogen infects the Malpighian able to sustain heavy infestations with little impact on bee
tubules of adult bees, and diagnosis of infection is generally health. However, in colder climates, the impact is much
based on the presence of cysts in the hindgut of adult bees. greater. Tracheal mites cause adult bees to leave the hive,
The pathogen is probably transmitted per os (Bailey and and affect their ability to fly and defecate, leading to large
Ball, 1991). losses of young adult bees in the winter and spring (Webster
and Delaplane, 2001).
It is possible that many of the treatments for varroa
12.3.6. Non-infectious Biotic Agents
mites also control tracheal mites. These mites are micro-
Varroa destructor (Fig. 12.2F) is easily the most serious scopic and infested bees can be asymptomatic; thus, the
invertebrate parasite of A. mellifera. Mated, adult female presence of this disease is easy to miss. A typical sign is
mites can be found as phoretic passengers on adult worker a weak colony in the early spring, where the returning
bees and drones. Typically, adults are found on the inter- foragers do not make it all the way back to the hive but must
segmental membrane on the ventral side of the abdomen, crawl the last few centimeters or so to the hive entrance,
particularly between tegula one and two, or two and three. resulting in large numbers of bees crawling on the ground
Mites probably feed on adult hemolymph during this time in front of the hive.
(Bowen-Walker and Gunn, 1998). When a colony is heavily Bumble bees are also susceptible to Locustacarus
infested, phoretic mites are also found on the dorsal side of buchneri, another parasitic mite that infests the trachea.
the thorax. Phoretic females eventually search for devel- This mite is thought to have originated as a parasite of
oping bee brood that are within a few hours of being capped B. terrestris in Europe but has increased its host range as
(honey bees cap over brood cells with wax when the brood a result of the importation of B. terrestris to other parts of
are ready to spin a cocoon) (Fig. 12.2G). The mites crawl the world, such as Japan (Goka et al., 2001)
inside the cell, wait for it be capped, then they lay a single Conopid flies are dipteran parasitoids in the family
female egg. Within the next couple of days, the mother mite Conopidae. Conopid flies have been collected from bumble
will lay another egg, usually a male. After that, the mother bees in both Europe and North America, including Bombus
mite may lay a few more female eggs, but no more than one pascuorum, B. bifarious, B. occidentalis, B. impatiens,
per day. The mites develop inside the capped brood cell, B. griseocollis, and B. fervidus (Gillespie, 2010; Otter-
feeding on the hemolymph of the developing honey bee statter, 2011). Several species of conopids probably occur
brood (Fig. 12.2F). The new female mites will mate with on bumble bees, but the two that are described are Sicus
the male before they emerge from the host cell. Varroa ferrugineus and Physocephala rufipes. Development time
mites typically emerge from the cell when the host emerges in the host for both parasitoids is about 11 days. The
as an adult, but if the host dies, other bees in the hive will parasitoids can sometimes occur together in a single host,
uncap the cell to remove the cadaver, and the mites may but then must compete with one another, as the host can
448 Insect Pathology

only support the complete development of one. The first the bees are exposed to subacute doses, but recent research
instar larvae have strong pointed mandibles that are prob- indicates that it can have serious consequences. Until
ably used to kill other eggs found in the same host (Schmid- recently, for example, the insect growth regulator nova-
Hempel and Schmid-Hempel, 1996). luron was considered safe for bees because it lacked acute
Another dipteran group of parasitoids is the phorid flies, toxicity to adults and so was approved in the USA for use in
in the family Phoridae. The phorids that attack bees belong alfalfa seed fields to control lygus bugs during the polli-
to three genera: Melaloncha, Styleta, and Apocephalus. nation season. However, applications of this pesticide
Melaloncha were last revised by Brown and Smith (2010) corresponded with very poor reproduction of M. rotundata.
and currently include 167 species. Melaloncha species are It was later determined that M. rotundata eggs are very
neotropical and attack most tropical genera of Apidae sensitive to novaluron. In addition, when reproducing
(bumble bees, honey bees, and stingless honey bees) females are fed novaluron, the progeny produced by these
(Disney, 1994; Brown and Smith, 2010). Apocephalus spp. bees have poor survivorship (Hodgson et al., 2011).
attack Bombus in North America and stingless bees in the Another source of pesticide exposure can be the
neotropical region (Brown, 1996, 1997). Overall, phorid beekeepers themselves. Until very recently, the two most
flies do not pose any real threat to beekeeping operations. commonly used pesticides to control varroa mite in the
Several hymenopteran parasitoids are a more serious USA were coumaphos and tau-fluvalinate. Both are rela-
problem for beekeeping, including Leucospis, Monoto- tively persistent acaricides that contaminate the wax, pollen
ntomeris, Mellitobia, and Chrysura species. These para- and bees in honey bee hives. Furthermore, varroa mites
sitoids enter nests through cracks and crevices, locate have developed resistance to both of these, rendering these
a developing larva, and then lay their eggs on this host. The products less useful to most beekeepers. Some beekeepers
parasitoids can be identified by the presence of a large have tried to compensate for pesticide resistance by
number of small larvae in a cell where there should be only increasing the concentration they put into the hive, espe-
one developing bee larva. These parasitoids are most cially with regard to tau-fluvalinate, which is readily
common in the managed solitary bees, especially available in liquid formulations for field crops (in both the
M. rotundata and O. lignaria, becoming a problem when USA and Europe); however, this practice is not a legal
the bees are managed in large, dense populations. Some of pesticide use. Contamination levels in the wax in honey bee
these parasitoids also attack Apidae. For example, Melli- hives are greater for these two insecticides than for any
tobia acasta has a long list of known bee hosts, including other insecticide, typically by 10-fold or more (Mullin
Megachile, Osmia, Chalicodoma, Anthophora, Anthidium, et al., 2010). Although the oral toxicities to honey bees
Bombus, and Apis (De Wael et al., 1993, 1995). have been quantified for these pesticides, it is not clear what
the real exposure levels are for contaminated wax, since the
pesticide may be bound to the wax in such a manner that
12.3.7. Abiotic Agents
bee exposure is minimal.
Bees are susceptible to many insecticides and, as pollina-
tors, they fly about visiting a large number of flowers to
12.3.8. Colony Collapse Disorder
collect pollen and nectar. This activity can expose them to
pesticides that are on or in flowering plants. This route of In around the year 2005, commercial honey beekeepers in
exposure usually occurs when pesticides are applied to the USA began suffering large losses of bees, and these
agricultural crops or home gardens for pest control. Many colony losses recurred annually after that. One beekeeper
countries have environmental regulations restricting the use once described the situation as “The law of thirds: one
of insecticides on flowering plants, especially insecticides third dead, one third weak, and one third fine.” This high
known to be acutely toxic to bees. However, bees still mortality has been attributed to a new disease (not
continue to be exposed to these chemicals, and pesticides necessarily an infectious disease) named colony collapse
can accumulate in the hive (Mullin et al., 2010). A typical disorder (CCD). The disease has been defined by the
sign of pesticide poisoning in honey bees is a large number following symptoms: (1) a rapid loss of adult worker bees,
of dead adult bees found in and around the hives, or large i.e., adult numbers decline over a few weeks, but not with
numbers of adult bees found writhing on the ground, often the sudden mortality that is commonly seen with pesticide
on their dorsum. Typical symptoms of pesticide poisoning poisonings; (2) the colony is rapidly dying, but with only
in M. rotundata include a loss of bee activity at the nesting a few or no dead bees found in or around the hive; (3) a
shelters and large numbers of dead bees around these small cluster of adult bees is present with an egg-laying
shelters, especially males (which tend to remain at the queen and brood; or (4) the colony is dead, but with brood
shelters to mate). This occurs when the pesticide has acute present and honey and pollen stores intact, but no invasion
toxicity, causing rapid bee mortality (Riedl et al., 2006). by hive pests such as wax moths (Cox-Foster et al., 2007).
Little is known about the chronic affects of pesticides when At the time of writing, no single pathogen, parasite, or
Chapter | 12 Silkworms and Bees 449

toxin has yet been found that fully explains the cause of all colonies die during the winter every year (vanEn-
of CCD. gelsdorp et al., 2008, 2010, 2011). However, CCD is not the
In 2006, a metagenomic survey of bees from several most prominent cause. Beekeepers most frequently repor-
states of the USA was undertaken to determine whether any ted queen failure, starvation (this is especially true among
pathogens or parasites could be correlated with CCD hobbyist beekeepers), varroa mites, and weather as the
prevalence. The only pathogen that occurred in all the CCD causes for their colony losses during the winter. Thus,
samples and none of the non-CCD samples was a virus that although honey bee colony losses may be on the increase
had recently been described in Israel, the Israeli acute worldwide (IBRA, 2010), the cause for this increase is not
paralysis virus (IAPV, Table 12.2) (Cox-Foster et al., clearly CCD, but CCD is certainly a factor.
2007). At the time of that survey, IAPV was not known to As shown above, CCD is a complex disorder that is not
be present in the USA, but it has since been shown to be well defined. Although a group of authors has agreed on the
present since at least 2002 (Chen and Evans, 2007). It has symptoms associated with CCD (as described above), the
not yet been confirmed whether IAPV is responsible for symptoms are vague and could result from many different
CCD. European beekeepers have also experienced large causes. For this reason, not all bee researchers are in
losses of honey bees, and some scientists have proposed agreement that CCD is actually a specific disease, sug-
that those losses are due to the invasion of N. ceranae (see gesting instead that it is a collection of symptoms with
Section 12.3.4), while others think that multiple factors, many possible causes, including N. ceranae infections,
including chronic exposure to pesticides, may be to blame. varroa mite infestation, and abiotic factors such as chronic
Indeed, N. ceranae is now widespread in the USA pesticide exposure (Anderson and East, 2008). From
(Chen and Huang, 2010), but the presence of this pathogen personal experience (R. R. James), commercial beekeepers
was not strongly tied to CCD in the metagenomic survey. It and apiary inspectors use the term loosely to describe any
has also been proposed that CCD is due to a co-infection of large losses they experience, even when they are certain of
N. ceranae and a new undescribed iridovirus (Bromen- the cause (such as varroa mite infestation or pesticide
shenk et al., 2010). An iridovirus has previously been exposure). For example, one apiary inspector said that CCD
described from A. cerana, but not from A. mellifera (Bailey in his area was caused mainly by varroa mites, and
et al., 1976; Bailey and Ball, 1978; Verma and Phogat, a commercial beekeeper in the same area expressed that
1982), and further confirmation on its presence in this host, “his” CCD was due to pesticide poisonings (R. R. James,
and in the USA, is still needed. unpubl.).
In 2009, a large epidemiological approach was taken in The loose manner in which CCD is defined by
the USA to identify the cause of CCD, and although more beekeepers makes records and surveys taken from
than 200 variables were quantified, no single factor could beekeepers difficult to interpret. The situation may be
be identified as the cause of this disorder. However, low similar to that described by Bailey and Ball (1991) for the
coumaphos levels in the hive did correspond with the Isle of Wight disease in the British Isles: “There are all
presence of CCD, although the intensity of varroa mite kinds of possible reasons for the death of bees, apart from
infestation was not a good predictor (vanEngelsdorp et al., infections, and there is little doubt that bees dying of non-
2009, 2010). Two other pesticides were also found to be infectious diseases were often included in the casualties
predictors for the prevalence of CCD, but the authors did attributed to the Isle of Wight disease”. At that time, the
not indicate whether CCD prevalence was related to high or conviction that Isle of Wight disease was indeed an infec-
low levels of these pesticides (vanEngelsdorp et al., 2010). tious disease led to the determination that it was caused by
A very thorough and extensive survey of pesticide tracheal mites, a conclusion that was disputed by some
contamination levels in honey bees and honey bee hives scientists (Bailey and Ball, 1991). It can be very difficult to
was conducted in the USA in the 2000s (Mullin et al., determine the reason why a colony dies, and therefore,
2010). Coumaphos and tau-fluvalinate were found to occur many kinds of mortalities are often included as CCD,
at high concentrations in nearly every honey bee colony in complicating the discovery of the real cause.
the USA. As previously discussed (see Section 12.3.7), low
levels of pesticides can have unexpected chronic or delayed 12.3.9. Disease Control Methods for
effects on bees, and this survey revealed that honey bees are
Managed Bees
exposed to a very long list of insecticides, miticides, and
fungicides. Therefore, the role of pesticide exposure, Disease control for bees is challenging. Bees are suscep-
especially exposures that are below the acute toxicity tible to many diseases, and they are kept outdoors where it
levels, merits further investigation, but it is not clear is difficult to keep the colonies isolated from other bees that
whether poisonings play a role in CCD. may be carriers of pathogens. Often, chemical treatments
Large annual surveys of commercial and hobbyist can reduce parasites and pathogens, but rarely are they
beekeepers in the USA have found that approximately 30% completely effective at eliminating them, and the parasites
450 Insect Pathology

and pathogens can become resistant. In addition, no treat- The second component of disease management is
ments exist for many insect diseases, especially viruses, sanitation and involves regular cleaning and disinfection to
and sanitation can be difficult to accomplish. prevent the spread of disease. Pathogen transmission can
Thus, a multifaceted approach is needed for disease occur when nesting materials are reused without sanitation,
control. Disease management can be broken down into six such as when A. mellifera hives are reused for different
main components: (1) quarantine; (2) sanitation; (3) colonies without first being disinfected, or when tools used
monitoring; (4) resistance; (5) control disease vectors; and by workers (e.g., the hive tools used by beekeepers or
(6) medication (chemical and/or antibiotic) treatment. All grafting tools used in queen production) are not disinfected
of these components are equally important and all can be between uses. Another measure that could be taken to
applied to A. mellifera, but they are not always used by prevent human workers from spreading disease is the
beekeepers. Efforts to develop integrated disease and pest decontamination of their outer clothing (e.g., gloves and
control in beekeeping have not been widely adopted by bee suits).
beekeepers. Furthermore, methods for disease manage- Apis mellifera beekeepers prefer to reuse old combs
ment are poorly developed for the other managed bees, because it takes the bees more energy to produce wax than
and more research is needed to provide beekeepers with honey; therefore, the comb is quite valuable in terms of the
effective tools. Below, currently available methods are honey production capacity of the colony. However, old
discussed and areas where more development is needed combs can also harbor pathogens, such as the causal agents
are identified. for AFB and chalkbrood, and can also accumulate pesti-
The first two components of disease management, cides. Thus, old comb and hive bodies should be regularly
quarantine and sanitation, are employed to avoid disease replaced or sanitized. Unfortunately, the methods for
establishment and prevent its spread. Quarantines are sanitation are somewhat limited. Sanitation methods for
forced isolations or restrictions on movement to prevent the wax combs include gamma radiation (Studier, 1958;
spread of contagious disease, and are usually established by Shimanuki et al., 1984), ethylene oxide (Shimanuki, 1967),
governments. For example, many countries have regula- heat (Cantwell and Shimanuki, 1970), and high concen-
tions that restrict the importation of bees and bee pollen. In trations of ozone (James, 2011a).
addition, when a new pathogen or parasite is found, Another approach is to replace the comb regularly,
beekeepers may be restricted from moving bees out of the perhaps as frequently as every two years. In practice, this
area where it has established. Thus, the state of Wyoming could mean replacing half the comb every year, as is done
in the USA requires not only A. mellifera, but also in Denmark (P. Kryger, pers. comm.). Pathogenic organ-
M. rotundata colonies to be inspected for disease. If more isms have been shown to spread on combs, especially brood
than 5% of the overwintering cocoons in a batch of combs, where they can contaminate the pollen fed to larvae
M. rotundata show signs of chalkbrood, the producer (see Sections 12.3.2 and 12.3.3). Beekeepers often transfer
cannot sell that batch of bees. Wyoming is the only state brood from strong colonies to weaker colonies in an effort
with a chalkbrood quarantine for M. rotundata. to bolster the bee population in the smaller colony;
Quarantines can also be implemented by beekeepers however, this can lead to the direct transfer of contaminated
themselves, although this is unusual. The main premise to combs. This transmission can also be reduced if measures
such a quarantine would be to prevent bringing diseased are taken to keep the brood out of the honey supers. Honey
bees into an apiary. One method to do this is to keep the combs are typically exchanged between hives/colonies by
bees in an isolated area for a few weeks and watch for signs beekeepers, and if the brood is not kept out of the honey
and symptoms of disease that may not have been apparent comb, then the brood comb will also be exchanged between
at first. This type of quarantine is critical for bumble bee colonies. Brood can be kept out of the honey comb using
producers, where the bees are produced in isolation, queen excluders, which are screens that have holes large
indoors. Before any new stock is introduced into a rearing enough to allow worker bees to pass through but too small
facility (such as field-caught queens), the beekeeper should to allow queens and drones to pass through. Queen
verify that those bees are disease free. With queens, this can excluders should be placed between the brood chamber and
be accomplished by allowing the queen to produce 20e30 the honey supers to confine the queen to desired parts of the
workers in isolation, then sampling the workers and testing hive, restricting where she can lay eggs. In addition,
them for the presence of N. bombi. This method allows time beekeepers should avoid the practice of transferring mate-
for the pathogen to build up to levels where infections can rial from the brood chamber of one colony to another
be detected. In addition, bumble bee colonies that have colony.
been used for field or greenhouse pollination should not be Megachile rotundata nesting boards and cocoons are
returned to the rearing facility. This self-imposed quaran- sanitized with formaldehyde gas in Canada to kill
tine method remains the primary means of disease control A. aggregata spores and prevent chalkbrood transmission
in commercial bumble bee production. (Goerzen and Watts, 1991). Formaldehyde is not registered
Chapter | 12 Silkworms and Bees 451

for this use in the USA; therefore, chlorine dips have been recently been replaced by powdered sugar (confectioners’
recommended (Stephen, 1982; Richards, 1984). However, sugar). Powdered sugar disrupts the ability of the mites to
sanitizing a large number of nesting boards with chlorine is stay attached to the bees, but the bees live and can be
messy and hazardous, and is not often done. Methyl released after sampling. The mites are then counted in the
bromide has been used by some farmers (Mayer et al., powdered sugar (Macedo et al., 2002). However, the
1991), but this has poor efficacy against A. aggregata measurement is more accurate if water is added to the jar
spores (James, 2005a). This compound is still legal in the after the bees are released, to dissolve the sugar and make
USA for treating wood, but it causes a deterioration of the the mites more visible. Powdered sugar can also be applied
ozone layer in the Earth’s upper atmosphere and a complete directly to the bee colony, and then the residue collected on
ban will eventually come into effect. Heat can be used to a board or tray at the bottom of the hive.
kill the spores on wooden nesting boards (Kish, 1983), but Varroa mites can also be sampled using a sticky board
melts polystyrene boards. Nesting board sanitation is trap at the bottom of a hive. A piece of stiff cardboard is cut
widely used in M. rotundata rearing, but when it is used to fit inside the bottom of the hive. The top of this board is
alone, it is not effective because the nesting boards are not covered with a thin film of cooking oil, or some other type
the primary source of pathogen transmission for of adhesive, which is in turn covered with hardware cloth or
chalkbrood (James, 2005a, 2011b). some other mesh screening (using a 3  3 mm mesh). This
Hygiene is also important in bumble bee rearing. All apparatus is placed at the bottom of the hive. The mesh
used bumble bee nesting materials should be sanitized keeps the bees from sticking to the board, but mites that fall
before disposal, or removed to places where bumble bees in the hive are trapped in the adhesive. This sampling
cannot reach them (e.g., buried or burned). Other sanitation method can be used to measure the natural fall rate of the
measures include cleaning all equipment (e.g., forceps, mites or it can be used in conjunction with a miticide
nesting boxes, food containers, mating chambers, etc.) with (Branco et al., 2006). Evaluating varroa mite infestation
a disinfectant after every use; never transferring food from levels allows beekeepers to determine when to use treat-
one colony to another; never transferring food containers ments for this parasite and disease vector (Delaplane and
between colonies without first sanitizing them; storing food Hood, 1999; Strange and Sheppard, 2001).
in the refrigerator, and disposing of any that spoils; and Disease monitoring methods are used in M. rotundata
maintaining a neat and clean rearing facility. beekeeping for chalkbrood. Larvae are sampled during the
The third component of disease management is moni- winter when they are in the cocoon stage. Typically,
toring. Bees should be regularly monitored for disease and cocoons are removed from the nesting boards in December
general overall health. Many diseases can go undetected in and stored at 4 C during the winter and spring. Cocoons are
a colony for long periods if they are not watched for checked for mortality and signs of chalkbrood using X-ray
carefully and regularly, and then once an outbreak is analysis (Stephen and Undurraga, 1976) or by cutting the
obvious, losses can be catastrophic. An example of this is cells open. Monitoring for chalkbrood is used to evaluate
Nosema in bumble bees. Once Nosema becomes estab- the quality of the bees when they are sold and purchased,
lished in a facility, it becomes necessary to destroy all the and before they are incubated for adult bee release. As
bees, sterilize the equipment and rearing room, and start stated earlier, it can also be used for quarantine purposes.
production all over again. Therefore, it is much more cost- Bumble bees in rearing facilities are monitored most
effective to monitor for the disease so that appropriate often for N. bombi. Several methods have been developed
prevention measures can be taken. Accurate monitoring of for detecting spores in the host and pollen, including
bee diseases, especially the viruses and microsporidia, may immunofluorescent staining and electron microscopy, as
require laboratory analyses, which are not practical for well as antigenic, biochemical, and molecular analyses
some beekeepers. However, many countries provide (Weber et al., 1999; Klee et al., 2006). Bumble bee colonies
disease-testing laboratories where beekeepers can send should be sampled regularly during production to verify
samples. In addition, simple, economical testing strips are that they are disease free, and infected colonies should be
now available for use to test for the foulbrood diseases. immediately destroyed. Pollen should also be monitored
Many sampling methods have been developed for for the disease to determine that it is safe before it is used as
detecting and quantifying varroa mite infestations. One of food.
the earliest methods was an ether roll, where a sample of Resistance is the fourth disease management compo-
approximately 300 bees is placed in a 1-liter (quart) jar with nent. Bee resistance to disease can be maintained through
a small amount of ether and shaken vigorously for 1 min. both genetics and good rearing practices. It is possible to
The sampled bees and mites are killed by this method. The select for disease resistance in a breeding program, but
ether causes the mites to fall off the bees and stick to the maintaining genetic resistance also means being careful not
side of the jar, which has become wet from the ether to select for susceptibility. Insects are often bred in
(Delaplane and Hood, 1999). The use of ether in the jar has production facilities to optimize production traits, and
452 Insect Pathology

breeders must be careful not to inadvertently select for and Reuter, 2001a, b) or USDA Russian queens
deleterious traits linked to the desired trait. For example, (Rinderer et al., 2001). These breeds are especially
A. mellifera are commonly bred for gentleness and honey capable of identifying diseased and dead brood and
production, yet little to nothing is known about how these removing them from the colony. The Russian and VSH
traits are linked to disease resistance or overwintering breeds are also resistant to varroa mites and may be
hardiness. In addition, inbreeding depression can be resistant to tracheal mites.
a challenge in bee breeding programs owing to the ease l When producing queens, avoid inbreeding, use hybrid
with which several reproductive females can be produced vigor, mate with many drones, and limit the use of
by a few parents (Rinderer, 1986). artificial insemination.
Environmental factors can also play a role in disease l In mid-summer, locate bees to an area with a strong
resistance. For example, nutrition, temperature, and nectar and pollen flow. This will increase both brood
humidity are all known to affect the rate of infectious and honey production (Rinderer and Rothenbuhler,
diseases in insects. Sometimes environmental conditions 1974), and pollen resources reduce viral infections
may enhance the ability of a pathogen to infect a host (Hoffman et al., 2010).
(e.g., high humidity might improve fungal spore germina-
tion; see Chapter 6), and some environmental conditions Little research has been done regarding the use of resis-
enhance insect immunity (e.g., slightly stressful tempera- tance for disease control in other bees.
tures can increase resistance to infection in bees and lady The fifth component in disease management is to
beetles) (James et al., 1998; James, 2005b). For A. melli- control disease vectors. That is, measures need to be taken
fera, nutritional supplements can enhance bee health in the to control infestations by pests, parasites, and parasitoids
spring, when adequate floral resources are scarce (Hoffman that may spread disease within the colony. Controlling
et al., 2008). For more information on resistance, see such pests is not always easy. The most notable bee
Chapter 13. vectors are the varroa mites in A. mellifera. Varroa mites
The goal is to keep disease resistance high in the bee transmit viruses, especially deformed wing virus (see
colonies through a combination of proper care and proper Section 12.3.1). Varroa mites should be monitored for as
genetics. For instance, population models have shown that discussed above, and treated with medications when
an A. mellifera colony can withstand varroa mite infesta- needed.
tions for many years if the queen lays a sufficient number of The sixth and last disease management component is
eggs. It is during the fall and winter, when brood production medication and chemical treatment. Medications should be
declines, that varroa mites often cause the most damage, reserved as a last resort because most do not completely
and similarly, they can quickly devastate a colony with eliminate the targeted pathogen or parasite and, conse-
a weak queen (e.g., poor egg layer). Varroa mites also quently, when treatments are stopped, the disease may
directly inhibit the immune system in A. mellifera (Yang recur. Furthermore, if this cycle is repeated often enough,
and Cox-Foster, 2005, 2007). The following general prac- the pathogen or parasite may develop resistance to the
tices can help to maintain brood health and disease treatments. Resistance to medication has been a serious
resistance. problem in A. mellifera. Paenibacillus larvae, the pathogen
causing AFB, has developed resistance to the antibiotic
l Keep the hives out of standing water; if they flood, move oxytetracycline hydrochloride in the USA (Kochansky
them to higher ground as soon as possible. If the bees et al., 2001) and varroa mites have developed resistance to
are kept in an area where the ground gets saturated, several miticides in the USA and Europe, rendering these
place the hives on a hive stand or concrete pad, above treatments useless (Elzen et al., 1999a, b; Elzen and
the water level. Use screened bottom boards to increase Westervelt, 2002).
ventilation in humid or wet environments. Controlling Methods for medicating bees other than A. mellifera
excessive humidity can greatly reduce the incidence of have not been developed. James (2011b) describes
chalkbrood. a method for treating M. rotundata with a fungicide to
l Avoid opening the hives when it is cold outside (below control chalkbrood, but the results are not consistent. No
18 C), especially on cloudy or rainy days, to avoid medications are currently available for viral diseases in
exposing the brood to the cold. If colonies must be A. mellifera either, but a new product for treatment is
worked during cool weather, minimize the time the hive currently being developed for IAPV (Maori et al., 2009)
is open. Again, this is especially important for and has been field tested with good results (Hunter et al.,
controlling chalkbrood. 2010). The product is based on gene silencing by RNA
l Use queens from a hygienic breed, such as bees with the interference (RNAi). If this technique works, it will
VSR (varroa sensitive hygiene, sometimes referred to as undoubtedly lead to the development of new treatments for
SMR, suppression of mite reproduction) trait (Spivak other RNA viruses and possibly other pathogens.
Chapter | 12 Silkworms and Bees 453

Bacterial diseases in A. mellifera, such as the foul- with each product, and some products must be applied
broods, can be treated with antibiotics. Currently, two multiple times at two-week intervals.
antibiotics are registered in the USA for controlling AFB, A few different methods are also available for control-
oxytetracycline hydrochloride (TerramycinÒ ) and tylosin ling tracheal mites. Vegetable shortening mixed with white
(TylanÒ ). In the USA, Terramycin has traditionally been granulated sugar is formed into patties and placed on top of
used as a prophylactic. The concept behind its use was to the frames in the brood chamber. Vapors from the short-
keep the pathogen load low enough that disease never ening are thought to disrupt the life cycle of the mites and
develops. Beekeepers typically treat in the spring or fall thus suppress mite populations. Menthol is another tracheal
(before or after the honey flow), or both. To prolong the mite control, and it is essentially a fumigation method.
presence of the antibiotic in the hive, a method called Unfortunately, no effective control methods are avail-
“extender patties” was developed, where beekeepers would able for some bee diseases. The extent of some major bee
mix the antibiotic with vegetable shortening to slow the rate health issues in managed bees, such as CCD in A. mellifera,
at which the bees consumed it (Wilson and Elliott, 1971; chalkbrood in M. rotundata, and nosema disease in bumble
Wilson et al., 1973). Unfortunately, the prophylactic bees, demonstrates that better disease management
approach to using Terramycin probably promoted the methods are needed. The integrated approach to disease
development of antibiotic resistance (Kochansky et al., management described here highlights some of the areas
2001). Terramycin was used prophylactically because it where more research is needed to improve disease control
was relatively inexpensive and preventive, but more of managed bees.
importantly, this antibiotic is not effective in colonies with
apparent infections. Terramycin cannot eliminate foul-
12.4. FUTURE RESEARCH DIRECTIONS
brood as it only reduces the pathogen load. Tylan is
effective after the disease symptoms are present in a colony. Although we often think of insects as pests, understanding
A few chemical-free methods exist for controlling an their pathogens and diseases is important to human survival
outbreak of AFB in an A. mellifera colony. All these and well-being. Many insects are beneficial, providing
methods are probably best summarized by the “shaking critical ecological services in wild and agricultural
colony method”, which is used in Denmark and France. In ecosystems, and the health of these beneficial insects is
brief, adult bees (only) are moved to a new hive and forced important. The importance of diseases in the population
to build new comb for two weeks; then they are moved to dynamics of insects, especially beneficial insects, has
another new hive and forced to build new comb again. The received very little attention, and now that bees (both
purpose of removing the brood and moving the adult bees managed bees and wild bees) are experiencing dramatic
to a new hive is to reduce the spore load in the hive and population declines for no apparent reason, it is clear that
colony, taking advantage of the fact that the pathogen this area of research has been neglected for too long. A
reproduces only in the brood. This is an organic method better understanding of the diseases present in native pop-
for foulbrood control, but it is labor intensive, and honey ulations of insects and their ability to move to new hosts is
yield will be severely reduced in the year of the treatment. needed, and this information can then be used to improve
This method was first recommended in 1895 (McEvoy, importation and quarantine regulations, with the protection
1895). A more recent study found this method insufficient of beneficial insects in mind.
to eliminate the pathogen; however, in that study, the bees More emphasis needs to be placed on utilizing an
were transferred to a new hive only once (Del Hoyo et al., integrated approach to disease management for bees. An
2001). emphasis on avoidance and prevention, as has been made in
Fumagillin-B has been the drug of choice for treating disease control efforts for silkworms, and some of the
N. apis infections in A. mellifera, but it is still unclear how principles learned could be better applied to bee manage-
effective this medication is for treating N. ceranae infec- ment. Past research efforts on disease control in A. mellifera
tions. Fumagillin-B is also ineffective in controlling have overly emphasized the use of medications to treat
N. bombi in bumble bees (Whittington and Winston, 2003) diseased bees. The problem with this approach is that it
and no other medications are available to control this cannot be attained for all pathogens and it does not guard
pathogen. against new pathogens and parasites. Every time a new
Several chemical treatments for varroa mites have honey bee pest invades a region, it causes serious declines
entered the market in the past several years. The current in bees until a new chemical control is developed, as has
compounds include formic acid (Mite-Away IIÒ ), thymol been experienced with AFB, varroa mites, and CCD.
(ApiguardÒ ), and sucrose octanoate esters (SucrocideÒ ). Furthermore, when chemicals are used in a hive, they have
Tau-fluvalinate and coumaphos can still be purchased. the potential to be harmful to the bees themselves and care
Chemical treatments should be applied in the fall or spring, must be taken to reduce contaminating hive products
but not during honey flow. The method of application varies intended for human use, such as honey, wax, and pollen.
454 Insect Pathology

The multifaceted, integrated approach described here Ball, B. V. (1999). An introduction to viruses and techniques for their
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