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The Physiology of Fishes

Edited by
Suzanne Currie and David H. Evans
Fifth edition published 2021
by CRC Press
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 Dedication
This book is dedicated to all past, present, and future students of fish
physiology.
Contents
The Physiology of Fishes: Fifth Edition
Preface for the Fifth Edition of The Physiology of Fishes
Contributors

Chapter 1 Evolution and Phylogeny

Terry C. Grande and Mark V. H. Wilson

Chapter 2 Locomotion and Biomechanics

Emily Standen

Chapter 3 Gas Exchange

Jodie L. Rummer and Colin J. Brauner

Chapter 4 The Cardiovascular System

Erika J. Eliason and Jonathan A. W. Stecyk

Chapter 5 Iono- and Osmoregulation

Dietmar Kültz and Kathleen M. Gilmour

Chapter 6 The Digestive System

Carol Bucking and W. Gary Anderson

Chapter 7 Thermal Biology

Nann A. Fangue, Anne E. Todgham and Patricia M. Schulte

Chapter 8 Endocrinology: An Evolutionary Perspective on

Neuroendocrine Axes in Teleosts
Sylvie Dufour and Karine Rousseau

Chapter 9 Reproduction
 Deborah MacLatchy

Chapter 10 Metabolism
Tommy Norin and Ben Speers-Roesch

Chapter 11 Hearing
Arthur N. Popper and Anthony D. Hawkins

Chapter 12 Active Electroreception and Electrocommunication

Vielka L. Salazar

Chapter 13 Vision
Ron Douglas

Chapter 14 Olfaction
Sigrun I. Korsching

Chapter 15 Aquaculture and Fisheries

Hsin-Yiu Chou and Guan-Chung Wu

Chapter 16 Epigenetics
Paul Craig

Chapter 17 Behaviour and Learning

Shaun S. Killen

Index
The Physiology of Fishes: Fifth Edition
EDITORS
Suzanne (Suzie) Currie, PhD Professor of Biology, Acadia University,
Wolfville, Nova Scotia, Canada
David H. Evans, PhD Professor Emeritus of Biology, University of
Florida, Gainesville Florida

DESCRIPTION
This Fifth Edition of the series continues the tradition of chapters on the
cutting edge of the latest advancements in fundamental areas of fish
physiology written by the leaders in these areas. The main goal of this book
is to provide a single-volume, comprehensive, accessible review of core
topics in fish physiology. The chapters integrate physiology with
environmental science, ecology, evolution, and molecular cell biology. The
book is intended for comparative animal physiologists, ichthyologists,
fisheries biologists, aquaculturists, and those interested in expanding their
knowledge base in fish biology. It is written for the fish physiologist
specialist as well as graduate and undergraduate students who have some
background in the field.
Preface for the Fifth Edition of The
Physiology of Fishes
Suzie Currie and David H. Evans

Fishes are the most speciose of vertebrates, and their diversity, behaviour,
ecology, and performance capture our imaginations. Since there is still
much to discover about these intriguing aquatic vertebrates, we are
delighted to present the fifth edition of The Physiology of Fishes. It has
been 27 years since the first edition of this book, and after three more
editions and variations on the theme, we feel we have returned to the
original ethos – a one-volume resource of the fundamentals and latest
developments in this fascinating area of biology.
For both of us, mentoring our students is and has been the most
rewarding and important aspect of our careers. With that in mind, the goal
of the fifth edition is to provide an introduction to fish physiology for
newcomers to the field. These learners may be undergraduate or graduate
students or, indeed, more seasoned scholars interested in pivoting in a new
and exciting research direction. We hope the readers share our enthusiasm
for this fifth edition, where you will read about fresh perspectives on the
basics of fish functioning as well as the latest advances and discoveries.
You will see that while each of our 17 chapters is focused on a specific
area of fish physiology, our authors have taken an integrative approach, as
is appropriate in this diverse field. The chapters also collectively highlight
the importance of fishes as models to understand essential aspects of animal
functioning, ecology, and evolution. We thought it important to begin the
book with an introduction to fish phylogenetics and evolution (Evolution
and Phylogeny), as these relationships are critical to our understanding of
the how and the why. Some of our chapters take a systems approach (Gas
Exchange, The Cardiovascular System, Iono- and Osmoregulation, The
Digestive System, Endocrinology, Reproduction), and others cover
sensation (Hearing, Active Electroreception and Electrocommunication,
Vision, Olfaction), performance (Locomotion and Biomechanics,
Metabolism, Behaviour and Learning), and the latest in integrative themes
of Thermal Biology, Aquaculture and Fisheries, and Epigenetics.
We are finishing this edition while physically distancing and self-
isolating in the middle of an astonishing global health crisis and pandemic.
By the time you read this book, we hope that we will have an effective
vaccine and that life will have returned to a new normal. This event gives us
perspective and gratitude for our lives, families, friends, and colleagues,
and we appreciate the opportunities to study fish biology. It has been an
absolute delight to work together on this project with our diverse and
brilliant authors and with our patient, responsive, and creative publishing
team. Thank you for your shared enthusiasm for all things fish!
Contributors
W. Gary Anderson
University of Manitoba
Winnipeg, Canada

Colin J. Brauner
University of British Columbia
Vancouver, Canada

Carol Bucking
York University
Toronto, Canada

Hsin-Yiu Chou
National Taiwan Ocean University
Keelung, Taiwan

Paul Craig
University of Waterloo
Waterloo, Canada

Ron Douglas
City University of London
London, UK

Sylvie Dufour
Muséum National d’Histoire Naturelle
Paris, France

Erika J. Eliason
University of California
Santa Barbara, California
Nann A. Fangue
University of California
Davis, California

Kathleen M. Gilmour
University of Ottawa
Ottawa, Canada

Terry C. Grande
Loyola University
Chicago, Illinois

Anthony D. Hawkins
Loughine Ltd
Aberdeen, Scotland

Shaun S. Killen
University of Glasgow
Glasgow, Scotland

Sigrun I. Korsching
Universität zu Köln
Köln, Germany

Dietmar Kültz
University of California
Davis, California

Deborah MacLatchy
Wilfred Laurier University
Waterloo, Canada

Tommy Norin
Technical University of Denmark
Kongens Lyngby, Denmark

Arthur N. Popper
University of Maryland
College Park, Maryland

Karine Rousseau
Muséum National d’Histoire Naturelle
Paris, France

Jodie L. Rummer
James Cook University
Townsville, Australia

Vielka L. Salazar
Cape Breton University
Sydney, Canada

Patricia M. Schulte
University of British Columbia
Vancouver, Canada

Ben Speers-Roesch
University of New Brunswick
Saint John, Canada

Emily Standen
University of Ottawa
Ottawa, Canada

Jonathan A. W. Stecyk
University of Alaska
Anchorage, Alaska

Anne E. Todgham
University of California
Davis, California

Mark V. H. Wilson
University of Alberta
Edmonton, Canada

Guan-Chung Wu
National Taiwan Ocean University
Keelung, Taiwan
 1 Evolution and Phylogeny
Terry C. Grande and Mark V. H. Wilson

CONTENTS
1.1 General Introduction
1.2 Jawless Vertebrates (Agnathans)
1.2.1 Order Myxiniformes (Hagfishes)
1.2.2 Order Petromyzontiformes (Lampreys)
1.3 Superclass Gnathostomata
1.4 Class Chondrichthyes (Ratfishes, Sharks, and Rays)
1.4.1 Subclass Holocephali (Chimaeras)
1.4.2 Subclass Euselachii, Infraclass Elasmobranchii (Neoselachii)
1.4.2.1 Division Selachii (Sharks)
1.4.2.2 Division Batomorphi (Rays)
1.5 Class Osteichthyes (Bony Fishes Including Tetrapods)
1.5.1 Subclass Sarcopterygii (Lobe-Finned Fishes and Tetrapods)
1.5.2 Subclass Actinopterygii (Ray-Finned Fishes)
1.5.2.1 Early-Branching Actinopterygii
1.5.2.2 Division Teleostei
1.5.2.3 Cohort Elopomorpha (Tarpons, Tenpounders, Bonefishes,
Eels)
1.5.2.4 Cohort Osteoglossomorpha (Bony-Tongues)
1.5.2.5 Cohort Otocephala
1.5.2.6 Cohort Euteleostei
1.5.2.7 Unranked Clade Neoteleostei
1.5.2.8 Unranked Clade Acanthomorpha (Spiny-Rayed Fishes)
1.5.2.9 Series Percomorpha
1.6 Conclusion
Literature Cited
1.1 GENERAL INTRODUCTION
Over half of the world’s living vertebrates are fishes (more than 85 orders,
536 families, 5000 living genera, 34,000 species, and counting; Nelson et
al. 2016). Fishes arose and began to radiate almost 500 million years ago
and now exhibit incomparable diversity (Figure 1.1) in morphology,
physiology, ecology, and behaviour.

FIGURE 1.1 Diversity among jawless fishes, chondrichthyans, and early-branching

osteichthyans. See Figures 1.3, 1.4, and 1.6 for phylogenies.

To understand biotic diversity, we employ phylogenetic systematics.

Phylogenies provide hypotheses of relationships among species to better
understand character evolution within and between groups, visualized as
phylogenetic trees or branching diagrams, and are the foundation for
comparative studies, including comparative physiology.
 The purpose of this chapter is to present an overview of the current
hypotheses of fish phylogeny as a framework for understanding the
remarkable diversity of fishes. We concentrate on the arrangement of large,
important clades (Figure 1.2) to illustrate broad patterns. Information
presented mainly follows Nelson et al. (2016). Another recent overview is
that of Hastings et al. (2014). Phylogenetic studies based on molecular data
include those of Near et al. (2012), Betancur-R. et al. (2013), Miya and
Nishida (2015), and Hughes et al. (2018).

FIGURE 1.2 Phylogenetic relationships among craniates. Letters indicate the sequence
of groups discussed in the text.

1.2 JAWLESS VERTEBRATES (AGNATHANS)

Only two orders (Myxiniformes or hagfishes and Petromyzontiformes or
lampreys) remain today from what was a very diverse radiation of jawless
fishes in the Early and Middle Paleozoic (Janvier 1996; Long 2012). Like
Long (2012), we place hagfishes outside Vertebrata. Phylogenetic analyses
of molecular sequence data, however, suggest that the two agnathan groups
may belong in a single clade after all (e.g., Mallatt and Sullivan 1998). This
controversy is far from settled, because placing them together requires
massive reinterpretation of their very different anatomical and physiological
features.
1.2.1 ORDER MYXINIFORMES (HAGFISHES)
The hagfishes (Figure 1.2 clade A) are an eel-like marine group of about 78
species with many adaptations to their scavenging lifestyle (they emerge
from burrows to scavenge on dead or dying animals) and many other
apparently archaic traits (Jørgensen et al. 1998), such as one pair of
semicircular canals (argued by some to be a fusion of two canals), one pair
of maculae in the inner ear rather than two or three, no lateral-line system or
neuromasts in adults, no rudimentary vertebral structures along the
notochord, and body fluids isosmotic with seawater.
1.2.2 ORDER PETROMYZONTIFORMES (LAMPREYS)
The lampreys (Figure 1.2 clade B) are a group of about 43 species with
anadromous or freshwater life histories (Renaud 2011). They have two
semicircular canals in the inner ear, whereas hagfishes have one and jawed
vertebrates have three. Lampreys pass through an “ammocoete,” filter-
feeding larval stage before metamorphosis. There are seven pairs of
external gill openings, a single nasohypophyseal opening and a pineal eye
on top of the head, a spiral fold in the intestine, a notochord that is
combined with rudimentary neural arches, and two dorsal fins but no paired
fins. Lampreys have a simple electroreception system in their skin, whereas
hagfishes do not. Petromyzon marinus is a model organism for spinal cord
research (e.g., Herman et al. 2018; Hanslik et al. 2019).

1.3 SUPERCLASS GNATHOSTOMATA

All other vertebrates have jaws and are thus gnathostomes (Figure 1.2). The
origin of the Gnathostomata was one of the most transformative events in
vertebrate history, and the list of their novel features (e.g., Nelson et al.
2016) is long, including jaws possibly homologous with an anterior gill
arch; hyomandibular supporting the jaw articulation; two or three inner ear
maculae (saccular, lagenar, and utricular) and three semicircular canals;
myelinated nerve fibers; and trunk muscles in epaxial and hypaxial blocks.
1.4 CLASS CHONDRICHTHYES (RATFISHES, SHARKS,
AND RAYS)
The Chondrichthyes or cartilaginous fishes (Figure 1.2 clade C) collectively
are the extant sister group to the Osteichthyes (including tetrapods). The
roughly 1200 species of chondrichthyans live in all major oceans and seas
but are rare in fresh waters (Lucifora et al. 2015) and virtually absent in the
deepest parts of the ocean (Treberg and Speers-Roesch 2016). An important
resource for information about chondrichthyans is Carrier et al. (2004).
The scales of chondrichthyans are denticles called placoid scales. The
teeth are arranged in families or whorls, vary in shape, and are usually shed
and replaced throughout life. Some sharks shed thousands of teeth in a
lifetime.
All chondrichthyans have a well-developed electroreceptive system in
the form of blind tubes called ampullae of Lorenzini, mostly on the head.
An innervated ampulla at the inner end of each tube is filled with a jelly-
like substance and opens externally via a pore. The tubes’ orientations
permit sensing of the directions and intensities of weak electric fields
generated by the heart or respiratory muscles of prey.
Chondrichthyans have internal fertilization, with males having
intromittent organs in the form of pelvic-fin claspers. Females either retain
young in their oviduct(s) until birth or lay large-yolked eggs enclosed in
leathery egg capsules. In some sharks, the unborn young feed in utero by
oophagy (eating of eggs) or by cannibalism (eating of unborn embryos).
Gestation periods can be up to 2 years (e.g., Musick 2010).
Separate groups of chondrichthyans, including chimaeras, horn sharks,
dogfish sharks, and stingrays, can deliver venom via dorsal spines or tail
spines, primarily for defense (Smith et al. 2016).
1.4.1 SUBCLASS HOLOCEPHALI (CHIMAERAS)
The Holocephali (Figure 1.3) include three families, six genera, and nearly
50 species. They live mostly in deeper marine environments and have a
dentition of flattened plates for crushing mollusks and crustaceans. Lateral-
line canals on the head are in deep grooves. Paired fins are usually large;
the first dorsal fin has an erectile spine, while the caudal fin is whip-shaped.
Chimaeras are oviparous; males have a clasper on the head in addition to
pelvic claspers. The chimaerid Callorhinchus milii, the elephant fish or
Australian ghostshark, has a compact genome and has become a model
organism for physiological and genomic studies (e.g., Hyodo et al. 2007;
Venkatesh et al. 2014).

FIGURE 1.3 Phylogenetic relationships within Chondrichthyes. Holocephali are sister

to Elasmobranchii, which in turn are divided into the Selachii (sharks) and the
Batomorphi (skates and rays). Triangle sizes are correlated to species diversity.

1.4.2 SUBCLASS EUSELACHII, INFRACLASS ELASMOBRANCHII

(NEOSELACHII)
The Elasmobranchii include all sharks (Selachii) and rays (Batomorphi).
Today, we accept that sharks and rays are sister groups (Figure 1.3); their
fossils are of about equal antiquity in the Early Jurassic (175–200 million
years ago; Maisey et al. 2004).
1.4.2.1 Division Selachii (Sharks)
The extant sharks (Figure 1.3) include two superorders, nine orders, 34
families, 106 genera, and at least 561 species. In contrast to rays, sharks
have gill slits that are mainly lateral, and the anterior margin of the pectoral
fin is not attached to the side of the head. Left and right pectoral girdles are
not joined dorsally. There are two main clades of selachians (Maisey et al.
2004): the Galeomorphi and the Squalomorphi.
1.4.2.1.1 Superorder Galeomorphi
Galeomorph sharks (Figure 1.3) comprise four orders, 23 families, 74
genera, and about 400 species (e.g., Compagno 2005). All have an anal fin
and a closed lateral-line canal.
The four orders of galeomorphs include the Heterodontiformes (horn
sharks), which are oviparous, and the Orectolobiformes (carpet sharks),
which include the nurse sharks (Ginglymostomatidae), wobbegongs
(Orectolobidae), and the whale shark (Rhincodontidae), the largest extant
fish, a large-gape filter feeder that often swims near the surface.
Lamniformes (mackerel sharks) have a distinctively coiled intestinal
spiral valve. Many embryos are nourished by oophagy (egg eating) or
cannibalism of other embryos before birth. Lamnids can maintain elevated
temperatures in their swimming muscles using a counter-current exchange
system of blood vessels. Examples include the filter-feeding basking shark
(Cetorhinidae) and megamouth shark (Megachasmidae) as well as the
thresher sharks (Alopiidae), goblin shark (Mitsukurinidae), and sand tigers
(Odontaspididae). The white shark (Lamnidae) is a top predator with a
global marine distribution at temperate latitudes. It can travel thousands of
kilometers in search of mates, food, or nursery grounds (Bonfil et al. 2005;
Jorgensen et al. 2009).
Carcharhiniformes (ground sharks) have a variety of developmental
modes, including oviparity and viviparity. Examples include cat sharks
(Scyliorhinidae), hound sharks (Triakidae), requiem sharks
(Carcharhinidae; some enter fresh water), and hammerhead sharks
(Sphyrnidae) with hydrofoil-shaped heads that widely separate the eyes,
nostrils, and ampullae of Lorenzini and might enable superior sensory
abilities. It has been reported that embryonic yolk sacs of requiem and
hammerhead sharks develop placenta-like attachments to the wall of the
mother’s oviduct (Hamlett 1989).
1.4.2.1.2 Superorder Squalomorphi
The squalomorph sharks (Figure 1.3) share a unique jaw articulation
(Maisey 1980) and lack an anal fin. The Hexanchiformes (six-gill sharks)
have one spineless dorsal fin, a very elongate body, a large mouth, and six
or seven gill slits. Eggs hatch from their egg cases inside the mother’s
oviduct. The Chlamydoselachidae (frilled sharks) have six pairs of gill slits,
and the Hexanchidae (cow sharks) have six or seven pairs.
Squaliformes (dogfish sharks) have two dorsal fins and retain spiracles.
Examples include the Centrophoridae (gulper sharks), Etmopteridae
(lantern sharks) known for their light-emitting organs, and Somniosidae
(sleeper sharks). The Squalidae (dogfish sharks) include the usual
comparative anatomy species, Squalus acanthias, which is viviparous and
has a long gestation of up to 2 years.
Echinorhiniformes (bramble sharks) have unusually large, widely
scattered scale denticles. Squatiniformes (angel sharks) are viviparous,
benthic sharks with a body shape convergent with that of rays. They are
ambush predators that use strong suction feeding. Pristiophoriformes (saw
sharks) have a pair of long barbels on the ventral side of their elongate
rostrum, which has laterally projecting “teeth” that are enlarged dermal
denticles (Welten et al. 2015); the mouth is ventral. Prey are detected by
ampullae of Lorenzini and/or by the barbels, exposed, and then killed or
disabled by sideways slashing of the rostrum.

1.4.2.2 Division Batomorphi (Rays)

The Batomorphi (Figure 1.3) are the rays, with 632 species in four orders.
They lack an anal fin. Pectoral fins are greatly enlarged and attached to the
head dorsal to the gill slits but ventral to the eyes and spiracle. The pectoral
girdles are joined ventrally as well as dorsal to the vertebral column. Rays
are viviparous, except for skates (see later).
Torpediniformes (electric rays) have electric organs in the pectoral fins
that can generate potentials with ventro-dorsal polarity of up to 220 volts or
currents of up to 50 amperes for feeding or defense. Eyes are small and
sometimes non-functional. The rays can ambush prey that swim near their
head (Lowe et al. 1994). For defense, they can roll themselves up in a
defensive posture.
Rajiformes (skates) are a single diverse family with 32 genera and about
288 species and a near-global marine distribution. Subfamilies divide
hardnose skates from softnose skates. Unlike other rays, skates are
oviparous, laying eggs in egg cases.
Pristiformes (guitarfishes and sawfishes) have a prominent elongate
rostrum, which in sawfishes (family Pristidae) is a blade armed with
socketed “teeth.” Like those of saw sharks, the rostral “teeth” of sawfishes
are modified dermal denticles (e.g., Welten et al. 2015).
Myliobatiformes (stingrays, etc.) are a diverse group, most with a
serrated and often venomous spine on their tail, which can inflict a painful
and sometimes fatal wound. Examples are the Platyrhinidae (thornback
rays), Zanobatidae (panrays), Dasyatidae (whiptail stingrays, including one
that ranges into fresh water), Potamotrygonidae (river stingrays of South
America), and Myliobatidae (eagle rays, cownose rays, devil rays, and
manta rays).

1.5 CLASS OSTEICHTHYES (BONY FISHES INCLUDING

TETRAPODS)
The Osteichthyes (Figure 1.2) comprise the remaining two monophyletic
groups: Sarcopterygii, lobe-finned fishes plus tetrapods (Figure 1.2 clade
D), and Actinopterygii, ray-finned fishes (Figure 1.2 clade E), all having
well-ossified internal skeletons of endochondral or membrane bone.
Lepidotrichia (bony fin rays) are now present and are formed as shafts of
bone around bundles of collagen fibers called actinotrichia. In addition, a
premaxilla is present, and the skull has sutures. Lungs, functioning as either
air-breathing organs or as a buoyancy-controlling swimbladder, are present
in all osteichthyans unless secondarily lost.
1.5.1 SUBCLASS SARCOPTERYGII (LOBE-FINNED FISHES AND
TETRAPODS)
Notable within Sarcopterygii are the coelacanths (e.g., Latimeriidae, with
one extant genus Latimeria and two species) and the lungfishes (Dipnoi)
containing the Neoceratodontidae (Australian lungfish), Lepidosirenidae
(South American lungfish), and Protopteridae (African lungfishes), the
latter two families sometimes placed together in Lepidosireniformes.
Lungfishes have true lungs that develop from an evagination of the
pharynx, a partially divided atrium and ventricle in the heart, and a
separation of pulmonary and systemic blood vessels with newly evolved
pulmonary arteries. Although Neoceratodus (Australian lungfish) possesses
one lung positioned dorsal to the gut, it respires mostly through gills.
Protopterus (African lungfishes) and Lepidosiren (South American
lungfish), on the other hand, possess paired lungs and are essentially
obligate airbreathers. They can also estivate (remain dormant) in burrows
during periods of heat or desiccation.
1.5.2 SUBCLASS ACTINOPTERYGII (RAY-FINNED FISHES)
The remainder of this chapter concerns the Actinopterygii or ray-finned
fishes (Figure 1.4). Basic features include dermal bones, a layer of ganoine
on the dermal bones and scales, a peg-and-socket articulation between trunk
scales, a well-developed endoskeleton, and fin muscles that do not extend
into the paired fins but still precisely control the fin rays. Actinopterygii are
divided among 67 orders, 469 families, over 4000 genera and 30,500
species (Nelson et al. 2016). Almost half of these species inhabit fresh
water.
 FIGURE 1.4 Phylogenetic relationships among early-branching actinopterygians.
Polypteridae form the sistergroup to Chondrostei (sturgeons and paddlefishes) +
Holostei (gars and bowfins) + Teleostei. Triangle sizes are correlated to species
diversity.

1.5.2.1 Early-Branching Actinopterygii

The three earliest branches from the actinopterygian stem that still have
extant members (Figure 1.4) are 1) the order Polypteriformes (bichirs) from
fresh waters of Africa; 2) the infraclass Chondrostei, comprising the order
Acipenseriformes (paddlefishes and sturgeons) from fresh waters of the
Northern Hemisphere (Grande and Bemis 1998)—both paddlefishes and
sturgeons possess ampullary electroreceptors concentrated in the rostrum
and oral area; and 3) the infraclass Holostei, containing the order
Lepisosteiformes (gars) of North and Central America plus Cuba, together
with the order Amiiformes (only the bowfin Amia calva) of North America
(Grande and Bemis 1998; Grande 2010).

1.5.2.2 Division Teleostei

All remaining extant actinopterygians are members of the Teleostei.
Teleosts have a rich fossil record. They first appeared in the Late Triassic
fossil record (201–237 million years before present) and radiated in the
Jurassic, Cretaceous, and Cenozoic, eventually replacing most other clades
of fishes (e.g., Long 2012). Their monophyly is strongly supported by both
morphological and molecular data (e.g., de Pinna 1996; Near et al. 2012,
2013). The teleostean caudal fin primitively includes two ural centra, ural
neural arches elongated to form paired, strap-like uroneurals, and an
externally homocercal shape.
Teleosts are the most speciose and diversified group of all vertebrates
(Figures 1.1 and 1.5). With about 30,000 extant species (95% of all extant
fishes), they dominate the world’s lakes, rivers, and oceans (Nelson et al.
2016).
 FIGURE 1.5 Diversity of fishes within Otocephala and Euteleostei (details are in all
remaining phylogeny figures) representing over 96% of all extant fishes.

1.5.2.3 Cohort Elopomorpha (Tarpons, Tenpounders, Bonefishes, Eels)

Elopomorphs (Figure 1.6) are known for their unique leptocephalus (slim-
headed, transparent, ribbon-like) pelagic larval stage. There are two major
body types of elopomorphs: the “fish-like” bodies of Elopiformes (tarpons,
ladyfishes) and Albuliformes (bonefishes) and the “eel-like” body types of
Notacanthiformes (deep-sea spiny eels) and Anguilliformes (marine and
freshwater eels). Among elopomorphs, Anguilliformes (eels) are by far the
most diverse order, with at least 938 species. Species within the family
Megalopidae (e.g., Tarpon, Megalops) exhibit coelomic extensions of the
swimbladder that expand anteriorly into bullae. Although these bullae make
no connection with the inner ear, hearing enhancement has been
hypothesized (Braun and Grande 2008) but apparently not experimentally
tested.

FIGURE 1.6 Phylogeny of Teleostei. Early-branching clades are Elopomorpha,

Osteoglossomorpha, and Otocephala (Clupeomorpha + Alepocephali + Ostariophysi),
the latter being sister to the Euteleostei. Triangle sizes are correlated to species
diversity.

1.5.2.4 Cohort Osteoglossomorpha (Bony-Tongues)

Osteoglossomorphs (Figure 1.6) are known as the bony-tongues because of
their unique feeding mechanism: teeth on the gill arches shear food against
teeth on the parasphenoid bone in the roof of the mouth. Anterior
extensions of the swimbladder that approach the inner ear are common
among osteoglossomorph taxa (e.g., Hiodon, Notopterus; Braun and Grande
2008). All living species inhabit fresh water and are divided into two
orders: Hiodontiformes (mooneyes, sister to all other osteoglossomorphs)
and Osteoglossiformes, comprising Pantodontidae (butterflyfishes),
Osteoglossidae (arowanas), Mormyridae (elephantfishes), Notopteridae
(featherfin knifefishes), and Gymnarchidae (the aba, Gymnarchus).
Arapaima gigas, native to the Amazon river basin, is an obligate airbreather
and is among the largest of extant freshwater fishes, growing to about 3
meters in length and at least 200 kilograms in weight. Mormyrids exhibit
electrosensory ability correlated with an enlarged cerebrum for processing
electroreceptive information. Gymnarchus niloticus can both produce and
detect electric fields. The electric fields can be used for navigation as well
as for species and individual recognition (e.g., Arnegard et al. 2010).

1.5.2.5 Cohort Otocephala

The Otocephala and its sistergroup the Euteleostei comprise all remaining
fishes (Figures 1.5 and 1.6). The cohort Otocephala contains fishes with an
otophysic connection between the swimbladder and the inner ear (Braun
and Grande 2008). The group contains three superorders: Clupeomorpha
(herrings, sardines, and anchovies), Alepocephali (slickheads), and
Ostariophysi (e.g., carp, minnows, characins, catfishes, and Neotropical
knifefishes) (Lecointre and Nelson 1996; Nelson et al. 2016).
1.5.2.5.1 Superorder Clupeomorpha
Clupeomorpha (Figure 1.6) are diagnosed by a unique otophysic connection
comprising a pair of diverticula extending from the swimbladder through
the exoccipitals and expanding into bullae within the prootics and often the
pterotics of the skull (Grande 1985). The prootic bullae are closely
associated with the utriculus of the inner ear. This connection enhances
hearing in these fishes. Taxa belonging to order Clupeiformes are diagnosed
by the presence of a recessus lateralis.
Clupeiforms comprise over 405 species, with about 79 of them being
primarily freshwater forms. Their diversity is concentrated in the Indo-
Pacific and the western Atlantic. Members of the families Engraulidae
(anchovies; 17 genera and about 146 species) and Clupeidae (herrings,
sardines, shads; 64 genera and about 218 species) contain the majority of
species and are of great economic value throughout the world.
1.5.2.5.2 Superorder Ostariophysi
Ostariophysans (Figure 1.6) are the largest clade of mostly freshwater
fishes. Breeding tubercles composed of keratin are diagnostic. There are
two monophyletic series: Anotophysi, consisting of the order
Gonorynchiformes (extant members mostly marine), and Otophysi, with
four primarily freshwater orders, all four of them having a Weberian
apparatus, which is a unique series of modified pleural ribs, neural arches,
and supraneurals of the anterior-most vertebrae that conduct sound
vibrations from the swimbladder to the inner ear.
The anotophysan order Gonorynchiformes contains three families
(Poyato-Ariza et al. 2010): Chanidae (milkfish) from marine waters of the
Indian and Pacific Oceans; Gonorynchidae (beaked sandfish) from marine
waters of the Indo-Pacific; and Kneriidae (knerias and snake mudheads),
from fresh waters of tropical Africa.
The Otophysi total over 10,000 species (Nelson et al. 2016) and are
divided into four orders: Cypriniformes (carps and minnows including the
goldfish and zebrafish, loaches, and suckers), Characiformes (characins
such as the tetras and piranhas), Siluriformes (catfishes), and
Gymnotiformes (Neotropical knifefishes).
Cypriniformes are very diverse in novelties, ecologies, and taxa
(Hernandez and Cohn 2019), with at least 4200 species (Nelson et al. 2016).
Their Weberian apparatus is relatively unspecialized (Bird and Hernandez
2007). Many are important food and aquarium fishes (e.g., Cyprinus carpio,
common carp). The goldfish (Carassius auratus) is an experimental animal
for fish physiology, and the zebrafish (Danio rerio), of course, is the model
organism for fish developmental biology.
Characiformes, according to most morphological studies, are the
sistergroup to gymnotiforms and siluriforms. All extant characiforms are
restricted to fresh waters; about 200 species occur in Africa, and the
remainder of the 2300 species are found in North, Central, and South
America.
Siluriphysi (catfishes—3725 species + Neotropical knifefishes—207
species) exhibit morphological and ecological specializations too numerous
to list here. Novelties of some catfishes (Siluriformes) include venom
glands (e.g., Heteropneustidae), electric organs (Malapteruridae—their
giant electromotor neurons are of special interest), armor (Callichthyidae,
Loricariidae), and dwarfism (Scoloplacidae). A few are parasitic
(Trichomycteridae: Vandellia cirrhosa). The Gymnotiformes (knifefishes)
are restricted to Neotropical regions. Many (e.g., Electrophorus electricus)
have electric organs capable of potentials of up to 600 volts.

1.5.2.6 Cohort Euteleostei

The Euteleostei contain all the remaining teleost fishes (Figures 1.5 and
1.7). The monophyly of this group is unquestioned and is supported by a
unique pattern of supraneural development, the presence of a stegural with
an anterodorsal membrane outgrowth, and the presence of caudal median
cartilages (Wiley and Johnson 2010).

FIGURE 1.7 Phylogeny of Euteleostei. Lepidogalaxiiformes are sister to

Protacanthopterygii (salmon and pikes) + Zoroteleostei (Osmeromorpha and
Neoteleostei). Triangle sizes are correlated to species diversity.
 Within Euteleostei, Lepidogalaxias (salamanderfish) is the sistergroup to
all other extant euteleosts (Li et al. 2010; Campbell et al. 2017). The
Protacanthopterygii (Salmoniformes plus Esociformes) are the sistergroup
to all the rest (Figure 1.7). Nelson et al. (2016) gave totals of 50 orders, 351
families, 3160 genera, and over 19,000 species for Euteleostei.
1.5.2.6.1 Superorder Protacanthopterygii
A very restricted membership of Protacanthopterygii is accepted here
(Wilson and Williams 2010, Campbell et al. 2013; Hughes et al. 2018):
Salmoniformes + Esociformes (Figure 1.7).
Salmoniformes (trout, salmon, whitefishes) contain only Salmonidae,
with 10 genera and at least 225 species. They are of high economic
importance because of their exploitation in commercial, sport, and
subsistence fisheries. Species of Salmo and Oncorhyncus are intensively
farmed and/or widely introduced. The 17 species of Oncorhyncus (Pacific
salmon) are of particular interest for their long-distance, anadromous
migrations and their homing ability to their natal streams for spawning.
Many of them are also semelparous (dying after just one spawning
episode).
Esociformes (pikes and mudminnows) contain two families, four genera,
and about 12 species (Grande et al. 2004). Esocidae comprise three of the
four genera (Novumbra, Dallia, and Esox). Species within the genus Esox
are well known for their characteristic duck-billed snout, elongated body,
mouth of sharp depressible teeth, and voracious feeding behaviour. Pikes
have recently been advocated as model organisms for developmental
studies (Pospisilova et al. 2019). Umbridae (mudminnows) contain only the
genus Umbra (López et al. 2004). At least two species of Umbra are
adapted to tolerate low oxygen levels (Currie et al. 2010). Umbra limi is
known to breathe from bubbles trapped under ice in ice-covered lakes
(Magnuson et al. 1983).
1.5.2.6.2 Unranked Clade Zoroteleostei
This clade (Figure 1.7) includes all remaining Euteleostei, united by a
partially or completely unroofed preopercular sensory canal in primitive
members (Wilson and Williams 2010; Nelson et al. 2016) and by genome-
scale molecular phylogenetic analysis (Hughes et al. 2018).
1.5.2.6.3 Superorder Osmeromorpha
The first group of Zoroteleostei (Figure 1.7) includes the orders
Argentiniformes (marine smelts), Galaxiiformes (galaxiiforms),
Osmeriformes (Northern Hemisphere smelts), and Stomiiformes
(dragonfishes) (Hughes et al. 2018).
Galaxiiforms of the Southern Hemisphere are predominantly in fresh
waters, while osmeriforms mostly spawn in fresh waters of the Northern
Hemisphere. Both groups contain anadromous members (Nelson et al.
2016). Termed dragonfishes for the nightmarish teeth on their premaxilla
and maxilla, stomiiforms are of particular interest because of
bioluminescent organs (photophores) and a mostly deep-sea lifestyle
(Kenaley 2009; Davis et al. 2016). Stomiiforms undergo daily feeding
migrations from deep waters during the day to surface waters around
sunset, returning to deep waters at sunrise, a behaviour typical of organisms
of the “deep scattering layer.”

1.5.2.7 Unranked Clade Neoteleostei

Neoteleostei (Figure 1.8) comprise the superorders Ateleopodomorpha
(Ateleopodiformes: jellynose fishes), Cyclosquamata (Aulopiformes:
lizardfishes), Scopelomorpha (Myctophiformes: lanternfishes),
Lamprimorpha (Lampriformes: opahs), Paracanthopterygii (five orders),
and Acanthopterygii (the many spiny-finned fishes). The last three clades
belong to the Acanthomorpha (spiny-rayed fishes discussed later). The
clade Neoteleostei is strongly supported by numerous molecular studies
(Davis 2010; Betancur-R. et al. 2013; Hughes et al. 2018) and is diagnosed
by the presence of a retractor dorsalis muscle (RAB) (also found in
stomiiforms), type 4 tooth attachment, insertion of the third levator on the
fifth upper pharyngeal toothplate, and presence of a transverse epibranchial
(Wiley and Johnson 2010).
 FIGURE 1.8 Phylogeny of Neoteleostei. Included as early branches are the jellynoses,
lizardfishes, and lanternfishes, the last being sister to the Acanthomorpha (spiny-rayed
fishes). Triangle sizes are correlated to species diversity.

Several interesting groups of fishes branch from the stem of the

neoteleost tree (Figure 1.8). The Ateleopodiformes (jellynoses) of
circumtropical seas are a small group of deep-water bottom dwellers.
Although they are teleosts, their skeletons are mostly cartilaginous, and
their bulbous snouts are filled with a jelly-like substance of unknown
function.
The order Aulopiformes (lizardfishes) includes about 260 mostly benthic
species, many of which are hermaphroditic. Extinct forms were very
diverse and important in Mesozoic Era seas (Davis and Fielitz 2010).
Telescopefishes (family Giganturidae) are two species known for their
bizarre tubular eyes with large lenses. They undergo an amazing
developmental transformation in which the larval stage is so different that it
was described as a completely separate fish, Rosaura rotunda, in the family
Rosauridae (Konstantinidis and Johnson 2016).
The order Myctophiformes (lanternfishes), of two families, about 36
genera, and at least 2534 species, is an essential part of the marine
ecosystem (Bray 2019). It accounts for more than half of all deep-sea
biomass and is the main component of the “deep scattering layer.” The
phylogeny of lanternfishes was studied by Martin et al. (2018).

1.5.2.8 Unranked Clade Acanthomorpha (Spiny-Rayed Fishes)

Acanthomorpha (Figure 1.8) have undergone an explosion of
morphological diversity and body plans since the mid-Cretaceous, 100
million years ago (Friedman 2010). The group was originally recognized by
Rosen (1973) to include teleosts with true spines in the dorsal, anal, and
pelvic fins. The group has since been supported as monophyletic by both
morphological and molecular studies (e.g., Wiley et al. 2000; Chen et al.
2014; Hughes et al. 2018).
1.5.2.8.1 Superorder Lamprimorpha
The sole order Lampriformes, sister to all other acanthomorphs (Figure
1.8), contains six families and about 22 species. These fishes have one of
two body types, the deep-bodied members (Veliferidae and Lampridae) and
the long, ribbon-bodied members (Lophotidae, Radiicephalidae,
Trachipteridae, and Regalecidae). The amazing Regalecidae (oarfishes) are
scaleless fishes with no anal fin and elongated pelvics that are reduced to
only one ray. The extremely long dorsal fin has 260–412 rays. Regalecus
glesne reaches a length of about 8 meters, the longest of all bony fishes, and
dead oarfish are sometimes mistaken for sea serpents. For relationships
within this group, see Olney et al. (1993) and Davesne et al. (2014).
1.5.2.8.2 Superorder Paracanthopterygii
The superorder Paracanthopterygii (Figure 1.8) was first proposed by
Greenwood et al. (1966) as a morphologically equivalent group to
Acanthopterygii. Currently included within Paracanthopterygii are
Polymixiiformes (beardfishes), Percopsiformes (trout-perches), Gadiformes
(cods), Stylephorus, and Zeiformes (dories) (Miya et al. 2007; Grande et al.
2013; Borden et al. 2013, 2019; Davesne et al. 2014; Hughes et al. 2018).
We follow Nelson et al. (2016) and Borden et al. (2019) in placing
Polymixia (beardfishes) as sister to all other members, and percopsiforms as
sister to the clade Zeiformes + [Stylephorus + Gadiformes]. See Tyler et al.
(2003) and Grande et al. (2018) for relationships among Zeiformes and
Borden et al. (2019) for relationships within Polymixiiformes.
The Gadiformes are of particular interest and include over 600 species. A
recent molecular phylogenetic analysis was done by Roa-Varón and Ortí
(2009). Examples of gadiforms include Steindachneriidae (luminous hakes),
Macrouridae (rattails), and Moridae (deep-sea cods) in the Antarctic,
Arctic, North Pacific, and North Atlantic oceans (Cohen 1984; Dunn 1989;
Borden et al. 2013). The circumpolar Lota lota (burbot) is the only one that
inhabits fresh waters. The cold-water cods are of immense historical
importance and still contribute over one-quarter of the world’s marine fish
catch; however, the Atlantic cod Gadus morhua has been over-fished in the
Western North Atlantic almost to extinction, and a cod-fishing moratorium
has now lasted over 20 years. Cod populations, now challenged by global
climate change and warming ocean waters, are projected to move farther
north in coming decades (Morley et al. 2018).
1.5.2.8.3 Superorder Acanthopterygii
Acanthopterygii (Figures 1.8 and 1.9) comprise all remaining fish species.
Their diversity in terms of morphology, ecology, physiology, and
distribution patterns is greater than that of any other group. At least 15,000
species are recognized (Nelson et al. 2016), divided among 34 orders, 287
or more families, and at least 2433 genera. About a quarter of these fishes
inhabit fresh waters.
 FIGURE 1.9 Phylogeny of higher Percomorpha. Scombrimorpharia (seahorses and
tunas) are sister to Ovalentaria (e.g., cichlids, killifishes, livebearers), Carangaria (e.g.,
jacks, remoras, barracudas, flatfishes), and Eupercaria (e.g., wrasses, perches,
butterflyfishes, scorpionfishes, anglerfishes, boxfishes, puffers, molas). Triangle sizes
are correlated to species diversity.

Three orders are early-branching clades (Figure 1.8): (1) Beryciformes

(e.g., Rondeletiidae, whalefishes; Berycidae, alfonsinos); (2)
Trachichthyiformes (e.g., Anomalopidae, flashlight fishes, which have
luminous organs with symbiotic bacteria beneath the eye; Monocentridae,
the heavily armored pinecone fishes; Trachichthyidae, including the slow-
growing and easily over-fished orange roughy); and (3) Holocentriformes
(squirrelfishes). These orders are sometimes grouped as series Berycida
(Nelson et al. 2016).

1.5.2.9 Series Percomorpha

Percomorphs are the most diverse and derived fishes within
Acanthopterygii. Here, we divide percomorphs into groups whose sequence
of taxa suggests phylogenetic hierarchy. However, the relationships are far
from settled. Groups recognized here (Figures 1.8 and 1.9) are Ophidiida,
Batrachoidida, Gobiida, Scombrimorpharia, Ovalentaria, Carangaria, and
Eupercaria (Betancur-R. et al. 2013; Nelson et al. 2016; Hughes et al.
2018).
1.5.2.9.1 Subseries Ophidiida
This sistergroup to all other Percomorpha (Figure 1.8) contains the cusk-
eels (Ophidiiae), pearlfishes (Carapidae), viviparous brotulas (Bythitidae),
blind cusk-eels (Aphyonidae), and false brotulas (Parabrotulidae).
1.5.2.9.2 Subseries Batrachoidida
This group contains the toadfishes in the single family Batrachoididae.
Most are drab and can survive out of water for several hours. Males make
nests or dig dens and then produce humming sounds with their swimbladder
to attract females, which attach their eggs to the walls of the nest to be
defended by the male. Their sound-producing “superfast” swimbladder
muscles can operate at over 200 Hz (Rome 2006). Members of the
subfamily Thalassophryninae have hollow dorsal and opercular spines
connected to venom glands.
1.5.2.9.3 Subseries Gobiida
The Gobiida (Figure 1.8) contain two orders; Kurtiformes (nurseryfishes
and cardinalfishes) and Gobiiformes (gobies). Male nurseryfishes have
occipital hooks for carrying eggs on the head (Berra and Neira 2003). The
cardinalfishes are mouth-brooders; a few have luminous organs. The gobies
number over 2000 species, including freshwater, cave-dwelling, and marine
forms with diverse ecological and life-history traits.
1.5.2.9.4 Subseries Scombrimorpharia
This group (Figure 1.9) includes Syngnathiformes (seamoths, pipefishes,
and seahorses), Scombriformes (tunas, mackerels), and three other orders
(Nelson et al. 2016). Evidence for grouping these orders is accumulating
from molecular sequence studies (e.g., Near et al. 2012; Wainwright et al.
2012; Betancur-R. et al. 2013; Hughes et al. 2018), but the full membership
of this clade has not been settled, and its unifying novelties remain
unknown.
1.5.2.9.5 Subseries Ovalentaria
This diverse group (Figure 1.9), named by Smith and Near in Wainwright et
al. (2012), includes Mugiliformes (mullets), Cichliformes (cichlids),
Blenniiformes (blennies), Gobiesociformes (clingfishes), Atheriniformes
(silversides), Beloniformes (needlefishes, flyingfishes, halfbeaks), and
Cyprinodontiformes (killifishes and livebearers). These fishes are mostly
small and have sticky demersal eggs. Reproductive strategies include
substrate brooding, mouth brooding, and live bearing. The Japanese ricefish
Oryzias latipes (medaka) is used in reproductive biology and toxicology.
Nothobranchius furzeri (turquoise killifish) is used in studies of ageing
because of its very short lifespan (Platzer and Englert 2016). Fundulus
heteroclitus (mummichog) is used extensively in genomic, osmoregulatory,
and toxicological studies (Reid and Whitehead 2016; Reid et al. 2017).
Cichlids (about 1700 species), so familiar to aquarists, behaviourists, and
evolutionary biologists, employ mouth brooding and provide notable
examples of adaptive radiations, rapid evolution, and species flocks,
especially in African rift lakes.
1.5.2.9.6 Subseries Carangaria
This sistergroup to Ovalentaria was suggested by Near et al. (2012),
Wainwright et al. (2012), and Betancur-R. et al. (2013). Its members
(Figure 1.9) include Synbranchiformes (swamp eels), Anabantiformes
(labyrinth fishes, gouramies, fighting fishes), Carangiformes (jacks,
dolphinfishes, remoras), Istiophoriformes (barracudas, billfishes,
swordfishes), and Pleuronectiformes (flatfishes).
1.5.2.9.7 Subseries Eupercaria
The sister to the preceding two orders contains all remaining fish diversity.
Included are Labriformes (wrasses and parrotfishes), Perciformes (perches
and darters, freshwater sunfishes, sea basses, butterflyfishes and
angelfishes, snappers, icefishes), Scorpaeniformes (scorpionfishes,
searobins, eelpouts, sticklebacks, sculpins), Acanthuriformes (drums,
surgeon fishes), Caproiformes (boarfishes), Lophiiformes (anglerfishes),
and Tetraodontiformes (puffers, boxfishes, triggerfishes, filefishes,
porcupinefishes, and molas).
Notothenioids (icefishes) are famous for their antifreeze glycoproteins
and their lack of glomeruli, red blood cells, and hemoglobin, allowing many
of them to survive sub-zero (˚C) temperatures in Antarctic waters (Eastman
2004).
Of particular interest are the scorpionfishes (Scorpaeniformes),
exhibiting many novelties, including armor, spines, and venom as anti-
predator defenses. They include some of the most deadly marine fishes
(Smith et al. 2016). The reef stonefish Synanceia verrucosa, for example,
has 13 dorsal-fin spines, each of which has two venom sacs filled with a
neurotoxin that can cause shock, paralysis, and tissue death (Smith et al.
2016). A new phylogeny by Smith et al. (2018) revises ideas about the
evolution of viviparity via a transition from external to internal fertilization,
with retention of developing eggs or embryos within the female.
Sticklebacks of the suborder Gasterosteoidei are important model
organisms for developmental and behavioural biologists and for studying
rapid evolution in changing environments (Ahmed et al. 2017).
Lophiiformes (anglerfishes) have large mouths, macrophagous habits,
and a cephalic spinous dorsal fin modified into a lure to attract prey. A
possible adaptation in some ceratioids to rare deep-sea encounters between
small males and larger females is extreme reduction in the size of males,
which become embedded as sexual attachments on the body of the female
(Pietsch 2005).
Finally, the incredible Tetraodontiformes (pufferfishes, boxfishes,
triggerfishes) have over 435 species, only 14 restricted to fresh water. They
were reviewed by Santini and Tyler (2003), and molecular phylogenetic
studies were done by Holcroft et al. (2008). The pufferfish genus Takifugu
has an extremely small genome and is a model organism for genomics.
Perhaps the most iconic tetraodontiform is the large-bodied ocean sunfish,
Mola mola (e.g., Nelson et al. 2016).

1.6 CONCLUSION
The survey presented here of the phylogeny and diversity of fishes is a
small fraction of what is known about these fascinating aquatic animals.
The bibliography included with Nelson et al. (2016) contains a wealth of
resources for learning more, planning comparative studies, and accessing
specialist literature. Our understanding of the fish tree of life has improved
rapidly in the last few decades, so that we are now more confident than ever
about the relationships presented here. Nevertheless, much remains to be
learned about the ecological, physiological, developmental, genetic,
morphological, and behavioural traits that have allowed these remarkable
animals to achieve such large numbers of species and often large
populations of individuals.

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 2 Locomotion and Biomechanics
Emily Standen

CONTENTS
2.1 History of Fish Locomotion
2.1.1 Classification of Swimming
2.1.2 Body Caudal Fin Locomotion
2.1.3 Median and Paired Fin Locomotion
2.1.4 Gait Changes
2.2 Complexity of Fish Forces
2.2.1 General Biomechanics: Force, Power and Thrust
2.2.2 A Little about Muscle: Motor, Spring or Break?
2.2.3 Muscle Anatomy
2.2.4 Diversity of Fin Anatomy and Structure
2.3 Muscle Activity and Neurocontrol
2.3.1 Muscle Activity
2.3.2 BCF Swimming
2.3.3 Labriform Locomotion
2.3.4 Unsteady Swimming
2.3.5 Escape Response
2.3.6 Swimming in Unsteady Flow
2.3.7 Neuro Control
2.4 Amphibious Locomotion in Fishes
2.4.1 Diversity of Terrestrial Locomotion
2.5 Conclusion
References

2.1 HISTORY OF FISH LOCOMOTION

2.1.1 CLASSIFICATION OF SWIMMING
Since Aristotle, the movement of fishes has fascinated scientists. Fish
movements are fast, and the first classifications of fish swimming, made
without high-speed video, are often criticized as being over generalized
(Figure 2.1; Breder 1926; Webb 1975; Lindsey 1978). Although these early
classifications are valuable as a general guide to the diversity of fish
swimming styles, as technology improves and we study more animals, we
find the distinction between groups becoming blurred.

FIGURE 2.1 Diversity of fish swimming. (a) Spectrum of swimming types: a –

anguilliform, b – subcarangiform, c – carangiform, d – ostraciiform, e – thunniform, f –
rajiform, g – diodontiform, h – mobuliform, i – labriform, j – amiiform, k –
gymnotiform, l – balistiform, m – tetraodontiform. Red dashed line denotes the fin or
body portion used in the stereotypical form of each locomotor mode. (b) Fish
swimming kinematic parameters. Outline of Polypterus senegalus swimming at two
points in a locomotor cycle. Tail amplitude (red), is measured from tail tip at its most
lateral position to the fish’s midline (approximated by dashed grey line). Body
 wavelength (λ) is the distance between two body wave peaks. Specific wavelength is
body wavelength divided by total body length (green).
(Panel A modified from Lindsey, C. C., in Fish Physiology, Academic Press, 1978, pp.
1–100.)

Fishes exhibit two broad categories of locomotion: body caudal fin

(BCF) locomotion and median-paired fin (MPF) locomotion (Breder 1926).
In the former, waves pass along the fish’s body, while in the latter, median
(dorsal, anal) and paired (usually pectoral) fin motion provide propulsion.
Fin or body motions can be oscillatory or undulatory as defined by the
motion’s resultant wave amplitude, wave speed and specific wavelength
(body wave length/body length [BL]) (Figure 2.1).
2.1.2 BODY CAUDAL FIN LOCOMOTION
Despite large species variation, generally, body wave amplitude is smallest
at the fish nose and increases posteriorly to a maximum at the tail tip (Gray
1933; Gillis 1996). For most species, the body wave moves at a constant (or
slightly accelerating) velocity towards the tail (Gillis 1997), and the specific
wavelength changes between species depending on their body length,
stiffness and general shape. Although the functional significance of specific
wavelength is debated, it is used to help define sub-types of BCF
locomotion (calculated as wavelength/body length) (Figure 2.1; Webb
1975).
Anguilliform locomotion, used by long, thin fishes, usually has a specific
wavelength of less than one (meaning that more than one wave is on the
body at a time) with a relatively large lateral amplitude along most of the
body (max 0.3–0.4 BL at the tail; e.g. Anguilla anguilla; Lindsey 1978).
Subcarangiform swimmers can have a specific wavelength of less than
one; however, body waves occur along the posterior half or third of the
body, increasing towards the tail (e.g. rainbow trout – Onchorynchus
mykiss; Webb 1988; Bainbridge 1963; Webb 1992). Carangiform fishes
usually have a specific wavelength greater than one, with waves confined to
the posterior third of the fish (e.g. mackerel – Scomber scombrus; Lindsey
1978). Thunniform swimmers (named after the Thunnus) have a specific
wavelength of 1–2 BL. This relatively long propulsive wavelength is likely
due to the specialized morphology of these fishes. Since they have a rigid
vertebral column with myotomes (groups of muscle fibers) that insert
across a larger number of vertebrae, they are less able to bend locally,
thereby increasing specific wavelength (Bainbridge 1958; Webb et al. 1984;
Dewar and Graham 1994). Ostraciform swimmers, named after the
boxfishes, tend to be armored anterior to the caudal fin, and so often only
the caudal fin undulates (Lindsey 1978). These fish can use synchronous
movements of their pectoral, dorsal, anal and caudal fins depending on their
speed (Hove et al. 2001). For example, pectoral and anal fins power
swimming at low speeds (<1 BL/s), anal and dorsal fins power swimming
between 1 and 5 BL/s, and a caudal, kick-and-glide gait is used above 5
BL/s.
Many of the variables used to describe and quantify swimming are in two
dimensions. Three-dimensional data on fishes is complex, and although
relatively easy to acquire with modern high-speed video, remains difficult
to quantify meaningfully. Recent work shows that the three-dimensional (3-
D) motion or twist of fishes may also be important, particularly in elongate
fishes (Donatelli et al. 2017). Computational fluid dynamists who integrate
the 3-D shape and motion of fins and body to describe predicted flow
patterns produced by swimming fishes are showing that a 3-D approach to
fish swimming is essential to determine how unsteady movements influence
fish locomotion (Ming et al. 2018; Liu et al. 2017). While the body and/or
caudal fin are likely responsible for producing the majority of the thrust
during BCF swimming, the fins are under active muscle control to help
stabilize against perturbations that might otherwise cause roll, tilt or yaw
(Standen and Lauder 2007; Drucker and Lauder 2005; Maia et al. 2017;
Tytell et al. 2008).
2.1.3 MEDIAN AND PAIRED FIN LOCOMOTION
There is a diverse spectrum of median and paired fin (MPF) use, from
undulatory motion (passing a wave along the fin surface) to oscillatory
motion (rowing the fin back and forth). Fin motions often change
depending on swimming speed, and individuals can switch between
pectoral fin, median fin, and body and caudal fin use as required. MPF
swimming modes are generally classified by the group of animals that tend
to demonstrate the behaviour.
 Labriform locomotion is powered by oscillatory pectoral fin flapping
(Figure 2.2) and can be drag-based or lift-based (Walker and Westneat
2002b; Drucker and Jensen 1997; Bellwood and Wainwright 2001). The
drag-based form consists of a power stroke followed by a recovery stroke
and is used across a narrow range of swim speeds by a variety of fishes,
from short-bodied Centrarchidae (Gibb et al. 1994) and Cichlidae (Blake
1979) to long-bodied Nototheniidae (Archer and Johnston 1989). During
the power stroke, fins adduct through the water with their maximum surface
area perpendicular to the axis of propulsion. During recovery, fins rotate
and abduct with the maximum surface area parallel to the direction of travel
to minimize drag. In contrast, lift-based labriform swimming orients the fin
parallel to the flow and oscillates it in a dorso-ventral motion to create lift
(Figure 2.2; Drucker and Jensen 1996a, 1996b; Webb 1993). Labridae
(Westneat 1996; Walker and Westneat 2002a) and perciform fishes (i.e.
Embiotocidae; (Webb and Weihs 1994; Drucker and Jensen 1997) use this
form of motion; however, drag and lift-based modes are often used in
conjunction with each other depending on the needs of the animal (Webb
1973; Westneat et al. 2017). The oddly shaped pectoral fins of skates and
rays can also oscillate in what looks like underwater flapping flight
(Mobuliform locomotion; Fish et al. 2018, 2016). Dorsal and anal fins can
also be used in an oscillatory fashion (Tetraodontiform locomotion), as
seen in the pufferfishes that flap their dorsal and anal fins from side to side
in concert to power locomotion.

FIGURE 2.2 Pectoral fin swimming. Labriform swimming. (a) Lift-based, dorso-
ventral fin motion acts like a flapping foil to produce lift and thrust. (b) Drag-based: fin
 adduction during the power stroke creates thrust (steps 1–3); during the recovery stroke,
the fin is abducted parallel to the flow, minimizing drag (steps 3–5). (c) Rajiform
pectoral fin undulation passes a wave anterior to posterior along the fin edge.
(Panels a and b modified from Walker and Westneat 2002a.)

Undulatory fin motion involves passing waves along the surface of the
median fins (Gymnotiform – anal fin, Amiiform – dorsal fin, and
Balistiform – both anal and dorsal fins) or the pectoral fins (Rajiform and
Diodontiform locomotion)(Figure 2.1; Blake 1978; 1983, Jagnandan et al.
2014). Generally, during these forms of steady swimming, the body is
straight, and additional fins are not used or are entirely absent. One notable
characteristic of this form of swimming is the ability to swim both forward
and backward with fluidity (Youngerman et al. 2014). Many species
combine oscillatory and undulatory fin movements, the locomotion style
correlating with the ecology and morphology of the fish (Rosenberger
2001).
2.1.4 GAIT CHANGES
A fish may exhibit one type of swimming at slow speeds and transition into
another at higher speeds (Hale 2006; Drucker and Jensen 1996a). For
example, the bluegill sunfish oscillates its pectoral fins at speeds under 1.0
BL/s and changes to BCF swimming at higher speeds (Standen and Lauder
2005). In fact, most fishes (especially BCF swimmers) will use the “kick-
and-glide” mode of locomotion when swimming at very high speeds. This
behavior is composed of short bursts of caudal fin oscillation interspersed
with glides where the body is straight, presumably to reduce drag (Jayne
and Lauder 1996; Hove et al. 2001). At high swimming speeds, this gait
appears to be more energy efficient than other modes of steady swimming
(Wu et al. 2007). Different swimming modes may also be more or less
efficient depending on the environment. For example, in an experiment
where fishes were exposed to water flows that mimic coral reef wave-
surges, it appeared that MPF swimmers were better able to reduce costs
associated with increased wave surge frequency compared with BCF
swimmers (Marcoux and Korsmeyer 2019).

2.2 COMPLEXITY OF FISH FORCES

2.2.1 GENERAL BIOMECHANICS: FORCE, POWER AND THRUST
Before understanding how fishes produce forces and the consequences of
those forces, it is important to clarify the basic relationship between force,
work and power. Force is the product of an object’s mass and its
acceleration (F = ma with units kgm/s2 or N); therefore, the size and
acceleration of an animal will determine the amount of force it must
produce. The amount of work an animal performs, which may be
considered a more important evolutionary metric, is the force produced
times the distance travelled; W = F*d with units kgm2/s2 or J. Finally,
power is the amount of work done per unit time; P = W/t with units J/s or
W. From an organismal perspective, you can consider that an animal
experiences mechanical forces from the environment and produces
biological forces with its muscles. Muscles can produce positive, negative
or no work depending on the resultant motion of the muscles and/or animal,
and finally, the speed with which the animal can perform the work will
determine the power it can produce. Using a whole animal as an example, a
fish accelerating from zero velocity in still water produces a force with its
tail (mass × acceleration). The distance it travels with that amount of force
determines the work it produces (F × d). The time it takes for it to cover that
distance or complete the work tells you the power it took (W/t). This type of
motion analysis can also be done at the muscle level, thinking about how
far and how fast myosin and actin move relative to each other while
producing force. With force, work and power concepts firmly in our minds,
we can now discuss how fishes produce forces and what it means in
evolutionary terms from the tissue level to the whole organism.
2.2.2 A LITTLE ABOUT MUSCLE: MOTOR, SPRING OR BREAK?
All animals have excitable muscle tissue. Muscle cells are specialized with
carefully organized myosin and actin filaments aligned such that they can
move relative to one another within the cell to produce forces. The
combination of fiber types and arrangement (muscle fiber architecture) on
the skeleton impacts muscle fiber performance, dictating how fast they can
contract, how long they can sustain a contraction, and where the force is
applied on the animal’s skeleton. Although all muscle contractions involve
the action of myosin and actin, muscles can act as motors (shortening
during activation, concentric contraction), brakes (elongating during
activation, eccentric contraction) or struts (remaining the same length
during activation, isometric contraction (Figure 2.3; for a general
biomechanics review see Vogel 2003; Dickinson et al. 2000). During each
of these phases, the myosin and actin are busy ratcheting relative to one
another and producing forces within the muscle cells, but the resistance of
the external environment dictates the resultant muscle movement.
Interestingly, the physiological performance, in this case measured as how
much force a muscle can produce, changes depending upon the length of
the muscle fiber; whether it is shortening or lengthening during activation
and whether it is stretched prior to activation (Figure 2.3). The complexities
of muscle performance are still being clarified, but it is important to keep
this variable performance in mind when considering how fishes power
swimming.
 FIGURE 2.3 Muscle functional diversity. Work loops (a, b and c plots) visualize
muscle length change and force production during cyclical activities. (a) Positive work
is produced by the pectoralis muscle during flight. (b) Leg muscles act as brakes in
running cockroaches. (c) The turkey gastrocnemius changes function between uphill
and level ground running. (d) Fish body muscle can act as a motor or a strut, depending
on cycle timing. Body muscle actively bends the fish (motor) or actively resists
bending, permitting the transmission of force to more posterior positions on the fish
(strut). (e) The amount of force a muscle can produce depends on its length and the
optimal overlap of myosin and actin fibers (highlighted in red). (f) Muscle force
production and power output vary with contraction velocity. The faster the contraction
speed, the less force is produced, since actin and myosin cannot ratchet properly. Force
continues to increase at negative velocities (active muscle lengthening). Power, the
product of force and velocity, peaks near the middle of the force–velocity curve. Force
in red, power in purple.
(Panels a–d modified from Dickinson, M. H. et al., Science, 288, 100–106, 2000).

2.2.3 MUSCLE ANATOMY

How the muscle fibers attach to the skeleton is also exceptional in fishes
and leads to an interesting passing of forces along the body. Fish body
musculature is divided by muscle fiber type; the red muscle is located in a
very small band along the side of the fish, and it is used for routine,
continuous swimming (Hickman et al. 2018); Figures 2.4 and 2.5). The bulk
of the body is white muscle, used for powerful maneuvers, burst swimming
or escape response (Syme 2006), and consists of a series of nested units.
Each unit is called a myomere, and they attach intricately with one another
in a V or W nested cone configuration (Figure 2.4). A series of inter-
myomere tendons and fascial sheets allow the contracting muscle to pass
forces between the spinal vertebrae and the fish skin, as well as along the
cones of the myomeres themselves, resulting in body stiffening and passing
forces to augment tail motion (Gemballa and Vogel 2002; Gemballa et al.
2003). The unique structure of the myomeres in fishes, as well as the curved
trajectories of the individual muscle fibers within the myomeres, has been
explained via the uniform strain hypothesis, which suggests that this
orientation allows all muscle fibers of a given type to contribute equally to
force production during swimming (Alexander 1969). Because a swimming
fish is essentially a bending beam, and because beam theory states that
forces are greater the further you are from the neutral axis of the beam, if
fish muscle ran parallel with the spine, the fibers located furthest from the
spine would provide the most force, and those close to the spine would
provide none. The uniform strain hypothesis states that for a fish to
effectively use all of its muscle fibers, they must alter their orientation with
respect to the spine. Subsequent modelling supports this idea by accurately
predicting the trajectories seen in fish muscle fibers based on achieving
mechanical stability of the fish (van Leeuwen 1999).
FIGURE 2.4 Fish muscle anatomy. (A) The arrangement of myomeres in a
gnathostome. (B) Diagram showing how myomeres (pink) and myosepta (grey) nestle
into one another. Tendons as part of the myosepta (grey) are indicated: epineural tendon
(ENT), epipleural tendon (EPT), myorhabdoid tendons (MT) and lateral bands/tendons
(LT). The attachment line of the myosepta to the skin is indicated by the red line.
(Tendon alignment adapted from Gemballa, S., et al., Journal of Evolutionary Biology,
16, 966–975, 2003; trout myomere image adapted from Hickman, C. L., et al., 2018.
Laboratory Studies in Animal Diversity, 8th ed, McGraw-Hill Publishing, 2018.)
 FIGURE 2.5 Muscle recruitment during swimming. Red muscle is active along the
length of a fish at all swim speeds and increases contraction frequency with speed.
White muscle is recruited at higher swimming speeds.
(Modified from Moyes, C.D. and Schulte, P.M., Principles of Animal Physiology, 2nd
ed., Pearson, 2016.)

The anatomy of the fish musculature, in combination with the tuning

capabilities of the muscle fibers themselves (white, red and intermediate
fibers; for review see Syme 2006), allows fishes to engage all of their
muscle mass for powerful burst swimming or only part of their muscle mass
for moderate endurance swimming (Moyes and Schulte 2016; Figure 2.5).
White and red fibers are more heterogeneously mixed in fin muscles;
however, there is often a general pattern of centrally located aerobic fibers
with surrounding white fibers (Du and Standen 2017). The functional
implication of fiber regionalization in fins is not clear.
2.2.4 DIVERSITY OF FIN ANATOMY AND STRUCTURE
Most fishes have two sets of paired fins, the pectoral and the pelvic fins,
and three median fins, the dorsal, anal and caudal fin (Figure 2.1). During
the evolution of fishes, immense variation in fin number, shape and size has
occurred. Many of the functions of ornate and elongate fins are for purposes
other than locomotion, such as sexual selection (i.e. swordtails) or crypsis
(i.e. sea dragons), and there is often a tradeoff between alternate functions
and locomotion. Among the fins selected for locomotory performance, there
are many shapes, sizes and levels of stiffness, and each is used in a different
style of locomotion (Lauder and Drucker 2004). Although there are
differences in the degree of fin element stiffness or fusion, they are all
controlled in a similar way. Fins are composed of segmented boney
elements, or fin rays, that are joined together by a flexible membrane
(Figure 2.6). Each fin ray, termed a lepidotrichia, is made up of two
hemitrichia. These bones are joined by elastic elements, which allows them
to slide relative to one another and facilitates bending of the fin surface
(Videler and Geerlink 1986; Alben et al. 2007). A set of muscles at the base
of each fin ray controls the motion of the ray, moving it relative to the body
but also moving hemitrichia relative to each other, allowing the fin to bend.
In general, fish fins are incredibly deformable surfaces that are highly
controlled. Fishes can increase or decrease fin surface area depending on
the required force production and function. Fins are critical for stabilization
and destabilization during locomotion and maneuvering.

FIGURE 2.6 Fish fin rays are under active muscle control to change fin shape. (a) Fin
rays are segmented and branch near the tip, as can be seen in a cleared and stained fin.
At rest (b), the two hemitrichia align, and the lepidotrichia is straight. (c) Under unequal
muscle tension, the hemitrichia slide relative to each other, thereby bending the ray.
(Modified from Alben, S., et al. Journal of The Royal Society Interface, 4, 243–256,
2007.)

2.3 MUSCLE ACTIVITY AND NEUROCONTROL

2.3.1 MUSCLE ACTIVITY
Swimming speed increases with tailbeat frequency, driven by an increase in
muscle recruitment and an increase in the frequency of the muscle
activation wave that passes along the body (Figure 2.5; Coughlin 2002).
The timing of muscle strain cycles (the lengthening and shortening of
muscle fibers) relative to muscle activity is essential to the functional
performance of a muscle during locomotion (Altringham et al. 1993;
Altringham and Ellerby 1999; Rome et al. 1993; Curtin and Woledge 1996).
The state of the muscle fiber (its length and motion) before activation will
influence the amount of force it can produce (Figure 2.3). Thus, the
difference in timing of muscle activation within a strain cycle (i.e. the phase
lag) is critical and varies both with species and along the length of a fish
(Grillner and Kashin 1976; Williams et al. 1989; van Leeuwen et al. 1990;
Wardle et al. 1995). At low speed, locomotion is powered exclusively by
red muscle, and white muscle is progressively recruited as fish swim faster
(Figure 2.5; Rome et al. 1993).
2.3.2 BCF SWIMMING
How red muscle is used during different swimming modes is still debated.
The larger-amplitude wave that is found all along the body in anguilliform
swimmers appears to be accompanied by more uniform head to tail muscle
activity patterns (Wardle et al. 1995). In contrast, the anterior body muscle
contractions of non-anguilliform swimmers do not accompany an anterior
body wave; they produce forces that pass via stiffened muscle and tendon to
the posterior body wave and tail to be subsequently turned into thrust
(Wardle et al. 1995; Altringham and Ellerby 1999). The suggested
functional outcome of this, which is supported by the hydrodynamic forces
produced in the wake of fishes (Lauder and Tytell 2006), is that
anguilliform swimmers produce power and thrust along the entire length of
their bodies.
The passing of muscle effort posteriorly in non-anguilliform swimmers
results in differences in muscle timing and behavior along the fish length
that are much more noticeable. For example, as you approach the tail of the
fish, there is a decrease in the duty cycle, the amount of time the muscle is
on during a tailbeat (anguilliform swimmers have a constant duty cycle
along their length; Gillis 1998). The amount by which a muscle shortens
(strain) and the resultant body amplitude increase towards the tail. The
phase lag in timing between muscle activation and resultant body amplitude
also increases towards the tail (posterior muscles come on earlier relative to
peak wave amplitude) (Figure 2.7; Altringham and Ellerby 1999; Rome et
al. 1993; Shadwick et al. 1998). In essence, the wave of muscle activity
passes anterior to posterior faster than the wave of body deflection. This is
taken to an extreme in tunniform swimmers, where the wave of muscle
activity passes along the anterior of a tuna so quickly that it is near
synchronous, straightening and stiffening the anterior body and passing all
of its force to caudal thrust production (Shadwick et al. 1999). Changes in
muscle function along the fish body can be related to swimming speed as
well as the ecology of the fish (Coughlin 2002).

FIGURE 2.7 Phase lag between muscle activation and body kinematic event.
Maximum body amplitude occurs sooner following muscle activation at the fish’s
 anterior than at its posterior. Muscle position is denoted as a blue dot on the fish outline.

2.3.3 LABRIFORM LOCOMOTION

Much less work has been done on the muscle activation patterns in fin
musculature during swimming. This is partly due to the very small size of
many fin muscles and the difficulty in accurately implanting electrodes
without damaging muscle function. Most work is on the anatomical
function or free-swimming activation patterns in the pectoral fins of
labriform swimmers (Williamson 1893; Danforth 1913; Westneat 1996).
Although the large abductor and adductor muscles tend to be responsible
for the larger-scale fin motions, a variable number of smaller fin muscles
also contribute to the locomotor stroke (Drucker and Jensen 1997; Westneat
and Walker 1997). The gait transition from pectoral fin swimming to BCF
swimming is likely due to aerobic fin muscles reaching a limit in their
power output, necessitating the recruitment of body muscle to swim faster
(Drucker and Jensen 1996a).
2.3.4 UNSTEADY SWIMMING
All the above has been concerned with fishes swimming steadily, but of
course, most fishes do not swim steadily, and most of their behaviour in the
wild is composed of turns, escapes from predators, swimming through
turbulent flow, etc. Limiting studies to steady swimming simplifies the
system and allows basic observations of muscle patterns, kinematics and
neural control; however, understanding the full range of kinematics and
muscle activity that fishes use on a regular basis requires thorough
quantification of unsteady swimming.
2.3.5 ESCAPE RESPONSE
One of the best-studied unsteady behaviours that fishes perform is the fast
start. Fast starts can be initiated either by the reticulospinal neurons (a
slower response) or by the Mauthner neurons (a faster response) to escape
predators and catch prey. This behaviour is composed of three kinematic
stages: preparatory stage 1, propulsive stage 2 and variable stage 3 (Eaton
and Hackett 1984). Stage 1 is generally defined as the formation of the
initial starting position (either a “c” shape or an “S” shape). Stage 2 can be
defined in a variety of ways but generally corresponds to the change in
direction of the anterior body (Kasapi et al. 1993; Domenici and Blake
1991, 1993), the onset of forward propulsion (Foreman and Eaton 1993),
and the onset of contralateral muscle activity (Jayne and Lauder 1993)
(though each of these occur at slightly different times). Stage 3 is highly
variable and loosely defined. Unlike steady swimming, the neural control
that leads to a Mauthner fast start is relatively well established. Stage 1 is
initiated by the triggering of the Mauthner neurons (Sillar 2009), which are
connected to each other via gap junctions, direct cell–cell electrical
connections that allow rapid transmission of neural signals. This
neuroanatomy facilitates extremely rapid initiation of escape responses via
sensory feedback.
2.3.6 SWIMMING IN UNSTEADY FLOW
While it might seem as though swimming through water with increased or
turbulent flow would pose an inherent problem to fishes, in fact, they can
gain some energetic advantage by swimming strategically through vortices
in water. When objects obstruct flowing water, the water bends around the
object and creates vortical flow that sheds in a characteristic pattern (the
von Karman street). A series of experiments that swam fishes in flow
behind a D-cylinder have demonstrated that, rather than fighting against the
structure’s wake, fishes can actually use the regular vortical flow to reduce
their energy expenditure, decreasing muscle activity while holding station
(Liao 2003, 2002). In fact, slaloming between the vortices that form behind
a cylinder is so effective at capturing energy from the flow that even a dead
trout, pointing into the flow, can passively “swim” upstream in the wake of
a structure (Liao 2004).
2.3.7 NEURO CONTROL
Motor neurons send signals to the muscles to coordinate muscle
contraction, which leads to locomotion. The pattern of the motor signals can
be the result of a “top-down” signal from the brain and/or rhythmical
signals from local central pattern generators (CPGs), both of which can be
influenced by sensory feedback experienced by the fish as it swims through
its environment. Our understanding of the importance and control of CPGs
has improved since the initial descriptions that locomotion may be
controlled by the spinal cord (Brown 1914). It is now known that CPGs are
responsible for controlling a variety of behaviours, including breathing,
chewing, swallowing and locomotion (Garcia-Campmany et al. 2010;
Marder and Calabrese 1996). Locomotor CPGs are neural centers found in
the spinal cord that can provide rhythmic motor output to the muscles,
which is tuned via sensory and top-down input from a heterogeneous
environment.
Much of our knowledge of CPG function is from work done on the
lamprey (Grillner 1985). The swimming CPGs of the lamprey are along the
entire length of the spinal cord, and an isolated spinal cord can produce
motor patterns that closely resemble that of an intact lamprey (Cohen and
Wallén 1980). This motor pattern is only created in response to an
excitatory drive (e.g. brainstem stimulation, sensory stimulation or
excitatory amino acids) (Grillner 1985). The presence of CPGs has also
been demonstrated in spiny dogfish (Gray and Sand 1936), stingray
(Leonard et al. 1979), carp (Kashin et al. 1974) the zebrafish (Downes and
Granato 2006; Gabriel et al. 2008). While these CPGs create the intrinsic
rhythm of swimming, they need additional input in order to adjust the fish’s
behaviour appropriately. Usually, the excitatory drive to turn on the CPGs is
provided by the mesencephalic locomotor region (MLR) and passed via the
reticulospinal neurons to the spinal CPGs (Grillner et al. 1997). The MLR
also receives top-down inputs, such as vision or hearing, from the cerebral
cortex (pallium in fishes) (Le Ray et al. 2011; Daghfous et al. 2016). While
these inputs historically are poorly characterized, recent work has shed
some light on the pathway that carries olfactory information to the MLR.
This sensory input appears to be able to influence the MLR output (and thus
the resulting locomotor behaviour) in a graded way, thereby fine-tuning an
animal’s behaviour (Ménard et al. 2007).
Proprioception in fishes has only recently started to become known,
showing clear sensory feedback associated with fin use in fishes (Aiello et
al. 2020).

2.4 AMPHIBIOUS LOCOMOTION IN FISHES

2.4.1 DIVERSITY OF TERRESTRIAL LOCOMOTION
There are over 200 different species of extant amphibious fishes
representing at least 17 genera. The diversity and range of morphological
and behavioural phenotypes found among these fishes are large. There are
many challenges associated with moving from an aquatic to a terrestrial
environment: desiccation, gas exchange, pH balance and disposal of
nitrogenous wastes are just a few (Wright and Turko 2016). Each of these
affects locomotion indirectly by influencing muscle and tissue performance.
Here, we will focus on the impacts a transition from an aquatic to a
terrestrial environment has on the locomotor performance of an animal.
The mechanical environments of water and land are very different.
Viscosity and drag are the major forces experienced by fishes in water,
whereas gravity and friction become the major challenges on land. Fish
body and fin function must be flexible in order to produce appropriate
forces across environments. Different fishes have different strategies when
moving about on land. There are three main categories of fish terrestrial
locomotion (Pace and Gibb 2014, 2009): axial-based locomotion, where
motion is produced entirely by the body (i.e. snake-like locomotion in
elongate ropefish or eel, or jumping locomotion in blennies and rivulus);
appendicular-based locomotion, where motion is derived entirely from the
fin (i.e. mudskipper); and axial-appendicular-based locomotion, where the
animal uses both fins and body to produce thrust (i.e. walking catfish and
Polypterus). Some amphibious fishes have totally different strategies, such
as climbing perch that use modified opercula to walk (Davenport and
Abdulmatin 1990) or the waterfall climbing gobies that use their mouths as
suckers to cling to rockfaces under rushing flow (Blob et al. 2006).
Often, fishes exhibit very different behaviours in one environment
relative to the other (Pace 2017). For example, fin range of motion
increases during walking in both bichir (Polypterus senegalus) and
mudskipper (Periophthalmus argentilineatus); however, body oscillation
increases for bichir and decreases for mudskipper (Figure 2.8; Standen et al.
2016, 2014; Kawano and Blob 2013). These differences between species
illustrate that there are multiple ways to modify strategies for locomotion
across environments.
 FIGURE 2.8 Aquatic vs. terrestrial locomotion. (a) Polypterus senegalus swimming in
water keeps the body relatively straight and uses fins to provide thrust; ventral view. (b)
P. senegalus walking on land uses exaggerated body movements and alternating fin
plants; dorsal view. (c) Periophthalmus argentilineatus swimming in water incorporates
body bending with reduced fin motion; ventral view. (d) P. argentilineatus walking on
land keeps body straight, and fins are planted simultaneously; dorsal view. (Panels a and
b modified from Standen, E. M., et al., Nature, 513 54–58, 2014; c and d modified from
Pace, C. M. and Gibb, A. C., Journal of Experimental Biology, 212, 2279–2286, 2009.)

2.5 CONCLUSION
Fishes and their broad diversity of form and locomotory function are an
excellent example of vertebrate musculoskeletal flexibility. Both from an
evolutionary perspective (many thousands of species) and an individual
performance perspective (the ability to walk and swim) they provide insight
into how to use anatomical form for different and diverse functions. As
technological advances increase our ability to quantify animal motion,
fishes will continue to provide important animal models to understand and
mimic the performance of the natural world.

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 3 Gas Exchange
Jodie L. Rummer and Colin J. Brauner

CONTENTS
3.1 Introduction
3.2 From Environment to Gill Branchial Gas Transfer
3.2.1 Ventilation
3.2.2 Morphology
3.2.3 Diffusion across Membranes
3.2.4 The Osmorespiratory Compromise
3.3 Circulatory Transport of Respiratory Gases
3.3.1 Blood
3.3.1.1 Oxygen
3.3.1.2 Carbon Dioxide
3.3.2 Blood Flow and Perfusion
3.4 Diffusion at the Tissue Level
3.5 Conclusion
Acknowledgements
References

3.1 INTRODUCTION
Oxygen is a prerequisite for life for all fish species with no known
exceptions. Oxygen (O2) uptake from the environment, transport across
respiratory surfaces and through the circulatory system, and ultimately,
delivery to metabolizing tissue, with the reverse for carbon dioxide (CO2),
which is produced in approximately equal amounts, have been topics of
interest for fish physiologists for centuries. Yet, gas exchange is not just
restricted to O2 and CO2. Ammonia (NH3) excretion, also collectively part
of gas exchange, is the key pathway for nitrogenous waste elimination for
both marine and freshwater fishes. Therefore, gas exchange includes O2,
CO2, and NH3 and, at least in most adult fishes, primarily occurs at the
gills. The skin and air-breathing organs can also be used for gas exchange,
depending on life stage and species, and will be discussed briefly here but
elaborated upon in other chapters. As gas exchange in fishes has been
reviewed relatively recently in detail (Evans et al., 2005; Randall et al.,
2014; Harter and Brauner, 2017), only the fundamentals are reviewed here
(focusing on O2 and CO2) along with more recent advances in the field. In
this chapter, we focus on the role of the gill in gas exchange, reviewing
aspects related to ventilation, morphology, contact with the external
environment, diffusion across membranes, blood flow and perfusion, and
diffusion at the tissue level (Figure 3.1). Cellular metabolism, the next
logical step in this cascade, is discussed at both the cellular and the whole-
organism level in Chapter 10: Metabolism. Interactions between gases and
effects on transport will be discussed here. Inter- and intra-specific
differences and physiological and morphological adaptations to stress will
also be examined throughout this chapter, especially with respect to
contemporary issues such as pollution, climate change, and other
anthropogenic disturbances, as these are significant areas for future
research.
 FIGURE 3.1 The respiratory gas transport cascade for O2 and CO2 in a model fish,
where the steps represent: 1, ventilation; 2, gill diffusion; 3, circulation; 4, tissue
diffusion; and 5, cellular metabolism. Flows and gas flux can be calculated at each step
by modifying the Fick equation as shown for O2, where V. W = ventilatory water flow
rate, f = frequency, VS= stroke volume, ṀO2 = oxygen uptake rate, K = Krogh’s
permeation coefficient, A = gill functional surface area, ∆PO2 = O2 partial pressure
gradient, D = diffusion distance, CaO2 and CvO2 = O2 content of arterial and mixed-
venous blood, respectively. See text for further information.

3.2 FROM ENVIRONMENT TO GILL BRANCHIAL GAS

TRANSFER
The external environment and lifestyle of the fish will dictate efficiencies or
inefficiencies in gas exchange, which can be elaborated upon by discussing
each step of the O2 or respiratory gas transport cascade (Figure 3.1).
Oxygen diffuses down its partial pressure gradient from the highest value in
the environment to the tissues. Metabolically produced CO2 is highest in
the tissues and also diffuses down its partial pressure gradient to the
environment for elimination. In both gases, a drop in partial pressure at
each of the five steps of the gas transport cascade represents resistance due
to convection and diffusion, and transport at each level of the gas transport
cascade can be quantified by modifications of the Fick equation (Figure
3.1), as discussed later.
3.2.1 VENTILATION
Most species, as adults, possess four to seven gill arches on each side of the
head that are collectively referred to as the branchial basket. The
developmental timeline for gill formation and, in some species, the external
to internal migration of the structures depend on myriad factors; as well as
species and temperature, other abiotic factors may be influential. Water
must pass over the gills via ventilatory flow rate (V. w), which is largely the
same process among species. For most teleost fishes, ventilation involves
asynchronous buccal (mouth) and opercular (gill cover) pumping to
generate pressure (Hughes, 1960). In contrast, ram-ventilators, such as
some pelagic teleosts and many elasmobranch species (Emery and
Szczepanski, 1986), generate dynamic pressure while swimming, which
drives water over the gills. Some species may transition from buccal
pumping to ram ventilating, with a change in swimming speed and/or
environmental O2 level likely due to the energetic savings (Steffensen,
1985) associated with gill water flow being powered by skeletal muscles
rather than by the buccal pump muscles. Ventilatory flow is determined by
the product of the ventilatory stroke volume (Vs) and breathing frequency
(f) (Figure 3.1, step 1), which can vary by species, developmental stage,
activity, and environmental conditions. For example, the stargazer
(Genyagnus monopterygius) is a successful ambush predator because it can
bury itself in the sand and remain largely undetected by exhibiting
extremely shallow ventilatory movements (i.e., branchiostegal movements)
even when water becomes hypoxic (Forster and Starling, 1982). Red drum
(Sciaenops ocellatus) hyperventilate in response to relatively low levels of
hypercapnia (elevated water CO2; 1000 and 5000 µatm; Ern and Esbaugh,
2016), with many fishes responding to higher CO2 levels (Gilmour, 2001).
Increases in ventilatory flow during exposure to gill irritants, such as
harmful algal blooms or toxicants, are often indicative of a reduction in O2
uptake efficiency in conjunction with an increased metabolic rate (Bradbury
et al., 1989; Svendsen et al., 2018). Moreover, some fishes can even appear
to hold their breath, which may be an adaptation to fend off would-be
predators. The deep-sea coffinfishes (Lophiiformes: Chaunacidae) use their
unique buccal and gill anatomy to slowly ventilate and even cease
ventilatory movements for as long as 245 s (Long and Farina, 2019). This
strategy may save energy, as ventilation can consume as much as 15% of
the oxygen budget of slow-moving fishes (Steffensen, 1985), and may also
increase their body volume by 30%, thus deterring would-be predators.
Once the water flows over the gills, with a ventilatory frequency averaging
from 30 to 70 min−1 for most adult fish species (Roberts, 1975) and as low
as 10 min−1 for newly hatched larvae with poorly developed gills
(McDonald and McMahon, 1977), gases must equilibrate between the water
and the blood. Thus, the structure and function of the gill filaments and
lamellae greatly influence the efficiency of gas exchange.
3.2.2 MORPHOLOGY
From a physical perspective, gill filaments and lamellae can act like a sieve,
being numerous or even fused at the tips to maximize gas exchange with the
water. Indeed, the morphology of the gill filaments and lamellae and how
they contact the external environment have been well studied across
species, lifestyles, habitat, and developmental stages, as histological and
microscopy preparations have been long established. On each filament,
lamellae are comprised of two epithelia separated by pillar cells for
structural integrity and to enable blood flow through the lamellae (Figure
3.1). Lamellae are often very thin, sometimes only one or two cell layers
thick. As a result, the potentially short diffusion distances between the
environment and the blood and the large surface area facilitate effective O2
uptake from the environment and gas exchange in general (Hughes and
Morgan, 1973). For this reason, it was long accepted that fast-swimming
marine fishes (e.g., mackerel, tuna) exhibit high lamellar densities of 26–31
per millimetre of filament and total lamellae approaching six million,
whereas slow swimmers exhibit lower densities (e.g., European sea bass:
21–26 lamellae per millimetre of filament) (Hughes, 1984; Gray, 1954).
However, tropical coral reef fishes exhibit lamellar densities ranging from
28 to 34 lamellae per millimetre of filament and are not necessarily
considered fast-swimming, active species (Bowden et al., 2014). It may be
that species with high O2 needs have the greatest surface areas, which then
would include not only the top performers but also hypoxia-tolerant species
(Chapman et al., 2000). While it was once thought that gill (lamellar)
surface area scaled predictably with body mass (Hughes, 1984) and activity
(Roberts, 1975), that may no longer be the case, as suggested here.
Moreover, while it was once thought that the gill anatomy for adult fishes
was relatively fixed, it is becoming apparent that a great degree of plasticity
can exist in some species and environmental conditions (Nilsson et al.,
2012; Gilmour and Perry, 2018); we elaborate on this later in discussions of
the osmorespiratory compromise. Lamellar spacing along the filament (and
thus, interlamellar distances) was also thought to scale with activity (Piiper
and Scheid, 1982). However, we now know that interlamellar distances are
uniform regardless of fish body mass, indicating optimal interlamellar
distances for maximizing gas exchange (Park et al., 2014).
3.2.3 DIFFUSION ACROSS MEMBRANES
Diffusion across the gills is directly proportional to the functional surface
area of the gills (A) and inversely proportional to the diffusion distance
between the blood and the environment (D; i.e., the diffusion distance;
Figure 3.1, step 2). In addition, water flows across the lamellae, counter-
current to blood flow (Figure 3.1; Randall and Daxboeck, 1984),
maximizing the partial pressure gradient between the environment and the
blood for gas exchange (i.e., ∆PO2 for O2; Figure 3.1, step 2). Indeed,
counter-current flow is so efficient that it enables 80–90% O2 extraction
from the water compared with approximately 25% extraction in humans
breathing air (Schmidt-Nielsen, 1997). It is thought that this combination
helps to overcome the slower diffusion rates (up to 300,000 times slower)
of O2 in water and the lower O2 content and higher viscosity (840 times)
and density (60 times) of water compared with air (Schmidt-Nielsen, 1997).
These three elements – functional surface area, diffusion distance, and the
gas partial pressure gradient between the water and blood – as well as a
fourth, the permeation coefficient (K), collectively dictate the diffusion of
gases (e.g., O2 transfer; ṀO2) across these membranes via the Fick equation
(Figure 3.1, step 2). The permeation coefficient (K) describes the mobility
of the gas in question and takes into consideration the diffusion coefficient
and the solubility of the gas. K varies with temperature; values are available
in the literature. The K for O2 moving through tissues, for example, is
estimated to be approximately one-third the K (i.e., slower) of O2 moving
through water (Randall and Daxboeck, 1984).
Functional surface area (A) is referred to as such because it is unlikely
that the entire surface area of the fish’s gill is used for gas exchange at any
given time. The area can be estimated using morphometric measurements to
sum the areas of all lamellae. However, it must be considered that the area
functioning for gas exchange may be only 70% of the total anatomical area
(Piiper et al., 1986). Not all lamellae will be perfused, especially in resting
fishes (Booth, 1978). Moreover, the bases of the lamellae have greater
diffusion distances, and the tips of the filaments receive reduced water flow
when compared to the rest of the lamellae, making both regions (i.e., base
and tips) minimally involved in gas exchange, if not largely non-respiratory
(Tuurula et al., 1984). The partial pressure gradient of the gas between the
environment and the blood (e.g., ∆PO2) is the driving force for diffusion.
For O2, this can be estimated as the difference between the average of
inspired and expired water PO2 and the average of arterial and venous blood
PO2 (Wood and Perry, 1985), all of which can be measured using
microelectrodes or fibre optic sensors. However, it should be noted that
unstirred layers cannot be accounted for in this calculation; this caveat, in
combination with differential lamellar recruitment and changes in blood
flow, makes estimates of ∆PO2 challenging. Finally, D describes the
diffusion distance or thickness of the tissue separating the environment
from the blood and can also be calculated from morphometric
measurements. It is important to consider that increases in ventilation and
blood pressure, differential dilation of arterioles, and lamellar recruitment
or shunting will alter this relationship. All of these variables may change
with exposure to environmental stress and/or changes in activity.
3.2.4 THE OSMORESPIRATORY COMPROMISE
As discussed, the functional surface area and the gas partial pressure
gradient between the environment and the blood are the primary drivers for
gas exchange, but alterations to increase the efficiency of gas exchange can
come with trade-offs to other gill functions, such as ion- and osmo-
regulation. Maximizing functional surface area while enhancing gas
diffusion promotes ion loss and water influx for freshwater teleosts, and ion
gain and water efflux for marine teleosts. For example, rainbow trout lose
Na+ to the environment with increases in ṀO2 (Gonzalez and McDonald,
1992); calculations suggest that Na+ efflux can increase by 70% in
freshwater rainbow trout during exercise (Wood and Randall, 1973). The
opposite (i.e., Na+ influx and water loss) occurs for marine teleosts (e.g.,
Coho salmon, Oncorhynchus kisutch; Brauner et al., 1992). It may be that
the osmorespiratory compromise underpins improved recovery in fishes
upon exercising in brackish or freshwater conditions (i.e., because of
maintained or decreased plasma ion levels) compared with fishes
recovering from exercising in seawater (e.g., Atlantic salmon, Salmo salar;
Hvas et al., 2018), an area worthy of further investigation. Given that
increased activity, especially under elevated temperatures, necessitates
increased perfusion and potentially lamellar recruitment, but ion- and osmo-
regulation processes may require between 5% and 50% of a fish’s energy
budget (Bæuf and Payan, 2001); balancing gas exchange with ion- and
osmo-respiratory functions represents a potentially profound compromise
(Randall et al., 1972). One of the most thorough examples to date where the
trade-offs associated with gill remodelling and the osmorespiratory
compromise have been examined has been in the cyprinids (reviewed in
Nilsson et al., 2012 and Gilmour and Perry, 2018). During winter months
and near-freezing temperatures, Crucian carp fill their interlamellar spaces,
reducing functional gill surface area and ultimately, safeguarding ion- and
osmo-regulation when energetic needs are low. When waters warm and/or
during hypoxic conditions or exercise, the interlamellar cell mass (ILCM)
disappears, revealing up to a sevenfold increase in functional surface area
and a reduction in blood–water diffusion distance to support an elevated
ṀO2 associated with changing conditions (reviewed in Nilsson et al., 2012).
However, the loss of the ILCM is also associated with a large reduction in
plasma ion levels (Matey et al., 2008). Indeed, the osmorespiratory
compromise is a key consideration when investigating physiological
constraints on fish performance and the role of environment, stress, activity,
species, and life stage.

3.3 CIRCULATORY TRANSPORT OF RESPIRATORY

GASES
While gill ventilation and diffusion represent important limitations in the
respiratory gas transport cascade (Figure 3.1, steps 1 and 2), gill perfusion
and blood convection represent the next important step in gas transport and
exchange (Figure 3.1, step 3). The convective transport of blood is
determined by cardiac output, the product of heart rate (f) and stroke
volume (V S; Figure 3.1, step 3 and see Chapter 4: Cardiovascular System),
and the ability of the blood to transport and exchange O2 and CO2 is largely
associated with the characteristics of haemoglobin (Hb), which is
encapsulated within red blood cells (RBCs), as described in the following
section.
3.3.1 BLOOD
3.3.1.1 Oxygen
Most O2 carried by the blood is bound to the respiratory pigment Hb, which
is encapsulated within RBCs. As a tetrameric protein, Hb consists of two α
(141 amino acid) and two β (146 amino acid) subunits (globins), each
containing a porphyrin ring with an iron (Fe2+) haem centre, the site of O2
binding (Perutz et al., 1960; Nikinmaa, 1990). Exceptions to this molecular
structure of Hb are found in the lampreys and hagfishes, which both possess
monomeric Hbs. In fishes in general, less than 5% of O2 is transported
physically dissolved in the blood; the remainder is bound to Hb (Nikinmaa,
1990). The only exception is the Antarctic icefishes, which lack both Hb
and RBCs (Suborder Notothenioidei, Family Channichthyidae; Sidell and
O’Brien, 2006). However, these Hb-less species are thought to compensate
via large blood volumes and increased nitric oxide, thereby influencing
vasodilation/constriction, angiogenesis, and mitochondrial biogenesis
(Sidell and O’Brien, 2006). Compensation also occurs via increased cardiac
stroke volume and power output (Pellegrino et al., 2003) and low metabolic
rates, all of which may only be possible due to the stable, cold environment
they inhabit. But for all other fishes, under specific conditions, Hb binds O2
at the respiratory surface, releases it to tissues in exchange for CO2, and
transports CO2 back to the respiratory surface for removal.
Due to its key role in both O2 and CO2 transport, Hb has become one of
the most well-studied proteins to date. Hoppe-Seyler (1866) was the first to
determine that Hb could reversibly bind O2. Then, during the early 1900s,
the reversible binding relationship between Hb and CO2 was investigated
by Bohr, Hasselbalch, and Krogh (1904), thus linking this molecule to the
respiratory gas cascade. Bohr et al. (1904) determined that the extent of Hb-
O2 binding was reduced in the presence of CO2. This is because CO2
decreases blood pH due to H+ formation from CO2 hydration, and Hb was
discovered to be sensitive to pH, which is now generally known as the Bohr
effect. The key to Bohr and colleagues’ early studies was that the effects of
CO2 on Hb–O2 binding were investigated at low and high O2 tensions,
whereas earlier studies had only investigated atmospheric O2 tensions.
Several decades later, Perutz et al. (1960) examined the binding properties
of these two respiratory gases and Hb and determined that binding was
largely determined by the structural and conformational changes incurred
by the protein upon exposure to O2, CO2, and other ligands. When ligands
break salt bridges, shifting the structural conformation of Hb from a tense
(T) to a relaxed (R) state, binding sites for oxygenation are revealed.
Breaking salt bridges is energetically costly, especially for the number of
salt bridges that must be broken to reveal the first O2 binding site. However,
after the first O2 molecule binds, subsequent O2 binding at other haem
groups is easier, because fewer salt bridges have to be broken to reveal
binding sites. This is termed cooperativity and ultimately results in an
oxygenated, high-affinity Hb (Bonaventura et al., 2004). While there were
seminal studies that linked O2 and CO2 transport to the Hb protein, Perutz
(1960) linked the physiochemical structure of the Hb protein with
respiratory function.
An O2 equilibrium curve (OEC) represents O2 binding to Hb (expressed
as % Hb-O2 saturation), which depends on the PO2 to which the system is
equilibrated. The shape and position of the OEC (Figure 3.2) have been
topics of intense investigation (Kobayashi et al., 1994; Rummer and
Brauner, 2015). The OEC shape is dictated by the way in which individual
Hb subunits interact upon binding. Cooperative binding is described by the
Hill coefficient (nH) and calculated from the slope of the line when [log(Hb-
O2)]/[1 − (Hb-O2)] is plotted against logPO2 (Hill, 1910). A sigmoidal OEC
has a higher nH and therefore a higher degree of cooperative Hb subunit
binding than a hyperbolic OEC. With high cooperativity, substantial
decreases in Hb-O2 can occur during capillary blood transit with only very
small decreases in PO2, maintaining a relatively constant driving force for
O2 unloading/delivery (Lapennas and Reeves, 1983; Nikinmaa, 1990). The
affinity of Hb for O2 is generally quantified by the PO2 at which 50% of Hb
is oxygenated (P50). The P50 differs markedly between and within
organisms and therefore has important implications for gas exchange
(Figure 3.2).
 FIGURE 3.2 Theoretical oxygen equilibrium curve (OEC) depicting the relationship
between haemoglobin oxygen saturation (% Hb-O2) and blood partial pressure for O2
(PO2) at a routine pH value (black line) and at a reduced pH, resulting in either a Bohr
effect (blue dashed line) right-shift or a combined Bohr–Root effect (red dashed line)
rightward and downward shift. Thin white drop lines depict P50 values, the PO2 at
which 50% of Hb is saturated with O2, for each of the three OECs.

The Bohr effect can be illustrated by the change in the position of the
OEC due to an increase in H+ concentration and/or CO2 (Figure 3.2). In the
presence of an acidosis (e.g., metabolic CO2 production at the tissues), a
low-affinity Hb conformation is favoured, and the OEC shifts to the right
(Figure 3.2), thus enhancing O2 delivery to tissues. An increase in blood pH
(e.g., at the gill due to CO2 removal) shifts the OEC back to the left, where
a lower PO2 is required to saturate Hb with O2 (Figure 3.2), thus enhancing
O2 uptake. The Bohr effect is quantified by the Bohr coefficient (Φ), which
describes the degree to which the OEC shifts for a given decrease in pH
(Figure 3.2; Bohr et al., 1904; Nikinmaa, 1990). This is usually calculated
for a single point (typically P50) on the OEC using the following equation:

If the Φ is known, the equation can be rearranged such that the

magnitude of the right-shift in the OEC (i.e., ΔPO2, in mmHg) at a constant
Hb-O2 saturation (e.g., P50) and a proposed ΔpH (e.g., the arterial and
venous blood pH change) can be determined. Intuitively, for a given ΔpH, a
large Φ results in a greater ΔPO2. However, the ΔPO2 can only be
calculated in this manner if the Φ is linear over a wide range of Hb-O2
saturations (20–80%) across the OEC, which is really only the case for air-
breathing vertebrates. Teleost fishes exhibit a non-linear Bohr effect over
the OEC (Jensen, 1986; Brauner and Randall, 1998), which makes
modelling O2 release from the Hb (i.e., to the tissues) in a teleost at
different blood oxygenation levels easiest by directly interpolating ΔPO2
from OECs, as has been done in model species such as the rainbow trout,
Oncorhynchus mykiss (Rummer and Brauner, 2015).
Of all vertebrates, teleosts possess Hbs that are typically the most pH-
sensitive. A decrease in pH reduces not only Hb–O2 affinity but also the
maximum carrying capacity of Hb for O2 (Figure 3.2; Root, 1931). Thus,
even at atmospheric O2 tensions, Hb will not be fully saturated with O2 at
low pH. Although it is possible that the Root effect is an extension of a
unique, non-mammalian Bohr effect (Berenbrink et al., 2005), it cannot be
regarded simply as an exaggerated Bohr effect. Structure–function analyses
have demonstrated specific locations where amino acid substitutions exist,
supporting distinct molecular differences between Bohr and Root effects in
fishes and the Bohr effect in other vertebrates, including human Hb-A
(Berenbrink et al., 2005; Bonaventura et al., 2004; Mylvaganam et al.,
1996). Conserved in Root effect Hbs are three elements: the βN-terminus,
an arginine for lysine at 21β, and histidine at 3β. Removal of any of these
can result in a decrease in the magnitude of the Root effect by 50%. It is
also thought that a serine to cysteine substitution at the β93 position allows
salt bridges to form with the C-terminal histidine residue to stabilize the T-
state. However, additional substitutions have since been identified that may
be involved in stabilizing the T-state at low pH (Berenbrink et al., 2005;
Brittain, 2005).
Until the early 21st century, Root effect Hbs were only really discussed
in terms of enhancing O2 delivery to the eye and swimbladder of fishes. At
both of these locations, dense capillary networks – retia – localize and
magnify an acidosis that greatly elevates arterial PO2 (Scholander and Van
Dam, 1954; Wittenberg and Wittenberg, 1974). In the eye, the high arterial
PO2 serves to overcome great diffusion distances to oxygenate the
metabolically active, yet poorly vascularized, retinal tissue (Wittenberg and
Wittenberg, 1974). At the swimbladder, the gas gland acidifies incoming
blood, and the high arterial PO2 inflates the swimbladder against large
pressure gradients (>50 atm) associated with depth, thus providing fish with
precise buoyancy regulation (Scholander and Van Dam, 1954). Across the
evolutionary trajectory of Percomorpha, one of the most advanced lineages
among teleost fishes, as the magnitude of the Root effect increased, Hb
buffer values decreased to a low plateau in the last common ancestor of
Amia (Figure 3.3). This was also the time when the choroid rete appeared
(Figure 3.3) and later, the swimbladder rete mirabile (Berenbrink et al.,
2005). The liability of the Root effect, however, is that during a generalized
acidosis, as occurs following exhaustive exercise or exposure to
environmental hypoxia or hypercapnia, O2 uptake at the gills could be
greatly impaired through a pH-induced reduction in Hb-O2 carrying
capacity. Fishes that possess a large Root effect generally possess RBC β-
adrenergic Na+–H+ exchangers (βNHE; Nikinmaa, 1990) (Figure 3.3). The
βNHE is activated by catecholamines released during stress. The
stimulation of RBC βNHE leads to extrusion of H+ that exceeds the rate of
the associated HCO3 −/Cl− exchange and results in a large disequilibrium
state across the RBC membrane, tightly regulating intracellular RBC pH
(pHi) despite the generalized reduction in blood pH (Figure 3.4). Thus, O2
uptake at the gills is secured. This system depends upon the lack of plasma-
accessible carbonic anhydrase (CA), which would otherwise short-circuit
the response by permitting the rapidly catalysed conversion of plasma H+
and HCO3 − to CO2, which would back-diffuse into the RBC, thus reducing
pHi (Figure 3.4). Teleosts in general are thought to lack plasma-accessible
CA at the gills and possess circulating plasma CA inhibitors (e.g., with the
strongest degree of inhibition in salmonids; Henry et al., 1997) to inhibit
CA upon RBC lysis (Randall et al., 2014; Harter and Brauner, 2017). Yet,
according to an evolutionary reconstruction, the Root effect evolved long
before the retia of the swimbladder or eye (Berenbrink et al., 2005). Given
this evolutionary trajectory, it was proposed that Root effect Hbs may have
evolved to enhance general O2 delivery in teleosts (Rummer et al., 2013;
Randall et al., 2014).

FIGURE 3.3 Evolutionary changes in specific Hb buffer value, Root effect, and βNHE
across the trajectory of the Percomorpha lineage. Beginning from the left of the figure
with Chondrichthyes, Sarcopterygii, and Polypteriformes, Hb buffer values were
highest (ancestral state), and both the magnitude of the Root effect (maximal %
decrease in O2 saturation of Hb) and βNHE activity were minimal. While Hb buffer
values decreased, Root effect magnitude increased in Polypteriformes to plateau in
Amia, Osteoglossomorpha, Elopomorpha, and Otocephala, only to increase again and
plateau in Oncorhynchus, Esox, Gadus, and Smegmamorpha. Activity of βNHE first
increased between Osteoglossomorpha and Elopomorpha. Shaded, labeled fields
indicate the presence of a choroid or swimbladder rete, respectively (the latter has been
secondarily lost in the Oncorhynchus lineage).
(Adapted from Berenbrink, M., et al. Science, 307, 1752–1757, 2005.)

FIGURE 3.4 During stress, catecholamine release in a model fish activates the sodium
and proton (Na+/H+) exchanger (e.g., the βNHE) on the RBC membrane through a G-
protein-activated cascade including adenylate cyclase and 3′,5′-cyclic monophosphate
(cAMP). The βNHE removes H+ (produced via carbonic anhydrase; CA-catalysed CO2
hydration) from the cell in exchange for Na+. With H+, bicarbonate (HCO3 −) is rapidly
produced inside the cell via the CA-catalysed reaction and removed via anion exchange
for Cl− but at a slower rate than H+. The H+ removed from the RBC acidifies the
plasma, resulting in a decrease in extracellular pH (pHe) without plasma-accessible CA.
Subsequent elevation of intracellular Na+ and Cl− results in RBC swelling through the
movement of osmotically obliged water and activation of Na+,K+,ATPase. If CA is
plasma-accessible, the βNHE is short-circuited by the catalysed conversion of plasma
H+ with HCO3 − to form CO2, which back-diffuses into the RBC. The RBC is then re-
acidified when CO2 is hydrated with CA inside the RBC to form H+, which displaces
 O2 from the Hb, thus enhancing O2 release to the tissue.
(Adapted from Rummer, J.L. and Brauner, C.J., Journal of Experimental Biology, 214,
2319–2328, 2011.)

3.3.1.2 Carbon Dioxide

CO2 transport in fishes has recently been reviewed (Brauner et al., 2019;
Morrison et al., 2015) and thus, is only briefly summarized here.
Metabolically produced CO2 diffuses from the tissues down its partial
pressure gradient through the gas transport cascade for ultimate elimination
at the gills (Figure 3.1). Each step of the cascade is facilitated by the
enzyme CA, which rapidly catalyses the conversion of CO2 to HCO3 −
(CO2 hydration) and vice versa (HCO3 − dehydration) through the
simplified equation

In the absence of CA, this reaction proceeds very slowly relative to blood
transit times, and thus, CA is crucial for CO2 transport and excretion. When
CO2 enters the blood, it diffuses into the RBC, where it is rapidly hydrated
in the presence of RBC CA, and HCO3 − is transported out of the RBC in
exchange for Cl− via an anion exchanger (AE). Some of the resulting H+ is
then either buffered by Hb or bound to Hb as O2 is delivered to the tissues
(Haldane effect). Any remaining H+ then reduces pH and induces the Bohr
effect, further facilitating O2 unloading, and thus, there is an interaction
between O2 and CO2 through the Bohr–Haldane effects (Figure 3.5;
Brauner and Randall, 1998). At physiological pH, 90–95% of the total CO2
is transported as HCO3 − – the majority in the plasma – with the remainder
transported as physically dissolved CO2 (Perry, 1986; Brauner and Randall,
1998).
 FIGURE 3.5 The transfer of oxygen (O2), carbon dioxide (CO2), protons (H+), and
bicarbonate (HCO3 −) between the plasma and RBCs as blood flows through the gills,
arteries, tissues, and venous circulation. The approximate transit time (seconds) for
blood through these portions of the circulation is in brackets. There is no CA available
to plasma in the gills and veins, but CA is available in the arteries and arterioles.
(Adapted from Randall, D.J., et al. Journal of Experimental Biology, 217, 1205–1214,
2014.)

At the gills, the reverse occurs; counter-current exchange of blood and

water facilitates CO2 removal into the ventilated water. The reduction in
PCO2 promotes intracellular HCO3 − dehydration in the presence of RBC
CA. Protons released upon Hb oxygenation further facilitate CO2 formation
and excretion. Depletion of RBC HCO3 − promotes plasma HCO3 − entry
into the RBC in exchange for Cl−, and this is thought to be the rate-limiting
step in CO2 excretion (Figure 3.5). In teleosts, the lack of plasma-accessible
CA at the gills restricts all HCO3 − dehydration to the RBC and due to the
large Bohr–Haldane effect in teleosts, results in a tight interaction between
O2 uptake and CO2 removal (Brauner and Randall, 1998). One exception to
this pattern in teleosts is in icefish (Champsocephalus gunnari), which, as
mentioned earlier, lack Hb and RBCs. To compensate, they possess plasma-
accessible CA in the gill, a trait thought to be absent in most teleosts, which
represents yet another interesting adaptation that permits all CO2 to be
excreted directly from the plasma compartment (Harter et al., 2018b). In
elasmobranchs and hagfishes that also possess plasma-accessible CA, some
CO2 excretion can occur directly from the plasma compartment (Morrison
et al., 2015; Nikinmaa et al., 2019).
3.3.2 BLOOD FLOW AND PERFUSION
After the blood leaves the gills and is circulated to the rest of the body (see
Chapter 4: Cardiovascular System), factors such as the proportion of RBCs
in circulation (i.e., haematocrit; Hct) and the mean cell (corpuscular) Hb
concentration (MCHC) are important considerations in matching gas
exchange to the metabolic requirements of the fish. Hct varies from 15% to
40% across species and is thought to be highest in the most active species
(e.g., tuna and mackerel) and lowest in sedentary species (e.g., hagfish), but
even this has been debated (Brill and Bushnell, 1991). Some studies have
demonstrated that Hct is often higher in male compared with female fishes,
increases incrementally with fish size up to a certain size, and is higher in
pre-spawning compared with spawning fishes (e.g., Indian shad, Tenualosa
ilisha; Jawad et al., 2004). Theoretically, a higher Hct should confer a
greater capacity for O2 transport. However, with increasing Hct comes
increasing blood viscosity, which at some point, is thought to limit
performance due to the subsequent increase in cardiac output needed to
pump viscous blood. Therefore, there must be an Hct where O2 carrying
capacity is maximized but without compromising cardiac output (i.e., the
optimal Hct hypothesis; Richardson and Guyton, 1961). However, studies,
including those where Hct is experimentally manipulated, suggest that other
factors, such as a species’ capacity for vasodilation and regulating cardiac
output, plasma viscosity, and levels of activity, will also influence this
relationship (Gallaugher et al., 1995). Moreover, stress can change Hct via
splenic contraction (i.e., releasing more RBCs into circulation; Perry and
Kinkead, 1989), RBC swelling (e.g., via adrenergic stimulation of RBC
βNHE; Nikinmaa, 1990), and plasma skimming and/or use of secondary
circulation (Steffensen and Lomholt, 1992; Rummer et al., 2014). Neither
splenic release of RBCs nor plasma skimming will, in theory, change
MCHC, but RBC swelling will. It is important to consider both changes in
Hct and Hb concentration, both of which can be used to calculate MCHC,
when determining responses at the level of the blood relative to O2
transport.

3.4 DIFFUSION AT THE TISSUE LEVEL

Gas exchange at the tissues is determined by perfusion of the tissues with
blood and diffusion across capillary membranes to and from the
metabolizing tissues. As at the gills, tissue diffusion is enhanced by an
increase in total capillary surface area, by a reduction in diffusion distance,
and by maximizing the partial pressure gradient between the blood and the
cytosol (Figure 3.1, step 4). It has been known since the beginning of the
20th century that in vertebrates, metabolically produced CO2 diffuses from
the tissues to the blood, inducing the Bohr effect, elevating venous PO2, and
enhancing O2 unloading (Bohr et al., 1904). The magnitude of this response
is the product of the pHa–v difference and the Bohr coefficient. However,
recently, it has been shown that the latter may be especially important in
some teleosts, such as salmonids, and perhaps more generally among
teleosts relative to other vertebrates. As discussed earlier, during stress,
catecholamines are released to protect RBC pHi during a generalized
acidosis to secure O2 binding at the gills. While it is generally thought that
plasma-accessible CA is absent at the gills, there is evidence for its
presence in some tissues, such as the white (Wang et al., 1998) and red
muscle (Rummer et al., 2013) of rainbow trout, the atrium of coho salmon
(Alderman et al., 2016, reviewed in Harter and Brauner, 2017), and
probably other species (Figure 3.5). When adrenergically stimulated RBCs
come into contact with plasma-accessible CA, this rapidly short-circuits the
βNHE and acidifies the RBC. This, combined with the high pH sensitivity
of Root effect Hbs, results in a large increase in PaO2, which has been
proposed to double O2 unloading with no change in tissue perfusion
(Rummer et al., 2013). When the RBC leaves the tissues and enters the
venous system, there is sufficient time (60–90 s) for RBC pHi to recover
through RBC βNHE (Randall et al., 2014; Harter et al., 2018a; Figure 3.5)
prior to gill perfusion. In Atlantic salmon forced to sustain different
swimming speeds, injection of a membrane-impermeable CA inhibitor
(C18) either impaired exercise or was associated with a 30% increase in
cardiac output to maintain a given exercise intensity and metabolic rate
(Harter et al., 2019). This mechanism of enhanced O2 unloading in fish
depends on three criteria: 1) a Hb with a high pH sensitivity, such as a Root
effect; 2) RBC pHi regulation, such as RBC βNHE or possibly other
transporters; and 3) a heterogeneous distribution of plasma-accessible CA
(i.e., presence in the tissues and absence in the gills). All three criteria may
apply to salmonids specifically and may be central to their tremendous
athletic ability. However, this concept may apply to teleosts in general, and
it has been proposed that this mechanism for enhanced O2 unloading may
have played an important evolutionary role in the adaptive radiation of
teleosts (Harter and Brauner, 2017; Randall et al., 2014; Rummer at al.,
2013), which is clearly an area worthy of further investigation.

3.5 CONCLUSION
As aerobic respiration is a prerequisite for life among the fishes, it follows
that selective pressures on the molecular, biochemical, physiological, and
behavioural aspects of gas exchange have been of key interest among
researchers. Here, we reiterate some of the fundamental principles
associated with gas exchange in the fishes and highlight where previous
assumptions have been tested and revised. We also note interesting caveats,
knowledge gaps, and areas for future research. Representing over half of all
extant vertebrates and over 400 million years of evolutionary history, the
fishes, indeed, provide a fascinating group of organisms in which to
investigate the array of adaptations at the level of gas exchange.

ACKNOWLEDGEMENTS
The authors thank Jacelyn Shu and Erin Walsh for preparing figures (Figure
3.1 and Figures 3.2–3.5, respectively). J.L.R. is supported by the Australian
Research Council Centre of Excellence for Coral Reef Studies, and C.J.B. is
supported by a Natural Sciences and Engineering Research Council of
Canada Discovery grant.

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 4 The Cardiovascular System
Erika J. Eliason and Jonathan A. W. Stecyk

CONTENTS
4.1 General Introduction
4.2 General Features of the Fish Cardiovascular System
4.2.1 Blood
4.2.2 Heart Morphology and Blood Flow Patterns
4.2.3 Cardiac Excitation–Contraction Coupling and Cardiovascular
Parameters
4.2.4 Vasculature
4.2.5 Control Systems
4.3 Integrative Cardiovascular Function
4.3.1 Exercise
4.3.2 Digestion
4.3.3 High Temperature
4.3.4 Low Temperature
4.3.5 Limiting Oxygen Levels
4.4 Conclusion and Future Cardiovascular Research
Acknowledgements
References

4.1 GENERAL INTRODUCTION

The cardiorespiratory system of fishes, as for other vertebrates, is designed
to efficiently transport a plethora of substances and molecules, including
respiratory gases, hormones, metabolites, electrolytes, nutrients, plasma
proteins, immune factors, signaling molecules, and wastes, among the
tissues. The fish cardiovascular system is composed of four fundamental
components: blood; heart(s); vasculature; and a control system. This
chapter broadly describes the general features of the fish cardiovascular
system, highlighting the latest ideas, advances, and research in the field, and
details some of the integrated responses of this system to changes in
ambient and internal conditions.

4.2 GENERAL FEATURES OF THE FISH

CARDIOVASCULAR SYSTEM
4.2.1 BLOOD
Blood serves to transport substances throughout the body (Fänge 1992;
McDonald and Milligan 1992; Olson 1992). It is composed of red blood
cells (RBCs, erythrocytes), white blood cells (WBCs, leukocytes), and
plasma (extracellular fluid that contains proteins, ions, hormones, nutrients,
metabolites, and wastes). The total blood volume is in the range of 3–4% of
body mass in most teleost fishes and 5–8% of body mass in most
elasmobranchs (Olson 1992). Antarctic icefishes (Nototheninoid fishes
from the family Channichthyidae) do not have RBCs, and their blood
volume is approximately four times higher than that of typical fishes (Wells
2005). Hagfishes top all known fishes, with a blood volume of ~18% of
body mass (Farrell 2007b).
RBCs serve to transport oxygen and carbon dioxide and regulate acid–
base balance (Nikinmaa 1990; Fänge 1992). In contrast to mammals, fish
RBCs are nucleated and thus, can synthesize proteins in the circulation.
Fish blood is red (except in icefishes) because their RBCs contain
hemoglobin. The hemoglobins of fishes are tetrameric (built of four
polypeptide chains), with the exception of hagfishes and lampreys, which
have monomeric and dimeric hemoglobins. Hematocrit (% of blood volume
that is occupied by RBCs) varies widely across fishes, from 0% to >50%,
though in most teleosts, it tends to range from 20% to 40%. Blood viscosity
increases with hematocrit and cold temperatures, which results in high
vascular resistance and high cardiac workload. Accordingly, fishes in cold,
stable environments often have a lower hematocrit, which serves to
decrease blood viscosity (Wells 2005).
The primary role of WBCs is in immunity and cellular defense (Fänge
1992). Leucocrit (% of blood volume that is occupied by WBCs) constitutes
a tiny proportion of blood volume (0.3–1.0%). There are four major types
of WBCs: lymphocytes, thrombocytes, granulocytes, and monocytes (Fänge
1992). Lymphocytes are common and are involved in immuno-competence
and antibody production. Thrombocytes are small and play a role in blood
clotting. The function of granulocytes is unknown, but they are thought to
play a diversity of roles, including in the inflammatory response, blood
coagulation, tissue repair, and phagocytizing microorganisms. The largest
and rarest WBCs are the monocytes, which are phagocytic (ingesting
harmful cells, particles, and bacteria). Infection and parasitic infestation
typically result in an increase in circulating WBC levels, while stress and
environmental toxicants can modulate levels and thus influence disease
resistance.
The components of the blood are tightly regulated; thus, blood sampling
is a common technique to assess the physiological status of a fish.
Numerous hormones are transported in the plasma (e.g., gonadotropins and
sex steroids, growth hormone, thyroid hormones, and cortisol). Metabolite
levels can be highly variable in the plasma. Lactate, the end product of
anaerobic metabolism, accumulates in the blood following strenuous
activity (e.g., exhaustive exercise) and in response to oxygen limitation
(hypoxia: decreased oxygen; anoxia: no oxygen). Anoxia-tolerant fishes
such as crucian carp (Carassius carassius), and its close relative the
goldfish (Carassius auratus), produce ethanol in response to severe hypoxia
or anoxia (Fagernes et al. 2017). The major plasma electrolytes in fishes are
Na+ and Cl−, whereas plasma Mg2+, K+, and Ca2+ are found at much lower
levels. Amino acids are transported within the RBCs and in the plasma,
while lipoproteins transport lipids in the plasma. Several plasma proteins
are present in the blood (including albumin, Ca2+-binding proteins, and
immunoglobulins). In addition, some teleosts in polar regions can produce
antifreeze proteins or antifreeze glycoproteins to inhibit ice crystallization
within body fluids that would otherwise be fatal (DeVries and Cheng 2005).
4.2.2 HEART MORPHOLOGY AND BLOOD FLOW PATTERNS
The fish heart is composed of morphologically distinct regions (segments).
Classically, the fish heart was considered to be composed of four chambers
arranged in series: the sinus venosus, atrium, ventricle, and bulbus (in
teleosts; or conus in elasmobranchs) arteriosus. However, the current
consensus is that the fish heart consists of six components (or segments)
arranged in series within the pericardium. These are the sinus venosus,
atrium, atrioventricular (AV) segment, ventricle, conus arteriosus, and
bulbus arteriosus, the latter two of which are collectively termed the
outflow tract (OFT) (Icardo 2017) (Figure 4.1). The Cyclostomata (hagfish
and lampreys) present an exception. In these species, all components of the
OFT are absent (Farrell 2007b).

FIGURE 4.1 Anatomical organization of the teleost heart. Direction of blood flow is
from right to left.

The thin-walled sinus venosus serves as a reservoir that receives venous

blood from the ducts of Cuvier and hepatic veins and subsequently conveys
it to the atrium through the sino-atrial (SA) valve (Icardo 2017) (Figure
4.1). The wall of the single atrium is formed by myocardial trabeculae that
generally converge to the AV orifice, thereby enabling blood to be expelled
to the single ventricle through the AV valves of the AV segment. Ventricular
contraction is responsible for generating the heart’s output and central
arterial blood pressure. As such, the ventricle wall is comprised almost
entirely of myocardial cells. Generally, but not exclusively, relative
ventricular mass (i.e., ventricular mass/body mass × 100) is positively
correlated with higher blood pressure generation and athletic performance
and ranges from ~0.03% to ~0.3%–0.4% across species (Farrell and Jones
1992).
Ventricular myocardial architecture is highly variable among species.
Inter-specific variation is classified into two broad categories: fish having a
completely trabeculated ventricle (termed spongiosa or spongy
myocardium) and those having a ventricle with an outer compact layer in
addition to an inner spongy layer. Spongy myocardium is typically
avascular (but see later), so oxygen and nutrients are supplied to the spongy
myocardium via the venous blood being pumped through the lumen of the
ventricle. As such, the spongy myocardium is the last tissue to receive
oxygen before the blood is re-oxygenated at the gills. In contrast, compact
myocardium has a coronary circulation (see Section 4.2.4), so it receives a
reliable source of oxygen. Historically, a completely spongy ventricular
myocardium was thought to be associated with sluggish fish with a low
cardiac performance, whereas the presence of a compact layer was believed
to be associated with very active fishes, such as salmonids (Icardo 2017).
However, this distinction has been blurred by studies showing that some
fish with an entirely trabeculated ventricle (e.g., the gilthead seabream;
Sparus auratus) are able to maintain high heart rates and cardiac output
similar to those recorded in highly athletic fish (Icardo 2017). Fish
ventricles are further divided into four morphological categories (Types I–
IV) according to the presence of compact myocardium and cardiac
vascularity (Farrell and Smith 2017; Icardo 2017). Type I ventricles, which
are found in all cyclostomes and approximately 50% of adult teleost
species, are completely composed of spongy myocardium and lack a
coronary circulation. Type II ventricles are composed of spongy and
compact components, but a coronary circulation is only associated with the
compact myocardium (e.g. salmonids). Type III and Type IV ventricles are
characterized by vascularization of both the compact and spongy
myocardium (e.g. sturgeon, elasmobranchs, some tuna), and the original
distinguishing morphological feature between the groupings was the
proportion of compact myocardium. However, due to the realization that
inter-specific phenotypic plasticity exists for the proportion of compact
myocardium, that the function of the compact myocardium is consistent
regardless of its proportion, and that the various methodologies utilized to
quantify the proportion of compact myocardium can return different results,
it has been suggested that the Type III and Type IV division is unnecessary
(Farrell and Smith 2017; Icardo 2017).
Ventricle contraction propels blood through the OFT, which contains
components of both the conus arteriosus and the bulbus arteriosus in
different proportions depending on taxa (Icardo 2017). In basal species such
as rays, sturgeons, and bichirs, the conus arteriosus constitutes the major
component of the OFT, and the bulbus arteriosus is a short ring-like portion
that establishes a structural connection to the ventral aorta. In contrast, in
more advanced teleosts such as the European eel (Anguilla anguilla) and
yellowfin tuna (Thunnus albacares), the bulbus arteriosus predominates in
the OFT, whereas the conus arteriosus is a short muscular ring situated
between the ventricle and the bulbus arteriosus (Figure 4.1). The bulbus
arteriosus of advanced teleosts has a structure like that of an artery and
contains collagen, elastin, and smooth muscle cells. The cellular
composition of the bulbus arteriosus allows it to be highly compliant,
thereby dampening the fluctuation in blood pressure that occurs with
ventricular contraction (termed the Windkessel effect), sheltering the gill
vasculature from high systolic blood pressure, and leading to a more
uniform perfusion of the gill vasculature (Farrell and Smith 2017; Icardo
2017).
After transiting the OFT, blood flows through the ventral aorta and then
on to the gills (branchial circulation), where gas and ion transfer occurs.
Blood flow then continues via the dorsal aorta to the peripheral tissues
(systemic circulation) before returning to the heart (Figure 4.2). Thus, in
fishes, the branchial and systemic circulations are in series (i.e., single
circuit circulation). In air-breathing fishes, the branchial and systemic
circulations are still in series, but the circulatory system is modified to
accommodate blood flow to and from the air-breathing organ (or lung, in
the lungfishes) (Figure 4.2). Although considerable variation exists among
air-breathing fishes in terms of the type and specific location of the air-
breathing organ within the circulation, in most species, the air-breathing
organ serves to increase the partial pressure of oxygen in the blood
supplying the heart (Figure 4.2) when venous oxygen return to the heart
might otherwise become limiting, including during exposure to aquatic
hypoxia, swimming, digestion, and elevated temperature (Graham 1997;
Stecyk 2017).
 FIGURE 4.2 Generalized circulatory patterns found in (a) water-breathing fish, (b) air-
breathing fish, and (c) lungfish. The lungfish circulation shows aspects of the double
circulation (i.e., respiratory and systemic circulations in parallel) found in mammals.
Deoxygenated blood leaves the right side of the heart and travels to the gills and lung to
be oxygenated, whereas oxygenated blood exits the left side and flows to the tissues via
modified gill arches. Morphological modifications of the heart serve to separate
oxygenated and deoxygenated blood.

4.2.3 CARDIAC EXCITATION–CONTRACTION COUPLING AND

CARDIOVASCULAR PARAMETERS
The coordinated pumping action of the fish heart is initiated by action
potentials (APs) generated by the pacemaker cells located in a
morphologically distinct ring of tissue at the base of the sinoatrial valve
(Vornanen 2017). The pacemaker cells set the spontaneous rhythm of
cardiac contraction, and this results in a synchronized propagation of
excitation throughout the atrium and ventricle. The generation of the AP,
and its spread throughout the heart, requires the integrated activities of
several sarcolemma (SL) ionic currents that pass through pore-forming ion
channel proteins, namely voltage-gated Na+ channels (INa), L-type Ca2+
channels (ICaL), T-type Ca2+ channels (ICaT), delayed-rectifier K+ channels
(including rapid [IKr] and slow [IKs] components), and background inward-
rectifier K+ channels (IK1) (Vornanen 2017). In contrast to mammals, no
specialized ventricular conducting system has been morphologically
identified in fish hearts, although optical mapping of ventricular excitation
suggests the presence of a conducting pathway that regulates the rate and
direction of impulse spread across the ventricle (Vornanen 2017).
The linkage of excitation of the cardiac myocyte SL to the contraction of
the myofilaments is termed excitation–contraction (E–C) coupling (Shiels
2017). The same processes of E–C coupling that occur in mammalian
cardiomyocytes occur in fish cardiomyocytes (Figure 4.3), but some notable
distinctions exist. Fish cardiomyocytes have a large surface area to volume
ratio compared with mammals. Consequently, trans-SL Ca2+ influx is the
predominant (>85%) contributor to the intracellular Ca2+ transient [Ca2+]i,
with the majority of Ca2+ entering the cardiomyocyte through L-type Ca2+
channels, although reverse-mode Na+–Ca2+-exchanger (NCX) activity also
contributes significantly to cardiac contractile Ca2+ in a number of species.
Considerable variability in the role of the sarcoplasmic reticulum (SR) in
cardiac contraction exists among and within fish species (Shiels and Galli
2014). In general, the contribution of SR Ca2+ to cardiac contraction is
minimal to none in slow and sluggish fishes, whereas it plays a more
important role in athletic fishes with a comparatively high fH and PVA (see
Table 4.1 for definitions). Within a species, the contribution of SR Ca2+ to
cardiac contraction is modulated by changes in temperature, contraction
frequency, and adrenergic stimulation (e.g., Kubly and Stecyk 2019).
FIGURE 4.3 Excitation–contraction (E–C) coupling in fish cardiomyocytes. The
primary source of the Ca2+ required for myofilament (i.e., cardiac) contraction is
through L-type Ca2+ channels (LTCC). LTCCs open upon the depolarization of the
sarcolemmal (SL) membrane, which is caused by the opening of voltage-gated Na+
channels (NaV) and influx of Na+ (INa). In some species and under certain conditions,
sarcoplasmic reticulum (SR) Ca2+ contributes to cardiac contraction. The opening of the
SR Ca2+ release channels (the ryanodine receptors [RyR]) is triggered by Ca2+ entry
into the cell. Additionally, Ca2+ influx via reverse-mode Na+/Ca2+-exchanger (NCX)
activity and T-type Ca2+ channels (TTCC) can contribute significantly to cardiac
contraction in some species. Myocardial relaxation occurs with the removal of Ca2+
from the cytosol by forward-mode NCX activity, the uptake of Ca2+ into the SR via the
SR Ca2+-ATPase (SERCA), and supposedly less importantly, by the efflux of Ca2+ by
the SL Ca2+-ATPase. Binding of adrenaline (Ad) to β-adrenergic receptors (β-AR)
increases force of contraction by leading to increased influx of Ca2+ through LTCCs.
Ad also decreases relaxation times in fish hearts, indirectly indicating that Ad stimulates
increased uptake of Ca2+ into the SR by SERCA (via the phosphorylation of the
accessory protein phospholamban [PLB] which causes it to lose its ability to inhibit
SERCA). Repolarization of the SL membrane occurs by the efflux of K+ through
various K+ channels, including the rapid (IKr) and slow (IKs) delayed-rectifier K+
channels and the background inward-rectifier K+ channel (IK1). Na+/K+-ATPase (NKA)
exchanges two K+ for three Na+ to maintain ion gradients.
 The cardiovascular performance of fish is quantifiable through the
measurement (and calculation) of numerous parameters. Table 4.1
summarizes and defines these parameters and provides the formulas to
calculate them. Here, it is important to note that the formulas presented for
calculating the vascular resistances (branchial, systemic, and total) and
cardiac power output assume that central venous pressure (PCV) is
negligible, which is not always true (see Sandblom and Gräns 2017).
Additionally, fish hearts have a remarkable ability to increase their force of
contraction in response to increased venous return and filling pressure,
which is termed the Frank–Starling response (Farrell and Smith 2017).
Accordingly, venous filling pressure is directly related to stroke volume
(Keen et al. 2017).
TABLE 4.1
Measured and Calculated Parameters Utilized to Quantify the Cardiovascular Performance of
Fishes
4.2.4 VASCULATURE
Fish vasculature consists of five basic vessel types: arteries, arterioles,
capillaries, venules, and veins. Arteries are conductance vessels distributing
blood to the tissues. Composed of elastin, collagen, and smooth muscle,
they also serve to dampen the pulse pressure with each heartbeat. Thus,
they are also termed elastance or compliance vessels. The arterial system
must balance tissue blood supply with tissue metabolic demands.
Regulation of blood flow distribution primarily occurs at the level of small
arteries and arterioles, termed resistance vessels, via dilation or constriction
of the smooth muscle in the vessel wall (see Section 4.2.5). The capillaries
(and some small arterioles and venules) are where gas and metabolites are
exchanged. Capillaries are thin (4–10 μm in diameter) vessels composed of
a single layer of endothelial cells with high surface area and low blood
velocity to facilitate diffusion. Veins and venules return blood to the heart.
These capacitance vessels act as a low-pressure, high-volume reservoir to
store the blood volume. The venous system profoundly affects cardiac
output by regulating blood flow returning to the heart and cardiac filling
pressure (see Section 4.2.2). Comprehensive descriptions of fish vasculature
anatomy have been provided elsewhere (Bushnell et al. 1992; Satchell
1992; Sandblom and Gräns 2017).
From the heart, all blood travels via the ventral aorta to the gills, which
function in gas exchange (Chapter 3), osmo- and ionoregulation (Chapter
5), acid–base balance, nitrogen excretion, hormone metabolism, and the
sensing of internal and external environments (Figures 4.4 and 4.5). Blood
flows into several pairs of afferent branchial arteries (ABA), which enter
the gill arches. Afferent filamental arteries (AFA) branch off the ABA to
supply blood to the gill filaments. Blood travels from the AFA through the
afferent lamellar arteriole, into the respiratory lamellae for gas and ion
exchange, and then back out via the efferent lamellar arteriole through the
efferent filamental artery to the efferent branchial artery (EBA). This
arterio-arterial (i.e., respiratory) pathway of blood flow in fish gills is a
highly effective countercurrent system, where blood flows in the opposite
direction to water to maximize oxygen uptake into the lamellae (Figure
4.5). A small portion (<10%) of blood that exits the lamellae re-enters the
filament and is directed to the nutrient circulation via nutrient arteries or
into the intralamellar vasculature. The coronary arteries typically originate
from the EBA and transport blood back to the myocardium of the conus
arteriosus, ventricle, and atrium, depending on the type of heart (see Section
4.2.2) (Icardo 2017). The EBA pairs converge with the dorsal aorta and
carotid arteries to supply oxygenated blood to the systemic circulation
(Figure 4.4). Blood is distributed to the systemic tissues via arteries that
branch from the dorsal aorta, which runs along the vertebrae towards the
tail (Figure 4.4). Once the dorsal aorta exits the peritoneal cavity, it is
known as the caudal artery. For the most part, a corresponding vein is
closely associated with each artery. Veins draining the head and skin deliver
blood to the sinus venosus and ducts of Cuvier. All other blood travels
through portal systems, whereby blood travels first through one capillary
bed in the tissues and then through another capillary bed before returning to
the heart.

FIGURE 4.4 Schematic of the water-breathing teleost circulatory system. See Section
4.2.4 for details.
(Adapted from Sandblom, E. and Gräns, A., in Fish Physiology, Volume 36, Part A, The
Cardiovascular System: Morphology, Control and Function, Academic Press,
Cambridge, MA, 2017.)
 FIGURE 4.5 Schematic of blood flow through the gill. The countercurrent arrangement
of the capillary bed and water flow (blood flows in the opposite direction to water)
allows highly efficient oxygen uptake.
(Adapted from Olson, K.R., J. Electron. Microsc. Tech., 19, 389–405, 1991.)

Fish have two portal systems: the hepatic portal system and the renal
portal system (Figure 4.4). As in other vertebrates, the hepatic portal system
drains blood from the stomach, intestines, spleen, and liver through the
hepatic portal vein into the sinusoidal capillaries of the liver and back out
through the hepatic veins to the sinus venosus. The renal portal system is
unique to fishes; blood draining the trunk muscles, tail, and skin via the
caudal vein, as well as the post-abdominal region (e.g. bladder and gonads),
travels through the kidney before returning to the heart via the ducts of
Cuvier.
Some fishes have evolved accessory pumps to help propel blood back to
the main heart (Farrell 2011). Hagfish have a portal heart associated with
the hepatic portal vein to assist blood flow from the intestines to the liver
(Farrell 2007b). The portal heart is comprised of true cardiac muscle and is
myogenic; thus, it generates an independent electrocardiogram. Caudal
pumps have been discovered in hagfish, eels, and a few species of sharks
(Farrell 2011). Though evolutionarily and morphologically distinct across
groups, in all three groups, skeletal muscle serves as an accessory pump to
enhance venous return. Branchial pumps take advantage of the gill muscles
and rhythmic ventilation cycle to improve venous return to the heart.
A variety of fishes have a specialized structure termed a vascular rete,
where arterial vessels are arranged in the opposite direction to venous
vessels to allow countercurrent exchange of heat or gas (Figure 4.6)
(Wittenberg and Wittenberg 1974; Stevens 2011). The swimbladder
vascular retia secrete oxygen into the swimbladder to contribute to
buoyancy. Fish eyes are avascular; thus, the eyes of most fish have a
vascular rete (termed a choroid rete mirabile) that secretes oxygen into the
eye to support aerobic metabolism. Heat-exchanging vascular retia are rarer.
They are found in only ~30 species of fish, including tunas, some billfishes,
some sharks, and the opah (Lampris guttatus) (Bushnell et al. 1992;
Graham and Dickson 2001; Wegner et al. 2015). These heat exchangers can
be found in the eyes, brain, muscles, gills, and viscera and function to
conserve metabolic heat. As a consequence, fish with heat-exchanging retes
are regional endotherms (part of their body is warmer than the
environment).
 FIGURE 4.6 Vascular retia showing countercurrent exchange of heat or gas. (a) A
typical circulation pattern for most fishes. Metabolic heat produced in the tissues is lost
at the gills. (b) The circulation pattern for countercurrent heat exchangers where
metabolic heat is conserved in the tissues where it is produced. (c) Countercurrent gas
exchanger serving the swimbladder. (d) Countercurrent gas exchangers serving the eye.
(Adapted from Wittenberg, J.B. and Wittenberg, B.A., Biol. Bull., 146, 116–136, 1974.)

The secondary circulation system is unique to fishes (Steffensen and

Lomholt 1992; Rummer et al. 2014). It consists of a vascular network of
arteries, capillaries, and veins that is derived from and runs in parallel with
the primary circulation but contains few RBCs. Bony (teleost) fishes are
considered to have two secondary circulations. In the first, secondary
vessels typically derive from the dorsal aorta or segmental arteries, and the
vessels are associated with the skin, scales, fins, mouth, and peritoneum.
Notably, the secondary system is not associated with skeletal muscle or the
gastrointestinal system, kidney, liver, swimbladder, brain, or eye. The other
secondary circulation is located in the gills and is composed of two distinct
vascular pathways: the nutrient pathway and the interlamellar pathway
(described earlier). The function of the secondary circulation is poorly
understood. Though morphological and molecular similarities between the
vertebrate lymphatic system and the secondary system in zebrafish have
been noted (Yaniv et al. 2006), the secondary vascular system does not
appear to function like a mammalian lymphatic system (i.e., it does not
assist with fluid balance). Historically, the secondary circulation system was
assumed to play a negligible role in gas exchange given the paucity of
RBCs, but it has been suggested to play a role in skin oxygen uptake,
immune protection, acid–base regulation and ion transport, and reduction in
cardiac work and RBC turnover (Steffensen and Lomholt 1992; Rummer et
al. 2014).
4.2.5 CONTROL SYSTEMS
Regulation of the fish cardiovascular system occurs primarily through the
action of the autonomic nervous system, with the exception of hagfishes, in
which nervous control of the circulatory system appears to be absent
(Sandblom and Axelsson 2011; Farrell and Smith 2017; Sandblom and
Gräns 2017). The autonomic nervous system consists of two opposing
pathways: a parasympathetic vagal inhibitory component and a sympathetic
(adrenergic) excitatory component. Cardioinhibitory parasympathetic
control via the cardiac branch of the vagus nerve is the predominant form of
fH regulation in teleosts. The sympathetic component is of paramount
importance for the regulation of blood pressure, cardiac contractility, and
distribution of blood flow among vascular beds. Adrenergic stimulation of
the cardiovascular system in teleosts occurs through both neuronal and
humoral means. The catecholamines adrenaline and noradrenaline, liberated
from the post-ganglionic terminals of sympathetic nerves near their site of
action, evoke rapid responses in effector tissues. A more widely distributed
adrenergic response occurs through catecholamine release directly into the
bloodstream from chromaffin cells. These cells are localized within the
walls of the posterior cardinal vein and in close proximity to the lymphoid
tissue in the region of the anterior (head) kidney.
Since signaling molecules such as catecholamines cannot penetrate the
cell membrane, a transduction pathway is needed for transmembrane
signaling. Transmembrane signaling begins with the binding of
catecholamines to cell-surface adrenoceptors, of which two main types, α
and β, have been identified in teleosts. However, the location of each type
varies among species (Sandblom and Axelsson 2011). Usually, α-
adrenoceptors are in the vascular smooth muscle of arterioles, and β-
adrenoceptors are found in the heart, vascular smooth muscle, and RBCs.
Adrenergic stimulation typically results in an α-adrenergically mediated
systemic vasoconstriction and β-adrenergically mediated increases in
cardiac inotropy (force) and chronotropy (rate). A β-adrenergic vasodilatory
effect can also occur in the arterioles of the systemic circulation but is often
masked by the more potent α-vasoconstriction. In addition, in some fish,
stimulation of cardiac α-adrenoceptors mediates negative chronotropy and
inotropy (Stecyk 2017). Adrenergic stimulation of the branchial vasculature
also exists in teleosts (Sandblom and Axelsson 2011). In contrast to the
systemic circulation, the β-adrenergic-mediated vasodilatory response
dominates in the gill respiratory (i.e., arterio-arterial) vasculature
(Sandblom and Gräns 2017). In RBCs, the β-adrenergic response is crucial
for maintaining or even enhancing oxygen delivery to tissues in time of
stress. Briefly, catecholamines stimulate the activation of Na+/ H+ exchange
across the erythrocyte membrane, which increases RBC intracellular pH
relative to the plasma, thereby facilitating oxygen binding.
In addition to autonomic control, a plethora of circulating (humoral) and
local (autocoid and paracrine) biochemical factors, hormones, and
gasotransmitters play fundamental roles in fish cardiovascular control
(Imbrogno and Cerra 2017; Sandblom and Gräns 2017). These include
angiotensin II and natriuretic peptides, which participate in homeostatic
circuits that integrate myocardial stretch and ion, water, and hemodynamic
homeostasis; the derived peptides from the prohormone chromogranin-A
(i.e., vasostatin and catestatin), which serve as cardiovascular stabilizers;
and the endogenously produced membrane-permeable gases carbon
monoxide, hydrogen sulfide, and nitric oxide, which exhibit neuroactive
and vasoactive properties and also play a role in oxygen sensing (Perry and
Tzaneva 2016). Finally, metabolic end-products, namely lactate ions, have
been recently shown to elicit cardiorespiratory responses via stimulation of
oxygen-sensing cells in the gills (Thomsen et al. 2019).

4.3 INTEGRATIVE CARDIOVASCULAR FUNCTION

4.3.1 EXERCISE
Cardiovascular responses to swimming in fishes have been well studied
(Farrell and Smith 2017). During aerobic swimming, steadily increases
with increasing swimming speeds, reaching 60–300% higher than resting
values. The relative contribution of fH and VS to the increased is species
specific. For example, tuna rely primarily on fH to increase during
swimming (Farrell 1996), whereas salmonids increase VS ~twofold but fH
usually only increases by ~50% (Eliason et al. 2013). During swimming, fH
increases primarily via a release of vagal cholinergic cardiac tone (i.e.
removal of the cardiac brake) and also via increasing sympathetic
adrenergic stimulation (i.e. pressing on the cardiac accelerator) (Farrell and
Smith 2017). Stroke volume increases via increased adrenergic stimulation
via the Frank–Starling effect (see Section 4.2.3) as well as via mobilization
of blood from capacitance vessels (Farrell and Smith 2017; Sandblom and
Gräns 2017). The consequence of the increased is increased O2 delivery
to the swimming muscles. Additionally, because the heart does not have the
capacity to simultaneously perfuse all capillary beds, blood flow is
redistributed during swimming to prioritize the perfusion of aerobic
muscles and the coronary circulation (when present; see Section 4.2.2). An
exciting area of current research includes the examination of how the Root
effect (pH-dependent reduction in the O2 carrying capacity of teleost
hemoglobins) enhances O2 delivery to metabolically active tissues (Harter
and Brauner 2017) and the role of plasma accessible carbonic anhydrase in
enhancing oxygen delivery to actively respiring tissues, such as the
avascular spongy myocardium, during swimming (Alderman et al. 2016).
Other current active areas of research consider how the cardiovascular
system in swimming fishes responds to external stressors such as hypoxia,
temperature changes, and toxicants.
4.3.2 DIGESTION
During digestion, oxygen consumption (termed specific dynamic action
[SDA] or heat increment of feeding; see Chapter 6) increases substantially
to support nutrient digestion, absorption, distribution, and processing.
Concomitantly, gut blood flow (GBF) increases 40–150% from resting
levels (30–40% of ; Farrell et al. 2001) to support nutrient, hormone, and
waste transport and to supply oxygen to the respiring intestinal tissues. The
increased GBF is achieved via an increase in and/or a redistribution of
regional blood flow to the stomach and intestinal tissues (Seth et al. 2010).
Although several studies have focused on the mechanisms of GBF control
and coordination (Seth et al. 2010), in general, regional blood flow and
tissue prioritization remains an understudied area. However, the influence
of exercise on GBF has been examined in a few species. In unfed fish, GBF
decreases during swimming, presumably to prioritize oxygen delivery to the
swimming muscles. However, in some species, GBF in fed fish is spared to
maintain blood flow to the intestines (e.g. salmonids; Thorarensen and
Farrell 2006). In contrast, GBF decreases during swimming in fed sea bass
(Altimiras et al. 2008; Dupont-Prinet et al. 2009). Several studies have
examined how environmental factors (e.g. temperature, hypoxia,
hypercapnia, salinity, and acute stress) influence GBF (Farrell et al. 2001;
Seth et al. 2010). Given the importance of digestion for a fish’s ability to
grow and thrive, the influence of climate change on the integrated
cardiovascular response during digestion is likely to be a fruitful area of
future research.
4.3.3HIGH TEMPERATURE
An extensive literature has considered how increasing temperature (across
both acute and acclimation time scales) influences cardiovascular variables
in fishes (Eliason and Anttila 2017). Broadly speaking, increases with
warming temperatures, which is almost exclusively driven by an increase in
fH (Eliason and Anttila 2017). In recent years, the heart has emerged as a
potential mediator of thermal tolerance in fishes (Wang and Overgaard
2007; Farrell et al. 2009). Accordingly, one of the most active research
areas in the field today seeks to determine why fish hearts fail at high
temperature. One leading possibility is that the ion channels involved in E-
C coupling become functionally impaired at elevated temperatures, which
would compromise the spread of the action potential across the heart and
reduce fH (Vornanen 2017). One study with brown trout (Salmo trutta fario)
found that Na+ channels were the most temperature sensitive (Vornanen et
al. 2014). Another possibility is that mitochondrial function could become
impaired at high temperature, causing a limitation in ATP supply for the
heart (Eliason and Anttila 2017). Mitochondria become more permeable at
elevated temperatures, reducing the efficacy of ATP production (Iftikar et
al. 2014). Another hypothesis suggests that insufficient oxygen supply to
the myocardium at high temperature may lead to cardiac collapse. As
temperatures increase, partial pressure of oxygen in the venous blood
decreases (Eliason and Anttila 2017), which could compromise oxygen
delivery to the avascular spongy myocardium. In species with a coronary
circulation, coronary blood flow has been demonstrated to play a role in
thermal tolerance (Ekström et al. 2017). Coronary blood flow increases
during warming, which would enhance cardiac oxygenation (particularly
when oxygen delivery to the spongy myocardium is compromised by
reduced venous oxygen levels) (Ekström et al. 2017). Moreover, cardiac
myoglobin levels were correlated with upper thermal tolerance in Atlantic
salmon (Anttila et al. 2013), which further supports the notion that oxygen
supply does play a role in cardiac thermal tolerance. Finally, a noxious
venous blood environment at high temperature could compromise cardiac
contractility. As temperatures rise, blood pH decreases (acidosis), and
plasma K+ increases (hyperkalemia). Acidosis and hyperkalemia (and
hypoxia) have negative ionotropic and chronotropic effects on the heart
(Hanson et al. 2006). Notably, adrenergic stimulation is critical to stimulate
and protect the heart under these noxious conditions (Hanson et al. 2006),
but the sensitivity of the heart to adrenaline decreases with warming in
some species (Keen et al. 1993). Indeed, cardiac thermal performance
varies across populations of sockeye salmon, which has been linked to the
density of adrenaline-binding ventricular β-adrenoceptors (Eliason et al.
2011) and potentially Ca2+ cycling (Anttila et al. 2019). Thus, the role of
the adrenaline signal transduction pathway in cardiac thermal tolerance
remains a dynamic area of future research (Ekström et al. 2014; Gilbert et
al. 2019).
4.3.4 LOW TEMPERATURE
For freshwater fish that experience near-freezing temperatures in winter,
cardiac physiology must be adjusted to accommodate the cold temperature–
driven effects on cardiac excitability and contractility, blood viscosity, and
associated vascular resistance (Vornanen 2016, 2017). The physiological
strategy employed by fishes in response to cold temperature acclimation (or
acclimatization) varies among species and is reflective of their
overwintering strategy (Vornanen 2016; Stecyk 2017). As summarized in
Table 4.1, some species, such as rainbow trout (Oncorhynchus mykiss) and
burbot (Lota lota), exhibit physiological compensation that allows the
continuation of an active lifestyle at cold temperature. By comparison,
species that experience prolonged periods of oxygen deprivation during the
winter months, such as the crucian carp (Carassius carassius), must prime
physiological processes to conserve ATP, making positive compensatory
changes maladaptive. Accordingly, for these species, cold exposure serves
as an important cue to reduce activity, metabolic rate, and subsequently
cardiac activity in anticipation of winter hypoxic and/or anoxic conditions
(Vornanen et al. 2009; Stecyk 2017). The phenomenon is termed inverse
thermal acclimation. Despite these general distinctions, some
cardiophysiological responses to cold are shared across overwintering
strategies. For example, both rainbow trout and crucian carp exhibit
increased density of K+ currents in the heart with cold acclimation, and the
Alaska blackfish (Dallia pectoralis) displays a combination of down-
regulatory and cold-compensatory responses to cold acclimation (Kubly
and Stecyk 2015, 2019). Finally, it is becoming increasingly recognized that
naturally occurring seasonal acclimatization induces more pronounced
changes in cardiac function than laboratory-based thermal acclimation
(Filatova et al. 2019).
4.3.5 LIMITING OXYGEN LEVELS
Hypoxia and anoxia are prevalent in a wide range of aquatic systems, both
naturally and due to anthropogenic causes (Diaz and Breitburg 2009). For
many water-breathing fish, the premier cardiovascular response to aquatic
hypoxia is a cholinergically mediated reflex slowing of fH (termed hypoxic
bradycardia). Hypoxic bradycardia is mediated through the activation of O2
chemoreceptors located in the gills; in most species, these chemoreceptors
sense and respond to changes in oxygen in the inspired water (Milsom
2012). Although hypoxic bradycardia is hypothesized to have several direct
benefits to the heart (reviewed by Farrell 2007a), variation exists among
species with regard to the water oxygen level at which hypoxic bradycardia
is initiated as well as in the magnitude of fH depression. In fact, some
species do not always exhibit hypoxic bradycardia (e.g., rainbow trout,
winter flounder, Pseudopleuronectes americanus, and Atlantic cod, Gadus
morhua; see Stecyk 2017), whereas others do not exhibit hypoxic
bradycardia at all (e.g., red- and white-blooded Antarctic fishes) even if
exposed to anoxia (e.g., crucian carp) (Stecyk et al. 2004; Stecyk 2017).
The variability relates to differences in ecology, lifestyle, and hypoxia
tolerance, but intra-specific differences could also reflect differences in
experimental design (Gamperl and Driedzic 2009). The typical cardiac
response of air-breathing fish with biomodal respiration when exposed to
aquatic hypoxia is a cycling of bradycardia prior to an air breath and a
subsequent transient tachycardia (increase in heart rate) that commences
with or just after each air breath (Graham 1997; Stecyk 2017). In addition
to bradycardia, typical cardiovascular responses to hypoxia exposure
include increased Rgill, PDA, and Rsys in water-breathing teleosts but
unchanged or decreased PDA and Rsys in elasmobranchs (see Table 4.1 for
definitions) (Stecyk 2017). In air-breathing fish, the prevailing response to
aquatic hypoxia exposure with continued access to atmospheric air is an
increase in , augmented blood flow to the air-breathing organ, and
decreased blood flow to the gills (Stecyk 2017). Nevertheless, despite these
general trends, it is becoming increasingly clear that the cardiovascular
responses to oxygen deprivation are highly variable among and even within
species.
Table 4.2 Comparison of the Cardiac Phenotypic Changes in Response to Cold Acclimation in
Cold-Active and Cold-Dormant Fishes
4.4 CONCLUSION AND FUTURE CARDIOVASCULAR
RESEARCH
Given the essential role the fish cardiovascular system plays in gas
exchange, osmo- and ionoregulation, reproduction, endocrinology,
digestion, immune function, locomotion, and in some fish species, thermal
regulation, it is unsurprising that this critical organ system has received an
enormous amount of research attention over the last century. In the current
era of climate change and anthropogenic stressors, there is an intense focus
on discovering the mechanisms that determine environmental tolerance
limits (e.g. temperature, hypoxia, and toxicants). Moving forward,
technological advances (e.g. smaller, cheaper, and longer-lasting biologgers
and telemetry for cardiovascular measurements) will enable more research
to transition from the laboratory to the field. The fish cardiovascular system
is also increasingly being used as a model for human cardiac development
and function as well as a tool to investigate questions relevant to human
health and various oxygen deprivation–related diseases.

ACKNOWLEDGMENTS
The research of E.J.E. is currently supported by the University of
California, Santa Barbara and the Hellman Fellows Fund. The research of
J.A.W.S. is currently supported by the National Science Foundation,
Division of Integrative Organismal Systems. The authors gratefully
acknowledge Paul Parsons for creating all the figures.

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 5 Iono- and Osmoregulation
Dietmar Kültz and Kathleen M. Gilmour

CONTENTS
5.1 General Introduction
5.2 Evolutionary Strategies
5.2.1 Hagfish
5.2.2 Lamprey
5.2.3 Elasmobranchs
5.2.4 Teleosts
5.3 Physiology of Iono- and Osmoregulatory Tissues
5.3.1 Skin
5.3.2 Gills
5.3.2.1 Freshwater Fishes
5.3.2.2 Marine Fishes
5.3.3 Kidney
5.3.3.1 Freshwater Fishes
5.3.3.2 Marine Fishes
5.3.4 Gastrointestinal Tract
5.3.4.1 Marine Fishes
5.4 Euryhalinity
5.4.1 When Does Natural Selection Favour Euryhalinity?
5.4.2 Cellular Mechanisms of Osmosensing and Signal Transduction
5.5 Conclusion
Acknowledgements
References

5.1 GENERAL INTRODUCTION

With the exception of some prokaryotes (e.g. halobacteria), metabolism in
all cells has evolved to function optimally in a particular ionic milieu.
Intracellular Na+ is low (~10 mM), K+ is high (~130 mM), and
concentrations of divalent cations (Ca2+, Mg2+, etc.) are in the micro- to
low millimolar range. Maintaining this ionic environment is critical because
natural selection has optimized macromolecular structure and function
under these conditions. Different strategies of achieving intracellular ion
homeostasis have evolved to support cell function in environments with
variable ion concentrations. Cells of fishes and other multicellular animals
are isolated from the environment not only by their plasma membrane but
also by an epithelial cell layer that separates the body fluids from the
external milieu (Figure 5.1). This additional layer buffers environmental
salinity fluctuations at the level of body fluids, shifting some of the
energetic burden of maintaining intracellular ion homeostasis from
individual cells to the level of specialized epithelial tissues that are in direct
contact with the external milieu. Most fishes, except primitive taxa, pursue
this strategy of osmoregulation to maintain intracellular ion homeostasis.
 FIGURE 5.1 The osmotic and ionic milieu is controlled at two barriers. (a) The outer
barrier (blue line) reflects epithelia that, in most fishes, maintain an osmotic gradient
between the external environment and the extracellular fluid (ECF). (b) The inner
barrier (red line) is the plasma membrane surrounding each cell, which maintains strong
ionic (but not osmotic) gradients (e.g. [Na+] and [K+]) between ECF and intracellular
fluid (ICF).

5.2 EVOLUTIONARY STRATEGIES

Fishes encompass a spectrum of strategies ranging from osmoconformers to
osmoregulators (Table 5.1; Figure 5.2). In osmoconformers, ionic
homeostasis is regulated primarily at the level of cells. This strategy is
evolutionarily primitive and employed by hagfish. However, lamprey (the
other clade of jawless fishes) are osmoregulators, a more advanced strategy,
as are teleosts (bony fish). Elasmobranchs use a third approach, being
osmoconformers but strong ionoregulators. These strategies are briefly
discussed in the following subsections.
Table 5.1
Ionic and Osmotic Composition of Aquatic Environments and the Body Fluids of
Representative Fishes
.

FIGURE 5.2 Osmotic and ionic strategies of (a) hagfish, (b) lamprey, (c) elasmobranch
and (d) teleost fishes in sea water, and (e) lamprey, (f) elasmobranch and (g) teleost
 fishes in fresh water. Grey arrows indicate obligate ion and water movements; black
arrows indicate regulated responses to maintain ionic and/or osmotic homeostasis.

5.2.1 HAGFISH
The body fluids of these primitive marine fish are approximately isosmotic
with seawater (SW), whereas their ionic composition is similar but not
identical to that of SW (Robertson 1976). Hagfish represent an early
evolutionary stage at which the burden of maintaining intracellular ion
homeostasis has started to shift from the plasma membrane of cells to the
level of specialized epithelia. These epithelia provide a barrier between the
fish and the environment, allowing the ionic composition of the body fluids
to be controlled. However, ion differences across hagfish external epithelia
are small because ionoregulatory mechanisms are at a primitive
evolutionary stage. Specialized epithelial cells (ionocytes) probably evolved
to support acid–base homeostasis and/or nitrogenous waste excretion rather
than ionoregulation (Evans 1984). Hagfish have ionocytes in their
osmoregulatory epithelia and may represent an evolutionary transition in
which ionocytes start to be adopted for ionoregulation beyond their other
roles.
5.2.2 LAMPREY
Unlike hagfish, lamprey have conquered freshwater (FW), with some
species remaining in FW throughout their life cycle, while others develop in
FW but spend most of their life in SW. The evolutionary transition from
SW to FW requires effective ionoregulation because FW ion concentrations
are too low to be tolerated in the body fluids. Thus, FW invasion required
the evolution of mechanisms to maintain significant ionic and osmotic
gradients across external epithelia. As in teleosts and tetrapods, the body
fluid osmolality of FW lamprey is 300–350 mosmol/kg. To achieve
hyperosmotic body fluids relative to FW, lamprey actively accumulate NaCl
and excrete water, processes that are energy dependent. Active NaCl uptake
counteracts passive diffusional NaCl loss from the body fluids to FW and is
achieved using ionocytes in the branchial epithelium. Thus, one
evolutionary advance that appeared in the ancestor of extant lamprey was
the functional adoption of branchial ionocytes for iono- and
osmoregulation. A second, equally important advance was the adoption of
renal glomeruli and distal tubules for selective filtration and excretion of
water. These renal novelties were critical for maximizing NaCl reabsorption
and retention (Beyenbach 2004).
5.2.3 ELASMOBRANCHS
Elasmobranchs include sharks, skates, and rays, and most species are
marine, although several euryhaline species complete at least part of their
life cycle in FW. Marine elasmobranchs are strong ionoregulators while still
being osmoconformers. They maintain an NaCl concentration of about one-
third SW yet a total osmolality of ~1000 mosmol/kg, slightly hyperosmotic
to SW, in their body fluid. Organic osmolytes, primarily urea and
trimethylamine oxide (TMAO), bridge this ‘osmotic gap’. The passive
inward diffusion of NaCl is countered by active NaCl excretion by
ionocytes in the rectal gland. Marine elasmobranchs must also actively
retain elevated urea and TMAO. To counter diffusional loss of these organic
osmolytes to SW, they are constantly produced as by-products of nutrient
metabolism.
Euryhaline elasmobranchs are iono- and osmoregulators in FW, with high
body fluid osmolality (>600 mosmol/kg) due to urea and TMAO
concentrations that are reduced in FW but remain considerable. The high
osmolality of elasmobranch body fluids relative to FW presents a
significant energetic burden; that is, in FW, elasmobranchs would have to
either greatly tighten their epithelia to minimize diffusion and/or actively
absorb NaCl against huge concentration and osmotic gradients. Unlike for
teleosts, it is currently unknown whether euryhaline elasmobranchs regulate
the leakiness of epithelial tight junctions when entering FW. Moreover, the
number of euryhaline elasmobranchs able to enter FW for extended periods
is small relative to their strictly marine counterparts. It appears that the
energetic burden associated with maintaining elevated body fluid osmolality
prevents widespread evolutionary expansion of elasmobranchs into FW
habitats. The few stenohaline FW elasmobranchs (e.g. the Amazonian
stingray Potamotrygon sp.) have low urea levels similar to those of FW
teleosts (Table 1, Evans and Claiborne 2009).
5.2.4 TELEOSTS
All teleosts are osmoregulators, with body fluid osmolality of ~300–350
mosmol/kg in both FW and SW. In an evolutionary sense, this strategy
shifted much of the burden of maintaining intracellular ion homeostasis
from the level of the cell to that of specialized epithelia. For instance,
instead of a Na+ gradient across the plasma membrane of ~500 mosmol/kg
in SW versus 10 mosmol/kg intracellular, it becomes ~150 mosmol/kg in
body fluids versus 10 mosmol/kg intracellular. However, maintaining body
fluid osmolality below that of SW represents a formidable task for marine
teleosts. Osmotic water loss across the external epithelia is countered by
ingestion of SW. Water absorption across the intestine is driven by NaCl
absorption, which adds to the NaCl load gained by diffusion. Excess NaCl
is actively excreted by gill ionocytes, while the absorbed water is actively
retained by reabsorption in the renal tubule, keeping urinary volume to the
minimum necessary for waste and divalent ion excretion. This process
yields a net gain of water that compensates for the dehydration resulting
from osmosis. Drinking rates of euryhaline fishes vary with environmental
salinity, being virtually zero in FW and high in SW. Maintaining body fluid
osmolality above that of FW similarly represents a challenge for FW
teleosts. Water gain by osmosis across the external epithelia is countered by
copious production of dilute urine, which is not completely devoid of ions
and aggravates ion loss. Thus, gill ionocytes in FW teleosts actively take up
NaCl and Ca2+.

5.3 PHYSIOLOGY OF IONO- AND OSMOREGULATORY

TISSUES
The main iono- and osmoregulatory tissues are skin (especially during early
development), gill, renal, and gastrointestinal epithelia. Other epithelia (e.g.
those of the operculum, oral cavity, and pyloric caeca) may also have
osmoregulatory functions but largely represent structural and functional
extensions of gill or gastrointestinal epithelia and are not considered
separately in the following overview. The elasmobranch rectal gland is an
extension of the gastrointestinal tract (GIT) but functions like the gill of a
marine teleost and will be discussed briefly in that context.
5.3.1 SKIN
In most adult fishes, ion transport across the body surface is dominated by
the gill (Evans et al. 2005), and the skin serves as a permeability barrier.
The exceptions include specific skin regions that are ionocyte rich, such as
the opercular epithelium (Degnan et al. 1977), and a few marine and
amphibious species that have ionocytes across the body surface (e.g. Martin
et al. 2019). The opercular epithelium was used as a model to investigate
NaCl secretion in marine teleosts because, unlike the gill, it provided a flat
epithelium that could be used for Ussing chamber analysis of
electrophysiology, and ionocytes are readily accessible to electrodes
(Marshall and Bellamy 2010).
In embryonic and larval fishes, ion transport is strictly cutaneous until the
gill develops. Systemic regulation of ion and water balance begins during
the early embryonic stages, when ionocytes appear in the skin and yolk sac
epithelium. These cutaneous ionocytes increase in abundance as
development proceeds, peaking as ionocytes first appear in the developing
gill. As the gill develops, the abundance of branchial ionocytes increases,
while that of cutaneous ionocytes falls (Figure 5.3). Correspondingly, ion
transport activity shifts from the skin and yolk sac epithelium to the gill.
Although the timing of this ontogenetic trajectory is species specific,
cutaneous ionocyte density peaks during larval development, and by the
juvenile stage, fishes are reliant on branchial ion transport (Varsamos et al.
2005; Rombough 2007). The transition from cutaneous to branchial ion
transport reflects surface area-to-volume constraints (Rombough 2007). As
the fish grows, the relative ionoregulatory capacity of the skin falls due to
the declining surface area-to-volume ratio. Exacerbating this situation are
two additional factors. First, the skin becomes thicker, making it more
difficult for surface ionocytes to achieve transepithelial transport. Second,
the period of rapid growth after hatching greatly increases the demand for
ions that, for FW fishes, must be acquired from the environment. The
transfer of ionoregulatory function from the skin to the gill overcomes these
limitations. Indeed, the gill is required for ionic regulation earlier in
development than it is required for gas transfer (Fu et al. 2010), which led
to the hypothesis that early gill development is driven by requirements for
ionic regulation (‘ionoregulatory hypothesis’) rather than gas transfer
(‘oxygen hypothesis’; Rombough 2007). Importantly, the cutaneous
ionocytes of larval fishes, particularly those of larval zebrafish (Danio
rerio), have emerged as a valuable model for investigating mechanisms of
active ion uptake in FW fishes (Evans 2011; Guh et al. 2015).

FIGURE 5.3 (a) Light micrograph of the lateral body and yolk sac of a larval zebrafish
(Danio rerio) at 4 days-post-fertilization. Immunofluorescent markers identify three
ionocyte types: green cells take up Na+ and excrete H+, red cells take up Na+ and Cl−,
and blue cells take up Ca2+. Scale bar = 1 mm. (b) Changes in cutaneous versus
branchial ionocyte abundance during early development of tilapia (Oreochromis
mossambicus).
(Panel a: image courtesy of E. Kunert. Panel b: figure modified from Rombough, P.,
Comp. Biochem. Physiol. A, 148, 732–742, 2007.)

5.3.2 GILLS
The fish gill is a structurally complex, multifunctional organ that serves as
the primary site of gas transfer as well as body fluid ion, water and pH
regulation and nitrogenous waste excretion. Anatomically, the gills consist
of paired arches on the pharynx that support rows of filaments, from which
arise small, plate-like lamellae (Evans et al. 2005). Blood flowing through
the lamellae is separated from water flowing over them by a branchial
epithelium that provides the extensive surface area, high permeability and
thin diffusion barrier necessary for efficient gas transfer. However, the
structural features that enhance gas transfer also favour diffusive ion and
water movements. Gill morphology therefore represents an
‘osmorespiratory compromise’ in which lamellar surface area and the
thickness of the lamellar epithelium have been selected through
evolutionary time to balance the minimum rate of O2 transfer needed for
metabolism against the maximum rate of passive ion and water transfer that
can be tolerated (Gonzalez 2011). To manage the osmorespiratory
compromise, some fish species use reversible gill remodelling. With this
strategy, the lamellar epithelium is covered by a cell mass to reduce passive
ion and water movement when O2 availability is high and O2 demand is
low, and the cell mass is shed to increase O2 uptake in response to exercise,
hypoxia or warm temperatures (Gilmour and Perry 2018).
Remodelling of the branchial epithelium itself can occur to meet
increased requirements for active ion transport or to respond to acid–base
challenges (Perry and Laurent 1993). The ionocytes of the branchial
epithelium are mitochondrion rich and relatively sparse, accounting for
<10% of the epithelial surface area. Because of their spherical shape,
increases in ionocyte abundance can cause thickening of the epithelium
that, in accordance with the osmorespiratory compromise, impairs gas
transfer and hypoxia tolerance (Perry 1997). Ionocytes exhibit a highly
folded basolateral membrane enriched in Na+,K+-ATPase (NKA), and more
generally, the apical and basolateral membranes of ionocytes express
distinct complements of ion-transporting proteins. Broadly, the branchial
ionocytes of marine fishes carry out NaCl secretion, whereas those of FW
fishes actively take up Na+, Cl− and Ca2+ from the dilute environment.
Ionocytes also play a key role in acid–base regulation. The structural
features of branchial ionocytes differ among teleost, elasmobranch and
agnathan fishes (Evans et al. 2005). Teleost ionocytes have been studied
most extensively and therefore are the focus of the following discussion.
5.3.2.1 Freshwater Fishes
Classic studies of ion uptake in FW fishes (reviewed by Evans et al. 2005;
Evans 2011; Gilmour and Perry 2009) indicated that Na+ uptake is linked to
H+ secretion and Cl− uptake is linked to HCO3 − secretion. As CO2 crosses
the gill by diffusion, some portion is hydrated to H+ and HCO3 −, which
serve as counter-ions in ion uptake mechanisms. Due to this coupling of
NaCl uptake to secretion of an acid–base equivalent, the FW gill is essential
for acid–base regulation as well as ion balance (Evans et al. 2005; Gilmour
and Perry 2009). Ionocyte contributions to ion uptake have been recognized
since the early 1960s (Dymowska et al. 2012), but the last 15 years have
yielded major advances. Through immunohistochemistry and in situ
hybridization, transporters have been localized to specific cell populations.
Techniques such as scanning ion-selective electrodes (Lin et al. 2006) and
isolation of specific cell populations by magnetic separation (Galvez et al.
2002) or laser capture microdissection (Leguen et al. 2015) provided insight
into ionocyte function. The coupling of such approaches with loss-of-
function technologies proved particularly powerful (Horng et al. 2007).
Across the handful of species examined to date, there are common features
but also marked differences. What is clear, however, is that multiple
ionocyte types differing in transporter complement and hence function are
present in the branchial epithelium (Figure 5.4).
 FIGURE 5.4 Generalized ion uptake mechanisms for (a) four types of ionocyte found
in the gill of freshwater teleost fish and (b) the NaCl-secreting ionocyte of the marine
teleost fish gill. ATPases are indicated by black fill and facilitated diffusion mechanisms
by white fill.
NHE: Na+,H+ exchanger; NCC: Na+,Cl− cotransporter; CA: carbonic anhydrase.

An ionocyte capable of Na+ uptake linked to H+ excretion is present in all

species studied to date and expresses apical Na+/H+ exchanger (NHE)
and/or vacuolar-type H+-ATPase (VHA). This ionocyte increased in
abundance in fish experiencing low Na+ or acidosis (Chang et al. 2013;
Brannen and Gilmour 2018), suggesting a role in both Na+ uptake and acid–
base regulation. A second ionocyte that contributes to Na+ uptake is present
in several species. This ionocyte expresses an apical Na+,Cl− cotransporter
(NCC) and therefore also contributes to Cl- uptake but probably not acid–
base regulation (Kumai and Perry 2012). Knowledge of ionocytes involved
in Cl− uptake linked to HCO3 − secretion (and hence acid–base regulation)
remains incomplete. Ionocytes expressing anion exchangers are present in
zebrafish (Bayaa et al. 2009; Perry et al. 2009). In rainbow trout
(Oncorhynchus mykiss), an ionocyte type increased in response to alkalosis,
suggesting a role in HCO3 − secretion (Brannen and Gilmour 2018).
Knowledge of Ca2+-transporting ionocytes is similarly sparse, except in
zebrafish, where an ionocyte that expresses apical and basolateral Ca2+
transporters and increases in abundance in low-Ca2+ environments was
identified (Liao et al. 2007). In short, despite considerable progress in
identifying ionocytes, their transport proteins and their responses to ionic
and acid–base challenges (Evans 2011; Dymowska et al. 2012; Kumai and
Perry 2012; Takei et al. 2014; Guh et al. 2015), much remains to be learned,
particularly on the molecular mechanisms of inwardly directed ion transport
from the dilute external environment.

5.3.2.2 Marine Fishes

The branchial epithelium of marine teleosts contains an NaCl-secreting
ionocyte. Unlike FW ionocytes, which generally occur singly in the
branchial epithelium and form extensive, multi-stranded tight junctions with
neighbouring cells, SW ionocytes occur in multicellular complexes with
accompanying accessory cells. The apical membranes of these cells form a
recessed crypt, which is a distinctive feature of the marine teleost branchial
epithelium (Evans et al. 2005). Shallow tight junctions occur between
ionocytes and accessory cells within the complex and are selectively leaky
to cations, a structural feature that is of functional importance for Na+
secretion (Chasiotis et al. 2012).
Salt secretion is achieved by secondary active transport of Cl− and
passive diffusion of Na+ (Evans et al. 2005). The mechanism (Figure 5.4)
relies on basolateral NKA, which keeps intracellular Na+ levels low to
create a gradient for Cl− entry coupled to Na+ via basolateral Na+, K+, 2Cl−-
cotransporter (NKCC). Chloride ions exit the ionocyte apically via a cystic
fibrosis transmembrane conductance regulator (CFTR) Cl− channel, while
Na+ leaves paracellularly via the cation-selective leaky tight junction. The
final component of the mechanism is the recycling of K+ ions that entered
the cell via NKA and NKCC by a K+ channel. In addition to the suite of
transporters for NaCl secretion, these ionocytes express apical NHE for
acid–base regulation – Na+ entry down its electrochemical gradient drives
H+ excretion. At the whole-animal level, acid and base secretion in marine
fishes are linked to Na+ and Cl− absorption, respectively, despite the
resulting ionic and osmotic burden (Evans et al. 2005). However, the
cellular and molecular mechanisms of acid–base regulation remain poorly
defined, especially for base secretion. Recent progress focused on
elasmobranch fishes, where two ionocytes contribute to acid–base
regulation (Figure 5.5): an acid-secreting cell that expresses NHE and is
enriched in NKA, and a base-secreting cell that expresses an apical anion
exchanger (pendrin) and is enriched in VHA (see Wright and Wood 2015).
Both cell types also express soluble adenylyl cyclase (sAC), an
evolutionarily conserved cellular acid–base sensor (Roa and Tresguerres
2017). In fish exposed to alkalosis, activation of sAC by HCO3 − ions
triggers translocation of VHA from cytosolic vesicles to the basolateral
membrane, thereby recycling H+ into the blood to counter the alkalosis,
while HCO3 − is exported across the apical membrane (Tresguerres et al.
2010). Whether comparable mechanisms are present in teleost gills remains
to be investigated.

FIGURE 5.5 Mechanisms of base and acid secretion by ionocytes of the elasmobranch
gill. White arrows indicate the sequence of events. Although sAC is present in the acid-
 secreting cell, its role in activating cell responses remains to be determined. sAC:
soluble adenylyl cyclase; CA: carbonic anhydrase; NHE: Na+,H+ exchanger.

Key to NaCl secretion is the elaboration of the basolateral membrane,

which houses the NKA that is the driving force for NaCl secretion.
Similarly, some FW ionocytes exhibit an elaborate basolateral membrane
enriched in NKA that helps drive ion uptake. At the molecular level,
however, there are differences in NKA between marine and FW ionocytes
(McCormick et al. 2009) and even among FW ionocytes (Guh et al. 2015).
When euryhaline teleosts move between FW and SW, not only does NKA
isoform switching occur, but so also must the complement of branchial
ionocytes. How this conversion occurs is not yet well understood, although
it seems to involve both transformation of ionocytes from one type to
another and differentiation of new ionocytes (Hiroi and McCormick 2012;
Inokuchi et al. 2017).
5.3.2.2.1 The Elasmobranch Rectal Gland
The branchial ionocytes of elasmobranch fishes contribute to acid–base
regulation, to active ion uptake in species that tolerate or inhabit FW
(Piermarini and Evans 2001), and probably to NaCl secretion in SW (see
Wright and Wood 2015). In marine elasmobranchs, the rectal gland also
secretes NaCl by producing a fluid that is iso-osmotic with plasma but only
contains NaCl (Wright and Wood 2015). The rectal gland is a small,
digitiform organ near the distal end of the intestine. It consists of secretory
tubules that drain into a central canal and from there, to the intestine.
Making up the secretory tubules are ionocytes that secrete Na+ and Cl−
using the mechanism described earlier. Although loss of the rectal gland
under normal conditions may have little impact on ion balance, rectal gland
NaCl secretion contributes to clearing salt and/or volume loads such as
those that accompany ingestion of SW during feeding. The secretory
activity of the rectal gland is under both nervous and hormonal control
(Anderson 2015).
5.3.3 KIDNEY
In fishes, the gill is responsible for the majority of active transepithelial ion
transport, but the kidney contributes to osmoregulation as well as waste
excretion, detoxification, hormone production and acid–base regulation.
The two main osmoregulatory functions of fish kidneys are water excretion
(urine production) and the secretion of divalent ions (chiefly Mg2+, Ca2+,
PO4 3− and SO4 2−).

5.3.3.1 Freshwater Fishes

FW fishes actively counter the passive diffusion of water into the body by
excreting large volumes of hypoosmotic urine. For instance, FW-acclimated
European eels (Anguilla anguilla) excrete 1.1–3.5 ml urine/kg/h compared
with only 0.25–0.6 ml/kg/h in SW-acclimated conspecifics (Hickman and
Trump 1969). FW lampreys have even higher urinary excretion rates of 15–
20 ml/kg/h (Logan et al. 1980). Urine production reflects high rates of
glomerular filtration and ion reabsorption. Glomerular filtration is size
selective, with larger molecules such as organic ions being retained. This
property affords a mechanism for selectively retaining organic osmolytes to
balance diffusional loss of ions. In addition, FW fishes actively reabsorb
most of the smaller ions that enter the glomerular filtrate; for example, only
5–20 mM sodium is lost in the urine (Evans and Claiborne 2009).
Reabsorption across the epithelial cells of the nephron (and the urinary
bladder) relies on basolateral NKA, which generates a Na+ gradient that
drives Na+-coupled transepithelial movement of Cl−, divalent ions and
organic solutes. For example, glucose is reabsorbed via Na+-glucose
cotransporters.

5.3.3.2 Marine Fishes

Hagfish are essentially isosmotic to SW but regulate the concentration of
divalent ions. In particular, Mg2+ and SO4 2− are actively excreted by
myxinid kidneys (Beyenbach and Liu 1996; Beyenbach 2004). Hagfish
have well-developed renal glomeruli used for selective filtration of water
and small molecules and retention of larger, energy-rich molecules. Marine
lamprey (P. marinus, euryhaline species in SW) and teleosts, which
osmoregulate in SW, have kidneys that are functionally similar. Divalent
ions are excreted, and water is reabsorbed along with NaCl to minimize
urinary water loss. Water conservation is facilitated by reduced glomerular
filtration rates. For example, when the euryhaline Lampetra fluviatilis was
acclimated to SW, urine volume fell by 95% compared with FW-acclimated
conspecifics; this was accomplished through increased water (and NaCl)
reabsorption and decreased glomerular filtration rate (Logan et al. 1980).
The kidney of marine elasmobranchs reabsorbs almost all urea, TMAO,
and other organic osmolytes (e.g. free amino acids) lost from the plasma by
glomerular filtration (Hickman and Trump 1969). Such reabsorption is
critical to maintain the high concentrations of these compounds needed to
achieve isosmolality with SW at a greatly reduced plasma NaCl
concentration. Urea transporters and presumably sodium-coupled amino
acid transporters facilitate the reabsorption of these organic osmolytes
(Schmidt-Nielsen and Rabinowitz 1964; Friedman and Hebert 1990).
Marine teleosts lack much of the distal tubule that is responsible for NaCl
reabsorption in FW fish. The functional priority of the kidney in marine
teleosts is water retention, which has promoted the evolutionary loss of the
glomerulus in several species. Unexpectedly, some aglomerular species are
euryhaline, such as the oyster toadfish Opsanus tau (Lahlou et al. 1969),
although these species may not tolerate dilute environments for extended
periods. Because hagfish exhibit glomerular kidneys, the aglomerular
kidney of some marine teleosts represents a secondary, derived trait rather
than an ancestral condition (Ditrich 2005). Water reabsorption is regulated
by aquaporins (water channels), with salinity-dependent changes occurring
in the expression of aquaporin isoforms in specific nephron segments
(Lignot et al. 2002). The kidney in marine teleosts must also excrete excess
divalent ions. This is accomplished by active transport processes driven
primarily by NKA and VHA with secondary active Na+- and H+-coupled
exchangers facilitating the movement of divalent cations (Evans and
Claiborne 2009). These transporters concentrate divalent ions into the small
volume of urine excreted by marine teleosts; for example, 80 mM for SO4
2− and 140 mM for Mg2+ (Marshall and Grosell 2006).

5.3.4 GASTROINTESTINAL TRACT

The fish GIT consists of the oesophagus, stomach and various segments of
the intestine, each of which contributes to osmoregulation. The function of
the GIT differs between FW and marine fishes. In FW fishes, which avoid
drinking, the GIT functions to absorb ions along with nutrients taken up
with the diet. The dietary availability of osmolytes is variable, and the
uptake of essential ions via the diet probably represents a feedback
parameter for controlling appetite in FW fishes. Thus, feeding and
ionoregulation are intimately linked in FW fishes because the diet
represents a major source of ions in the dilute aquatic environment; dietary
uptake of ions reduces the need for branchial ion uptake. Dietary ion uptake
may be particularly important in FW carnivores that ingest prey rich in
organic and inorganic ions.

5.3.4.1 Marine Fishes

Marine osmoregulators (lamprey and teleosts) drink up to 1% of their body
weight in SW per hour, with about 75% of the ingested water being
absorbed via GIT epithelia to counteract passive dehydration (Rankin
1997). Active absorption of NaCl from ingested SW generates an osmotic
gradient that is harnessed to move water from the GIT into the body.
Basolateral NKA generates a gradient for Na+ entry into the cell, and
secondary active transporters use this gradient to shuttle NaCl from the GIT
lumen into the epithelial cells (apical NKCC, NCC), and then to the plasma
(basolateral K,Cl-cotransporter, Cl− channels). Water follows NaCl
passively by osmosis (Figure 5.6). Aquaporins are expressed in GIT
epithelia, including oesophagus and intestine, to increase the (transcellular)
water permeability of these epithelia (Lignot et al. 2002). In addition,
paracellular water movement occurs across tight junctions (Evans and
Claiborne 2009). An apical anion exchanger also contributes to Cl− uptake,
and the HCO3 − secreted into the GIT lumen combines with divalent cations
(Ca2+, Mg2+) to form insoluble carbonate precipitates. Precipitate formation
in the GIT lumen is enhanced by increases (~4×) in divalent cation
concentrations, as water is absorbed but the divalent cations are not.
Excretion of these carbonates by marine teleosts is sufficient to contribute
substantially to the global ocean carbon cycle (Wilson et al. 2009).
 FIGURE 5.6 Ion transport mechanisms involved in water absorption across the
intestine of the marine teleost. NCC, Na+,Cl− cotransporter; CA, carbonic anhydrase;
NBC, Na+,HCO3 − cotransporter; KCC, K+,Cl− cotransporter.

5.4 EURYHALINITY
It is evident that FW and marine fishes face different osmoregulatory
challenges. Energy-dependent water and salt transport rely on disparate sets
of proteins, molecular transport mechanisms and cellular adaptations in FW
versus marine fishes. Ionocytes differentiate into distinct populations
depending on the external salinity. Furthermore, intercellular junctions,
epithelial morphology, blood circulation, drinking rates and endocrine
functions are markedly different in FW and marine fishes. Stenohaline
fishes are limited in their ability to overcome these differences; they are
adapted to either FW or SW and cannot tolerate salinity deviations that
exceed 15 g/kg from the norm (Figure 5.7). In contrast, euryhaline fishes
tolerate both FW and SW. These species are fewer in number than
stenohaline species and have multiple independent evolutionary origins
(Schultz and McCormick 2013). Even within families and genera, a mosaic
phylogenetic pattern of euryhalinity is evident (Kültz 2015).
 FIGURE 5.7 Tolerance limits of stenohaline marine (blue line), stenohaline freshwater
(FW, green line) and euryhaline fishes (red line). Euryhaline species typically exceed
the upper salinity tolerance limit of marine fishes, tolerating up to 2–4× sea water.

How did euryhalinity evolve, and what are the key physiological
innovations that permitted elaborate switches from active water
retention/salt secretion in SW to active water excretion/salt absorption in
FW? The multiple independent evolutionary origins of euryhalinity suggest
that fishes are inherently equipped with the molecular mechanisms to
support both hyper- and hypo-osmoregulation. These mechanisms are likely
pleiotropic, and other functions (e.g. acid–base regulation, nitrogenous
waste excretion, cellular and neuroendocrine stress responses) originally
drove their evolution. To understand the evolutionary and physiological
processes that facilitate euryhalinity, we must ask how fishes perceive
changes in environmental salinity, and how these osmotic signals are
processed to direct active ion transport according to the external salinity.
5.4.1 WHEN DOES NATURAL SELECTION FAVOUR EURYHALINITY?
Euryhaline fishes evolved in environments that experience large temporal
or spatial salinity gradients. Whereas stenohaline species are limited to
behavioural avoidance, euryhaline fishes can respond physiologically to
salinity fluctuations rather than being forced out of particular environments.
If behavioural avoidance is not possible, then extreme selection pressure for
euryhalinity may arise. For example, rainfall during low tide can trap
marine fishes in tide pools, where they must cope with low salinity. Desert
fishes that live in FW creeks and ponds may encounter the opposite
scenario. If the underlying bedrock or sediment contains large amounts of
salt, then evaporation during the warm season can temporarily increase
water salinity even above SW. In these examples, salinity fluctuations are
stochastic and can be sudden. Fishes must rapidly sense, process and
respond to salinity fluctuations in these environments.
Contrasting with such temporal salinity fluctuations are the spatial
salinity gradients encountered by migratory species. In this case, euryhaline
fishes voluntarily migrate between FW and SW habitats (diadromy) and can
anticipate and pre-acclimatize to salinity changes via dedicated
developmental processes (e.g. smoltification; see McCormick 1994). Two
forms of diadromy are common. Anadromous fishes begin their life cycle in
FW, migrate to SW as juveniles and return to FW as adults to reproduce.
Examples include salmonids (Salmo sp. and Oncorhynchus sp.), some
populations of three-spined sticklebacks (Gasterosteus aculeatus) and
several sturgeon (Acipenser sp.). Catadromous fishes hatch and complete
early development in SW. They enter FW or low-salinity brackish water as
juveniles to grow out before returning to the ocean to reproduce. Examples
include eels (Anguilla sp.), mullets (e.g. Mugil cephalus) and milkfish
(Chanos chanos).
Both temporal and spatial salinity gradients exist in estuaries, especially
those that are subject to large tidal currents. The salinity gradient can shift
horizontally, depending on whether incoming tidal flow or river-runoff
prevails, and seasonally with rainfall, floods and droughts. Vertical
gradients also exist because water density is affected by salinity.
Stenohaline fishes in these environments are restricted to habitat within
their physiological capacity, whereas euryhaline species inhabit a much
larger realm. For example, the Salou estuary (Senegal) exhibits an
extremely large and dynamic salinity gradient, ranging from FW to 130
g/kg, and euryhaline species such as the blackchin tilapia (Sarotherodon
melanotheron) use habitat over this entire salinity gradient (Tine et al.
2011).
Despite the evolutionary advantage of increased habitat use provided by
euryhalinity, most fish species are stenohaline. The energetic cost of
keeping osmosensory surveillance and signalling mechanisms poised to
trigger salinity acclimation processes when needed may select against
euryhalinity. The following section briefly reviews these osmosensory and
signalling processes.
5.4.2 CELLULAR MECHANISMS OF OSMOSENSING AND SIGNAL
TRANSDUCTION
Transitions between FW and SW environments necessitate a switch in
osmoregulatory mode that is completed in several stages (Foskett et al.
1981). The first stage occurs only with acute transfer between salinity
extremes and is limited in duration to approximately 1 day. It consists of
rapid termination of the prevailing active ion transport mechanisms, which
are now operating in the wrong direction, tightening of epithelia to
minimize passive water and ion movements, and activation of cell volume
regulatory and cellular stress responses to overcome temporary
dysregulation of ionic/osmotic homeostasis. Such dysregulation is evident
in transiently altered plasma osmolality when euryhaline fishes are acutely
transferred between salinities (Pavlosky et al. 2019). In the second stage,
effector mechanisms (e.g. secondary active ion transporters) that are
constitutively expressed at a minimum level are activated rapidly to operate
in the proper direction and support the new osmoregulatory demand. This
stage is completed within approximately 3 days and is characterized by
altered expression and posttranslational regulation of osmoregulatory genes
and their corresponding proteins as well as adjustments of epithelial and
ionocyte morphology (Foskett et al. 1981). The third stage takes up to a
week and consists of changes in cell proliferation and differentiation, giving
rise to distinct populations of ionocytes and altered leakiness of tight
junctions. The extent to which each of these stages is activated depends on
the magnitude and acuteness of the salinity change encountered by the fish
(Kültz 2015).
Euryhaline fishes must be able to sense environmental salinity to induce
qualitatively and quantitatively appropriate responses. This is achieved
using osmosensors, which trigger signalling cascades that activate
appropriate effector mechanisms (Figure 5.8). Fishes monitor external
salinity using both direct and indirect osmosensory input from multiple
proteins (Fiol and Kültz 2007). With indirect input, environmental salinity
changes are recognized by monitoring corresponding changes in plasma
osmolality. Osmotic imbalances in body fluids are detected at the level of
individual cells, by the epithelial cells that face the external environment
apically and the internal milieu basolaterally and by pituitary cells and
neurons. The epithelial cells of osmoregulatory tissues also use apical,
basolateral and intracellular proteins as direct molecular osmosensors.
Plasma membrane tension, plasma membrane ion transporters and water
channels, cell volume and cytoskeletal organization, protein and chromatin
structure, and macromolecular crowding all may serve as osmosensors
because they are directly impacted by changes in intracellular ionic strength
(Kültz 2001). In some cases, activation of osmoregulatory effectors is very
direct. For instance, the enzymes myo-inositol phosphate synthase (MIPS)
and inositol monophosphatase 1.1 (IMPA1.1) combine osmosensory and
effector functions in a single molecule. Abnormally high intracellular
inorganic cation levels stimulate MIPS and IMPA1.1 activity and induce a
feedback loop that replaces excess cations with the compatible organic
osmolyte myo-inositol (Villarreal and Kültz 2015). However, this very short
chain of events from sensory input to effector seems to be the exception,
and most effector mechanisms depend on signalling networks.

FIGURE 5.8 Combinatorial input into osmosensory signalling networks of fishes.

Multiple molecular osmosensors perceive osmotic stress in fish cells, including steroid
 hormone receptors (SHR), arachidonic acid-regulated Ca2+ selective channels (ARC),
annexin A11 (ANXA11), arachidonic acid (AA), phospholipase A2 (PLA2), calcium
sensing receptor (CaSR), adenylate cyclase (AC), aquaporins (AQP), transient receptor
potential vanilloid 4 cation channel (TRPV4) and cytokine receptors (CR).
(Reproduced from Kültz, D., Physiology, 27, 259–275, 2012.)

Examples of the osmosensory proteins of euryhaline fishes are depicted

in Figure 5.8 (Kültz 2012). Osmotic and ionic changes alter the
conformation and activity of these proteins, in turn activating common
intracellular signalling pathways. The combination and relative degree of
activation of these signal transduction pathways forms a signalling network
that adjusts osmoregulatory effector mechanisms to the extent needed. The
activity of osmoregulatory effector proteins may be adjusted directly
through posttranslational regulation. For example, both NKCC and CFTR
are phosphorylated by focal adhesion kinase in euryhaline killifish
(Fundulus heteroclitus) exposed to salinity stress (Marshall et al. 2009).
Increasing mRNA and protein abundance is another common mechanism
for regulating ion transporters and organic osmolyte-producing enzymes.
For example, increased salinity induces transcription of MIPS and IMPA1.1
in tilapia and eels (Gardell et al. 2013; Kalujnaia et al. 2010). These
enzymes produce the organic osmolyte myo-inositol, which compensates
for disturbances of plasma osmolality, cell volume and intracellular ion
homeostasis during a switch from FW to SW.
Recently, the cis-regulatory element (OSRE1) responsible for induction
of MIPS and IMPA1.1 was identified (Wang and Kültz 2017). However, the
transcription factor that binds to OSRE1 and links the osmosensory
signalling network to transcriptional induction of effector genes is not yet
known. Two candidates include nuclear factor of activated T cells 5
(NFAT5), which binds to a similar cis-element in osmoresponsive genes of
mammals (Lee et al. 2011), and osmotic stress transcription factor 1
(OSTF1), which has been identified in euryhaline fishes (Fiol and Kültz
2005; Tse 2014). Linking osmoresponsive transcription factors to effector
genes, on the one hand, and osmosensory signalling networks, on the other
hand, remains a formidable task for the future.

5.5 CONCLUSION
Natural selection has optimized the macromolecular structures and function
of cells to operate in an ionic milieu in which intracellular Na+
concentrations are low, K+ concentrations are high, and the concentrations
of divalent ions (e.g. Ca2+, Mg2+) are in the micro- to low millimolar range.
In multicellular animals, the cells are exposed to extracellular fluids that are
isolated from the external milieu by an epithelial cell layer. Different
strategies of achieving intra- and extracellular ionic and osmotic
homeostasis have evolved to support cell function across a wide range of
environmental ion concentrations. Fishes exhibit a spectrum of strategies
ranging from iono- and osmoconforming to iono- and osmoregulating. The
present chapter presented an overview of the ionic and osmotic strategies of
the major fish groups, reviewed the structure and function of iono- and
osmoregulatory tissues, and considered the challenges faced by fishes that
encounter environmental salinity fluctuations in space and/or time.

ACKNOWLEDGEMENTS
Research of the authors was supported by NSF grant IOS-1656371 (DK)
and NSERC DG RGPIN-2017-05487 (KMG).

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 6 The Digestive System
Carol Bucking and W. Gary Anderson

CONTENTS
6.1 Overview
6.2 Primary Function of the Digestive System
6.3 Digestive System Morphology
6.3.1 Buccal Cavity, Pharynx, and Associated Structures
6.3.2 Oesophagus
6.3.3 Stomach
6.3.4 Intestine
6.3.5 Colon and Rectum
6.3.6 Associated Organs
6.3.7 Microbiome
6.4 Future Perspectives
Acknowledgements
References

6.1 OVERVIEW
The primary function of the digestive system is to deliver fuels and building
blocks to sustain life and to eliminate wastes. This occurs via anatomical
structures that reflect the specific function they provide to the organism.
From a broad perspective, the digestive system is a simple tube connecting
the organism to the environment, delivering supplies brought in via the
buccal cavity and eliminating wastes via the anus, most simply illustrated
by the gross anatomy of the hagfish digestive tract (Figure 6.1). This
conduit can have several names, including the gastrointestinal tract (GIT),
the alimentary canal, the digestive tract, or more colloquially, the gut. The
connection to the environment and essential provisioning of fuels to the
body drive adaptations within the digestive system across many biological
levels, from molecular and cellular to morphological, much of which will
be the focus of this chapter.

FIGURE 6.1 Gastrointestinal tract of the Pacific hagfish, Eptatretus stoutii. FG:
foregut; HG: hindgut.
Photo credit Alyssa Weinrauch.

6.2 PRIMARY FUNCTION OF THE DIGESTIVE SYSTEM

The primary function of the digestive system, digestion, can be defined as
the mechanical, chemical, and enzymatic breakdown of food into
metabolizable substances that are subsequently absorbed and distributed for
use by the body. The type of food consumed can be used to distinguish and
categorize fish as detritivores, herbivores, carnivores, or omnivores. These
distinctions are often extremely informative, as the type of food consumed
frequently drives structural and biochemical adaptations.
The mechanical breakdown of food is primarily achieved by the action of
specialized teeth and/or other grinding or muscular surfaces, and is a
species-specific occurrence that is largely shaped by dietary niche. Indeed,
the morphology of teeth can provide an indicator of dominant diet and can
be used to model trophic diversification (Hellig et al., 2010). Teeth in fishes
are not restricted to the dentary or oral jaw but can also be found on
pharyngeal jaws and/or the vomer or palatine bones, such as in the lingcod,
Asemichthys taylori (Galloway et al., 2016). In the case of stomachless fish
(discussed shortly), compensation in the form of pharyngeal teeth or a
gizzard is often present to provide mechanical breakdown (Fänge and
Grove, 1979).
Hydrochloric acid (HCl), one of the chemical components of digestion
and produced in the stomach, is necessary for activating the zymogen
pepsinogen into the active proteolytic enzyme pepsin, responsible for the
cleavage and breakdown of proteins. The strong acid also lowers the pH of
the stomach (e.g. Nikolopoulou et al., 2011), providing acidic catabolism
and denaturation of ingested material. The genes encoding pepsinogen as
well as the H+-ATPase responsible for HCl formation are expressed in
gastric glands of the fish stomach, specifically the singular acid (oxyntic)-
and pepsinogen (peptic)-secreting oxynticopeptic cells (e.g. Gawlicka et al.,
2001). The secretion of both HCl and pepsinogen appears to be species
specific (e.g. Bucking and Wood, 2009 vs. Nikolopoulou et al., 2011) and is
likely modified by abiotic factors such as feeding patterns and/or circadian
rhythms (e.g. Yúfera et al., 2012). Further along the GIT, bile is required for
efficient lipid digestion (Rørvik et al., 2000), providing emulsifiers for
hydrophobic nutrients, forming micelles for transport, and activating
enzymatic catabolism.
Digestion represents the catabolism of carbohydrates, proteins, and/or
lipids into easily absorbed nutrients (i.e. sugars, amino acids, and fatty acids
and glycerol, respectively), and each type of macromolecule has specific
enzymes designed for efficient catabolism. A review of enzyme locations
and functions in the fish GIT is found in Bakke et al. (2011). Not
surprisingly, the enzyme expression profile found in the gut is often diet
specific, with herbivorous and omnivorous fishes tending to have higher
ratios of protease:amylase activities compared with carnivorous fishes
(Hidalgo et al., 1999). However, caution is urged against forming overly
broad generalizations, as a lack of correlation with diet has also been
detected (Chakrabarti et al., 1995), and phylogeny has been presented as a
major driver of enzyme expression patterns over diet (German et al., 2004).
Finally, the enzymatic components of digestion can be endogenously
produced by the enterocytes, liver, and/or pancreas, or in some cases
obtained from exogenous sources (e.g. cellulase; Kuz’mina, 2008; Castillo
and Gatlin, 2015).
 Once catabolized, the nutrients are then transported across the
gastrointestinal epithelium. Transcellular transport (across the apical and
basolateral membranes of the enterocytes lining the GIT) is supported by a
diverse and plentiful array of transporters. Paracellular diffusion is possible
but considered to be insignificant (Oxley et al., 2007). Generally speaking,
sugars are transported by Na+-linked transporters such as SGLTs or
channels such as GLUTs, while amino acids and di- and tri-peptides are
transported through specific amino acid and peptide transporters. Lipid
transport is less well studied but is assumed to progress as in mammals,
with diffusion or facilitated transport of fatty acids released from micelles
upon contact with the intestinal mucosa. Nutrient transport is a well-
covered field with numerous reviews and books (e.g. Bakke et al., 2011;
Halver, 2013), and thorough discussion is beyond our scope here.
Digestion is affected by numerous factors beyond the explicit enzyme
and transport activities. The rate at which food is captured will determine
the rate at which it is provided to the system for processing in both teleosts
(e.g. Higham, 2011) and elasmobranchs (e.g. Wilga and Ferry, 2015). Once
it is captured, the rate of processing (g of digesta g−1 of body mass−1 h−1)
can be controlled by the animal and/or set by the food (e.g. Jackson et al.,
1987). Gastric evacuation rate investigations have revealed a variety of
patterns, including exponential, linear, and logarithmic models (e.g. Jobling,
1987). Using these models alongside physiological experiments, GIT
evacuation rates were shown to be inversely proportional to predator size
(e.g. reviewed by Bromley, 1994; Gillum et al., 2012). The anatomy of the
GIT also affects digestion rates, with narrow sphincters restricting and
controlling digesta movement (Edwards, 1971; Kionka and Windell, 1972).
Further, the length and/or diameter of the intestine can control digestion
times (MacDonald et al., 1982); indeed, a larger intestine diameter or length
may represent an adaptation to increase nutrient assimilation in many
herbivores for more efficient digestion (Sibly and Calow, 1986; Munoz and
Ojeda, 2000).

6.3 DIGESTIVE SYSTEM MORPHOLOGY

The functions of the digestive system are supported through various
anatomical organs that combine to form the organ system with a ‘front end’
– mouth and buccal cavity, which is responsible for capture and mechanical
processing of food, and a ‘back end’ – oesophagus to rectum, which is
associated with digestion, assimilation of nutrients, and removal of waste. It
is important to note that evolution of ‘back-end’ function would necessarily
influence ‘front-end’ form and function, and vice versa. Here, we will focus
on the back end of the gut, summarizing centuries of investigation in fish
gut anatomy and physiology. We refer the reader to more in-depth reviews
of the physiology of the fish gut (e.g. Al-Hussani, 1949; Harder, 1975;
Clements and Raubenheimer, 2005; Wilson and Castro, 2011) and include
recent examinations of functional relevance and evolution for some of the
more intriguing modifications of the fish gut.
With more than 30,000 species of fishes (Nelson et al., 2016), there is an
amazing array of modifications related to environment, trophic niche,
dominant diet, and life stage, with a number of examples of convergent
evolution (a trait that has evolved independently in different species) of key
structures within the GIT (Wilson and Castro, 2011; Agyriou et al., 2016).
Teleosts occupy a variety of trophic levels and ecological niches, thus
demanding a variety of specific functions from their digestive systems
(Kapoor et al., 1975; Fänge and Grove, 1979; Wilson and Castro, 2011).
Conversely the gut of elasmobranchs is relatively anatomically uniform
despite a broad range in dietary preferences across species (Bucking, 2015).
That said, where gross morphology may not reveal specializations to the
naked eye, it is safe to assume that differences must exist. Several accessory
organs, such as the liver, gallbladder, and pancreas, are associated with the
GIT, completing the digestive system (Table 6.1). With exciting
advancements being made regarding the contribution of the bacterial
communities in the gut to the physiology of fishes, more recently under
consideration as an accessory organ is the GIT microbiome (Table 6.1).
Table 6.1 Overview of Structure and Function of the Digestive System

Structure Function
Buccal Cavity and Filtration, mechanical digestion, mucus production, nervous signalling,
Oesophagus osmoregulation, respiration
Stomach Absorption, chemical and enzymatic digestion, hormonal and nervous
signalling, pathogen protection, respiration
Intestine Absorption, chemical and enzymatic digestion, hormonal and nervous
 signalling, respiration
Rectum and Colon Defecation, final processing of contents
Accessory Organs

Liver Chemical and enzyme production and secretion

Gallbladder Storage and release of enzyme and chemical components for breakdown of
macromolecules
Pancreas Exocrine – enzyme production and secretion Endocrine – production of
hormones involved in regulation of carbohydrate, amino acid, and lipid
uptake

Intestinal Enterocyte proliferation, enzyme production, nutrient

Microbiome transport and storage, gene expression regulation,
immunity

6.3.1 BUCCAL CAVITY, PHARYNX, AND ASSOCIATED STRUCTURES

There is a vast array of information on the anatomy of the head and
cranium, and detailed reviews are available on the morphology and feeding
mechanics in fish (e.g. Higham, 2011; Wilga and Ferry, 2015). Mucus
produced in the buccal cavity may play a role in trapping food particles in
suspension-feeders (Sanderson et al., 1996), but there is little evidence of
enzymatic catabolism for digestion (Al-Hussani, 1949) as seen in higher
vertebrates. Posterior to the buccal cavity is the pharynx, containing the gill
slits located in the ventrolateral walls. There are nutrient digestion
processes supported here by anatomical adaptations in the form of gill
rakers for filtering and trapping food particles. These structures are of
particular importance in ram or filter feeding. For example, the pharyngeal
epithelium of the basking shark contains papillae that may additionally aid
in food particle capture and movement (Matthews and Parker, 1950).
Finally, the buccal cavity contains taste buds, acting as peripheral sense
organs and transmitting information to the central nervous system, which
show ultrastructural heterogeneity among taxa (Reutter et al., 2000).
6.3.2 OESOPHAGUS
The oesophagus is a short tube-like structure connecting the buccal cavity
with the rest of the intestinal tract (see Figures 6.2 through 6.4). Entry into
the oesophagus is controlled by a sphincter that is closed except when
passing food. There is usually a proximal and a distal portion in the
oesophagus, which serve differing functions, often related to diet and/or life
history. One universal feature of the oesophagus in fishes is numerous
mucus-secreting cells, which probably lubricate the mucosa, aiding the
passage of food in addition to protecting the oesophageal epithelium from
chemical and/or physical damage as food passes (Yamamoto and Hirano,
1978). The oesophagus also plays a role in desalination of imbibed seawater
in marine teleosts, reducing the osmolality from ~1000 to 500 mOsm.L−1,
absorbing NaCl at an approximate ratio of 1:1 (reviewed in Grosell, 2014).
The transport mechanisms of either sodium or chloride in this region of the
marine teleost intestine are not well understood, but recent identification of
sodium binding by the mucus (Wong et al., 2018) has revealed a potential
novel mechanism for the removal of sodium from imbibed seawater. This
substantial contribution to the desalination of imbibed seawater in marine
teleosts is clearly linked to the overall osmoregulatory function of the gut in
marine teleosts (reviewed in Grosell, 2014).

FIGURE 6.2 Gastrointestinal tract of the ratfish, Hydrolagus colliei. C: colon; E:

oesophagus; SV: spiral valve.
Photo credit Alyssa Weinrauch.
FIGURE 6.3 Gastrointestinal tract of the North Pacific spiny dogfish, Squalus suckleyi.
C: colon; CS: cardiac stomach; E: oesophagus, P: pylorus; PS: pyloric stomach; RG:
rectal gland; SV: spiral valve.
Photo credit Alyssa Weinrauch.
 FIGURE 6.4 Gastrointestinal tract of the lake sturgeon, Acipenser fulvescens. AI:
anterior intestine; CS: cardiac stomach; E: oesophagus, P: pylorus; PC: pyloric caeca;
PS: pyloric stomach; SV: spiral valve.
Photo credit Alyssa Weinrauch.

The oesogastric region connects the oesophagus to the stomach and can
be a valve-like structure, as observed in some elasmobranchs, but in most
actinopterygians (ray-finned fishes), there is an absence of any valve
structure; there is simply an abrupt transition from non-glandular tissue to
gastric glandular tissue (Kapoor et al., 1975; Fänge and Grove, 1979).
6.3.3 STOMACH
Posterior to the oesophagus is a gastric region, which may or may not
contain a stomach. The agastric phenotype is found throughout the fishes
and is one example of convergent evolution, as it is found in lampreys and
hagfish (Agnathans; Figure 6.1), ratfish and rabbitfish (Chimera); Figure
6.2), lungfish (Lipidosireniformes), and close to 20% of the ray-finned
fishes (Wilson and Castro, 2011). Using genomic and transcriptomic
mapping, almost all genes associated with gastric gland function were
found to be absent in the stomachless ballan wrasse, Cheilinus undulatus
(Lie et al., 2018). Why stomach loss has repeatedly occurred in the fish
lineage is at present unknown. Possible hypotheses were put forward by
Wilson and Castro (2011); however, in each of their hypotheses, Wilson and
Castro (2011) provide examples of species and/or mechanisms that are
counter to their arguments, underscoring the need for continued research to
understand the driving factors in the evolution of stomach (and indeed, gut)
form and function in the fishes. Importantly, the agastric phenotype is
absent in the sharks, skates, and rays (Elasmobranchii) and non-teleostean
ray-finned fishes: bichir, gar, sturgeons, paddlefish, and bowfin. Curiously,
all these clades possess a spiral valve as a major, or minor (Figures 6.3 and
6.4), component of their intestine.
When present, the stomach is used for storage of ingested food as well as
the initial chemical and enzymatic breakdown of food and absorption of
ions, as introduced earlier. It is the first real glandular component of the fish
gut and is a uniquely vertebrate innovation, distinguished by secretion of
gastric juice containing HCl and pepsinogen (Smit, 1968). The anatomy of
the stomach in those species that possess one varies between three forms: U
(or J or siphon shaped), Y (or caecal shaped), and straight (Wilson and
Castro, 2011). The U shape appears to be the most prevalent across the ray-
finned fishes and sharks, skates, and rays; it will normally accommodate a
large meal size and therefore could be considered a site for food storage.
There are two primary sections: the anterior/cardiac or fundic stomach,
immediately posterior to the oesophagus, is followed by the posterior or
pyloric stomach (see Figure 6.3). A Y-shaped stomach has been reported for
a number of species, including the teleosts Regalecus (oarfish) and Anguilla
(eel). The blind-ended sac or caecum is thought to act as a food storage site
(Barrington, 1957), which makes sense in the highly active predators
Scomber (mackerel), which consume large amounts of prey over a short
period when encountering shoals of bait fish (Suyehiro, 1941). The straight
or sac-like stomach is rare in fishes but has been described in the Esocidae
(pike) and the Tetraodontiformes (ocean sunfish/pufferfish) (Suyehiro,
1941).
The lumen of the gut is basically an opening to the environment, and
with the ingestion of food and/or water, there is the potential for invasion of
aquatic pathogens across the mucosa and into the fish. Mucus in fish is
recognized as a major chemical and physical barrier (reviewed in Cain and
Swain, 2011); however, gastric secretions in the stomach also serve as a
barrier to pathogenic invasion in vertebrates that may damage or interfere
with the existing biome community in the intestine (see later) or indeed, the
host itself (Martinsen et al., 2005).
The stomach may also function as an air-breathing organ. This requires
increased vascularization of the stomach wall and a substantial reduction in
the thickness of the stomach wall in comparison to non-air-breathing
species (reviewed in Nelson and Dehn, 2011). The majority of, and
certainly the most well-studied, air-breathing fishes are found in neo-
tropical zones and belong to the family Loriicaridae (Graham, 1999). The
stomach as an air-breathing organ is often accompanied by a reduction in
gastric gland activity in favour of oxygen uptake. Interestingly, the intestine
in many of these species, such as the armoured catfishes, is exceptionally
long and thus may be a compensation for the reduced chemical digestion
available in the stomach (Nelson et al., 2007).
6.3.4 INTESTINE
Posterior to the stomach (if present) is the intestinal portion of the GIT,
separated from the pyloric stomach by a circular band of muscle fibre
forming the pyloric valve or sphincter (see pylorus in Figures 6.3 and 6.4),
where the majority of chemical and enzymatic catabolism and nutrient
absorption takes place.
In species lacking a stomach, an obvious anatomical and functional
demarcation between the two may be lacking (see Figure 6.2). The portion
immediately posterior to the stomach is often referred to as the small
intestine or the anterior intestine. In most fishes studied to date, the entire
length of the post-gastric region of the intestine (intestine + caeca) is
capable of active nutrient transport (Ferraris and Ahearn, 1984; Bakke-
McKellep et al., 2000). Even the enterocytes of the most distal region
contain a brush border (Murray et al., 1996); however, such studies have not
extended to elasmobranchs. The absorption of nutrients seems to occur at a
greater rate in proximal intestinal regions as opposed to distal regions in
both in vitro (Collie, 1985; Buddington and Diamond, 1987; Jutfelt et al.,
2007) and in vivo (Diaz et al., 1997; Hernandez-Blasquez et al., 2006)
studies. Certainly, most proteinaceous-derived material appears to be
absorbed across the proximal region of the intestine (Fänge and Grove,
1979; Stroband and van der Veen, 1981), but there are, as always,
exceptions, such as in the white sturgeon, where the posterior spiral valve
(Figure 6.4) seemed to play a greater role in nutrient acquisition in
comparison to anterior regions of the intestine (Buddington et al., 1987).
The optimization of enzyme degradation of food particles and absorption
of nutrients in the intestine is largely dependent on surface area; this is most
easily achieved by simply increasing the length of the organ and/or the
surface area of the mucosa. The latter component is central to nutrient
absorption with primary, secondary, and tertiary folds in the intestinal
mucosa and the presence of microvilli at the brush border on the apical
surface of intestinal epithelial cells that can contribute up to 90% of the
total surface area in some tilapia (Frierson and Foltz, 1992). It may seem
intuitive to suggest that the length of the fish intestine should be related to
dominant diet type. For example, the lower digestibility of herbivorous
diets tends toward a longer intestine in herbivorous fishes (Clements and
Raubenheimer, 2005), with the high nutrient availability in carnivorous
diets generally resulting in a shorter intestine in carnivorous fishes.
However, fish intestinal length and mass are also related to other key
factors, such as physical space available in the abdominal cavity, allowing
for a large liver and the development of embryos in viviparous
elasmobranch species (Wetherbee and Cortés, 2012); meal size/type;
satiation state (fasted vs. satiated); fish size for some species but not others
(reviewed by Barrington, 1957); activity levels: tunafish are known to have
particularly long intestines relative to less active ambush predators such as
pike (Suyehiro, 1941); and development stage (Clements and
Raubenheimer, 2005). Thus, the gross morphology of the intestine cannot
immediately be correlated with dietary habits or trophic niche; indeed, there
are examples of strictly carnivorous, agastric species (cyprinid Aspius
aspius) (Dabrowski, 1993) or short intestine length in herbivorous species
(Horn, 1989), nor does intestinal length necessarily correlate with gut
evacuation time (Hofer, 1988).
One structure worthy of further discussion is the spiral, scroll, or roll
valve that occurred early in the vertebrate lineage (Argyriou et al., 2016). It
is absent in the derived ray-finned fishes (teleosts) but is found in non-
teleostean ray-finned fishes (Acipenseriformes, Amiiformes,
Polypteriformes, and Lepisosteiformes) and is ubiquitous in the sharks,
skates, rays, and chimeras (Harder, 1975; Wilson and Castro, 2011). In the
non-teleostean ray-finned fishes, the valve forms the posterior region of the
intestine (Figure 6.4), varying in size (Argyriou et al., 2016), whereas in the
elasmobranchs, it is the intestinal region of the gut (Figure 6.3), and in the
agastric Holocephali, the spiral portion begins approximately one-third
down the length of the gut from the oesophagus (Figure 6.2). While the
spiral valve is considered a primitive form of the intestine, its absence in the
hagfish (Figure 6.1) but presence in the lampreys, combined with recent
fossil records, provides evidence that the spiral valve is another example of
convergent evolution in the fish intestine (Argyriou et al., 2016). In the non-
teleostean actinopterygians, the anterior intestine links the stomach to the
spiral valve through a series of loops that can be long (bowfin and gar),
medium (sturgeon and paddlefish; Figure 6.4), or short (bichir). From a
functional perspective, the spiral valve shortens the total length of the
intestine; however, the potential reduction in surface area with the loss in
length is offset by the spiral, scroll, or roll arrangement of the mucosa,
resulting in a substantial increase in surface area, up to threefold in ring-
type valve structures or sixfold in conicospiral configurations (Bertin,
1958).
Naturally, the more turns in the spiral, the greater the surface area, and
attempts have been made to correlate the number of turns with the ecology
of the animal. An increase in the number of turns was historically
considered to be largely influenced by diet (Qingweng and Yuanding, 1985;
Holmgren and Nilsson, 1999; Wetherbee and Cortés, 2012). Indeed, in large
elasmobranchs that may consume considerable meal sizes, such as the great
white, Carcharodon carcharius; thresher sharks, Allopidae; whale shark,
Rhinchodon typus; and basking shark, Cetorhinus maximus, the spiral valve
has a tight ring-type structure with a large number of folds (Qingwen and
Yuanding, 1985) compared with the less compact spiral shape found in
smaller predatory sharks such as Squalus suckleyi (Figure 6.3). With
correction for relatedness, larger animals tend to have an increase in the
number of turns in the spiral valve, but there is reasonable consistency in
the number of folds within a species regardless of size (Agryriou et al.,
2016).
 The driving factors causing the evolution of the spiral valve are unclear.
One notion in elasmobranchs is a conservation of space in the coelomic
cavity to accommodate embryonic development and liver size (Moss, 1984;
Wetherbee and Cortés, 2012). It seems that space constraints for embryonic
development would not be relevant for males, and there is no evidence of
sexual dimorphism in spiral valve morphology in elasmobranchs. What is
uniform in elasmobranchs and chimeras is a large liver. The high fat content
of the liver makes it a buoyancy aid in elasmobranchs and holocephalans
that lack a swim bladder (Baldridge, 1972), and there is no evidence
indicating sexual dimorphism of liver size in these fishes, so similar
restrictions on available space in the coelomic cavity would apply
regardless of sex. However, similar space constraints (at least in terms of
liver size) do not exist in the non-teleost ray-finned fishes and therefore
cannot be used to explain the retention of a spiral valve in those fishes.
Additional structures related to digestive function include the pyloric
caecae found in many teleosts (~60% of all species; Hossain and Dutta,
1996) and a few elasmobranchs (Buddington and Diamond, 1987;
Holmgren and Nilsson, 1999). The number of caecae is species dependent
and ranges from a single blind-ended structure (Figure 6.4) to over 1000
(Suyehiro, 1941; Rahimullah, 1945). The length, diameter, and shape of
caecae vary greatly across the fishes; however, prevailing evidence suggests
that function is similar (absorption of nutrients and ions) across those
species that have them (Buddington and Diamond, 1987). Investigation into
the role of caecae in rainbow trout revealed functional heterogeneity both
along the gut (from anterior to posterior) as well as along the individual
caecae from proximal to the intestinal lumen to the distal tip (Williams et
al., 2019). Pyloric caecae are another example of convergent evolution in
the fish gut.
6.3.5 COLON AND RECTUM
The colon and rectum connect to the anus, which forms the posterior valve
to the environment and controls the elimination of wastes through
defecation. The function of this region of the intestine is not as clear as in
mammals, but roles in water absorption (e.g. Bucking and Wood, 2006) and
assimilation of nutrients by endocytosis (e.g. Stroband and van der Veen,
1981) are the most clearly studied. Extensive muscular layers are found in
this section, clearly distinguishing it from the intestine. The ileorectal valve
separates the intestine from the rectum and is usually linked with a
thickening of the muscle layer and/or a reduction of the diameter of the
intestine (Harder, 1975; Kapoor et al., 1975). Further, the epithelium in the
rectum is stratified compared with the columnar cells observed in the
intestine (Holmgren and Nilsson, 1999). In elasmobranchs, there is clear
separation between the spiral valve and the rectum, and it was recently
shown that the rectum may have a role in solute balance in the little skate,
Leucoraja erinacea, prior to evacuation of gut contents into the
environment (Anderson et al., 2010).
6.3.6 ASSOCIATED ORGANS
The anterior intestine is connected to the liver and pancreas, which are
responsible for producing numerous enzymatic and chemical components
of digestion. These components are then secreted into the GIT lumen,
where they exert their effects via several ducts. The liver produces bile,
which is stored in the gallbladder before secretion into the anterior intestine
via the biliary duct. Bile production rates are generally similar across
teleosts (Grosell et al., 2000) and elasmobranchs (Boyer et al., 1976).
Control of bile secretion may be via a gastrin- or CCK-like peptide, as in
other vertebrates (Andrews and Young, 1988). Gastrin and CCK-like
peptide have been detected in elasmobranch (Hansen, 1975; Vigna, 1979)
and teleost (Kurokawa et al., 2003) gastric mucosa.
The pancreas in fishes has both endocrine and exocrine functions and
varies from a discrete, easily identifiable organ in elasmobranchs and
chimeras to a diffuse network of cells embedded in the wall of the digestive
tract in many teleosts. The exocrine pancreas produces digestive enzymes
for protein (Holmgren and Nilsson, 1999) and fat (Sternby et al., 1983)
digestion and occasionally chitinases when insects and crustaceans are a
large dietary component (Fänge et al., 1979). These secretions are
stimulated by gastric acid additions to the anterior intestine (Babkin, 1929,
1933), although limited work has occurred since these early studies.
6.3.7 MICROBIOME
A microcosm of bacteria, fungi, and archaea exists in the digestive tract of
fishes. And while it is often considered to be its own system, its intimate
ties to the digestive tract in terms of both location and function necessitate
its inclusion here as an associated organ. We will focus on bacteria here, but
be aware that there are more contributors to the microbiome, such as fungi
and other unicellular organisms. The GIT microbiome bacteria can be
classified as autochthonous or allochthonous. Autochthonous bacteria
adhere to the intestine and colonize the surface and are generally thought of
as the functional microbiome. Allochthonous bacteria are transiently found
in the gut, likely due to ingestion, and do not form permanent colonies.
Work on the microbiome has exploded in the last 10–15 years, driven
mainly through technological advances in DNA sequencing. These new
studies show that there are species-specific microbiomes in fishes that are
environmentally dependent (Sullam et al., 2012) and shaped by unknown
host factors as well as age, starvation, pollutants, and diet (reviewed by
Nayak, 2010; Ghanbari et al., 2015; Wang et al., 2018). Further, it appears
that the microbial communities display heterogeneity along the tract in
terms of both number of bacteria (e.g. Navarrete et al., 2009) as well as
their identity (e.g. Zhou et al., 2009). Generally speaking, deep sequencing
has revealed that the autochthonous bacterial communities found in the fish
intestine contain an enormous variety of microorganisms (e.g. Nayak,
2010). This variety can be not only between host species but also within
host species gathered from the same geographical location (e.g. Star et al.,
2013). The fish gut can contain an average of 109 bacteria per gram of
intestinal content (Nayak, 2010), and the predominant phyla are
Proteobacteria, Fusobacteria, Firmicutes, Bacteroidetes, and Actinobacteria
(e.g. Wang et al., 2018).
We are just beginning to understand the functional importance of the fish
gut microbiome, including roles such as epithelial renewal and cell
proliferation (Rawls et al., 2004; Cheesman et al., 2011), nutrient transport
(Rawls et al., 2004; Bates et al., 2006) and storage (Semova et al., 2012;
Camp et al., 2012), as well as immunity (Galindo-Villegas et al., 2012). An
important function of the microbiome is probably to aid in nutrient
absorption by supplementing or providing digestive enzymes (Nayak, 2010;
Ray et al., 2012), and the contribution of exogenous sources of digestive
enzymes to fish has been studied for decades. For example, early studies
have revealed cellulase activity in the stomachs (Stickney and Shumway,
1974) and intestines of vertebrates (e.g. Das and Tripathi, 1991); however,
it is more than likely that this is contributed by endosymbionts like bacteria
(Saha and Ray, 1998). In fact, it appears that the microbiome is capable of
contributing numerous enzymes, including amylase, lipase, and proteases
(Ray et al., 2012).

6.4 FUTURE PERSPECTIVES

Without question, the vast majority of functional and morphological
information regarding the digestive system in fishes has come from species
of commercial importance. Understandably, describing basic gut physiology
in these species has huge financial implications for the aquaculture industry
given the need for sourcing viable food types and creating sustainable
aquaculture resources. Additionally, the advent of modern molecular
techniques has led to significant advances in understanding the role of the
microbiome. By restricting research to this small proportion of species, we
are limiting our knowledge and potentially obscuring important
evolutionary drivers and patterns. More needs to be done in terms of
understanding the molecular triggers for development of key gut structures
in non-model fishes as well as their functional relevance. For example,
three illustrations of convergent evolution of gross morphological features
of the fish gut have been highlighted: gastric vs. agastric; presence vs.
absence of pyloric caecae; and presence vs. absence of a spiral valve. As
investigation of non-commercially relevant species continues, we will be
able to unravel the functional evolution of these structures in vertebrates.
Additionally, understanding the importance of the microbiome in fish
physiology promises to yield novel and intriguing lines of research that
undoubtedly will feed into innovative applications in the aquaculture
industry, promoting nutrient uptake and growth in food fishes.

ACKNOWLEDGEMENTS
Research conducted by CB and WGA is supported by funding from the
Natural Sciences and Engineering Research Council of Canada Discovery
Grant Program. The authors thank Dr. Alyssa Weinrauch for the pictures
and associated artwork presented in this chapter and staff at Bamfield
Marine Science Centre (BMSC) for assistance with the collection and
maintenance of the marine fishes displayed in this chapter, in particular Dr.
Eric Clelland for facilitating research conducted at BMSC.

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 7 Thermal Biology
Nann A. Fangue , Anne E. Todgham and
Patricia M. Schulte

CONTENTS
7.1 Introduction
7.1.1 Thermal Strategies
7.1.2 Mechanisms of Endothermy in Fishes
7.2 Characterizing the Thermal Niche of a Fish
7.2.1 Thermal Tolerance
7.2.2 Thermal Performance
7.2.3 Thermal Compensation
7.3 Cellular and Molecular Effects of Temperature
7.3.1 Cellular Stress Response
7.3.2 Effects on Cellular Metabolism
7.3.3 Effects on Membranes
7.3.4 Temperature and Oxidative Stress
7.4 Effects on Whole-Organism Performance
7.4.1 Effects on Metabolism
7.4.2 Effects on the Cardiorespiratory System
7.4.3 Effects on Swimming Performance and Behaviour
7.5 Developmental Plasticity
7.5.1 Epigenetic Effects of Temperature
7.6 Thermal Adaptation
7.6.1 Adaptations to Constant Cold in Antarctic Fishes
7.7 Thermal Biology in a Changing World
Acknowledgements
References
7.1 INTRODUCTION
Temperature has profound effects on biological systems because it alters the
rate of chemical reactions and the stability of weak chemical bonds
(Schulte, 2015). The direct effects of temperature on the biochemical and
physiological functions of fish scale up through multiple levels of
biological organization to affect key fitness-relevant rate functions such as
population growth (Schulte, 2015). Indeed, for fishes, temperature has long
been recognized as a critical abiotic factor shaping their natural history,
distribution and abundance (Fry, 1971). Thus, it is not surprising that fishes
have evolved a diverse array of strategies to either avoid or compensate for
any detrimental effects of temperature.
In this chapter, we provide an overview of the various strategies that
fishes use to mitigate the effects of temperature change, summarize how
physiologists characterize the thermal niche of fishes, examine the
mechanisms fishes use to compensate for the effects of temperature across
levels of biological organization, and discuss how we interpret the effects of
temperature in a changing world.
7.1.1 THERMAL STRATEGIES
The thermal strategies employed by animals can be classified using four
inter-related terms (Table 7.1). All fishes have relatively low metabolic
rates compared with mammals and birds, they have little heat-retaining
insulation, and their gills, which are in direct contact with the external
environment, act as a major site of heat exchange. As a result, it is
challenging for fishes to maintain a body temperature different from that of
the environment, and the majority of fishes are poikilothermic ectotherms
(Hazel and Prosser, 1974). A few species, such as Antarctic fishes, are
considered homeothermic ectotherms because they live in environments
that are exceptionally thermally stable. Fewer than 0.1% of described fish
species are considered to be homeothermic endotherms, and most of these
species (e.g. tuna, billfish, marlin and some sharks) are regional
endotherms, with only certain parts of the body maintained above ambient
temperatures. The one known exception to this pattern is the opah (Lampris
guttatus), which is a whole-body endotherm (Wegner et al., 2015).
Table 7.1 Thermal Strategies Employed by Fishes

7.1.2 MECHANISMS OF ENDOTHERMY IN FISHES

Endothermy requires that fish increase their capacity to produce
metabolically derived heat and have mechanisms to conserve this heat by
reducing convective heat loss. In tunas and lamnid sharks, for example, red
slow-twitch aerobic swimming muscle is located close to the body core, as
opposed to laterally along the skin where heat loss is high. These fishes also
have a specialized arrangement of blood vessels that form retia mirabilia or
‘wonderful nets’ associated with locomotory muscle, viscera, brain and eye
muscles. Retia mirabilia are countercurrent heat exchangers with arterioles
and venules arranged in opposing directions, such that the heat from warm
venous blood from heat-producing tissues is transferred to the cold arterial
blood arriving from the gills (Stevens, 2011). Only the opah, a relatively
recently discovered endotherm (Wegner et al., 2015), is capable of elevating
its whole-body temperature 3–6 °C above ambient depending on body
location (Figure 7.1a). The opah uses heat-conserving retia, similarly to
other endothermic fishes, but what is unique is that the retia are located
inside fat-insulated gill arches (Figures 7.1c and 7.1d). The result is a more
uniform and warm body core (Figure 7.1b) that includes a warm heart.
 FIGURE 7.1 Body temperature, gill anatomy and vasculature in the opah, L. guttatus.
(a) In situ internal temperature profile (measurements taken ~4 to 5 cm below the skin)
for a 98.0 cm fork length (40.0 kg) opah with an ambient reference temperature of 10.5
°C. (b) In vivo pectoral muscle temperature for a 96.4 cm (39.0 kg) opah swimming at
depth. (c) Fixed left gill arch (GA) highlighting the blood vessels of the rete mirabile,
enlarged in (d) showing the alternating afferent (blue, deoxygenated) and efferent (red,
oxygenated) filament arteries forming the rete. (Modified from Wegner et al., 2015.)

The repeated evolution of endothermy in highly active fishes such as

tunas and lamnid sharks indicates that this energetically costly strategy is
probably under strong selection. The advantages conferred on these
specialized fishes through endothermy include increased biochemical
reaction rates, elevated muscle power output and capacity for sustained
swimming performance, enhanced neural conductance for the eye and
brain, and increased rates of digestion and assimilation. It is thought that
endothermy allowed these fishes to expand their thermal niche to include
colder waters (Block et al., 1993). Unlike tunas and lamnid sharks, the
independent evolution of whole-body endothermy in opah is likely tied to
the evolutionary history of the mesopelagic Lampridiformes lineage,
allowing opah to regularly exploit cold, deep waters while maintaining
elevated performance (Wegner et al., 2015).

7.2 CHARACTERIZING THE THERMAL NICHE OF A

FISH
The fundamental thermal niche of a fish species is defined as the range of
environmental temperatures over which that species can survive, grow and
reproduce (i.e. where its fitness is positive). It is challenging, however, to
measure fitness in most species, and physiologists typically characterize the
thermal niche by examining tolerance or performance across temperatures.
7.2.1 THERMAL TOLERANCE
The ability of a fish to tolerate a particular range of temperatures can be
quantified, and there are a number of excellent reviews highlighting the
advantages and disadvantages of the various methods used to assess thermal
tolerance (e.g. Beitinger and Lutterschmidt, 2011). While these methods
quantify thermal tolerance in slightly different ways, they can ultimately be
used to generate thermal tolerance polygons (Figure 7.2). Thermal tolerance
polygons display the maximum and minimum tolerated temperatures, the
effects of thermal acclimation on these temperatures, and the maximum and
minimum temperatures that can be tolerated for long periods. A thermal
tolerance polygon is thus a complete characterization of the zone of
tolerance of an organism, and polygons derived using standardized methods
can be compared directly across species.
 FIGURE 7.2 Graphical representations of the relationship between temperature and
thermal tolerance. Panels (a) and (b) show the thermal tolerance polygons for different
species (a) or biological processes (b), which are obtained by plotting the temperatures
at which a fish begins to demonstrate a specific behaviour (which are termed the critical
thermal maximum or minimum) during an acute thermal ramp for fish acclimated at a
variety of temperatures. Typical endpoints include the onset of opercular spasms or loss
of the ability to maintain equilibrium.

Species living in different environments differ in the size, shape and

position of their thermal tolerance polygons. Fishes living in variable
environments are typically eurythermal – able to tolerate a wide range of
environmental temperatures. In contrast, fishes living in environments with
relatively constant temperatures can often tolerate only a small range of
temperatures and are termed stenothermal. For example, the highly
stenothermal fishes of the Southern Ocean (such as the Antarctic icefish of
the sub-order Notothenioidei) have small thermal tolerance polygons that
are centred at very low temperatures (Beitinger and Lutterschmidt, 2011).
Most tropical warm water fishes are also stenothermal and tend to have
small thermal tolerance polygons, but they are centred at high temperatures
(Figure 7.2a).
Thermal tolerance polygons summarize the maximum and minimum
tolerance temperatures at which fishes can survive for relatively short
periods of time, but the thermal zones over which organisms can grow and
reproduce effectively are usually smaller (Figure 7.2b). Temperatures
outside these regions but within the bounds of the thermal tolerance
polygon are temperatures at which animals can survive but must divert
energy away from growth, reproduction and other activities in order to
recruit processes that allow them to survive.
7.2.2 THERMAL PERFORMANCE
The effects of temperature on performance within the range of tolerated
temperatures can be summarized in the form of a thermal performance
curve (TPC) or reaction norm (Figure 7.3) (Sinclair et al., 2016). Thermal
performance curves can be used to identify a number of important
characteristics of an organism (Figure 7.3), including the thermal optimum,
the thermal breadth (or the range of temperatures over which performance
is near optimal) and the thermal limits (or the threshold temperatures at
which performance declines below some specified level). For example,
Pörtner and colleagues (e.g. Pörtner and Farrell, 2008) have defined the
pejus (Latin for ‘getting worse’) temperature (Tp) as the temperature at
which performance declines below the maximum level (usually arbitrarily
set at ~80–90% of maximum performance) and the critical temperature (Tc)
at which performance is minimal. These parameters were originally defined
in the context of the performance curves for aerobic scope (difference
between minimum and maximum metabolic rates) (see Chapter 10), but
similar parameters can be defined in the context of any organismal
performance trait.
 FIGURE 7.3 Typical thermal performance curve. Performance curves can be plotted
for any biological rate process, including cellular processes such as enzyme activity,
organismal processes such as metabolic rate or swimming performance, or population
processes such as the intrinsic rate of population growth. Temperature (on the x-axis)
may be acute exposure temperature or acclimation temperature. The optimal
temperature (Topt) is the temperature at which performance is maximized. The pejus
temperature (Tp) is the temperature at which performance declines below a specified
percentage of the optimal performance. The critical temperature (Tc) is the point at
which performance declines to zero.

At all levels of biological organization, empirically determined

performance curves typically have a skewed normal distribution (Figure
7.3), with a much steeper slope at high temperatures than at low
temperatures. As a result of this skewed shape, many fishes die at
temperatures only a few degrees above their optimal operating temperature
(Somero, 2010). In contrast, most species can tolerate larger declines in
temperature below the optimal temperature (Topt) before reaching the Tc.
However, as most TPCs have been determined for north-temperate fishes,
the extent to which this generalization holds for tropical fishes is unclear.
The ability of fishes to maintain physiological performance in the face of
thermal variation differs among species and is largely reflective of the range
of temperatures experienced in their natural environment. Stenothermal
species, such as tropical and Antarctic fishes, can perform well across
relatively narrow thermal ranges and thus have a narrow TPC, while
eurythermal species (such as species found in salt marshes or in the
intertidal zone) can maintain performance across a much wider range of
temperatures and have much broader TPCs. But for both stenothermal and
eurythermal fishes, temperatures that are higher than the upper pejus
temperature, or lower than the lower pejus temperature, result in thermal
stress and reduced performance.
7.2.3 THERMAL COMPENSATION
If possible, fishes avoid temperatures falling outside their ideal thermal
range or thermal niche. If avoidance is not possible, there is a suite of
behavioural, physiological, biochemical and molecular responses that are
initiated in an attempt to allow the animal to ‘adjust’ to the new thermal
environment. These compensatory mechanisms result in changes to the
shape of the TPCs (e.g. a shift in the thermal optimum), which eventually
restores homeostasis. Ultimately, if a fish is unable to induce such
mechanisms or if the temperature change is too great, performance will
decline, and the fish may die.
For ectothermic fishes, exposure to temperature change causes changes
in biochemical processes and metabolism that must be compensated for in
order to maintain performance. Thus, many ectothermic fishes, particularly
those living in thermally variable environments, have biochemical
compensatory mechanisms that allow them to maintain performance across
a range of environmental temperatures. Different compensatory
mechanisms are recruited at different time-scales of exposure. Acute
thermal exposures occur because of hourly or daily fluctuations in
temperature, such as those encountered in the intertidal zone. The
compensatory adjustments associated with these exposures are part of a
generalized stress response and are particularly important for survival and
recovery from brief, near-lethal temperature exposure. Longer-term
exposure to a particular temperature regime results in the processes of
acclimation (i.e. in the lab) or acclimatization (i.e. in a natural setting),
which occur over a period of weeks or months, for example in response to
seasonal changes in temperature. The adjustments due to acclimation or
acclimatization are usually, but not always, reversible. In contrast,
developmental plasticity occurs due to thermal exposures early in
development and often results in irreversible changes in phenotype that
persist in the adult organism. These adjustments may even be passed on to
subsequent generations – a process that is termed transgenerational
plasticity. In contrast, genetic changes in populations due to thermal
selection may constitute an adaptive response, which occurs over many
generations.
Many fish species have some capacity to acclimate to environmental
temperatures that are stressful during acute exposures, but there is
substantial variation among species in the extent of this capacity. In general,
north temperate fishes have greater capacity to acclimate (Beitinger and
Bennett, 2000) than tropical and Antarctic stenotherms (e.g. Nilsson et al.,
2010; Bilyk and DeVries, 2011, although see Seebacher et al., 2005).

7.3 CELLULAR AND MOLECULAR EFFECTS OF

TEMPERATURE
Exposure to altered temperatures causes multiple effects at the cellular and
molecular level, including effects on DNA, RNA, proteins and biological
membranes (Schulte, 2015), and fishes use a wide range of mechanisms to
compensate for these effects.
7.3.1 CELLULAR STRESS RESPONSE
The cellular stress response (CSR) is an early (i.e. minutes–hours) defence
reaction at the level of the cell that is triggered by strain or damage to
macromolecules, such as DNA and proteins. There is a large suite of
proteins involved in orchestrating the CSR, aimed at sensing, repairing and
minimizing protein damage arising as a result of an environmental insult.
These cellular and molecular responses to temperature change have been
reviewed in detail by Kültz, (2005), Somero (2011) and Somero et al.
(2017).
A class of highly conserved and ancient proteins termed ‘molecular
chaperones’, particularly the heat shock proteins (HSPs), are clearly and
consistently induced at high levels in response to an acute heat stress. HSPs
are key to the CSR by interacting with other proteins to promote productive
protein folding or refolding, to stabilize cellular proteins and membranes, to
aid in the repair of damaged proteins, and to prevent deleterious protein
aggregation (Feder and Hofmann, 1999; Basu et al., 2002). If thermally
denatured proteins are unable to be remodelled to their native state, the
proteins will be degraded by proteases, primarily by the ubiquitin (Ub)-
proteasome pathway (Wickner et al., 1999). Protein degradation by the Ub-
proteasome pathway involves tagging of damaged proteins by multiple Ub
molecules and then degradation by the 26S proteasome complex. Levels of
Ub-conjugated proteins have been shown to be a good indicator of the
integrity of the cellular protein pool, with high levels of Ub-conjugated
proteins representing significant protein damage (e.g. Todgham et al.,
2017). Interestingly, variation in the E3 ubiquitin ligase gene, which
encodes one of the proteins involved in this pathway, is associated with
variation in CTMax in Atlantic killifish (Fundulus heteroclitus; Healy et al.,
2018).
The CSR is dependent on the thermal history of the cell and/or animal
(e.g. Dietz and Somero, 1992; Fangue et al., 2011). Acclimation
particularly influences the onset temperature of the CSR, and the responses
of the CSR to acclimation may be tissue specific, but together these data
point to the important effects of acclimation on the CSR. The thermal
plasticity of the CSR, and particularly of the heat shock genes and proteins,
is probably related to plasticity in the activation of the heat shock
transcription factor HSF1 (Buckley and Hofmann, 2002).
7.3.2 EFFECTS ON CELLULAR METABOLISM
A picture of the molecular and cellular response to temperature has
emerged from transcriptomic studies (e.g. Gracey et al., 2004; Lewis et al.,
2010; Logan and Somero, 2010; Windisch et al., 2011). The nature of the
gene expression response to thermal acclimation is strongly dependent on
acclimation time and the intensity of the thermal challenge (Podrabsky and
Somero, 2004). After several weeks of thermal acclimation, the primary
adjustments at the transcriptomic level appear to be shifts in the expression
of metabolic genes consistent with alterations in fuel sources (Windisch et
al., 2011) and other adjustments to central metabolism (Seebacher et al.,
2012). Alterations in the expression of genes associated with ion transport
and protein biosynthesis have also been observed (Logan and Somero,
2010). In addition, targeted studies of specific genes suggest that there are
substantial changes in RNA processing and translation between warm- and
cold-acclimated fish (Alvarez et al., 2007; Healy and Schulte, 2019).
In comparison to transcriptomic studies, comprehensive studies of the
acclimation response using proteomics are rare. The general message from
these few studies is that proteomic changes appear to be less extensive than
transcriptomic changes (e.g. Kültz and Somero, 1996; Klaiman et al.,
2011); however, transcriptomic and proteomic techniques differ in
sensitivity, and thus the extent to which these different patterns are the
result of technical issues remains unknown. Responses to thermal
acclimation at the protein level have also been examined using biochemical
approaches and have revealed great variety in the responses of specific
enzymes to thermal acclimation (Hazel and Prosser, 1974; Pierce and
Crawford, 2012). Taken together, these data point to the importance of
adjustments in central metabolism for long-term thermal acclimation.
Thermal acclimation also causes changes in mitochondrial amount. In
particular, cold acclimation is often associated with an increase in the
density of mitochondria (O’Brien, 2011), which is thought to offset the
decreases in mitochondrial activity at low temperature and maintain energy
production. Moreover, increasing the number of mitochondria provides
additional pathways for oxygen diffusion through lipids, which is much
more efficient than diffusion of oxygen through the cytoplasm. The extent
of increased mitochondrial biogenesis in the cold varies among tissues,
among species and even among populations of a single species (O’Brien,
2011).
7.3.3 EFFECTS ON MEMBRANES
Plasma and intracellular membranes are substantially influenced by
temperature, with temperature affecting the fluidity and integrity of
membranes (Somero et al., 2017). Compensatory responses to these effects
include changes in membrane phospholipid composition and cholesterol
content (Hazel, 1995). These changes in plasma membrane saturation are
driven in part by changes in the expression of desaturase enzymes, in
particular the Δ9 desaturase (e.g. Gracey et al., 2004). Mitochondrial
membranes also show clear changes in lipid composition with thermal
acclimation that are thought to maintain membrane fluidity (Chung et al.,
2018).
7.3.4 TEMPERATURE AND OXIDATIVE STRESS
Acute exposure to temperatures above a fish’s thermal preference and niche
can lead to the generation of excess reactive oxygen species (ROS). ROS
are a natural byproduct of metabolism, but if allowed to accumulate, they
trigger oxidative damage to lipids, proteins and nucleotides (reviewed in
Crockett, 2008). In an attempt to protect against this oxidative damage, fish
synthesize and mobilize a suite of antioxidant enzymatic defences such as
superoxide dismutase, catalase and glutathione peroxidase.
Several lines of evidence suggest that exposure to cold may also result in
increased oxidative stress. For example, the change in mitochondrial
membrane fluidity that occurs with acute cold exposure may result in a
decrease in the efficiency of oxidative phosphorylation, causing an increase
in the production of ROS (Kammer et al., 2011). In addition, changes in
membrane composition that occur with cold acclimation may increase
susceptibility to oxidative stress (Crockett, 2008). Pathways that are
protective against oxidative stress are also upregulated in the cold (e.g.
Kammer et al., 2011).

7.4 EFFECTS ON WHOLE-ORGANISM PERFORMANCE

7.4.1 EFFECTS ON METABOLISM
Acute increases in water temperature increase the aerobic metabolic rate in
fishes up to a point, after which the metabolic rate declines rapidly.
Temperature affects both standard (basal) metabolic rate and maximum
metabolic rate in fishes, and the shapes of these curves are not necessarily
the same. Indeed, the mismatch between the effects of temperature on
standard and maximum metabolic rate can result in strong effects of
temperature on aerobic scope, defined as the difference between standard
and maximum metabolic rate (also see Chapter 10).
It has been suggested that the effects of temperature on aerobic
metabolism and aerobic scope are related to challenges with oxygen supply.
This idea, first formulated by Fry (see Fry, 1971), was more recently
formalized into the ‘oxygen limitation hypothesis’ or ‘oxygen and capacity-
limited thermal tolerance’ (OCLTT; Pörtner, 2010; detailed also in Chapter
10). This hypothesis suggests that beyond the upper pejus limits, oxygen
supply becomes limiting, while demand increases. This results in decreases
in blood oxygen (hypoxaemia) leading to declines in animal performance.
At critical temperatures (Tc), anaerobic metabolism takes over, and thus,
anaerobic processes are likely recruited as the animal approaches Tc when
temperatures become stressful and potentially damaging. It has been
suggested that oxygen limitation at extreme temperatures may be the main
determinant of the temperature-dependent geographical distribution of
marine fishes (Pörtner, 2010), but the generality of this hypothesis is highly
debated (Jutfelt et al., 2018).
7.4.2 EFFECTS ON THE CARDIORESPIRATORY SYSTEM
The role of the fish cardiovascular system during acute thermal challenge is
discussed in detail in Chapter 4, but it is thought that the circulatory system,
rather than ventilatory performance, may be the first process to cause
oxygen limitation during acute heat stress (e.g. Sandblom and Axelsson,
2007). As temperatures approach the thermal maximum for these fishes,
circulatory O2 uptake is limited at least partly because of reductions in
haemoglobin–O2 affinity and haemoglobin binding capacity. In swimming
Pacific sockeye salmon (Oncorhynchus nerka), oxygen unloading to the
tissues is favoured at high temperatures, and fatigue is due to cardiac
limitation resulting from insufficient delivery of oxygen to working muscles
and not oxygen uptake over the gills (e.g. Clark et al., 2011). There is
substantial evidence that thermal acclimation affects the cardiorespiratory
system in terms of changes in ventricular mass (e.g. Young and Egginton,
2011), cardiac action potentials and other aspects of cardiac excitation-
contraction coupling (e.g. Shiels et al., 2011). These changes in cardiac
performance with acclimation may help to restore aerobic scope at stressful
temperatures (Kassahn et al., 2009; Pörtner, 2010).
7.4.3 EFFECTS ON SWIMMING PERFORMANCE AND BEHAVIOUR
Acute temperature change has profound effects on swimming performance
in most species of fish, and thermal acclimation can offset these effects. For
example, acclimation has been shown to affect both the thermal optimum
for sustained swimming performance (Schnell and Seebacher, 2008) and the
level of performance (Fangue et al., 2008). The extent of the change,
however, varies depending on the species (Muñoz et al., 2012), which has
the potential to alter species interactions (Grigaltchik et al., 2012; also see
Chapter 2).
Acclimation to constant or variable environmental temperatures often
modulates fish thermal preference (see also Chapter 17), and the
behavioural selection (Podrabsky et al., 2008) of thermal microhabitats is
an important mechanism exploited by fishes to buffer the effects of changes
in environmental temperature (Fangue et al., 2009). In most species, there is
good agreement between performance optima of measures such as growth,
metabolism, swim performance and reproduction with preferred
temperatures. Thermal tolerance thresholds and preferred temperatures are
often positively correlated. Although the physiological mechanisms
underlying such behavioural changes remain largely unknown, a few
studies have examined the effects of thermal acclimation on neurobiological
processes underlying these responses (Szabo et al., 2008).

7.5 DEVELOPMENTAL PLASTICITY

Temperature can have long-lasting effects on the morphology and
physiology of fishes. Even a relatively short exposure to altered
temperatures experienced during early development can impact the
phenotype of the adult. Most of the early work in this area focused on
meristic traits such as vertebral number and fin ray counts (e.g. Lindsey and
Harrington, 1972). More recently, it has become clear that altered
temperatures experienced during early development can also affect body
shape (e.g. Georgakopoulou et al., 2007) and subsequent growth trajectories
(e.g. Carballo et al., 2018). Temperature during development also has
persistent effects on muscle structure and function (e.g. Schnurr et al.,
2014). These effects are not confined solely to skeletal muscle, as
development temperature also has effects on cardiac anatomy (Dimitriadi et
al., 2018). Early exposure to altered temperatures, particularly prior to
hatch, can have profound effects on adult life history traits, including
effects on sex ratio, egg size and number, spawning time and age at
maturity. The role of developmental temperature in shaping these long-
lasting life history effects has been the subject of a comprehensive recent
review (Jonsson and Jonsson, 2019).
There is also substantial evidence that the temperature experienced by
one generation of fish can affect the morphology and physiology of the
next. As is the case for developmental plasticity, there are reports of
transgenerational plasticity for many offspring traits, including body size
(Shama and Wegner, 2014), aerobic metabolism (Donelson et al., 2012),
swimming performance (LeRoy et al., 2017), growth (Salinas and Munch,
2012) and reproductive traits (Shama and Wegner, 2014).
7.5.1 EPIGENETIC EFFECTS OF TEMPERATURE
The molecular mechanisms that underlie developmental and
transgenerational plasticity are poorly understood but are thought to
ultimately involve epigenetic processes such as DNA methylation, histone
modification or alteration of non-coding RNAs (see Chapter 16).
Collectively, these mechanisms determine chromatin architecture, the
accessibility of regulatory elements to the transcriptional machinery of the
cell, and gene expression levels, which ultimately determine the phenotype.
There is evidence that exposure to altered developmental temperatures
affects whole genome methylation (Metzger and Schulte, 2017), and alters
methylation at specific loci (Burgerhout et al., 2017), which alters gene
expression and thus cellular and whole-organism phenotypes. In addition,
developmental temperatures have been shown to alter the expression of
genes regulating DNA methylation in fishes, including DNA
methyltransferases (dnmt) (Carballo et al., 2018). Similarly, there is
evidence that developmental temperature alters the expression of
microRNAs in fishes (Campos et al., 2014) and can alter histone protein
levels (Carballo et al., 2018).

7.6 THERMAL ADAPTATION

Many studies have documented inter- and intra-specific variation in
thermally relevant phenotypes that is correlated with environmental
temperature in fishes, including metabolic rate (Healy and Schulte, 2012),
growth rate (Baumann and Conover, 2011), development rate and juvenile
growth rate (Yamahira et al., 2007), the thermal dependence of swimming
performance (Eliason et al., 2011), thermal preference (Fangue et al., 2009)
and thermal tolerance (Fangue et al., 2006). These patterns suggest, but do
not prove, that these traits have been shaped by natural selection. There are
several complementary approaches that can be used to explicitly test for
signatures of natural selection in response to temperature, including (i)
comparative approaches such phylogenetically independent contrasts, (ii)
population genetic or genomic approaches to identify the signatures of
natural selection in DNA sequences, and (iii) experimental evolution
approaches. Although these studies are relatively rare in fishes, the
emerging consensus from this work is that thermal adaptation is pervasive
and that environmental temperature is a critical factor shaping the evolution
of fishes.
Phylogenetic comparative approaches have been used to convincingly
demonstrate the action of natural selection on gene expression and
metabolic enzymes in killifish (e.g. Whitehead and Crawford, 2006). The
decreasing cost of DNA sequencing has led to an increasing number of
studies using population genetic and genomic approaches for detecting
natural selection in fishes (Bernatchez, 2016). Population genomic
signatures of natural selection related to environmental temperature have
been detected in many species (e.g. Chen et al., 2018; Healy et al., 2018).
Studies using an experimental evolution approach are relatively rare in
fishes, because they are most conveniently performed on species with very
short generation times. Artificial selection has been shown to affect thermal
tolerance in least killifish (Heterandria formosa; Baer and Travis, 2000),
but artificial selection imposes much stronger selection than does
experimental evolution or natural selection. One study (Barrett et al., 2011)
has examined the response of cold-tolerance using experimental evolution
in threespine stickleback (Gasterosteus aculeatus), in which fish of a
marine origin were placed in a series of semi-natural freshwater ponds. This
study showed that within three generations, the genetic composition of the
populations in the ponds had shifted, as had their cold-tolerance. Although
it is not possible to eliminate the possibility that epigenetic effects caused
the change in phenotype in the pond population using this experimental
design, these data suggest that cold-tolerance can shift rapidly in
stickleback (through either genetic or epigenetic mechanisms) when they
colonize a novel environment and suggest the possibility of rapid adaptation
in this thermally relevant trait.
7.6.1 ADAPTATIONS TO CONSTANT COLD IN ANTARCTIC FISHES
The waters of the Southern Ocean that surrounds Antarctica are extremely
cold, with temperatures reaching as low as −1.9 °C, and these temperatures
remain relatively stable throughout the year (ranging approximately
between −1.9 °C and +1.5 °C). Low temperatures such as this would
represent a substantial thermal stress for most fishes; however, the Southern
Ocean contains a diverse fish fauna. In particular, there has been a
substantial radiation of fishes in the sub-order Notothenioidei, which make
up ~90% of the fish biomass in the waters of the continental shelf around
Antarctica (Eastman, 2005). Thus, the type and extent of potential
adaptations to sub-zero temperatures in Antarctic fishes have long intrigued
biologists (for reviews see Petricorena and Somero, 2007; Beers and
Jayasundara, 2015). Compared with their subpolar or temperate relatives,
which can be found in New Zealand and South America, Antarctic
notothenioids differ in a number of respects. Some of the best studied of
these putative adaptations include the production of antifreeze proteins, the
loss of haemoglobin and myoglobin in many species of this sub-order,
larger hearts and more extensive vasculature, high mitochondrial densities
in oxidative muscle, larger muscle fibres, low standard metabolic rates and
the existence of enzymes with properties that allow them to maintain
function at low temperatures (for review see Petricorena and Somero,
2007). Transcriptomic studies of the Antarctic notothenioid Dissostichus
mawsoni (Antarctic toothfish) have revealed almost 200 gene families
whose expression was highly upregulated compared with that in temperate
fishes. Many of these gene families also exhibited Antarctic-fish-specific
gene duplications, suggesting that transcriptional upregulation may have
been (at least in part) mediated by these duplications (Chen et al., 2008).
Recent transcriptomic studies of Antarctic fishes also suggest that there is
enrichment of genes associated with oxidative stress (Berthelot et al., 2018).
The lack of haemoglobin in Antarctic icefishes of the family
Channichthyidae may cause them to be particularly sensitive to temperature
elevation as a result of oxygen limitations at elevated temperatures (e.g.
Joyce et al., 2018). Further exacerbating the susceptibility of Antarctic
fishes to high temperatures is the loss of the normally highly conserved heat
shock response (Hofmann et al., 2000; Thorne et al., 2010). These fishes
possess the genes for the HSPs but have lost the capacity to increase hsp70
gene expression in response to acute heat stress (Hofmann et al., 2005).
Instead, these fishes have constitutively high levels of the (normally)
inducible HSPs, which is probably part of the adaptive strategy used to cope
with stable but extremely cold temperatures and maintain protein
homeostasis (Place et al., 2004). Interestingly, polar fishes still increase
whole-organism thermal tolerance following an acute thermal stress, a
phenomenon known as heat hardening (Bilyk et al., 2012), which suggests
that the heat shock response and heat hardening at the whole-organismal
level may not be closely associated.
The sub-zero environment likely places fundamental constraints on
maintaining protein homeostasis in Antarctic fishes. Polar fishes also have
elevated levels of ubiquitin-conjugated proteins compared with their
temperate zone relatives, indicative of higher levels of denatured proteins
(Place and Hofmann, 2005), which may also explain elevated basal levels
of HSPs in these fishes. Further examination of the ubiquitin-proteasome
pathway in Antarctic fishes compared with their temperate New Zealand
relatives demonstrated that 20S proteasome activity in Antarctic fishes has
undergone a high degree of temperature compensation, providing evidence
that inefficiencies in protein catabolism are probably not the cause of the
elevated levels of denatured proteins (Todgham et al., 2017). It may be that
protein folding is particularly inefficient at cold temperatures, resulting in
high levels of misfolded proteins, but this remains to be tested.

7.7 THERMAL BIOLOGY IN A CHANGING WORLD

While the physiological and biochemical characteristics of fishes will
certainly influence how they will respond to global change and rising
temperatures, consideration of these traits in a broader ecological context is
pivotal to our ability to make predictions. Sunday et al. (2012) provide a
meta-analysis showing that the distribution patterns of animals are
determined primarily by their intrinsic thermal sensitivities, and in marine
organisms, their distributions are well matched to their thermal niches
(though see also Stuart-Smith et al. (2015)). In addition to intrinsic
tolerance limits of fishes, their dispersal capabilities, allowing movement to
new areas where biochemical trait optima are retained or regained and
where these fish can survive and reproduce, will influence their persistence.
Species movements will also alter species interactions, and fishes will have
to contend with mismatches and decoupling of natural events in their life
cycles (altered phenologies). A loss of synchrony between critical
biological processes will ultimately lead to imbalances in energy needs and
energy supplies. Reviews that consider adaptive potential, population or life
stage specific trait sensitivities, multiple interacting climate-related
stressors, and the importance of studying the appropriate local, biologically
relevant spatiotemporal scale are fundamental readings for physiologists
who want to connect their studies to a broader understanding of the
ecological consequences of global change (e.g. Angert et al., 2011;
Helmuth et al., 2014, Sinclair et al. 2016).

ACKNOWLEDGEMENTS
The authors acknowledge funding from the Natural Sciences and
Engineering Research Council (NSERC) Canada, the National Science
Foundation and the California Agricultural Experimental Station of the
University of California Davis.

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 8 Endocrinology
An Evolutionary Perspective on
Neuroendocrine Axes in Teleosts
Sylvie Dufour and Karine Rousseau

CONTENTS
8.1 Neuroendocrine Axes in Vertebrates and Special Features in Teleosts
8.1.1 Control of Physiological Functions and Life Cycles
8.1.2 The Innovation of the Pituitary Gland in Vertebrates
8.1.3 Specific Aspects of Pituitary Functional Anatomy in Teleosts
8.2 Diversification of Neuroendocrine Actors via Gene Duplications
8.2.1 Ancient Origin of the Molecular Families of Neuroendocrine
Actors
8.2.2 Gene Duplications of Neuroendocrine Actors
8.2.3 Vertebrate- and Teleost-Specific Whole-Genome Duplications and
Impact on Neuroendocrine Actors
8.2.4 Conservation or Loss of Duplicated Paralogs and Species-Specific
Diversity of Neuroendocrine Actors
8.3 The Thyrotropic Axis and the Control of Development, Metabolism,
and Metamorphosis in Teleosts
8.3.1 Introduction to the Thyrotropic Axis
8.3.2 Specific Features of the Thyrotropic Axis in Teleosts
8.3.2.1 Teleost Metamorphosis and Role of the Thyroid Hormones
(TH)
8.3.2.2 Knowledge Gaps in the Teleost Thyrotropic Axis
8.3.2.3 Impact of Gene Duplication, Conservation, or Loss on
Teleost Thyrotropic Axis
8.4 The Somatotropic Axis and the Control of Growth and Pleiotropic
Functions in Teleosts
8.4.1 Introduction to the Somatotropic Axis
8.4.2 Specific Features of the Somatotropic Axis in Teleosts
8.4.2.1 Various Roles in Teleosts
8.4.2.2 Multiple Hypophysiotropic Controls Integrated at the
Pituitary Somatotroph Level in Teleosts
8.4.2.3 Impact of Gene Duplication, Conservation, or Loss on
Teleost Somatotropic Axis
References

8.1 NEUROENDOCRINE AXES IN VERTEBRATES AND

SPECIAL FEATURES IN TELEOSTS
The term “fish” covers multiple taxonomic groups of aquatic vertebrates
with a variety of phylogenetic positions, such as cyclostomes,
chondrichthyans, actinopterygians, and sarcopterygians. In the present
study, we mainly focus on one of the actinopterygian groups, the teleosts,
which is the largest group of vertebrates with more than 25,000 species. We
refer to other vertebrate groups for comparative and evolutionary
perspectives.
8.1.1 CONTROL OF PHYSIOLOGICAL FUNCTIONS AND LIFE CYCLES
Neuroendocrine axes are responsible for the control of major physiological
functions and life traits in vertebrates, such as growth, metabolism,
reproduction, stress, metamorphosis, and migration. They are composed of
brain neurohormones, pituitary hormones, and peripheral gland hormones
(Figure 8.1). Environmental and internal cues are integrated by brain
neuronal networks that control the activity of brain hypophysiotropic
neurons; the neurohormones secreted by the hypophysiotropic neurons
regulate the production of pituitary hormones, which are released in the
general blood circulation and control the activity of peripheral glands, such
as gonads, thyroid, adrenal glands, and liver. The neuroendocrine axes in
vertebrates thus operate as an integrated control system, with the pituitary
acting as a relay between the brain and peripheral glands/target tissues.
 FIGURE 8.1 Vertebrate neuroendocrine axes. Environmental and internal factors are
integrated by brain networks regulating the activity of hypophysiotropic neurons, which
control the production of pituitary hormones by the adenohypophysis. Pituitary
hormones control the activity of peripheral glands, which produce hormones acting on
target tissues.

8.1.2 THE INNOVATION OF THE PITUITARY GLAND IN VERTEBRATES

The pituitary gland is an anatomical and functional innovation in
vertebrates. Located at the base of the hypothalamus, the pituitary is
composed of the neurohypophysis and the adenohypophysis, which have
different embryogenic origins. The neurohypophysis originates from an
infundibular extension of the floor of the diencephalon, while the
adenohypophysis originates from the oral ectoderm (stomodeum) coming in
contact with the infundibulum.
The neurohypophysis (pars nervosa or posterior pituitary) contains the
axonal endings of diencephalic neurons producing two types of
neurohormones, belonging to the vasotocin/vasopressin family and the
oxytocin/isotocin/mesotocin family. These neurohormones are released into
the main blood circulation and are involved in various physiological and
behavioural functions.
 The adenohypophysis (pars distalis or anterior pituitary) comprises
various types of glandular cells producing pituitary hormones involved in
the control of peripheral glands (Figure 8.1). Based on their molecular
relationships, adenohypophyseal hormones belong to the following
families: the glycoprotein hormone family, including thyrotropin (TSH) and
gonadotropins (luteinizing hormone [LH] and follicle stimulating hormone
[FSH]); the growth hormone (GH)/ prolactin (PRL)/ somatolactin (SL)
family; and the proopiomelanocortin (POMC) family (adrenocorticotropic
hormone [ACTH] and melanocyte-stimulating hormones [MSH]). The
production of adenohypophyseal hormones is regulated by brain
hypophysiotropic neurons. In mammals, axonal endings of
hypophysiotropic neurons project to the median eminence at the basis of the
hypothalamus, and neurohormones are secreted into the portal vascular
system that links the median eminence with the adenohypophysis; in
teleosts, hypophysiotropic neurons directly innervate the adenohypophysis.
Even if the pituitary is considered to be an organ specific to vertebrates,
its possible ancestral origin in early chordates (Gorbman 1995) has been
reinforced by recent embryological investigations. It is in vertebrates,
together with the remarkable brain development, that the pituitary has
acquired its anatomical features and functional key role as the relay and
amplifier of the brain’s control of major physiological functions and life
cycle.
8.1.3 SPECIFIC ASPECTS OF PITUITARY FUNCTIONAL ANATOMY IN
TELEOSTS
The anatomical relationships between the brain neurohypophysiotropic
neurons and the adenohypophysis vary among vertebrates. Three modes of
connection are classically reported: via the portal vascular system linking
the median eminence to the adenohypophysis in tetrapods and some other
vertebrates; via a direct innervation of the adenohypophysis in teleosts, with
the axonal endings of the hypophysiotropic neurons terminating in close
proximity to the adenohypophyseal cells they control; and via diffusion in
agnathans (Gorbman 1995; Sower 2018).
The connection between hypophysiotropic neurons and
adenohypophyseal cells may be more complex with multiple modalities in
some species. A dual anatomical mode of regulation of gonadotropic cells
by gonadotropin-releasing hormone (GnRH) neurons was observed in
zebrafish: directly by GnRH fibers, as classically reported (neuroglandular
mode), and also indirectly via the pituitary vasculature (neurovascular
mode) (Golan et al. 2015). In addition to the diffusion mode, some
neurovascular connections were suggested in an agnathan, the hagfish, with
neurosecretory fibers terminating on blood vessels in the hypothalamus
(Nozaki and Sower 2015).
In addition to the direct innervation of the adenohypophysis, another
striking feature of the pituitary in teleosts as compared with mammals is the
spatial regionalization of the different types of pituitary cells (Schreibman
et al. 1973). Hypophysiotropic neurons thus project to specific regions of
the adenohypophysis where their target cells are located. This pituitary cell
regionalization and the direct pituitary innervation by hypophysiotropic
neurons likely result from a co-evolutionary process in the teleost lineage.
Furthermore, a variety of hypophysiotropic neurons may target a specific
adenohypophyseal region in teleosts. In mammals, the brain is the major
center of integration of various environmental and internal regulatory cues,
and this integrated information is conveyed via the production of a single or
a few neurohormones that are released in the pituitary portal system and
control the production of their corresponding target adenohypophyseal
hormone(s). In teleosts, in contrast, beside the brain, the pituitary cells
themselves play an important role in the integration of various cues
conveyed by multiple neurohormones. For instance, the possibility of
independent regulation of teleost pituitary gonadotrophs by more than 20
different neurohormones was reported (Zohar et al. 2010; Trudeau 2018).
As recently proposed by Trudeau (2018), the multiple, parallel, and
independent neurohormonal controls and their integration at the pituitary
cell level in teleosts may account for the surprisingly low impact of
knockout of key genes such as GnRH (Whitlock et al. 2019) on
reproductive function in some teleost species. This may result from
compensation between the independent neuroendocrine regulatory
pathways integrated at the pituitary cell level.
8.2 DIVERSIFICATION OF NEUROENDOCRINE ACTORS
VIA GENE DUPLICATIONS
8.2.1 ANCIENT ORIGIN OF THE MOLECULAR FAMILIES OF
NEUROENDOCRINE ACTORS
While the brain–pituitary–peripheral gland neuroendocrine axes are typical
of the vertebrate lineage, the molecular actors themselves of these
neuroendocrine axes, such as various neurohormones, hormones, and their
receptors, belong to molecular families with an ancient origin in metazoa.
An increasing number of molecular phylogeny investigations highlight the
ancient origin of vertebrate neuroendocrine actor families, tracing back
their origin not only in chordates and deuterostomes but also in a common
bilaterian ancestor of deuterostomes and protostomes, and even earlier, in a
non-bilaterian metazoan (Elphick and Mirabeau 2014). In parallel with the
anatomical innovations of the pituitary gland and its relationships with the
brain, vertebrate neuroendocrine axes have thus been established through
the recruitment of ancient molecular actors and by the diversification and
emergence of new actors via gene duplications expanding their molecular
families.
8.2.2 GENE DUPLICATIONS OF NEUROENDOCRINE ACTORS
Gene duplications are at the origin of the increased number of members of
molecular families. Among other genes, duplications have occurred in
genes coding for key actors of the vertebrate and teleost neuroendocrine
axes, such as peptidic neurohormones, peptidic hormones, receptors, and
biosynthesis enzymes of non-peptidic neuromediators and hormones. They
include intragenic duplications, gene-specific local duplications, and large-
scale, whole-genome duplications (WGD).
Intragenic duplications led to genes encoding for several homologous
peptides. A classic example is the thyrotropin-releasing hormone (TRH)
gene encoding multiple identical repeats of the three–amino acid TRH
sequence as seen in teleosts and in mammals (Galas et al. 2009). These
repetitions allow amplification of the synthesis and production of this
neurohormone.
 Gene-specific duplication, also referred to as local gene duplication,
involves the duplication of a given gene into two paralogous genes, which
remain located in a tandem position in some cases. Local gene-specific
duplications are, for instance, at the origin of the duplication of the
ancestral gonadotropin receptor into the LH receptor (LHR) and the FSH
receptor (FSHR) in vertebrates and of the further duplication of LHR into
LHR1 and LHR2 in the actinopterygian lineage, inherited by teleosts
(Maugars and Dufour 2015).
8.2.3 VERTEBRATE- AND TELEOST-SPECIFIC WHOLE-GENOME
DUPLICATIONS AND IMPACT ON NEUROENDOCRINE ACTORS
Beside intragenic and gene-specific duplications, WGD events have been
the major source of the expansion of gene numbers. Comparative genomics
led to the hypothesis that two events of WGD, referred to as “1R” and “2R”
for “first and second rounds” of WGD, occurred in ancestral vertebrates
(Figure 8.2) (Dehal and Boore 2005). These two rounds of WGD, which
have provided new molecular tools for natural selection, are considered to
have largely contributed to the innovations and success of the vertebrate
lineage. They are at the origin of various actors of the vertebrate
neuroendocrine axes. This is the case, for instance, for the hormone-specific
beta subunits of the two gonadotropins, LH and FSH, and their sister
hormone, the thyrotropin TSH (see earlier in this section).
 FIGURE 8.2 Vertebrate phylogeny and events of whole-genome duplication. Two
rounds (1R, 2R) of whole genome duplication occurred early in the vertebrate lineage, a
third round (3R) in the teleost lineage, and an additional fourth round (4R) in some
teleost groups, such as salmonids.

Sequencing of teleost genomes indicated that an additional WGD event

has occurred in ancestral teleosts, referred to as “3R” for “third round” of
WGD or as “teleost-specific WGD” (TSGD or TSWGD) (Meyer and
Schartl 1999) (Figure 8.2). This additional WGD is considered to have
favored the remarkable radiation and diversification of teleosts, the largest
vertebrate group. Concerning the neuroendocrine axes, the 3R is at the
origin of additional paralogs for a number of actors. One striking example is
the 3R-duplicated tsh beta subunit gene (Maugars et al. 2014), with one
paralog (tshβb and not tshβa) specifically involved in salmon smoltification
metamorphosis (Fleming et al. 2019).
A further WGD has occurred more recently in some teleost groups, such
as salmonids (Lien et al. 2016) and some carps (Wang et al. 2012). These
events are referred to as “4R” (“fourth round” of WGD) or “SGD”
(salmonid genome duplication) and “CGD” (carp genome duplication)
(Figure 8.2). If some of the 4R-duplicated paralogous genes are still
potentially redundant as a result of this relatively recent tetraploidization,
some 4R-duplicated genes have been shown to play differential
physiological roles. This is the case, for instance, of the 4R-duplicated
paralogs of deiodinase2, an enzyme responsible for the conversion of
thyroid hormone, thyroxin (T4), into the more active triiodothyronin (T3),
which exhibits differential tissue expression and responses to environmental
challenges in salmon (Lorgen et al. 2015).
8.2.4 CONSERVATION OR LOSS OF DUPLICATED PARALOGS AND
SPECIES-SPECIFIC DIVERSITY OF NEUROENDOCRINE ACTORS
Duplicated paralogs, resulting from gene-specific duplication or from
WGD, are initially identical and therefore redundant. In many cases, one of
the redundant paralogs is eliminated by the accumulation of mutations in its
promoter and/or coding region, leading to pseudogenization and ultimately,
gene loss.
Inversely, the conservation of duplicated paralogs may be related to
positive selection. Duplicated paralogs, even if redundant, may allow a
favorable amplification of expression of their encoded product, as
mentioned earlier for the intragenic duplicated TRH sequence. This may
also account for duplicated genes. However, in most cases, duplicated
paralogous genes are conserved in relation to divergences in their promoter
and/or coding sequences, resulting in differential tissue distribution,
expression regulation, and physiological roles. One driving force for the
conservation of divergent duplicated paralogs is referred to as
“subfunctionalization”, with the various functions of the ancestral gene
being divided into the duplicated paralogous genes. The other driving force
is referred to as “neofunctionalization”, with one paralogous gene
conserving the major physiological roles of the ancestral gene while the
other paralog develops a novel function. Neofunctionalization and
subfunctionalization have underlined the innovation of the diversification of
the actors of the neuroendocrine axes in vertebrates and teleosts.
In the following sections, we shed light on some specificities of the
teleost neuroendocrine axes from an evolutionary perspective, as illustrated
with the examples of two major neuroendocrine axes involved in the
regulation of development and growth, the thyrotropic and somatotropic
axes. For information on the gonadotropic axis and the control of
reproduction, see Chapter 9 of this book. On the corticotropic axis and the
regulation of stress, see Flik et al. (2006).

8.3 THE THYROTROPIC AXIS AND THE CONTROL OF

DEVELOPMENT, METABOLISM, AND
METAMORPHOSIS IN TELEOSTS
8.3.1 INTRODUCTION TO THE THYROTROPIC AXIS
The brain–pituitary–thyroid axis is involved in the regulation of
development and metabolism in all vertebrates (Figure 8.3). It is also
responsible for the thyroid hormone–induced larval metamorphosis, a key
step in the life cycle of anuran amphibians, which has been widely
investigated (Sachs and Buchholz 2019). Metamorphosis is defined as a
post-embryonic developmental event, which includes drastic
morphological, physiological, and behavioral changes allowing the
transition from one developmental stage and habitat (ecophase) to the next
stage/ecophase of the life cycle. Typical larval metamorphosis, also referred
to as “primary metamorphosis”, occurs in the life cycle of some teleost
species, such as elopomorphs (e.g., eels) and pleuronectiforms (e.g.,
flatfishes) (Inui and Miwa 2012; Yamano 2012).
 FIGURE 8.3 The thyrotropic axis in teleosts. Few data are available on brain
stimulatory (green) and inhibitory (red) controls of pituitary thyrotrophs in teleosts.
CRH: corticotropin-releasing hormone; DA: dopamine; SRIH: somatostatin; TRH:
thyrotropin-releasing hormone. Duplicated thyrotropins (TSH) result from teleost
whole-genome duplication. T4: thyroxine; T3: triiodothyronine.

As characterized in mammals, the thyrotropic axis is composed of brain

TRH neurons, pituitary TSH cells, and thyroid hormones (TH), T4 and T3.
The TRH gene encodes a pre-prohormone including multiple repeats of the
TRH peptide (Galas et al. 2009). TRH acts via its cognate membrane class
A GPCR receptor, TRHR, expressed by the pituitary thyrotrophs, and
stimulates the synthesis and release of pituitary TSH.
TSH is a glycoprotein hormone constituted of two subunits, the alpha
subunit (Gpα), common to gonadotropins, and the hormone-specific TSHβ
subunit, paralogous to the LH and FSH beta subunits. TSH binds to its
cognate class A GPCR receptor TSHR, paralogous to gonadotropin
receptors LHR and FSHR. TSH activates the synthesis, by thyroid follicular
cells, of thyrogobulin, a large protein containing many tyrosine residues,
precursors of the thyroid hormone T4. T4 is converted by deiodinase into
the more active hormone T3 in the thyroid gland or in target tissues. TH
acts via nuclear receptors TRα and TRβ on various target tissues, including
the brain and pituitary, where they exert feedback on the thyrotropic axis.
The composition of the thyrotropic axis is similar in amphibians, with a
notable exception: at the time of metamorphosis in anurans, the master
control of TSH and subsequent thyroid gland activation is exerted not by
TRH neurons but by corticotropin-releasing hormone (CRH) neurons. CRH
is classically known as the master controller of the corticotropic axis in
vertebrates by stimulating the production of pituitary corticotropin (ACTH),
which in turn stimulates the production of cortisol by the adrenal gland or
the interrenal gland in teleosts. The dual involvement of CRH in the
regulation of both thyrotropic and adrenocorticotropic axes at the time of
metamorphosis supports the synergistic role exerted by TH and cortisol on
frog metamorphosis (Sachs and Buchholz 2019), a synergism also reported
in teleost metamorphosis (de Jesus et al. 1990).
8.3.2 SPECIFIC FEATURES OF THE THYROTROPIC AXIS IN TELEOSTS
8.3.2.1 Teleost Metamorphosis and Role of the Thyroid Hormones (TH)
As in other vertebrates, TH play a major role in teleosts in the regulation of
development, metabolism, and behavior (for instance, locomotor and
migratory behaviors) as well as in the modulation of other physiological
functions, such as reproduction. As for anuran amphibians, TH are also
involved in the control of metamorphosis in some teleosts.
Histological studies of thyroid follicle activity and assays of TH body
levels, as well as experimental approaches, support the role of TH in larval
metamorphoses of elopomorphs (Yamano 2012) and pleuronectiforms (Inui
and Miwa 2012). The role of TH in the control of metamorphosis in
teleosts, as in amphibians, may reflect either a conserved or a convergent
regulatory process among osteichthyans.
Some other remarkable life transitions, the “smoltification” in
anadromous salmonids and the “silvering” in catadromous eels, allow the
transition from a juvenile ecophase in freshwater to the next ecophase in the
ocean; they encompass striking morphological, physiological, and
behavioral changes, including similar changes in salmon and eel, such as
silvering of the skin resembling that of pelagic fishes, preparation of the gill
for osmoregulation in seawater, and induction of downstream migratory
behavior (Rousseau et al. 2012a, 2012b). These events are described by
some authors as “secondary metamorphosis” to distinguish them from
typical larval metamorphosis (or “primary metamorphosis”) (Youson 1988).
While thyroid follicle histology, hormone assays, and experimental
treatments clearly indicated an important role of TH in salmon
smoltification (Boeuf 1993; Rousseau et al. 2012a), investigation in the eel
revealed the major role of the gonadotropic axis, namely sexual steroids, in
the induction of silvering changes (Rousseau et al. 2012b). This highlights
the recruitment of different neuroendocrine axes, the thyrotropic axis in
salmon versus the gonadotropic axis in eel, likely related to the distinct
position of this transition in their respective life cycle: between two juvenile
growth phases in salmon, and between the juvenile growth phase and the
initiation of puberty in eel. This remarkable evolutionary plasticity in the
recruitment and role of neuroendocrine axes in teleost life transitions may
have favored the diversity of their life cycles.

8.3.2.2 Knowledge Gaps in the Teleost Thyrotropic Axis

Compared with tetrapods, much less is known concerning the brain
component of the thyrotropic axis in teleosts. Early studies showed that
ectopic transplantation of the pituitary gland (Ball et al. 1963) or stereotaxic
brain lesion (Peter 1970) induced a long-term activation of pituitary
thyrotrophs and the thyroid gland in some teleost species. This suggested
that TSH cells may be under a major brain inhibitory control, unlike in
tetrapods, where they are mainly under the stimulatory control of TRH (De
Groef et al. 2006). The role of inhibitory neurohormones such as
somatostatin and dopamine, which may counteract the stimulatory effect of
TRH on TSH production in mammals and other tetrapods, is largely
unexplored in teleosts. A few studies have addressed the stimulatory control
of TSH in fish. In vitro, CRH stimulates TSH release in coho salmon
(Oncorhynchus kisutch), while TRH stimulates (Japanese eel Anguilla
japonica, bighead carp Aristichthys nobilis) or has no effect (common carp
Cyprinus carpio, coho salmon) on tshβ transcript levels or TSH release
(MacKenzie et al. 2009). TH exert an inhibitory effect on TSH transcript
levels in teleosts (MacKenzie et al. 2009) as in mammals, supporting the
conservation of the negative feedback of TH on the thyrotropic axis among
vertebrates.

8.3.2.3 Impact of Gene Duplication, Conservation, or Loss on Teleost

Thyrotropic Axis
As mentioned before, the genes encoding the beta subunits of the two
gonadotropins LH and FSH and of TSH are paralogous genes that likely
arose from the two successive 1R/2R WGD in ancestral vertebrates.
According to this origin, a fourth paralogous gene would have been
expected. In agreement with this hypothesis, recent studies based on
genome data mining and phylogeny and synteny analyses revealed the
presence of a second TSH beta subunit (TSHβ2) in some representative
species of early vertebrates, such as chondrichthyans and basal
sarcopterygians (Maugars et al. 2014). The presence of a single TSHβ (the
“classical” TSHβ, also now named TSHβ1) in mammals and other
tetrapods, and in actinopterygians, suggests that the gene coding for tshβ2
was lost twice independently during osteichthyan evolution, in a tetrapod
ancestor and in an actinopterygian ancestor. tshβ2 could not be retrieved in
the genome of a holostean (spotted gar) or of any teleosts, supporting an
early loss of tshβ2 in the actinopterygian lineage, leading to inheritance of
only tshβ1 by the teleost lineage (Maugars et al. 2014).
Recent studies revealed that two tshβ paralogs are present in teleosts,
resulting from the 3R duplication of tshβ1 (Maugars et al. 2014; Fleming et
al. 2019). They are named here tshβ1a and tshβ1b according to the
consensual “a” and “b” nomenclature for 3R-paralogs. The conservation of
3R-duplicated tsh beta subunit paralogs across teleost radiation opens new
research avenues on their respective roles according to species and the
diversity of life cycles. Strikingly, in the Atlantic salmon, the discovery of
TSHβ1b led to a large peak of expression of tshβ1b being revealed during
smoltification, at the time of the initiation of the downstream migration,
with no change in the expression of the previously investigated paralog
tshβ1a. This provided the first demonstration of the involvement of TSH in
the smoltification metamorphosis (Fleming et al. 2019). In stickleback
(Gasterosteus aculeatus), a role in migration of the tshβ1b paralog (named
by the authors tshβ2) was also proposed by comparing its expression in
different ecotypes, those migrating to the sea versus stream-residents
(Kitano et al. 2010). Remarkably, the two tshβ1 paralogs are expressed in
different pituitary cells, as investigated in the Atlantic salmon (Salmo
salar): tshβ1a in the anterior adenohypophysis and tshβ1b near to the
pituitary stalk (Fleming et al. 2019), a location comparable to the pars
tuberalis TSH cells involved in seasonal physiology and behavior in birds
and mammals. As known for pars tuberalis TSH in birds and mammals, it
is hypothesized that the second TSH in salmon may act in a retrograde way
on the hypothalamus to stimulate deiodinase 2, leading to the conversion of
T4 into T3 and activation of brain neuronal networks (Fleming et al. 2019).
After the salmonid-specific WGD (4R), a differential conservation of
additional tshβ paralogs was observed according to species. While only two
tshβ1 paralogs (tshβ1a and tshβ1b) issued from 3R are present in Atlantic
salmon, as in other teleosts, up to three tshβ1 paralogs were identified in
Oncorhynchus species due to the conservation of the duplicated 4R-
paralogs of tshβ1a (Fleming et al. 2019).
TSH receptors (TSHR) have been duplicated via the 3R in teleosts (tshra
and tshrb). Analysis of the tissue distribution of their transcripts in the
European eel, Anguilla anguilla (Maugars et al. 2014), showed that tshra is
expressed by the thyroid follicles in agreement with the “classical” role of
TSH in the activation of the thyroid gland. In contrast, tshrb is expressed in
other tissues, including brain and peripheral organs. The expression of tshrb
in the brain is in agreement with the potential role of TSHb in brain
deiodinase 2, as mentioned earlier. Another remarkable expression of tshrb
is in the gonads, also expressing the tshβ1b paralog, which may support an
important and still unexplored endocrine and paracrine role of TSH in
reproduction in teleosts (Maugars et al. 2014).
Thyroid hormone receptors belong to the nuclear receptor superfamily.
Two homologous receptors, TRα and TRβ, encoded by thra and thrb genes,
respectively, have been characterized in tetrapods. These receptor genes
have been duplicated via the 3R in teleosts, leading to the presence of two
trα paralogs and two trβ paralogs in basal teleosts, such as conger and eel.
One of the trβ duplicated paralogs would have been lost in the course of
teleost evolution, as two trα genes but a single trβ gene have been retrieved
in other teleosts, such as zebrafish, Danio rerio (Lazcano and Orozco
2018). In tetrapods, alternative splicing of thyroid hormone receptors leads
to various protein isoforms with different binding activities and biological
functions. Similarly, in addition to the 3R-duplicated genes, alternative
splicing, including sequence insertion or deletion, may lead to a large
variety of thyroid hormone receptor isoforms in teleosts, with species-
specific variations. Differential expression of thyroid hormone receptors,
according to paralogs and/or isoforms, has been reported during
development and metamorphosis in some teleost species, such as eel,
zebrafish, and flatfishes, supporting differential roles of these variants
(Lazcano and Orozco 2018).

8.4 THE SOMATOTROPIC AXIS AND THE CONTROL OF

GROWTH AND PLEIOTROPIC FUNCTIONS IN
TELEOSTS
8.4.1 INTRODUCTION TO THE SOMATOTROPIC AXIS
The somatotropic axis plays an essential role in the regulation of body
growth in all vertebrates (Figure 8.4). It can also influence metabolism,
reproduction, and immunity in most vertebrates, as well as osmoregulation
in teleosts.
 FIGURE 8.4 The somatotropic axis in teleosts. Multiple brain neurohormones are
involved in the stimulatory (green) or inhibitory (red) controls of pituitary somatotrophs
in teleosts. 5-HT: serotonin; CRH: corticotropin-releasing hormone; DA: dopamine;
GH: growth hormone; GHRH: growth hormone-releasing hormone; GnRH:
gonadotropin-releasing hormone; IGF: insulin-like growth factor; NE: norepinephrine;
PACAP: pituitary adenylate cyclase-activating peptide; SRIH: somatostatin.

In mammals, brain growth hormone-releasing hormone (GHRH)

stimulates the synthesis and release of growth hormone (GH) from the
pituitary, while somatostatin (SRIH) acts as an inhibitor. In non-mammalian
vertebrates, there have been misleading data, as GHRH was originally
considered to be encoded by the same gene as another neuropeptide,
pituitary adenylate cyclase-activating polypeptide (PACAP), and in vitro
experiments in some fish species failed to show that this GHRH was a
potent releaser of GH, while PACAP was highly efficient (Montero et al.
2000). In 2007, the “true” non-mammalian GHRH was finally identified on
a gene paralogous to the PACAP gene, and the originally named GHRH
was re-classified as PACAP-related peptide, PRP (Lee et al. 2007). The
“true” GHRH was shown to have potent GH-releasing activity in vitro in
teleosts. GHRH and PACAP are both members of the secretin/glucagon
superfamily, and they act via receptors belonging to the class B/class II
GPCR (Tam et al. 2014).
The gh gene belongs to the same family as the prolactin (prl), prolactin 2
(prl2), and somatolactin (sl) genes within the large superfamily of class-1
helical cytokines (Huising et al. 2006). gh and prl are present in all
vertebrates. prl2 was identified in 2009 in non-mammalian vertebrates,
including chondrichthyans (Huang et al. 2009), and was suggested to have
been lost twice in tetrapods: at the base of amphibians, and early in
mammalian evolution (Ocampo Daza and Larhammar 2018a). sl was
initially isolated from coho salmon (Rand-Weaver et al. 1992) and thought
to be teleost specific, but its identification in a non-teleost actinopterygian,
the white sturgeon (Acipenser transmontanus), and a sarcopterygian, the
African lungfish (Protopterus amnectens), demonstrates that, like gh and
prl, sl was present before the divergence of sarcopterygians and
actinopterygians. sl was recently identified in two species of salamander,
suggesting that sl had been lost independently in an amniote ancestor and in
the lineage leading to frogs within amphibians (Ocampo Daza and
Larhammar 2018a). GH receptors (GHR) are members of the type I
cytokine receptor family together with PRL receptors.
GH, via its receptor, stimulates the hepatic synthesis and release of
insulin-like growth factors (IGF), which act via specific receptors (IGFR)
on various target tissues, including muscle and bone, and negatively feed
back to the brain and pituitary to regulate the somatotropic axis. In
vertebrates, two IGF (IGF1 and IGF2) exist (Caruso and Sheridan 2011). In
teleosts, a third peptide, termed IGF3, has been first identified in zebrafish,
with its expression confined to the gonads (Wang et al. 2008). All three
IGFs share five domains (A–E) with a similar tertiary protein structure and
are structurally related to insulin and relaxin in vertebrates and to insulin-
like peptides in invertebrates (Caruso and Sheridan 2011). IGFs bind to two
types of receptors in vertebrates, type 1 (IGFR1) and type 2 (IGFR2), which
belong to a family that includes insulin and relaxin receptors.
GH and IGF actions, in addition to being mediated by their respective
receptors, are modulated by binding proteins (GHBP and IGFBP,
respectively), which extend their half-life and prevent their signaling
(Reindl and Sheridan 2012).
8.4.2 SPECIFIC FEATURES OF THE SOMATOTROPIC AXIS IN TELEOSTS
8.4.2.1 Various Roles in Teleosts
GH is perhaps the most pleiotropic pituitary hormone, as in vertebrates it is
involved in the control not only of growth but also of reproduction,
metabolism, and immunity, as well as osmoregulation in teleosts.
Compared with mammals, fish present a unique feature, in that their body
growth is continuous during life. The growth-promoting effects of GH may
be direct or mediated by IGF (Bergan-Roller and Sheridan 2018). In
teleosts, the action of GH on post-embryonic growth may be mediated by
both IGF1 and IGF2, in contrast to mammals, in which only IGF1 is
involved (Caruso and Sheridan 2011).
As in mammals, the somatotropic axis also modulates reproduction in
teleosts (Le Gac et al. 1993; Reinecke 2010). GH-deficient zebrafish lines
present complete arrest of folliculogenesis in females and delay of
spermatogenesis in males (Hu et al. 2019). IGF3 mediates the action of
gonadotropins on gametogenesis in zebrafish, revealing a new interaction
between the somatotropic and gonadotropic axes (Nóbrega et al. 2015). As
in mammals, GH and IGF are also involved in the regulation of metabolism
(Bergan-Roller and Sheridan 2018) and immunity (Yada 2007). In addition
to all these functions, GH is involved in osmoregulation in teleosts, a
crucial adaptative function allowing change of habitat during the life cycle.
A striking example is the role of GH in the acclimation to seawater during
smoltification in salmonids (Sakamoto and McCormick 2006).

8.4.2.2 Multiple Hypophysiotropic Controls Integrated at the Pituitary

Somatotroph Level in Teleosts
In relation to its pleiotropic actions in teleosts, regulation of GH release is
multifactorial and varies according to species, developmental stage, and
environmental conditions. SRIH is the major inhibitor of GH at the
somatotroph level in teleosts as in mammals, while various neuropeptides
are involved in its direct stimulatory control, depending on species. SRIH
direct action on GH pituitary production and release has been well
documented in all teleost species studied so far (Sheridan and Hagemeister
2010). SRIH is also able to inhibit the stimulatory pituitary effects of other
neurohormones such as GHRH, dopamine (DA), or GnRH (Gahete et al.
2009). Norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT)
can also inhibit basal and stimulated GH release in teleosts (Gahete et al.
2009).
In addition to GHRH and PACAP (Gahete et al. 2009), already
mentioned in Section 8.4.1, various other neurohormones have direct
stimulatory effects on GH synthesis and release. These include DA, GnRH,
TRH, and CRH (Rousseau and Dufour 2007), whose direct stimulatory
actions on GH are not reported in mammals (Gahete et al. 2009). Ghrelin, a
brain and gastrointestinal hormone that is the natural endogenous ligand for
GH secretagogue receptor, possesses a direct GH-releasing effect in teleosts
as in mammals (Unniappan and Peter 2005).

8.4.2.3 Impact of Gene Duplication, Conservation, or Loss on Teleost

Somatotropic Axis
Six SRIH paralogous genes (SST) have been identified in extant
gnathostomes: SST1, SST2 (also named cortistatin in mammals), SST3,
SST4, SST5, and SST6 (Tostivint et al. 2014). A recent study revisited the
complex evolution of SRIH genes (Tostivint et al. 2019). SST1, SST2, and
SST5 likely arose from 1R/2R in ancestral vertebrates; SST3 and SST6 from
local gene duplications of SST1 and SST2, respectively, in ancestral
gnathostomes; and SST4 from SST3 in the actinopterygian lineage. Lineage-
and species-specific SRIH gene losses led to the presence of only SST1 and
SST2 in the human and a number of SST in teleosts (six in zebrafish; five in
medaka, Oryzias latipes) no higher than in the spotted gar, Lepisosteus
oculatus (six SST), despite the 3R (Tostivint et al. 2019). Concerning SRIH
receptors, evolutionary studies propose that six genes (SSTR) would have
been present in ancestral vertebrates as a result of local gene duplication
followed by 1R/2R. After gene loss of SSTR6, the human possesses five
receptors, while zebrafish possesses a total of eight SSTR, subsequently to
SSTR4 gene loss and conservation of 3R-duplicated SSTR2a/b, SSTR3a/b,
and STTR5a/b (Tostivint et al. 2014).
The ancestral PRP-PACAP was generated by exon duplication from a
PACAP-like gene. After 1R/2R, four paralogous genes were generated, but
only three (PRP-PACAP, PHI-VIP, and GHRH) persisted in later lineages
(Lee et al. 2007). Teleosts possess 3R-duplicated PACAP genes: adcyap1a
and adcyap1b (Cardoso et al. 2007). Up to now, three PACAP receptors
have been identified in mammals (adcyap1r; vipr1; vipr2). In spotted gar
and coelacanth, a single copy of each receptor gene was found (Cardoso et
al. 2015), while teleosts possess duplicates for each of them as a result of
3R (adcyap1r1a; adcyap1r1b; vipr1a; vipr1b; vipr2a; vipr2b) (Cardoso et
al. 2007). Thus, PACAP and its receptors emerged early in the evolution of
vertebrates and were duplicated by 3R and conserved during teleost
radiation.
Gh, ancestral prl, and sl genes arose from local gene duplications in a
vertebrate ancestor. 1R/2R gave rise to prl and prl2 from ancestral prl,
while the other potential 1R/2R duplicates of gh and sl would not have been
conserved (Ocampo Daza and Larhammar 2018a). In teleosts, 3R resulted
in duplicated sla and slb with no impact on the other gh, prl, and prl2
genes, due to gene losses after 3R. Zebrafish possesses both sla and slb,
while slb may have been lost in acanthomorpha, since only sla is present in
medaka and stickleback. Conservation of salmonid-4R duplicated paralogs
except for slb led to the presence of up to nine members (duplicated-gh, -
prl, -prl2, -sla, and a single slb) (Ocampo Daza and Larhammar 2018a). An
increase of gill expression of 4R-duplicated gh2 but not gh1 was reported
during the smoltification process in rainbow and steelhead trout,
Oncorhynchus mykiss (Hecht et al. 2014). This suggests that GH2 may be
responsible for the important role played by GH in osmoregulation during
this crucial metamorphic process in salmonids. Recent study on the origin
and evolution of the GH/PRL receptor superfamily proposes that ghr and
prlr arose from local gene duplication of an ancestral gene in early
gnathostomes (Ocampo Daza and Larhammar 2018b). Both were duplicated
via 3R, leading to the presence of two ghr (ghra and ghrb) and two prlr
(prlra and prlrb) in extant teleosts. Conservation of 4R-duplicated ghr led
to the presence of four ghr in salmonids, while due to gene loss, only two
prlr are present in salmonids as in other teleosts (Ocampo Daza and
Larhammar 2018b).
Two IGF genes (igf1 and igf2) are present in extant gnathostomes,
including chondrichthyans (Duguay et al. 1995), and likely arose from a
common ancestor via the 2R. Four igf genes have been evidenced in some
teleosts such as zebrafish; they are proposed to originate from 3R (igf1a,
igf1b, igf2a, and igf2b) (Zou et al. 2009). Further studies may be required,
as some authors classified igf1b as a special type named igf3 (Wang et al.
2008) or highlighted that the “IGF3” sequence closely resembles that of
relaxin (Caruso and Sheridan 2011). In zebrafish, igf2b, but not igf2a, has
been shown to be involved during embryogenesis in the regulation of
primordial germ cell (PGC) migration (Sang et al. 2008) and in the control
of the development of the kidney (White et al. 2009).
Two receptors for IGF, igf1r and igf2r, are present in mammals. IGF1R,
like insulin receptor, belongs to the tyrosine kinase receptor family, while
IGF2R, like relaxin receptor, belongs to the mannose-6-phosphate receptor
family. Duplicated igf1r have been identified in some teleosts, including
zebrafish, likely resulting from 3R (Schlueter et al. 2006). In zebrafish,
functional divergence of the paralogs was investigated by knockdown
experiments, which revealed that igf1rb, but not igf1ra, disrupts PGC
migration (Schlueter et al. 2017). Analysis of IGF1R repertoires in Atlantic
salmon identified the conservation of 4R-duplicated igf1ra1 (two paralogs)
but a single igf1rb (Alzaid et al. 2016). To our knowledge, there has been
little investigation concerning the evolution and role of IGF2R in teleosts.
In conclusion, the actors of teleost neuroendocrine axes are the results of
multiple gene and genome duplications followed by lineage- and species-
specific paralog conservation or losses. As spotlighted by some of the
preceding examples, this has contributed to the remarkable diversification
among teleosts in their regulatory processes, life cycles, and habitat
adaptation. Teleost species currently provide various models for
biodiversity conservation, aquaculture, or biomedical research, with a large
diversity in their regulatory processes. This strengthens the necessity for
comparative and evolutionary approaches in order to highlight common
features as well as species-specific innovative adaptations and to understand
the evolutionary origin of this diversity across teleost radiation.

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 9 Reproduction
Deborah MacLatchy

CONTENTS
9.1 General Introduction
9.2 Neuroendocrinology of Reproduction
9.2.1 GnRH
9.2.2 Other Neural Factors
9.2.2.1 Dopamine
9.2.2.2 KiSS
9.2.2.3 Additional Factors
9.3 Pituitary-Gonadal Axis
9.3.1 Steroids and Steroid Receptors
9.3.2 Oocyte Development and Maturation
9.3.3 Spermatogenesis
9.3.4 Sexual Determination and Sexual Differentiation
9.4 Environmental Effects on Fish Reproduction
9.4.1 Environmental Cues
9.4.1.1 Photoperiod
9.4.1.2 Temperature
9.4.2 Environmental Endocrine Disruption
9.5 Conclusion
Acknowledgement
References

9.1 GENERAL INTRODUCTION

Spawning is the most metabolically demanding activity in the lives of
fishes, whether it occurs as a single (semelparous) spawning event or over
multiple (iteroparous) spawning sessions and/or seasons. Regulation of the
physiology of maturation, spawning, and gonadal differentiation, primarily
by the hypothalamo-pituitary-gonadal (HPG) system (Figure 9.1), allows
fishes to be reproductively successful under a variety of conditions. At its
most simplistic, gonadotropin-releasing hormone (GnRH) from the
hypothalamus controls release of the gonadotropins follicle-stimulating
hormone (FSH) and luteinizing hormone (LH) from the pituitary, which
regulate gonadal development and maturation, respectively. Produced by
the gonads under FSH and LH control, the sex steroids, for example 17β-
estradiol (E2) and 11-ketotesterone (11-KT), regulate the development of
gametes and activities in target cells in support of reproduction and
metabolic processes. This chapter synthesizes our understanding of HPG
regulation of reproduction in teleosts and includes a section on
environmental cues and endocrine disruption. Further coordination occurs
through cross-talk of additional endocrine systems, too numerous to include
in this chapter, including corticosteroids during times of stress and
maturation (Milla et al., 2009) and thyroid hormones during development
and maturation (Duarte-Guterman et al., 2014).

FIGURE 9.1 The hypothalamo-pituitary-gonadal (HPG) axis and its neurohormonal

controls in teleosts. 11-ΚT: 11-ketotestosterone; E2: 17β-estradiol; FSH: follicle-
stimulating hormone; GABA: γ-aminobutyric acid; GnIH: gonadotropin-inhibitory
hormone GnRH: gonadotropin-releasing hormone; KiSS: kisspeptin; LH: luteinizing
 hormone; NPY: neuropeptide Y; Τ: testosterone. Positive feedback represented by solid
green arrow (—>), negative feedback represented by a dashed red arrow (—>), and a
dual role of positive or negative feedback represented by a solid and dotted blue arrow
(---►).
(Modified from Trudeau, V., Rev. Reprod., 2, 55–68, 1997 and DuFour, S., et al., J. Fish
Biol., 76, 129–160, 2010.)

9.2 NEUROENDOCRINOLOGY OF REPRODUCTION

In vertebrates, the hypothalamus and pituitary connect via a short stalk
containing neurosecretory axons passing from cell bodies in the
hypothalamus to axon terminals in the adenohypophysis, the glandular part
of the pituitary, and the neurohypophysis, the neural part of the pituitary.
Gonadotropes (FSH- and LH-secreting cells) are located in the
adenohypophysis. Uniquely among vertebrates, there is no hypothalamo-
portal system in teleosts. Hypothalamic neurohormones are released
directly by nerve endings terminating in close proximity to their pituitary
target cells. Transgenic zebrafish (Danio rerio) lines expressing fluorescent
proteins in their LH and FSH gonadotropes, crossed with fluorescently
labelled pituitary vasculature or GnRH neuron lines, demonstrate a
neurovascular mode of delivery of GnRH to the gonadotropes, especially
those releasing LH (Golan et al., 2015).
9.2.1 GNRH
Sherwood et al. (1983) first described GnRH, a neural decapeptide, in chum
salmon (Oncorhynchus keta). GnRH gene expression and secretion vary
with physiological state (Zohar et al., 2010). Vertebrates express two or
three forms of GnRH, and GnRHs exert pleiotropic actions via several
classes of receptors (Kah et al., 2007; Zohar et al., 2010; Xia et al., 2014).
GnRH forms include GnRH1 (mainly found in tetrapods and some species
of fish), GnRH2 (general across vertebrates except some mammals;
previously GnRH-II or chicken GnRH-II), and GnRH3 (previously salmon
GnRH; in many but not all fishes) (Zohar et al., 2010). In two-form fishes
that have lost either GnRH1 or GnRH3 genes, the remaining form assumes
the functions of the lost one (Xia et al., 2014). Marvel et al. (2019)
generated a knockout zebrafish line, using TALEN (transcription activator-
like effector nucleases)-mediated technology, in which a targeted mutation
in the gnrh2 gene caused a frameshift coding disruption of the GnRH2
protein. Although slight decreases in oocyte quality were detected, the main
changes in the knockout line were feeding (ate more) and growth (grew
more). The expression of hypocretin (a neuropeptide that increases food
craving) was increased in the knockout females, supporting the hypothesis
that GnRH2 downregulates hypocretin to modulate feeding behavior
(Hoskins et al., 2008; Marvel et al., 2019).
The GnRH receptors (GnRHRs) in the pituitary are G-protein-coupled
receptors (GPCRs/GPRs). GnRH1 is considered a “widowed” ligand in
teleosts in which the type 1 GnRHR has been lost (Roch et al., 2014);
GnRH1 retains its function due to release in sufficient quantities and
location to activate type 2 GnRHRs (Kah et al., 2007; Roch et al., 2014).
Differences in effectiveness of the GnRH variants occur due to reproductive
state, indicating coordination of effects of the GnRH variants on pituitary
function (Zohar et al., 2010).
9.2.2 OTHER NEURAL FACTORS
9.2.2.1 Dopamine
Additional neurons innervating the adenohypophysis contain transmitters
and neurohormones that modulate gonadotropin release (Figure 9.1).
Formative studies in Richard Peter’s laboratory in the late 1970s and early
1980s identified dopamine, a monoamine catecholamine, as a strong
inhibitor of gonadotropin release in goldfish (Carassius auratus) in vitro
and in vivo (Chang and Peter, 1983); it also acts directly on gonadotropes
through D2 receptors of the GPCR family to inhibit LH release (Chang et
al., 1990). Dopamine also reduces neuronal release of GnRH at the
gonadotropes in goldfish (Yu et al., 1988). Although significant roles of
dopamine in reproduction occur in some fish species, other species, such as
Atlantic croaker (Micropogonias undulates), show no involvement of
dopamine control (Copeland and Thomas, 1989; Dufour et al., 2010). When
present, dopamine inhibits the stimulatory actions of GnRH during pre-
maturational or non-optimal times for reproduction; for example, in male
goldfish, dopamine prevents spermiation prior to ovulation of potential
female partners (as cited in Zohar et al., 2010). In fishes exposed to stress,
dopamine levels remain high during the prespawning phase, LH activity is
decreased, and ovarian maturation is suppressed, indicating a role of
dopamine in stress-induced depression of reproduction (Chabbi and
Ganesh, 2015). Down-regulation of GnRHRs in the pituitary has also been
demonstrated (Levavi-Sivan et al., 2004). Because of the role of dopamine
in inhibiting fish ovulation, including in many fish species used in
aquaculture, ovulating/spermiating agents such as Ovaprim® by Syndel
contain both a GnRH analogue and a dopamine inhibitor to promote timed
reproduction in aquaculture operations.

9.2.2.2 KiSS
In vertebrates, the KiSS system links environmental cues and
neuroendocrine signals to the HPG axis (Figure 9.1). Originally discovered
as a metastasis-suppressor gene in 1996, kisspeptin (KiSS1) was named for
its role as a suppressor sequence (ss); the letters “Ki” were appended to the
“SS” in homage to the location of its discovery, Hershey, Pennsylvania,
home of the famous “Hershey Chocolate Kiss” (Oakley et al., 2009).
Teleosts have two genes each encoding kisspeptin (kiss1 and kiss2) and
KiSSR (kissr1 and kissr2); the presence of two kisspeptin systems suggests
independent roles in the teleost brain (Gopurappilly et al., 2013). In Morone
(bass) sp., Kiss1 and Kiss2 peptides alternate between stimulation and
inhibition, depending on reproductive stage; mechanisms include both
kisspeptin effects on GnRH release and GnRH-independent actions at the
pituitary (Zmora et al., 2012; 2014). In other species, GnRH neurons do not
co-express kisspeptin receptors, for example in European seabass
(Dicentrarchus labrax; Escobar et al., 2013), increasing the complexity of
the kisspeptin system in teleosts. More recently, Tang et al. (2015)
developed the zebrafish kiss1 −/−, kiss2 −/−, and kiss1 −/−;kiss2 −/− mutant
lines as well as the kissr1 −/−, kissr2 −/−, and kissr1 −/−;kissr2 −/− mutant
lines using TALEN methodology. They showed that gonadal development
and reproductive capability (number of eggs, fertilization rate, and healthy
embryos) were not impaired in any of the mutant lines lacking functional
kiss genes, perhaps indicating system redundancy (Trudeau, 2018).

9.2.2.3 Additional Factors

Additional factors play a role in teleosts in control of the upper part of the
HPG axis, including neuropeptide tyrosine (NPY), GABA (gamma-
aminobutyric acid), and GnIH (gonadotropin-inhibitory hormone) (Figure
9.1). NPY is a highly conserved 36–amino acid peptide that has multiple G-
protein receptor subtypes and centrally regulates feeding by increasing food
intake (de Pedro et al., 2000). In goldfish, NPY acts on the pituitary to
enhance LH and neuroendocrine GnRH release; sex steroids potentiate the
effect on LH release (Trudeau, 1997).
GABA is a ubiquitous inhibitory amino acid neurotransmitter in
vertebrates. GABA’s effects are mediated by two major membrane-bound
GABA receptor classes; inotropic GABAA receptors conduct Cl−, while the
GABAB G-protein receptors are slower acting and responsible for
prolonged signaling through K+ and Ca2+ channels (Popesku et al., 2008).
Counter-intuitively in view of its generally inhibitory action, GABA
stimulates LH release by stimulating GnRH; it also inhibits dopamine
neurons in the goldfish brain (Trudeau et al., 2000). The stimulatory effect
of GABA on LH is abolished in E2-treated female goldfish, while
testosterone-treated males continue to be stimulated by GABA through
effects on LH (Trudeau, 1997). Season-dependent sex differences in the
effects of sex steroids on GABA synthesis indicate that the GABAergic
system transduces both external environmental and internal hormonal
feedback signals to exert control over LH release (Popesku et al., 2008).
The neuropeptide GnIH acts directly on the pituitary via a novel G-
protein-coupled receptor (GnIHR; GPR147) to inhibit gonadotropin release
(Tsutsui et al., 2012). In vivo, goldfish GnIH peptides significantly decrease
GnRH mRNA levels (Qi et al., 2013), and GnIH levels are dependent on
maturational status (Moussavi et al., 2012).
Additional modulators of the neuroendocrine system in vertebrates
associated with the HP axis include a significant number of metabolic and
growth factors, such as growth hormone (GH), orexin, galanin, ghrelin,
cholecystokinin, neurokinin B, and serotonin (Trudeau, 1997; Shahjahan et
al., 2014). Local production of steroids in the brain regulates reproduction
and sexual behavior as well as neurogenesis, brain plasticity, and
regeneration; steroids and neurotransmitters/neuropeptides themselves
regulate neurosteroid biosynthesis (Diotel et al., 2011). Trudeau (2018)
convincingly argues that the possibility of independent and redundant
regulation of the HP by >20 neurohormones supports diversification and
adaptation to new ecological niches and that the ability to positively link
function based on absence (e.g., in knockout studies; see Section 9.2.2.2)
remains challenging given such extensive overlap.

9.3 PITUITARY-GONADAL AXIS

Fishes have two distinct (FSH and LH) gonadotropic cell types; combined
with direct hypothalamic innervation, this simplifies differential regulation
of the gonadotropins by GnRH (Golan et al., 2015). Activation of the
GnRH system and subsequent synthesis and release of pituitary
gonadotropins that stimulate gonadal development are key events during
puberty, because during juvenile stages one or more of the components of
the axis are non-functional (Nocillado et al., 2007). FSH and LH are
glycoprotein polypeptide dimers with a common α-subunit and differing β
subunits that determine receptor-mediated roles. FSH is present in the
pituitary and circulating in the bloodstream in immature fishes and at early
developmental stages and is most associated with gametogenesis
(production of gametes) and vitellogenesis (production by the liver of
vitellogenin, the primary lipoprotein required for developing oocytes as
nutrition for the embryos). LH increases at more advanced stages of
gametogenesis and causes increases in sex and maturation-inducing steroids
from the gonads.
LH and FSH receptors are G-protein-coupled receptors (Levavi-Sivan et
al., 2010). FSH and LH stimulate E2 production at different stages; LH is
also a potent inducer of the maturation-inducing steroid (MIS; 17α,20β-
dihydroxy-4-pregnen-3-one or 17α,20β-P/DHP in most species,
17α,20β,21-trihydroxy-4-pregnen-3-one or 20β-S in others, such as
perciforms) at final maturation and spawning (Tokarz et al., 2015). In most
teleosts, but not all, the switch from ovarian E2 production to MIS is
dramatic (Section 9.3.2) immediately prior to oocyte maturation in what is
termed the two-cell model (Nagahama and Yamashita, 2008). However, this
shift has not been described in Atlantic killifish (Fundulus heteroclitus);
rather, all stages of developing follicles secrete high levels of E2 in
response to gonadotropin, and only fully grown pre-maturational follicles
produce 17α,20β-P (Lister et al., 2011).
9.3.1 STEROIDS AND STEROID RECEPTORS
In addition to the gonads, the brain and interrenal gland produce sex
steroids in fishes (Tokarz et al., 2015). Steroids are synthesized de novo
from cholesterol after transfer by StAR (steroidogenic acute regulatory
protein) across the barrier of the outer and inner mitochondrial membranes.
A series of cleavage (P450 side-change cleavage; P450scc or cyp11a1), ∆5/
∆4-isomerization, hydrogenation, aromatization, and functional group
modifications results in the terminal steroids, which are classified as
estrogens (18-carbon steroids), androgens (19 carbons), and progestins (21
carbons) (Figure 9.2). Estrogens are responsible for liver production of
vitellogenin and zona radiata proteins (part of the ova vitelline envelope);
positive and negative feedback at the level of the brain and pituitary;
inhibition of oocyte maturation; and reproductive behavior in female fishes
(Nagler et al., 2012). Androgens are associated with general metabolic
functions and masculinization, including differentiation of male
reproductive tracts, male secondary sexual characters, male reproductive
behavior, and spermatogenesis (Borg, 1994). Progestins regulate the
reproductive axis, play a role in sex determination, brain and sex
development, and are the major oocyte and sperm maturation-inducing
steroids (Fent, 2015).

FIGURE 9.2 The steroidogenic pathway within teleost gonadal cells. 11-KT: 11-
ketotestosterone; 17,20β-Ρ: 17,20β-dihydroxy-4-pregnen-3-one; DHEA:
 dehydroepiandrosterone.
(Modified from Tokarz, J., et al., Steroids, 103, 123–144, 2015.)

All the associated genes in the steroidogenic pathway (as well as

additional transcription factors) are cloned, and their expression has been
analyzed in a wide number of teleost species (for a review, see Tokarz et al.,
2015). Key enzymes and genes in the pathway include P450scc (cyp11a1),
the entrance point for cholesterol into the pathway via conversion to
pregnenolone; 17α-hydroxylase/lyase (cyp17a), the only enzyme that
converts C21 to C19 steroids; 17β-hydroxysteroid dehydrogenases
(hsd17b3 and hsd17b1), which are highly responsive to androgen feedback;
and aromatase (cyp19a1), which forms the C18 steroids and importantly,
converts testosterone to E2. Distinct androgenic pathways in teleosts
finalize in 11-KT, not 5α-dihydrotestosterone as in mammals. Other
intracellular messengers, such as prostaglandins (PGs), act in coordination
with the steroids as signals in developing and maturing fish gonads (Lister
and Van Der Kraak, 2008).
Steroids can exert their actions via cellular genomic or membrane-located
non-genomic mechanisms. The nuclear steroid receptors in fishes are well
characterized concerning their expression patterns and ligand binding
properties (for a review, see Tokarz et al., 2015). The nuclear steroid
receptors are members of the nuclear receptor superfamily; they contain
DNA binding and ligand binding domains as well as areas for binding co-
repressor, co-activator, and heat shock proteins (Nelson and Habibi, 2013).
Actinopterygii (ray-finned fishes) have at least three ER subtypes (ERα
[esr1 gene], ERβ1 [ERγ], and ERβ2 [esr2b and esr2a genes, respectively]),
with ERα being homologous across vertebrates, ERβ2 similar to ERβ in
mammals, and ERβ1 without homology to mammals and likely from a gene
duplication in fishes (Nelson and Habibi, 2013).
In the absence of a ligand, nuclear ERs are mostly found in the cytoplasm
with inhibitory heat shock proteins (HSPs); when ligands bind, the HSPs
are lost, and the ligand–ER complex moves to the nucleus and interacts
with estrogen responsive elements of the DNA. The ER/DNA recruits co-
regulator (repressor and activator) proteins required for transcription. The
liver is a critical E2 target organ in fishes. ERs mediate E2 stimulation of
mRNA levels of hepatic ER and vitellogenin (Garcia-Reyero et al., 2018).
Hepatic nuclear ER subtypes vary by sex and reproductive state. As ERβ
affinity for E2 is greater than that of ERα, it is hypothesized that ERβ acts
as an initial sensor as E2 levels begin to increase early in vitellogenesis.
ERβ binding to E2 initiates increasing vitellogenin and ERα gene
expression, and as E2 increases and directly stimulates ERα synthesis, E2 is
able to further stimulate vitellogenin (Nelson and Habibi, 2010; Nagler et
al., 2012).
The tissue distribution and androgen binding affinity of the two nuclear
AR subtypes (α and β or 1 and 2) vary in fishes (Golshan and Alavi, 2019).
For example, Atlantic croaker AR1, which binds testosterone, is located
only in brain tissue, and AR2 is expressed in gonad and brain and binds
various androgens (Sperry and Thomas, 2000). Progesterone nuclear
receptors (PRα and PRβ) have high homology across vertebrates and are
abundant in the ovary, testis, pituitary, and brain of adult teleosts (Fent,
2015). Similarly to AR, they show variation between tissues and
reproductive stages; in European eel (Anguilla anguilla), pgr1 transcripts in
the brain and pgr1 and pgr2 transcripts in the pituitary increase during
spermatogenesis (Morini et al., 2017).
Membrane-bound receptors for estrogens, androgens, and progestins
provide a contrasting mechanism of action for steroids in comparison to the
nuclear receptors. While nuclear receptor activation results in a relatively
slow response via gene transcription and translation, membrane receptors
provide the opportunity for non-genomic responses via rapid activation of
intracellular signal transduction pathways. In teleost oocytes (not follicles),
a G-protein-coupled estrogen membrane receptor (GPER or previously
GPR30) is hypothesized to mediate the estrogen maintenance of oocyte
meiotic arrest through increases in cAMP production (Thomas, 2017).
Membrane AR, a G-protein-coupled receptor, mediates androgen responses
via intracellular second messengers and protein kinase activation (Golshan
and Alavi, 2019). A novel AR (ZIP9) in Atlantic croaker ovaries is one of
14 members of the ZIP (ZRT-and Irt-like Protein, SLC39A) family that
regulates zinc homeostasis by transporting zinc across cell and organelle
membranes into the cytoplasm. In croaker granulosa cells, testosterone
induces apoptosis (cell death) through ZIP9 by a unique mechanism
involving increases in both second messengers and intracellular free zinc
concentrations (Thomas et al., 2018). Progestin membrane receptors are
receptors coupled to G-proteins of the progestin and adipoQ receptor family
and a single transmembrane progesterone receptor membrane component
(Morini et al., 2017). Perhaps the most studied of the membrane PRs are
mPRα and mPRβ, located on the oocyte plasma membranes. LH induces
the expression of oocyte mPR and the steroidogenic transition from E2 to
MIS; MIS then mediates oocyte maturation through activation of the mPRs,
which trigger intracellular signaling, thereby initiating germinal vesicle
breakdown (GVBD) corresponding to the first meiotic cell division, spindle
formation, chromosome condensation, and formation of the first polar body
(Figure 9.3; Lubzens et al., 2010).

FIGURE 9.3 Five phases of oogenesis and the main molecular mechanisms involved.
See text for specific details. E2: 17β-estradiol; FSH: follicle-stimulating hormone; LH =
luteinizing hormone; Vtgs: vitellogenins; ZPPs: zona pellucida proteins. Solid black
line: local transport; dotted black line: vascular transport.
(Redrawn from Cerdà et al., Rev Fisheries Sci, 16, 56–72, 2008).

Steroids also act as exogenous behavioral signals, synchronizing

reproduction and other social activities. Sex pheromones produced by
female and male fishes are molecular cues, released into the aquatic
medium, composed of reproductive steroids or PGs (or modified versions),
for which conspecifics have sensitivity via specific olfactory receptors
(Kobayashi et al., 2002; Stacey, 2003).
9.3.2 OOCYTE DEVELOPMENT AND MATURATION
The ovary consists of two basic cell types: somatic cells forming the
supportive ovarian structure and germ cells that ultimately become the
haploid cells of the gametes (oocytes). The primordial germ cells develop
first into diploid oogonia, which differentiate and undergo the first meiotic
division after a number of mitotic cell divisions. An inner continuous
granulosa follicular layer surrounds developing oocytes, and an outer thecal
layer formed from the outer stromal connective tissue surrounds the
granulosa (Lubzens et al., 2010; Kagawa, 2013). As detailed in Kagawa
(2013) and Lubzens et al. (2010), the process of oogenesis is divided into
four phases: proliferation, primary growth, secondary growth, and
maturation (Figure 9.3). In proliferation, after prescribed times of mitotic
cell division particular to a fish species, oogonia become primary oocytes at
diplotene of the first meiotic division. There is incomplete understanding of
the endocrine role of proliferation and oocyte recruitment, but there is some
evidence that gonadotropins have a role. Prior to the cortical alveoli stage,
the follicles are able to proceed through development in the absence of
pituitary gonadotropins; however, there is evidence that both FSHβ and
LHβ mRNA transcripts of local origin may have a role (Wong and Zohar,
2004), as may other growth factors (Lubzens et al., 2010). During the
secondary growth phase, massive growth of the oocyte occurs through
FSH-stimulated vitellogenesis, in which oocytes accumulate the nutritional
reserves needed for embryo development (Lubzens et al., 2010).
Maturation occurs when meiosis returns, GVBD occurs, and the cortical
alveoli forms. The first meiotic division results in a polar body (which
degenerates) and a large secondary oocyte that is expelled into the ovarian
or abdominal cavity at metaphase II from the follicular layers and,
following the second meiotic division, becomes an ovum (female gamete)
and a second degenerating polar body. Maturational competency is induced
by the gonadotropins and occurs through the increase of MIS receptors on
the oocyte cell membrane; roles for IGF (insulin-like growth factor),
activin, and other ovarian factors have been described (Lubzens et al.,
2010). In ovulation, the MIS-induced increase in oocyte nuclear progestin
receptors is an important step in the ovulatory process (Nagahama and
Yamashita, 2008). In addition to MIS and progestin, ovulation involves
arachidonic acid and its metabolites, including PGs, and other factors acting
in cooperation, such as proteases, anti-proteases, catecholamines,
vasoactive peptides, etc., to separate the oocyte from the granulosa layer
and cause the localized rupture of the follicle (Lubzens et al., 2010).
9.3.3 SPERMATOGENESIS
Vertebrate testes are composed of two main compartments: the intertubular
(or interstitial) compartment, made of steroidogenic Leydig cells, blood,
and nervous and structural tissue, and the tubular compartment, containing
a germinal epithelium that includes somatic Sertoli cells and the germ cells.
During spermatogenesis, diploid spermatogonia multiply and differentiate
into spermatozoa in three phases: proliferation (proliferation of different
generations of spermatogonia); meiotic (primary and secondary
spermatocytes); and maturation (production of haploid spermatids and
motile spermatozoa) (Figure 9.4; Nóbrega et al., 2009; Schulz et al., 2010).

FIGURE 9.4 Four stages of teleost spermatogenesis and the major mechanisms in each.
See text for specific details. 11-KT: 11-ketotestosterone; 17.20β-Ρ: 17α,20β-dihydroxy-
4-pregnen-3-one; AMH: anti-Müllerian hormone;·E2: 17β-estradıol; FSH: follicle-
stimulating hormone; LH: luteinizing hormone; SRS34: spermatogenesis stem cell
renewal factor 34. Solid black line: local transport; dotted black line: vascular transport.
(Updated from Cerdā et al. 2008).

Receptor-mediated Leydig cell steroidogenesis is regulated by both FSH

and LH, while FSH regulates Sertoli cells. High LH concentrations, such as
occur during spawning, can stimulate FSHRs (Schulz et al., 2010). In
salmonids, FSH levels increase at the time of spermatogonia multiplication
and decrease before the spawning season, while LH levels are low or
undetectable during the start of testis development, increase marginally
during meiosis, and increase significantly close to the spawning season
(Schulz et al., 2010). Direct roles of FSH on Sertoli cells have not been
identified in fishes; a variety of other growth factors, such as TGFβ
(transforming growth factor β), AMH (anti-Müllerian hormone or Müllerian
inhibiting substance) produced locally in the germ cells under steroid
control, and IGF-1 induced by pituitary GH appear to modulate
spermatogenesis through differential expression of hormone and growth
factor receptors (Schulz et al., 2010). AMH inhibits spermatogonial
proliferation in teleosts (Pfennig et al., 2015). Testosterone and 11-KT
increase gradually during spermatogenesis and decrease at spermiation.
Two peaks of MIS occur, a small one during mitotic proliferation of
spermatogonia and a large one during spawning (Scott and Sumpter, 1989).
Estrogens also play a receptor-mediated role in fish testes. For example, in
Japanese eel (Anguilla japonica), E2 induced spermatogonial mitosis, and
tamoxifen (an estrogen receptor antagonist) depressed it (Miura et al.,
1999).
9.3.4 SEXUAL DETERMINATION AND SEXUAL DIFFERENTIATION
Fish genetic sex determination includes monofactorial regulation in
Japanese medaka (Oryzias latipes), a female (XX) and male (XY) system,
with the DMY (DM-domain gene) on the Y chromosome (Nagahama,
2005); multifactorial regulation in the swordtail (Xiphophorus madulatus),
which has three different genetically well-defined sex chromosomes,
resulting in females (XX, XW, and YW) and males (XY or YY) (Franchini
et al, 2018); and polyfactorial (polygenic) regulation, as hypothesized in
zebrafish, which do not have a chromosomal sex determination system
(Liew et al., 2012), and the European sea bass (Dicentrarchus labrax), a
species whose sex determination is influenced by temperature (Vandeputte
et al., 2007). Gonadal sex, however, is determined not just genetically in
fish species but also environmentally (e.g., by temperature, population
structure, or endocrine-active contaminants, or through their interaction);
sex differentiation describes the processes that result in gonadal sex (Devlin
and Nagahama, 2002). While the sex-determining genetic profiles vary in
teleosts, the subsequent molecular cascades leading to differentiation in
males and females are relatively conserved; synthesized steroids,
transcription factors, and receptors are critical for sex differentiation
(Devlin and Nagahama, 2002; Nagahama, 2005). Gonadal sex can be
reversed from genetic sex by exposure to exogenous steroids (Paul-
Pransanth et al., 2011) and by temperature changes (Ospina-Álvarez and
Piferrer, 2005; see Section 9.4.1.2) in some species. Fish gonadal
differentiation is classified by the order of phenotypic gonadal development
(as detailed in Devlin and Nagahama, 2002).

9.4 ENVIRONMENTAL EFFECTS ON FISH

REPRODUCTION
9.4.1 ENVIRONMENTAL CUES
Reproductive development and spawning readiness in fishes are generally
signaled by environmental cues, including photoperiod (Norberg et al.,
2004) and temperature (Ospina-Álvarex and Piferrer, 2008), as well as other
variables such as (semi-)lunar/tidal cycles (Takemura et al., 2010), rainfall
and water level (de Magalhães Lopes et al., 2018), and social conditions
(Juntti and Fernald, 2016). These “proximal cues” (Bromage et al., 2001)
stimulate signal transduction in the endocrine system to regulate
reproduction so that it occurs at the most beneficial time of the year, season,
or day for offspring survival (Migaud et al., 2010). Other “permissive
cues”, such as food availability/nutritional status (Vrtilek and Reichard,
2014), hypoxia (Landry et al., 2007), and habitat (Able and Hagen, 2003),
can modify entrained environmental cues. This may result in alteration of
the response to the proximate cue(s) for the purpose of encouraging
reproduction at its most advantageous or discouraging it when conditions
are less than favorable.

9.4.1.1 Photoperiod
Photoperiod (solar day length) is the principal environmental cue of
reproduction in teleosts; it delivers an unambiguous “date signal” (Bromage
et al., 2001; Pankhurst and Porter, 2003; Wang et al., 2010; Migaud et al.,
2010) and can entrain both seasonal and daily cycles. Seasonality, as related
to photoperiod and temperature, is most marked at higher latitudes
(Pankhurst and Porter, 2003); photoperiod and temperature combine in
many fish species as the proximate seasonal environmental cues (for details,
see Wang et al., 2010). Tropical species, in which seasonality plays a
limited role, inhabit environments in which conditions for eggs and young
support reproductive success over much of the year and in which other
factors, such as food availability and lunar cycle, take on additional
importance (Shima et al., 2017). Even when photothermal cycles are out of
phase, reproduction remains entrained to photoperiod (Norberg et al., 2004;
Migaud et al., 2010).
In brief, photoperiod operates as a reproductive signal because light is
inhibitory on the synthesis of the monoamine neurotransmitter melatonin by
the pineal gland (Pankhurst and Porter, 2003), and the melatonin signal is
transduced into a reproductive endocrine signal (see Saha et al., 2019 for a
detailed review). Melatonin is both a clock (duration of plasma levels
correspond to length of the night) and a calendar (seasonal changes in
photoperiod and temperature affect the duration and amplitude of the
nocturnal pulse) (Servili et al., 2013). The seminal study by Khan and
Thomas (1996) demonstrated that melatonin stimulated in vitro LH release
from pituitary cells taken from mature Atlantic croaker; additionally, in
vivo, melatonin injected late in the day into the diencephalon increased
circulating LH levels. Melatonin signals may be interpreted differently
depending on species, gender, photoperiod, reproductive status, and
endpoints assessed (Migaud et al., 2010; Servili et al., 2013). Differential
tissue distribution and expression variations of three G-protein-coupled
transmembrane melatonin receptors (MT1 or mel1a, MT2 or mel1b, and
mel1c) underlie melatonin function in vertebrates (Chai et al., 2013). In the
orange-spotted grouper (Epinephelus coidoides), seasonal breeding appears
to be controlled by endocrine cascades in which a decrease in MT1
expression may increase the expression of gnrh1 by upregulating the
expression of kiss2 and/or direct upregulation of gnrh1 (Chai et al., 2013).

9.4.1.2 Temperature
In addition to its cueing role, temperature may also play a modulating role
(Wang et al., 2010). For example, a sudden decrease in temperature during a
normally induction-cueing period of warming in spring may act as a
stressor and/or affect metabolism, ultimately altering oogenesis (Wang et
al., 2010). Elevated temperature has been shown to have a range of
signaling effects in teleosts, including decreased LH secretion, reduced
and/or delayed ovulation, reduced steroid production, reduced vitellogenin,
and reduced aromatase (for a summary, see Pankhurst and Munday, 2011).
 Fishes have an extraordinary degree of interaction between genetic sex
and temperature (Devlin and Nagahama, 2002). Temperature-dependent sex
determination occurs in teleosts (e.g., Menidia menidia, Atlantic silverside;
Ospina-Álvarez and Piferrer, 2008), as does temperature alteration of
genotypic sex determination (e.g., European sea bass; Piferrer et al., 2005).
Rising temperatures due to climate change have the potential to impact fish
reproduction across all habitats by changing sex ratios (Ospina-Álvarez and
Piferrer, 2008) and impacting recruitment and generational population
structures (Pankhurst and Munday, 2011).
9.4.2 ENVIRONMENTAL ENDOCRINE DISRUPTION
Anthropogenic sources of compounds capable of interacting with the HPG
axis in fishes are a modern environmental challenge. Endocrine disrupting
compounds (EDCs) affect fish reproduction, for example, through
interfering with HP signaling pathways in the brain (Duarte-Gutterman et
al., 2014), reducing gonadal hormone levels (Schug et al., 2011), and
causing intersex (feminization or masculinization that differs from genetic
sex; Marjan et al., 2017). EDCs are introduced into the environment from a
variety of anthropogenic sources and may act as receptor agonists or
antagonists (Fent, 2015). Once bound, these complexes can activate or
inhibit the translation of responsive genes, initiating a signaling cascade that
can result in negative effects of exposure. Some EDCs alter the activity of
steroidogenic enzymes, changing the overall synthetic capacity for sex
hormones (Rutherford et al., 2015), which may result in changes in mating
behavior, offset of gamete maturation, or decreased fecundity (Söffker and
Tyler, 2012). Changes in clearance and transport are common impacts of
EDCs on the reproductive axis in teleosts (Parks and LeBlanc, 1998;
Saxena et al., 2014). EDC research has enhanced our understanding of
reproductive endocrine biology in teleosts, especially in regard to the
mechanistic links between molecular modes of action and population-level
effects.

9.5 CONCLUSION
Successful reproduction is critical for population maintenance and growth.
The reproductive endocrine system controls reproduction in fishes for times
most beneficial for spawning and offspring survival. Evolutionarily, the
HPG system is highly conserved and very robust; however, climate change
and anthropogenic contaminants have the potential to endanger fish
populations through adverse effects on the reproductive endocrine system.

ACKNOWLEDGEMENT
The development of this chapter was skillfully assisted by Robert
Rutherford, PhD student in my research laboratory at Wilfrid Laurier
University.

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 10 Metabolism
Tommy Norin and Ben Speers-Roesch

CONTENTS
10.1 Introduction
10.2 Levels of Metabolic Rate
10.3 Modulators of Metabolic Rate
10.3.1 Body Mass
10.3.2 Temperature
10.3.3 Hypoxia
10.4 Variation in Metabolic Rate among and within Species
10.5 Ecological and Evolutionary Relevance of (Varation in) Metabolic
Rate
10.6 Conclusion
References

10.1 INTRODUCTION
All living things, including fishes, must harness energy and materials from
their environment in order to maintain homeostasis, grow, and reproduce.
Metabolism is the sum of the chemical reactions within the body that
convert food to energy molecules (e.g. ATP) and building blocks for
growth, and which use these compounds to sustain the cellular energy
demands that support life (e.g. protein synthesis, molecular movement, and
generation of membrane electrochemical gradients). To thrive, organisms
must modulate their rates of energy supply to match varying energy
demands, or vice versa (Somero et al. 2017). This energy turnover is
metabolic rate, which is most easily thought of as the amount of energy
used by an animal per unit of time (i.e. energy expenditure). While different
cells, tissues, and organs have their own metabolic rates, we will focus on
whole-animal metabolic rate, which reflects the integrated, overall energy
expenditure of an ecologically relevant unit, the individual. Metabolic rate
determines an animal’s demands for environmental resources and its
capacities for biological activities (Brown et al. 2012). Consequently,
metabolic rate fundamentally influences the ecology and evolution of
animals and their responses to environmental change, which can affect
energy supply or impose energy demands.
Metabolic rate is sustained by aerobic or anaerobic energy supply
(Ballantyne 2014). Aerobic metabolism occurs in the mitochondria, where
ATP is generated from ADP using food energy (lipids, carbohydrates, and
proteins) and oxygen delivered by the cardiorespiratory system. Anaerobic
metabolism produces ATP without oxygen by directly transforming ADP
into ATP using high-energy compounds (e.g. creatine phosphate) or
anaerobic glycolysis (conversion of carbohydrates to lactate) in the
cytoplasm. Although anaerobic metabolism produces ATP more rapidly
than aerobic metabolism, it does so far less efficiently (i.e. less ATP per unit
of fuel) and is accompanied by metabolic acid and/or other deleterious by-
products (Somero et al. 2017). Thus, animals rely on anaerobic metabolism
only under special circumstances, typically of shorter duration, such as
intense exercise or daily hypoxic events.
Under normal, well-oxygenated conditions where aerobic metabolism
predominates, metabolic rate can be estimated indirectly as oxygen uptake
rate (ṀO2). ṀO2 reflects the rate of oxygen flow from the environment to
the mitochondria to sustain aerobic metabolic rate. This oxygen transport
cascade consists of a series of diffusion and convection steps as oxygen
diffuses into the animal at the respiratory surface, is transported to tissues
bound to haemoglobin in blood flowing via the cardiovascular system (with
the exception of some haemoglobin-less Antarctic icefish, where oxygen is
transported dissolved in the blood plasma), and diffuses into cells and
finally, their mitochondria. Because of the primacy of aerobic metabolism
and the relative ease of oxygen measurement via respirometry (Steffensen
1989; Clark et al. 2013), the vast majority of work on metabolic rate of
fishes focuses on ṀO2, and we will too. However, it is worth noting that
ṀO2 may underestimate metabolic rate under conditions where the
contribution of anaerobic metabolism becomes great (e.g. hypoxia or
exhaustive exercise). The anaerobic contribution to metabolism can be
evaluated as the excess ṀO2 occurring after the anaerobic period (Cox et al.
2011) or from the accumulation rate of anaerobic end-products (e.g.
lactate), which can be used to estimate ATP production using oxycalorific
equivalents (Regan et al. 2017).
A calorimeter can be used to estimate total metabolic rate (aerobic plus
anaerobic metabolic rate) via measurement of whole-animal heat
production (Regan et al. 2017), but this is challenging in fishes, as they
have low heat production. Nonetheless, this approach has shown, for
example, that metabolic rate can become decoupled from ṀO2 during rapid
hypoxia exposure (Regan et al. 2017).

10.2 LEVELS OF METABOLIC RATE

The metabolic rate of an individual fish can run at a variety of levels
bounded by minimum and maximum limits, depending on the ecological
and physiological circumstances. The lower limit is the minimum metabolic
rate required to maintain homeostasis to stay alive (i.e. the basic cost of
living). In fishes and other ectotherms under normal environmental
conditions, this is referred to as the standard metabolic rate (SMR), which is
the energy expenditure of a post-absorptive (not digesting), non-
reproducing, and quiescent animal at a defined temperature (Chabot et al.
2016). The SMR of fishes generally is slightly lower than that of
amphibians and reptiles and around 25 times lower than that of endothermic
mammals and birds when compared at a standard body temperature (White
et al. 2006).
Despite its importance as a baseline of maintenance requirements, fish
spend little time at their SMR, because essential life cycle processes (such
as physical activity, digestion, growth, or reproduction) require elevations
in energy expenditure above SMR. A fish undergoing voluntary activity in
the laboratory is said to be at a routine metabolic rate (RMR), while the
average metabolic rate of an animal in the wild is referred to as its field
metabolic rate (FMR). The upper limit on aerobic metabolic rate is the
maximum metabolic rate (MMR), which typically occurs when a fish is
exercising maximally (Norin and Clark 2016). The level of MMR that can
be achieved is determined by the maximum capacity of the
cardiorespiratory system to supply oxygen to tissues (Norin and Clark
2016), though an even higher metabolic rate fuelled anaerobically may be
briefly possible (Treberg et al. 2016). SMR and MMR may be subtly
linked, because organ systems required to sustain a high MMR are more
expensive to maintain at rest (Norin and Clark 2016). The difference
between MMR and SMR is the aerobic scope (AS), which is the aerobic
metabolic capacity to sustain critical performance activities including
exercise, digestion, and reproduction (Clark et al. 2013).
When faced with a significant environmental constraint on energy supply
(e.g. food scarcity or hypoxia), some fish species can temporarily lower
their baseline energy expenditure via metabolic rate depression, which is a
reversible downregulation of cellular energy turnover that decreases whole-
animal metabolic rate to a new, temporary level below SMR (Guppy and
Withers 1999; Richards 2010; Mandic and Regan 2018). A metabolically
depressed animal thus sacrifices normal function to save energy and survive
in an adverse environment.
Because the various levels of metabolic rate set the basic cost of life and
performance capacities of organisms, and are influenced by environmental
variation, they are highly plastic, are under selective pressure, and have
great ecological relevance.

10.3 MODULATORS OF METABOLIC RATE

Metabolic rate is influenced by numerous physiological and environmental
factors that affect energy supply and/or demand. For example, digestion,
swimming, and reproduction elevate metabolic rate (Masonjones 2001;
Secor 2009; Fu et al. 2011), but because of the upper aerobic limit set by
MMR, energy must be allocated between maintenance (i.e. SMR) and these
various activities (Figure 10.1). Environmental challenges such as salinity
changes (Ern et al. 2014) and food availability can similarly influence
energy demands or energy supply, requiring shifts in metabolic rate to
ensure energy balance (Richards 2010).
 FIGURE 10.1 Example of elevations in aerobic metabolic rate above SMR (dotted
black line and orange bars) caused by digestion and swimming in Southern catfish
(Silurus meridionalis) at ~25 °C. Panel (a) shows the post-prandial elevation of ṀO2
(termed ‘specific dynamic action’, SDA; see Secor, 2009) in catfish digesting meals
from 2 to 24% of their body mass (BM) relative to an unfed control (0% BM). Panel (b)
shows how digestion competes with swimming for the available aerobic scope (grey
area in a and b) in catfish that were either fasting (blue bars) or fed 16% of their BM
(pink bars) and swum in a flume at different speeds. While the cost of SMR was
necessarily paid at all times, the energetic cost of swimming increased with increasing
speed until the critical swimming speed (U crit) was reached at 42 cm s−1 in fasting fish
(corresponding to ~3.1 body lengths [BL] s−1). Fish that were both swimming and
digesting reached a higher MMR (dashed black line) than fish that were swimming
while fasting, but U crit was reduced with increasing meal size (c), reflecting a trade-off
in energy allocation.
(Data from Fu, S.J., et al., J. Exp. Biol., 212, 2296–2302, 2009; Fu, S.J., et al., Comp.
Biochem. Physiol. A, 158, 498–505, 2011.)

In the following, we focus on three factors that pervasively affect the

metabolic rates of fishes: body mass, temperature, and hypoxia.
10.3.1 BODY MASS
As for all animals, the metabolic rate (MR) of fishes changes with body
mass (BM) according to a power relationship: MR = a BM b, where a is the
scaling coefficient and b the scaling exponent (Figure 10.2). If log10–log10
transformed, the relationship between metabolic rate and body mass
becomes linear: log10 MR = b log10 BM + log10 a, with a being the
intercept of the line and b the slope. The scaling exponent (b) thus
represents how steeply metabolic rate changes (scales) with body mass.
This metabolic scaling has fundamental importance for understanding how
resource demands change within and among animal communities, which
are composed of individuals and species that grow and vary in size.
Metabolic scaling also informs predictions of the impact of environmental
factors that affect animal size. For example, a reduction in body size of
fishes is occurring as a result of climate warming, with consequences for
energy demand relative to supply (Cheung et al. 2013; Lefevre et al. 2018).

FIGURE 10.2 Scaling of metabolic rate with body mass (BM) for SMR (diamonds)
and MMR (circles) of 140 and 115 species of teleost fish, respectively. Each point
represents a species, and eight species with relatively low (Cyprinus carpio, Solea
solea, Anguilla rostrata), average (Salmo trutta, Perca fluviatilis, Gadus morhua), or
high metabolic rates (Thunnus albacares, Oncorhynchus nerka) are illustrated with
coloured points and associated fish silhouettes. Data are from the literature, and the
body mass of each species is the mean for fish used in the original study (i.e. it does not
necessarily reflect the species’ average mass). All data have been adjusted to a
 temperature (T) of 15 °C using residuals from the regression equation for SMR (log10
2
SMR = −1.426 + 1.013 log10 BM + 0.018T; r = 0.909) or MMR (log10 MMR =
−0.717 + 0.953 log10 BM + 0.023T; r 2 = 0.899). The lines are least-squares regression
lines for SMR (dashed black line) and MMR (solid black line) surrounded by 95%
confidence bands in grey. Scaling equations for the temperature-adjusted data are
presented on the figure. The 95% confidence intervals for scaling exponents are 0.958–
1.068 for SMR and 0.890–1.017 for MMR.

After accounting for variation in temperature among studies, the SMR of

fishes is found to scale with exponents around 0.9 to 1 (White et al. 2006;
Killen et al. 2016; Figure 10.2). These exponents are significantly higher
than those found for mammals (b ≈ 0.7), birds (b ≈ 0.7), and reptiles (b ≈
0.8), but not necessarily amphibians (b ≈ 0.9) (White et al. 2006).
The MMR of mammals has been suggested to scale with an exponent
higher than that for SMR, approaching a value of 1 due to volume-related
processes (muscle work) dominating metabolic rate during activity (Weibel
and Hoppeler 2005). However, in fishes, the scaling of temperature-
adjusted MMR with body mass across species (i.e. inter-specifically)
appears to be similar to that for SMR (b SMR = 0.95 vs. b MMR = 0.94 in
Killen et al., 2016 and b SMR = 1.01 vs. b MMR = 0.95 in Figure 10.2].
Scaling relationships can also differ at different taxonomic levels. For
example, SMR has been found to scale with an average exponent of 0.89
within species of fish (i.e. intra-specifically; Jerde et al. 2019), which is
shallower than inter-specific scaling (Figure 10.2, b SMR = 1.01). Moreover,
there is evidence that intra-individual scaling exponents (b SMR = 0.74, b
MMR = 0.83) are lower than intra-specific ones (b SMR = 0.89, b MMR = 0.94)
(Norin and Gamperl 2018). This decrease in scaling exponents with
taxonomic level could potentially result from high selection on fast growth
during early life stages (i.e. strong selection to grow out of the risky larval
or juvenile stage in fish), where metabolic rate generally scales more
steeply than later in life (fish usually exhibit a biphasic scaling pattern,
where both SMR and MMR of larvae scale more steeply than those of
juveniles or adults; Post and Lee 1996; Killen et al. 2007).
10.3.2 TEMPERATURE
Temperature is a ‘master factor’ influencing the distribution and abundance
of animals (see also Chapter 7), in part because of its fundamental effect on
the biochemical reaction rates that underlie metabolic rate (i.e. slower with
cooling, faster with warming; Schulte, 2015). Ectotherms, including almost
all fishes, are particularly temperature sensitive, because their body
temperature matches the surrounding environment. The effect of
temperature on metabolic rate (or other biological rates, e.g. growth) can be
described with a thermal performance curve (TPC; Figure 10.3). Lethal
temperatures (critical thermal minima or maxima; CTmin or CTmax,
respectively) occur at the extremes of the TPC, resulting from adverse
thermal effects on multiple physiological processes. At high temperatures,
there is probably a species-specific role for a thermal limitation on the
cardiorespiratory system to deliver enough oxygen to sustain a high
metabolic rate (Schulte 2015). Thus, temperature profoundly affects
metabolic rate and consequently, fitness-linked performance capacity (e.g.
swimming, reproduction, or growth) or even survival.

FIGURE 10.3 Conceptual thermal performance curves (TPCs) for SMR (orange line)
and MMR (green lines) on either absolute (a) or ln-transformed (b) scales. SMR
increases exponentially with increasing temperature (panel a), thus becoming linear
when ln-transformed (panel b). The thermal dependence of MMR is less known and
appears to be species specific (e.g. eurythermal vs. stenothermal species), with a plateau
at intermediate temperatures (dashed green line) in some species and an increase until
near-lethal limits in others species (solid green line; also see Figure 10.4 and Lefevre,
2016).

To cope with thermal challenges, fishes may compensate their metabolic

rate to maintain normal capacities. Compensation at evolutionary scales
(specifically, adaptive change among species) is shown by TPCs of cold-
and warm-water species being shifted to cold and warm temperatures,
respectively (Johnston et al. 1991; Payne et al. 2016). However, the extent
to which species adapt to compensate for the effects of temperature on
metabolic rate is controversial. For example, the ‘metabolic cold
adaptation’ hypothesis proposes that species that evolved in cold
environments have comparatively higher metabolic rates than warm-water
species, when evaluated at a common temperature, to compensate for the
slowing effects of cold on metabolism. Conversely, warm-water species can
benefit from evolving lower metabolic rates to avoid unsustainable resting
metabolic demands at warm temperatures. Historically, the evidence for
metabolic cold adaptation in fishes was considered robust, indicating near-
complete compensation of the effects of cold on metabolism. However, this
conclusion was probably an artefact of unaccounted influences of elevated
activity and stress levels in early measurements of the metabolic rate of
polar fishes (Steffensen 2002). Currently, there is some evidence supporting
the hypothesis, but the compensation across species is only partial (White et
al. 2011); adaptation has not fully compensated for the slowing effects of
cold.
The breadth and height of metabolic rate TPCs also respond to
adaptation. Some species are thermal generalists and have broad TPCs
reflecting a more thermally tolerant phenotype, usually at a trade-off with
maximum performance, whereas other species are specialists that prioritize
higher metabolic performance across a narrower temperature range
(Angilletta 2009).
Within individual fish, thermal plasticity of metabolic rate is a common
adaptive response to match the phenotype to the environment. Plasticity
may occur during development, typically irreversibly. For example, there
are persistent beneficial effects of development temperature on the
metabolism and swimming performance of zebrafish (Scott and Johnston
2012). During an adult fish’s lifetime, reversible thermal plasticity of
metabolic rate is well known and is termed thermal acclimation (when in
the laboratory) or acclimatization (when in the wild) (Seebacher et al.
2015). Reversible metabolic rate plasticity is a common response to
seasonal temperature change, whereby metabolic rate is increased at cold
temperatures and decreased at warm temperatures relative to the metabolic
rate response to more rapid thermal changes (Figure 10.4). This metabolic
plasticity comes about via coordinated changes in tissue mitochondrial
abundance, tissue enzyme activities, and properties of the oxygen transport
cascade (Angilletta 2009).

FIGURE 10.4 Thermal responses of SMR (orange) and MMR (green) on either
absolute (a) or ln-transformed (b) scales for the eurythermal barramundi (Lates
calcarifer) acclimated to 29 °C or 38 °C for 5+ weeks (triangles) and for the 29 °C-
acclimated fish acutely exposed to 23, 35, and 38 °C (circles). Acute exposure to 41 °C
was intolerable (i.e. CTmax was somewhere between 38 and 41 °C). The thermal
sensitivity (Q 10) of metabolic rate is presented in panel (b) and can be calculated from
the slopes of the linear (ln-transformed) regressions as Q 10 = e(10 · slope). Acclimation to
38 °C reduced SMR and MMR by 14% and 32%, respectively, relative to acutely
exposed fish, as reflected by the reduced Q 10 values.
Data are from Norin, T., et al., J. Exp. Biol., 217, 244–251, 2014.)

The compensation associated with thermal plasticity of metabolic rate is

rarely complete (i.e. the thermal sensitivity coefficient, Q 10, is larger than
1; Figure 10.4), possibly because acclimation costs energy (Seebacher et al.
2015). Certain species do not show thermal plasticity of metabolic rate (e.g.
Speers-Roesch et al. 2018), and may even enter a dormant state, possibly
because in some situations, having a slowed SMR is useful (e.g. a food-
poor winter). There is, however, little evidence for metabolic rate
depression in dormant fish, in particular at cold temperatures, where
reductions in activity, combined with passive physicochemical slowing of
SMR, accrue substantial energy savings (Speers-Roesch et al. 2018). In
such cases, reduced metabolic capacity is presumably an acceptable trade-
off for the benefit of having low energy demands, at least temporarily.
10.3.3 HYPOXIA
Hypoxia, a common stressor in aquatic environments, has profound effects
on metabolic rate because it can constrain aerobic metabolism, increase
reliance upon anaerobic metabolism, and in some species, cause a
metabolic rate depression.
Fishes are oxyregulators: as water oxygen declines from an oxygen
partial pressure (PO2) of 100% air saturation (i.e. atmospheric oxygen
level), fish initially defend their ṀO2 until a certain PO2 is reached, below
which ṀO2 declines (conforms) with further decreases in water PO2
(Mandic et al. 2009; Rogers et al. 2016). This inflection point between the
zones of oxyregulation and oxyconformation is the critical oxygen tension
(P crit), which indicates the ability of an animal to extract oxygen from its
environment in order to maintain its metabolic rate (Figure 10.5). Below P
crit, the animal transitions to unsustainable anaerobic metabolism or
metabolic rate depression. P crit is determined by the characteristics of the
steps in the oxygen transport cascade, in particular gill surface area and
haemoglobin–oxygen binding affinity (Nilsson 2007; Mandic et al. 2009;
Speers-Roesch et al. 2012; Lau et al. 2017).

FIGURE 10.5 Conceptual (a) and empirical (b) relationships between aerobic
metabolic rate and environmental oxygen availability (oxygen partial pressure, PO2).
The critical oxygen tension (P crit) is the PO2 below which SMR can no longer be
sustained. A P crit can also be envisioned for RMR (P crit, RMR) and MMR (P crit, MMR),
with the latter found to occur at around 21 kPa (normoxia) in a majority of species
(Seibel and Deutsch 2020). The grey area represents aerobic scope, which decreases
with increasing hypoxia (i.e. decreasing PO2) and becomes zero at P crit.
 The empirical data in panel (b) are from Ern, R., et al., J. Exp. Biol., 219, 3376–3383,
2016 for lumpfish (Cyclopterus lumpus) at 10 °C.

Variation in P crit has evolved among fish species and populations. Fishes
with lower P crit are, in general, more hypoxia tolerant and more likely to
live in hypoxia-prone habitats (Mandic and Regan 2018). Within
individuals, development or acclimation in hypoxia may also result in lower
P crit (Rogers et al. 2016). These evolutionary and plastic adjustments of P
crit occur via changes in the characteristics of the oxygen transport cascade
(Nilsson 2007; Mandic et al. 2009; Speers-Roesch et al. 2012; Borowiec et
al. 2015). While P crit has a robust theoretical basis and ecological
relevance, it should be measured and interpreted carefully, and ideally
alongside other measures of hypoxia tolerance (Speers-Roesch et al. 2013;
Ultsch and Regan 2019).
As water PO2 falls below P crit, aerobic energy supply becomes
constrained, and anaerobic energy supply is increasingly relied upon to
meet energy demand and maintain cellular energy balance. Consequently,
lactate and metabolic acid accumulate in the body, which can interfere with
normal physiological function and ultimately limit the magnitude and
duration of hypoxia that are survivable (Speers-Roesch et al. 2013). Thus,
for longer or more severe hypoxia exposures, other strategies have evolved
to mitigate the harmful side effects of anaerobic metabolism. Certain
species of hypoxia-tolerant fish, including cyprinids that overwinter in
anoxic freshwaters and myctophids that live in the oxygen minimum zone
of ocean midwaters, have evolved a capacity to produce ethanol as an
anaerobic end-product rather than lactate (Somero et al. 2017). However,
even if anaerobic metabolism is made more tolerable, hypoxic survival will
be limited by available fuel stores, specifically glycogen (Speers-Roesch et
al. 2013). The ability to reduce energy demand then becomes important to
maintain energy balance without rapidly exhausting anaerobic fuel stores or
creating a fatal acidosis.
Decreases in energy demand during hypoxia exposure can be achieved
via behavioural reductions in feeding and movement to attain SMR and/or
via a metabolic rate depression below SMR (Richards 2010). Metabolic rate
depression has evolved repeatedly in hypoxia- and anoxia-tolerant fishes
(Mandic and Regan 2018). The mechanisms underlying metabolic rate
depression include modulation of inhibitory neurotransmitter levels, protein
phosphorylation cascades that downregulate energy demand pathways such
as protein synthesis, and modulation of mitochondrial energy supply
(Somero et al. 2017). Metabolic rate can be depressed by 85% or more in
fishes (Richards 2010; Regan et al. 2017), decreasing reliance on anaerobic
metabolism and prolonging hypoxia survival time. However, a consequence
is greatly inhibited metabolic performance (e.g. feeding and reproduction).
Indeed, the constraints that hypoxia places on metabolic rate help explain
the effects of hypoxia on the distribution and ecological function of fishes
(Deutsch et al. 2015).

10.4 VARIATION IN METABOLIC RATE AMONG AND

WITHIN SPECIES
Metabolic rates of fishes vary widely and in association with both lifestyle
and trophic level. After accounting for variation in body mass and
temperature, metabolic rate adjusted to a 1 kg fish at 15 °C ranges from ~13
to 442 mg O2 h−1 for SMR and from ~55 to as high as 2259 mg O2 h−1 for
MMR across a variety of teleost species (Figure 10.6; also see Killen et al.,
2016 for a similar analysis). This corresponds to a 33- and 41-fold variation
in SMR and MMR, respectively, with pelagic species often having higher
metabolic rates than benthic ones, and benthopelagic species being
intermediate (Killen et al. 2010, 2016; Figure 10.6). Generally, relatively
sluggish groups such as cyprinids and anguillids fall at the lower end of the
metabolic continuum, with both low SMRs and MMRs, and the high-
performing groups such as salmonids and scombrids fall at the other end,
with high SMRs and MMRs (Killen et al. 2016; Norin and Clark 2016;
Figure 10.6). Interestingly, the scaling of metabolic rate with body mass (cf.
Section 10.3.1) appears to show opposite patterns, with negative
correlations between scaling exponents and metabolic rates across fishes
with different lifestyles and swimming modes (Killen et al. 2010).
 FIGURE 10.6 Metabolic rates of 105 species of teleost fish for which both SMR
(diamonds) and MMR (circles) values were available from the literature. All data have
been adjusted to a body mass of 1 kg and a temperature of 15 °C using residuals from
the regression equations for SMR and MMR presented in the caption for Figure 10.2.
Each species’ scientific name is given on the x-axes, and data points are ranked
according to MMR. The dashed black line is zero. The eight species highlighted in
Figure 10.2 are illustrated again here, and data points for all species are colour coded
according to the species’ lifestyle (pelagic, benthopelagic, or benthic; cf.
www.fishbase.org). The grey area represents the aerobic scope of each species.

The evolution of differences in metabolic rates may be caused by two

differing selection pressures: one acting to reduce SMR in order to improve
either tolerance to environmental hypoxia or low food availability, and
another acting to increase MMR to improve locomotor performance (Killen
et al. 2016). Similarly, an observed decline in metabolic rate with ocean
depth may result from relaxed selection for high locomotory capacity in the
deep sea, because the perpetual darkness means that predator–prey
interaction distances are minimized (Drazen and Seibel 2007).
Compared with the ~30- to 40-fold variation in metabolic rates across
species of fish, metabolic rates vary up to two- or three-fold among
individuals of the same species after accounting for variation in body mass
(Burton et al. 2011; Metcalfe et al. 2016). This among-individual variation
is real (as opposed to measurement noise), as evidenced by significant
repeatability of metabolic rate (i.e. consistency in the measured rate),
although repeatability does tend to decrease over time (White et al. 2013).
Repeatability of metabolic rate is also found to decrease across context in
fish, for example in response to food deprivation as well as changing
temperature and hypoxia (Norin et al. 2016; Metcalfe et al. 2016; Auer et
al. 2018a). The reduced cross-context repeatability can be due to different
individuals responding differently, yet predictably, to changes in the
environment (Norin and Metcalfe 2019). For example, individual
barramundi (Lates calcarifer) with relatively high SMR and MMR are less
responsive to an increase in temperature compared with their low-
metabolic-rate conspecifics, but high-MMR individuals are faced with a
much stronger reduction in metabolic performance (MMR and AS) in
response to hypoxia (Norin et al. 2016). Variation and flexibility in SMR
are also related to variation in growth and food availability, with a general
positive relationship between SMR and growth rate if food is readily
available and intake is high (potentially due to a greater capacity of high-
SMR fish to process food) but a negative or no relationship between SMR
and growth rate if food is unpredictable and intake is low (Metcalfe et al.
2016). Differences in metabolic rate flexibility, and associated
consequences for growth performance, may determine which individuals
will be selected for under rapidly changing environmental conditions, such
as those occurring with climate change (Norin and Metcalfe 2019).
Metabolic rate can also be linked with behaviour; individual fish with
high SMR are often more aggressive, dominant, and bold relative to
conspecifics with a lower SMR (Metcalfe et al. 2016). This is possibly due
to higher energy requirements motivating behaviours that facilitate energy
acquisition, such as being bold and aggressive to gain preferential access to
food or being more active in foraging. The causal direction could, however,
also be the opposite (or consist of a feedback loop), whereby the elevated
SMR is caused by higher food intake (Van Leeuwen et al. 2012), and
possibly higher stress levels (Øverli et al. 2007), resulting from being
aggressive and bold.
The underlying physiological causes of intra-specific variation in
metabolic rates are not well understood, but differences in organ size
(Boldsen et al. 2013) and enzyme activities (Norin and Malte 2012) are
involved. Mitochondrial (in)efficiency, evaluated as proton leak across the
mitochondrial inner membrane, also helps explain variation in both SMR
and MMR (Salin et al. 2016). Since high proton leak results in less ATP
generated per amount of oxygen consumed, such uncoupling (inefficiency)
of the mitochondria of high-metabolic-rate individuals appears maladaptive.
However, fish with higher SMR have lower levels of potentially damaging
reactive oxygen species (Salin et al. 2015), so they may live longer.
Overall, the costs and benefits of possessing a certain metabolic rate are
context dependent (Burton et al. 2011; Metcalfe et al. 2016), which is most
likely what maintains the two- to three-fold variation in metabolic rate
among individuals of the same species.

10.5 ECOLOGICAL AND EVOLUTIONARY RELEVANCE

OF (VARATION IN) METABOLIC RATE
The metabolic rates of fish are usually measured in the laboratory. We know
little about the metabolic levels at which fish operate in nature or the fitness
consequences of variation in metabolic rate. This is largely due to the
difficulty of estimating metabolic rates of fish in the field.
Nonetheless, laboratory studies indicate that the growth advantage of
having a high SMR disappears in more complex environments
characteristic of the field, where food availability can be unpredictable
(Metcalfe et al. 2016). This has indeed been observed, as either no or a
negative relationship is found between metabolic rates of both brown trout
(Salmo trutta) and Atlantic salmon (Salmo salar) measured in the
laboratory and the fish’s subsequent growth after a period of living in the
wild (Álavarez and Nicieza 2005; Robertsen et al. 2014).
Intra-specific variation in metabolic rate is also related to shoaling
behaviour and swimming performance. In a laboratory setting, juvenile
golden grey mullet (Liza aurata) with relatively high AS swim at the front
of the shoal, where they would be more likely to encounter food first in the
wild, while individuals with low AS swim at the rear (Killen et al. 2012).
Individuals with low AS likely swim at the rear because of the
hydrodynamic advantage of a trailing position, as reflected by the fish at the
back using fewer tail beats to swim at the same speed as fish at the front
(Killen et al. 2012). However, feeding elevates metabolic rate due to
specific dynamic action (SDA) and thus reduces the AS available for
swimming (cf. Figure 10.1). This can cause a predictable shift in shoaling
dynamics, whereby fish that initially eat a lot at the front of the shoal
subsequently move further back in the shoal as their AS becomes occupied
by SDA (McLean et al. 2018). However, among-individual variation in
metabolic rate measured in the laboratory does not necessarily explain
variation in swimming activity in the wild (Baktoft et al. 2016).
Variation in metabolic performance can also affect the vulnerability of
fish to capture by fishing gear. Fish with a high anaerobic metabolic
capacity are less likely to be caught in a trawl in a scaled-down fishing
scenario in the laboratory (Killen et al. 2015; Hollins et al. 2019). Similarly,
higher anaerobic energy expenditure, as well as higher aerobic metabolic
rates, is associated with higher vulnerability to recreational angling in
largemouth bass (Micropterus salmoides) (Redpath et al. 2010). These
effects could lead to fisheries-induced evolution and a shift in the metabolic
phenotypes within wild populations (Hollins et al. 2018), although this will
depend on the heritability of metabolic rate.
Very little is known about the heritability of metabolic rates in fish, but in
animals in general, the so-called narrow-sense heritability ranges from 0 to
0.72 with an overall mean of 0.19 (Pettersen et al. 2018). Although
relatively low, this level of heritability indicates that metabolic rate can
evolve under selection. Indeed, SMR has been found to evolve rapidly in
Trinidadian guppies (Poecilia reticulata) in the wild after invasion into low-
predation environments with a slower pace of life (Auer et al. 2018b). Low-
food environments have also been found to select for higher MMR in
Atlantic salmon, possibly due to a greater competitive ability of high-MMR
fish (Auer et al. 2018c). Evidence from three-spined stickleback
(Gasterosteus aculeatus) also indicates that MMR has a genetic basis and
has undergone an evolutionary reduction alongside swimming performance
in non-migratory populations (relative to ancestral migratory populations)
where a high aerobic capacity and high swimming performance are
unnecessary (Dalziel et al. 2012).
Although this indicates that variation in metabolic rate is ecologically
and evolutionarily important, the metabolic levels of fish in the wild remain
poorly understood. Advances in aquatic telemetry are, however, providing
new insights (Hussey et al. 2015). For example, by calibrating heart rate
with ṀO2 in the laboratory and using heart rate telemetry as a proxy for
metabolic rate, Northern pike (Esox lucius), which is a relatively sedentary
ambush predator, has been estimated to operate at a daily average metabolic
rate of ~1.5 times SMR in the wild, with physical activity accounting for
only 5–10% of AS, while SDA accounts for 15–25% (Lucas et al. 1993).
Heart rate measurements of Atlantic salmon (albeit uncalibrated with ṀO2)
during their spawning season indicate that they spend much of their time
operating near their MMR because of active upstream movement, fighting
with rivals, and redd (nest) building (Lucas et al. 1993). This mirrors
findings for Pacific sockeye salmon (Oncorhynchus nerka), where
populations migrating the farthest upriver to reach their spawning grounds
have the highest AS, indicating that metabolic rate is adapted to local
conditions (Eliason et al. 2011). Metabolic adaptation to the environment is
also indicated by tropical damselfish (Acanthochromis polyacanthus) living
in wave-exposed, high-flow habitats having higher MMR and AS compared
with conspecifics from sheltered areas (Binning et al. 2014). A few studies
have used accelerometers (calibrated with ṀO2) to estimate field metabolic
rates of fish. Using this approach, lake trout (Salvelinus namaycush) were
found to spend most of their time cruising at relatively low metabolic rates
(within ~two times their SMR) but intermittently reaching eight to nine
times their SMR during burst swimming events (Cruz-Font et al. 2016). A
similar study on bonefish (Albula vulpes) found that these fish routinely
occupy 40–60% of their AS and intermittently exceed 90% of AS in the
wild (Murchie et al. 2011). Measurement of the isotopic composition of
carbon embedded in otoliths (‘ear stones’) is also emerging as a promising
technique to retrospectively estimate field metabolic rates of fishes (Chung
et al. 2019).
Together, the available evidence from free-swimming fishes in the field
indicates that metabolic rates measured in the laboratory and associated
with a certain lifestyle (cf. Killen et al. 2010, 2016) are mirrored in nature;
for example, sit-and-wait predators such as Northern pike exhibit a slow
pace of life and spend little time at metabolic rates much above resting,
whereas athletic and faster-paced species like Atlantic salmon and bonefish
routinely use their full aerobic metabolic capacity.

10.6 CONCLUSION
With the exception of a few relatively large, highly active fishes (e.g. some
tunas, lamnid sharks, and billfishes) that can maintain certain body regions
at temperatures above that of the ambient water (due to high regional heat
production and retention; Block et al. 1993; Wegner et al. 2015), fishes
operate at some of the lowest metabolic rates among the vertebrates.
Nonetheless, as illustrated in Figure 10.6, metabolic rates vary profoundly
among fish species with different ecology and lifestyles. Even among
individuals within species, there is inherent and consistent variation in
metabolic rate that is associated with variation in growth, behaviour, and
life history. While metabolic rate is a property of all organisms, it is itself
subject to change and can be up- or down-regulated in fishes in response to
changes in food availability, physical activity, temperature, hypoxia, and
many other factors. Due to the fundamental importance of energy intake
and expenditure in biology, metabolic rate has received immense attention
from physiologists and ecologists alike. However, we are only just starting
to understand the ecological significance of variation in metabolic rate of
fishes in the wild, largely due to technological advancements in aquatic
telemetry. Integration of laboratory and field measurements of metabolic
rate is an exciting research direction that will improve our understanding of
the metabolic physiology of fishes and how it is affected by (changes in) the
environment.

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 11 Hearing
Arthur N. Popper and Anthony D. Hawkins

CONTENTS
11.1 Introduction
11.2 How and Why Hearing?
11.3 The Importance of Sound to Fishes Today
11.4 Primer on Underwater Sound
11.4.1 Underwater Sound and Fishes
11.5 How Do Fishes Hear?
11.5.1 The Inner Ear
11.5.2 Response of the Ear to Sound Stimulation
11.5.3 Ancillary Structures
11.6 Diversity of Fish Ears
11.7 What Do Fishses Hear?
11.7.1 Other Aspects of Hearing by Fishes
11.8 What Don’t We Know about Fish Hearing (Future Directions)?
11.9 Anthropogenic Sound and Fishes
References

11.1 INTRODUCTION
It has been known since the days of Aristotle that fishes make and detect
sounds (reviewed in Moulton, 1963). The fish ear was perhaps first
described by E. H. Weber (1820) and G. Retzius (1881) (see Figure 11.1 for
illustrations from the original publications). However, it was not until the
experimental work of G. H. Parker (1903), and then K. von Frisch (1938)
(who later went on to win the Nobel Prize for his work on the dance
language of bees) and his students (e.g., von Frisch and Stetter, 1932; von
Frisch and Dijkgraaf, 1935), that hearing in fishes came to be better
understood in terms of sensitivity to different frequencies and overall
capabilities.

FIGURE 11.1 Illustrations from Weber (a) and Retzius (b) with original labeling (note,
only labels relevant to this discussion are defined). (a) A portion of plate 20 from Weber
(1820) showing a dorsal view of a carp (Cyprinus carpio) to illustrate the relationship
of what are now known as the Weberian ossicles (30, 31, 32), the inner ear (19), and the
swim bladder (33). (b) Diversity in inner ear structures for three teleost species as
shown in this partial plate VII from Retzius. Each drawing is a medial view of the right
ear, with anterior to the left and dorsal to the top. Top: Lucioperca sandra (pike-perch);
middle: Mullus barbatus (goatfish); bottom: Pegellus centrodontus (seabream).
(Scientific names used by Retzius are not updated.) In each case, note that the shape of
the saccule and lagena differ rather substantially, and the distinct shapes of the otoliths.
The following key gives the structures related to hearing. Other structures are associated
with the vestibular sense: l: lagena; ms: macula sacculi; o: otolith; rl, rs, ru: rami of
eighth cranial nerve to the otolith end organs; s: saccule; u: utricle.
11.2 HOW AND WHY HEARING?
Critical questions are when, how, and why did hearing evolve in
vertebrates? Hearing evolved in the earliest vertebrates (e.g., Pumphrey,
1950; Van Bergeijk, 1967; Popper et al., 1992; Popper and Fay, 1997). Van
Bergeijk (1967), following Pumphrey (1950), proposed that the ear evolved
from surface receptors, perhaps ancestral to the lateral line, that dropped
below the surface and eventually became covered. Van Bergeijk
hypothesized that this provided a system that would not be directly affected
by water motion and that, over time, the frequency range of this
accelerometer-like system would have served a number of functions,
including both auditory and vestibular senses.
But why? Consider that the most primitive vertebrates lived in water.
Other senses these animals may have had – light and chemical receptors –
had limited ranges over which they could detect stimuli. Vision would be
restricted by the light levels and murkiness of the water, and so the “visual
field” for the animals was likely measured in centimeters or, perhaps, in
meters. Chemical reception is at the mercy of currents and is a very slow
way to glean information.
Sound, on the other hand, is less restricted by most environmental
conditions, and in water, sound travels very fast and is directional (e.g.,
Urick, 1983; Rogers and Cox, 1988; also www.dosits.org). Thus, by
evolving a system to detect sound, fish ancestors developed a means to get
information about predators, prey, and their environment from long
distances and very quickly. In other words, the evolution of hearing resulted
in fishes sensing the “acoustic scene,” or soundscape, which provided them
with a far larger waterscape than any other sense (Popper and Fay, 1997;
Fay and Popper, 2000; Fay, 2009).

11.3 THE IMPORTANCE OF SOUND TO FISHES TODAY

All fishes, including the bony fishes, elasmobranchs, lampreys, and
hagfishes, have ears. While we have data on hearing of relatively few
species, it is highly likely that all fishes use the soundscapes to glean
information important for survival. Even though all fishes hear, a lower
number of fishes have been shown to make and use sounds for purposes of
reproduction, territorial behavior, and other behaviors (e.g., Hawkins and
Myrberg, 1983; Hawkins, 1993; Ladich, 2019).

11.4 PRIMER ON UNDERWATER SOUND

In order to understand fish hearing, it is critical to have a basic
understanding of underwater sound. A fuller discussion can be found at
www.dosits.org and in several recent papers that specifically deal with
sound and fishes (Hawkins and Popper, 2018a; Popper and Hawkins, 2018,
2019).
Sound is generated by the vibration of a source and depends upon the
elasticity of the medium. As the source vibrates, it imparts energy, which
passes on as a propagated wave within which the medium’s particles move
back and forth over the same location and do not, themselves, travel. The
waves that travel result from the fact that the particles oscillate along the
line of transmission, thereby transmitting their oscillatory motion to their
neighbors. This particle motion is accompanied by waves that include
increases (compression) and reductions (rarefaction) in pressure. Together,
the compressions and rarefactions that propagate from the sound source are
referred to as the sound pressure.
In air, due to the low density of the medium, particle motion does not
travel far from the source, and terrestrial hearing is primarily sound
pressure detection. However, in water, which is far denser, particle motion
continues to be a significant part of the acoustic environment for
considerable distances from the source (Urick, 1983). Indeed, in open
water, distant from any boundaries (e.g., surface or bottom), the sound
pressure radiated from a simple acoustic source falls off as 1/r, where r is
the distance from the source (van Bergeijk, 1964; Ainslie and de Jong,
2016). Far from the source (“far field”), the energies associated with
acoustic pressure and acoustic particle velocity are equal, whereas close to
the source (“near field”), the particle velocity component of the field
contains more energy. The distance of the transition point is related to the
nature of the source as well as to the frequency of the signal, with the
distance greater for lower frequencies (van Bergeijk, 1964).
It is critical to appreciate that particle motion is inherently directional
along its axis of transmission. In essence, particle motion, whether
considered in terms of particle displacement, velocity, or acceleration, is a
vector quantity. Indeed, if properly constructed, a particle motion detector
(based on three mutually perpendicular accelerometers) can detect the axis
of sound propagation. Moreover, as will be discussed later, the ears of
fishes are ideally “designed” to serve as accelerometers that detect the
directional component of the particle motion, thus determining sound
source direction.
In contrast, the sound pressure is a scalar quantity and acts in all
directions. Thus, a single sound pressure detector cannot determine the axis
of propagation of the sound. Several detectors are necessary, spaced well
apart, to achieve this.
11.4.1 UNDERWATER SOUND AND FISHES
It is critical to understand that the inner ear of fishes responds only to
particle motion and not to sound pressure (Hawkins and Popper, 2018a;
Popper and Hawkins, 2018), although as discussed in Section 11.5.3, some
fishes can also detect sound pressure using ancillary structures. The
importance of particle motion for fish hearing was recognized decades ago
by Dijkgraaf (1960) and others, but with few exceptions, investigators, even
to this day, focus on sound pressure as a stimulus in measuring underwater
sound, which includes reporting hearing in terms of sound pressure rather
than particle motion. While this is somewhat understandable due to the
difficulties in measuring particle motion and the complexities of defining
sound fields in experimental tanks (Rogers et al., 2016; Popper and
Hawkins, 2018), it also means that much of the earlier and even some
recent data are difficult, if not impossible, to interpret in terms of detection
capabilities or the sound fields to which fish were exposed (Popper and
Hawkins, 2018; Popper et al., 2019b). Moreover, this also makes defining
the soundscapes detected by fishes in the wild difficult, since most field
recordings of sound use hydrophones sensitive to sound pressure, whereas
most fish species are only detecting particle motion.

11.5 HOW DO FISHES HEAR?

External auditory structures are not needed by fishes because of the density
similarities between the water and the body. Thus, an impinging sound
passes right through the body and cannot be detected unless there are
structures of different densities with which the sound can interact. Most
bony fishes have two such discontinuities. The first are the otoliths of the
inner ear (Figures 11.1b and 11.2), which are about three times denser than
the rest of the body. The second may be a bubble of gas, such as the swim
bladder, that is much less dense, and more compressible, than the water
(Figure 11.1a).

FIGURE 11.2 Medial view of the left inner ear of an Atlantic cod, Gadus morhua. The
position of the saccular macula is illustrated, but it would not be visible from this
direction.
(Figure © 2019 Anthony D. Hawkins, all rights reserved.)

11.5.1 THE INNER EAR

The inner ear of fishes consists of three semicircular canals for detection of
angular motion of the body (Platt, 1983) and three otolith organs, the
saccule, lagena, and utricle, that respond to linear acceleration and gravity
as well as sound (Figures 11.1b and 11.2). In most bony fishes, the otolith
organs contain a single, solid mass of calcium carbonate, the otolith, that
grows year by year, creating “rings” that can be used to determine the age
of the fish. The shape and orientation of the otolith differ between the three
otolith organs in fishes. All other vertebrates, including non-teleost bony
fishes, sharks, and terrestrial vertebrates, have similar calcium carbonate
material in their otolith organs but in the form of crystals, otoconia,
embedded in a gelatinous mass. The functional basis for having an otolith
rather than otoconia is as yet unknown (Popper et al., 2005; Schulz-Mirbach
et al., 2019).
Each otolith organ has a sensory epithelium (or macula) that is overlain
by the otolith (Figure 11.3) (Popper and Hawkins, 2019; Schulz-Mirbach et
al., 2019). The sensory epithelium contains sensory hair cells that are very
similar to those found in the ears of terrestrial vertebrates (Coffin et al.,
2004). The sensory cells are surrounded by supporting cells and are
innervated by the eighth cranial nerve (Figure 11.4). They have a ciliary
bundle consisting of a single true cilium, the kinocilium, and a series of
stereocilia, on their apical ends (e.g., Dale, 1976; Popper and Hoxter, 1981).
The otolith overlies the epithelium and is separated from it by a thin
otolithic membrane (Figure 11.4) that connects microvilli on the surface of
the supporting cells and the rough surface of the otolith itself (e.g.,
Dunkelberger et al., 1980). While this has never been demonstrated
experimentally, the otolithic membrane probably limits the relative motion
between the otolith and the epithelium.
FIGURE 11.3 Frontal section of the right side of the head of a fish. Dorsal to the top
and right side to the right. Note the position of the ear relative to the brain and presence
of the saccular otolith in close proximity to the sensory epithelium and the innervation
by the auditory (eighth) cranial nerve.
(Figure © 2019 Anthony D. Hawkins, all rights reserved.)
 FIGURE 11.4 Schematic of a dissection of the saccule showing the epithelium with the
sensory hair cells, each of which is innervated by one or more fibers from the eighth
cranial nerve (see text for more details). On the top (apical) end of the hair cells are
ciliary bundles, which project into the lumen of the end organ. The ciliary bundles are
surrounded by a fibrous matrix of the otolith membrane, which is made up of several
layers, on top of which sits the otolith. The membrane physically connects the otolith to
the macula.
(Figure © 2019 Anthony D. Hawkins, all rights reserved.)

Hearing results from the fish’s body moving in the sound field with the
water, while the otolith moves with a different amplitude and phase due to
its very different density. This relative motion between the otolith and the
underlying sensory epithelium results in bending of the ciliary bundles and
opening of ion channels in the cilia, which leads to chemical changes in the
cells and the release of a neurotransmitter that stimulates the innervating
afferent nerve fibers (e.g., Hudspeth and Corey, 1977).
Moreover, the sensory cells are both morphologically and physiologically
polarized, so that bending of the bundle in different directions results in
different responses from each cell, as shown in Figure 11.5, with maximum
response when the stereocilia bend in the direction of the kinocilium (e.g.,
Flock, 1964). In effect, each sensory cell is a directional detector!
 FIGURE 11.5 Physiological response of sensory hair cells. As the ciliary bundle (A) is
bent, there are different levels of nerve impulses (B). The actual response level is
proportional to the direction of stimulation, with maximum depolarization when
bending is from the stereocilia to the kinocilium.
(Figure © 2019 Anthony D. Hawkins, all rights reserved.)

The sensory epithelium contains large numbers of hair cells that continue
to be added as a fish grows (e.g., Lombarte and Popper, 1994). Thus, a
small fish may have hundreds of hair cells in the saccular epithelium, while
a larger fish may have hundreds of thousands. Most importantly, the hair
cells are organized into “orientation groups,” as shown in Figure 11.6,
where all of the cells in a particular region are oriented in the same general
direction.
 FIGURE 11.6 Hair cell orientation patterns on lagenar and saccular maculae (top) and
utricular maculae (bottom) in different teleost species. For the saccule and lagena,
anterior is to the right and dorsal to the top. For the utricle, anterior is to the right and
lateral to the top. The arrows point in the direction of the orientation of the sensory cells
in each region (separated by dotted lines), with the kinocilium at the tip of the arrow in
each cell. There is particular inter-specific variation in the saccular hair cell orientation
patterns. In most species, the anterior end of the saccular epithelium has hair cells
oriented horizontally, while those on the caudal end are oriented vertically. The only
exception is found in the otophysan fishes, represented here by Arius felis, a group of
species that have lost the horizontal group of cells. Note that not all maculae are to the
same scale.
(Figure © 2019 Arthur N. Popper, all rights reserved.)

11.5.2 RESPONSE OF THE EAR TO SOUND STIMULATION

The otolith organ responds to the particle motion component of the sound
field. Since particle motion is directional, sound from a particular direction
will maximally stimulate sensory cells oriented in that direction, while cells
oriented in other directions will give a different level of response, as shown
in Figure 11.5.
Considering that each fish has six otolith organs, each oriented on a
somewhat different plane, and that even within one organ the cells may be
oriented in different directions (e.g., see the orientation on the utricles in
Figure 11.6), it is clear that fishes have sensory cells that will give
maximum response to signals from any direction (Figure 11.7). Once this
information from sensory cells oriented in different directions is combined
in the central auditory system, fishes can derive the direction of a sound
source with some accuracy (reviewed in Walton et al., 2017; Hawkins and
Popper, 2018a; Schulz-Mirbach et al., 2019). At the same time, as pointed
out by Schuijf and his colleagues, information from just particle motion is
the same from opposite directions, and they proposed that some fishes may
additionally use sound pressure to resolve this 180 degree ambiguity (e.g.,
Schuijf, 1975; Schuijf and Hawkins, 1983).

FIGURE 11.7 Directional sensitivity of a single nerve unit from the saccule of the cod
ear. See text for discussion. Note that the fish is viewed from the left-hand side, from a
ventral position, and from the tail.
(Unpublished data from Hawkins and King. Figure © 2019 Anthony D. Hawkins, all
rights reserved.)

The response is most clearly seen in Figure 11.7, which shows the
directional sensitivity of a single neuron in the anterior saccular branch of
the left auditory nerve of the Atlantic cod, Gadus morhua, obtained using a
shaking table to apply particle velocity stimuli from different directions in
three orthogonal planes at 125 Hz. The radial axis is the level of nerve
response.
The level of response shown in the graphs reflects the direction in which
the nerve showed maximum output, suggesting that the hair cells had
maximum response from that direction (e.g., Figure 11.5). This is
particularly seen as the two peaks in the directional responses within each
plane, separated by 180 degrees. Thus, in the top two graphs, maximum
response was when stimulation was from anterior to posterior (along the
axis of the fish), while there was minimal response to stimulation from right
and left. This result is as expected, since the nerve branch that was recorded
primarily innervates hair cells oriented anteriorly and posteriorly (Figure
11.6). At the same time, when the fish was turned sideways in the lower
graph, there were no sensory cells in the anterior region of the saccule
oriented in that direction, and so there is minimal response. If, however, the
recording had been done from the nerve innervating the utricle, there would
have been cells oriented to the side (see Figure 11.5).
11.5.3 ANCILLARY STRUCTURES
By being a particle motion detector, the fish ear can only detect a limited
frequency range, and sensitivity to sounds at any particular frequency is
also limited. At the same time, some fishes can detect a wider frequency
bandwidth and have better sensitivity than others, and this is accomplished
by adding the detection of sound pressure, as occurs in species that hear
above about 500 Hz (Figure 11.8). However, in order to detect sound
pressure, fishes needed ancillary structures that are responsive to pressure
changes. In all cases, such a structure is a bubble of gas, most often in the
form of the swim bladder.
FIGURE 11.8 Hearing sensitivity (thresholds) determined using behavioral methods
for several species of fish. (Note, these curves are called audiograms.) In both figures,
the lower the threshold (y-axis), the more sensitive the fish is to a sound at a particular
frequency (x-axis). (a) Species that were tested under open sea, free-field conditions in
response to pure tone stimuli at different frequencies (see text for citations and more
information). Thresholds for Atlantic cod and Atlantic herring at some frequencies may
actually be below natural ambient noise levels. In the presence of higher levels of noise,
the thresholds are masked. (b) Hearing thresholds of diverse fish species measured with
animals in test chambers.
Species and data sources: (a) Common dab (Limanda limanda) (Chapman and Sand,
1974); Atlantic salmon (Salmo salar) (Hawkins and Johnstone, 1978); Atlantic cod
(Gadus morhua) (Chapman and Hawkins, 1973); herring (Clupea harengus) (Enger,
1967). (b) European perch (Perca fluviatilus) (Wolff, 1967); blue-striped grunt
(Haemulon sciurus) (Tavolga and Wodinsky, 1965); damselfish (Eupomacentrus
partitus) (Myrberg Jr and Spires, 1980); squirrelfish (Adioryx xantherythrus) (Coombs
and Popper, 1979); goldfish (Carassius auratus) (Jacobs and Tavolga, 1967); soldierfish
(Myripritis kuntee) (Coombs and Popper, 1979.)(Figures ©2019 Anthony D. Hawkins,
all rights reserved.)
 The swim bladder’s role in hearing is simple. When the gas is subject to
compression and rarefaction by a sound wave, the walls of the chamber
move, and this serves as a secondary sound source that reradiates the energy
as both sound pressure and particle motion. This particle motion has the
potential to stimulate the inner ear.
However, for the reradiated particle motion to stimulate the ear, it must
not attenuate as it propagates from the air chamber to the ear (Alexander,
1966). Presumably, the degree of attenuation depends on the distance
between the air bubble and the ear, and the nature of intervening tissues
such as bones and muscle, although this has never been investigated. At the
same time, it is clear, as will be discussed in Section 11.7 on hearing
capabilities, that fishes with close proximity between the gas bubble and the
ear, as in the Atlantic cod (Gadus morhua), or where there is some direct
link between the gas bubble and the inner ear, as in the goldfish (Carassius
auratus) and the soldier fish (Myripristis berndti), hear a wider range of
frequencies and show higher sensitivity than do fishes in which the gas
bubble and inner ear are further apart, such as salmonids (Figure 11.8)
(hearing data reviewed in Ladich and Fay, 2013).
Fishes with direct links between the inner ear and an air bubble appear to
hear better than all other species, since the movements of the swim bladder
caused by sound pressure are directly tied to the ear. The best-known
example of this connection is found in the otophysan fishes (goldfish,
catfishes, etc.), where a series of bones, the Weberian ossicles, connect the
swim bladder to the ear (Figures 11.1a and 11.9) (Weber, 1820), so that the
motion of the walls of the swim bladder are carried directly to the fluids of
the inner ear (Poggendorf, 1952; Alexander, 1964) in a manner analogous to
the function of the mammalian middle ear bones.
 FIGURE 11.9 Dorsal view of the Weberian ossicles (anterior at the top). The ossicles
(tripus, intercalarium, and scaphium) are set into motion when the swim bladder walls
move in response to sound pressure. The rostralmost ossicle, the scaphium, makes up
the outer walls of a fluid-filled perilymphatic sac. Fluid movements connect to a
transverse canal that connects the left and right saccules. Movement of the fluid causes
direct movement of the saccular otoliths and stimulation of the sensory hair cells on the
epithelium.
(Figure © 2019 Anthony D. Hawkins, all rights reserved.)

Outside of the otophysans, many other species, from diverse taxonomic

families, have evolved specialized connections that bring the swim bladder
into close, or intimate, proximity to the inner ear. These include, but are not
limited to, swim bladder extensions that terminate on the walls of the
saccule, as in the soldierfish (Coombs and Popper, 1979), swim bladder
extensions that are within the ear, as in the clupeid fishes (O’Connell,
1955), or gas bubbles that are totally unconnected to the swim bladder and
actually make up one wall of the saccule, as in the mormyrids (McCormick
and Popper, 1984).

11.6 DIVERSITY OF FISH EARS

A perusal of Retzius (1881) and more recent work (e.g., Deng et al., 2013)
shows remarkable diversity in the morphology of the ears of fishes. While
this diversity leads to obvious questions about the functional significance of
different ears, we actually know very little about the relationship between
form and function. Indeed, this is an area open to considerable speculation
(e.g., Hawkins and Popper, 2018a; Popper and Hawkins, 2018; Schulz-
Mirbach et al., 2019).
The diversity is seen in several distinct ways. First, there is a wide range
in the size and shape of the otolith organs themselves, and particularly of
the saccule, the main hearing end organ (Figure 11.1b). Second, there is
substantial diversity in the size, shape, and overall structure of the otoliths
in different species, and particularly in the saccular otolith (Figures 11.1a
and 11.2) (e.g., Popper et al., 2005; Lychakov et al., 2006). And finally,
there is wide variation in the hair cell orientation patterns on the saccules
(and to a lesser degree the lagena and utricles) of different species (Figure
11.6). And this does not include the diversity in other aspects of the inner
ear, such as the lengths of ciliary bundles on hair cells in different epithelial
regions (and in different species) (e.g., Platt and Popper, 1984) and
potentially, variation in the nature of the connection between the epithelium
and the otolith (the otolith membrane).
Unfortunately, we still have very little functional data on the inner ears of
fishes (Popper and Hawkins, 2018; Schulz-Mirbach et al., 2019). Thus, we
do not know whether the diversity in structure relates to differences in the
way sounds are processed in the ear in different species, but with all
structures giving the same information, or whether the different structures
suggest that different species are extracting different information from the
sounds.

11.7 WHAT DO FISHSES HEAR?

There is great diversity in hearing capabilities among various species
(Figure 11.8). At the same time, the majority of studies to date (reviewed in
Ladich and Fay, 2013) must be treated with caution for several reasons
(Popper et al., 2019b). First, hearing data were most often determined in
terms of detection of sound pressure, whereas, as discussed earlier, particle
motion is the predominant hearing stimulus for most species.
Second, most earlier work has been done in tanks, the vast majority of
which have significant acoustic problems (e.g., Duncan et al., 2016; Rogers
et al., 2016). A third problem with studies is that hearing was studied using
physiological methodologies that only give hearing in terms of what the ear
can detect, as opposed to behavioral studies in which the responses depend
not only on what is detected by the ear but also on analysis by the central
nervous system (Sisneros et al., 2016; Popper and Hawkins, 2019). Thus,
while physiological methods give some sense of the hearing range of fishes
as well as some indication of sensitivity at each frequency, results from
behavioral studies are of far greater value.
Despite these issues, it is possible to develop some understanding of what
fishes can hear (Figure 11.8). It is, for example, clear that all fishes studied
to date (perhaps 100 species) can hear, and that the hearing range extends
from below 50 Hz to perhaps 10–30 Hz, or even lower in some species
(Sand and Karlsen, 2000), and up to 300–500 Hz. Moreover, species that
detect sound pressure may hear up to approximately 1000 Hz, while fishes
with some of the aforementioned specializations can detect sounds to 3–4
kHz.
Measurement of the lowest levels of sound that fishes can detect at any
particular frequency is problematic since results vary even for the same
species when determined in different laboratories, as shown for the goldfish
(Ladich and Fay, 2013; Popper et al., 2019b), and with the method of
determination. Moreover, studies may have calibrated sound levels in terms
of sound pressure when the fish was actually responding to some unknown
level of particle motion.
Perhaps the most useful data are from studies where hearing has been
determined under well-defined acoustic conditions, either in open bodies of
water or in very specialized tanks that allow careful sound calibration of
both pressure and particle motion. Such data are shown in Figure 11.8a for
studies done in the open sea. In this figure, the common dab (and the
Atlantic salmon) are only sensitive to particle motion, and so they only have
a relatively narrow bandwidth of hearing, whereas species in which the gas-
filled swim bladder is close to the ear, such as the Atlantic cod, also detect
sound pressure, and so they have an increased hearing bandwidth. Finally,
the Atlantic herring has the widest bandwidth because of the presence of an
air bubble within the ear cavity. Many other species have been tested in
various types of chambers (Figure 11.8b) and must be viewed with a
number of caveats (Section 11.4.1) (reviewed in Popper et al., 2019b).
Despite these caveats, however, these data show the range of hearing
capabilities of different species, depending on factors such as connections
between the swim bladder and the ear. In particular, goldfish and soldierfish
have connections between the swim bladder and the inner ear and so hear to
over 3 kHz. In contrast, the closely related squirrelfish does not have
connections to the ear, so it is unlikely to be stimulated by the swim bladder
and therefore, has a narrower hearing range.
11.7.1 OTHER ASPECTS OF HEARING BY FISHES
While hearing sensitivity and sound detection are important, it is more
important that the auditory system of fishes extract far more information
about a sound than just its presence. Indeed, for sound to be useful, animals
must be able to discriminate between sounds to know “friend from foe,” to
determine the direction of a sound source so that the fish can move towards
or away from the signal, and to detect a biologically relevant signal in the
presence of other ambient noises that make up every acoustic environment
(e.g., sound from waves, other biological sources, and wind).
While there are as yet few studies on sound detection capabilities of
fishes, it is clear that at least the species studied (and likely all species) are
capable of signal analysis that provides a good deal of information about
sounds detected, including discrimination of sounds that are close to one
another in frequency and intensity (reviewed in Fay and Megela Simmons,
1999), and determining the direction of a sound source (reviewed in
Hawkins and Popper, 2018a). And, while studies on topics other than
threshold determination have mainly been done on a single species, the
goldfish, it is likely that fishes have far more complex sound processing
capabilities, which are similar to those of terrestrial vertebrates, including
mammals.
One other critically important capability is detection of a biologically
relevant signal in the presence of background noise. Indeed, hearing in
normal environments always occurs in the presence of background noise. If
that background noise is below the hearing sensitivity of a fish, it has no
impact on hearing. However, if the background (ambient) noise is higher, it
may be above the hearing sensitivity of the fish and thus, as shown in
Figure 11.8, increase the lowest detectable sound level for the species. This
means that the lowest sound levels detectable by fishes that hear well, such
as Atlantic cod and Atlantic herring, are limited by their ability to detect
and discriminate biologically important sounds in the presence of the
ambient noise background. In such conditions, the level of noise limits the
lowest sound level that an animal can detect. Interference in detection of
biologically relevant sounds by other sounds (including natural or human-
made sound) is referred to as masking (Fay and Megela Simmons, 1999).

11.8 WHAT DON’T WE KNOW ABOUT FISH HEARING

(FUTURE DIRECTIONS)?
While we know a reasonable amount about fish hearing capabilities and
mechanisms, it is also clear that we know far less about hearing by fishes
than we do for other vertebrate taxa. This was dealt with in papers that
focused on the major unanswered questions about fish hearing (Hawkins et
al., 2015; Hawkins and Popper, 2016). This list for further study related to
fishes is extensive but includes, among other things:

The functional significance of diversity in ear structure, otolith shape,

hair cell orientation patterns, length of ciliary bundles on hair cells,
etc. Do these reflect different species extracting different information
from sounds detected or different evolutionary approaches to doing the
same acoustic tasks?
The function of the ear itself and the interactions between sound, the
otoliths, and the sensory epithelia – including hair cells oriented in
different directions.
 Mechanisms of central processing of sounds in the brain and the role
of the brain in extracting signals from noise, sound direction, etc.
The specific function and role of the swim bladder and other ancillary
structures that, presumably, enhance hearing capabilities. Are they, for
example, frequency specific? How do their roles change with fish
depth?
Hearing capabilities of a more diverse group of fishes, with particular
emphasis on detection and processing of particle motion. This includes
determination of particle motion thresholds as well as developing a
better understanding of processing of complex sounds.
While not discussed in this chapter, there are many questions on the
acoustic behavior of fishes in the wild, including how fishes use their
soundscape. There are also questions on how fishes actually use sound
for communication.

11.9 ANTHROPOGENIC SOUND AND FISHES

There is a growing concern about the potential effects of anthropogenic
(human-made) sound on fishes as well as other aquatic life. The sources of
such sounds are quite diverse and range from sounds of vessels, to sounds
used in exploration for off-shore oil and gas, to sounds resulting from the
construction of wind farms. Importantly, as the number and variety of
anthropogenic sounds increase, they have the potential to have a broad
impact on aquatic life that ranges from death, to physiological effects such
as increased stress levels, to loss of hearing sensitivity to behavioral
changes – all of which can potentially cause significant impacts on fitness
(Popper and Hawkins, 2016; Hawkins and Popper, 2018b; Harding et al.,
2019; Hawkins et al., 2020).
While there is concern about a wide range of potential effects, perhaps
two are most important, since they can happen as long as fishes are located
near enough to an anthropogenic sound that it can be heard or that the
sound can interfere with hearing (masking).
The first of these are related to behavioral changes that result from fishes
changing patterns of behavior, such as migratory routes, feeding sites, or
breeding sites (to name just a few). Behavioral changes in fishes exposed to
anthropogenic sounds have been shown in a number of cases with wild
animals, and in each case the behavioral changes could have a broad impact
on the daily activities of animals (e.g., Engås et al., 1996; Hawkins et al.,
2014).
The second, though related, major effect is when the presence of
anthropogenic sound masks the ability of fishes to detect biologically
relevant sounds such as those produced by conspecifics or those produced
by predators or prey. Thus, the results of masking mean that the
“communication distance” over which a fish can detect a sound of
relevance decreases, thereby reducing fitness (Dooling and Leek, 2018;
Popper and Hawkins, 2019).
In effect, concerns about potential effects of anthropogenic sounds on
fishes becomes a “driving force” for studies of fish hearing today. This is
because it is imperative to know much more about hearing and acoustic
behavior if we are to understand potential effects of different sounds and
sound levels on fishes and make efforts to either mitigate these sounds, if
they are too loud, or develop regulatory actions that will protect fish from
harm while still allowing human activities in the water. These and other
issues regarding potential impacts of anthropogenic sound have been
reviewed a number of times recently (e.g., Popper et al., 2014; Andersson et
al., 2017; Carroll et al., 2017; Popper and Hawkins, 2019; Popper et al.,
2019a; Putland et al., 2019).

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 12 Active Electroreception and
Electrocommunication
Vielka L. Salazar

CONTENTS
12.1 Introduction to Electroreception and Electrogenesis
12.2 Classification of Electric Fishes Based on Electric Signal Type
12.3 Electrocommunication
12.4 Generalized Anatomy of the Electro-Sensory-Motor Pathways in
Gymnotiform Weakly Electric Fishes
12.5 Structural Organization and Premotor Neural Regulation of the
Pacemaker Nucleus
12.6 Endocrine Regulation and Neuromodulation of the Premotor and
Motor Brain Centers
12.7 Endocrine Regulation of the Peripheral Electric Organ
12.8 Conclusion
Acknowledgements
References

12.1 INTRODUCTION TO ELECTRORECEPTION AND

ELECTROGENESIS
Electroreception, an ancestral vertebrate trait, refers to the ability of an
organism to sense electrical stimuli from its environment. Electroreceptive
organisms can detect electrical stimuli passively or actively. In fishes,
passive electroreception is more common and involves using ampullary
electroreceptors (with the exception of the marine uranoscopid stargazers)
to detect weak, low-frequency electric sources (Moller, 1995). The usual
electric sources in the surrounding environment are bioelectric outputs from
surrounding living organisms, such as muscle contraction during swimming
and feeding. Passive electroreception was lost in the ancestor of all teleost
fishes but independently re-evolved in the ancestor of the lineages
Notopteridae and Mormyroidea and in the ancestor to the lineage
Siluriphysi (which includes the orders Siluriformes and Gymnotiformes)
(Crampton, 2019).
Active electroreception is less common; it involves organisms emitting
an electric field that interacts with the surrounding environment and then
detecting the distortions that the environment causes to this electric field
(Lissmann, 1951) (Figure 12.1). To achieve this, active electroreceptive
fishes activate an electric organ (EO) that discharges the electric field
(electrogenesis) and use specialized tuberous electroreceptors to detect this
electric field (Heiligenberg and Bastian, 1984). Subtraction of their own
field composition from the distorted field allows these electric fishes to
produce an electric image of their surroundings (Lissmann and Machin,
1958) (Figure 12.1). In teleost fishes, active electroreception and
electrogenesis evolved independently in two freshwater fish groups: one
Osteoglossiformes clade, the African superfamily Mormyroidea (known as
mormyroids), which includes the families Mormyridae (elephant nose fish)
and Gymnarchidae, and one Ostariophysi clade, the Neotropical order
Gymnotiformes (known as gymnotiforms or knifefish) (Crampton, 2019).
 FIGURE 12.1 Snapshot representation of the electric field of a weakly electric fish. (a)
The electric organ (EO) (horizontal black line within the fish’s silhouette) generates an
electric signal due to the separation of positive (+) and negative charges (−) along the
fish’s body (lines of current flow). The fish can emit a continuous, non-propagating
electric field (equipotential lines) for several centimeters away from its body and use
this electric field to navigate its environment or locate objects in the dark. (b) A close-
up showing how the conductive properties of objects distort the fish’s own electric
signal differently. The current lines are attracted and concentrated by a conductive
object (gray circle) and are repelled by a nonconductive object (black circle). These
electrical differences can be detected by the tuberous electroreceptors in the skin surface
closest to the object.
(Based on Lissmann, H.W., J. Exp. Biol., 35, 156–191, 1958; Lissmann, H.W. and
Machin, K.E., J. Exp. Biol., 35, 451–486, 1958.)

12.2 CLASSIFICATION OF ELECTRIC FISHES BASED ON

ELECTRIC SIGNAL TYPE
Electric fishes can be subdivided on the basis of voltage output (field
strength) into strongly and weakly electric fishes (Moller, 1995). Strongly
electric fishes can generate episodic bouts of high-voltage electric signals
up to 860 V and include some marine torpediniform electric rays, marine
uranoscopid stargazers, the African freshwater malapterurid electric
catfishes, and three species of freshwater gymnotiform electric eels in the
genus Electrophorus (Moller, 1995; Crampton, 2019; de Santana et al.,
2019). These fishes use their large voltage discharges mainly to hunt prey
and protect themselves (Moller, 1995). Weakly electric fishes produce low-
voltage (lower than 10 V), continuous electric organ discharges (EODs) that
project the electric field for several centimeters around the fish’s body
(Bullock, 1982) (Figure 12.1). Although skates, stingrays, electric rays, and
some catfishes generate weak electric signals, only gymnotiforms and
mormyroids use their weak electric signals for active electroreception.
Since many of these weakly electric fishes are nocturnal or live in murky
waters, active electroreception is believed to be advantageous by acting to
complement or even replace the visual system (Carr, 1990). With the
exception of the gymnotiform electric eels (Electrophorus), all
gymnotiforms and mormyroids generate only weak electric signals. The
gymnotiform genus Electrophorus is the only active electroreceptive clade
that produces both strong and weak electric signals due to the segregation
of function of its three EOs (Figure 12.2). Electric eels activate the main
organ and the anterior portion of the Hunter’s organ to produce strong
electric signals, while activation of the Sach’s organ and the posterior
portion of the Hunter’s organ generates weak electric signals (de Santana et
al., 2019) (Figure 12.2).

FIGURE 12.2 Summary of the most recent proposed phylogenetic relationships for the
order Gymnotiformes, also showing the outgroup Siluriformes (catfishes). The
gymnotiform fish clade is composed of five families: Apteronotidae, Sternopygidae,
Gymnotidae, Hypopomidae, and Rhamphichthyidae. The family Apteronotidae is the
only one with a neural-derived (neurogenic) electric organ (EO). The pulse-type EOD
seems to be a derived trait, with all pulse-type species forming a monophyletic group.
This phylogenetic tree includes a sketch of at least one representative member of each
family (black horizontal lines within fish silhouettes show the position of the EOs; all
three EOs for Electrophorus electricus are labeled) and the characteristic EOD of each
species shown (amplitudes are not to scale). Also, the different electrical outputs are
shown (s = strong and w = weak) for each family.
(Based on Moller, P., Electric Fishes: History and Behavior, Chapman & Hall, London,
1995; Betancur-R, R., et al., BMC Evol. Biol., 17,162, 2017; Crampton, W.G.R.,
Neuroscience, 140, 491–504, 2019.)

Gymnotiforms and mormyroids display one of two types of weak electric

signal patterns: a wave-type EOD or a pulse-type EOD (see Figures 12.2
and 12.3 for gymnotiform examples). Each EOD pattern is taxon specific
and represents a different strategy for characterizing the fish’s electrical
environment (Carr, 1990). The majority of mormyroids, the only exception
being the family Gymnarchidae, produce wave-type EODs. Gymnotiforms
constitute a clade of five families (Figure 12.2): Apteronotidae,
Gymnotidae, Hypopomidae, Rhamphichthyidae, and Sternopygidae
(Betancur-R et al., 2017). The families Apteronotidae and Sternopygidae
produce wave-type EODs, while the families Gymnotidae, Hypopomidae
and Rhamphichthyidae produce pulse-type EODs (Figure 12.2).

FIGURE 12.3 Differences between wave-type and pulse-type gymnotiform weakly

electric fishes. (a) The wave-type gymnotiform species Apteronotus leptorhynchus
produces a regular, quasi-sine wave EOD at high frequencies. (b) The pulse-type
gymnotiform species Brachyhypopomus gauderio produces a continuous train of
biphasic EOD pulses. Consecutive EOD pulses are separated by a period of silence,
known as the interpulse interval (IPI). B. gauderio breeding males display a pronounced
EOD circadian rhythm; at night, males increase the EOD repetition rate and the
amplitude and duration of the EOD waveform. (c) Under breeding conditions, the EOD
biphasic (P1, first [positive] phase and P2, second [negative] phase) waveform and the
EOD circadian rhythmicity of B. gauderio are sexually dimorphic.
(Modified from Salazar, V.L. and Stoddard, P.K., J. Exp. Biol., 211, 1012–1020, 2008;
Salazar, V.L., et al., J. Exp. Biol., 216, 2459–2468, 2013.)

Wave-type EODs may be an ancestral trait for gymnotiforms and

mormyroids, while pulse-type EODs may represent a derived strategy
within these clades (Betancur-R et al., 2017; Crampton, 2019) (Figure
12.2). In gymnotiforms, wave-type fishes discharge their EODs in an
extremely regular rhythmic pattern (EOD frequency) with a relatively
simple sinusoidal waveform when compared to the signals of pulse-type
fishes (Moortgat et al., 1998) (Figures 12.2 and 12.3a, b). Pulse-type fishes
discharge brief pulse EOD waveforms separated by electrically silent
periods known as interpulse intervals (IPIs) (Figures 12.2 and 12.3). This
configuration allows pulse-type fishes to generate more plastic and dynamic
EODs, whereby EOD repetition rate (=1/IPI) can be altered independently
of the duration of the pulse waveform (Hopkins and Bass, 1981) (Figure
12.3b). In addition, pulse-type electric signal complexity is further
enhanced in those species that generate EOD waveforms with two or more
phases by encoding different pieces of information in each phase. For
instance, in the family Hypopomidae, the first phase (P1) of the EOD pulse
waveform is believed to be species specific, while the second phase (P2),
the sexually dimorphic phase (Figure 12.3b, c), is specific to each
individual (Hagedorn and Carr, 1985; Zakon et al., 1991). Therefore, pulse-
type EODs appear to encode more information than wave-type EODs
(Hopkins, 1974b).
In this chapter, I will briefly describe the repertoire of
electrocommunication behaviors and the generalized anatomy and
connections of the electro-sensory-motor system of weakly electric
gymnotiforms. Then, I will describe in more detail our current knowledge
of how connectivity and neuro-endocrine modulation at different levels of
the electromotor pathway account for the diversity in electrical behaviors
observed in gymnotiforms, with a focus on the best-documented
representative genera: Apteronotus, Brachyhypopomus, Eigenmannia,
Gymnotus, and Sternopygus.

12.3 ELECTROCOMMUNICATION
One of the most fascinating events in the evolutionary history of fishes is
that the re-emergence of electroreception in gymnotiforms and mormyroids
was accompanied by the additional ability to electrocommunicate among
conspecifics by encoding social information (such as sex and dominance
status) within the EODs (Lissmann, 1958; Westby, 1988; McGregor and
Westby, 1992). In gymnotiforms, sexual dimorphism has been observed in
the EOD waveform in the pulse-type family Hypopomidae, while sexually
dimorphic EOD frequencies have been documented in the wave-type
families Sternopygidae and Apteronotidae (Hagedorn and Heiligenberg,
1985; Stoddard et al., 2007). For instance, during the breeding season,
Brachyhypopomus gauderio (formerly Hypopomus or Brachyhypopomus
pinnicaudatus) mature males produce an EOD waveform with longer
duration and higher amplitude than females; these males further enhance
this sexual dimorphism during the nighttime hours of courtship, following a
circadian rhythm (Stoddard et al., 2007) (see Figure 12.3b, c). In wave-type
species with sexually dimorphic EOD frequencies, it is more common for
males to display lower EOD frequencies when compared with females, as
seen in Sternopygus macrurus, Eigenmannia virescens, and Apteronotus
albifrons (Hopkins, 1974a, 1974b; Zakon and Smith, 2002). Yet,
Apteronotus leptorhynchus displays the opposite pattern, with males having
higher EOD frequencies than females (Dunlap et al., 1998).
Superimposed on these sexually dimorphic EOD traits are sex differences
in EOD frequency changes (EOD modulations) observed during courtship
and aggressive interactions. In the context of these social interactions,
gymnotiforms exhibit a wide behavioral repertoire of EOD modulations,
including gradual EOD frequency increases (accelerations) and decreases
(decelerations), rapid and brief EOD frequency increases with a change in
the EOD waveform (chirps), and periods of EOD silences (interruptions)
(Hagedorn and Heiligenberg, 1985; Hagedorn, 1986). These EOD
modulations have been characterized in more detail in A. leptorhynchus and
B. gauderio. For instance, during male–female and male–male dyadic
interactions, A. leptorhynchus displays two types of chirps (types 1 and 2;
although six different types have been categorized), gradual accelerations,
and “abrupt frequency rises” or AFRs (Zupanc, 2002; Tallarovic and Zakon,
2005; Hupe and Lewis, 2008). Males chirp more than females, and their
dominance status correlates with chirping output (Hagedorn and
Heiligenberg, 1985; Zakon and Smith, 2002). In B. gauderio, Perrone and
her colleagues (2009) performed an in-depth characterization of EOD
modulations across differences in sex and behavioral contexts. During
courtship, males produced mostly accelerations and three different types of
chirps (A, B, and C), and females generated interruptions (Perrone et al.,
2009). In aggressive interactions, males produced a different chirp (type
M), and submissive males generated interruptions (Perrone et al., 2009). In
addition, during social interactions, gymnotiforms are susceptible to signal
jamming from nearby conspecifics emitting EODs at similar frequencies.
To avoid this, many gymnotiforms perform the jamming avoidance
response (JAR), whereby they shift their EOD frequency away from the
jamming source (Heiligenberg et al., 1996). The JAR differs across species;
S. macrurus does not have the JAR, E. virescens can shift its frequency up
or down during a JAR, while A. leptorhynchus can only shift its frequency
up during the JAR (Heiligenberg et al., 1996). Interestingly, during
aggressive interactions, A. leptorhynchus males with low EOD frequency
will increase it to be within the ‘jamming range’ of a male opponent with a
higher EOD frequency (Tallarovic and Zakon, 2005). A similar behavior
has been observed in Hypopomus artedi during aggressive interactions,
where males display EOD ‘discharge synchrony,’ potentially jamming each
other (Westby, 1975).

12.4 GENERALIZED ANATOMY OF THE ELECTRO-

SENSORY-MOTOR PATHWAYS IN GYMNOTIFORM
WEAKLY ELECTRIC FISHES
The evolution of any communication system requires co-adaptation
between a sensory pathway that analyzes the environmental cues and a
motor pathway that controls different behavioral responses. Gymnotiform
weakly electric fishes perceive electric stimuli via their ampullary and
tuberous electroreceptors. Their tuberous electroreceptors are tuned to their
own electric signal and as such, perceive any distortions to the fish’s own
electric signal. Information from the electroreceptors is conveyed to the
electrosensory lateral line lobe (ELL) (Krahe and Maler, 2014) (Figure
12.4). The ELL conveys electrosensory information mainly to the torus
semicircularis (TS), which then passes it on to the nucleus electrosensorius
(nE) (Heiligenberg and Bastian, 1984) (Figure 12.4). The nE is a sensory-
motor integration center, a transition place where sensory information is
translated into motor commands and conveyed to the EOD motor network
(Heiligenberg et al., 1991).
 FIGURE 12.4 Sagittal view of the brain of a gymnotiform fish showing the generalized
electro-sensory-motor pathway. In brief, electroreceptors detect electrical stimuli and
convey this information via their afferents to the electrosensory lateral line lobe (ELL).
The ELL sends projections via two main pathways, one to a major processing center,
the torus semicircularis (TS), and the other to an electrosensory feedback loop made up
of the nucleus praeminentialis (nP) and the eminentia granularis posterior (EGp). The
TS sends projections to the optic tectum (TeO) and the nucleus electrosensorius (nE), a
sensory-motor interface. The nE sends projections to two premotor areas, the central
posterior/prepacemaker nucleus complex (CP/PPn) and the sublemniscal prepacemaker
nucleus (SPPn). These two premotor areas control the intrinsic activity of the motor
command center, the pacemaker nucleus (Pn). The Pn sends axons to innervate the
spinal cord’s electromotoneurons (EMNs). The EMNs form or innervate the peripheral
electric organ (EO).
(Based on Heiligenberg, W., Neural Nets in Electric Fish, MIT Press, Cambridge, MA,
1991; Zupanc, G.K. and Maler, L., J. Comp. Physiol. A, 180, 99–111, 1997.)

Gymnotiform weakly electric fishes have an EOD motor network

composed of a chain of hierarchically arranged nuclei located at the
diencephalic, midbrain, and medullary levels (Heiligenberg et al., 1981;
Zupanc and Maler, 1993; Juranek and Metzner, 1997; Zupanc and Maler,
1997) (Figures 12.4 and 12.5). The premotor areas, collectively called the
diencephalic central posterior/prepacemaker nucleus (CP/PPn) and the
midbrain sublemniscal prepacemaker nucleus (SPPn), send direct input to
the medullary pacemaker nucleus (Pn), the EOD control center (Juranek
and Metzner, 1998) (Figures 12.4 and 12.5). The Pn directs the activity of
the spinal electromotoneurons (EMNs). Subsequently, spinal EMNs
innervate, via cholinergic (ACh-R) synapses, the electrocytes that compose
the peripheral EO (Bennett, 1971). Electrocytes within the EO fire
synchronously to produce the EOD (Bennett et al., 1967). Each action
potential of the electrocytes corresponds to an individual EOD event on a
one-to-one basis (Mills and Zakon, 1991). In gymnotiforms, the
contributions of the central and peripheral motor components to the
communication signal can be compartmentalized: EOD frequency or
repetition rate is controlled by the medullary Pn, while the EOD
waveform’s duration and amplitude are determined by the ionic
conductances of the electrocytes (Zakon, 1998).

FIGURE 12.5 Diagram of the synaptic connections between the premotor areas (PPn
and SPPn) and the electromotor command center (Pn) for a representative gymnotiform
family, Hypopomidae. The different PPn and SPPn projections to the pacemaker
neurons (P) and relay neurons (R) are shown, indicating the type of synaptic connection
(e.g., AMPA-R, NMDA-R, and GABA-R). R axons project to the spinal cord’s EMNs,
which innervate the electrocytes in the electric organ (EO) via cholinergic (ACh-R)
synapses.
(Based on Kawasaki, M. and Heiligenberg, W., J. Neurosci., 10, 3896–3904, 1990;
Kennedy, G. and Heiligenberg, W., J. Comp. Physiol. A, 174, 267–280, 1994; Quintana,
L., et al., J. Comp. Physiol. A, 197, 75–88, 2011.)
12.5 STRUCTURAL ORGANIZATION AND PREMOTOR
NEURAL REGULATION OF THE PACEMAKER
NUCLEUS
In all gymnotiform weakly electric fishes, the EOD is controlled by the Pn,
an unpaired midline nucleus in the brainstem (Bennett et al., 1967;
Kawasaki and Heiligenberg, 1989, 1990). The Pn is composed of two
electrically coupled cell groups, the pacemaker neurons and relay neurons
(Bennett et al., 1967; Ellis and Szabo, 1980). The pacemaker neurons fire
synchronously and locally excite the relay neurons (Kennedy and
Heiligenberg, 1994) (Figure 12.5). The intrinsic bursting activity of
pacemaker neurons drives the rhythm of the EOD frequency or repetition
rate, while the electrotonic connections between pacemaker and relay
neurons contribute to the regularity of the output (Bennett et al., 1967;
Moortgat et al., 2000). The relay neurons innervate the spinal EMNs in all
gymnotiforms; EMNs innervate a muscle-derived (myogenic) EO or, as
observed only in Apteronotidae, have their axons end in specialized
processes, which constitute the neural-derived (neurogenic) EO (Bennett et
al., 1967; Bennett, 1971; Pappas et al., 1975; Elekes and Szabo, 1981). A
third population of Pn neurons, the parvocell interneurons, has been
identified in A. leptorhynchus (Smith et al., 2000). Parvocells are connected
to pacemaker and relay neurons via chemical and electrical synapses, yet
their function remains unclear (Smith et al., 2000).
The association, synapse type, degree of electrotonic coupling, relative
size and organization, and ratio between pacemaker and relay neurons vary
among gymnotiform genera (Tables 12.1 and 12.2). These differences
correlate with the repertoire of electric signal modulations displayed during
social interactions. For instance, the pulse-type gymnotiform fish B.
gauderio, which arguably produces a more complex repertoire of electric
signal modulations, has the pacemaker and relay neurons densely packed in
a 1:1 ratio in the Pn and arranged in two distinct populations (Quintana et
al., 2011) (Figure 12.5 and Table 12.1). The pacemaker and relay neurons
are not only dorso-ventrally segregated but also rostro-caudally segregated,
a pattern so far only documented in this genus (Quintana et al., 2011)
(Figure 12.5). In addition, the relay neurons can be categorized into three
size classes, displaying size class dorso-ventral segregation (larger cells
more dorsal and smaller cells more ventral) (Quintana et al., 2011).
Quintana and her colleagues (2011) proposed that differential activation of
different size classes of relay neurons and uncoupling of pacemaker and
relay neurons can account for the diversity of electric social signals (such as
chirps) in B. gauderio.
TABLE 12.1
Main Anatomical Characteristics of the Pacemaker Nucleus (Pn) of Five Gymnotiform Genera

TABLE 12.2
Modulation of Premotor Areas to the Pacemaker Nucleus by Differential Synaptic Connectivity
 The activity of the Pn is modulated by three distinct diencephalic CP/PPn
subpopulations, consisting of the PPnG, the PPnC, and the PPnI, and one
midbrain premotor area, the SPPn (Heiligenberg et al., 1981; Kawasaki and
Heiligenberg, 1989, 1990; Keller et al., 1991; Kennedy and Heiligenberg,
1994) (Figures 12.4 and 12.5). Different premotor areas send differential
neurotransmitter signals to the Pn, which modify the electrical properties of
the pacemaker and relay neurons and produce a diversity of electrical
behaviors (Heiligenberg, 1994; Kennedy and Heiligenberg, 1994; Juranek
and Metzner, 1998) (Figure 12.5 and Tables 12.2 and 12.3). For instance,
experimental stimulation by current injection to different CP/PPn
subpopulations and SPPn yields temporal patterns in the EOD rhythm
similar to the fish’s natural behaviors (Kawasaki and Heiligenberg, 1989).
TABLE 12.3
EOD Frequency or Repetition Rate Modulations Observed as a Result of Direct Input of the
Different Premotor Areas to the Pn

In all gymnotiforms studied to date, the PPnG sends axons to the

pacemaker neurons, but not the relay neurons, and excites them via
glutamatergic synapses involving NMDA receptors (NMDA-R), AMPA
receptors (AMPA-R), or both (Dye and Heiligenberg, 1987; Dye et al.,
1989; Kennedy and Heiligenberg, 1994) (Figure 12.5 and Table 12.2). The
PPnG has the common function of generating gradual accelerations (i.e.
EOD frequency or repetition rate increases) (Dye and Heiligenberg, 1987;
Kawasaki et al., 1988). In the wave-type genus Eigenmannia, gradual
accelerations via PPnG activation of pacemaker neurons are complemented
by gradual decelerations resulting from disruption of the SPPn tonic input
to relay neurons via NMDA-Rs (Heiligenberg et al., 1996). The combined
effect of these connections allows Eigenmannia to have a more dynamic
control of its EOD frequency. Brachyhypopomus implements a different
strategy to generate gradual accelerations and decelerations. In common
with all other gymnotiforms, PPnG controls the gradual accelerations, but a
CP/PPn subpopulation only documented in this species, PPnI, controls the
gradual decelerations (Kawasaki and Heiligenberg, 1989) (Figure 12.5 and
Tables 12.2 and 12.3). The PPnI sends axons to the pacemaker neurons and
inhibits them via activation of GABA receptors (GABA-R) (Kawasaki and
Heiligenberg, 1990) (Figure 12.5). It remains unclear why the PPnI is not
present in other gymnotiform species. These gradual accelerations and
decelerations are used in courtship and aggressive interactions and to
execute the JAR (Juranek and Metzner, 1997; Zakon et al., 2002; Perrone et
al., 2009). Interestingly, the SPPn–relay connection plays a different role in
other gymnotiform genera. In the wave-type genus Apteronotus, which
displays co-expression of NMDA-R and AMPA-R receptors at this synaptic
connection, SPPn is not tonically active, and its excitatory input on the
relay neurons generates gradual accelerations (Heiligenberg et al., 1996;
Juranek and Metzner, 1997). As such, Apteronotus can only shift up when
performing a JAR (Heiligenberg et al., 1996). In the wave-type genera
Eigenmannia and Sternopygus and the pulse-type genus Brachyhypopomus,
SPPn input to relay neurons via NMDA-R causes interruptions, temporarily
silencing the EOD (Keller et al., 1991; Heiligenberg, 1994).
The PPnC seems to be the “chirp” control in all the genera displaying this
behavior. It innervates dendrites of the relay neurons and excites them via
AMPA-R, producing chirps (rapid, short accelerations with a decrease in
EOD waveform) (Dye and Heiligenberg, 1987; Kawasaki et al., 1988; Dye
et al., 1989; Heiligenberg, 1994) (Figure 12.5 and Tables 12.2 and 12.3).
Connectivity of the Pn and modulation of the Pn by the PPnC seem to play
a role in explaining the diversity of chirps (e.g. courtship chirps versus
agonistic chirps) observed in gymnotiforms (Zupanc and Maler, 1993).
PPnC input to relay neurons can desynchronize the Pn network as well as
briefly increase the firing rate of relay cells (Kawasaki and Heiligenberg,
1989). As such, in species where the Pn is more compact, and the
pacemaker and relay connections are heterogeneous, this PPnC-driven
desynchronization can increase the diversity of chirps produced across
species (Lucas et al., 2019). In B. gauderio, a different model has been
proposed, whereby differential PPnC stimulation (i.e. brief, low strength
versus long, high strength) recruits different size class populations of relay
neurons, resulting in the production of different chirp types (Quintana et al.,
2011). Another premotor input to the Pn, the medullary Mauthner cells (M-
cells), has been identified in the pulse-type genus Gymnotus (Trujillo-Cenoz
and Bertolotto, 1990). M-cells send connections to the pacemaker neurons,
but not to the relay neurons, and their input is excitatory via NMDA-R and
metabotropic glutamatergic synapses (Curti et al., 2006). M-cell activation
generates a “Mauthner-initiated abrupt increase rate” or M-AIR (Falconi et
al., 1995). Unlike the pattern observed in chirps, the M-AIR consists of a
rapid, longer-lasting, and smaller increase in the EOD rate without a change
in the EOD waveform, and as such, it is believed to improve sampling of
electrical information in moving fishes (Falconi et al., 1995). This
connection has not been found in any other gymnotiform genera.

12.6 ENDOCRINE REGULATION AND

NEUROMODULATION OF THE PREMOTOR AND
MOTOR BRAIN CENTERS
As discussed in the previous section, the effects of the premotor inputs on
the pacemaker and relay neurons vary among species (Table 12.3).
Connections between the CP/PPn and brain areas associated with mating
behavior and neuroendocrinological control (e.g. ventral telencephalon,
hypothalamus, and preoptic nucleus) have been documented (Zupanc,
2002). In addition, the rhythmic output of the Pn can also be altered due to
neuromodulation targeting any of the PPn subpopulations or the Pn. In
Apteronotus and Eigenmannia, studies using immunohistochemical labeling
and brain site-specific injection techniques have shown the presence of
many neuromodulators in the CP/PPn, including serotonin, noradrenaline,
dopamine, somatostatin, galanin, substance P, met-enkephalin, and
corticotropin-releasing hormone (Zupanc, 2002). Interestingly, in A.
leptorhynchus, substance P-labeling in fibers innervating the PPnC is
sexually dimorphic (females’ fibers have no label), and androgen treatment
in females not only masculinized chirping pattern but also masculinized
substance P-labeling associated with the PPn (Weld and Maler, 1992; Dulka
et al., 1995). In addition, in B. gauderio and G. omarorum, arginine
vasotocin (AVT)–positive cells in the preoptic area (POA) send projections
nearby to the Pn, and treatment of Pn-containing brain slices with AVT
increased the Pn’s firing rate (Perrone et al., 2014; Pouso et al., 2017). In B.
gauderio, the number of AVT-positive cells in the anterior POA positively
correlated with the number of chirps recorded in social males (Pouso et al.,
2019). In A. leptorhynchus, AVT injections in intact fish increased male
chirping but had no effect in females, while electrical stimulation of the
POA in paralyzed fish produced chirp-like responses (Wong, 2000; Bastian
et al., 2001).
Sex-specific and seasonal changes in behavior (e.g. courtship) are
typically associated with physiological changes due to hormones and
peptides. For instance, in Sternopygus, androgens such as
dihydrotestosterone (DHT) decrease the firing frequency of the Pn,
masculinizing the EOD frequency, yet androgen receptor (AR) expression
was found in the PPn and SPPn but not in the Pn (Zakon, 1996, 1998). In
contrast, in B. gauderio, breeding males have a higher expression of ARs in
both pacemaker and relay neurons when compared with nonbreeding males
(Pouso et al., 2010). When nonbreeding males were treated with
testosterone implants, AR expression was upregulated in the Pn (Pouso et
al., 2010). AR upregulation could lead to increased expression of glutamate
receptors such as NMDA-R and AMPA-R.

12.7 ENDOCRINE REGULATION OF THE PERIPHERAL

ELECTRIC ORGAN
EMNs’ organization and sensitivity to steroid effects can contribute to EOD
waveform plasticity. For instance, in Gymnotus carapo, two EMN
populations have been described: small EMNs, which innervate the rostral
portion of the EO and tend to fire first, and large EMNs, which innervate
the highest number of electrocytes (caudal region of the EO) and fire last,
with a mixture of the two populations prevailing at the intermediate
segment of the EO (Caputi and Trujillo-Cenoz, 1994). In addition, steroid
hormones can also modulate the electromotor network directly at the level
of the EMNs. In spinally transected A. leptorhynchus treated with either the
androgen 11-ketotestosterone (11-KT) or estrogen, EMNs display an
increase or a decrease (respectively) in their firing rate (Zakon, 1996).
Since, with the exception of Apteronotus, all gymnotiforms have a
myogenic EO (Bennett, 1971), this section will focus on the anatomy and
physiology of myogenic EOs. Electrocyte architecture constitutes one of the
most diversified structures in gymnotiforms (Mills et al., 1992). The
anatomical distribution, pattern of innervation, and ionic conductances of
the electrocytes determine the shape, the complexity, and the generation of
the EOD waveform (Bennett, 1971). For instance, the wave-type
monophasic Sternopygus and Eigenmannia have only one excitable
membrane (single innervation) in the electrocytes (Bennett, 1971). The
pulse-type biphasic Brachyhypopomus has two excitable membranes (single
innervation of only one of these membranes) (Bennett, 1971). And in the
pulse-type multiphasic G. carapo, its two EMN populations differentially
innervate electrocytes depending on their location in the EO; rostral and
mid-body electrocytes are innervated on both rostral and caudal sides, while
caudal electrocytes are only innervated caudally (Caputi, 1999). The
differential distribution of the EMNs (as described earlier) plus their
differential innervation of the electrocytes generates a highly complex EOD
waveform while maintaining synchronicity of the EOD.
The duration of the EOD waveform is controlled by the kinetics of the
electrocytes’ ionic currents (Zakon, 1996). The excitable membranes of the
electrocytes are composed of ion channels, which control the inward and
outward flow of Na+, K+, Cl−, and other ions (Ferrari and Zakon, 1993;
Zakon, 1996). Steroid hormones can target the EO directly to modify the
EOD waveform (Heiligenberg and Bastian, 1984). The EO can be described
as an androgen-target tissue. This can be readily observed in the hormone-
dependent sex differences in the EOD (Mills and Zakon, 1991). In
Sternopygus, the effects of androgens in the Pn are independent of the
effects of androgens in the electrocytes (Few and Zakon, 2001). Mills and
Zakon (1991) showed that androgens affect the EO directly by changing the
ionic currents responsible for the action potential generation. DHT broadens
the EO’s action potential, which is mainly controlled by the Na+ current
(Zakon, 1998).
 Sex steroid regulatory effects on electrocytes’ action potential duration
could be better assessed by understanding the processes of expression,
diversity, and kinetics of the Na+ and K+ channels (Ferrari and Zakon,
1993). In S. macrurus, recordings from electrocytes after treatment with
DHT showed a decrease in the Na+ channels’ closed state, and this effect
correlated with an increase in the duration of the electrocytes’ action
potential (Ferrari et al., 1995). Ferrari and colleagues (1995) offered a few
possible ways by which DHT could exert the variation observed:
differential expression of Na+ channel α subunit genes or transcripts that
generate kinetically distinct Na+ channels, and/or differential expression of
Na+ channels’ accessory β subunits. Subsequent studies have provided
evidence in support of both these mechanisms. In S. macrurus, two different
Na+ channel α subunits have been identified in electrocytes, Nav1.4a and
Nav1.4b, as well as an accessory β1 subunit (Zakon et al., 2006; Liu et al.,
2007, 2008). In addition, Nav1.4b occurs as two splice variants, short and
long transcripts. Androgen treatment decreases the expression of the
Nav1.4b long transcript and the β1 subunit, and the expression of these Na+
channel subunits negatively correlates with the duration of the electrocyte’s
action potential (i.e. EOD duration) (Liu et al., 2007, 2008).
Finally, beyond these sex differences, hormones can also regulate the
electrocyte Na+ current in a shorter timeline to account for the day–night
EOD waveform changes that have been observed in some gymnotiforms.
For instance, in a series of elegant studies, Markham and his colleagues
(2009) showed that Na+ channels can be trafficked in and out of the
electrocyte membrane in response to treatment with the melanocortin
hormones, α-melanocyte hormone (α-MSH) and adrenocorticotropic
hormone (ACTH) (Markham et al., 2009b). Injections of these hormones in
intact fish mimicked the timescale and magnitude of the observed day-to-
night increase in the EOD waveform (Markham et al., 2009a). Based on
electrophysiological recordings and pharmacological manipulations of
electrocytes in vitro, the effect of α-MSH and ACTH seems to be mediated
by binding to the melanocortin receptor 5, a G-protein coupled receptor, and
activation of a cyclic AMP second messenger pathway, resulting in the
insertion of Na+ channels in the membrane from a cytoplasmic vesicular
pool and an increase in the Na+ current (Markham and Stoddard, 2005;
Markham et al., 2009b).

12.8 CONCLUSION
All the electric behaviors observed in gymnotiform weakly electric fishes
(JAR, agonistic and courtship chirps, gradual accelerations and
decelerations, interruptions, and daily and seasonal EOD changes) have an
unifying underlying neural circuitry, which, depending on the behavioral
context, can be modified by the action of a wide range of neural synaptic
connections, neuromodulators, and hormones (Zakon, 1996). Neural
modulation is observed at the premotor and motor areas of the CNS, and it
tends to mediate rapid and brief responses.
The premotor areas (i.e. CP/PPn and SPPn) are the principal neural
modulators of the Pn. They briefly and rapidly change the basic firing
properties of the same group of neurons (pacemaker and relay neurons) by
sending connections with different neurotransmitters. This process increases
the repertoire of signals expressed, allowing a fish to produce a complex set
of behaviors. The premotor areas and the Pn are sensitive to steroid
hormones. Steroid hormones have been shown to exert slower, long-term
changes to the electrical properties of the neurons in the premotor areas and
the Pn (Zupanc and Heiligenberg, 1989). In the periphery, the electrocytes
are major targets of hormonal modulation. Different studies have shown
that treatment with steroid hormones affects the kinetics of the ionic
channels of the excitable membranes of the electrocytes (Zakon, 1996).
These ionic changes in the electrocytes seem to be responsible for sex
differences in the EODs (Zakon, 1998). Fast EOD waveform circadian
changes are mediated by melanocortins via the activation of second
messenger systems and circadian cycling of Na+ channels. Although the
basic architecture of the electromotor pathway is fairly well conserved
across gymnotiforms, a diversity of electrical behaviors and electric signal
plasticity has emerged across the clade as a result of a rich repertoire of
neural and endocrine modulatory inputs.

ACKNOWLEDGEMENTS
Supported by the Natural Sciences and Engineering Research Council of
Canada, the Canada Foundation for Innovation, and the Nova Scotia
Research and Innovation Trust.

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 13 Vision
Ron Douglas

CONTENTS
13.1 Introduction
13.2 The Eye
13.2.1 Adnexa
13.2.2 Sclera/Cornea
13.2.3 Uvea
13.2.4 Lens
13.2.5 Aqueous and Vitreous Humour
13.2.6 Retina
13.2.6.1 Rods and Cones
13.2.6.2 Light/Dark Adaptation
13.2.6.3 Regional Variation in Retinal Structure
13.2.6.4 Visual Pigments
13.3 Visual Optics
13.3.1 Eye Shape
13.3.2 Image Formation
13.3.2.1 Resting Refractive State and Accommodation
13.3.2.2 Amphibious Vision
13.3.3 Pupil
13.3.4 Tapeta
13.3.5 Intraocular Filters
13.4 Visual Abilities
13.4.1 Absolute Sensitivity
13.4.2 Contrast
13.4.3 Spatial Resolution
13.4.4 Temporal Vision
 13.4.5 Colour Vision
13.4.6 Polarization
13.4.7 Depth Perception
References

13.1 INTRODUCTION
‘Does a human see better than a fish?’ is an ill-advised question. What does
‘better’ mean? Is one asking who can see most colours, resolve the finest
spatial detail, detect movement optimally, sense polarized or UV light, or
see in the lowest light levels? No animal is ‘better’ than any other; they are
just ‘different’.
The major visual stimulus for most fishes is sunlight, although in the
ocean, bioluminescence is the major light source in deeper water during the
day and at all depths at night. Fluorescent emissions might also have
significance in some species. The major challenge for vision underwater is
scatter and absorption of light by particles suspended in the water, which
degrade the image, limiting the range at which objects can be detected.
Pure water transmits light most effectively at 460–480 nm. Thus, clear
blue oceanic waters, which contain the least particulate matter, transmit
about 96–98% of light per metre at 480 nm. With depth, the amount of
sunlight decreases, and the spectrum becomes more restricted, so that
eventually, only a narrow window around 470–480 nm remains. Closer to
the coast, water is often green, and much freshwater appears red, as longer
wavelengths are preferentially transmitted but over much shorter distances
(Figure 13.1).
 FIGURE 13.1 Visible spectrum as a function of depth in (a) open ocean, (b) coastal
waters and (c) freshwater.

Given the many stimulus attributes that make up vision, and the diversity
of the fish visual system resulting from adaptation to different optical
habitats and lifestyles, this review is not exhaustive. It concentrates on
teleosts, highlighting species that either demonstrate general principles or
are visually exotic.

13.2 THE EYE

The fish eye is structurally and functionally similar to that of other
vertebrates (Figure 13.2).
 FIGURE 13.2 The teleost eye. (a) Simplified schematic transverse section. (b) Cryostat
transverse section from Gnathonemus petersii.
((a) taken from Douglas, RH. 2006. Biological Sciences Review Magazine 19(1), 36–41.
(b) provided by Prof HJ Wagner.)

13.2.1 ADNEXA
The adnexa comprises eyelids, conjunctiva, extraocular muscles and the
lacrimal system. Fishes have no need for tears and have no moveable
eyelids, but the eyes of some are partially or wholly covered by a thin,
usually static and transparent continuation of the skin. Some elasmobranchs
have a mobile protective nictitating membrane. Most fishes, like other
vertebrates, have six extraocular muscles that rotate and tilt the eye,
producing voluntary and reflex eye movements that serve to keep the image
stable on the retina or to shift the point of fixation.
13.2.2 SCLERA/CORNEA
The sclera and cornea form the eye’s outer tunic. While the sclera of
mammals consists mostly of dense irregular connective tissue and is
opaque, that of most fishes additionally contains variable amounts of
cartilage and bone and is often transparent. Anteriorly, it merges with the
cornea at the limbus. The cornea consists of an outer stratified epithelium
that is continuous with the surrounding epidermis or conjunctiva (Collin
and Collin, 2001). In some fishes, this epithelium rests on a homogeneous
basal membrane composed of collagen (Bowman’s layer). The corneal
stroma contains regularly arranged collagen lamellae, running parallel to
the corneal surface but alternating in orientation by about 90°, embedded in
glycosaminoglycans and separated by isolated fibrocytes. At variable
depths within the stroma, the corneas of some fishes contain structures that
produce iridescence by constructive interference, while others have
chromatophores. Some teleosts have an additional outer dermal stroma that
is a continuation of the skin, often separated from the main stroma by
granular or mucoid material, allowing the eye to rotate below a clear
protective layer. Internally, the cornea is separated from the aqueous
humour by a simple squamous endothelium, usually resting externally on a
basement membrane (Descemet’s membrane).
13.2.3 UVEA
The uvea, the globe’s middle tunic, consists of the posterior choroid, a
central ciliary region and anteriorly, the iris.
The choroid is highly vascular, the innermost layer forming a capillary
bed, the choriocapillaris, supplying nutrients to the retina. The outer choroid
frequently contains a reflective argentea lying internal to the transparent
sclera, giving the eye a silvery sheen. The argentea continues anteriorly,
traversing the ciliary region before forming a reflective layer in the iris.
While in many vertebrates the ciliary zone is large and muscular, in
fishes it is narrow, consisting of little more than a thin stroma covered
internally by a double layered epithelium. The inner epithelial layer is
unpigmented and secretes aqueous humour, while the melanized outer
epithelial layer reduces internal reflections.
 Anteriorly, the ciliary zone bends inwards, forming the iris, whose
posterior surface is covered by a continuation of the double ciliary
epithelium. Anterior to this epithelium, most vertebrates have one or two
muscles: a circular sphincter muscle encircling the pupillary rim that
constricts the pupil is present in almost all vertebrates, while a radial dilator
muscle is present in many. Surprisingly, many teleosts also have these
muscles despite the fact they have no pupillary movements, the muscles
probably serving to maintain the shape of the pupil. The stroma of the iris
consists of loosely arranged collagen and nerve fibres, blood vessels,
chromatophores and the argentea, while the anterior surface is covered by a
simple epithelium.
13.2.4 LENS
In fishes, the lens is usually close to spherical and protrudes through the
pupil into the anterior chamber. Its entire external surface is composed of an
elastic capsule overlying a simple epithelium covering the anterior lens.
Internally, the lens stroma is composed of elongated, flattened, hexagonal
cells running from the anterior to the posterior lens surface. These lens
fibres are formed throughout life from mitotically active epithelial cells
near the lens equator, which migrate into the lens stroma before elongating.
Fibres lose their nucleus and contain virtually no organelles but are packed
with crystallin proteins, which give them their high refractive index, which
is greatest centrally and reduces towards the periphery, minimizing
spherical aberration. To maintain this gradient throughout life, the
concentration of crystallins must increase in central fibres. As these lack the
cellular machinery to make proteins, new crystallins are apparently
transported centrally from peripheral, synthetically active cells (Kozlowski
and Kröger, 2019).
13.2.5 AQUEOUS AND VITREOUS HUMOUR
While aqueous humour fills the anterior chambers, the viscous, collagen-
containing vitreous humour occupies the space between the retinal inner
limiting membrane and the posterior lens (Figure 13.2). Although the
composition of the vitreous is relatively static and its function largely
structural, the aqueous is continually circulated through the anterior
segment, providing nutrients to the avascular lens and cornea. It also
produces intraocular pressure, which helps to maintain the shape of the eye
and removes potentially damaging metabolites and debris. In mammals and
fishes, aqueous is secreted by the ciliary epithelium into the posterior
chamber, entering the anterior chamber through the pupil before being
drained at the iridocorneal angle. However, while in mammals aqueous is
produced and drained circumferentially, in zebrafish (Danio rerio) at least,
it is only secreted dorsally and drained ventrally (Gray et al., 2009).
13.2.6 RETINA
The retina, the posterior inner coat of the eye terminating anteriorly at the
ora serrata, consists of 10 layers in all vertebrates (Figure 13.3a). The
outermost, the single-layered retinal pigment epithelium (RPE), serves a
multitude of functions, including regulation of the passage of substances
between the retina and the choroidal blood supply, removal of old
photoreceptor outer segment discs, stray light absorption and regeneration
of visual pigments. The remaining nine retinal layers are composed of six
regularly arranged nerve cell types and glia. Photoreceptors (rods and
cones) involved in image-forming vision make up the bulk of the outer
retina. Visual pigments within photoreceptor outer segments absorb light,
converting it into neurobiological signals (phototransduction). Cells of the
inner retina process these signals, and action potentials leave the retina
along the axons of retinal ganglion cells (RGCs), which in most species
leave the eye via a single scleral foramen, forming the optic nerve.
FIGURE 13.3 Retinal histology. (a) Tinca tinca, a typical diurnal retina. (b)
Kryptopterus bicchris, with enlarged rod outer segments (ros) adapted to dim light. (c)
Xenodermichthys copei, deep-sea species with elongated ros. (d) Notacanthus
chemnitzii, deep-sea species with multiple banks of ros. (e) Gnathonemus petersii, with
grouped photoreceptors. (f) Oncorhynchus mykiss, showing single and double cones.
(a)–(e) are transverse sections (light hitting the retina from ‘below’), while (f) is cut
tangentially at the inner segments. Retinal pigment epithelium (rpe); cone outer
segments (cos); external limiting membrane (elm) formed by tight junctions between
Müller cells and photoreceptors; outer nuclear layer (onl) composed of photoreceptor
nuclei; outer plexiform layer (opl) including synapses between photoreceptors, bipolar,
horizontal and interplexiform cells; inner nuclear layer (inl) comprising horizontal,
bipolar, amacrine, interplexiform and Müller cell bodies; inner plexiform layer (ipl)
consisting of synapses between bipolar, amacrine, interplexiform and ganglion cells;
ganglion cell layer (gcl) composed primarily of ganglion cell bodies but also containing
some interplexiform and ‘displaced’ amacrine cell nuclei; nerve fibre layer (nfl)
containing ganglion cell axons; internal limiting membrane (ilm). (Parts (a)–(d) and (f)
are from Douglas, RH. 2008. Encyclopedia of Ocean Sciences, 445–457. Part (e) was
supplied by Prof HJ Wagner.)
 Apart from forming a detailed representation of the environment, eyes
are responsible for measuring overall light levels, which allows light to, for
example, synchronize circadian rhythms and influence hormone levels. The
regulation of these and many other processes requires photoreceptors with
very different physiological properties from rods and cones, such as long
integration times. They are therefore controlled by visual pigments within
retinal cells other than rods and cones. Such non-visual photoreceptors will
not be discussed further here (see Davies et al., 2015 for review).

13.2.6.1 Rods and Cones

The light levels experienced by fishes can vary by 16 log units, ranging
from those in shallow tropical waters on a cloudless day at noon to the near
darkness experienced in deep and turbid waters at night. As this is beyond
the dynamic range of a single photoreceptor type, the retinae of most adult
fishes contain two, differentially sensitive photoreceptors: rods, employed
at low (scotopic) levels of illumination, and cones, active in higher
(photopic) light levels. While cones are relatively insensitive, they are able
to mediate colour vision and provide high temporal and spatial resolution.
Rods, on the other hand, maximize absolute sensitivity but provide poor
temporal and achromatic spatial resolution.
The reasons for the differences in chromatic and temporal vision
provided by rods and cones will be discussed elsewhere. The higher
sensitivity of rods is partially due to the greater gain of their visual
transduction cascade and the large size of their outer segments. However,
the main reason for their high sensitivity is the extensive convergence of
their output onto post-receptoral neurons. Such spatial summation is
required because photoreceptors are occasionally activated in the absence of
light. To ensure that the visual system does not respond to such noise,
RGCs require several independent signals indicating photon absorption for
activation. The likelihood of this in low light levels is increased by many
rods feeding into one RGC. However, this same convergence results in poor
spatial resolution. Most simply, for two objects to be distinguished, they
must be imaged on individual photoreceptors separated by one that is
unstimulated, and the output of these photoreceptors must be processed by
different RGCs. As cones show far less convergence than rods, they are
better suited for high acuity.
Although most fishes can see something over a large range of light
levels, by switching between their rods and cones, species are usually
specialized for vision within a more limited intensity range, reflected in the
relative abundance and morphology of the two photoreceptor classes.
Surface-dwelling fishes have relatively small rods and multiple cone types
(Figure 13.3a). Species living in more turbid waters have reduced cones and
enlarged rods (Figure 13.3b). Deep-sea fishes, which never leave an
extremely low light-level environment, rarely have cones, while the outer
segments of rods are either very long or banked into several layers (Figure
13.3c, d).
The photoreceptor inner and outer segments of some turbid freshwater
and deep-sea teleosts are grouped into bundles, usually surrounded by
reflective crystals forming a ‘tapetal cup’ (Figure 13.3e). As each bundle
acts as a functional unit, they provide a detector with high sensitivity but
poor spatial resolution, which may also enhance motion detection (Francke
et al., 2014).
In most fishes, all rods are structurally similar and contain only a single
visual pigment. However, cones are more variable both morphologically
and spectrally. Cones can be ‘single’ or ‘double’, the latter consisting of
two individual cones fused together (Figure 13.3f). While cones normally
mediate colour vision, double cones may principally be involved in
achromatic tasks involving luminance, motion or polarized light detection.
However, in one species at least, the lagoon triggerfish (Rhinecanthus
aculeatus), double cones are involved in colour vision (Pignatelli et al.,
2010). While double cones are the most common cone type in most teleosts,
they are absent from elasmobranchs.
The cones of some, highly visual, teleosts are arranged into regular arrays
(Figure 13.3f). The form of these ‘mosaics’ can change during
development, with retinal region and during light/dark adaptation. Although
the function of such patterns is unclear, they may enhance acuity and aid
polarization sensitivity.

13.2.6.2 Light/Dark Adaptation

Most vertebrates cope with the large range of light levels experienced
during 24 h by switching between rod- and cone-based vision. As the
dynamic range of each photoreceptor is only ca. 3 log units, their sensitivity
is further adjusted by a variety of morphological, biochemical and
physiological changes within the retina, resulting in a classic dark
adaptation curve (Figure 13.4a).

FIGURE 13.4 Dark adaptation. (a) Sensitivity change in zebrafish during dark
adaptation, using the escape response to determine threshold. For the first 8 min,
sensitivity is determined by the cone system (dotted line). Thereafter, retinal thresholds
are determined by rods (solid line; from Li and Dowling, 1997). (b) Dark- (right) and
light (left)-adapted outer retina of a perch (Perca fluviatilis) (from Douglas, 2008).
Abbreviations as for Figure 13.3.

In fishes, the most prominent structural changes during the light/dark

cycle are retinomotor movements (RMM). On light exposure, cone myoids
contract, placing cone outer segments near the inner retina, while rod
myoids elongate, burying the rod outer segments behind a screen of
melanosomes that are dispersed within the apical processes of the RPE
(Figure 13.4b). In lower light levels, the rods are positioned closer to the
inner retina, while the cone outer segments lie nearer the choroid along with
the melanosomes aggregated at the base of the RPE cells. Such movements
make optimal use of the retinal space and protect the rods in high light
levels. RMM are triggered by both light and endogenous signals and are the
result of actin, myosin and tubulin-associated mechanisms, controlled
antagonistically by melatonin (dark signal) and dopamine (light signal)
regulating cAMP levels (Burnside and Kingsmith, 2010).

13.2.6.3 Regional Variation in Retinal Structure

The structure of the retina is rarely uniform across its entire area (Collin,
1999). Some regions are characterized by a high density of cones and
ganglion cells, leading to a finer sampling of the retinal image and
decreased convergence, enhancing spatial resolution. Fishes will often
move their eyes to place images on such ‘areas’, the shape and location of
which vary and sample visual space most effectively for a given species.
For example, a roughly circular retinal area of maximal ganglion cell
density in Aulosioma chinensis is situated temporally, providing high-acuity
frontal binocular vision (Figure 13.5a). A similar area is located ventro-
temporally in archerfish (Toxotes spp.), aligning it with the location of prey
positioned dorso-nasally on overhanging branches. Bottom-dwelling
Liposarcus pardalis, on the other hand, have two areas of increased acuity
nasally and temporally, allowing detailed sampling of the visual field both
behind and in front of the animal (Figure 13.5b). Species inhabiting less
complex visual environments, such as sandy bottoms, have elongated
horizontal ‘visual streaks’ of increased cone and ganglion cell density,
allowing detailed sampling of the horizon (Figure 13.5c).
 FIGURE 13.5 Iso-density contour map of cells within the ganglion cell layer. Densities
for (a) and (c) are ×104 cells/mm2 and for (b) ×102 cells/mm2. d: dorsal; n: nasal; t:
temporal; v: ventral.
(From Collin, S.P. and Pettigrew, J.D., Brain Behav. Evol., 31, 283–295, 1988; Douglas,
R.H. et al., J. Exp. Biol., 205, 3425–3433, 2002.)

13.2.6.4 Visual Pigments

Visual pigments consist of a chromophore bound to a protein (opsin) within
photoreceptor outer segment disc membranes. The chromophore is either
retinal, derived from vitamin A1, or the vitamin A2-derived 3,4-
dehydroretinal, forming, respectively, rhodopsin and porphyropsin visual
pigments. Opsins are chains of ca. 350 amino acids traversing the
membranes seven times as an alpha helix, forming a cluster that surrounds a
central ‘binding pocket’. In darkness, the chromophore is in an 11-cis
configuration and attached within this pocket to a lysine residue in the
seventh transmembrane loop. When activated by a photon, it isomerizes to
the all-trans form, altering the conformation of opsin and separating from it.
An intermediate stage in this process, metarhodopsin, initiates a G-protein-
coupled enzyme cascade that closes cation channels, hyperpolarizing the
photoreceptor and decreasing synaptic glutamate release.
Visual pigments have bell-shaped absorption profiles (Figure 13.6), the
point of maximum absorption, the λmax, indicating the wavelength of
maximum sensitivity. A pigment’s spectral characteristics are determined
by both the nature of the chromophore and the structure of the opsin.
Changing the identity of just a few key opsin amino acids can produce
major shifts in the pigment’s spectral sensitivity. In vertebrates, visual
pigments are coded for by five different opsin gene families. All rods
contain an RH1 pigment (λmax 460–530 nm), while there are four types of
cone pigment: LWS (λmax 490–570 nm), RH2 (λmax 480–535 nm), SWS1
and SWS2 (λmax 410–490 and 355–440 nm). These absorption maxima are
for retinal-based rhodopsin visual pigments, the λmax of porphyropsins
being shifted to longer wavelengths.
 FIGURE 13.6 Visual pigment absorption spectra. Rod pigments are shown as dashed
lines and cone pigments as solid. (f) shows the two rod pigments of Malacosteus niger,
the emission spectra of its longwave (red) and shortwave (blue) bioluminescence, and
the absorption spectrum of its retinal chlorophyll-derived photosensitizer (green).

Of all vertebrates, fishes have the greatest range of visual pigments, with
λmax values from ca.350-630 nm, reflecting the spectral diversity of the
aquatic habitat. As deep-sea fishes live in a world of few photons, cones
would serve no function, and most have only rods containing a single visual
pigment (Figure 13.6a). No adult fishes have only cones, although this is
common in larval stages. In addition to rods, most teleosts have variable
numbers of cones. Although some possess only a single class of cone
(Figure 13.6b), the retinas of most express visual pigments from either two,
three or four opsin families (Figure 13.6c through e). Examples of
dichromatic animals include species inhabiting turbid coastal waters.
Trichromats and tetrachromats, on the other hand, are common in shallow,
clearer waters. Among elasmobranchs, skates possess only rods, most
sharks are cone monochromats, and rays have up to three different spectral
cone classes (Collin, 2018). As described later, the possession of two or
more spectral photoreceptor types is required for colour vision.
Some teleosts have multiple opsin genes from the same family, although
not necessarily all expressed at the same time. Astonishingly, a single deep-
sea fish, Diretmus argenteus, has 38 rod opsins in its genome,14 of which
are expressed (Musilova et al., 2019). Expressing multiple opsins
simultaneously in a deep-sea fish broadens the photoreceptor’s spectral
absorption, thereby increasing absolute sensitivity, while in other species it
might underlie regional differences in retinal spectral sensitivity. Expressing
different opsin genes sequentially allows ontogenetic changes in an
animal’s visual pigment complement.
The adaptive significance of a visual pigment’s λmax is rarely obvious,
and a single explanation is unlikely to apply to all species or even account
for all pigments within a given retina (Douglas, 2001). Most simply, the
pigment maximizes photon catch by matching the spectral characteristics of
the available light (‘sensitivity hypotheses’). Therefore, the λmax of most
deep-sea fish rods is shortwave-shifted (470–490 nm) compared with more
shallow-water species (ca. 500 nm), both approximating the spectrally
restricted spacelight and matching the spectra of most bioluminescent
emissions. Maximization of photon catch also explains why many species
inhabiting ‘red’ freshwater have longwave-sensitive porphyropsins,
sometimes only expressing longwave opsins (Figure 13.6b), which in
animals from blue/green oceanic waters are replaced by opsins maximally
sensitive at shorter wavelengths within rhodopsin pigments. In catadromous
species, both the nature of the chromophore, and in eels (Anguilla anguilla)
the opsins expressed, change during migration to shift the animal’s
sensitivity to shorter wavelengths.
Perhaps the best example of visual pigments matching a specific signal is
three genera of deep-sea dragonfishes (Aristostomias, Pachystomias and
Malacosteus), which in addition to blue bioluminescence similar to that of
other mesopelagic inhabitants, also produce far-red bioluminescence with
peak emissions above 700 nm. This red bioluminescence would not be
visible using ‘normal’, shortwave-sensitive, deep-sea visual pigments.
However, the visual pigments of dragonfish are longwave-shifted compared
with other deep-sea fishes, and one species, M. niger, enhances its
longwave sensitivity with a unique, chlorophyll-derived photosensitizer
(Figure 13.6f).
Although in some instances the spectral sensitivity of the visual
pigments, especially for rods and double cones, matches the spectral
characteristics of the available light, this will not always be the most
appropriate strategy when detecting specific targets. A visual pigment
‘offset’ from the spacelight, for example, would enhance the contrast of
objects with a different spectral radiance from the background.

13.3 VISUAL OPTICS

13.3.1 EYE SHAPE
Most fishes have approximately spherical, symmetrical eyes (Figures 13.2
and 13.7a). The most obvious exceptions are the upwardly-directed, tubular
eyes of some mesopelagic fishes (Figure 13.7b). These enhance an animal’s
sensitivity by providing a large pupil and a short focal-length lens, which
can nevertheless be accommodated by the small bodies of mesopelagic
animals. Their large binocular overlap will further enhance sensitivity and
possibly provide a cue to object distance. They also aid the detection of
dark objects seen from below, which appear as dark silhouettes against any
residual sunlight. A major drawback of these eyes, however, is their limited
visual field, which is restricted to ca. 50° above the animal. Consequently,
such eyes will miss much of the bioluminescence occurring all around an
animal. Therefore, several species with tubular eyes have developed devices
that extend their limited visual fields. For example, several
Opisthoproctidae have diverticula on their lateral walls, which range from
simple retina-containing outpouchings in some species, which do little more
than sense unfocused light, to more complex structures producing better-
focused images using either refraction or reflection (Wagner et al., 2009)
(Figure 13.7c). Alternatively, the Evermanellidae and Scopelarchidae have
lateral lamellar structures that channel ventro-lateral illumination into the
eye (Wagner et al., 2019) (Figure 13.7d).
 FIGURE 13.7 Tubular eyes. (a) The symmetrical eye of shallow-water Astatotilapia
burtoni, showing the retina (r) lining the posterior globe. (b) The tubular eye of
Argyropelecus affinis, whose large spherical lens (l) produces a well-focused image on a
complex main retina (mr) lining the base of the tube. The medial wall is lined with a
rudimentary accessory retina (ar), which is too close to the lens to receive a focused
image. (c) The tubular eye of Dolichopteryx longipes has a diverticulum that focuses
ventrolateral illumination on a lateral diverticular retina (dr) using a mirror (m). (d) The
tubular eye of Evermanella balbo has a lateral lamellar optical fold (of), which most
likely directs ventrolateral illumination onto the dorsal ar. d: dorsal; l: lateral; m:
medial; v: ventral. Dotted lines indicate the presumed path of ventrolateral illumination.
(Photographs in parts (a) and (b) courtesy of HJ Wagner. Part (c) taken from Wagner HJ
et al., 2009. Current Biology 19(2), 108–114. Part (d) taken from Wagner HJ et al.,
2019. Cell and Tissue Research, 378(3), 411–425.)

13.3.2 IMAGE FORMATION

In most vertebrates, a focused image is produced by refraction from a single
lens and cornea. In terrestrial animals, most refraction occurs at the cornea,
as its refractive index is higher than that of air, the relatively flattened lens
accounting for little of the eye’s dioptric power. Underwater, however, the
cornea is optically ineffective, as it is thin, has parallel surfaces and
separates the surrounding water from aqueous humour, which have similar
refractive indices. Most fishes, therefore, have optically more powerful,
near-spherical lenses, which form their only refractive element. Only in the
sandlance (Limnichthyes fasciatus) does the unusually thick cornea
contribute significantly to the eye’s dioptric power.
As the lens is the only refractive surface in most fishes, the optical
quality of the whole eye is dictated by it. Image quality is degraded by two
main imperfections: spherical and chromatic aberration. However, visual
performance in fishes is little affected by these, as they are minimized by
refractive index gradients and multifocal lenses (Kröger et al., 1999), so
that the lens provides a better image than can be resolved by the retina.

13.3.2.1 Resting Refractive State and Accommodation

The resting refractive state is the distance at which an eye is focused
without having to exert any accommodative effort. Most vertebrates are
emmetropic, so that parallel light rays, which approximate those from a
distant object, are focused on the retina. As most eyes have a degree of
tolerance to blur, such eyes will produce clear images of everything beyond
a few metres. To focus closer objects, emmetropes either increase the
curvature of their lens and/or cornea or relocate their lens further from the
retina.
However, given the limited range underwater, it is unsurprising that most
teleost fishes are myopic along the lines of sight most important to them.
Parallel light rays are focused in front of the retina, and this focuses light
coming from closer objects. Teleosts, thus, usually accommodate to see
more distant objects by moving their lens towards the retina using a ventral
retractor lentis muscle (Figure 13.2a). A complex series of ligaments holds
the lens in place, ensuring no unwanted lens movements during
accommodation (Khorramshahi et al., 2008).
Nocturnal, less visual fishes may not accommodate much, while actively
hunting diurnal species have accommodative amplitudes of over 20 D.
Once more, the sandlance is unusual, as its accommodative range is
extremely large (180 D) and involves a flattening of the cornea to focus at
distance (Pettigrew et al., 1999).
The largest accommodative lens movement in many teleosts is in a naso-
temporal direction, in a plane roughly parallel to the pupil. Since the lenses
of most teleosts protrude through the pupil, many teleosts have a rostral
aphakic gap in their pupils (Figure 13.8a) allowing such lens movement.

FIGURE 13.8 Pupil shape.

(Photos (a)–(d) come from Douglas RH, 2018. Progress in Retinal and Eye Research,
66, 7–48. (e) comes from Douglas RH et al., 2002. Journal of Experimental Biology,
205(22), 3425–3433. (f)–(g) come from Douglas RH et al., 1998. Vision Research,
38(18), 2697–2710.)

The resting refractive state of freely swimming lemon sharks (Negaprion

brevirostris) is emmetropic (Hueter et al., 2001). Accommodation is not
well understood in elasmobranchs but is less extensive than in teleosts and
involves a protractor lentis muscle that moves the lens closer to the cornea
on contraction to focus close objects.

13.3.2.2 Amphibious Vision

Amphibious animals would benefit from seeing both in air and underwater.
However, sharp vision in both media is optically complex, as the cornea
acts as a refractive surface in air but not when submerged. Thus, an eye
with a powerful lens suitable for focused vision underwater will become
very myopic in air, while a terrestrial eye with a more flattened lens
becomes hyperopic underwater.
Many amphibious mammals, reptiles and birds compensate for the loss of
a refractive cornea underwater either by extreme accommodation (50–80 D)
or by constricting their pupils to increase depth of field. The few
amphibious fishes use neither of these tactics as far as we understand to
date. Instead, the intertidal blenny (Dialommus macrocephalus) and the
flying fish (Cheilopogon heterurus) minimize the cornea as a refractive
surface in air using flattened corneal facets (Baylor, 1967).
Most impressively, the so-called four-eyed fish (it has only two),
Anapleps anapleps, achieves simultaneous aerial and aquatic vision while
half of each eye is above the water and half below. Its elliptical lens is tilted
so that light from above, which encounters a refractive cornea, traverses the
optically less powerful short axis of the lens, while light from below, which
traverses the optically neutral cornea, goes through its long axis.
Although not amphibious, archerfish deserve mention, as they can ‘shoot
down’ insects sitting on branches overhanging the water, or even flying
above its surface, using jets of water projected from their mouths, whose
volume and force is adjusted depending on the target’s size and location.
This ability is especially impressive as they compensate for the distorting
effect of refraction by the water surface when calculating the location of
their prey (Schuster, 2018).
13.3.3 PUPIL
Light-evoked constriction of the pupil decreases the amount of retinal
illumination and improves image quality by decreasing spherical aberration
and increasing the depth of field. It is widespread among vertebrates and
occurs in most shallow-water elasmobranchs. However, among teleosts, it is
restricted to a few, largely bottom-dwelling species, in whom it probably
camouflages the otherwise very visible large dark round pupil (Douglas,
2018).
In most fishes, dilated pupils are close to round. In skates and rays,
constricted pupils often have a prominent dorsal operculum (Figure 13.8b
through d), as do armoured catfish (Figure 13.8e). The constricted pupils of
the plainfin midshipman consist of two, almost independent pinholes
(Figure 13.8f), while those of sharks vary from circular to elliptical and
various orientations of slits (Figure 13.8g). Dorsal opercula camouflage the
eye when seen from above and smooth out the unevenly distributed
underwater light field. Slit pupils allow a greater range of pupillary areas
than circular ones, aid in alleviating chromatic aberration by allowing the
whole width of a multifocal lens to be used, and may facilitate the
perceptions of objects in specific orientations. Pinholes aid depth
perception.
13.3.4 TAPETA
As much incident light is not initially absorbed by the photoreceptors,
animals inhabiting low light levels often have a ‘tapetum lucidum’ external
to their photoreceptors that reflects light back towards the photoreceptors,
giving them another opportunity to absorb it. As tapeta scatter light,
inevitably decreasing spatial resolution, the tapeta of some shallow-living
elasmobranchs are occlusable, a layer of pigment migrating to cover the
reflective structures in brighter light, when enhanced sensitivity is not
required.
In teleosts, most tapeta are within the RPE, although in some, they are in
the choroid. Choroidal tapeta are more common in elasmobranchs (Nicol
and Somiya, 1989). Either tapeta reflect a large part of the spectrum,
producing a white or silvery appearance, or reflection is more spectrally
restricted. The tapeta of deep-sea species usually reflect short wavelengths,
as these are most readily transmitted by sea water, although the tapetum of
the red-light-sensitive Malacosteus niger reflects long wavelengths. Turbid
freshwater fishes also often have orange or red tapeta, reflecting the
predominant wavelengths. The sandbar shark, Carcharhinus plumbeus,
‘tunes’ the reflectance of its tapetum to match the environment, clear-water
populations having blue tapeta while those of turbid water, estuary
populations are orange-green. Frequently, the tapetum is best developed in
the dorsal retina, which receives the relatively dimmer upwelling
illumination, and in some species, the tapetum isolates bundles of
photoreceptors (Figure 13.3e).
13.3.5 INTRAOCULAR FILTERS
Animals maximizing photon capture have ocular media transmitting all
wavelengths from the near infrared down to ca.300–320 nm, the structural
proteins absorbing shorter wavelengths. However, the corneas and lenses of
diurnal fishes, for which photon capture is not a primary concern,
frequently contain a variety of pigments that remove short wavelengths
(Figure 13.9b). Since these are the most damaging to ocular tissue and also
prone to chromatic aberration and small particle (Rayleigh) scatter, their
removal is beneficial in high light levels. Depending on the identity and
concentration of the pigments, variable amounts of short wavelengths are
removed by high pigment concentrations resulting in visibly coloured
structures.
 FIGURE 13.9 Ocular media pigmentation. (a) Central transmission spectra and
schematic representation of the occlusable cornea of Torquigener pleurogramma in the
dark (solid/left) and light (dotted/right). (b) Transmission spectra of fish lenses: the
unpigmented Protomyctophum germanium (black), the carotenoid-containing
Argyropelecus sladeni lens (blue), and the pigmented lenses of Gobiusculus flavescens
(green) and Benthalbella macropinna (red). (c) Although both downwelling sunlight
(black) and bioluminescence (orange) are maximal between 450 and 500 nm,
bioluminescent maxima can occur at longer wavelengths, and emission spectra are often
broader at longer wavelengths than the downwelling sunlight. The yellow lenses of
mesopelagic fish (grey) therefore remove more of the veiling background light than the
bioluminescence, increasing the contrast of the bioluminescence. The inset shows the
head of the tube-eyed mesopelagic Scopelarchus analis from above, highlighting its
yellow lenses.
(Panel (a): data from Siebeck, U.E. et al., 2003. J. Exp. Biol., 206, 2177–2190. Inset
photo (c) from Douglas RH, 2006. Biological Sciences Review Magazine 19(1), 36–41.)

As removing short wavelengths is only beneficial in high light levels,

pigmentation is often restricted to the dorsal cornea, which receives the
more intense downwelling illumination. Furthermore, the corneas of some
species are occlusable. In low light levels, pigmentation is aggregated
around the edge of the cornea. Only in bright light do the pigments disperse
throughout the cornea (Figure 13.9a).
As yellow lenses are characteristic of animals inhabiting high light levels,
it is surprising to find shortwave-absorbing pigments in the lenses of several
mesopelagic species (Figure 13.9 b, c). Light levels will be so low, and the
acuity of the rod-dominated retina so poor, that protection and acuity
enhancement are unlikely functions of such filters. Instead, they probably
serve to increase the visibility of bioluminescence.

13.4 VISUAL ABILITIES

Although the anatomy and electrophysiology of visual structures give some
insight into what an animal can see, they rarely reflect the true visual
potential of the animal. This can only be obtained by assessing the
behavioural response of an intact animal to visual stimuli (Douglas and
Hawrhyshyn, 1990).
13.4.1 ABSOLUTE SENSITIVITY
The absolute sensitivity is the minimum amount of light an animal can
detect when fully dark adapted. Eyes with high sensitivity have
enlarged/plentiful rods connected to few ganglion cells, large pupils,
reflective tapeta and short focal-length lenses. These attributes are all well
developed in deep-sea fishes, and while in ideal conditions humans can see
sunlight underwater to a depth of ca. 850 m, deep-sea fishes may perceive
vestiges of sunlight down to 1000 m, making them 100× more sensitive
than man (Denton, 1990). Psychophysical measurements on a few
shallower-water species such as goldfish (Carassius auratus) and lemon
sharks (Negaprion brevirostris) indicate that they too are more sensitive
than humans. The delayed development of rods in most fishes means that
sensitivity will increase in older animals.
13.4.2 CONTRAST
Underwater, contrast is degraded by both light absorption and scattering.
The greater the sensitivity of a fish to any contrast between an object and
the background, the greater the distance at which it can be perceived.
Although this can involve differences in texture, pattern, colour and
polarization, brightness contrast usually determines the visibility of objects
underwater. The Port Jackson shark (Heterodontus portusjacksoni) can
detect contrast differences of around 1% (Ryan et al., 2016), which is
comparable to humans. Other fishes have only slightly lower sensitivity to
contrast.
13.4.3 SPATIAL RESOLUTION
A fish with good spatial resolution will, for example, be able to discern the
detailed body markings of others and spot prey and potential mates from
greater distances. Maximum resolution is expressed by the angle subtended
at the nodal point of the eye by two points an animal can just distinguish,
the minimum resolvable angle (MRA).
A black and white square-wave grating can theoretically be resolved if
two white bars fall on separate photoreceptors and the black bar is imaged
on one in between, as long as the outputs of the photoreceptors are analysed
by separate RGCs. The low convergence of cones onto RGCs makes them
well suited to providing high spatial resolution. Resolution is low in dim
light, when rods, with their high convergence ratios, are employed. Spatial
resolution is increased by sampling the image with a large number of cones,
which requires a long focal-length lens and a high density of cones, often
restricted to small regions of the retina.
A measure of an animal’s theoretical ability to resolve detail is the
highest density of cones and/or RGCs within its retina. Sometimes, as in the
archerfish, the MRA determined by such morphological criteria is close to
that determined psychophysically (Temple et al., 2013). Frequently,
however, the behavioural acuity is not as good as that predicted by cone
spacing due to a degree of convergence of cone output, the use of
inappropriate behavioural assays or not all spectral cone types being
involved in spatial resolution.
Many fishes, when placed in a circular container surrounded by rotating
stripes, will either swim after the stripes or follow them with their eyes. The
width of the stripes can be decreased until the animal no longer follows
them. This optomotor response is a convenient method for determining
spatial resolution as it requires no training. However, it measures the
response of the retina to global movement, which is assessed primarily by
the peripheral retina rather than by the area of highest spatial resolution.
Alternatively, animals can be trained to distinguish two targets, either
composed of stripes of different orientations or one composed of stripes and
the other of an equiluminant grey. Once more, the grating frequency at
which the discrimination breaks down indicates the limits of resolution
(Douglas and Hawrhyshyn, 1990).
Not surprisingly, the MRAs measured in fishes vary, depending in part on
the methods used but also indicating species differences. Those with the
best spatial resolution include tuna (Katsuwonus pelamis), archerfish
(Toxotes chatareus) and bluegill (Lepomis macrochirus), with MRAs of 5.5,
9.3 and 5.5 minutes of arc, respectively. The acuities of other teleosts, less
reliant on the perception of fine detail, are substantially lower, and
resolution in sharks is extremely poor. No fish comes close to the maximal
spatial resolution of humans (ca. 1 minute of arc).
Behavioural acuities are usually determined using high-contrast gratings.
At lower contrast, which is closer to the natural world, acuities will be
lower. The contrast sensitivity function (CSF) measures contrast sensitivity
at different spatial frequencies. In all animals, it peaks at a given spatial
frequency and falls off at higher and lower frequencies (Figure 13.10).
CSFs in teleosts and sharks peak at around 0.1–0.4 cycles/degree, which is
substantially lower than the frequency of maximal human contrast
sensitivity (5 cycles/degree). This is not surprising, as light scatter
underwater blurs the edges of objects, causing a loss of high spatial
frequencies.
 FIGURE 13.10 Spatial contrast sensitivity functions. Human (black solid), macaque
(dotted black), Carassius auratus (green), Lepomis macrochirus (red), Tripterygion
delaisi (blue), Heterodontus portusjacksoni (brown), Chiloscyllium punctatum (yellow),
Danio rerio (purple).
(Data from Northmore, D.P.M. et al., Visual Neurosci., 24, 319–331, 2007; Santon, M.
et al., J. Vision, 19, 1–10, 2019; Tappeiner, C. et al., Front. Zool., 9, 10, 2012; Ryan,
L.A. et al., J. Exp. Biol., 219, 3971–3980, 2016.)

13.4.4 TEMPORAL VISION

Often, it is only when something moves that it is perceived. Movement is
also part of many sexual and aggressive displays, and without being able to
predict the movements of prey, many predatory species would go hungry. A
measure of an animal’s temporal resolution is the frequency at which a
flashing light appears as a steady glow, the ‘critical flicker fusion
frequency’ (CFF).
The physiology of the rod system is sluggish, resulting in low CFFs in
dim light. Cones have higher temporal resolution, and light-adapted CFFs
in teleosts inhabiting warm, clear surface waters that forage on fast-moving
prey are comparable to those of humans (60 Hz). Some tuna have CFFs
close to 100 Hz. However, more sedentary species or those inhabiting
turbid, deeper, colder environments have significantly lower temporal
resolution. Although the CFFs of only a few elasmobranchs have been
studied, they appear to be lower (10–44 Hz) than those of teleosts.
 As temporal resolution is temperature-dependent, swordfish (Xiphias
gladius) raise the temperature of their retina 10–15 °C above that of the
water, ensuring a temporal resolution up to 10× greater than that of their
cold-eyed prey (Fritsches et al., 2005). Other open-ocean predators such as
billfish, tunas and some sharks also have the ability to raise the temperature
of their eyes and probably have similarly elevated CFFs.
The temporal CSF (tCSF) relates the luminance difference between the
light and dark phase of a flickering stimulus that an animal can just detect
to the frequency of its temporal modulation. While the tCSFs of most
vertebrates peak at 10–15 Hz, peak sensitivity of goldfish and some sharks
is at lower frequencies (4–5 Hz).
13.4.5 COLOUR VISION
Colour is an important social signal, allowing individual recognition,
facilitating mate choice and signalling dominance, and is used in aggressive
interactions. Its perception can also be beneficial in discriminating items of
interest from the background and other objects.
The human visible spectrum is restricted, wavelengths shorter than 400
nm being absorbed by the lens and the most longwave-sensitive visual
pigment (λmax 563 nm) absorbing little light beyond 750 nm. Many fishes,
however, have more transparent ocular media and visual pigments with
peak sensitivity ranging from the UV to the far-red, giving them a potential
spectral sensitivity from ca. 300 to around 850 nm; the biggest spectral
range of any animal. However, being sensitive to a range of wavelengths
does not equate to a sensitivity to colour.
As the likelihood of visual pigment isomerization depends on both the
wavelength and the brightness of the light, the degree of hyperpolarization
of a single class of photoreceptor cannot be used as a basis for colour
vision. Animals with only one spectral class of photoreceptor, such as most
deep-sea fishes (Figure 13.6a), will not see colour. For this, an animal must
have at least two types of spectrally distinct photoreceptors, whose output is
compared. Although rudimentary colour vision based on rod–cone
interaction is theoretically possible, sharks possessing only rods and a
single cone type are unable to distinguish stimuli using their colour
(Schluessel et al., 2014). True photopic colour vision requires at least two
spectrally distinct cone types.
However, showing that an animal has two or more cones of differing
spectral sensitivity is still not proof of colour vision. If the output of the
different cone types were simply summed rather than compared, although
the sensitivity and spectral range of vision would be extended, information
about wavelength would be lost. To confirm colour vision, one must show
that an animal can discriminate wavelength independently of brightness.
For example, one could train a fish to discriminate two differently coloured,
but equiluminant, targets. However, since it is difficult to produce stimuli
perceived as equally bright by non-humans, the ability to distinguish
coloured targets with a variety of brightnesses or the capacity to choose a
coloured target from among a variety of similar grey ones can be examined.
Colour vision in fishes was first shown behaviourally over a century ago
in minnows (Phoxinus laevis); it has more recently been demonstrated in a
variety of other marine and freshwater species with multiple cone pigments.
Although the total number of fishes in which colour vision has been
definitively proved is less than 10, it would be surprising if most species
with multiple cone pigments were not able to discriminate colour (Marshall
et al., 2018).
13.4.6 POLARIZATION
Light is a wave with an electric field perpendicular to the light’s direction of
travel. The axis of this field is the ‘e-vector’. Light emanating from most
sources is unpolarized, with the e-vectors in all possible orientations, but
becomes polarized to varying degrees after being scattered or reflected.
Although humans cannot detect the e-vector, some fishes can.
Light scattering by air molecules produces a band of polarized light 90°
from the sun. Sensitivity to polarized light therefore allows the sun to be
located and used in navigation by many invertebrates and birds. The sky’s
polarized light field could also be used for orientation in fishes, especially
near the surface. However, the main use of e-vector detection underwater is
probably to enhance the contrast of specific objects with differing
polarization properties from the background. Polarized light reflected from
areas of the body might also serve as intra- or inter-specific signals.
 Just as an animal with one visual pigment cannot perceive colour, being
sensitive to a single plane of polarized light is not the same as polarization
vision. This requires distinguishing e-vector orientations using at least two
receptors with different angles of maximal e-vector sensitivity. The
orthogonal orientation of invertebrate photoreceptor microvilli forms an
ideal substrate for mediating polarization sensitivity. However, with the
exception of anchovies (Engraulis mordax), vertebrate photoreceptors lack
such organization (Novales Flamarique, 2017).
13.4.7 DEPTH PERCEPTION
Many fishes live in complex three-dimensional environments and need
information about the distance and size of the things they see. Without this,
archerfish could not hit their targets, a territorial reef fish could not find its
refuge to avoid a passing predator, and even pelagic species, living in an
environment where there are no obvious landmarks, need to be aware of the
distance and size of other animals in order to react appropriately to them.
The angle an object subtends on the retina depends on both its size and
its distance. Goldfish can be trained to find food hidden at a given distance
from a landmark using only its visual angle, as the size of a landmark is
usually constant. However, using visual angle alone would lead to
potentially fatal confusion when trying to differentiate a nearby prey item
from a more distant, larger predator. Goldfish can be trained, however, to
distinguish two different-sized discs at different distances but subtending
the same visual angle. Thus, they are determining the true size of the object
using both its visual angle and its distance. This is known as ‘size
constancy’ and may be widespread in fishes, but perhaps only in certain
conditions (Frech et al., 2012).
If an object is within an animal’s binocular visual field, the image is
formed in different parts of the retina in the two eyes. The closer the object,
the greater this ‘retinal disparity’. Either the disparity or the vergence eye
movements made to image the object on retinal areas of high acuity specify
object distance. However, the laterally placed eyes of most fishes result in
them seeing much of the world with only one eye, suggesting that they use
one or more monocular cues to object distance. It is likely that some fishes
monitor their accommodation or use motion parallax, and looming stimuli
probably enable archerfish to rapidly gauge the height of falling prey they
have dislodged.

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4.
 14 Olfaction
Sigrun I. Korsching

CONTENTS
14.1 Introduction
14.2 Morphology of the Olfactory System
14.3 Olfactory Circuits
14.4 Fishes Recognize at Least Six Different Classes of Odors
14.5 Five Distinct Classes of OSNs Detect Odors in Fishes
14.6 Molecular Basis of Odor Detection: Families of Olfactory Receptors in
Fishes
14.7 Model Fish Species for Developmental, Behavioral and Genetic
Studies
14.8 Olfaction in Relation to Systematic Position and Ecological
Environment
Acknowledgement
References

14.1 INTRODUCTION
Bony, cartilaginous and jawless fishes all possess a well-developed sense of
smell (Buchinger et al., 2015; Korsching, 2016). In fact, the ability of
sharks to smell blood has made it into a figure of speech. Although extant
lobe-finned fishes are but a shadow of their former relevance in the
Devonian, two families with a handful of species have survived: the ‘living
fossil’ coelacanth and the lungfishes. Genomes of many ray-finned fishes,
lampreys, sharks and coelacanth have now become available. Thus, we can
study the sense of smell at the molecular level not only in ray-finned but
also in lobe-finned, cartilaginous and jawless fishes. Comparing lobe-finned
fishes with tetrapods will provide a much clearer picture of the
reorganization of the sense of smell necessitated by the water-to-land
transition than would be possible by comparing tetrapods with ray-finned
fishes such as Danio rerio (zebrafish), a recently emerging vertebrate model
system (Dahm and Geisler, 2006; Holtzman et al., 2016).

14.2 MORPHOLOGY OF THE OLFACTORY SYSTEM

Fishes, like all vertebrates, possess separate smell and taste systems
(olfaction and gustation, respectively) as well as solitary chemosensory
cells (SCC) distributed over the skin surface (Daghfous et al., 2020). Taste
buds of fishes are not limited to the tongue but are also found on gills, head
and body surface to varying degrees in different species (Kasumyan, 2019).
In contrast, odor detection occurs in a single specialized organ, the nose.
The structure of the olfactory organ exhibits huge diversity in bony fishes
(Hansen and Zielinski, 2005) (Figure 14.1). The nose of bony and
cartilaginous fishes consists of two separate organs, often situated close to
and medial to the eye (Yamamoto, 1982), whereas the earliest diverging
vertebrates, the jawless fishes, possess a single circular olfactory organ
(Daghfous et al., 2016). Noses in most ray-finned fishes, sharks and
lampreys form a blind sac, which is not connected to the pharynx, that is,
the respiratory system. Thus, breathing does not result in increased odor
exposure in these species, unlike tetrapods, which increase the sensitivity of
odor detection by sniffing. However, lungfishes, chimaeras (sister group to
sharks) and hagfishes (sister group to lampreys; see Chapter 1) possess a
direct connection between nose and pharynx (Hara, 1975; Holmes et al.,
2011), so that odor sniffing is possible. Some ray-finned fishes have
evolved a similar opening into the pharynx, attesting to the diverse
morphology of the olfactory organ in this clade (Yamamoto, 1982).
 FIGURE 14.1 Morphology of the olfactory organ. (a) In normosmic species, such as
zebrafish, the olfactory epithelium is folded into several lamellae, which rise with
bilateral symmetry from a non-sensory median raphe. (b) Horizontal cross section
through an epithelium of type (a). (c) In microsmatic species, such as medaka, the
olfactory epithelium consists of a simple sensory patch without further substructure. (d)
Secondary folding in lamellae of some macrosmatic species, such as sharks, increases
the sensory surface considerably. (a) to (d) OSNs expressing a particular receptor (red)
are scattered throughout the epithelium.

In the absence of sniffing, fishes have several solutions to increase the

sensitivity of odor detection. Water flow through the nose is unidirectional,
with a specialized inlet nostril and a separate outlet nostril, which can be
either concentrically arranged or distant, with the inlet nostril anterior of the
outlet. Several detailed reviews of naris shape and formation across many
fish species have been published (Hara, 1975; Hansen and Zielinski, 2005).
Water flow through the nose is often generated by active swimming
(Theisen et al., 1986; Hansen and Zielinski, 2005). For some fishes, the
beating of motile cilia of non-sensory cells also plays a role in increasing
convection (Hara, 1975; Hansen and Zielinski, 2005). Fish species that live
in habitats with little turbulence, that is, sedentary, bottom-dwelling fishes,
often use active pumping to increase flow-through in the nose by indirectly
contracting a nasal sac that is connected to the olfactory organ, for example
pleuronectid flounders (Olivares and Schmachtenberg, 2019). A similar
mechanism is found in lamprey (Thornhill, 1967).
Sensitivity can also be increased by enlarging the area of the sensory
surface (Figure 14.1). In microsmatic species, the sensory surface may be a
simple patch (e.g. Oryzias latipes, medaka) or interspersed with non-
sensory ridges or spots (Yamamoto, 1982). In many species, the sensory
surface is folded to increase the surface and presents as several to many
dozens of lamellae (Hara, 1975; Hansen and Zielinski, 2005). Eels (genus
Anguilla), which are macrosmatic species, possess up to 120 lamellae
(Teichmann, 1959; Yamamoto, 1982). In sharks and chimaeras, the sensory
surface often is further enlarged by the formation of secondary lamellae
emanating from the primary lamellae like twigs emanating from branches
(Hara, 1975; Theisen et al., 1986; Ferrando et al., 2019). Such secondary
folding is also observed in some teleost fishes (Hara, 1975). Each olfactory
sensory surface has bilateral symmetry, and the nasal and lateral
hemispheres are separated by a non-sensory central region (Hara, 1975;
Yamamoto, 1982).
The olfactory nerve connects the sensory surface to the olfactory bulb. In
many Cypriniformes and Siluriformes, the olfactory nerve is very short, but
the olfactory tract connecting the olfactory bulb to the telencephalon is
rather long. In the majority of teleosts, the inverse situation is found: a long
olfactory nerve and a short olfactory tract (Hara, 1975).

14.3 OLFACTORY CIRCUITS

Odor-mediated behavior requires multi-step processing, from odor binding
in the sensory surface, to signal transduction via the olfactory bulb and
telencephalic and diencephalic centers to the mesencephalic locomotor
region, and finally, to command neurons for locomotion situated in the
brainstem, which give rise to coordinated movements (Figure 14.2).
 FIGURE 14.2 Olfactory circuits. Schematic representation of a fish brain and olfactory
processing pathways.
Red: OSNs; blue: mitral cells; green: subsequent projections. Dp: dorsoposterior
telencephalon; Hb: habenula; HT: hypothalamus; IPN: interpeduncular nucleus; MLR:
mesencephalic locomotor region; OB: olfactory bulb; OE: olfactory epithelium; PT:
posterior tubercle; RST: reticulospinal tract; Vv: ventral nucleus of ventral
telencephalon.

The sensory surface contains the olfactory sensory neurons (OSNs).

OSNs generally express a single olfactory receptor gene (Yoshihara, 2009),
and OSNs with different receptors are broadly intermingled, although their
spatial patterns are not completely random (Korsching, 2001). OSNs send
their axons into an olfactory nerve that terminates in the olfactory bulb,
where they synapse onto mitral cell dendrites. The number of mitral cells is
three orders of magnitude smaller than the number of OSNs (Hara, 1975).
Axons of OSNs expressing the same receptor converge in single glomeruli
in the olfactory bulb (Korsching, 2001). Mitral cells of jawless,
cartilaginous and bony fishes possess several primary dendrites but no basal
dendrites (which first occur in amphibians) (Dryer and Graziadei, 1994).
Mitral cells of jawless and cartilaginous fishes converge these dendrites
onto a single glomerulus, whereas mitral cells of bony fishes may contact
more than one glomerulus (Dryer and Graziadei, 1994). A second type of
projection neuron, the ruffed cells, found only in teleosts, including
zebrafish, obtains input only from granule cells but not from OSNs
(Kermen et al., 2013). Several classes of interneurons provide lateral
inhibition in the bulbar network, most prominently the granule cells.
Reciprocal inhibition between neurons with similar tuning in the olfactory
bulb may serve to decorrelate neuronal representations of similar odors
(Wanner and Friedrich, 2020).
 Odorant responses in the olfactory bulb of fishes generally form a coarse
chemotopic map, such that chemically related odorants are represented in
neighboring areas of the olfactory bulb (Korsching, 2001; Yoshihara, 2009;
Kermen et al., 2013). Amino acids are often represented in a lateral region
of the olfactory bulb, whereas bile acids and steroids are represented in
more medial regions. Such chemotopic maps in the olfactory bulb appear to
be a phylogenetically ancient feature, since a coarse segregation of
responses is already found in the olfactory bulb of a lamprey (Petromyzon
marinus, sea lamprey) (Green et al., 2017).
The olfactory tract contains the axons of the mitral cells and is split in all
vertebrates into a medial and a lateral bundle. The medial tract conveys
pheromone information, whereas the lateral tract carries food odor (Kermen
et al., 2013). The mitral cells innervate two telencephalic and three
diencephalic centers (Figure 14.2; Kermen et al., 2013). Whether the
olfactory telencephalic centers show chemotopic representation similar to
that observed in the olfactory bulb remains controversial (Kermen et al.,
2013). The right habenula appears – via a projection to the interpeduncular
nucleus – to be involved in odor-mediated food-seeking responses; a role in
processing of aversive odors is controversial (Chen et al., 2019).
In sea lamprey, it was recently demonstrated that neurons in the posterior
tubercle, one of the diencephalic target regions of mitral cells, project
directly to the mesencephalic locomotor region, which itself gives input to
reticulospinal neurons, which serve as command neurons for locomotion in
all vertebrates (Daghfous et al., 2016). This olfacto-motor pathway
constitutes the first demonstration of a functional connection between the
olfactory system and the spinal locomotor network in vertebrates.

14.4 FISHES RECOGNIZE AT LEAST SIX DIFFERENT

CLASSES OF ODORS
Fishes use odors to detect food or mating partners, to evade predators and to
mediate social interactions (Figure 14.3). These odors need to be soluble in
water, whereas terrestrial species need odors to be volatile, resulting in a
partially different odor space for fishes compared with that of terrestrial
species. In particular, fish can smell charged molecules such as amino acids,
whereas tetrapods cannot. At least six classes of odorants can be detected
by fishes: amines, amino acids, bile acids, nucleotides, steroids and
prostaglandins; isolated reports of olfactory detection of further substances
exist (Mathuru et al., 2012; Hinz et al., 2013; Olivares and Schmachtenberg,
2019). Individual OSNs detect either pheromones (bile acids, steroids and
prostaglandins), usually with high specificity, or food odors (amines, amino
acids or nucleotides). The latter OSNs often are more broadly tuned and
may detect more than one compound, also from different classes, such as
amino acid and nucleotide (Carassius auratus, goldfish; Sato and Sorensen,
2018).

FIGURE 14.3 Main functions for the sense of smell. Fish use olfaction to (a) enable
mating, (b) flee from predators (alarm reaction), (c) detect food or prey, or (d) avoid
aversive and potentially dangerous odor sources.

Free amino acids result from the breakdown of proteins, and amino acids
have been extensively studied as attractive odorants in diverse fish species
(Hara, 1975, but see (Vitebsky et al., 2005). Zebrafish and catfish
(Ameiurus nebulosus, brown bullhead catfish) are able to distinguish many
amino acids in pairwise comparisons (Miklavc and Valentincic, 2012).
Another important odor class is nucleotides, which also serve to signal the
presence of food. Polyamines are known as fish odors, but more recently,
mono- and diamines have also been recognized as odorants (Michel et al.,
2003; Hussain et al., 2013; Li et al., 2015; Olivares and Schmachtenberg,
2019). Diamines such as cadaverine and putrescine, the bacterial
breakdown products of lysine and arginine, respectively, have been reported
as attractive substances for goldfish but elicit an aversive response in
zebrafish (Hussain et al., 2013).
A second class of odors is constituted by pheromones (Figure 14.3).
These include reproductive hormones that double as odorants to coordinate
male and female reproductive behavior. This is a necessity in those fish
species with external fertilization, where both eggs and sperm may only
stay active for very short time periods, such as in zebrafish. Known
reproductive pheromones include several steroids and prostaglandins
(Stacey et al., 2003; Scott et al., 2010). F-prostaglandins have been shown
to act as post-ovulatory releaser pheromones stimulating male sexual
behavior in Cypriniformes and function as odors in several cyprinid species
but not in other Otomorpha or more distantly related ray-finned fishes
(Kitamura et al., 1994). Bile acids, which are steroids produced in the liver,
constitute another class of odors for many fish species. Two unique bile
acids have been identified as components of a migratory pheromone for sea
lamprey (Li et al., 1995).
Alarm substances that are produced in the skin and signal the injury of
conspecifics constitute another class of pheromones. The chemical nature is
not resolved, but the glycosaminoglycan chondroitin can trigger fear
responses in zebrafish, which partially mimic the reaction to alarm
substance (Mathuru et al., 2012).
Olfactory cues may mediate imprinting to home streams (migratory
species) or kin (swarm fishes). The recognition of home streams after
migration into the open ocean is known to be odor guided for salmonids and
can in fact be influenced by spiking the home river with an artificial
chemical at the sensitive time period (Nevitt et al., 1994). The natural odors
recognized for salmon homing are unknown. Social interactions of swarm
fishes can be at least partially mediated by olfaction, including imprinting
on kin (some reef fishes and zebrafish; Gerlach et al., 2019). The odorants
responsible for imprinting on kin may include peptide ligands for major
histocompatibility complex molecules (MHC) (Hinz et al., 2013), which
would provide a straightforward mechanism to select genetically more
similar individuals, that is, kin (Gerlach et al., 2019). MHC peptides also
seem to underly the odor-mediated mate choice of three-spined sticklebacks
(Gasterosteus aculeatus). Female sticklebacks prefer males with somewhat
dissimilar MHC alleles (Milinski et al., 2005).
Sensitivities for odorants vary by many orders of magnitude between
macrosmatic, normosmic and microsmatic fish species. Within a species,
pheromones generally are detected at much lower concentrations than food
components such as amino acids. Zebrafish, a normosmic species, require
amino acid concentrations above 10−6 M (Friedrich and Korsching, 1998),
whereas sharks detect amino acids down to 10−10 M (Tricas et al., 2009).
Eels, another macrosmatic species, detect several odorants at thresholds of
10−18 to 10−15 M (Teichmann, 1959). A reproductive pheromone of
zebrafish, prostaglandin F2α, can be detected at 10−9 M (Friedrich and
Korsching, 1997), and its receptor has been identified (Yabuki et al., 2016).
A bile acid serving as a reproductive pheromone in sea lamprey can elicit a
behavioral response in female lamprey down to 10−14 M (Johnson et al.,
2009).

14.5 FIVE DISTINCT CLASSES OF OSNS DETECT ODORS

IN FISHES
Odors are perceived by olfactory sensory neurons (OSNs), which are
sensory receptor cells expressing olfactory receptors and at the same time
bona fide neurons that make synapses in the central nervous system to
propagate odor information. The two major populations of OSNs present in
the olfactory epithelium of bony fishes are the same as those that occur in
terrestrial animals, ciliated and microvillous neurons, named for the
properties of their dendritic specializations (Figure 14.4) (Yoshihara, 2009).
Ciliated OSNs possess a dendritic knob, from which a ring of non-motile
cilia emanates that makes contact with the odors of the external world.
Microvillous receptor neurons possess instead much shorter microvilli.
 FIGURE 14.4 Five different populations of OSNs. (a) Ciliated OSN, long slender
dendrite; (b) crypt neuron, globose, with cilia and microvilli; (c) microvillous OSN,
short microvilli; (d) pear and kappe neuron, apical location, with cilia and microvilli,
respectively.

The olfactory signal transduction of ciliated and microvillous receptor

neurons is distinctly different. Ciliated neurons express Golf, a Gs protein,
adenylate cyclase III and cyclic nucleotide-gated channels. These signal
transduction components have been associated with detection of
pheromones and bile acids – in some cases, also amino acids (Sato and
Sorensen, 2018). Microvillous receptor neurons express Go, Gi and in some
species Gq (Hansen et al., 2003; Oka and Korsching, 2011). Microvillous
neurons are expected to detect amino acids and possibly nucleotides
(Lipschitz and Michel, 2002; Koide et al., 2009). Signal transduction for
polyamines may differ from each of these cascades (Michel et al., 2003).
Other molecular markers are TRPC2 for microvillous receptor neurons and
OMP for ciliated neurons – unlike the mammalian situation, OMP only
labels ciliated neurons in fishes (Yoshihara, 2009; Korsching, 2016).
Ciliated and microvillous neurons are intermingled in a shared sensory
surface in both ray-finned (Hansen and Zielinski, 2005) and lobe-finned
fishes (Derivot et al., 1979), in contrast to the situation in mammals, where
(nearly) all microvillous receptor neurons are segregated into a separate
olfactory organ, the vomeronasal epithelium. Interestingly, both lungfishes
and lampreys possess accessory sensory regions situated at the base of the
lamellae of the main olfactory system (Gonzalez et al., 2010; Green et al.,
2017). In African lungfish (Protopterus dolloi), Go antibodies label the
crypts, and Gi antibodies label the main lamellae (Gonzalez et al., 2010),
which would be consistent with a partial segregation of microvillous
neurons into the accessory olfactory subsystem. Thus, the unitary sensory
surface in ray-finned fishes might be a derived feature.
Cilia and microvilli are covered in mucus, which provides mechanical
stabilization and adaptive immunity among other functions (Yu et al.,
2018), similarly to the situation in air-breathing, terrestrial species.
However, the mucus layer is comparatively thinner (4 µm in some
cyprinodonts; Yamamoto, 1982), perhaps reflecting the lack of a
requirement to protect against desiccation.
Interestingly, in sharks and rays, no ciliated receptor neurons have been
found, and only microvillous receptor neurons appear to be present
(Schluessel et al., 2008; Sharma et al., 2019). The earlier-diverging
lampreys seem to possess both cell types, judged by morphology and
position in the olfactory lamella (Laframboise et al., 2007), although an
earlier ultrastructural study had reported ciliated neurons only (Thornhill,
1967). Follow up studies with molecular markers for both cell types may be
required to decide this question. In any case, the presence of ciliated
neurons in lampreys is well supported on the ultrastructural and light
microscopic level, which suggests that the absence of ciliated neurons in
sharks is a derived feature.
Several additional minor OSN populations have been described for
zebrafish – crypt, kappe and pear neurons (Figure 14.4). Kappe and pear
neurons have only been described fairly recently (Ahuja et al., 2014;
Wakisaka et al., 2017), and their occurrence in other fish species has not
been investigated so far. Crypt neurons, on the other hand, have been
identified in many bony fish species based on their peculiar morphology
(Hansen and Zielinski, 2005; Korsching, 2016). Crypt neurons are found in
sharks (Korsching, 2016) and may even be present in lamprey (Laframboise
et al., 2007), suggesting that this cell population is evolutionarily ancient
and may already have been present in the most recent common ancestor
(MRCA) of jawless and jawed fishes. Crypt neurons have not been detected
in lungfishes or tetrapods so far, consistent with an early loss in the lobe-
finned lineage. Crypt neurons have a globular shape and possess both
microvilli and cilia, the latter emerging from the bottom of the eponymous
crypt. Crypt neurons may be involved in kin recognition in zebrafish
(Olivares and Schmachtenberg, 2019). They have been shown to express a
single olfactory receptor from the V1R-related ORA family (Korsching,
2016).

14.6 MOLECULAR BASIS OF ODOR DETECTION:

FAMILIES OF OLFACTORY RECEPTORS IN FISHES
Bony fishes possess four main olfactory receptor families, all of them G
protein-coupled receptors (GPCRs), which are homologous to the
respective mammalian receptor families (Figure 14.5). Odorant receptors
(ORs) belong to the rhodopsin subgroup of GPCRs. They form generally
the largest family, with well over 100 receptors in zebrafish and some
cichlids but many fewer in several other teleosts, such as salmon (Salmo
salar) and neoteleostei (medaka and pufferfishes – Tetraodon nigroviridis
and Takifugu rubripes) (Korsching, 2016). Thus, the fish OR repertoires are
much smaller than that of many mammals, which can reach 1,000–2,000
genes. Nevertheless, the sequence divergence of fish ORs is much larger
than that of mammals. Zebrafish possess six different OR subfamilies, of
which only two have mammalian orthologs (Niimura, 2012). ORs are the
oldest family of olfactory receptors and predate the evolution of vertebrates,
as they are already found in a cephalochordate (Branchiostoma floridae, a
lancelet) (Niimura, 2012). In mammals, ORs are known to have very
diverse odor ligands, but in fishes, no systematic ligand mapping studies
have been performed so far. In sharks and chimaeras, the family is very
small (Venkatesh et al., 2014; Sharma et al., 2019; Marra et al., 2019),
consistent with the absence of ciliated neurons in these species (see earlier)
– ciliated neurons are expected to express OR (and trace amine-associated
receptor [TAAR]) genes (Korsching, 2016).

FIGURE 14.5 Molecular structure of olfactory receptors. (a) OR, TAAR and the
adenosine receptor A2c belong to the rhodopsin family of GPCRs, with short N- and C-
termini. (b) ORA/V1R receptors belong to a rhodopsin-associated family of GPCRs. (c)
V2R-related OlfC receptors belong to GPCR family C, characterized by a large N-
terminal extracellular domain. (d) Membrane-bound guanylate cyclase, the single non-
GPCR olfactory receptor so far reported in fish.

The second largest family of olfactory receptors in many fishes is the

TAARs, with well over 100 genes in zebrafish and again many fewer in
medaka and pufferfishes (Korsching, 2009, 2016). This gene family belongs
to the rhodopsin subgroup of GPCRs as well. Several TAAR genes have
been deorphanized and, like mammalian TAARs, have been found to be
activated by various amines, sometimes with high specificity (Hussain et
al., 2013). TAAR genes show particularly rapid evolution, with frequent
gene birth and death events resulting in a high degree of species specificity
in the TAAR repertoires (Korsching, 2016). TAAR genes also show
positive selection, that is, a selective pressure towards diversification of the
gene repertoire (Korsching, 2016). TAAR families of bony fishes are much
larger than those of tetrapods, which is unusual, since all the other olfactory
receptor families tend to be (much) larger in tetrapods.
Another large receptor family is homologous to tetrapod vomeronasal
receptor type 2 (V2Rs) but in fishes is also called OlfC (Korsching, 2016),
since fishes do not possess a segregated vomeronasal organ and since these
receptors belong to the C family of GPCRs, which is characterized by a
large extracellular N-terminal domain containing the binding domain (ORs
and TAARs only have short N- and C-termini). This family is also different
from the two previous ones in that it appears to require hetero-dimerization
of a unique OlfC with a common co-receptor, termed OlfCc1 in zebrafish
(DeMaria et al., 2013). The co-receptor is highly conserved and in fact is
the only V2R/OlfC gene for which orthologs exist in both the lobe-finned
and the ray-finned lineage (Korsching, 2016). Zebrafish possess 60 OlfC
genes, and some other Ostariophysan fishes have similarly large repertoires,
whereas many neo-teleost fish species tend to have smaller OlfC families
(Korsching, 2016; Yang et al., 2019). A lineage-specific expansion of OlfC
genes in cichlids may contribute to diversification of amino acid detection
(Korsching, 2016).
This family is already well developed in sharks and chimaeras but
appears to be absent in lampreys (Venkatesh et al., 2014; Korsching, 2016;
Sharma et al., 2019; Marra et al., 2019), which makes it the youngest of all
olfactory receptor gene families. OlfC could be a candidate for mediating a
potential MHC peptide detection (Milinski et al., 2005; Hinz et al., 2013) –
the related V2Rs comprise peptide receptors – but their main function in
fishes seems to be as amino acid receptors (Koide et al., 2009; Korsching,
2016). Many behavioral experiments have been performed with amino
acids, either singly or as mixtures, but the results are not easily summarized.
Often, amino acid mixes serve as attractants, including in zebrafish (Koide
et al., 2009), but several experiments show aversion at least to some amino
acids, notably cysteine (Vitebsky et al., 2005). Knockdown experiments for
the common co-receptor in zebrafish showed decreased attraction to amino
acid mixes (DeMaria et al., 2013), consistently with OlfC receptors in
fishes mediating the behavioral response to amino acids. However, there
could also be additional amino acid receptors in the other receptor families,
since ciliated neurons also show responses to amino acids (channel catfish,
Ictalurus punctatus, Hansen et al., 2003; goldfish, Sato and Sorensen,
2018).
In mammals, there is a second large family of vomeronasal receptors,
named V1R. Fishes do possess orthologs of these V1Rs, often named ORAs
– for olfactory receptor class A-related, according to their phylogenetic
position (Nordström et al., 2011). Interestingly, the evolutionary
characteristics of the ORA family are very different in fishes compared with
tetrapods. The family is already present in jawless fishes (Korsching, 2016)
and thus the second-oldest family. In all ray-finned fish species
investigated, it has been found to be rather small, with a canonical set of six
highly conserved genes in teleost species and only occasional gene birth or
death events, very unlike the situation for the three other receptor families
discussed earlier. In several teleost species, strong negative selection on the
sequence of these six genes was observed, but in the rapidly evolving
cichlids from the East African Great Lakes, an atypical positive selective
pressure was observed (Korsching, 2009, 2016). In contrast, the coelacanth
(Latimeria chalumnae), one of the very few remaining lobe-finned fishes,
already possesses 20 V1R genes, including the same six canonical genes as
well as some gene expansions (Korsching, 2016). In other words, lobe-
finned fishes show evolutionary dynamics more similar to those of
tetrapods than to those of ray-finned fishes. When comparing with sharks,
we can conclude that the small, highly conserved family appears to be the
ancestral feature, because sharks possess orthologs for the same six genes
(catshark, Scyliorhinus canicula, Sharma et al., 2019). The genomic
arrangement is also rather peculiar. ORs, TAARs and V2R/OlfCs are
mainly arranged in large clusters, whereas V1R/ORAs show pairwise
arrangement (Korsching, 2009, 2016). These pairs must have originated
from local gene duplications in the MRCA of sharks and bony fishes, since
lampreys exhibit single orthologs. ORA receptors are activated by
pheromones and some bile acids (Korsching, 2016; Cong et al., 2019).
 Two olfactory receptors for zebrafish do not belong to any of these four
families (Figure 14.5): the adenosine receptor A2c, a GPCR of the
rhodopsin subfamily (Wakisaka et al., 2017), and a membrane-bound
guanylate cyclase, gucy2f (Saraiva et al., 2015), whose ligands are not
known as yet. A soluble guanylate cyclase, gucy1b2, is expressed in a
sparse population of OSNs in zebrafish, which is the expected expression
pattern for an olfactory receptor (Saraiva et al., 2015). However, as an
intracellular molecule, gucy1b2 is more likely to be a component of a signal
transduction cascade than a bona fide olfactory receptor. This would be
consistent with an observation that increased sensitivity to an artificial
imprinting odor during homing of salmon is accompanied by an increase in
guanylate cyclase activity (Dittman et al., 1997).

14.7 MODEL FISH SPECIES FOR DEVELOPMENTAL,

BEHAVIORAL AND GENETIC STUDIES
Zebrafish, or Danio rerio (obsolete name Brachydanio rerio), has in recent
decades come to the forefront of genetic vertebrate model organisms
(Holtzman et al., 2016) and in particular for aquaculture fish species (Dahm
and Geisler, 2006). Zebrafish are small subtropical swarm fish that can be
held cost-efficiently and can provide hundreds of progeny every 1–2 weeks.
Their early development is very rapid, and the transparency of the early
developmental stages allows their direct observation under the microscope.
Their reproductive cycle is about 3 months, very similar to that of mouse.
Their genome is sequenced and annotated, and many methods are
established to introduce transgenes and to knock out endogenous genes.
Their olfactory system has been well characterized at the morphological,
molecular and physiological level (Korsching, 2001, 2009, 2016;
Yoshihara, 2009; Kermen et al., 2013; Olivares and Schmachtenberg,
2019).
A second subtropical fish species, medaka or Oryzias latipes, has in
recent years become an alternative to zebrafish as a genetic fish model
system (Kirchmaier et al., 2015). The size of their clutches is much smaller,
and the chorion is very rigid, which makes injection into the fertilized egg
more difficult, but they are very easy to keep and breed, and their
phylogenetic position as much more ‘modern’ fish compared with zebrafish
makes for an interesting evolutionary comparison. Their genome is
sequenced and annotated, and methods to introduce transgenes and
eliminate endogenous genes are well established (Grabher and Wittbrodt,
2008). The olfactory system of medaka appears morphologically less well
developed than that of zebrafish, and the olfactory epithelium presents as a
single patch on the nasal tube (Figure 14.1) (Yamamoto, 1982). Individual
olfactory receptor genes are expressed in sparsely distributed neurons, very
similar to the spatial expression patterns observed in other fish species with
a more developed olfactory epithelium, such as zebrafish (Korsching,
2001). Several comparative genomic studies including medaka olfactory
receptor gene families have been performed (Korsching, 2009, 2016;
Niimura, 2012).
Other fish species serve as more specialized model systems. Three-
spined stickleback (Gasterosteus aculeatus), a neo-teleost, has been
extensively used in behavioral studies, in particular concerning odor-
mediated mating preferences. Female stickleback prefer mating partners
with a moderately divergent repertoire of secreted MHC peptides (Milinski
et al., 2005). Olfactory receptors mediating this response are unknown to
date.
Recently, the African turquoise killifish (Nothobranchius furzeri) has
been established as a vertebrate model system to study aging. This killifish
has an annual lifestyle, and for vertebrates, an extremely short life span of
few months. It is used to study aging and aging-related diseases. The only
olfactory study performed so far shows that killifish mate recognition
depends on olfaction (Austrolebias reicherti, Reyes Blengini et al., 2018).
However, annual killifish would seem to present an excellent model system
to study aging-related functional impairment of the sense of smell. In
humans, such impairment is well known and results in a severely reduced
quality of life and increased health hazards.

14.8 OLFACTION IN RELATION TO SYSTEMATIC

POSITION AND ECOLOGICAL ENVIRONMENT
The relevance of olfaction for particular fish species is not generally
correlated to their systematic position (Betancur et al., 2013), nor is there a
general correlation to the size of the olfactory receptor repertoire.
Macrosmatic fishes are found in several branches of bony fish systematics
(Betancur et al., 2013), for example bichir (Polypterus bichir, an archaic
ray-finned fish), eels (Anguilla), a Gadiformes (Lota) and anglerfishes
(Ceratioidei) (Hara, 1975). Some species with rather limited olfactory
receptor repertoires, such as sharks (catshark, Sharma et al., 2019; white
shark, Carcharodon carcharias, Marra et al., 2019), have, nevertheless, a
keen olfactory sense (Tricas et al., 2009).
It may be expected that the olfactory receptor repertoire will be adapted
to the particular ecological needs of the respective species. In the world’s
deepest-living fish, the Mariana snailfish (Pseudoliparis swirei), many OR
genes have been pseudogenized or completely lost, whereas the TAAR gene
repertoire shows the same size as that of a closely related surface relative
(Jiang et al., 2019). The deep-sea environment is notoriously food scarce,
and amines (TAAR ligands), as bacterial breakdown products of amino
acids, may indicate the presence of food most sensitively under these
circumstances.
Some fish species perform far-ranging migrations between freshwater
habitat and the ocean (eel, salmon and sea lamprey). Sea lampreys imprint
as juveniles on their own pheromones and use this memory as adults to
select previously successful breeding grounds when they come back from
the open sea (Li et al., 1995). It is known that juvenile salmon imprint on
odors of their home river and return to this river (homing) with high
probability. Homing is guided by olfactory cues in the coastal and river
region (Bett and Hinch, 2016). It might be expected that the olfactory
receptor repertoire would require adaptation to the ocean vs. freshwater
habitat. A recent study found some differences in olfactory receptor gene
expression from three different receptor families (OR, OlfC and ORA)
when comparing smolting and nonsmolting salmon (Madsen et al., 2019).
More knowledge about the ligands of such receptors will be required before
their link to a change in habitat can be assessed.
Ostariophysan fishes, the largest group of freshwater fishes (Betancur et
al., 2013), have evolved a fright or alarm reaction that is caused by the
smell of alarm substance. Alarm substance is produced by non-secretory
club cells, which are located in the skin; that is, it can only be released by
mechanical injury (Figure 14.3). The alarm reaction involves freezing
and/or darting. Alarm substance from related species elicits a stronger
response than that from more distantly related species (Pfeiffer, 1966),
suggesting that alarm substances are not common metabolites but somewhat
species-specific compounds.
The chemical nature of the alarm substance has not been determined
despite many efforts, but see Mathuru et al. (2012). Correlation of the OlfC
subfamily size with the presence of the fright reaction could suggest alarm
substances as ligands for OlfC receptors (Yang et al., 2019). Interestingly, in
zebrafish, the alarm response can be suppressed by mating signals,
suggesting dominance of mating over flight behavior, an inverse strategy to
that observed in mammals (Diaz-Verdugo et al., 2019).

ACKNOWLEDGEMENT
I am very grateful to Kai R. Korsching for creating the artwork for Figures
14.1 through 14.3.

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 15 Aquaculture and Fisheries
Hsin-Yiu Chou and Guan-Chung Wu

CONTENTS
15.1 General Introduction
15.2 World Fisheries and Aquaculture Status
15.2.1 Global Fisheries Development
15.2.2 History of Aquaculture
15.2.3 The Current State of World Aquaculture
15.3 Fish in Culture Environment
15.3.1 Food Digestion
15.3.2 Energetic Transformation
15.3.3 Temperature Tolerance and Oxygen Demand
15.3.4 Osmoregulation and Salinity Tolerance
15.3.5 Carbon Dioxide and Alkalinity in Pond
15.4 Sustainable Fishery and Aquaculture
15.4.1 Co-Management: A Key to Sustainable Future Fisheries
15.4.2 Sustainable Aquaculture
15.4.3 Threats to Sustainability and the Application of Biotechnology
15.5 Conclusion
Acknowledgements
References

15.1 GENERAL INTRODUCTION

The earth’s oceans are rich in biological resources, and they have always been
regarded as the “second granary” of mankind. In addition to their provision of
everyday consumption for people, approximately one billion humans rely on marine
species as the primary or sole source of protein. However, as time passes, damaged
marine ecology, over-exploited natural resources, anthropogenic pressures, and
irreversible climate change have caused the gradual exhaustion of the world’s fishery
resources (Free et al., 2019). At this point in time, aquaculture has become one of the
most promising avenues for providing fish production. Aquaculture, both
technologically and in the rate of production, has been continuously upgraded, has
become an important source of high-quality protein and nutritious food for humans,
and will continue to provide food in the future.
Reay (1979) defined aquaculture as “Man's attempt, through inputs of labor and
energy, to improve the yield of useful aquatic organisms by deliberate manipulation of
their rates of growth, mortality and reproduction” based on a biological perspective
(Beveridge and Little, 2002). In 1988, adding the concept of ownership or the
extension of access and exploitation rights, the revised definition of aquaculture used
by the UN Food and Agricultural Organization (FAO) stated:
Aquaculture is the farming of aquatic organisms, including fish, mollusks, crustaceans
and aquatic plants. Farming implies some form of intervention in the rearing process
to enhance production, such as regular stocking, feeding, protection from predators,
etc. Farming also implies individual or corporate ownership of the stock being
cultivated. For statistical purposes, aquatic organisms which are harvested by an
individual or corporate body which has owned them throughout their rearing period
contribute to aquaculture, while aquatic organisms which are exploitable by the public
as a common property resources, with or without appropriate licenses, are the harvest
of fisheries.
During the past half century, aquaculture has become one of the most rapidly growing
food sectors globally, which is in sharp contrast with stagnant capture fisheries
production. The global use of fishmeal represents a shift from poultry farming to
aquaculture. In this context, aquaculture is essential for reducing the demand for
fishmeal and the oil from caught fishes. Thus, establishing optimal environments and
appropriate food for farming fish is an important issue. In aquaculture, the general goal
is to maximize the rate of growth and minimize the cost of feed. Achieving this goal
requires the manipulation of environmental factors such as temperature, dissolved
oxygen level, salinity, and pH in aquaculture systems.
According to the FAO (2018), since the 1970s, global aquaculture production has
maintained an average annual growth rate of 8.8%, which is significantly higher than
the 1.2% growth rate of the capture fisheries. In 2012, farmed fish reached 66.6
million tonnes, providing approximately 46% of the food fish for humans. By 2030,
aquaculture production is expected to outpace capture fisheries, accounting for 62% of
the world’s edible fish supply (World Bank, 2013). The aquaculture sector is probably
the fastest-growing food-producing sector of the century. Especially as marine
resources begin to deplete and market demand for aquatic products continues to rise,
aquaculture is recognized as an important industry for supplementing the shortage of
capture fisheries. The sustainable aquaculture industry will make a lasting contribution
to global food security and economic growth. The World Bank defines sustainable
aquaculture as “a dynamic concept and the sustainability of an aquaculture system will
vary with species, location, societal norms and the state of knowledge and technology”
(2014). Moreover, it must have environmental, economic, social, and community
sustainability. In this chapter, we will focus on three aspects of aquaculture: 1) the
status of world fisheries and aquaculture; 2) the fish in aquaculture environments; and
3) sustainable fisheries and aquaculture.

15.2 WORLD FISHERIES AND AQUACULTURE STATUS

The Food and Agriculture Organization of the United Nations published “The State of
World Fisheries and Aquaculture 2018” in Rome. The report is cautiously optimistic
about the development of global fish production. Even so, wild fishing tonnage has
been relatively stable since 1990 or has even decreased. Aquaculture production
continues to grow and had an annual growth rate of 3.3% to 7.4% from 2011 to 2016
(FAO, 2018) (Figure 15.1). The combined production from marine captures and
aquaculture will continue to increase by the next decade, increasing by approximately
18% to reach 201 million tonnes by 2030.

FIGURE 15.1 World fisheries and aquaculture production in millions of tonnes for the period of
2011–2016, as reported by the FAO (2018).

15.2.1 GLOBAL FISHERIES DEVELOPMENT

Global fish production peaked at approximately 171 million tonnes in 2016, with
aquaculture representing 47% of the total (FAO, 2018). However, marine captures
have been stagnant since the late 1980s, especially in developed countries/regions,
such as the EU, the United States, and Japan. On the other hand, fisheries production
by developing countries, including emerging Asian countries, such as Indonesia and
Vietnam, continues to increase, and China accounts for 17% of the world’s production
at 15.58 million tonnes (2005–2014; average until 2016).
By type of fish, herring and sardines make up the largest share of the global catch at
16.62 million tonnes, accounting for 18% of the total. However, for abundant fish with
high fat content, the stock levels repeatedly fluctuate due to environmental changes.
The catch of thin sardines is also fluctuating. The catch of cod species has been
decreasing since the late 1980s. The overall catch trend has been increasing since the
late 2000s. Tuna, skipjack, swordfish, and shrimp catches have been increasing over
time (www.fao.org/fishery/statistics/global-capture-production/en).
FAO summarized the status of global marine fisheries’ resources based on the
results of resource assessments around the world. In 1974, 90% of fisheries’ resources
were at or below the appropriate level. Nevertheless, in 2015, the percentage dropped
to 67%, and the proportion of overfishing rose from 10% to 33%. Therefore, the
United Nations Convention on the Law of the Sea stipulates that coastal countries and
the countries that catch highly migratory fish species must cooperate with regional
fisheries management organizations within and outside the EEZ (exclusive economic
zone) to conserve and use marine resources. Regional fisheries management
organizations discuss effective resource management measures, such as catch
regulations, catch effort regulations, and technical regulations after examining the
level of compliance through stock assessments and stock management measures.
15.2.2 HISTORY OF AQUACULTURE
Aquaculture is an ancient industry. According to the historical data of cultural relics,
the practice of raising fish in ponds can be traced back to more than 3000 years ago in
China (eleventh century BC, Late Yin Dynasty). “The Book of Fish Farming” is the
earliest aquaculture literature in history, which was written by Fan Li, a famous
politician, strategist, and economist in the Spring and Autumn (770–476 BC) period
and the Warring States (475–221 BC) periods (Liao and Chao, 2009). In China, black
carp (Mylopharyngodon piceus), grass carp (Ctenopharyngodon idellus), bighead carp
(Aristichthys nobilis), and silver carp (Hypophthalmichthys molitrix) are the most
familiar food fishes for the Chinese and are mainly cultivated in freshwater
aquaculture.
Western aquaculture activities originated in Europe; the word “aquaculture”
originated from the Latin for “water-culture”. In ancient Rome, moray eels
(Muraenidae) and eels (Anguilliformes) were in high demand. Until 500 BCE, the
Romans farmed oysters and carp in Mediterranean lagoons. By the fifteenth and
sixteenth centuries, the cultivation of common carp (Cyprinus carpio) was very
common throughout southern Europe (Rabanal, 1988). Marine aquaculture started in
France when oyster farming began in the 1860s, but progressed slowly until 1960.
Norway began to farm salmon in 1982, and the production of farmed salmon and trout
rose from only 500 tonnes in 1971 to approximately 15,500 tonnes in1983 (Ford,
1984). During the nineteenth century, which was an era of rapid industrialization,
pollution caused the fish populations to diminish; industrial construction (dams and
irrigation canals) obstructed the migratory paths of some species, such as salmon. To
combat these dramatic population declines, artificial breeding research began in
Germany. Researchers managed to master all stages of the process, from fertilization
to egg storage and transportation, pond farming, and releasing fish into the wild.
Nordic countries have been farming salmon since the last century; at the same time,
the aquaculture industry has made tremendous progress through recent years. The total
revenue has increased by nearly 300% over the past 10 years and by approximately
18.5% in 2016. In particular, farmed salmon has become the main export commodity
of Norway, accounting for nearly 50% of the salmon in the international market (EY,
2017).
15.2.3 THE CURRENT STATE OF WORLD AQUACULTURE
Global aquaculture has grown dramatically over the past 50 years and reached a
production of approximately 110.2 million tonnes (including aquatic plants) in 2016,
which was worth US$243.5 billion and accounted for approximately 46% of the world
fish food supply in 2016 and is projected to reach 54% in 2030, or 21 times the
production in the 1970s (FAO, 2018). Asia is the home of aquaculture. Under the
influence of political, social, economic, technological, and cultural factors, there has
been rapid growth of the aquaculture sector in Asia (Figure 15.2). The current global
aquaculture production is still mainly in Asia, and this region dominates fish
production with 89.4% of the market by volume and approximately 79% by value.
China is by far the largest producer (64.36 million tonnes in 2017), and the second
largest is Indonesia, which contributed 15.896 million tonnes of aquaculture
production in 2017 (www.fao.org/figis/servlet/TabSelector).
 FIGURE 15.2 Global annual aquaculture productions in 2016 by continent and the ratio of main food
fish species.

In Europe (European Economic Area, EEA39), marine aquaculture production has

increased since the early 1990s, mostly due to Atlantic salmon (Salmo salar)
production in Norway. Other farmed species include rainbow trout (Oncorhynchus
mykiss), European sea bass (Dicentrarchus labrax), gilthead sea bream (Sparus
aurata), oysters (ostreidae), and carp, barbels, and other cyprinids. Norway
(approximately 46% of total European production), Spain, Turkey, the United
Kingdom, France, Italy, and Greece account for 90% of all aquaculture production in
Europe (FAO, 2018).
Aquaculture production in North America grew rapidly between 1988 and 1997,
increasing from 379,000 to 521,000 tonnes, which represented a 38% increase at an
APR (average percentage rate) of 4.0 but has stagnated since then. In 2008, the
production was 644,213 tonnes, which was valued at US$1.6 billion. Among the
countries that produce salmon, Canada is the fourth-largest producer of farmed salmon
in the world, and salmon represents an average of 70% of the total volume of Canadian
aquaculture (Olin, 2001).
Looking at aquaculture production by country, the increase in China and Indonesia
is remarkable. As of 2018, the annual aquaculture production in China was estimated
at 63.7 million tonnes, which was 58% of the global total; Indonesia produced 16.6
million tonnes, accounting for 14% of the global total. By fish species, carps, barbels,
and other cyprinids are the most common produced, at 28.35 million tonnes,
accounting for 25% of the total (www.fao.org/fishery/statistics/global-aquaculture-
production/en). In recent years, the increase in these species has been remarkable.
According to ASFIS (Aquatic Sciences and Fisheries Information System), the 10
most produced species in world aquaculture in 2017 were the following: three species
of seaweeds (Japanese kelp [#1], Eucheuma seaweeds nei (not elsewhere included)
[#2], and Gracilaria seaweeds [#7]); four freshwater fishes (grass carp [#3], silver carp
[#5], Nile tilapia [#9], and common carp [#10]); two mollusks (cupped oysters nei [#4]
and Japanese carpet shell [#8]); and one crustacean (whiteleg shrimp [#6]). The major
groups of finfish species (excluding aquatic plants) in world aquaculture production
from 2010 to 2016 are shown in Figure 15.3.

FIGURE 15.3 Major finfish species groups (excluding aquatic plants) in world aquaculture
production, 2010–2016.

15.3 FISH IN CULTURE ENVIRONMENT

Commercial aquaculture systems are highly intensive farming environments and
include water-based systems (cages and pens), land-based systems (ponds, tanks, and
raceways), and recycling systems. Unlike wild fishes, which must find an appropriate
environment and food for themselves, farmed fishes must adapt to the artificial
conditions of the farming environment and food supply. Thus, the establishment of an
optimal environment and appropriate feed for farmed fish is an important issue.
15.3.1 FOOD DIGESTION
Digestion is the process by which ingested foods are reduced to small size or into other
appropriate molecules for absorption (see Chapter 6). Indigestible chromium (III)
oxide (Cr2O3) is a commonly used external indicator for fish digestibility trials. The
digestibility of fish feed is determined by measuring the proportion of the caloric value
in the ingested feed that remains in the feces and assessing the replacement rate of
Cr2O3 in the feed (Chabot et al., 2016; Olsen and Ringø, 1997), which is calculated as
follows:

The digestibility of different foods is related to feeding habits (Olsen and Ringø,
1997). In general, the digestibility of most natural proteins reaches 80% in plant
protein and 90% in animal protein. The digestibility of most natural lipids may reach
90%. However, the digestibility ranges of different carbohydrates vary: glucose is
almost 100% digestible, while raw starch and plant fibers are less than 10%.
Furthermore, herbivorous and omnivorous fishes have greater ability to digest
carbohydrates than carnivorous fishes. Therefore, the optimal formula of feed is an
important factor for the digestibility for the fish and could enhance the digestibility
rate and reduce the amount of gases produced. Furthermore, marine fish and salmonids
require high levels of omega-3 essential fatty acids, eicosapentaenoic acids (EPA,
20:5ω3), and docosahexaenoic acids (DHA, 22:6ω3) (Naylor et al., 2000). However,
fish do not produce omega-3 fatty acids, so they get them either by consuming the
microalgae that produce these fatty acids or by eating prey fish that have accumulated
omega-3 fatty acids from the microalgae. Farming carnivorous species, such as
Atlantic salmon (S. salar), European sea bass (D. labrax), gilthead sea bream (S.
aurata), Atlantic flounder (Paralichthys dentatus), and grouper (Epinephelinae sp.),
leads to a high demand for coarse prey fish to match the nutrition that the predators get
in the wild (Naylor et al., 2000). To satisfy this requirement, most fish that are used for
fish oil production around the world are fed farmed fish. However, minimizing the use
of fish oil (from coarse fish) is important because it is one of the most expensive
components of feed (Naylor et al., 2000; 2009). Therefore, reducing the consumption
of fish oil in feed and shifting to plant-based feed is an important step. However, one
problem that still limits full replacement of fish oils with plant oils is the low level of
omega-3 fatty acids in these oils.
15.3.2 ENERGETIC TRANSFORMATION
To increase the body weight of farmed fishes, a positive energy balance must be
created, such that input of food energy is higher than the energy output for the standard
metabolic rate (SMR), activity energy, and diet-induced thermogenesis (DIT, also
called specific dynamic action, SDA); this balance is calculated as follows:

The SMR is not a constant with species; it can vary between individuals, life stages,
and sexes (see Chapter 10). The aerobic scope is an estimation of the metabolic energy
available for activity in a given environmental and physiological context, which is
calculated as the difference between the standard and maximal metabolic rates (SMR
and MMR). The SMR corresponds to the minimum metabolic rate required to sustain
life, and the MMR is the maximum rate of oxygen consumption. Activity energy is the
cost per unit time of neuromuscular efforts that exceed the SMR. SDA is an increase in
the metabolic rate above the SMR, which occurs as a consequence of food intake.
In aquaculture, the general goal is to maximize the rate of growth (Eproduction) while
minimizing the allocation of energy to other functions. Achieving this goal requires
the manipulation of environmental factors, such as temperature, dissolved oxygen
levels, salinity, and pH, in an aquaculture system.
15.3.3 TEMPERATURE TOLERANCE AND OXYGEN DEMAND
The physiological condition of fishes is strongly affected by temperature (see Chapter
7). Because fishes are ectothermic (poikilothermic), they maintain a body temperature
that is close to that of the environment. However, the temperature of water in most
aquaculture systems cannot be controlled and depends upon natural conditions. Based
on the seasonal changes in the natural habitats of different fish species, they have an
optimal range of water temperature for growth. Temperatures outside the optimal
range will result in slow growth or no growth and will even result in death at high or
low extremes. Thus, aquaculture operations must be carried out at appropriate
temperatures.
Increased temperatures reduce the oxygen levels in the water. Dissolved oxygen
availability strongly influences the MMR; as a result, hypoxia can reduce aerobic
scope independently of temperature. By limiting the availability of environmental
oxygen to fishes, hypoxia makes it more challenging to meet the increased metabolic
demands driven by higher temperature. In addition, fish body size is another factor that
is likely to affect oxygen-limited thermal tolerance. Small fish use more oxygen than
large fish per gram of body weight, but oxygen demand in large fishes is higher than in
small fishes. Therefore, thermal sensitivity is potentially higher in large fish, and the
optimal temperature for growth and the temperature preferences have been shown to
decline with body size (Pörtner and Peck, 2010). Furthermore, oxygen consumption by
microorganisms and microalgae may dramatically increase when the nitrogenous
waste level is high (Pörtner and Peck, 2010). Taken together, culturists need to monitor
dissolved oxygen levels and to supply enough oxygen in the aquaculture systems to
prevent hypoxia.
15.3.4 OSMOREGULATION AND SALINITY TOLERANCE
In anadromous (fish that are born in freshwater and then migrate to saltwater to grow)
and catadromous fish (fish that are born in saltwater and then migrate to freshwater to
grow), determining the timing of migration is an important event for aquaculture
manipulation. For anadromous salmonids such as the Atlantic salmon, individuals
grow faster in seawater than in freshwater (McCormick, 2012). However, the
migratory period of juvenile salmon from freshwater to saltwater has a critical period.
Early migration, or migration that misses this critical window, will cause high levels of
mortality. Furthermore, some saltwater fish, such as black porgy (Acanthopagrus
schlegelii) and Asian sea bass (Lates calcarifer), grow faster in brackish water than in
saltwater (Boeuf and Payan, 2001). These higher growth rates have been shown to
correlate with a lower SMR when the environmental salinity is similar to plasma
osmolality. Therefore, the manipulation of salinity can help culturists to increase
aquaculture production. In addition, such manipulation is used to treat ectoparasitic
infections (Diggles et al., 1993; Mueller et al., 1992). Copper sulfate (CuSO4) is one of
the most effective chemicals for treating protozoans and monogenoideans (Durborow
et al., 1998). However, at high concentrations, CuSO4 can damage the fish gill tissues.
It has been shown that the practice of salinity can replace chemical reagents to treat
monogenoideans.
15.3.5 CARBON DIOXIDE AND ALKALINITY IN POND
Carbon dioxide and pH concentrations may fluctuate daily in pond systems due to
varying respiration and photosynthesis levels (Wurt and Durborow, 1992). After
sunset, the dissolved oxygen concentration declines faster in microalgae-rich ponds, as
microalgae and fish consume oxygen for respiration. However, carbon dioxide rarely
causes direct toxicity in fish. In contrast, the high alkalinity increases the pH and
results in ammonia formation, while the less toxic form, ammonium, forms in acidic
water in freshwater ponds. In general, a freshwater pond should have a pH between 6.5
and 9 as well as moderate to high total alkalinity (between 75 and 200 mg per liter but
not lower than 20 mg per liter) (Wurt and Durborow, 1992).
Because of the buffering chemicals present in saltwater, fluctuations of pH levels in
freshwater ponds are higher than in saltwater ponds. Thus, unlike freshwater fishes,
saltwater fishes are more sensitive to acidic seawater. In general, compared with
heavily calcified organisms, such as corals and mollusks, saltwater fishes are less
sensitive to ocean acidification (Kroeker et al., 2013). However, acidification produces
neurological effects in saltwater fishes and has repercussions on their behavior, but
effects on fish growth have not been detected (Bamber et al., 2018; Kroeker et al.,
2013).

15.4 SUSTAINABLE FISHERY AND AQUACULTURE

As global demand for aquatic products continues to increase, sustainable resources and
aquaculture have become important issues in recent years (Little et al., 2016).
However, several challenges face future global fisheries development, including (1)
overfishing, (2) bycatch, (3) global warming, (4) ocean acidification, (5) marine
pollution (e.g. ship oil spills, marine debris, and plastic particles), (6) illegal fishing,
and (7) waste of resources (discard, feed, and culture fishery), and there is a limit to
the amount of fisheries resources. For this reason, aiming for the sustainable
development of the aquaculture industry, along with the appropriate management and
conservation of marine resources, is a particularly important issue in securing the
supply of fishery products (Kearns, 2015).
15.4.1 CO-MANAGEMENT: A KEY TO SUSTAINABLE FUTURE FISHERIES
Co-management refers to a system of resource management in which the government
and local fishermen share responsibility for managing fisheries resources and
formulate operational regulations through discussions. This process has the following
advantages: (1) the fishers’ sense of responsibility for resource management is
improved, and (2) the operational order is improved through mutual monitoring
between fishers. The effectiveness of this method has attracted attention in recent
years. According to Professor Ray Hilborn of the School of Aquatic and Fishery
Sciences, University of Washington, and his doctoral student Nicolas Gutiérrez, “the
majority of the world's fisheries are not – and never will be – managed by strong
centralized governments with top-down rules and the means to enforce them”.
Gutiérrez et al. (2011) published their results of an analysis of 130 co-management
fisheries in 44 countries around the world. They indicated that the success of resource
management is greatly influenced by the presence of regional leaders. The presence of
social solidarity and joint management can be an effective solution to problems in
fisheries around the world.
15.4.2 SUSTAINABLE AQUACULTURE
Aquaculture development plays an important role in food supply, as it has the
following advantages over the production of terrestrial animals, such as cattle, pigs.
and poultry:
(1) The feed efficiency of fish is better than that of other animals, and the highest protein conversion rate can be
obtained under the same feed conditions.
(2) Generally, fish have a higher net meat percentage ([total meat/carcass weight] × 100).
(3) The eutrophication potential of fish production is low.
(4) Fish farming poses a relatively low risk for contributing to global warming (carbon dioxide equivalent).
(5) Less land and water are needed for aquaculture.
(6) Many aquatic products have high nutritional value (for example, they contain certain omega-3 fatty acids).

However, with the development of global aquaculture, there have also been arguments
against it. Pérez et al. (2000) propose the concept “aquaculture: part of the problem,
not a solution”. In response to widespread complaints about the practice, in 2010, the
World Wide Fund for Nature (WWF) and the Dutch Sustainable Trade Initiative (IDH)
established the Aquaculture Stewardship Council (ASC) to promote responsible
aquaculture. The ASC has developed a stringent certification system for sustainable
and responsible aquaculture and has provided labeling schemes for producers. It
guarantees to consumers that the seafood they are purchasing is environmentally
sustainable and socially responsible.
The concept of “sustainability” is increasingly recognized to incorporate aspects of
environmental, economic, and social parameters. Practitioners of sustainable
aquaculture have found that adhering to this concept not only maximizes benefits but
also minimizes damage to the natural environment and negative impact on society.
Valenti et al. (2018) developed a series of quantitative indicators for the economy, the
environment, and society to assess the sustainability of different aquaculture systems.
These indicators were defined from 2003 to 2016 and combine top-down and bottom-
up approaches as well as practical observations from experimental and commercial
aquaculture facilities. A total of 56 indicators were proposed, including 14 for the
economy, 22 for the environment, and 20 for society. These indicators can be used to
evaluate production systems and to compare different experimental treatments in
research experiments. Simultaneously, they allow diagnostics to be performed,
strengths and weaknesses to be identified, goals to be set and actions to be determined,
and the effectiveness of actions and public policies to be assessed. Therefore, this set
of indicators can be used by certifying organizations, investors, and policy makers and
can provide fair assessment criteria for monitoring all aspects of sustainable
aquaculture (Valenti et al., 2018).
15.4.3 THREATS TO SUSTAINABILITY AND THE APPLICATION OF
BIOTECHNOLOGY
The world’s oceans not only provide sustainable natural resources for human beings
but also possess the capability to conserve heat energy and carbon dioxide, thus
effectively regulating the climate. However, as humans continue to release carbon
dioxide into the atmosphere, global warming has become an immediate result. As a
result, detrimental changes in the circulation of oceanic currents, including changes to
the marine ecosystem and CO2 cycling, have occurred and further aggravate the
extreme effects of climate change, such as ocean warming, sea level rise, ocean
acidification, the decline in CO2 absorption, overexploitation of fisheries’ resources,
forced migration of marine species facing extinction (Nagelkerken et al., 2015; 2016),
and the increased frequency of strong typhoons and heavy rainstorms. All these events
have seriously threatened the development and continuous existence of human beings
and the aquaculture industry.
In addition to extreme weather/environmental impacts, most current aquaculture
problems result from attempts to stretch a balanced ecosystem too far in the interests
of higher production or higher profits. Therefore, sustainable aquaculture can only be
achieved if the following challenges are addressed: improving water quality, nutrition,
and health and genetics; domesticating fish; increasing growth rates, productivity, and
disease resistance; and expanding ecological ranges. Biotechnology is the use of
biological processing methods to solve problems, which can help make aquaculture
sustainable. By using knowledge produced by nutrition and food science about the
physiology of nourishment and the production of suitable foods, biotechnology will
reduce the current cost of aquafarming feed, improve feed conversion ratios, and
provide adequate substitutes for protein ingredients. Sanitation in intensive aquaculture
systems will also be improved. The development of diagnostic kits for identifying
pathogens and the development of genomic vaccines and probiotics will be other
achievements of biotechnology. Genetic engineering has yielded transgenic organisms
with improved capabilities, such as fast-growing and cold-resistant varieties. The
application of biotechnology will revitalize aquaculture by improving the performance
of the industry.

15.5 CONCLUSION
Over the past decade, the biological and chemical diversity found in the marine
environment has made the oceans a sustainable source of healthy foods, desalinated
fresh water, marine renewable energy, and the development of new drugs. The green
economy driven by algae technology, with a wide range of applications including food
supplements, feed, and bioenergy and with environmental pharmacological effects, is
one of the emerging industries with great development potential (Manirafasha et al.,
2019). During the same period, the application of biotechnology in aquaculture has
promoted the industry from traditional farming to among the most competitive
emerging industries. Moreover, an increasing number of genetically improved
aquaculture species are now commercially available throughout the world. In 2015, the
U.S. Food and Drug Administration (FDA) approved the AquaAdvantage salmon, a
rapidly growing Atlantic salmon developed by inserting a transgene constructed from
the growth hormone gene of a related species, as a human food. In the future, this
technology could be used to produce genetically modified food fish with specific
beneficial characteristics, such as strong disease resistance, rapid growth of somatic
cells, increased muscle mass, and increased unsaturated and essential fatty acids. If the
public can acknowledge and understand the safety and progress of transgenic
technology by accepting inexpensive and high-quality genetically modified fish as
food, this will be the key to solving the food crisis for humanity in the near future.

ACKNOWLEDGEMENTS
The authors gratefully thank the editors for their constructive comments. We are
indebted to Professor Ching-Fong Chang, President, National Taiwan Ocean
University and Professor Pung-Pung Hwang, Distinguished Research Fellow, Institute
of Cellular and Organismic Biology, Academia Sinica for their valuable suggestions of
the manuscript. And we would also like to thank Miss Sz-Ching Wu, Miss Tzu-Chiao
Chou, and Miss Yu-Ting Tseng for their help in collecting information.

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 16 Epigenetics
Paul Craig

CONTENTS
16.1 Introduction
16.2 Epigenetic Mechanisms
16.2.1 DNA Methylation
16.2.2 Histone Modification
16.2.2.1 Histone Methylation
16.2.2.2 Histone Acetylation
16.2.2.3 Histone Phosphorylation
16.2.3 Non-Coding RNA
16.3 Abiotic and Biotic Effects on Epigenetics
16.3.1 Temperature
16.3.2 Hypoxia
16.3.3 Metabolism
16.3.4 Toxicological Effects
16.4 Conclusion and Future Directions
References

16.1 INTRODUCTION
The term ‘epigenetics’ was first coined by Conrad H. Waddington, a British
developmental biologist and geneticist, in 1942 (Waddington 1942).
Waddington explained the complex of developmental processes that link the
genotype to the phenotype and described the mechanism of these processes
as the ‘epigenotype’ (Waddington 1942). Since that time, the definition of
epigenetics has controversially changed due to the initial implication of
epigenetic support for Lamarckian inheritance; the now widely accepted
definition is: ‘an epigenetic trait is a stably heritable phenotype resulting
from changes in a chromosome without alterations in the DNA sequence’
(Berger et al. 2009). While this definition implies heritability, this term has
also been used to define the genetic machinery that responds to
environmental perturbations, which drives phenotypic plasticity within a
given cell line, tissue, or whole organism. In essence, epigenetics examines
how the environment interacts and affects phenotypic traits without altering
the underlying genetic code. The mechanisms driving epigenetic change
that have been studied most extensively in the fish literature are changes in
DNA methylation, histone modification, and the effects of non-coding
RNA. While the majority of work studying the mechanisms and functional
impacts of altering epigenetic machinery in the context of environmental
influence and pathologies (i.e. cancer, cardiac infarction) is focused in
model mammalian and plant literature, non-model species have become
prominent in the field of epigenetic study, particularly in the field of fish
physiology (Best et al. 2018). The aim of this chapter is to provide an
overview of epigenetic machinery present in fishes and provide examples of
how epigenetics can have functional consequences under a variety of biotic
and abiotic stimuli through both laboratory and naturally invoked
experimental approaches.

16.2 EPIGENETIC MECHANISMS

The following focuses on the best-studied areas of epigenetic mechanisms,
which include DNA methylation, post-translational modification of
histones, and non-coding RNA (Figure 16.1). It should be noted that these
mechanisms are not entirely mutually exclusive, and there is interplay that
exists between these mechanisms, especially with microRNA (miRNA),
which can target the post-transcriptional regulation of key chromatin- and
DNA-modifying enzymes (Kuc et al. 2017).
 FIGURE 16.1 Schematic of the major epigenetic marks that regulate both gene
expression and translation. For DNA methylation, methyl groups can be added by DNA
methyltransferases (DNMT1 and DNMT3) and removed by the ten-eleven translocation
(TET) methylcytosine dioxygenase family of enzymes. Histones can be modified in
several ways, including methylation (histone methyltransferases [KMT]; lysine-specific
demethylases [HLSD]), acetylation (lysine acetyltransferases [KAT]; lysine histone
deacetyltransferases [HDAT]), and phosphorylation. Translation repression via
microRNA occurs when pre-miRNA translocated from the nucleus is cleaved by the
Dicer enzyme, which forms the mature miRNA. This miRNA then combines with
RNA-induced silencing complex (RISC) proteins, which can bind to the 3′-untranslated
region (UTR) of an mRNA transcript and either inhibit ribosomal binding or target the
mRNA for degradation.

16.2.1 DNA METHYLATION

DNA methylation was one of the earliest forms of epigenetic marks to be
discovered (Holliday and Pugh 1975; Riggs 1975). DNA methylation
involves the enzymatic addition of a methyl group (CH3) onto a cytosine
residue located in cytosine-phosphate-guanine (CpG) nucleotides and
results in the complementary methylation of both DNA strands (Ramsahoye
et al. 2000). Methylation patterning plays a significant role in the structure
and function of chromatin, and when the promoter region of a gene
becomes heavily methylated, there is repression of transcription (Li and
Zhang 2014). Methylation is not solely confined to the promoter region and
can occur in a variety of contexts outside CpG islands (Ambrosi et al.
2017). However, the function of this type of methylation is not well
understood in fishes. In general, DNA methylation is found in numerous
eukaryotic genomes (Zemach et al. 2010; Suzuki and Bird 2008) and is
associated with long-term transcriptional repression that can be propagated
through mitotic division, suggesting a potential memory mechanism for
transcriptional regulation (Holliday and Pugh 1975; Riggs 1975; Kass et al.
1997). The enzymes that regulate methylation are well conserved through
the vertebrate lineage (Kuc et al. 2017; Best et al. 2018) and are known as
DNA methyltransferases (DNMTs); these can be subdivided into two
classes. The first is DNMT1, principally involved in maintenance and
memory of methylation, as it is located in the replication fork and copies
methylation patterns to the newly synthesized strand, maintaining a
methylation memory in mitotically dividing cells (Hermann et al. 2004).
The second is DNMT3, also known as the de novo DNMT, as its function is
linked to establishing new methylation marks. This enzyme plays a
significant role in regulating environmentally driven promoter region
methylation and gene silencing (Okano et al. 1999), since the methyl group
can act as a physical barrier to transcription factor binding (Bell and
Felsenfeld 2000). While methylation patterning can persist through somatic
cell division, meiotic and transgenerational transmission of DNA
methylation patterns has not been identified in the mammalian literature, as
there is complete erasure of epigenetic marks upon embryogenesis (Van
Otterdijk and Michels 2016). This erasure period during early
embryogenesis has also been identified in zebrafish (Fang et al. 2013) and
further represents a highly critical window of sensitivity to environmental
stressors (Dorts et al. 2016). In the self-fertilizing hermaphroditic mangrove
rivulus (Kryptolebias marmoratus), Fellous et al. (2019a) provided
convincing evidence that the demethylation period occurs later, with a
higher degree of demethylation, and persists for a longer period of time
when compared with zebrafish. This suggests that the unique reproductive
nature of mangrove rivulus provides an excellent model for the study of
epigenetic changes related to environmental perturbations. Methylated
DNA can also be demethylated through two different processes. One is
through passive demethylation, where there is a dilution of methylation
signals on DNA strands that do not receive methylation marks during
mitotic division (Inoue and Zhang 2011). The other is via active DNA
demethylation, mediated via 5-methylcytosine (5mC) oxidation catalysed
by members of the ten-eleven translocation (TET) methylcytosine
dioxygenase family (Wu and Zhang 2017). These demethylation pathways
have been identified in zebrafish and probably exist in other fish species
(Best et al. 2018).
16.2.2 HISTONE MODIFICATION
The covalent modification of histone protein tails plays a crucial role in
chromatin structure and function. In general terms, these covalent
modifications alter the structural interactions between positively charged
histones and negatively charged DNA, resulting in either compacted
heterochromatin or a relaxed euchromatin, which either suppresses or
promotes transcription, respectively (see later and Allis and Jenuwein
2016). Several enzymatic mechanisms, which can be generalized as
‘writers’ and ‘erasers’, exist that can add or remove covalent modifications
(methylation, acetylation, and phosphorylation) to the N-terminus of
histones (Allis and Jenuwein 2016). Recent studies have demonstrated that
these mechanisms are conserved and found within at least two teleost
species, namely zebrafish (Kim et al. 2014, 2015) and the mangrove rivulus
(Fellous et al. 2019b).

16.2.2.1 Histone Methylation

While DNA methylation can play a critical role in the expression of genes,
methylation can also act as a regulatory mechanism associated with histone
proteins, where methylation and demethylation of histone lysine residues
can alter the compactions of chromatin and its transcriptional status (Martin
and Zhang 2005). Furthermore, methylation of histones is not as
straightforward as DNA methylation. The number of residues, the position,
and the number of times each residue is methylated can result in either
heterochromatin compaction and silencing or methylation of specific lysine
residues, such as histone 3 lysine 4 (H3K4), H3K36, H4K20, and H3K79,
which can lead to a relaxed chromatin state and promote transcription
(Martin and Zhang 2005). This description appears straightforward;
however, it becomes inherently more complex when the cell type, tissue,
and species examined, such as Arabidopsis thaliana (Berr et al. 2011),
pacific oyster (Crassostrea gigas; Fellous et al. 2014, 2015), zebrafish
(Danio rerio; Kim et al. 2014, 2015), and most recently, the mangrove
rivulus fish (Kryptolebias marmoratus; Fellous et al. 2019b), all respond
differently to how histones are methylated and the mechanism of
methylation. On the other hand, histone lysine methyltransferase (KMT)
tightly regulates the methylation of specific lysine residues, and this
enzyme is highly conserved from yeast to throughout the vertebrate lineage
(Jenuwein et al. 1998; Best et al. 2018). Likewise, the same conservation
persists within the histone lysine-specific demethylases (HLSDs; Best et al.
2018; Fellous et al. 2019b).

16.2.2.2 Histone Acetylation

Much like histone methylation, histone acetylation, and deacetylation, is
regulated by a highly conserved group of enzymes known as lysine
acetyltransferases (KATs, to reflect substrate preference; Allis et al. 2007)
and lysine histone deacetyltransferases (HDATs; Roth et al. 2001). Through
acetylation, the positive charge of histones is removed, which decreases the
interaction of the negatively charged phosphate groups of DNA, resulting in
a relaxed state that is permissive to increased levels of transcription.
Conversely, HDATs can remove these acetyl groups, resulting in a more
condensed heterochromatin and preventing transcription (Kuo et al. 1998).
Of the limited studies, the majority have focused on medaka (Oryzias
latipes) and zebrafish (Labbé et al. 2017; Best et al. 2018), with
characterization of KATs/HDATs focused primarily on the zebrafish model
(Karmodiya et al. 2014). Most recently, Fellous et al. (2019a) identified and
characterized the conserved HDATs and KATs in the mangrove rivulus and
provided evidence to support histone acetylation as a crucial regulator
involved in early and late development, gametogenesis, and neurogenesis in
adults.
16.2.2.3 Histone Phosphorylation
Protein phosphorylation is one of the most common post-translational
modifications, and evidence has demonstrated that nuclear histones can be
phosphorylated and dephosphorylated (Lau and Cheung 2011). Currently,
there is little known regarding the phosphorylation of histones in fish
species; however, there is evidence to suggest that phosphorylation may
play a role in the activation of HDACs during mitotic division in zebrafish
embryonic development (Loponte et al. 2016).
16.2.3 NON-CODING RNA
There is a large portion of genomic material that does not code for protein
and was originally labelled ‘junk DNA’ (Britten and Kohne 1968; Ohno
1972). However, since the late 1990s and the discovery of RNA
interference by Andrew Fire et al. (1998), our knowledge of the
classification, characterization, and function of non-coding RNA has
drastically expanded. Non-coding RNA (ncRNA) is defined as RNA that is
not translated into a protein, and includes transfer RNA, ribosomal RNAs,
and a number of long and small non-coding RNAs (Alexander et al. 2010),
the last of which are the focus of this section. The defining features of small
ncRNAs are their short length (~20–30 nucleotides), their association with
the argonaut family of proteins, which are the essential components of the
RNA-induced silencing complex (RISC), and their ability to attenuate or
silence translation via binding to target transcripts (Ghildiyal and Zamore
2009). Outside these common characteristics, separate classes of small
ncRNAs have unique functions, locations, and characteristics that define
their mode of action, which include miRNAs, small interfering RNAs
(siRNAs), and Piwi-interacting RNAs (piRNAs; Choudhuri 2010).
Moreover, there is a growing body of evidence that small non-coding RNA
plays critical roles in immunity (Wang et al. 2018), development and
differentiation (Takacs and Giraldez 2010), metabolism (Mennigen 2016),
and response to environmental stimuli (Goodale et al. 2019) in various
teleost species. There is also evidence that long-non-coding RNAs play a
role in growth, development, cell differentiation, and hypoxic response in
teleosts (Basu et al. 2016; Wang et al. 2016; Liu et al. 2018). Since ncRNAs
contribute substantially to the regulation of genes and genomic expression,
it is presumed that their patterns of expression and dysregulation are linked
significantly to disfunction, disease, and environmental perturbation, and
there are data to demonstrate this (Choudhuri 2010; Bizuayehu and Babiak
2014; Metzger and Schulte 2016; Herkenhoff et al. 2018). The biogenesis
of miRNAs, piRNAs, and siRNAs has been extensively reviewed
(Choudhuri 2010; Bizuayehu and Babiak 2014; Best et al. 2018).
This chapter will focus more on miRNAs, as these have been well
characterized in fishes. Briefly, miRNAs are 22-nt-long non-coding RNAs
that regulate mRNA predominately through imperfect binding at the 5′
seed-region of the mature miRNA, which consists of about 5–8 nt, and are
complementary to target sites found in the 3′untranslated region (3′UTR) of
their predicted mRNA target (Bartel 2009). Furthermore, miRNAs have
been sequenced and their evolutionary conservation validated across a
number of fish species (Huang et al. 2015; Juanchich et al. 2016).

16.3 ABIOTIC AND BIOTIC EFFECTS ON EPIGENETICS

Since 2010, the fish literature has vastly expanded the study of the biotic
and abiotic affecters of epigenetic marks and the functional consequences.
The following sections will highlight the most current literature (<5 years)
to date regarding some of the major influencers of epigenetic marks in
fishes, such as temperature, oxygen, toxicant exposure, and metabolism.
16.3.1 TEMPERATURE
Temperature plays a major role in fish physiology. With the looming threat
of predicted increases in freshwater and marine surface temperatures, it
becomes essential to understand the epigenetic factors that affect the plastic
response of fishes to warming waters, driving the ability of fish to cope
with and respond to a changing global climate (Seebacher et al. 2015). One
of the most intriguing patterns to emerge in aquatic species is the
evolutionary conservation of methylation patterning between warm- and
cold-water fish species. There is a higher degree of global DNA
methylation exhibited by polar fish, such as the Channichthyidae
(icefishes), when compared with species found in temperate and tropical
regions (Varriale and Bernardi 2006). Furthermore, among all vertebrates,
birds and mammals have about 50% less global methylation compared with
fishes and amphibians, suggesting an inverse relationship between degree
of methylation and body temperature (Jabbari et al. 1997), although the
significance of this relationship has yet to be defined. This relationship does
suggest that temperature plays a crucial role in maintaining gene expression
stability over temperature gradients, and shifts in temperature, like those
predicted in association with climate change, may have significant
functional impacts. For example, in Atlantic cod (Gadus morhua), a 4 °C
increase during incubation of embryos resulted in significant changes in
DNA methylation pathways (Skjæryen et al. 2014). A 6 °C temperature
elevation in half-smooth tongue cod (Cynoglossus semilaevis) resulted in
almost three-quarters of the females undergoing sex reversal, and the testis
of these females displayed a 10% increase in genome-wide DNA
methylation compared with normal female ovaries (Shao et al. 2014).
Furthermore, temperature-dependent sex ratios have been linked to DNA
methylation in specific promoter regions. For example, in the European sea
bass, males have almost double the DNA methylation levels compared with
females in the promoter region of gonadal aromatase following a 6 °C
increase in ambient temperature during development (cyp19a; Navarro-
Martin et al. 2011). This phenomenon has been further demonstrated in Nile
tilapia (Oreochromis niloticus), where high-temperature epigenetic
regulation is critical for masculinization (Wang et al. 2017). While these
studies have focused on relatively large temperature increases, Anastasiadi
et al. (2017) took an environmentally relevant approach and demonstrated
in European sea bass (Dicentrarchus labrax) that even a 2 °C increase
during larvae (15 days post-fertilization) incubation resulted in an increase
in global methylation combined with significantly increased dnmt1 and
dnmt3 gene expression. However, the strength of this response was not
demonstrated in 60 dpf juveniles, suggesting a critical developmental
window of exposure that can result in more severe, long-lasting
consequences (Anastasiadi et al. 2017). While these studies represent
impacts associated with increasing temperatures, Metzger and Schulte
(2018) have shown that both increases and decreases in temperature during
development in threespine stickleback (Gasterosteus aculeatus) increased
global DNA methylation levels, with approximately 25% of the
differentially methylated regions responding to both temperature treatments.
Metzger and Schulte further demonstrated that differentially methylated loci
were specific to a particular thermal treatment and life stage (developmental
or adult), suggesting accumulation of epigenetic marks that can have long-
lasting effects across multiple time-scales. Combined, these studies have
suggested that DNA methylation plays a critical role in temperature-
dependent phenotypic responses, which are crucial in the resilience or
susceptibility of species responding to the threat of climate change.
While many studies have examined DNA methylation in response to
temperature in fish, fewer have examined miRNA. Using an acute heat
stress in rainbow trout (Oncorhynchus mykiss), Zhou et al. (2019)
characterized the miRNA-mRNA head kidney response and found several
differentially expressed miRNAs (miR-133 family, miR10c-5p, miR-27e,
miR16C-5p) that were predicted to have 31 negative miRNA-mRNA
pathways, implying an adverse phenotypic response to heat stress. These
pathways included those responsible for regulation of metabolism and
immune function, providing initial data for investigating the high-
temperature molecular mechanisms of rainbow trout heat tolerance (Zhou et
al. 2019). Interestingly, another study examined the liver miRNA profile in
the common carp (Cyprinus carpio) and included an increase and decrease
in temperature, comparing exposure regimes of 5 °C, 17 °C, and 30 °C (Sun
et al. 2019). This varying temperature approach revealed a more complex
picture of miRNA regulation of metabolic pathways, where the directional
increase or decrease in specific miRNA correlated with the increase or
decrease in temperature exposure.
In aquaculture, increased temperatures have been associated with
increased fish feeding and growth, although when a fish exceeds its optimal
temperature, a number of adverse consequences result (Ma et al. 2015).
Genetically improved farmed tilapia (Oreochromis niloticus), which can
tolerate higher temperatures up to 33 °C, although their mortality rate
drastically increases beyond 37 °C (Baras et al. 2001), are a model species
for the study of temperature-induced epigenetic regulation. Recent years
have seen dramatic increases in temperatures approaching the lethal range
for this bred species, and it is essential to understand the relationship
between heat stress signalling pathways and adaptation mechanisms to help
effectively breed high-temperature tolerant strains of tilapia and other
species. In 2017, Qiang et al. demonstrated that an acute 37.5 °C exposure
resulted in 38 target genes that were predicted from 21 differentially
expressed miRNAs. Bioinformatic prediction indicated negative impacts on
immune and oxidative stress pathways, energy homeostasis, oxygen
carrying capacity, and impaired membrane homeostasis. These changes in
miRNA were validated upon examining the circulating profile of miRNAs
in the blood of tilapia following 48 h of heat stress at 35 °C, suggesting that
there is a systemic, epigenetic response to heat stress (Bao et al. 2018).
With respect to histone modification, very few studies have examined the
relationship between histones and temperature in fishes. In the Senegalese
sole (Solea senegalensis), it was shown that histone 3, an important target
for epigenetic modifications, increased in protein abundance, as did the
expression of DNA methyltransferases when raised in 20 °C culture media
(Carballo et al. 2018). Several studies have also linked temperature-
dependent sex reversal to histone modification, including cyp19a regulation
in European sea bass (Navarro-Martin et al. 2011), Nile tilapia (Wang et al.
2017), and ricefield eel (Monopterus albus; Zhang et al. 2013).
16.3.2 HYPOXIA
There are numerous physiological adaptations to hypoxic exposure that
confer a plastic response that offers greater resistance, such as
modifications in gill morphology and surface area, among other
physiological, behavioural, and growth modifications (see review by Abdel-
Tawwab et al. 2019). Ho and Burrgren (2012) demonstrated that parental
influences are an important component of the transgenerational transfer of
phenotypes in zebrafish. When parents were exposed to a short (1 week)
hypoxic bout, offspring had a 15% reduction in hypoxia resistance.
However, resistance was increased by ~30% in larvae whose parents were
exposed to hypoxia for 2, 3, or 4 weeks. Using the marine medaka (Oryzias
melastigma), Wang et al. (2016) demonstrated that parental exposure to
25% hypoxia resulted in reproductive impairments in the F1 and F2
progeny despite these fish having never been exposed to hypoxia.
There is strong evidence to suggest that the DNA methylome of zebrafish
is reorganized in the maternal germline but remains stable in the paternal
germline (Jiang et al. 2013). Paternal inheritance was confirmed in the
marine medaka, where specific genes were hypomethylated and altered the
expression of genes and proteins related to spermatogenesis, resulting in the
reduction of sperm quality (Wang et al. 2016). There was also evidence to
suggest that the impairment in sperm quality was mediated by euchromatic
histone lysine methyltransferase 2, a methyltransferase that methylates the
lysine residues of histone 3 (Wang et al. 2016). More recently, the same
group has demonstrated that hypoxic exposure in female marine medaka
can result in reproductive impairments in ovarian follicular development in
the F0, F1, and F2 generations, although the mechanism of impairment was
related to hypomethylation of tumour protein 53 (Lai et al. 2019).
Collectively, this research demonstrates that while there are sex-specific
reproductive impairments, the transgenerational effects of hypoxia exposure
are regulated through DNA methylation (Figure 16.2).

FIGURE 16.2 Unlike in mammals, there is little evidence to suggest that the
methylome is reset during development. In fact, evidence suggests in zebrafish that
while the maternal methylome is reorganized, the paternal methylome remains constant.
Moreover, during development, it appears that the degree of methylation is parentally
driven; the methylation levels during development reach parity with the original degree
of parental methyl contribution.
(Adapted from Jiang, L. et al., Cell, 153, 773–784, 2013.)

miRNAs have a rapid turnover rate, ranging from less than an hour to
several hours (Rüegger and Großhans 2012), and rapid miRNA
deadenylation of target mRNA for degradation (Wu et al. 2006), making
them an ideal candidate for regulating the hypoxic response in fish. In Nile
tilapia, miR-204 is downregulated following hypoxic exposure, leading to
an upregulation in one of its targets, vascular endothelial growth factor
(VEGF; Zhao et al. 2014). In marine medaka raised for 3 months in hypoxia
(25% of control), Lau et al. (2014) showed that 55 miRNA were commonly
expressed in all tissues. However, there were some sex-specific and tissue-
specific differences in miRNA, such as let-7a, miR-122, and miR-9-3p,
which were downregulated in the brain of females, and miR-2184, which
was upregulated in the testis of males. Furthermore, Lai et al. (2016)
demonstrated that hypoxia alters the steroidogenesis pathway in female
marine medaka, which was mediated through miRNA. Conversely, in male
marine medaka, hypoxia exposure resulted in altered testicular function,
attributed to hypoxia-responsive miRNAs (i.e. miR-125-5p) that impaired
several key spermatogenesis pathways through targeting euchromatic
histone lysine methyltransferase 2 (Tse et al. 2016). In anoxia-tolerant
annual killifish (Austrofundulus limnaeus) embryos, there is a significant
alteration in the pattern of small ncRNAs during alternating bouts of anoxia
and reoxygenation, along with constitutively expressed stress-responsive
miRNAs (Riggs and Podrabsky 2017). These studies imply that miRNAs
are required for the functional responses attributed to hypoxia/anoxia
tolerance in a number of fish species.
16.3.3 METABOLISM
There are epigenetic mechanisms that respond and regulate metabolic
function, notably ncRNAs, which can affect mitochondrial respiration.
There is a great deal of interest in understanding the role that miRNAs play
in maintaining mitochondrial homeostasis and energy balance. It has been
shown that overexpression of miR-338 decreases cytochrome C oxidase IV
gene and protein expression, which translates into diminished mitochondrial
oxygen consumption, metabolic activity, and ATP production (Aschrafi et
al. 2008). The fact that numerous fish species demonstrate functional
suppression of mitochondria suggests that this would be a unique avenue to
explore the molecular mechanisms that drive this type of metabolic
suppression (Richards 2010). Mennigen (2016) reviewed the role that
miRNAs play in teleost metabolism and highlighted specific cases of
miRNA regulating hyperglycaemia in the glucose-intolerant rainbow trout
(Mennigen et al. 2014a, 2014b), with further evidence demonstrating
miRNA regulation of insulin growth factor 1 expression, which accelerated
muscle growth in Nile tilapia (Yan et al. 2013).
16.3.4 TOXICOLOGICAL EFFECTS
Studies examining epigenetic responses in fishes to toxicological insults
have greatly accelerated in the last few years. In particular, the majority of
research has focused on two main avenues of epigenetic mechanisms that
drive adverse phenotypic outcomes: DNA methylation and miRNA. In the
literature associated with DNA methylation, zebrafish are the top research
candidate, attributed to their short generation time and ease of laboratory-
based contaminant exposure (Aluru 2017). A recent review by Kamstra et
al. (2015) identified a number of DNA methylation specific responses in
zebrafish exposed to a variety of contaminants (arsenic, benzo[a]pyrene
[B[a]P], tris(1,3-dichloro-2-propyl)phosphate, and methyl mercury),
generally indicating that many contaminants resulted in an increase in
global hypomethylation, and established the zebrafish as a model in
environmental epigenetics. B[a]P is a ubiquitous and well-characterized
toxicant (Howsan and Jones 1998) that has dramatic impacts on DNA
methylation in several fish species. In zebrafish, it has been demonstrated
that fishes exposed to 5 and 10 μM B[a]P during a critical window of
development (6–120 hpf) and then raised in chemical-free water and bred
through to the F2 generation displayed hyper locomotor activity, decreased
heartbeat frequency, and mitochondrial dysfunction in the F1 and F2
generations, all attributed to hypomethylation (Knecht et al. 2017).
Conversely, Gao et al. (2018) demonstrated that B[a]P exposure (up to 50
nmol/L) resulted in an increased degree of methylation in gonadotropin
releasing hormone (GnRH) and the GnRH receptor, which resulted in
physiological changes along the brain–pituitary–gonad axis. In a study in
rainbow trout using environmentally relevant concentrations of B[a]P (1
and 10 ng/L), there was a significant increase in the degree of liver and
muscle hypomethylation, which was attributed to a decrease in the activity
of DNMTs, and a decrease in DNMT3 abundance (Kuc et al. 2017). What
was intriguing about this study was that a specific miRNA (mir-29a) was
implicated in the regulation of DNMT3a expression, suggesting that
miRNAs act as essential mediators between the environment and DNA
methylation patterns via DNMTs, and this was deeply conserved throughout
the evolution of vertebrates (Kuc et al. 2017). In Atlantic killifish (Fundulus
heteroclitus), exposure to high polychlorinated biphenyl (PCB)
concentrations also resulted in an increase in hepatic hypomethylation,
which was attributed to a decrease in DNMT1 and an increase in TET
expression, suggesting both reduced methylation and increased
demethylation (Glazer et al. 2018). Atrazine, one of the most abundantly
used herbicides and a common aquatic contaminant, was shown to induce
transgenerational reproductive effects in the form of decreased fertilization
rates through to the F2 generation in medaka, and this was linked to
changes in DNMT expression and increased global methylation (Oryzias
latipes; Cleary et al. 2019). Additionally, newly fertilized Atlantic cod
(Gadus morhua) embryos chronically exposed to 3.2 mg/L mining particles
(containing copper and iron) displayed increase in DNMT and TET gene
expression, further validating that toxicant exposure not only affects
methylation but also increases rates of demethylation (Reinardy et al.
2019). A similar response of increased TET expression but decreased
Dnmt1 expression has been demonstrated in Japanese medaka exposed to
Roundup (herbicide; glyphosate in an acid equivalent) during the first 15
dpf (Smith et al. 2019). What should be noted throughout all these studies is
that the methylation patterning is in response to an environmental insult,
permitting the upregulation of specific genes, such as those responsible for
detoxification, and that changes in epigenetic marks and mechanism are
part of the adaptive, plastic response to maintain homeostasis.
miRNAs also play a significant role at the interface between the
environment and the functional, phenotypic response in fishes. Numerous
studies have examined either the broad effects on the miRNAome or
specific miRNAs with respect to unique contaminants, such as aluminium
in Atlantic salmon (Kure et al. 2013), copper exposure in zebrafish (Wang
et al. 2013) and yellow catfish (Cui et al. 2018; Qiang et al. 2019), iron
accumulation in the turquoise killifish (Notobranchius furzeri; Ripa et al.
2017), cadmium exposure in tilapia (Qiang et al. 2017), and PCB (Ju et al.
2017) or triclosan (Liu et al. 2019) exposure in zebrafish. Of recent concern
are the emerging pharmaceutical and endocrine disrupting chemicals that
can have profound effects on fish physiology (Cameron et al. 2016).
Fluoxetine (FLX), a commonly prescribed antidepressant also known as
Prozac®, has been shown to cause transgenerational hypocortisolism and
behavioural disruptive effects in zebrafish to the F3 generation after the F0
embryos were exposed to relevant concentrations of FLX for 6 days (Vera-
Chang et al. 2018). An additional study implicated specific miRNAs that
targeted the stress axis (miR-740, miR-26, miR-30d, miR-92a, and miR-
103) from the maternal lineage as being responsible for this disruption
(Martinez et al. 2019). There were also pronounced changes in the
expression of DNA methyltransferases and genes involved in miRNA
biogenesis (Martinez et al. 2019). The studies of Vera-Chang et al. (2018)
and Martinez et al. (2019) suggest that miRNAs play a role in maintaining
homeostatic balance and that transmission of phenotypic effects of exposure
may be attributed to epigenetic mechanisms.
Very little research has examined histone modification with toxicant
exposure. There is evidence to suggest that wild yellow perch (Perca
flavescens) chronically exposed to metals (Cu, Cd) demonstrated an
increase of genes involved in histone modification, which were positively
correlated with liver metal concentrations (Pierron et al. 2011). A similar
study examined the effects of captive and wild populations of European
(Anguilla anguilla) and American (Anguilla rostrata) eels exposed to a
variety of contaminants and found that only captive eels displayed changes
in genes related to histone modification, suggesting that captive stress may
contribute to the epigenetic marks (Baillon et al. 2016).

16.4 CONCLUSION AND FUTURE DIRECTIONS

This chapter has focused on the epigenetic mechanisms common to fish
species and some of the major stressors that can affect epigenetic marks.
What becomes clear is that there are critical time windows during
embryogenesis and development in which the epigenome becomes
extremely vulnerable to environmental stressors, which can result in the
modification of epigenetic information within the developing organisms and
can be transferred across multiple generations. While the focus was on
some of the major environmental stressors that are currently threatening our
natural fish populations, there are many other stressors that have implicated
epigenetic mechanisms that regulate responses to environmental stimuli,
such as immune challenge, and these have been recently well reviewed
(Valenzuela-Muñoz et al. 2017; Andreassen and Høyheim 2017; Zhou et al.
2018; Zhang et al. 2019). With many new fish genomes being published
and annotated (Mennigen 2016; Best et al. 2018), studying epigenetic
responses to environmental perturbation in non-model species is becoming
more and more accessible, especially with new molecular techniques, such
as whole methylome and small-RNAome sequencing platforms. As we face
the consequences of global climate change, it becomes imperative to
understand the molecular, epigenetic mechanisms that maintain homeostasis
in fishes in order to protect future populations and communities from
collapse.

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 17 Behaviour and Learning
Shaun S. Killen

CONTENTS
17.1 Introduction
17.2 Foraging and Feeding
17.2.1 Control
17.2.2 Feedbacks
17.2.3 Plasticity
17.3 Risk and Predator Avoidance
17.3.1 Control
17.3.2 Feedbacks
17.3.3 Plasticity
17.4 Aggression
17.4.1 Control
17.4.2 Feedbacks
17.4.3 Plasticity
17.5 Sociability
17.5.1 Control
17.5.2 Feedbacks
17.5.3 Plasticity
17.6 Conclusion
References

17.1 INTRODUCTION
For decades, research into fish physiology and research into behaviour
existed in relative isolation from each other. More recently, however, there
has been a strong integration of research from different disciplines to give a
more complete view of how fish function and how their physiological
systems manifest in behavioural decisions. From an ecological perspective,
this holistic approach has provided insight into how physiology is related
not only to moment-to-moment decisions associated with foraging success,
predator avoidance, or among- and within-species competition but also to
broader-scale behaviours associated with habitat preferences, migrations,
and species distributions. Links between physiology and behaviour are also
important from an evolutionary perspective (Norin and Metcalfe 2019).
Behaviour is often considered the interface between an animal’s internal
physiology and the external world. When the environment changes,
behaviour acts as the first line of defence for readjustment or coping, and in
many cases, the expression of behaviour is where selection will ultimately
take place. Meanwhile, physiological traits also dynamically respond to the
environment, particularly in ectotherms, and often constrain the behavioural
options that are available to an individual animal. Physiological traits that
are correlated with behaviour, including neuroendocrine systems,
bioenergetic traits, and cardiovascular and musculoskeletal systems, will
therefore also be under direct or indirect selection (Killen et al. 2013;
Metcalfe et al. 2016).
This chapter reviews associations between physiology and several
classifications of fish behaviour. This categorization of behaviour is simply
for the purposes of discussion. In reality, the various forms of behaviour are
interconnected. These associations can occur functionally through common
physiological mechanisms or more generally via behavioural trade-offs that
occur across temporal scales. Critically, behaviour will feed back to have
direct effects on individual physiology (Sih et al. 2015). Therefore, I review
not only the physiological control of various types of behaviour but also the
ways in which behaviours affect fish physiology.

17.2 FORAGING AND FEEDING

17.2.1 CONTROL
Foraging behaviour in fish is a complex, integrated expression of numerous
physiological signals spanning various organ systems and levels of
organization within the animal. There are also many components of
foraging behaviour, including prey searching and food item identification,
selecting prey when options are available, and finally, prey pursuit and
ingestion (Figure 17.1).

FIGURE 17.1 Components of a typical foraging cycle in fish, beginning with the onset
of searching for prey and ending with prey consumption. Various physiological and
morphological factors (middle of circle) can influence the outcome at any phase of the
foraging cycle. Environmental factors and prey traits (outside the circle) may also
influence the outcome of each phase and the effort exerted by the predator. External
factors are listed beside phases where they will have the greatest effect. Note that
predation threat may have effects at numerous points along the foraging cycle.
(From Killen, Encyclopaedia of Fish Physiology, 2011.)

Perception of satiation and foraging motivation are controlled by several

hormonal signals, including those originating from within feeding centres
of the central nervous system, peripheral factors such as the gut, and
signalling between the two (Volkoff 2016). Within the brain, secretion of
factors such as agouti-related protein (AgRP), galanin, and orexin generally
increases appetite and feeding behaviour (Mensah et al. 2010), while
cocaine and amphetamine regulated transcript (CART) has a suppressive
effect (Volkoff and Peter 2000). The gastro-intestinal tract also secretes
various factors in response to feeding or fasting, releasing galanin and
ghrelin to increase appetite and CCK and leptin to decrease feeding
(Mensah et al. 2010). Leptin may play a particularly important role in
linking endocrine signalling with whole-animal energy stores and metabolic
rate, as it is secreted by adipose tissue, gut, and liver tissues in fish, and its
concentration is generally proportional to fat content (Dalman et al. 2013).
The hypothalamus–pituitary–thyroid (HPT) axis is also key in regulating
metabolism and feeding behaviour. Thyrotropin releasing hormone, thyroid
stimulating hormone, and the thyroid hormones all have varying roles in
regulating appetite and spontaneous locomotor activity and have complex
feedbacks with feeding state, nutritional history, and other hormonal signals
(Fekete and Lechan 2013). The hypothalamus–pituitary–interrenal (HPI)
axis also appears to modulate links between metabolism and foraging
motivation. Chronic stress increases cortisol expression in fish and appears
to have a suppressive effect on appetite, while fasting seems to decrease the
expression of corticotropin-releasing hormone (Bernier 2006).
Relationships between metabolic demand and foraging behaviour have
been difficult to observe and appear highly context dependent. Indirect
evidence for links between metabolism and foraging comes from the
observation that bolder sticklebacks (Gasterosteus aculeatus) consume
more food when allowed to feed until satiation (Jolles et al. 2016). This is
notable because boldness itself has been observed to be linked with
metabolic rate (Binder et al. 2016; Huntingford et al. 2010), although
results regarding this relationship are also mixed (Killen et al. 2013;
Väätäinen et al. 2018). Among-individual variance in energy stores will
also influence foraging motivation, perhaps obscuring links between
metabolism and foraging. Variance in metabolic expenditure above that
required for maintenance (e.g. energy spent on locomotion) will also affect
hunger levels and may not be incorporated in estimates of metabolic rates
during measurements of animals confined in respirometers. Finally,
estimates of metabolic rates via measurement of oxygen uptake may not
reflect true ATP conversion efficiency, further dampening the ability to
observe foraging–metabolism relationships (Salin et al. 2015).
17.2.2 FEEDBACKS
Foraging and feeding have an array of feedbacks on fish physiology. The
most obvious are effects on growth, body composition, and energy storage,
but there are numerous additional effects that occur over varying
timescales. Immediately after feeding, for example, fish experience an
increase in metabolic rate associated with the digestion of food and
assimilation of nutrients (Secor 2009; Fu et al. 2018; and Chapter 6).
Depending on meal size and nutritional content, peak SDA (specific
dynamic action) can even approach the maximum aerobic metabolic rate
(MMR) in some species, temporally restricting the aerobic scope available
for other physiological process and behaviours (McLean et al. 2018; Norin
and Clark 2017). Foraging and food intake can also have more protracted
effects on fish physiology. Interestingly, fish that regularly feed on larger
and more active prey can acquire a higher capacity for anaerobic
swimming. Feeding history also affects maintenance metabolic rates and
activity levels (Rosenfeld et al. 2015). Reduced food intake and starvation
can elicit increased activity and food searching behaviour in the short term,
but over the course of weeks, some species display metabolic depression
and a reduction in activity, presumably to reduce energy expenditure and
prolong survival as energy stores decline (García-Esquivel et al. 2002). This
process is also accompanied by a reduction in skeletal muscle mass as
proteins are catabolized for fuel (Bugeon et al. 2004), a decrease in muscle
aerobic and anaerobic enzyme activities in tissues (Martínez et al. 2000), a
reduction in fuel stores for anaerobic metabolism (Kieffer and Tufts 1998),
and reduced aerobic and anaerobic swimming ability during prolonged
fasting (Killen 2014; Martínez et al. 2004), which could compromise the
capacity to perform behaviours requiring locomotor capacity.
The quantity or quality of prey ingested can also affect the proportion of
essential fatty acids ingested by fish, which can in turn have important
effects on development, growth, and stress responsiveness in young fish
(Rocha et al. 2017). Dietary fatty acids are also known to be reflected in the
brain tissues of other vertebrates (Dyall 2015), with potential impacts on
learning ability, though this has not yet been studied in fishes.
17.2.3 PLASTICITY
Temperature has a profound effect on fish foraging behaviour, generally
increasing feeding rates with warming until the fish approaches its upper
lethal limits, where activity is reduced. Feeding activity often tracks with
AS (aerobic scope) during temperature increases, so reductions in the
available AS for locomotion and SDA at high temperatures could be at least
partially responsible for decreased feeding at higher temperatures (Zanuzzo
et al. 2019). Similarly, environmental hypoxia generally reduces appetite in
fish (Chabot and Claireaux 2008). This is probably due to a limited aerobic
scope to accommodate digestion and appears to be controlled by an
upregulation of leptin in hypoxia (Chu et al. 2010).
Learning is an important component of behavioural plasticity during
foraging in fish, and fish that can learn the location or timing of food
sources while minimizing the risk of predation and competition should have
an advantage. Evidence suggests that the stress coping style of individuals
can affect decision-making accuracy and the rate of learning while foraging
(Raoult et al. 2017). Specifically, low-stress-responsiveness individuals
(categorized by circulating cortisol levels) tend to be more active in a novel
environment, sample within their habitat more frequently, and learn more
quickly. In contrast, high stress responsiveness is linked with lower
exploration and rate of information collection and learning. Implanting
individual fish with cortisol-releasing mechanisms also seems to reduce
learning capacity and memory (Barreto et al. 2006).
Some fish species can switch between an ambush, “sit-and-wait”
foraging strategy and a more active strategy whereby individuals will be
more active in searching for food. There are trade-offs involved in the
choice of foraging mode, with more energetically costly, active foraging
only being used under specific circumstances. Juvenile salmonids, for
example, are more likely to use ambush foraging during high flow rates
(Grant and Noakes 1987), while juvenile lumpfish (Cyclopterus lumpus) are
more likely to actively forage for food only when planktonic prey densities
are very low (Killen et al. 2007a, 2007b). Sardines (Sardina pilchardus) can
switch between bite-feeding on discrete prey items and filter feeding when
only tiny planktonic prey are available. The switch to filter feeding appears
to be extremely costly and can potentially impair growth rates over
prolonged time periods (Queiros et al. 2019).

17.3 RISK AND PREDATOR AVOIDANCE

17.3.1 CONTROL
In general, there is a trade-off between increased foraging, particularly
active foraging, and predator avoidance. This is because movement will
increase the chances of being detected by a predator and may even reduce
the vigilance of the foraging animal, thus making them less likely to escape
an attack. For these reasons, many of the physiological mechanisms already
described for foraging behaviour are also important in the context of
predator avoidance. However, there are many other physiological factors
that can generally increase risk-prone behaviours, affect the ability to
perceive or respond to threats, or escape a predator once an attack has been
initiated.
Within species, individuals consistently vary in their behavioural
responses to potentially threatening situations, and these responses are
linked to more generalized physiological responses to stressful situations
referred to as stress coping styles (Vindas et al. 2017). Reactive individuals
are generally risk averse, being less active and exploratory, and are more
reliant on the HPI axis and cortisol secretion during exposure to stressful
situations. In contrast, proactive individuals tend to be more exploratory,
active, and therefore risk prone, but are more reliant on catecholamine
secretion while coping with a stressor. Manipulation of growth hormone
concentrations or receptivity also greatly increases risk-taking behaviour in
fishes, probably due to increases in foraging demand and activity (Johnsson
et al. 1996).
17.3.2 FEEDBACKS
The presence of a predatory threat can have various effects on fish
physiology. Upon detection of a predatory cue –via visual, auditory, or
chemical stimuli – there is an activation of the HPA axis in fish, with
increased circulating levels of adrenalin and cortisol (Romero et al. 2009).
This in turn results in a series of physiological effects that can increase
vigilance and enhance performance during escapes, including increased
blood glucose levels and increased heart rate and oxygen uptake. Although
beneficial in the short term, chronically elevated cortisol due to persistent
predation threat or other stressors can have a range of adverse physiological
effects, including reduced growth, reproductive investment, and immune
function (Romero et al. 2009). The exact effects of cortisol on the behaviour
of fish during a perceived threat are unclear, as studies in which individuals
have been administered cortisol via implants or injection have observed
little or no effect on antipredator behaviours (Lawrence et al. 2017, 2018).
The lost foraging opportunities and food intake due to frequent predation
threat can have a cumulative impact on growth rate, lipid stores, and
concentrations of specific fatty acids within tissues, particularly during the
early life-stages of fishes (Killen and Brown 2006; Killen et al. 2007b).
These effects may also occur across multiple trophic levels in trophic
cascades, whereby the presence of top predators suppresses the foraging
activity of mesopredators, thus freeing smaller prey fish from physiological
stress they would otherwise experience and allowing them to forage to an
extent they would otherwise not have been able to (Palacios et al. 2016;
Ripple et al. 2016). The absence of shelter can also induce a stress-related
increase in oxygen uptake in at least some fish species, which is also likely
related to the increased neural processing costs of an increase in vigilance
(Millidine et al. 2006).
17.3.3 PLASTICITY
As discussed previously, changes in temperature can affect fish activity and
foraging behaviour via effects on metabolism, and these behavioural
changes will also affect their risk of encountering a predator. Temperature
can also affect swimming performance during the fast-start escape response
that is employed by prey fish during an attack (Domenici et al. 2019).
Cooler temperatures tend to decrease the power produced during skeletal
muscle contraction, while warmer temperatures can increase contraction
power and swimming speed and acceleration during escapes. Temperature
also affects nervous system functioning in fish and so alters non-locomotor
components of the escape response, including threat perception and
response latency.
In response to hypoxia, fish can either decrease activity, to reduce energy
expenditure, or increase spontaneous swimming as they attempt to relocate
to an area with increased oxygen availability. These changes in activity will
alter the likelihood of encountering or drawing the attention of a predator. A
prime example of this is species that perform aquatic surface respiration
(ASR), during which they skim the surface of the water to respire using
water at the boundary with the air. Here, increased diffusion from the
atmosphere to the water increases oxygen availability (Kramer and
McClure 1982). This behaviour increases access to environmental oxygen
but also exposes individuals to aerial and terrestrial predators by making
them extremely visible at the surface of the water (Shingles et al. 2005).
Individual European seabass (Dicentrarchus labrax) with a higher
metabolic rate are more likely to engage in ASR in severe hypoxia,
probably because they have a correspondingly high oxygen demand (Killen
et al. 2012a). There are also many fish species that are capable of breathing
air during exposure to hypoxia, quickly surfacing to gulp a bubble of air,
which they can store within a specialized air-breathing organ (Graham
1997). Among individuals, the tendency to perform air-breathing during
hypoxia can be related to SMR and oxygen demand, but this relationship is
context specific (McKenzie et al. 2015). In the case of both ASR and air-
breathing, fish have been observed to surface in coordinated groups,
probably to reduce the risk to each individual within the shoal while
approaching the surface of the water (Chapman and Chapman 1994; Killen
et al. 2018).
The effects of dissolved CO2 and associated water acidification on
antipredator behaviours have received increasing research attention during
the last decade, and the evidence for the effects of acidification on
antipredator behaviours is mixed. Early work suggested a range of effects of
acute CO2 exposure on the ability to discriminate olfactory cues, altered
behavioural lateralization, and decreased survival (Munday et al. 2010;
Nilsson et al. 2012) that may persist across generations (Welch et al. 2014).
A suggested mechanism for these effects is interference with
neurotransmitter function in the fish brain, particularly the GABA-A
receptors (Nilsson et al. 2012; Tresguerres and Hamilton 2017). Additional
work, however, has observed little or no effect of elevated CO2 on a range
of antipredator behaviours (Clark et al. 2020; Sundin and Jutfelt 2019;
Sundin et al. 2017). Additional work is needed to verify the extent to which
GABA-A disruption is responsible for the reported effects, species- and
life-stage-specific responses to elevated CO2, specific effects of long-term
acclimation, and sources of methodological bias that may potentially
confound observations of behaviour (Sundin et al. 2017; Tresguerres and
Hamilton 2017).

17.4 AGGRESSION
17.4.1 CONTROL
Aggression is any agonistic interaction between animals and may take place
over competition for territory, social status, resources, or mating
opportunities. The physiological factors influencing aggression range from
neuroendocrine signalling and bioenergetic traits that determine the
motivation to be aggressive, to performance-related traits that determine
whether an animal is capable of winning an aggressive interaction.
Circulating levels of androgens (e.g. 11-ketotesteosteron in fishes) are
key in determining aggression and preparedness for agonistic interactions.
At finer timescales, nonapeptides (arginine vasotocin [AVT] and isotocin
[IT] in fishes) can modulate aggression and sociability in response to rapid
changes in stimuli and social cues. AVT and IT production and export to
peripheral tissues can occur via many modes of action, with various sites of
origin within the brain and receptor sites throughout the body, and so there
are an array of effects on the long-term expression of aggression and social
behaviour in general that are only beginning to be understood. AVT seems
to decrease sociability and increase aggression, with the exact effects
depending on species. IT increases sociability but has relatively little direct
effect on aggression, though it can elicit submission behaviour (Hellmann et
al. 2015). Despite these general observations, determining the exact effects
of these nonapeptides is difficult due to the many methods by which their
actions have been measured and is further complicated by the numerous
modalities by which nonapeptides can be excreted and received by tissues
(Reddon et al. 2015).
Some whole-animal metabolic traits appear to be linked with aggression
and dominance. In juvenile salmonids in particular, individuals with a
higher SMR tend to be most dominant (Metcalfe et al. 1995; Cutts et al.
1998; Yamamoto et al. 1998; McCarthy 2001), though this is not always the
case (Seppänen et al. 2009). Whether these links carry over to other species
is unclear, as SMR is not related to aggression in sharp-tooth catfish
(Clarias gariepinus) or juvenile Ambon damselfish (Pomacentrus
amboinensis), two species in which competition and aggression play a key
role in hierarchy formation and territoriality, respectively (Killen et al.
2014, 2018). There have been very few attempts to examine whether
aerobic or anaerobic capacity may be linked to aggression in fishes. In
juvenile Ambon damselfish, individuals with a higher aerobic scope are
more likely to win dyadic contests for coral territories (Killen et al. 2014).
In male Siamese fighting fish (Betta splendens), dominance is associated
with increased citrate synthase activity, an indicator of aerobic capacity,
while glycolytic enzymes show no link with the likelihood of becoming
dominant (Alton et al. 2013).
17.4.2 FEEDBACKS
The links between aggression and physiology are so intimately associated
with feedbacks that the causal directions of the relationships are extremely
difficult to disentangle (Sih et al. 2015; Metcalfe et al. 2016). For example,
although circulating androgens increase aggression, prior experience with
aggressive interactions can cause individuals to increase their androgen
production. This is believed to prepare individuals for future agonistic
interactions, and according to the “challenge hypothesis”, androgen levels
should increase in response to upward movements in social hierarchy
position or during times of general hierarchy instability (Teles and Oliveira
2016). This can produce “winner effects” whereby previous winners are
more likely to win additional competitive interactions in the future.
Relatedly, dominance in social hierarchies can often be accompanied by the
stress of hierarchy maintenance. Individuals may be repeatedly involved in
aggression, for example, if they hold prime territories with frequent
challengers (Killen et al. 2014). Subordinate individuals can experience
chronically increased metabolic rates and cortisol levels due to frequent
harassment and social stress (e.g. Sloman 2007).
The physical activity involved in aggression can also impose an energetic
cost. The increase in oxygen uptake during a fight is not necessarily related
to which individual wins or loses but is generally well correlated with the
occurrence of aggressive displays, bites, and chases. As fights progress and
continue without a winner, they tend to involve increasingly more violent
types of behaviour (e.g. biting and chasing), which can increase the
energetic costs of fighting (Castro et al. 2006; Seebacher et al. 2013). In
extreme cases, this may occupy a substantial portion of an individual’s
aerobic scope, possibly constraining them from performing additional
physiological functions above maintenance during recovery after a fight
takes place. In guppies (Poecilia reticulata), females are frequently the
receivers of aggression from males as they are chased and harassed for
mating opportunities (Magurran and Seghers 1994). Over prolonged
periods, however, females respond to this frequent chasing by increasing
the efficiency of their aerobic swimming (Killen et al. 2016a).
17.4.3 PLASTICITY
At least in theory, any factor that increases resource requirements – such as
an increase in temperature causing an increase in metabolic rate and food
requirements – could lead to increased aggression. In the African sharp-
tooth catfish, aggression in groups increases in hypoxia, particularly by
dominant individuals within groups, and this appears to elicit group
surfacing behaviour while air-breathing (Killen et al. 2018). It is also
possible that depending on individual reaction norms within species in
terms of their responses to a change in temperature or some other
environmental variable, differing individuals might be capable of becoming
dominant depending on the prevailing conditions. Daily fluctuations in
factors such as temperature or oxygen availability could destabilize social
hierarchies if different individuals have different environmental
sensitivities, possibly changing individual androgen expression and
increasing aggression in habitats with greater environmental fluctuations
(Morash et al. 2018).

17.5 SOCIABILITY
17.5.1 CONTROL
Sociability is defined as non-aggressive associations among conspecifics,
and in fish, this takes the form of schooling or shoaling behaviour (Réale et
al. 2007). Because sociability is defined in terms of being a non-aggressive
association between conspecifics, sociability and aggression are in some
sense functionally two sides of the same coin and are under the influence of
many of the same physiological systems and feedbacks (Kelly and Vitousek
2017). For example, the same nonapeptide signalling that modulates
aggression also influences sociability, but with largely reciprocal effects.
Notably, however, some individuals can be “socially indifferent” without
displaying aggression (Conradt et al. 2009).
The available evidence suggests that within species, individuals with a
higher energy demand (e.g. SMR) tend to be less social, preferring to
distance themselves from conspecifics (Killen et al. 2016b; Cooper et al.
2018). This may be due to the potential competition for food items when in
social groups. In an analogous fashion, fish that have been food deprived
for several days are also less social, presumably due to increased hunger
levels (Hansen et al. 2016; Krause et al. 2011). These links among feeding
history, metabolism, and sociability also suggest potential links between
endocrine factors that primarily affect feeding (e.g. leptins and ghrelin) and
sociability, but this has not been investigated.
Once a fish has joined a shoal, metabolic traits and swimming
performance can influence their spatial position within the group. When
moving at faster speeds, individuals with a higher capacity for aerobic
swimming tend to swim near the front of groups, while those with a lower
aerobic scope move toward the back of groups (Killen et al. 2012a), though
this situation may be reversed when freely roaming groups move at slow
speeds (Ward et al. 2018). While swimming at higher speeds, shifts toward
the back of groups would allow low-performance individuals to take
advantage of the vortices produced by groupmates to reduce their own costs
of locomotion or conserve aerobic scope (Hemelrijk et al. 2015; Marras et
al. 2015). Food-deprived individuals also tend to move toward the front of
moving groups, where they can access more food or higher-quality food
(Krause et al. 2011; Deblois et al. 2011). After fish have fed at the front of
groups, the metabolic costs of digestion can constrain their aerobic scope,
such that they can no longer remain in these leading positions if the group
continues to move (McLean et al. 2018). This implies that the individuals
that lead group movements may dynamically shift in response to the recent
feeding history of individual group members (Figure 17.2). Numerous
behavioural traits, including boldness, also appear linked to leadership
within moving fish schools (Balaban-Feld et al. 2018; Jolles et al. 2017;
Conradt et al. 2009), and so it is plausible that there may also be a range of
correlated physiological traits that underlie these relationships.

FIGURE 17.2 Feeding while swimming in a school causes fish to move to more
posterior spatial positions due to the metabolic costs of digestion. In the top panel, fish
(European minnows, Phoxinus phoxinus) swim against a moderate current and feed on
drifting prey items. Their position from the front of the school is quantified before and
during feeding and then 20, 40, and 60 min post feeding during their digestive period.
Fish that eat the most prey items (bottom left) showed the greatest backwards shift
within each shoal (each data point equals one fish within a school of six fish). In
separate trials, specific dynamic action was quantified after feeding. Values at each time
point were used to predict the approximate aerobic scope remaining for each individual
fish; fish with a lower aerobic scope after feeding moved furthest backwards within
each school.
(From McLean, S., et al., Curr. Biol., 28, 2018, pp. 1144–1149. With permission.)

17.5.2 FEEDBACKS
Non-aggressive social behaviour can also affect stress experienced by fish.
One way in which this can occur is via “calming effects”, whereby the
presence of conspecifics appears to reduce stress in an individual (Nadler et
al. 2016a; Queiroz and Magurran 2005). Predator attacks or extreme
weather events can break apart fish shoals or cause individuals to be
isolated from social groups. The presence of conspecifics can also increase
the rate of recovery from other stressors via “social buffering” (Culbert et
al. 2019).
By definition, animal social groups occupy the same location in time and
space, and so all individuals within the group will experience similar
environmental conditions. Therefore, the environment selected by the
group, or leaders within the group, will influence the physiology of all
group members via the effects of the environment they occupy. It has been
shown, for example, that individual three-spined sticklebacks depart from
their own preferred temperature to associate with conspecifics (Cooper et
al. 2018), and such deviations would cause their metabolic rate to shift from
what it would otherwise be at their own preferred temperature.
17.5.3 PLASTICITY
In general, fish schools become less dense during exposure to acute hypoxia
as individuals move away from one another (Killen et al. 2017). This could
be a means to increase the oxygen available per individual fish, especially
given that there is evidence that extremely dense fish schools can actually
reduce the oxygen content of the water they occupy (McFarland and Moss
1967). As hypoxia progresses, schools become less polarized,
synchronized, and coordinated, either due to neuromuscular impairment or
because individuals are attempting to relocate. In any case, to date, these
effects have only been studied in a small handful of pelagic species, and we
still know virtually nothing about how physiological acclimation to hypoxia
may help fish social groups compensate for any negative effects of hypoxia.
Elevated CO2 has been largely overlooked regarding its effects on fish
social behaviour. There is evidence that short-term exposure to elevated
CO2 in the damselfish (Chromis viridis) decreases the ability or willingness
of fish to distinguish between familiar and unfamiliar conspecifics (Nadler
et al. 2016b). It has also been observed that in the same species, elevated
CO2 can cause fish groups to engage in more risky behaviour, though group
cohesion appears unaffected (Cattano et al. 2019). In the only temperate
species to be examined for CO2 effects in groups, juvenile blacksmith
(Chromis punctipinnis) showed no notable effects of social behaviour
following CO2 exposure (Kwan et al. 2017).

17.6 CONCLUSION
The relationships between physiology and behaviour in fishes consist of a
complex array of control systems and feedbacks that determine the
phenotype that is exposed to the environment and ultimately, selection. This
chapter has summarized some of these interactions, but the
multidisciplinary approach to understanding the relationships among
physiology, behaviour, and ecology is still in its early phases. For example,
the study of neuroendocrine physiology and that of metabolism have
progressed largely independently within the context of ecophysiology and
behaviour. We therefore have relatively little understanding of the interface
between endocrine factors and whole-animal metabolism and the relevance
for fish behaviour. This is a major obstacle, because these two layers of
physiological control are both clearly relevant for fish behaviour, and yet
their direct interactions have been largely overlooked. Moreover, the
endocrine system and metabolic traits are also related to allostasis in the
face of environmental stressors. As such, any response or disturbance to
either of these systems or their components could set off a cascade of
effects that alter the expression of behaviour. This understanding will be
vital for not only the immediate responses to shifting or fluctuating
environmental conditions but also the long-term evolutionary responses to
environmental change via selection on behaviour and correlated traits.

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Index
ABA, see Afferent branchial arteries
Acanthomorpha, 10
Acanthopterygii, 12
Lamprimorpha, 11
Paracanthopterygii, 11–12
Acid–base regulation, 68–71
Acipenseriformes, 6
ACTH, see Adrenocorticotropic hormone
Actinopterygii, 6
Acanthomorpha, 10–12
Euteleostei, 9–10
Neoteleostei, 10, 11
Percomorpha, 12–14
Teleostei, 6–9
Action potentials (APs), 49–50, 168
Activity energy, 208
Adenohypophyseal hormones, 106
Adenohypophysis, 106–107
Adnexa, 174
Adrenergic stimulation, 55, 56
α-Adrenoceptors, 55
β-Adrenoceptors, 55, 57
Adrenocorticotropic hormone (ACTH), 110, 169
Aerobic scope (AS), 130
AFA, see Afferent filamental arteries
Afferent branchial arteries (ABA), 52
Afferent filamental arteries (AFA), 52
Aggression, 229
control, 229
feedbacks, 229–230
plasticity, 230
Agnathans, 1
Myxiniformes, 3
Petromyzontiformes, 3
Albuliformes, 6
Alkalinity in pond, 209
α-melanocyte hormone (α-MSH), 169
Amino acids, 48, 193, 194, 197
Ammocoete, 3
Amphibious locomotion, 28–29
Ancillary structures, 148–151
Androgens, 121
Anguilliformes, 6
Anguilliform locomotion, 19–20
Anoxia, 48, 57
Aquaculture, defined, 203
Aquaculture, sustainable fishery and, 209
co-management, 209
sustainable aquaculture, 209–210
threats to sustainability and application of biotechnology, 210
Aquaculture status, world fisheries and, 204
global fisheries development, 204–205
history of aquaculture, 205
world aquaculture, current state of, 205–207
Aquatic surface respiration (ASR), 228
Aqueous and vitreous humour, 176
Arginine vasotocin (AVT), 229
ASR, see Aquatic surface respiration
Atrazine, 219
Aulopiformes, 10
AVT, see Arginine vasotocin

Batomorphi, 5
Batrachoidida, 12
BCF, see Body caudal fin
Behaviour and learning, 225
aggression, 229–230
foraging and feeding, 226–227
risk and predator avoidance, 228–229
sociability, 230–232
Bile, 80
Blood, 47–48
Blood flow
through the gill, 52–53
and perfusion, 41
BM, see Body mass
Body caudal fin (BCF)
locomotion, 19–21
swimming, 22, 25–26
Body mass (BM), 130–131
Bohr effect, 37, 38, 40
Bohr–Haldane effects, 40
Bowman’s layer, 175
Branchial gas transfer, 33
diffusion across membranes, 35–36
morphology, 35
osmorespiratory compromise, 36
ventilation, 34–35
Buccal cavity, 81
 Calorimeter, 130
Carangaria, 13
Carangiform fishes, 20
Carbon dioxide, 39–41
and alkalinity in pond, 209
Carcharhiniformes, 4
Cardiovascular system, 47
future cardiovascular research, 58
general features of, 47
blood, 47–48
cardiac excitation–contraction coupling and cardiovascular parameters, 49–51
control systems, 55–56
heart morphology and blood flow patterns, 48–49
vasculature, 51–55
integrative cardiovascular function
digestion, 56
exercise, 56
high temperature, 56–57
low temperature, 57
oxygen levels, limiting, 57–58
CART, see Cocaine and amphetamine regulated transcript
Cellular stress response (CSR), 95–96
Central pattern generators (CPGs), 27
Central posterior/prepacemaker nucleus (CP/PPn), 163
CFF, see Critical flicker fusion frequency
Chimaeras, see Holocephali
Chlamydoselachidae, 5
Chondrichthyes, 3
Elasmobranchii, 3–5
Holocephali, 3
Chondrostei, 6
Choroid, 175
Choroid rete mirabile, 54
Ciliated neurons, 195–196
Clupeomorpha, 8
Cocaine and amphetamine regulated transcript (CART), 226
Cold temperatures, adaptations to, 99
Cold-tolerance, 99
Colon and rectum, 86
Colour vision, 188–189
Co-management, 209
Compact myocardium, 49
Compliance vessel, 51
Contrast sensitivity function (CSF), 187
Cornea, 174–175
Corticotropin-releasing hormone (CRH) neurons, 110
CPGs, see Central pattern generators
CP/PPn, see Central posterior/prepacemaker nucleus
 CRH neurons, see Corticotropin-releasing hormone neurons
Critical flicker fusion frequency (CFF), 187
CSF, see Contrast sensitivity function
CSR, see Cellular stress response
Culture environment, fish in, 207
carbon dioxide and alkalinity in pond, 209
energetic transformation, 208
food digestion, 207–208
osmoregulation and salinity tolerance, 208–209
temperature tolerance and oxygen demand, 208
Cypriniformes, 9
Cytosine-phosphate-guanine (CpG) nucleotides, 215

Descemet’s membrane, 175

Developmental plasticity, 98
Dietary fatty acids, 227
Diet-induced thermogenesis (DIT), 208
Digestive system, 56, 79, 207–208
future perspectives, 87
morphology, 81
associated organs, 86
buccal cavity, pharynx, and associated structures, 81
colon and rectum, 86
intestine, 84–86
microbiome, 86–87
oesophagus, 82
stomach, 82–84
primary function of, 79–80
DIT, see Diet-induced thermogenesis
DNA methylation, 214–215
DNA methyltransferases (DNMTs), 214, 219
Dopamine, 118–119

Ears
diversity of, 151–152
sound stimulation, response to, 148
EBA, see Efferent branchial artery
Echinorhiniformes, 5
EDCs, see Endocrine disrupting compounds
Efferent branchial artery (EBA), 52–54
Elasmobranchii, 3
Batomorphi, 5
Selachii, 4–5
Elasmobranch rectal gland, 71
Elasmobranchs, 63, 66
Elastance vessel, 51
Electric organ discharges (EODs), 159–160
Electric signal type, classification of electric fishes based on, 159–162
 Electrocommunication, 162–163
Electrogenesis, 159
Electromotoneurons (EMNs), 164, 168
Electroreception, 159
electric fishes, 159–162
electrocommunication, 162–163
and electrogenesis, 159
endocrine regulation, 167–169
pacemaker neurons, 164–167
Electrosensory lateral line lobe (ELL), 163
Electro-sensory-motor pathways in Gymnotiform weakly electric fishes, 163–164
ELL, see Electrosensory lateral line lobe
Elopiformes, 6
Elopomorpha, 6
EMNs, see Electromotoneurons
Endocrine disrupting compounds (EDCs), 125
Endocrine regulation
and neuromodulation of premotor and motor brain centers, 167–168
of peripheral electric organ, 168–169
Endothermy, 92
Energetic transformation, 208
EODs, see Electric organ discharges
Epigenetic mechanisms, 213
abiotic and biotic effects on, 216
hypoxia, 218–219
metabolism, 219
temperature, 216–218
toxicological effects, 219–220
DNA methylation, 214–215
histone modification, 215–216
Epigenotype, 213
Erythrocytes, see Red blood cells
Escape response, 27
Esociformes, 10
Eupercaria, 13–14
Euryhalinity, 72–75
cellular mechanisms of osmosensing and signal transduction, 74–75
natural selection favouring, 73
Euteleostei, 9
Protacanthopterygii, 10
Zoroteleostei, 10
Evermanellidae, 182
Excitation–contraction (E–C) coupling, 50, 51, 56
Exercise, 56
Eye, 174
adnexa, 174
aqueous and vitreous humour, 176
lens, 176
 retina, 176–182
sclera/cornea, 174–175
shape, 182
uvea, 175–176

Field metabolic rate (FMR), 130

Fin anatomy and structure, diversity of, 24
Fluoxetine (FLX), 220
FLX, see Fluoxetine
FMR, see Field metabolic rate
Food, mechanical breakdown of, 79
Foraging and feeding, 226
control, 226–227
feedbacks, 227
plasticity, 227
Forces, fish, 22–25
F-prostaglandins, 194
Frank–Starling effect, 51, 56
Freshwater (FW), 66
Freshwater fishes
gills, 68–69
kidney, 71
Functional surface area, 36
FW, see Freshwater

GABA (gamma-aminobutyric acid), 119

Gadiformes, 12
Gait changes, 22
Galaxiiforms, 10
Galeomorphi, 4–5
Gas exchange, 33
branchial gas transfer, 33–36
diffusion at tissue level, 41–42
respiratory gases, circulatory transport of, 36–41
Gasterosteoidei, 14
Gastrointestinal tract (GIT), 72, 79–80
GBF, see Gut blood flow
Gene duplications of neuroendocrine actors, 107–108
GH, see Growth hormone
Gh gene, 112, 114
GHRH, see Growth hormone-releasing hormone
Gills, 67–71
Ginglymostomatidae, 4
GIT, see Gastrointestinal tract
Gnathostomata, 3
GnRH, see Gonadotropin releasing hormone
GnRH receptors (GnRHRs), 118
Gobiida, 12
Gonadotropes, 117
Gonadotropin releasing hormone (GnRH), 219
Gonorynchiformes, 8
GPCRs, see G protein-coupled receptors
G protein-coupled receptors (GPCRs), 196, 197
Granulocytes, 48
Growth hormone (GH), 112–113
Growth hormone-releasing hormone (GHRH), 112
Gut blood flow (GBF), 56
Gymnotiforms, 160, 163–164

HDATs, see Histone deacetyltransferases

Head of fish, 147
Hearing, 143–145
ancillary structures, 148–151
anthropogenic sound and fishes, 154
diversity of fish ears, 151–152
future directions, 153–154
hearing range of fishes, 152–153
importance of sound to fishes today, 145
inner ear, 146–148
response of the ear to sound stimulation, 148
underwater sound and fishes, 145
Heart, 48
atrium, 48
bulbus arteriosus, 49
outflow tract (OFT), 48
sinus venosus, 48
ventricle, 48–49
Heat hardening, 99
Heat shock proteins (HSPs), 95
Hematocrit, 47
Hemoglobins, 47
Hepatic portal system, 54
Heterodontiformes, 4
Hexanchiformes, 5
Hiodontiformes, 7
Histone deacetyltransferases (HDATs), 215
Histone lysine-specific demethylases (HLSDs), 215
Histone modification, 215
histone acetylation, 215
histone methylation, 215
histone phosphorylation, 216
non-coding RNA, 216
HLSDs, see Histone lysine-specific demethylases
Holocephali, 3
Holostei, 6
HPG system, see Hypothalamo-pituitary-gonadal system
 HPI axis, see Hypothalamus–pituitary–interrenal axis
HPT axis, see Hypothalamus–pituitary–thyroid axis
HSPs, see Heat shock proteins
Hunter’s organ, 160
Hydrochloric acid, 79–80, 84
Hypophysiotropic neurons, 105–107
Hypothalamo-pituitary-gonadal (HPG) system, 117, 118
Hypothalamus–pituitary–interrenal (HPI) axis, 226
Hypothalamus–pituitary–thyroid (HPT) axis, 226
Hypoxia, 57
exposure, 218–219
metabolic rate (MR) and, 133–135
Hypoxic bradycardia, 57

IGF, see Insulin-like growth factors

ILCM, see Interlamellar cell mass
Inner ear, 146–148
Insulin-like growth factors (IGF), 113
Interlamellar cell mass (ILCM), 36
Interpulse intervals (IPIs), 161
Intestine, 84–86
Intraocular filters, 185
Inverse thermal acclimation, 57
Iono-and osmoregulation, 63
euryhalinity, 72–75
evolutionary strategies, 63
elasmobranchs, 66
hagfish, 64–66
lamprey, 66
teleosts, 66
gastrointestinal tract (GIT), 72
gills, 67–71
kidney, 71–72
skin, 67
Ion transport, 67
IPIs, see Interpulse intervals
Isotocin (IT), 229

Jamming avoidance response (JAR), 163

JAR, see Jamming avoidance response
Jawless vertebrates, see Agnathans

KATs, see Lysine acetyltransferases

Kidney, 71
freshwater fishes, 71
marine fishes, 71–72
KiSS system, 119
KMT, see Lysine methyltransferase
 Labriform locomotion, 21, 26–27
Lamniformes, 4
Lamprey, 66
Lamprimorpha, 11
Lens, 176
Lepidosireniformes, 5
Lepidotrichia, 5, 24
Leucocrit, 48
Leukocytes, see White blood cells
Light, 189
Local gene duplication, 107
Locomotion, 19
amphibious locomotion in fishes, 28–29
body caudal fin (BCF) swimming, 25–26
complexity of fish forces, 22
fin anatomy and structure, diversity of, 24
general biomechanics, 22
muscle anatomy, 22–25
muscle cells, 22
escape response, 27
history, 19
body caudal fin (BCF) locomotion, 19–21
gait changes, 22
median and paired fin (MPF) locomotion, 21
swimming, classification of, 19
labriform locomotion, 26–27
muscle activity, 25
neuro control, 27–28
swimming in unsteady flow, 27
unsteady swimming, 27
Lophiiformes, 14
Lymphocytes, 48
Lysine acetyltransferases (KATs), 215
Lysine methyltransferase (KMT), 215

Major histocompatibility complex molecules (MHC), 195

Marine fishes, 69
gastrointestinal tract (GIT), 72
gills, 69
kidney, 71–72
Mauthner cells (M-cells), 167
Mauthner neurons, 27
Maximum metabolic rate (MMR), 130–131, 135, 227
M-cells, see Mauthner cells
Medaka, 198
Median and paired fin (MPF) locomotion, 21
Melatonin, 124
Mesencephalic locomotor region (MLR), 27
 Metabolic rate, 129
ecological and evolutionary relevance of, 136–138
levels of, 130
modulators of, 130
body mass (BM), 130–131
hypoxia, 133–135
temperature, 131–133
variation in, 135–137
Metabolism, epigenetic mechanisms and, 219
Methylated DNA, 215
Methylation, 214
MHC, see Major histocompatibility complex molecules
Microbiome, 86–87
MicroRNA (miRNA), 213, 218–220
Microvillous neurons, 195–196
Minimum resolvable angle (MRA), 187
miRNA, see MicroRNA
Mitochondria, 57
MLR, see Mesencephalic locomotor region
MMR, see Maximum metabolic rate
Mobuliform locomotion, 21
Molecular chaperones, 95
Monocytes, 48
Mormyroids, 160
MPF locomotion, see Median and paired fin locomotion
MRA, see Minimum resolvable angle
Muscle activity, 25
Muscle anatomy, 22–25
Muscle cells, 22
Myctophiformes, 10
Myliobatiformes, 5
Myxiniformes, 1, 3

ncRNA, see Non-coding RNA

nE, see Nucleus electrosensorius
Neofunctionalization, 109
Neoselachii, see Elasmobranchii
Neoteleostei, 10, 11
Neuro control, 27–28
Neuroendocrine actors, 107
ancient origin of molecular families of, 107
conservation/loss of duplicated paralogs and species-specific diversity of, 108–109
gene duplications of, 107–108
vertebrate and teleost-specific whole-genome duplications and impact on, 108
Neuroendocrine axes, 105
control of physiological functions and life cycles, 105–106
innovation of pituitary gland in vertebrates, 106
pituitary functional anatomy in teleosts, 106–107
Neurohypophysis, 106
NFAT5, see Nuclear factor of activated T cells 5
Non-coding RNA (ncRNA), 216
Notacanthiformes, 6
Notothenioids, 14
Nuclear factor of activated T cells 5 (NFAT5), 75
Nucleus electrosensorius (nE), 163

O2 equilibrium curve (OEC), 37

OCLTT, see Oxygen and capacity-limited thermal tolerance
Odorant receptors (ORs), 196
Odor detection, molecular basis of, 196–198
Odors, recognition of, 194–195
OEC, see O2 equilibrium curve
Oesophagus, 82
OFT, see Outflow tract
Olfaction
model fish species for developmental, behavioral and genetic studies, 198–199
molecular basis of odor detection, 196–198
olfactory sensory neurons (OSNs) detecting odors, 193, 195–196
recognition of odors, 194–195
in relation to systematic position and ecological environment, 199
Olfactory circuits, 193
Olfactory organ, morphology of, 192
Olfactory system, morphology of, 191–193
Oocyte development and maturation, 122–123
Ophidiida, 12
Opisthoproctidae, 182
Orectolobiformes, 4
ORs, see Odorant receptors
Osmeromorpha, 10
Osmoregulation, 208–209
Osmosensing and signal transduction, cellular mechanisms of, 74–75
Ostariophysi, 8–9
Osteichthyes, 5
Actinopterygii, 6–14
Sarcopterygii, 5
Osteoglossiformes, 7
Osteoglossomorpha, 6–7
Otocephala, 7
Clupeomorpha, 8
Ostariophysi, 8–9
Otophysi, 8–9
Outflow tract (OFT), 48, 49
Ovalentaria, 12–13
Oxidative stress, temperature and, 96–97
Oxygen, 33, 37–39
Oxygen and capacity-limited thermal tolerance (OCLTT), 97
Oxygen demand, 208
Oxygen levels, limiting, 57–58
Oxygen limitation hypothesis, 97

PACAP, see Pituitary adenylate cyclase-activating polypeptide

Pacemaker nucleus (Pn), 164
Paracanthopterygii, 11–12
Pectoral fin swimming, 21
Percomorpha, 12
Batrachoidida, 12
Carangaria, 13
Eupercaria, 13–14
Gobiida, 12
Ophidiida, 12
Ovalentaria, 12–13
Scombrimorpharia, 12
Peripheral electric organ, endocrine regulation of, 168–169
Petromyzontiformes, 1, 3
PGC migration, see Primordial germ cell migration
Pharynx, 81
Photoperiod and reproduction, 124
Photoreceptors, 176
Phylogenetic systematics, 1
piRNAs, see Piwi-interacting RNAs
Pituitary adenylate cyclase-activating polypeptide (PACAP), 112
Pituitary gland
innovation of pituitary gland in vertebrates, 106
pituitary functional anatomy in teleosts, 106–107
Pituitary-gonadal axis, 120–124
Piwi-interacting RNAs (piRNAs), 216
Plasma, 48
Polypteriformes, 6
Prepacemaker nucleus–chirps (PPnC), 166–167
Primary metamorphosis, 109, 110
Primordial germ cell (PGC) migration, 114
Pristiformes, 5
Pristiophoriformes, 5
Prolactin (prl) gene, 112, 114
Prolactin 2 (prl2) gene, 112, 114
Protacanthopterygii, 10
Prozac, 220
Pupil, 184–185

Rajiformes, 5
RBCs, see Red blood cells
Reactive oxygen species (ROS), 96
Rectum, 86
Red blood cells (RBCs), 47
 Regalecidae, 11
Renal portal system, 54
Reproduction, 117
environmental cues, 124
photoperiod, 124
temperature, 124–125
environmental endocrine disruption, 125
neuroendocrinology of, 117
additional factors, 119–120
dopamine, 118–119
GnRH receptors (GnRHRs), 118
KiSS system, 119
pituitary-gonadal axis, 120
oocyte development and maturation, 122–123
sexual determination and sexual differentiation, 123–124
spermatogenesis, 123
steroids and steroid receptors, 120–122
Resistance vessels, 52
Respiratory gases, circulatory transport of, 36–41
blood flow and perfusion, 41
carbon dioxide, 39–41
oxygen, 37–39
Retia mirabilia, 92
Retina, 176
light/dark adaptation, 178
regional variation in retinal structure, 178
rods and cones, 176–178
visual pigments, 178–182
Retinal disparity, 189
Retinal ganglion cells (RGCs), 176–178, 187
Retinal pigment epithelium (RPE), 176, 178, 185
Retinomotor movements (RMM), 178
RGCs, see Retinal ganglion cells
Rhincodontidae, 4
Risk and predator avoidance, 228
control, 228
feedbacks, 228
plasticity, 228–229
RMM, see Retinomotor movements
RMR, see Routine metabolic rate
RNA-induced silencing complex (RISC), 216
Root effect, 38, 56
ROS, see Reactive oxygen species
Routine metabolic rate (RMR), 130
RPE, see Retinal pigment epithelium

sAC, see Soluble adenylyl cyclase

Sach’s organ, 160
Salinity tolerance, 208–209
Salmoniformes, 10
Sarcoplasmic reticulum (SR), 51
Sarcopterygii, 5
SA valve, see Sino-atrial valve
SCC, see Solitary chemosensory cells
Sclera/cornea, 174–175
Scombrimorpharia, 12
Scopelarchidae, 182
SDA, see Specific dynamic action
Seawater (SW), 64
Secondary metamorphosis, 110
Selachii, 4
Galeomorphi, 4–5
Squalomorphi, 5
Sensory hair cells, 146–148
Sertoli cells, 123
Sexual determination and sexual differentiation, 123–124
Siluriphysi, 9
Silvering, 110
Sino-atrial (SA) valve, 48
siRNAs, see Small interfering RNAs
Skin, 67
Small interfering RNAs (siRNAs), 216
Smell, sense of, 194
Smoltification, 110
SMR, see Standard metabolic rate
Sociability, 230
control, 230–231
feedbacks, 231
plasticity, 232
Solitary chemosensory cells (SCC), 191
Soluble adenylyl cyclase (sAC), 70
Somatolactin (sl) gene, 112, 114
Somatotropic axis, 112–113
impact of gene duplication, conservation, or loss on, 113–114
multiple hypophysiotropic controls integrated at pituitary somatotroph level, 113
various roles in teleosts, 113
Spatial resolution in fishes, 187
Spawning, 117
Specific dynamic action (SDA), 56, 208
Spermatogenesis, 123
Spongy myocardium, 49
SPPn, see Sublemniscal prepacemaker nucleus
Squaliformes, 5
Squalomorphi, 5
Squatiniformes, 5
SR, see Sarcoplasmic reticulum
 SRIH, 113
Standard metabolic rate (SMR), 130–131, 135–136, 208
Steroids, 122
and steroid receptors, 120–122
Stomach, 82–84
Stomiiforms, 10
Stress coping styles, 228
Subcarangiform swimmers, 20
Subfunctionalization, 109
Sublemniscal prepacemaker nucleus (SPPn), 164
Sustainable aquaculture, 209–210
SW, see Seawater
Swimming
classification of, 19
in unsteady flow, 27

TAAR, see Trace amine-associated receptor

Tapeta, 185
Teeth, 79
Teleostei, 6
Elopomorpha, 6
Osteoglossomorpha, 6–7
Otocephala, 7–9
Temperature
epigenetic regulation, 216–218
high, 56–57
low, 57
metabolic rate (MR) and, 131–133
and oxidative stress, 96–97
role in reproduction, 124–125
tolerance, 208
Temporal CSF (tCSF), 188
Temporal vision in fishes, 187–188
Terrestrial locomotion, diversity of, 28–29
Tetraodontiformes, 14
Tetraodontiform locomotion, 21
Thermal biology, 91
cellular and molecular effects of temperature, 95
cellular stress response (CSR), 95–96
effects on cellular metabolism, 96
effects on membranes, 96
temperature and oxidative stress, 96–97
in a changing world, 99–100
characterizing the thermal niche of a fish, 92
thermal compensation, 94–95
thermal performance, 93–94
thermal tolerance, 92–93
developmental plasticity, 98
epigenetic effects of temperature, 98
endothermy in fishes, 92
thermal adaption, 98–99
adaptations to constant cold in antarctic fishes, 99
thermal strategies, 91–92
whole-organism performance, effects on, 97
cardiorespiratory system, effects on, 97
metabolism, effects on, 97
swimming performance and behaviour, effects on, 97
Thermal performance curve (TPC), 93–94, 133
Thrombocytes, 48
Thunniform swimmers, 20
Thyroid hormone receptors, 111
Thyrotropic axis, 109–110
impact of gene duplication, conservation, or loss on, 111–112
knowledge gaps in, 110–111
teleost metamorphosis and role of thyroid hormones, 110
Thyrotropin (TSH), 110
TSH receptors (TSHR), 111
Thyrotropin-releasing hormone (TRH), 107, 109–110
Torpediniformes, 5
Toxicological effects, epigenetic mechanisms, 219–220
TPC, see Thermal performance curve
Trace amine-associated receptor (TAAR), 196–197, 199
Trans-generational plasticity, 95
TRH, see Thyrotropin-releasing hormone

Underwater sound and fishes, 145

Unsteady swimming, 27
Uvea, 175–176

Vascular rete, 54
Vasculature, 51–55
Ventricular myocardial architecture, 49
Vision, 173–178
Visual abilities, 185
absolute sensitivity, 185–186
colour vision, 188–189
contrast, 187
depth perception, 189
polarization, 189
spatial resolution, 187
temporal vision, 187–188
Visual optics, 182
eye shape, 182
image formation, 182
amphibious vision, 184
resting refractive state and accommodation, 182–184
 intraocular filters, 185
pupil, 184–185
tapeta, 185
Vomeronasal receptor type 2 (V2Rs), 197

Water-breathing teleost circulatory system, 53

WBCs, see White blood cells
Weberian apparatus, 8
Weberian ossicles, 150, 152
WGD, see Whole-genome duplications
White blood cells (WBCs), 48
Whole-genome duplications (WGD), 107–108

Y-shaped stomach, 82

Zoroteleostei, 10