Primitive Fishes (Fish Physiology, Vol. 26)

PRIMITIVE FISHES

This is Volume 26 in the FISH PHYSIOLOGY series Edited by Anthony P. Farrell and Colin J. Brauner Honorary Editor: William S. Hoar and David J. Randall A complete list of books in this series appears at the end of the volume

PRIMITIVE FISHES Edited by

DAVID J. MCKENZIE

Institut des Sciences de l’ Evolution UMR 5554 CMRS‐Universite´ de Montpellier II Station Me´diterrane´enne de l’ Environnement Littoral Se´te, France

ANTHONY P. FARRELL Department of Zoology University of British Columbia Vancouver, British Columbia, Canada

COLIN J. BRAUNER

Department of Zoology University of British Columbia Vancouver, British Columbia, Canada

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Front Cover Photograph: The cover shows various present‐day reputedly primitive fishes pointing downwards and some of their earliest fossil relatives pointing upwards. The tails of two examples of extinct fish taxa, Palaeozoic in age, are also just visible at the bottom. Drawing by P. Janvier

Academic Press is an imprint of Elsevier 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK

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CONTENTS CONTRIBUTORS

ix

PREFACE

xi

1.

Living Primitive Fishes and Fishes From Deep Time Philippe Janvier

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

2.

Introduction Primitive Characters, Primitive Taxa, and Ancient Taxa Living Fossils Living Primitive Fishes in Vertebrate Phylogeny Living Primitive Fishes and Their Fossil Relatives: Naming and Dating Taxa Extinct Major Fish Taxa and Their Position in Vertebrate Phylogeny How Stable is Vertebrate Phylogeny? Fossils and Physiology The Environment of Early Fishes: Marine Versus Freshwater Vertebrates Conclusions References

2 4 6 9 16 28 38 39 41 45 45

Cardiovascular Systems in Primitive Fishes Anthony P. Farrell 1. 2. 3. 4. 5. 6. 7.

Introduction An Overview of Evolutionary Progressions Details of the Cyclostome Circulatory Systems Details of the Sarcopterygii (Lobe‐Finned Fishes) Circulatory Systems Details of the Circulatory Systems in Polypterids, Gars, and Bowfins Details of the Sturgeon Circulatory Systems Conclusions References

v

54 57 64 86 105 109 111 112

vi

CONTENTS

3.

Nervous and Sensory Systems Shaun P. Collin 1. 2. 3. 4. 5. 6. 7. 8. 9.

4.

Introduction Development of the CNS The Brains of Primitive Fishes Functional Classification of Cranial Nerves in Fishes The Visual System Chemoreceptive Systems Octavolateralis System Electroreception Concluding Remarks References

122 123 124 129 132 144 152 160 165 166

Ventilatory Systems Emily Coolidge, Michael S. Hedrick, and William K. Milsom 1. 2. 3. 4. 5. 6.

5.

Introduction Respiratory Strategies Respiratory Organs Ventilatory Mechanisms Respiratory Control Conclusions References

182 183 184 189 196 206 206

Gas Transport and Exchange C. J. Brauner and M. Berenbrink 1. 2. 3. 4. 5.

6.

Introduction Partitioning of O2 and CO2 Exchange Across the Respiratory Surfaces Blood O2 Transport Transport and Elimination of CO2 Synthesis References

214 214 230 253 262 270

Ionic, Osmotic, and Nitrogenous Waste Regulation Patricia A. Wright 1. 2. 3. 4.

Introduction Ionic and Osmotic Regulation Nitrogen Excretion Concluding Remarks References

284 285 291 309 310

vii

CONTENTS

7.

Locomotion in Primitive Fishes D. J. McKenzie, M. E. Hale, and P. Domenici 1. 2. 3. 4. 5. 6.

8.

Introduction Swimming Modes and Associated Morphological Adaptations Locomotor Muscles Neuromotor Coordination Locomotor Performance and Physiology Conclusions References

320 321 328 331 338 368 370

Peripheral Endocrine Glands. I. The Gastroenteropancreatic Endocrine System and the Thyroid Gland John H. Youson 1. 2. 3. 4.

9.

Introduction Endocrine Pancreas and Related Gastrointestinal Endocrine System Thyroid Gland Summary and Conclusions References

382 383 405 440 442

Peripheral Endocrine Glands. II. The Adrenal Glands and the Corpuscles of Stannius John H. Youson 1. 2. 3. 4.

10.

Introduction Adrenal Glands Corpuscles of Stannius Summary and Conclusions References

458 459 487 500 502

Why Have Primitive Fishes Survived? K. L. Ilves and D. J. Randall 1. 2. 3. 4. 5. 6.

Introduction Life During the Early Phanerozoic The Teleosts Primitive Fishes: Relationships Between Groups Why Have These Primitive Fishes Survived? Conclusions References

537

INDEX OTHER VOLUMES

516 516 518 520 530 532 533

IN THE

SERIES

561

CONTRIBUTORS The numbers in parentheses indicate the chapter(s) that the authors have written.

MICHAEL BERENBRINK (213), Integrative Biology Resarch Division, School of Biological Sciences, The University of Liverpool, Liverpool, United Kingdom COLIN J. BRAUNER (213), Department of Zoology, University of British Columbia, Vancouver, Canada SHAUN P. COLLIN (121), Vision, Touch and Hearing Research Centre, School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia EMILY COOLIDGE (181), Department of Zoology, University of British Columbia, Vancouver, Canada PAOLO DOMENICI (319), IAMC-CNR, Localita’ Sa Mardini, Torregrande (Oristano), Italy ANTHONY P. FARRELL (53), Department of Zoology, University of British Columbia, Vancouver, Canada MELINA HALE (319), Department of Organismal Biology & Anatomy, University of Chicago, Chicago, IL, USA MICHAEL S. HEDRICK (181), Department of Biological Sciences, California State University, Hayward, CA, USA K. L. ILVES (515), Department of Zoology, University of British Columbia, Vancouver, BC, Canada PHILIPPE JANVIER (1), UMR 5143 du CNRS, Muse´um National d’Histoire Naturelle, De´partement Histoire de la Terre, Paris, France D. J. MCKENZIE (319), UMR 5554 du CNRS, Institut des Sciences de l’Evolution, Station Me´diterrane´enne de L’Environnement Littoral, Se`te, France ix

x

CONTRIBUTORS

WILLIAM K. MILSOM (181), Department of Zoology, University of British Columbia, Vancouver, Canada D. J. RANDALL (515), Department of Zoology, University of British Columbia, Vancouver, BC, Canada PATRICIA A. WRIGHT (283), Department of Integrative Biology, University of Guelph, Guelph, ON, Canada JOHN H. YOUSON (381, 457), Department of Life Sciences, University of Toronto, Scarborough, Toronto, Ontario, Canada

PREFACE We had many discussions about the most appropriate title for this volume of the Fish Physiology series. “Primitive fishes” is a loose denomination that is typically used to describe species from taxonomic groups which appeared in vertebrate evolution earlier than the modern elasmobranchs and the teleosts. In this context, the term “primitive” is synonymous with the more scientifically correct “plesiomorphic,” which indicates the possession of primitive morphological characters, hence characters that occurred earlier in the fossil record than those by which dominant modern groups are defined. In most cases, primitive fishes are the extant remnants of taxa that dominated periods of the fossil record but comprise a limited number of species today. This has led them also to be described as “living fossils,” “evolutionary relics,” or “ancient fishes.” Therefore, by selecting primitive fishes, we elected for a simpler descriptor, rejecting the more scientifically robust or more emotive terms. The primitive fishes that this book focuses on include the jawless agnathans (hagfishes and lampreys), the lobe-finned sarcopterygians (coelacanth and lungfishes), and the primitive ray-finned actinopterygian fishes (the sturgeons, the bichirs and the ropefish, the gars, and the bowfin). This is, therefore, a rather diverse collection of taxa whose only universal feature is being of ancient lineage. The primitive fishes, so defined, are all interesting because they are representative of stages in the evolution of physiological systems in fishes, and in some cases also of the tetrapods. This book reviews what is known about the physiology of these unusual animals, by comparison with the two fish groups that dominate today, the modern elasmobranchs and the teleosts. The book takes a systems approach, with chapters that review and summarize what is known about cardiovascular, nervous, and ventilatory systems, gas exchange, ion and nitrogenous waste regulation, locomotion, and the endocrine systems. The cardiovascular system is crucial by virtue of its role in transporting nutrients, respiratory gases, hormones, and waste products. A chapter focuses on circulatory form and function: cardiovascular anatomy, cardiac dynamics, and cardiovascular control. Unusual adaptations of primitive fishes that xi

xii

PREFACE

deviate from features common to elasmobranchs and teleosts are highlighted, and the chapter examines the evolutionary roots and evolutionary divergence of the piscine cardiovascular system. The nervous and sensory systems of the primitive fishes are reviewed and compared, with anatomical, physiological, molecular, and behavioral data discussed in relation to both ecological and phylogenetic relationships. The peripheral and central components of the sensory systems are examined in some detail highlighting the physiological basis for behavior wherever possible. Primitive fishes exhibit a tremendous adaptive radiation in their respiratory physiologies. A chapter reviews respiratory strategies, respiratory organs, ventilatory mechanisms, and the control systems that integrate multiple exchange sites and receptors into the overall ventilatory response to environmental perturbations such as hypoxia and hypercarbia. The following chapter reviews the physiology of gas transport and exchange, in particular, those adaptations for gas exchange, such as air breathing, which may have contributed to the survival of the primitive fishes. It also considers the evolution of the Bohr/Haldane eVect, Root eVect, hemoglobin buVer value, and the appearance of the choroid rete mirabile, in the primitive ray-finned fishes. Among the primitive fishes, there is a diversity of strategies that have evolved to cope with ion, water, and nitrogen balance. A chapter reviews the regulation of salt and water balance, from the ionic and osmotic conformation seen in hagfish to the strategies for regulation of body fluids distinct from the environment that characterizes most other fish groups. Strategies for nitrogen balance are also reviewed, including urea synthesis via the urea cycle in the coelacanth and estivating lungfish. Swimming is critical to the ecology of many fishes as it determines, for example, their ability to forage, to escape predators, and to migrate. A chapter reviews the information about swimming modes, locomotor muscles, and neuromotor coordination in primitive fishes. It then compares swimming endurance, prolonged exercise performance, fast start escape responses, and recovery from anaerobic burst exercise between these fish and the modern elasmobranchs and teleosts. The peripheral endocrine systems of plesiomorphic fishes have been the focus of quite significant research. A chapter focuses on the phylogenetic development of two elements of the peripheral endocrine system, the gastroenteropancreatic (GEP) system, and the thyroid gland. In particular, evidence is provided for clear phylogenetic patterns in distribution and structure of the GEP system from protochordates to the ancient agnathans through more generalized teleosts.

PREFACE

xiii

A further chapter reviews morphological and molecular data in a phylogenetic analysis of two other elements of the peripheral endocrine system in primitive fishes. A scheme is provided of the phylogeny of the steroidsynthesizing, adrenocortical homologue, and of the catecholamine-secreting, chromaYn tissue in agnathans (hagfish and lamprey) and in bony fishes of ancient lineage. The corpuscles of Stannius first appear in the primitive actinopterygians, and the subsequent phylogenetic trends of these glycoproteinsecreting glands are reviewed. These chapters are preceded by a chapter that places the primitive fish groups within their evolutionary context relative to other vertebrates, and the volume concludes with a chapter that ponders on how each primitive fish (or fish group) might have endured while their evolutionary contemporaries have gone extinct. We wish to thank the reviewers for their suggestions and criticisms and Kirsten Funk at Elsevier for publication advice and support. David J. McKenzie Anthony P. Farrell Colin J. Brauner

1 LIVING PRIMITIVE FISHES AND FISHES FROM DEEP TIME PHILIPPE JANVIER

1. 2. 3. 4.

5.

6.

7. 8. 9. 10.

Introduction Primitive Characters, Primitive Taxa, and Ancient Taxa Living Fossils Living Primitive Fishes in Vertebrate Phylogeny 4.1. The Hagfish‐Lamprey‐Gnathostome Node 4.2. The Gar‐Bowfin‐Teleosts Node 4.3. The Coelacanth‐Lungfish‐Tetrapod Node 4.4. Other Problematic Nodes Living Primitive Fishes and Their Fossil Relatives: Naming and Dating Taxa 5.1. Hagfishes and Lampreys 5.2. Chondrichthyans 5.3. Actinopterygians 5.4. Sarcopterygians Extinct Major Fish Taxa and Their Position in Vertebrate Phylogeny 6.1. Yunnanozoans and Myllokunmingiids 6.2. ‘‘Ostracoderms’’ 6.3. Placoderms 6.4. Acanthodians 6.5. ‘‘Paleoniscoids’’ and Basal Neopterygians 6.6. Extinct Sarcopterygian Taxa How Stable is Vertebrate Phylogeny? Fossils and Physiology The Environment of Early Fishes: Marine Versus Freshwater Vertebrates Conclusions

The notion of ‘‘primitive living taxon,’’ or ‘‘living fossil,’’ largely stems from the evolutionary concepts that have pervaded systematics for nearly a century, notably the view that paraphyletic taxa are real and ancestral. Certain living taxa are regarded as primitive because some of their characters remain in a plesiomorphic state relative to their homologues in other, closely 1 Primitive Fishes: Volume 26 FISH PHYSIOLOGY

Copyright # 2007 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(07)26001-7

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PHILIPPE JANVIER

related living taxa, and this assessment rests on both out‐group comparison and fossil data. The biology of ‘‘primitive living taxa’’ is thus supposed to mirror that of the related fossil taxa they resemble. Physiologists, therefore, bet that the physiological functions of a reputedly ‘‘primitive living fish’’ are the same as those of its fossil anatomical proxies, but paleontologists often infer those of the latter on the basis of ‘‘primitive’’ living models. In some cases, such circular reasoning can be avoided by considering paleoenvironmental data that are inferred preferably from geochemical parameters. An overview of living and fossil vertebrate phylogeny, however stable it may seem, shows that there are several ways of defining and naming taxa, and that shared physiological characters of a crown group may not be extrapolated to its stem group, the divergence of which may be much earlier. Physiological characters are probably no more and no less homoplastic than morphological characters. Like the latter, they can be decomposed into series of states that can be included in fractioned and combined parsimony analyses, and can contribute to patterning the trees, instead of being interpreted a priori as adaptive and mapped as attributes on trees based on other kinds of characters. 1. INTRODUCTION By comparison to that of the morphological or molecular characters, the question of the homology of physiological characters has been little debated during the last three decades of the twentieth century, which roughly correspond to the time of the ‘‘cladistic revolution’’ in comparative biology. The reason for this neglect is that physiology was long regarded as a discipline of ‘‘general biology’’: the biology of processes, as opposed to ‘‘comparative biology,’’ that is, the biology of patterns, as outlined by Nelson (1970, 1994). There is indeed an old tradition of considering physiological characters as highly ‘‘adaptive’’; that is, they are assumed to be commonly subject to homoplasy and thus their distribution, however hierarchical it may sometimes look, tells us little about their evolutionary history. There are multiple historical reasons for this deep‐rooted belief, some of which date back to the nineteenth century, possibly with a scent of Lamarckism, but there is no clear evidence that physiological characters are more ‘‘adaptive’’ than such anatomical structures, as the pattern of the skull bones or tail skeleton morphology. Nevertheless, already in the early twentieth century, some physiologists pointed out congruences between the ‘‘laws’’ based on morphological character distributions (or evolution) and the presumed history of physiological characters. For example, Needham (1938) came to the conclusion that, following the morphology‐based ‘‘Dollo’s Law,’’ losses of physiological functions are irreversible.

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PRIMITIVE FISHES THROUGH TIME

3

During the past two or three decades, there have been some attempts at exploiting the phylogenetic message of physiological characters, notably by Løvtrup (1977), who was the first to suggest cyclostome paraphyly on this ground (see below). More recently, Cunchillos and Lecointre (2005) demonstrated that metabolic pathways could be described as nested sets of character states and coded like any other kind of characters in a data matrix aimed at reconstructing phylogenetic relationships between taxa. Thus, there are multiple ways of including physiological traits in phylogenetic analyses, by considering either the distribution of a particular function (coded as absent/present) or that of diVerent states of a function (as a hierarchy of more and more complex pathways). A classical bias in physiology‐based phylogenies is perhaps that physiologists readily know (or think they can readily know) the selective advantage of a physiological character, notably by means of experiments. Therefore, they are tempted to first make inferences about the evolution of physiological characters on the basis of the supposedly known history of the environment or behavior of an organism. In contrast, morphologists generally can make only vague inferences about the selective advantage of morphological characters, not to speak of molecular phylogeneticists, whose nucleotide sequences tell little about their impact on the phenotype. Yet comparative functional genomics may soon provide information in this field. It is thus time to restore the consideration of physiological characters as a source of potential shared homologies, irrespective of the morphological characters they are inferred from, and stop considering that their interest essentially lies in their adaptive plasticity, that is homoplasy. Physiological characters are no worse, no better than any other legacy of evolution: they provide examples of both phylogenetic messages (synapomorphies) and adaptive convergences (homoplasies), but their assessment is always relative, in the light of parsimony. There are nevertheless certainly some very robust physiological ‘‘signatures’’ in phylogeny (e.g., uric acid excretion in sauropsid amniotes), which can be regarded as being just as good node supports as, for example, gnathostome jaws or tetrapod limbs. In this introduction, I should like first to make clear that the relationships between organisms (and thus the criteria on the basis of which we decide whether the latter are ‘‘primitive’’) are exclusively based on assumptions about homology relationships between parts of these organisms, be they anatomical or physiological characters, or even nucleotide sequences. Therefore, the phylogenetic trees from which physiologists may infer evolutionary patterns are mere theories based on most parsimonious character distributions, and in which fossils provide additional character combinations, as well as information about the minimum age of characters and taxa. Then, I shall briefly depict the lost world of the ancient fossil fishes, on which rests the notion of ‘‘living primitive fishes,’’ or, more generally, ‘‘living fossils.’’

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2. PRIMITIVE CHARACTERS, PRIMITIVE TAXA, AND ANCIENT TAXA Taxa reflect relationships between organisms, which are inferred from homology relationships between parts of these organisms (Nelson, 1994). Consequently, a theory of phylogenetic relationships (and thus a phylogenetic classification) reflects the most parsimonious distribution of congruent homology relationships at one particular time and for one particular sample of characters and terminal taxa. The structure of the vertebrate tree at the level of the higher terminal taxa has been relatively stable during the past two decades, despite some divergent theories that essentially arose from conflicts between morphological and molecular sequence data (see below). However, in detail, the numerous trees of each of the higher terminal taxa, expressed in the same graphic way (a branching diagram), are derived from several, entirely diVerent, conceptual backgrounds, such as genetic distance (phenetics), parsimony, or model‐based approaches such as maximum likelihood or Bayesian approaches. Although phylogeneticists now tend to provide, for the same data set, the trees that are yielded by these respective methods and generally consider that the diVerences are minimal, this often generates confusion because subsequent authors often compare trees that are not, in fact, comparable. Phylogenetic trees are doomed to remain theories forever, some of which will be less and less frequently refuted. Therefore, the unending quest of phylogenetics (i.e., to progressively tend toward a more and more stable pattern of relationship) requires ever more data, and, above all, diVerent kinds of data of approximately equal quality. Physiological characters are certainly underexploited to this end. Perhaps genomics (i.e., parsimony‐based analysis of the organization of the genome as a whole, rather than mere sequences of particular genes) may also provide one of these new sources of data. Vertebrates are currently regarded as a taxon because they are the only living beings that share such diverse characters as migrating neural crest cells, massive gene duplication, or a labyrinth with at least one semicircular canal with ampullae. The congruence of these character distributions is, to date, not contradicted by conflicting distributions of other characters, apart from minor examples currently regarded as homoplasies. Although phylogeneticists focus their interest on the search for shared derived characters (synapomorphies) and thus regard shared primitive characters (symplesiomorphies) as uninformative, other biologists whose main interest lies in biological or evolutionary processes consider living organisms as a functional assemblage of characters that can tell us something about the biology of a hypothetical ancestor at one particular node of the tree of life. Thus, the closer to that

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5

node, the more interesting the real organisms! This is particularly true for physiologists, who pay much attention to the so‐called primitive living taxa, that is, extant taxa that retain a large number of symplesiomorphies. For example, hagfishes have a number of characters, notably physiological ones, that are lacking in all other vertebrates (lampreys and gnathostomes, or jawed vertebrates), but shared with other chordates (cephalochordates, tunicates), and other deuterostomes (Jørgensen et al., 1998). These plesiomorphous characters are thus regarded as ‘‘primitive’’ and inherited from ‘‘invertebrates,’’ and tell us nothing about the particular relationships of hagfishes but their absence (or presumed modification) in lampreys and gnathostomes does as they exclude hagfishes. However, hagfishes also share some unique anatomical characters with lampreys (Yalden, 1985), and these homology relationships might also suggest that hagfishes and lampreys are a taxon, the cyclostomes, unless these characters are lost in jawed vertebrates. Relationships between parts of organisms almost inevitably conflict, and relationships between organisms are, in principle, never stable. They depend on the number and quality of characters that biological research progressively and endlessly pours into the bag of comparative biology. The principle of parsimony is currently considered the best way to choose one theory of relationships between organisms: the one that is supported by the largest number of congruent derived character distributions beyond the bounds of chance. However, this way of considering homology relationships (and thus relationships between taxa) developed only in the 1960s, with the rise of Hennig’s (1950) phylogenetic systematics. Before that time (and occasionally still now), evolutionary systematics defined taxa on the basis of the overall resemblance of the organisms they include, and a demarcation was arbitrarily drawn between groups of organism that possessed a character and others that did not, but both groups were named and thus were equally regarded as taxa. Therefore, we still find such names as protozoans (versus metazoans), invertebrates (versus vertebrates), fishes (versus tetrapods), anamniotes (versus amniotes), and so on, used by some biologists, not in a colloquial context but as real groups of organisms whose history has a beginning and an end. The beginning of fishes is that of vertebrates, but do fishes end with the first tetrapod? In all these cases, the group that lacks the character that defines the other group is generally said to be ‘‘primitive’’ or ‘‘ancestral.’’ The former is what Hennig (1950) called a paraphyletic group (or a grade), the latter is what he called a monophyletic group (or a clade). In a historical context, these notions are admittedly relative, and fishes were monophyletic before the rise of limbs (and thus tetrapods). Grades were very convenient to paleontologists for accommodating fossils whose relationships were unclear and whose anatomy displayed

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an overwhelming number of general (i.e., plesiomorphous) characters. Among vertebrates, agnathans (jawless fishes), crossopterygians (‘‘lobe‐ finned’’ fishes), or paleoniscoids (primitive ray‐finned fishes) were such groups that are only ‘‘defined’’ by the lack of the characters of their presumed descendants. ‘‘Primitive living fishes’’ are thus such extant taxa that have once been classified in a grade along with many extinct taxa. 3. LIVING FOSSILS The term ‘‘living fossil,’’ coined by Darwin in his Origin of Species, became widespread in the literature by the end of the nineteenth century. It has been applied to a wide range of modern taxa for a variety of reasons. The concept of ‘‘living fossil’’ is in general linked to evolutionary classifications and thus to the notion that grades are taxa. The case of the living actinistian (coelacanth) Latimeria is a good example of how a taxon became regarded as a ‘‘living fossil.’’ The status of ‘‘living fossil’’ assigned to Latimeria is essentially due to the fact that actinistians (which were known only as fossils for almost a century) have long been classified in a taxon Crossopterygii (crossopterygians), which also included a number of Paleozoic taxa and is now considered to be paraphyletic because it includes stem tetrapods (rhizodontids, ‘‘osteolepiforms,’’ elpistostegalians) and stem dipnoans (porolepiforms, youngolepidids; Figure 1.1). All the ‘‘crossopterygian’’ characters that Latimeria shares with these taxa are merely general sarcopterygian or even osteichthyan characters (e.g., monobasal‐paired fin skeleton, intracranial articulation, large notochord, Figure 1.1A), which have been modified or lost in most of the living descendants of ‘‘crossopterygians’’ (Figure 1.1C and D). Although actinistians in general show a relatively stable morphology during the past 380 million years (Myr), Latimeria is no more a ‘‘living fossil’’ than many other fish taxa, such as lungfishes, and probably less so, unless by the virtue of its retaining a few plesiomorphous characters, such as the intracranial joint (Figure 1.1B). The survival of ‘‘living fossils’’ is thus rather a survival of some characters that were once general, but have largely been lost in related extant taxa (Janvier, 1984). The concept of ‘‘living fossil’’ can also be seen in a diVerent way, that is when conspicuous specializations appear very early in phylogeny, and are retained during a long period of morphological stability. This refers to the ‘‘panchronic’’ taxa which, among fishes, are relatively rare. Example of such ‘‘panchronic taxa’’ are hagfishes and lampreys (Figure 1.2). The earliest fossil hagfishes are 305 Myr old, and the earliest lamprey 360 Myr, and, as far as it can be seen from fossils preserved as soft‐tissue imprints, looked practically identical to the living hagfishes and lampreys (Figure 1.2A, C, E, and G).

1.

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Sarcopterygians B

0 Myr

C

Latimeria

D

Lungfishes

Tetrapods

299 Myr

"Crossopterygians" A 443 Myr Fig. 1.1. Latimeria as a ‘‘living fossil.’’ In typical evolutionary classifications, Latimeria is often referred to as the only living ‘‘crossopterygian,’’ an essentially Paleozoic, paraphyletic group that includes stem lungfishes and stem tetrapods (in gray). ‘‘Crossopterygians’’ are in fact diagnosed on the basis of characters that have been lost or modified in their descendants, lungfishes and tetrapods. One of these characters is the intracranial joint (arrowheads), a general (plesiomorphous) character of the sarcopterygian braincase (A), conserved in Latimeria (B), but lost in lungfishes (C) and tetrapods (D). (A) Braincases of Nesides, (B) a late Devonian actinistian, Latimeria, (C) Neoceratodus, and (D) a primitive Carboniferous tetrapod. [Based on Janvier (1984, 1996a) by permission of Oxford University Press.]

Hagfishes already possessed the characteristic tentacles (Figure 1.2B and D) and lampreys already possessed a piston cartilage (hence a protractible and retractable ‘‘tongue’’), an annular cartilage surrounding the mouth (Figure 1.2F and H), and a sucker (Figure 1.2I and J; see also Gess et al., 2006 for new data). Such cases of panchronic taxa are perhaps the only ones that can actually be referred to as ‘‘living fossils’’ since it does not rest on the retention of a few plesiomorphous characters, but the conservation (or survival) of a large number of apomorphous characters that appeared early in time and have remained unmodified (Janvier, 1984). Large gaps in the fossil record, like in the case of Latimeria (for which there is no Tertiary record), are also regarded as a criterion to assess a living taxon as a ‘‘living fossil.’’

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A

Prenasal duct B Tentacles D C

Prenasal duct

Tentacles E

F Annular cartilage Piston cartilage G

H Annular cartilage Piston cartilage I

J

K

Fig. 1.2. Hagfishes and lampreys are regarded as ‘‘living fossils,’’ not because they retain characters that have once been general to a larger group but because they have gained some highly derived characters that remained extremely stable throughout time. The overall morphology (A) and skull structure (B) of living hagfishes strikingly resemble those of the late Carboniferous hagfish Myxinikela (C and D). Although Myxinikela is somewhat stouter in body shape than the living forms (C), it already displays the main unique characters of the living hagfish head skeleton (D), such as the tentacles and long prenasal duct. Similarly, the overall morphology (E) and head skeleton of the living lampreys (F) are almost identical to those of the late

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The notion of ‘‘living fossil,’’ in common with evolutionary systematics, remains rather vague and subjective. This is reflected in Eldredge and Stanley’s (1984) review of the diVerent taxa that may be regarded as such and which includes elopomorph teleosts but, strangely, not lampreys. Whatever the ground for regarding a taxon as a ‘‘living fossil,’’ it always rests on morphology since this is essentially what is known in fossils. It is, of course, tempting to consider that if the morphology of an extant taxon has undergone little change through time, the same may apply to its physiological characters that leave no fossil record. This postulate, which cannot be tested by direct evidence, is nevertheless often regarded as the only means for attempting a reconstruction of the history of physiological characters. Thus, the only possible test for theories about physiological character phylogeny is their congruence with molecular sequence‐based phylogenies of living taxa and/or morphological character distributions in living and fossil taxa. 4. LIVING PRIMITIVE FISHES IN VERTEBRATE PHYLOGENY Living fish taxa that are traditionally regarded as ‘‘primitive’’ are essentially the jawless vertebrates or agnathans [hagfishes (Hyperotreti) and lampreys (Hyperoartia)], and, among the gnathostomes, some chondrichthyans [batomorphs (sawfishes, torpedoes, skate, and rays), hexanchiform sharks, Chlamydoselachus, and chimaeriforms], cladistians (i.e., polypteriformes or bichirs), acipenceriforms (sturgeons and paddlefishes), ginglymods (or lepisosteiforms, i.e., gars), Amia calva (bowfin), actinistians (Latimeria), and lungfishes (or dipnoans). In addition, the osteoglossomorphs (e.g., bony‐ tongues) and elopomorphs (e.g., tarpons, eels) are sometimes regarded as examples of primitive teleosts. The trees in Figure 1.3 show the relationships of the major living vertebrate taxa, with special reference to the so‐called ‘‘primitive fishes.’’ The fully resolved tree on the left‐hand side shows the phylogeny that is most widely accepted by morphologists and paleontologists. The tree on the right‐hand side shows a consensus of the various trees based on either morphological or molecular sequence data, which may yield slightly diVerent topologies (hence, the debated Carboniferous Mayomyzon (G and H). Both share the characteristic annular and piston cartilages. The late Carboniferous Pipiscius (I), regarded as a fossil lamprey, also possessed an oral funnel (J) armed with horny plates, which strikingly resembles that of living lampreys (K). Scale bars ¼ 10 mm. [A–C, E–G, J, and K, from Janvier (1984, 1996a by permission of Oxford University Press); H, redrawn and modified from Bardack and Zangerl (1968); D, redrawn and modified from Bardack (1991); and I, redrawn and modified from Bardack and Richardson (1977).]

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Hagfishes Lampreys 1

Sharks 5 Batomorphs

4

A

2 Chimaeriforms Cladistians (Polypteriformes) 7

Acipenserids

9

3 Polyodontids 8 Ginglymods (lepisosteids) 10 Amia

B

11 Osteoglossomorphs

6 12 13

Elopomorphs Other teleosts

D

Latimeria 14 15

Neoceratodus C Protopterus

16 17

Lepidosiren

Tetrapods Fig. 1.3. Interrelationships of the living vertebrates, with particular reference to the reputedly primitive fishes. The tree on the right‐hand side shows the four major polytomies (A–D), which are essentially due to conflicts between morphological and molecular sequence data. The question of the relationships of sharks and batomorphs is regarded here as resolved as there is increasingly strong support for their sister‐group relationships (thus shark monophyly). The tree on the left‐hand side depicts the topology that is most widely accepted by morphologists

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nodes A–D for which diVerent data sets provide diVerent topologies for the more crownward taxa). There are, however, some widely diVerent, molecular sequence‐based phylogenies, notably that proposed by Arnason et al. (2001, 2004) and Rasmussen and Arnason (1999a,b), one of which is shown here in Figure 1.4. To date, these phylogenies are diYcult to reconcile with most of the currently available morphological data as they support fish monophyly but not osteichthyan, sarcopterygian, and actinopterygian monophyly. However, provided that they are merely noise due to inappropriate gene sequences, these ‘‘odd phylogenies’’ (Janvier, 1998) are not to be ignored and should stimulate a new look at certain conflicting character combinations displayed by some early fossil chondrichthyans and osteichthyans (Janvier, 1998; Zhu et al., 1999, 2006; Maisey, 2001), which, at any rate, suggest that some of the classical osteichthyan and sarcopterygian characters may be more general than currently believed. Current morphology‐based gnathostome phylogenies (basically that in Figure 1.3, left‐hand tree), notably the assumption that chondrichthyans are the plesiomorphous sister group of osteichthyans, stem from (or is consistent with) Huxley’s (1880) conception that chondrichthyans were ancestral to osteichthyans. Most molecular sequence‐based phylogenies have proven to be consistent with the monophyly of these two respective groups (Hedges, 2001; Zardoya and Meyer, 2001). Arnason et al. (2004) pointed out, however, that molecular (mitogenomic) sequence‐based osteichthyan phylogenies may be biased by the fact that they are generally rooted with chondrichthyans, rather than being rooted with either lampreys or hagfishes. When considering the current (or ‘‘conventional’’) consensus tree for higher vertebrate taxa, only three major ‘‘piscine’’ nodes remain controversial: and paleontologists. Main clades and selected characters applying to living taxa: 1, craniates (migrating neural crest cells, epidermal placodes, skull, olfactory, optic, and otic capsules); 2, vertebrates (arcualia, extrinsic eye muscles, radial muscles in unpaired fins); 3, gnathostomes (jaws, horizontal semicircular canal, paired fins, calcified or ossified endo‐ and exoskeleton, epicercal tail); 4, chondrichthyans (prismatic calcified cartilage, pelvic claspers); 5, elasmobranchs (posteriorly directed basibranchials, paired occipital condyles); 6, osteichthyans (endochondral bone, large dermal bones covering the head and shoulder girdle, lepidotrichs); 7, actinopterygians (only one dorsal fin, primitively ganoid scales, acrodin cap on teeth, everted telencephalon); 8, actinopterans (fringing fulcla on the leading edge of fins); 9, acipenseriforms (anterior symphysis of palatoquadrate); 10, neopterygians (unpaired fin lepidotrichs equal in number to their supports); 11, halecostomes (mobile maxillae); 12, teleosts (median tooth plate on basihyal, mobile premaxillae); 13, elopocephalans (two uroneurals); 14, sarcopterygians (monobasal paired fins, pulmonary vein, veina cava); 15, lungfishes (massive entopterygoid and prearticular tooth plates); 16, lepidosirenidae (reduced paired fins); and 17, rhipidistians, or choanates (alveolae in lungs, partially divided and sigmoid arterial cone in heart, incipient atrial septum). [Illustrations for terminal taxa from Janvier (1996a) by permission of Oxford University Press.]

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Lampreys

Tetrapods Cladistians (polypteriformes)

Sharks Chondrichthyans Batomorphs

Chimaeriforms

Latimeria Neoceratodus

Lungfishes

Protopterus Lepidosiren Acipenserids

Acipenseriforms

Polyodontids Ginglymods (lepisosteids)

Amia Teleosts Fig. 1.4. Mitochondrial DNA‐based vertebrate tree (hagfishes not considered) proposed by Arnason et al. (2004). This tree is strongly at odds with the current consensus (compare to Figure 1.3), notably by the breakdown of the osteichthyans, sarcopterygians, actinopterygians, and neopterygians. [Illustrations for terminal taxa from Janvier (1996a) by permission of Oxford University Press.]

the hagfish‐lamprey‐gnathostome node, the gar‐bowfin‐teleost node, and the coelacanth‐lungfish‐tetrapod node. However, one must concede, in agreement with Arnason et al. (2004), that the morphological support to the osteichthyan clade is relatively low and even more so when early fossil taxa are considered.

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4.1. The Hagfish‐Lamprey‐Gnathostome Node Since the early nineteenth century, lampreys and hagfishes have long been gathered in a group, the Cyclostomi (cyclostomes) characterized by horny ‘‘teeth’’ covering a protractible and retractable ‘‘tongue,’’ and pouch‐shaped gills. All other cyclostome features are either absence of gnathostome characters or characters whose state cannot be assessed on the basis of out‐group comparison (i.e., not applicable to nonvertebrate chordate taxa). Since the mid‐twentieth century, morphologists and physiologists had long been aware that lampreys were in many respects more similar to gnathostomes than to hagfish, but the apparently more ‘‘simple’’ or ‘‘invertebrate‐like’’ characters of hagfishes were regarded as a consequence of ‘‘degeneracy,’’ due to their legendary endoparasitic habits (already alluded to by Linnaeus; Jørgensen et al., 1998). Løvtrup (1977) was the first to suggest clearly that this appeal to ‘‘degeneracy’’ was groundless and that lampreys display an overwhelming number of morphological and physiological characters shared only with gnathostomes, which suggest sister‐group relationships between these two taxa (2, Figure 1.3). Then, cyclostome paraphyly became progressively accepted by many comparative biologists (Hardisty, 1982; Maisey, 1986; Janvier, 1996a,b). In contrast, molecular sequence‐based phylogenies tended to support cyclostome monophyly (Stock and Whitt, 1992; Delarbre et al., 2000; Hedges, 2001; Mallatt et al., 2001), although analysis of data sets partitioned into small and large subunit components provided conflicting support for both monophyly and paraphyly (Zrvav’y et al., 1998). Cyclostome monophyly in molecular sequence‐based phylogenies was first regarded as an artifact of long‐branch attraction, but it is still strongly supported by Bayesian methods, which are supposed to minimize this bias (Furlong and Holland, 2002). Some molecular sequence‐based data, however, rather support the position of lampreys as sister group to gnathostomes (cyclostome paraphyly) such as those based on small RNA units (Gursoy et al., 2000). Currently, the problem is still unresolved. To morphologists and physiologists, cyclostome monophyly would imply an impressive number of either reversions in hagfishes or convergences in lampreys and gnathostomes. 4.2. The Gar‐Bowfin‐Teleosts Node Gars and bowfins have long been grouped in a group called Holostei (holosteans), along with a number of fossil neopterygian actinopterygian taxa (e.g., semionotids, parasemionotids, macrosemiiforms). However, Patterson (1973) regarded bowfins as more closely related to teleosts than gars. Bowfins and teleosts were thus gathered in the clade Halecostomi (halecostomes), characterized notably by mobile maxillae (11, Figure 1.3). Apart from a

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paleontology‐based phylogeny proposed by Olsen and McCune (1991), very few anatomical data support a sister‐group relationship between gars and teleosts. Similarly, no anatomical data supports the topology proposed by Inoue et al. (2003) and Arnason et al. (2004), in which gars, bowfins, and acipenseriforms form an unresolved clade, sister to the teleosts (Figure 1.4). Conversely, a reconsideration of fossil neopterygians anatomy (notably for early gars, bowfins, semionotids, and parasemionotids) as well as molecular sequence‐based neopterygian phylogenies (Kikugawa et al., 2004) now strongly supports holostean monophyly again; that is, gars and bowfins would be sister groups. 4.3. The Coelacanth‐Lungfish‐Tetrapod Node Piscine sarcopterygians are only represented by two strongly depauperized living taxa: actinistians (coelacanths) and dipnoans (lungfishes). The living coelacanths are represented by Latimeria chalumnae from the Strait of Mozambique, and possibly a second species Latimerai menadoensis from Sulawezi (Indonesia). Lungfishes fall into three genera, the Australian Neoceratodus (one species), the African Protopterus (four species), and the South American Lepidosiren (one species). Neoceratodus is the sister group of the clade Lepidosirenidae, which includes the other two genera (15, Figure 1.3). Although long debated, the question of actinistian‐lungfish‐tetrapod relationships is now generally regarded as settled by morphologists and paleontologists (Rosen et al., 1981; Cloutier and Ahlberg, 1996; Zhu et al., 2006), with coelacanths being sister to lungfishes and tetrapods, despite a few anatomical data that may support an actinistian–tetrapod sister‐group relationship. In contrast, molecular data remain ambiguous on this issue (Zardoya and Meyer, 1997, 2001; Zardoya et al., 1998; Brinkmann et al., 2004; Takezaki et al., 2004). Molecular sequence‐based trees sometimes show coelacanths as sister to tetrapods and sometimes as sister to lungfishes. Only the molecular sequence‐based phylogeny proposed by Arnason et al. (2004) is strongly at odds with the current consensus, as it shows monophyletic living fishes, with coelacanths, chondrichthyans, lungfishes, and actinopterans (i.e., actinopterygians minus cladistians) as forming an unresolved clade, sister to cladistians (Figure 1.4). 4.4. Other Problematic Nodes The in‐group relationships of some other higher piscine gnathostome taxa are also the subject of controversies, yet to a lesser degree. Within elasmobranch chondrichthyans, the relationships of batomorphs (pristiophoriforms,

1.

PRIMITIVE FISHES THROUGH TIME

15

sawfish, torpedoes, skates, and rays) have long been debated, but, during the past 20 years, most morphologists agreed that batomorphs were nested within the squalomorph sharks as sister group to pristiophoroids (Maisey, 1984; Shirai, 1996). Molecular sequence‐based phylogenies do not clearly support this relationship and suggest, rather, that batomorphs are the sister group of all living sharks (Arnason et al., 2001). Recent consideration of this question, involving collaboration between morphologists, paleontologists, and molecular phylogeneticists, has provided much stronger support for this theory, which entails living shark monophyly (Maisey et al., 2004; Figures 1.3 and 1.4). It now appears that living shark monophyly is actually supported by a number of morphological characters, but previous parsimony analyses considered too many doubtful characters, which now turn out to be homoplastic. Cladistians (bichirs) are yet another taxon whose phylogenetic position has been much debated since the nineteenth century. Cladistians share some unique skeletal characters with actinopterans (i.e., acipenseriformes and neopterygians), such as the ganoid scale structure or the acrodin cap on teeth, and numerous soft‐tissue characters, notably in brain development (everted telencephalon) and muscles of the jaw and gill arches. Most other characters seen in cladistians are unique to them (pectoral fin and dorsal fin structures), but a few characters were once regarded as either shared only with sarcopterygians, notably actinistians, or supposedly primitive for osteichthyans in general (Jarvik, 1980). Cladistians are almost unanimously regarded as the sister group to actinopterans (7, Figure 1.3), but molecular sequence data remain ambiguous in this respect. Early sequence‐based gnathostome phylogenies generally ignored cladistians because they made trees either collapse or display very odd topologies, as in Arnason et al.’s (2004) tree (Figure 1.4), where they appear as the sister group of all other living fishes. Other molecular trees show a more conventional position for cladistians as sister to actinopterans (Noack et al., 1996). The question may not be entirely settled as long as the early phase of cladistian history, possibly in early Devonian times, remains undocumented by fossils. Within teleosts, osteoglossomorphs (osteoglossids, arapaimids, mormyrids, and hiodontids) and elopomorphs (elopids, megalopids, albuloids, nothacanthids, anguilloids, and saccopharyngids) are currently considered as the most inclusive living teleost taxa. However, the relationships of these two taxa to other teleosts (i.e., clupeocephalans) remain debated. A classical theory is that osteoglossomorphs are the sister group of elopomorphs and all other living teleosts (Patterson, 1977; Patterson and Rosen, 1977; 12 and 13, Figure 1.3), but this has been challenged over the last decade, notably by Arratia (2004), who favors the theory that elopomorphs are the sister group of osteoglossomorphs and all other living teleosts. The latter result is a

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consequence of the new inclusion of a large number of extinct Cretaceous and Jurassic teleost taxa in data matrices. Other phylogenies, either molecular and morphological, suggest that either the two groups form a clade, sister to all other living teleosts (Leˆ et al., 1993), or the relationships of osteoglossomorphs, elopomorphs, and the ensemble of all other living teleosts are unresolved (Li and Wilson, 1996) (D, Figure 1.3). Elopomorph monophyly has long been regarded as strongly supported by at least the leptocephalous larva, supposed to be a uniquely derived condition. However, by combining sequence‐based and morphology‐based analyses, Filleul and Lavoue´ (2001) have challenged elopomorph monophyly. Surprisingly, they suggest that the leptocephalous larva may be a primitive condition for teleosts and that eels are the sister group of osteoglossomorphs.

5. LIVING PRIMITIVE FISHES AND THEIR FOSSIL RELATIVES: NAMING AND DATING TAXA Most of the reputedly primitive fish taxa referred to above have a large number of fossil relatives. However, the fossil record for some of these taxa remains desperately poor, as is notably the case for hagfishes and lampreys because they lack an extensively mineralized skeleton and can only be fossilized under particular conditions. The principle of a molecular clock (which followed in the wake of phenetics) was based on the assumption of a constant mutation rate (now regarded as unfounded; Maisey et al., 2004) and required accurate, paleontology‐based calibrations of divergence times for taxa that have extant representatives. Bracketing divergence times thus became a raison d’eˆtre for paleontologists working on early vertebrates (see Donoghue et al., 2003 for a review of the question). The phylogenetic tree of any taxon that includes living and fossil representatives comprises a ‘‘crown group’’ and a ‘‘stem group’’ (Figure 1.5). The crown group includes the youngest common ancestor to all the living representatives of the taxon under consideration and their respective fossil relatives (A1, Figure 1.5). The stem group includes all the taxa that have diverged before the common ancestor of the crown group and after the youngest common ancestor it shares with its living sister group (A2, Figure 1.5). For example, assuming that lampreys are the sister group of gnathostomes, crown‐group gnathostomes include the youngest common ancestor to a shark and a tetrapod and all its other living and fossil descendants such as actinopterygians, piscine tetrapodomorphs, or extinct chondrichthyan taxa (A1, Figure 1.5). Stem gnathostomes include all the extinct taxa

17

Sa la m an s pi le

Eu st he no pt er on

s El op iro

st eo st O

Xe na ca nt hu s

ca tra er os et H

ra ca Pl ns ac od er m s

L

ns

t0

us

al

u Sq

he

s

ey

pr

am

dr

a

PRIMITIVE FISHES THROUGH TIME

C

1.

A1 Jaws A2

Bone

Pectoral fins

Fig. 1.5. The structure of taxa. When considering the living (above t0) and fossil (below t0) representatives of a taxon, there are various ways of considering its composition and definition, as exemplified here by some fossil and living gnathostomes. The crown‐group gnathostomes (gray) include the most recent common ancestor of the three major living gnathostome groups, namely chondrichthyans (e. g., Squalus), actinopterygians (e.g., Elops), and sarcopterygians (e.g., Salamandra), as well as all their respective extinct fossil relatives (e. g., Xenacanthus, Cheirolepis, Eusthenopteron) (A1). The stem‐group gnathostomes (light gray) include all the extinct taxa (e.g., heterostracans, osteostracans, and placoderms) that have diverged before the common ancestor to crown‐group vertebrates (A1) and after the most recent common to crown‐ group gnathostomes and lampreys (A2; assuming that lampreys are the sister group of gnathostomes). Some stem‐group gnathostomes are jawless (heterostracans, osteostracans), whereas others are jawed (placoderms). The total‐group gnathostomes (dark gray) include both the stem‐ and crown‐group gnathostomes, that is, all fossil or living vertebrates that are more closely related to any member of the crown‐group than to lampreys. An apomorphy‐based taxon is defined on the basis of at least one particular derived character that supports a node of the tree. Apomorphy‐based gnathostomes defined on the basis of the presence of jaws would include both the crown‐group gnathostomes and some stem gnathostomes (placoderms). [Illustrations for terminal taxa from Janvier (1996a), by permission of Oxford University Press.]

that diverged before the divergence between any crown‐group gnathostome but after the divergence between lampreys and the latter (A2, Figure 1.5). Some of these stem gnathostomes have jaws (e.g., placoderms), whereas others do not (e.g., heterostracans and osteostracans), but they all share at least one uniquely derived character with crown‐group gnathostomes (the most general one being the ability to produce bone). The ‘‘total group’’ includes the crown group and the stem group. The crown group and the total group are clades (monophyletic taxa), the stem group is a grade (paraphyletic taxon). The next question is how to name these diVerent segments of

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a tree. This is currently a much debated matter among certain systematicists, within the framework of the Phylocode debate (for information about the Phylocode, see http://www.ohiou.edu/phylocode). In brief, there are three ways of considering a clade: node‐based (e.g., a crown group), stem‐based (i.e., a total group), or apomorphy‐based (i.e., all organisms that display a particular, uniquely derived character, e.g. pectoral fins or jaws; Figure 1.5). Consequently and depending on which type of clade one considers for a taxon name, the range of this taxon in time (thus its earliest occurrence) may vary considerably. Biologists who are interested in the minimum age of characters that cannot be directly documented by fossils (e.g., gene sequences or physiological functions) generally prefer to use node‐based crown groups. However, one must keep in mind that a node is a matter of data set, methodological procedure, and options in phylogenetic softwares. Therefore, it may not always refer to any conspicuous character and is often labile. Examples are the node‐based definition of tetrapods (Laurin and Anderson, 2004), which does not refer to the presence of digits and limbs, or the node‐based definition of gnathostomes, which does not refer to jaws (Figure 1.5). Apomorphy‐ based clades may be of interest to ecophysiologists, as certain fossil taxa that are members of a stem group may provide indirect evidence for a particular function in the form of a unique combination of anatomical structures. An apomorphy‐based definition of gnathostomes would include the presence of jaws (Figure 1.5), which actually are ecologically important structures. In contrast, an apomorphy‐based definition of tetrapods would include limbs with digits, supposedly important for the conquest of land, but the taxon would also comprise such stem tetrapods as Acanthostega (see tree in Figure 1.12) in which limbs and digits had no role in terrestrial locomotion. The interest of stem‐based taxa is that they may provide information about a ‘‘ghost range,’’ that is a segment of the phylogeny of a taxon that is not documented by fossils but which must have existed because its sister group has earlier representatives (Figure 1.6). For example, assuming that the most general character of the total‐group gnathostomes is the presence of bone, which occurs first in the early Ordovician, the minimum age of this total group is about 475 Myr (Figure 1.6A). In contrast, the earliest record of lampreys is only 360 Myr (Figure 1.6). Now, assuming that lampreys alone are the sister group of the gnathostomes, one may infer that lampreys and/or hagfishes have a ghost range of at least 115 Myr. This ghost range may even be increased by 25 Myr if the character ‘‘bone’’ is extended to ‘‘mineralized dermal skeleton’’ in general, and if one considers that the denticles of euconodonts (‘‘conodonts’’; see Figure 1.15) actually are evidence for dermal skeleton and thus that euconodonts are stem gnathostomes (Figure 1.6B) (Donoghue and Sansom, 2002).

1.

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0

B

La m pr ey G na s th os to m es La m pr ey s G na th os to m es

A

299

488

475 500

542 Fig. 1.6. The length of the ‘‘ghost ranges’’ that are inferred from phylogenetic trees may vary considerably, depending on the topology of the latter. Assuming that lampreys are the sister group of the total‐group gnathostomes and that the earliest evidence for the latter are early Ordovician arandaspid remains, the minimum age for the divergence between the two taxa is about 475 Myr (A). The ghost range of the lampreys (in gray) is thus about 115 Myr. However, if euconodonts are regarded as stem gnathostomes, the minimum age for the lamprey‐gnathostome divergence would be about 500 Myr (B), and the ghost range of lampreys about 140 Myr.

5.1. Hagfishes and Lampreys Only two fossils, Myxinikela and Myxineides, both late Carboniferous (305 Myr) in age, are referred to hagfishes. Myxinikela (Figure 1.2C and D) is perhaps the most convincing fossil hagfish as it shows traces of the typical nasal basket, prenasal sinus, and tentacles, but its body is stouter than that of living hagfishes (Bardack, 1991). Myxineides shows no clear evidence for tentacles, but the internal cast of its oral cavity clearly shows the imprint of the two V‐shaped rows of horny teeth. Its body is eel‐shaped, like in modern hagfishes (Poplin et al., 2001). Myxinikela occurs in marine sediments, but Myxineides poses a problem, as it occurs in reputedly lacustrine sediments, whereas hagfish physiology supposedly precludes freshwater habits. Two fossil lampreys, Mayomyzon and Hardistiella, are known from the late Carboniferous (Bardack and Zangerl, 1968; Janvier and Lund, 1983). Mayomyzon (Figure 1.2G and H) is from the same locality as the hagfish Myxinikela, and its excellent preservation leaves little doubt about its assignment. Certain specimens, preserved in lateral view, display imprints of exactly

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the same cartilages as in modern lampreys, notably a piston cartilage, large tectal cartilages, and an annular cartilage (Figure 1.2F and H). Another form from the same locality, Pipiscius (Figure 1.2I and J), may also be a lamprey and possesses an oral funnel armed with horny plates that recall those of modern lampreys (Figure 1.2K; Bardack and Richardson, 1977). Hardistiella is about 20‐Myr older than Mayomyzon. It is quite similar to the latter but displays less distinct cartilage imprints. The recent discovery of the first Devonian (360 Myr) lamprey, Priscomyzon (Gess et al., 2006), also confirmed the remarkable conservatism of lamprey morphology through time. These fossil lampreys only diVer from the living ones by their lack of separate dorsal fins and their shorter branchial basket. It is diYcult to assign these fossil hagfishes and lampreys a particular position relative to the members of their respective crown groups, all the more so because there are no reliable phylogenies for the living representatives of these two taxa. Nevertheless, it is likely that the Carboniferous forms are stem hagfishes and stem lampreys, respectively. The apomorphy‐based minimum age of these two taxa is thus
300 Myr for hagfishes and 360 Myr for lampreys, and the stem‐based minimum age for both is from 475 to about 500 Myr (see above). 5.2. Chondrichthyans The morphological disparity of the living chondrichthyans is significantly lower than the late Paleozoic ones. The trees in Figures 1.7 and 1.8 show reconstructions of some representatives of the major Paleozoic elasmobranch‐ and chimaeriform‐related extinct chondrichthyan taxa. The overall morphology of some of them is grossly sharklike (e.g., ctenacanthiforms, eugeneodontids), but others, in particular stem chimaeriformes, display a most unusual aspect (e.g., petalodontids, iniopterygians, chondrencheliids). All living elasmobranchs (sharks and rays) are neoselachians, whose earliest undisputable representatives are only early Jurassic (199 Myr) in age (3, Figure 1.7). However, on the basis of their structure, some isolated scales and teeth suggest, albeit with great reservations, a Carboniferous and even Devonian neoselachian record. By the early Jurassic, several major living neoselachian higher taxa were already represented, notably batomorphs, hexanchoids, heterodontoids, and orectoloboids, and in the late Jurassic appear the earliest carcharinoids, lamnoids, and squatinoids (Maisey et al., 2004). Thus, the minimum age for crown‐group neoselachians is about 200 Myr, but a number of Triassic and even Permian taxa are regarded as stem neoselachians on the basis of various anatomical characters. Major extinct elasmobranch clades are the Carboniferous to Cretaceous hybodontiforms, the sister group of neoselachians, and the essentially Paleozoic ctenacanthiforms and xenacanthiforms (Figure 1.7). The earliest evidence for elasmobranch

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PRIMITIVE FISHES THROUGH TIME 542 Cam 488 Ord 443 Sil 416 Dev 359 Carb 299 Perm 251 Tr 199 Jur 145 Cret 65 Cen

1.

Cladoselache ? Symmoriids and stethacanthids Xenacanthiforms Ctenacanthiforms

1

Hybodontiforms Paleospinax Chlamydoselachus Hexanchoids 2 Pristiophoroids

Squatinoids Squaloids 3 Heterodontoids Orectoloboids Lamnoids

Carcharhinoids Batomorphs (rays, skates, torpedoes, and sawfishes) Fig. 1.7. Elasmobranch phylogeny (the out‐group being euchondrocephalans). Geologic timescale in Myr, with dates referring to the limits between periods (Cam, Cambrian; Carb, Carbonife`re; Cen, Cenozoic; Cret, Cretaceous; Dev, Devonian; Jur, Jurassic; Ord, Ordovician; Perm, Permian; Sil, Silurian; Tr, Triassic). Paleozoic in white, Mesozoic in light gray, Cenozoic in dark gray. Horizontal bars in the timescale indicate the distributions of the taxa through time. Interrelationships of batomorphs not detailed. Carcharhinoids include scylliorhinids. Symmoriids and

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based on articulated material is from the early Devonian (400–410 Myr) (Figures 1.7 and 1.15). Living holocephalans, or chimaeriforms, are the relicts of a very diverse ensemble of chondrichthyans, referred to as ‘‘euchondrocephalans’’ (Grogan and Lund, 2004) (1, Figure 1.8). Living chimaeriform overall morphology is certainly much derived, relative to the general chondrichthyan morphology, but it appeared early in time, probably in the late Paleozoic (e.g., Echinochimaera, Figure 1.8). The living chimaeriforms only consists of six genera distributed in three families: the Callorhinchidae, Rhinochimaeridae, and Chimaeridae, the former being sister group to the latter two (Didier, 2004) (3, Figure 1.8). The phylogeny of the crown‐group chimaeriforms is thus quite clear, but informative fossils referred to these three families are relatively rare, hence the diYculty to infer their minimum age. Nevertheless the earliest representatives of the crown‐group chimaeriforms are late Jurassic callorhinchids, and egg capsules referred to rhinochimaerids are known since the early Triassic (245 Myr) (Stahl, 1999). Stem chimaeriforms are known since the late Devonian (370 Myr) and include notably the ‘‘bradyodonts’’ (e.g., chondrenchelyids), a paraphyletic ensemble of Paleozoic taxa which display chimaeriform‐like features such as holostyly and tubular tooth structure. Apart from the in‐group phylogeny of elasmobranchs and chimaeriforms, chondrichthyan phylogeny remains tenuously supported, and the phylogenetic position of a number of fossil higher taxa, such as cladoselachids, edestids, petalodontids, symmoriids (including stethacanthids), inopterygians, or Pucapampella, remain extremely labile (Figures 1.7, 1.8, and 1.15). 5.3. Actinopterygians The fossil record of cladistians is very poor and the earliest evidence for this taxon are isolated scales, dorsal fin pinnules, and vertebrae from the early Cretaceous of Africa. Serenoichthys, from the late Cretaceous (98 Myr) freshwater deposits of Morocco (Dutheil, 1999), is the only fossil cladistian known from articulated specimens (Figure 1.9). It is remarkably similar to modern cladistians, except for its stouter body shape, but certainly represents a stem cladistian as it is not the sister group of one or the other living cladistian genera, Polypterus and Erpetoichthys. Considering stethacanthids are regarded as single clade, but their relationships remain debated, and it is not inconceivable that they are in fact basal euchondrocephalans (Janvier, 1996a). The clade that includes xenacanthiforms and all the more crownward taxa is better supported, and is therefore regarded as the most reliable total‐group elasmobranchs. 1, elasmobranchs; 2, euselachians; and 3, neoselachians. [Illustrations for terminal taxa from Janvier (1996a), by permission of Oxford University Press.]

23

Cen

Cret

65

PRIMITIVE FISHES THROUGH TIME 542 Cam 488 Ord 443 Sil 416 Dev 359 Carb 299 Perm 251 Tr 199 Jur 145

1.

Petalodontids

Helodontids 1 Iniopterygians

Eugeneodontids Chondrenchelyids 2 Echinochimaera

Callorhinchids 3 Chimaerids

Rhinochimaerids Fig. 1.8. Euchondrocephalan phylogeny (the out‐group being elasmobranchs). For abbreviations to the geologic timescale, see Figure 1.7. 1, euchondrocephalans; 2, holocephalans; and 3, chimaeriforms. [Illustrations for terminal taxa from Janvier (1996a), by permission of Oxford University Press.]

that (according to current morphology‐based actinopterygian phylogenies) cladistians are supposed to have diverged from actinopterans before such late Devonian taxa as Moythomasia (Coates, 1999; Cloutier and Arratia, 2004) and, at any rate, before the earliest crown‐group actinopterans (2 and 4, Figure 1.9), the rather late occurrence of the earliest cladistians remains a riddle. It is possible that earlier (Paleozoic) stem cladistians are in fact known, but we do not recognize them because they lack conspicuous cladistian characters. Lund (2000) suggested that the Carboniferous guildayichthyiform actinopterygians are the fossil sister group of cladistians,

24 542 Cam 488 Ord 433 Sil 416 Dev 359 Carb 299 Perm 251 Tr 199 Jur 145 Cret 65 Cen

PHILIPPE JANVIER

? Dialipina Cheirolepis Serenoichthys

3

1

Polypterus Moythomasia

2

Birgeria 6

4

Chondrosteus 7 Acipenserids Polyodontids

5

Australosomus 10

Semionotids

8

Ginglymods

Parasemionotids 11 9 Caturids

12

Amiids Pholidophorus

13

Crown-group teleosts Fig. 1.9. Actinopterygian phylogeny. For abbreviations to the geologic timescale, see Figure 1.7. The assignment of Dialipina to the actinopterygians still rests on tenuous characters, notably scale histology. In this tree, we regard as undoubted actinopterygians Cheirolepis and all more crownward taxa. 1, actinopterygians; 2, crown‐group actinopterygians; 3, cladistians

1.

PRIMITIVE FISHES THROUGH TIME

25

but this rests on a very small number of characters, which are frequently homoplastic among early actinopterygians (Cloutier and Arratia, 2004). The earliest crown‐group acipenseriforms are late Jurassic polyodontids and late Cretaceous acipenserids (Figure 1.9). The minimum age for the crown‐group acipenseriforms is thus about 150 Myr (Bemis et al., 1997). However, stem acipenseriformes, such as Chondrosteus (Figure 1.9), are known since the early Jurassic, and certain extinct taxa, such as the early Triassic (250 Myr) Birgeria (Figure 1.9) and saurichthyids, are possibly more closely related to the acipenseriforms than to any other actinopterygian group (6 and 7, Figure 1.9). Living gars (ginglymods) fall into two genera, Lepisosteus (four species) and Atractosteus (three species), both of which are known since the late Cretaceous (65–100 Myr) (Figure 1.9). The stem lepisosteid Obaichthys is early Cretaceous in age, and the minimum age of the crown‐group ginglymods is thus about 112 Myr. The earliest known gars display much the same morphology as the extant ones. Stem gars are represented by the early Triassic to late Cretaceous (250–65 Myr) semionotids, a probably paraphyletic taxon that includes large, ubiquitous fishes with thick ganoid scales (10, Figure 1.9). The bowfin (A. calva) is represented by a single living species, which is thus the crown group, and is recorded since the early Pleistocene (about 1 Myr). However, the more and more inclusive taxa Amiinae, Amiidae, Amiiformes, and Halecomorphi comprise a large number of fossil marine and freshwater species. The earliest Amiinae are known since the early Cretaceous and were already exclusively freshwater, but the earliest halecomorphs, the middle Triassic (230 Myr) paraseminotids, were exclusively marine (Grande and Bemis, 1999) (11 and 12, Figure 1.9). Osteoglossomorphs and elopomorphs are known since the late Jurassic (elopomorphs having a slightly older first occurrence than osteoglossomorphs) and include a large number of fossil species. The debate about their relationships to other teleosts arose with the interpretation of certain fossil representatives of those respective taxa, as well as that of some stem teleosts (Arratia, 2004). The minimum age for crown‐group teleosts is thus indicated by the earliest elopomorphs, that is, about 150 Myr. However, there is a large number of stem teleost taxa the earliest of which, such as Pholidophorus (Figure 1.9), are late Triassic (215 Myr) in age. (Polypteriformes); 4, actinopterans; 5, crown‐group actinopterans; 6, chondrosteans; 7, Acipenseriformes; 8, neopterygians; 9, crown‐group neopterygians; 10, Semionotiformes; 11, halecomorphs; 12, Amiiformes; and 13, teleosts. [Illustrations for terminal taxa from Janvier (1996a), by permission of Oxford University Press, except for the reconstruction of Dialipina, which is based on photographs in Schultze and Cumbaa (2001).]

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5.4. Sarcopterygians

542 Cam 488 Ord 443 Sil 416 Dev 359 Carb 299 Perm 251 Tr 199 Jur 145 Cret 65 Cen

Apart from the two living species of Latimeria, all other actinistians are fossils and thus stem actinistians (Figure 1.10). The earliest known actinistians were long believed to date from the middle Devonian only, but their position within sarcopterygian phylogeny entailed a rather long ‘‘ghost range’’ because their living sister group, the rhipidistians (i.e., dipnomorphs and tetrapodomorphs), are known since the base of the Devonian

Miguashaia

Holopterygius

1

Allenypterus

Hadronector

Rhabdoderma Coelacanthus

Mawsonia 2 Macropoma No fossil known Latimeria Fig. 1.10. Actinistian (coelacanth) phylogeny. For abbreviations to the geologic timescale, see Figure 1.7. 1, actinistians; 2, coelacanthiformes. [Illustrations for terminal taxa from Janvier (1996a) by permission of Oxford University Press, except for that of Holopterygius, redrawn and modified after Friedman and Coates (2005).]

1.

27

PRIMITIVE FISHES THROUGH TIME

542 Cam 488 Ord 443 Sil 416 Dev 359 Carb 299 Perm 251 Tr 199 Jur 145 Cret 65 Cen

(Figure 1.15). However, there is now some evidence for actinistian remains in the early Devonian (400 Myr) (Johanson et al., 2006). Although the group superficially shows relatively little morphological disparity (in particular since the beginning of the Mesozoic), some Paleozoic coelacanths display strongly divergent morphologies such as the eel‐shaped middle Devonian Holopterygius or the deep‐bodied Carboniferous Allenypterus (Friedman and Coates, 2005) (Figure 1.10). There is no fossil actinistian record between the late Cretaceous (70 Myr) and present. Lungfishes are among the earliest known osteichthyans (2, Figure 1.11), as they are recorded since the beginning of the Devonian (415 Myr) and show a spectacular radiation during the Devonian, with a majority of marine

Porolepiforms 1 Diabolepis

2

Dipterus

3

Gryphognathus

Scaumenacia

Phaneropleuron

Neoceratodus Protopterus 4 Lepidosiren Fig. 1.11. Dipnomorph phylogeny. For abbreviations to the geologic timescale, see Figure 1.7.1, Dipnomorphs; 2, dipnoiforms; 3, dipnoans; and 4, crown‐group dipnoans. [Illustrations for terminal taxa from Janvier (1996a, by permission of Oxford University Press, 2004b).]

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forms, often associated with coral reef environments. Lungfish diversity declines progressively during the Carboniferous, when most species occur in reputedly freshwater environments. After the Paleozoic, lungfishes undergo a considerable reduction of their dermal skeleton and are essentially known by poorly informative isolated tooth plates. Therefore, the relationships of the Mesozoic taxa to the living Neoceratodus and lepidosirenids remain unclear, except for some species. It is generally assumed that the earliest tooth plates of Neoceratodus type occur in the early Cretaceous, and tooth plates of lepidosirenid type are known in the late Cretaceous. The minimum age for crown‐group lungfishes is thus about 140 Myr. The earliest lungfish estivation burrows are from the late Permian. Although this book is about fishes, it must be kept in mind that tetrapods are only part of a large total group called tetrapodomorphs and which includes a number of piscine taxa (Figure 1.12). The paleobiology of the piscine tetrapodomorphs has little bearing on the biology of the living tetrapods, but these extinct taxa will be dealt with briefly in the next section. 6. EXTINCT MAJOR FISH TAXA AND THEIR POSITION IN VERTEBRATE PHYLOGENY Besides these living primitive taxa and their extinct closest relatives, vertebrates include a number of major extinct clades, which are shown in the trees in (Figures 1.13 and 1.15). Although these will not be discussed in detail here, they deserve some comment because they illustrate the most interesting property of fossils, that is they provide character combinations that no longer exist in present‐day nature and thus often help in resolving conflicting relationships between the major living taxa. 6.1. Yunnanozoans and Myllokunmingiids Strangely, there are very few stem vertebrates (i.e., fossil vertebrates that have diverged earlier than the most recent common ancestor to all living vertebrates, and after the divergence between vertebrates and either cephalochordates or tunicates). There is no fossil cephalochordate, despite certain claims (Blieck, 1992; Janvier, 1997), but there are undoubted tunicates in the lower Cambrian Chengjiang marine Lagersta¨tte of Yunnan, China (535 Myr) (Shu et al., 2001). Two taxa from the same fossil locality, Yunnanozoa and Myllokunmingiida, have been regarded as possible stem vertebrates that ‘‘filled’’ the morphological gap between cephalochordates and vertebrates. Yunnanozoans possess a distinct branchial apparatus with six pairs of filamentous gills, followed posteriorly by a series of vertical body

29

PRIMITIVE FISHES THROUGH TIME 542 Cam 488 Ord 443 Sil 416 Dev 359 Carb 299 Perm 251 Tr 199 Jur 145 Cret 65 Cen

1.

Kenichthys 1 Rhizodontids Osteolepids Gyroptychius Tristichopterids Panderichthys

Tiktaalik Acanthostega lchthyostega

2

Crown-group tetrapods Fig. 1.12. Tetrapodomorph phylogeny. For geologic timescale, see Figure 1.7. 1, Tetrapodomorphs; and 2, apomorphy‐based tetrapods. [Illustrations for terminal taxa from Janvier (1996a, by permission of Oxford University Press, and 2004b), except for Tiktaalik, redrawn and modified from Daeschler et al. (2006).]

muscle blocks. Despite Mallatt and Chen’s (2003) attempts to interpret other structures of the yunnanozoan’s head with regard to larval lamprey anatomy, there is no clear evidence that this taxon is more closely related to the vertebrates than to any other chordate or deuterostome taxon. Yunnanozoans have been assigned to a wide range of phylogenetic positions, notably stem vertebrates, stem cephalochordates, stem hemichordates, and finally stem deuterostostomes (see review in Janvier, 2003). It is possible that Pikaia, from the late Cambrian Burgess Shale Lagertsta¨tte, long popularized as a the earliest chordate (Conway Morris, 1998), is in fact a close relative of yunnanozoans (Janvier, 2003). In contrast, myllokunmingiids (Figure 1.13) are more likely to be stem vertebrates. Although preserved as imprints, they display most of the

30 542 Cam 488 Ord 443 Sil 416 Dev 359 Carb 299 Perm 251 Tr 199 Jur 145 Cret 65 Cen

PHILIPPE JANVIER

Myllokunmingiids Hagfishes Lampreys

1

? Euconodonts Euphaneropids

2

Anaspids Arandaspids

Astraspids

3

Heterostracans

Thelodonts Galeaspids Pituriaspids Osteostracans Jawed vertebrates Fig. 1.13. Vertebrate phylogeny. For abbreviations to the geologic timescale, see Figure 7. 1, vertebrates/craniates; 2, crown‐group vertebrates/craniates; and 3, pteraspidomorphs. The name ‘‘ostracoderms,’’ a grade of basal, jawless gnathostome‐related vertebrates, generally refers to anaspids, pteraspidomorphs, thelodonts, galeaspids, pituriaspids, and osteostracans. The monophyly of the thelodonts remains poorly supported. [Illustrations for terminal taxa from Janvier (1996a by permission of Oxford University press, and in press).]

vertebrate characters such as olfactory organs, eyes,and possibly otic capsules, which are acceptable evidence for neurogenic placodes, as well as six gill arches, which may be evidence for migrating neural crest cells. They also display W‐shaped myomeres that resemble more those of vertebrates than those of

1.

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31

cephalochordates (Shu et al., 2003). Although myllokunmingiids were initially regarded as a paraphyletic group (Shu et al., 1999), including stem lampreys (Myllokunmingia) and the sister group of lampreys and all other vertebrates except for hagfishes (Haikouichthys), they are now regarded as probably a monophyletic group; they lack endoskeletal fin radials, which are a character of crown‐group vertebrates (Janvier, 2003, in press; Zhang and Hou, 2004). 6.2. ‘‘Ostracoderms’’ The name Ostracodermi was coined by Cope (1889) for lower Paleozoic vertebrates (essentially heterostracans and osteostracans) that lack jaws but possess a heavily ossified dermal skeleton, like gnathostomes. These armored jawless vertebrates are regarded as stem gnathostomes (Figures 1.5 and 1.13), but the name ‘‘ostracoderm’’ is still used informally by some authors. Eight major taxa are commonly referred to as ‘‘ostracoderms.’’ In addition to heterostracans and osteostracans, ‘‘ostracoderms’’ include anaspids, galeaspids, arandaspids, eriptychiids, pituriaspids, and thelodonts. Even some partly or entirely soft‐bodied jawless vertebrate taxa (such as euconodonts and euphaneropids; Figure 1.13) are now sometimes referred to as ‘‘ostracoderms’’ as they are regarded as stem gnathostomes. At the beginning of the twentieth century, ‘‘ostracoderms’’ were thought to be ancestral to hagfishes and lampreys essentially because they were jawless and ancient. Stensio¨ (1927) was the first to unravel the details of the internal anatomy of osteostracans and pointed out some unique characters they (and also anaspids) shared with lampreys, notably the presence of a median, dorsal nasohypophysial opening. Stensio¨ also proposed that hagfishes were derived from yet another ‘‘ostracoderm’’ group, heterostracans, although this idea is now discarded. Stensio¨’s conception of vertebrate phylogeny was that agnathans are monophyletic and that ‘‘ostracoderms’’ are ancestral to the modern cyclostomes as a whole. This theory was also coherent with the old idea that hagfishes and lampreys were ‘‘degenerate’’ and had lost numerous characters such as the paired fins and mineralized skeleton. Some authors, however, suggested that certain ‘‘ostracoderms,’’ notably heterostracans, could be ancestral to the gnathostomes because they possessed paired olfactory organs (Halstead, 1973). During the last two decades, evidence has accumulated to indicate that ‘‘ostracoderms,’’ though undoubtedly lacking jaws, are in fact more closely related to the jawed vertebrates than to either hagfishes or lampreys (Gagnier, 1991; Forey and Janvier, 1993; Forey, 1995; Janvier, 1996b; Donoghue et al., 2000; Donoghue and Smith, 2001). As a consequence, the classical assumption that hagfishes and lampreys had lost many characters became pointless, as the presence of bone, along with other characters uniquely shared by ‘‘ostracoderms’’ and gnathostomes, came to

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be considered as synapomorphies. Although some characters, such as the strikingly similar condition of the nasohypophysial complex in lampreys and osteostracans (and probably anaspids), have to be regarded as homoplastic, the current tree of fossil and living vertebrates is, if not robust, at any rate clearly more parsimonious than any tree assuming agnathan monophyly (Donoghue et al., 2000). It shows ‘‘ostracoderms’’ as a grade of stem gnathostomes that fills the morphological gap between the living cyclostomes (or the living lampreys, if cyclostomes are not a clade) and the living gnathostomes (Figures 1.5 and 1.13). The most interesting implication of the current vertebrate tree is that what is often referred to as the so‐called ‘‘gnathostome body plan’’ is not the result of a ‘‘burst of anatomical innovations,’’ as frequently alleged, but a progressive accumulation of new characters that can be inferred at several nodes of the tree (Mazan et al., 2000; Janvier, 2001; Donoghue and Sansom, 2002). For example, this tree tells us that the dermal skeleton became ossified before the endoskeleton or that paired fins appeared before jaws. Osteostracans, which now appear as the closest jawless fossil relatives of the jawed vertebrates, show that such characters as cellular bone, perichondral bone, sclerotic ring, pectoral fins, shoulder girdle, and epicercal tail have also preceded jaws in vertebrate evolution (Figure 1.13). Of course, we know nothing of the evolution of physiology along this long segment of vertebrate phylogeny that extends between the divergence of lampreys (or the cyclostomes as whole) and crown‐group gnathostomes, and that is only documented by fossil taxa (apart from functions linked to calcified tissue development), but one might expect that some anatomical or histological data might inform insights in this field. The input is now expected from physiologists, rather than from paleontologists. During the last decade, considerable attention has been paid to ‘‘conodonts’’ (or, more restrictively, euconodonts; Figure 1.13), which are currently regarded by many paleontologists as the basalmost stem gnathostomes. From the mid‐nineteenth century, conodonts (including at least paraconodonts and euconodonts) were only known by minute comb‐ or tooth‐shaped denticles of unknown derivation. They always occur in marine sediments from the late Cambrian to the Triassic (500–220 Myr) and were widely used by stratigraphers for dating the rocks. Since the first discovery of the soft‐tissue imprint of articulated euconodonts from the Carboniferous and Ordovician (Briggs et al., 1983; Gabbott et al., 1995), it became widely accepted that euconodonts are vertebrates as they display a caudal fin supported by radials, a body musculature made up by chevron‐shaped myomeres, and a pair of large eyes. Their mouth is armed with denticles made of calcium phosphate, whose structure somewhat recalls the vertebrate bone, dentine, and enamel (though this homology is far from widely

1.

PRIMITIVE FISHES THROUGH TIME

33

accepted; Schultze, 1996). The phylogenetic position of euconodonts as stem gnathostomes remains tenuously supported, and they may turn out to be either more closely related to hagfishes or lampreys (or cyclostomes as a whole), or even stem vertebrates. Euconodonts are, however, interesting because of their extraordinary abundance and specific diversity during the Paleozoic. While most of the classical ‘‘ostracoderm’’ groups are relatively rare and have a relatively low specific diversity, the contemporary euconodonts comprise hundreds of species, despite their low morphological disparity. In a sense, they could compare to rodents among the living mammals. 6.3. Placoderms Placoderms, or armored jawed vertebrates (Figures 1.14 and 1.15), lived from the early Silurian to the very end of the Devonian (435–360 Myr). They are characterized by a massive armor made up by bony plates that cover the anterior part of the trunk and the head. The trunk armor articulates with the head armor at the level of the neck. The jaws are armed with bony, denticle‐ bearing, dermal plates. Placoderms were the most diverse and abundant fish group during the Devonian, and their extinction at the end of this period is as sudden and enigmatic as that of the nonavian dinosaurs at the end of the Cretaceous. Geologic events, such as an extensive marine regression at the end of the Devonian, may have been the coup de graˆce that caused their disappearance, but they may also have been outcompeted by the sudden burst of the chondrichthyans, notably the durophagous, chimaeriform‐ related ‘‘bradyodonts.’’ The diversity of placoderms during the Devonian may compare to that of living teleosts, with about 400 known species ranging from 10 mm to 6–7 m in total length (adult size), and morphologies that vaguely parallel those of rays, catfishes, boxfishes, tunas, or swordfishes (Figure 1.14). Placoderms were once regarded as either chondrichthyan relatives because of their neurocranial anatomy, or osteichthyan relatives because of their dermal bone pattern, but they are currently regarded as the sister group to all other jawed vertebrates, based on various morphological characters (Figure 1.15). 6.4. Acanthodians Acanthodians are a poorly understood group of late Ordovician to early Permian (445–295 Myr) gnathostomes (Figure 1.15), whose exoskeleton generally consists of minute, square‐shaped scales covered with overgrowing layers of dentine. They display significant specific diversity, but comparatively low morphological disparity. They have long been characterized by the presence of large fin spines in front of the paired and unpaired fins until the

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Arthrodires

Rhenanids

Antiarchs

Ptyctodonts Fig. 1.14. Placoderm diversity. Placoderms are one of the two major extinct jawed vertebrate clades and were among the most abundant fishes during the Devonian. These reconstructions of representatives of four main placoderm groups illustrate their morphological diversity. The antiarchs (e.g. Asterolepis) possessed extensive trunk and head armors, and their pectoral fins were covered with bony plates and articulated in a crab leg‐like manner. The rhenanids (e.g., Gemuendina) displayed a ray‐ or squatinoid‐like overall morphology. Arthrodires (e.g., Coccosteus), the most diversified placoderm group, possessed a massive dermal armor and could grow up to 7 m in total length. In contrast, ptyctodonts (e.g., Ctenurella) had a much reduced dermal armor, and their overall shape recalls that of living chimaeriformes. Scale bar ¼ 10 mm. [From Janvier (1996a) by permission of Oxford University Press, Janvier and Racheboeuf (2003), and Janvier et al. (2003).]

discovery of paired fin spines in early Devonian primitive chondrichthyans (Miller et al., 2003). Unfortunately, very little is known of acanthodian anatomy, in particular for the endoskeleton, except in the Permian genus Acanthodes, which is the last surviving taxon and shares some unique derived characters with osteichthyans, notably three otoliths. The status of acanthodians is debated, and they are increasingly suspected to be nonmonophyletic. If Acanthodes (and acanthodids as a whole) are still regarded as a potential

35

PRIMITIVE FISHES THROUGH TIME 542 Cam 488 Ord 443 Sil 416 Dev 359 Carb 299 Perm 251 Tr 199 Jur 145 Cret 65 Cen

1.

Placoderms

Pucapampella 1

3 Elasmobranchs 4 Euchondrocephalans (incl. chimaeriforms)

2

Acanthodians ? Dialipina

5 7

All other actinopterygians 6

Meemannia Psarolepis 8 Onychodontiforms

Actinistians

9

Styloichthys

Dipnomorphs 10 Tetrapodomorphs Fig. 1.15. Jawed vertebrate phylogeny. For abbreviations to the geologic timescale, see Figure 1.7. The monophyly of acanthodians is currently debated and the position of Dialipina is tenuously supported. Meemannia, Psarolepis, and Styloichthys are known from fragmentary material and are not reconstructed here. 1, Jawed vertebrates (apomorphy‐based gnathostomes); 2, crown‐group gnathostomes; 3, chondrichthyans; 4, crown‐group chondrichthyans;

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sister group to osteichthyans, there are increasingly numerous data, notably histological ones, suggesting that certain Silurian and early Devonian acanthodian‐like taxa are in fact stem chondrichthyans. Thus, acanthodians are likely to turn out to be a paraphyletic group, comprising both stem osteichthyans, stem chondrichthyans, and possibly stem gnathostomes (Janvier, 1996a). 6.5. ‘‘Paleoniscoids’’ and Basal Neopterygians The earliest known undoubted actinopterygians are early Devonian (410 Myr) in age, although certain late Silurian (420 Myr) remains (e.g., Andreolepis, Lophosteus) have been tentatively referred to actinopterygians, but may in fact be derived from stem osteichthyans. Basal actinopterygian and basal actinopteran relationships remain heatedly debated and extremely labile (see for review Cloutier and Arratia, 2004). During the past three decades, a number of trees have been proposed for the total‐group actinopterygians (Patterson, 1982; Gardiner, 1984; Long, 1988; Gardiner and SchaeVer, 1989; Gardiner et al., 1996; Coates, 1999; Cloutier and Arratia, 2004). These trees diVer in many respects, although the relationships between the extant taxa generally remain the same. A large number of taxa that have long been referred to as ‘‘paleonisciforms’’ or ‘‘paleoniscoids,’’ an obviously paraphyletic taxon composed of Devonian to Jurassic taxa, are in fact a mix of stem actinopterygians (e.g., Cheirolepis), stem actinopterans (e.g., Moythomasia), and stem neopterygians (e.g., Australosomus) (Figure 1.9). That is, they diverged before either cladistians, acipenseriforms, or crown‐group neopterygians. It is also probable that some of the ‘‘paleonisciforms,’’ such as the platysomids, may be gathered into large extinct actinopterygian clades. The diVerences between the various tree topologies published during the past three decades rest mainly on the minimum age of total groups. For example, depending on the earliest fossil sister group of the total‐group acipenseriforms, the minimum age for the divergence between acipenseriforms and neopterygians may be dated as either early Triassic or early Carboniferous (Coates, 1999). One of the most intriguing early actinopterygians is certainly the early Devonian genus Dialipina (Figures 1.9 and 1.15), which possesses typical actinopterygian ‘‘ganoid’’ scales but retains two dorsal fins (all other actinopterygians have a single dorsal fin—a derived character), possesses a diphycercal caudal fin that resembles that of coelacanths, and shows a dermal 5, osteichthyans (or teleostomes); 6, crown‐group osteichthyans; 7, actinopterygians; 8, sarcopterygians; 9, crown‐group sarcopterygians; 10, rhipidistians; and 11, crown‐group rhipidistians (or ‘‘choanates’’) [Illustrations for terminal taxa from Janvier (1996a) by permission of Oxford University Press, Janvier and Maisey (in press), and the reconstruction of Dialipina is based on photographs in Schultze and Cumbaa (2001).]

1.

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bone pattern that is strongly at odds with that of all other osteichthyans, notably because it lacks the classical osteichthyan cheek bones (opercular, preopercular jugal, and so on; Schultze and Cumbaa, 2001). The peculiar anatomy of Dialipina is interpreted as a mix of general gnathostome characters (e.g., two dorsal fins) and general osteichthyan or actinopterygian characters, with some unique highly derived features. However, it is possible that Dialipina tells us that some of the characters long used to define actinopterygians (e.g.,the ganoid scales) are in fact more general than currently believed. 6.6. Extinct Sarcopterygian Taxa Extinct nontetrapod sarcopterygian taxa are relatively numerous, notably in the Devonian (415–360 Myr). However, their relationships are comparably better elucidated than for extinct actinopterygian taxa. Most of these piscine sarcopterygians fall into two major clades of the crown‐group sarcopterygians, the dipnomorphs and tetrapodomorphs (Figure 1.15), which are currently regarded as sister groups (10, Figure 1.15). Dipnomorphs include dipnoans (lungfishes) and porolepiforms (Figure 1.11), as well as some monogeneric taxa (e.g., Powichthys, Youngolepis) that are variously regarded as either basal lungfishes or porolepiforms. The most generalized lungfish (or dipnoiform), Diabolepis, from the early Devonian of China already possesses the characteristic entopterygoid and prearticular tooth plates, but retains (at any rate ventrally) the characteristic intracranial joint of basal sarcopterygians and the living Latimeria (Figure 1.1), and two external nostrils. Tetrapodomorphs are notably characterized by the choana, which is assumed to be the posterior nostril that has migrated into the palate, though independently from the intrabuccal posterior nostril of lungfishes (Zhu and Ahlberg, 2004; Janvier, 2004b). They include Kenichthys, the rhizodontids, osteolepids, megalichthyids, tristichopterids, elpistostegalians, and tetrapods (Figure 1.12). Osteolepids, megalichthyids, and tristichopterids were formerly included in a taxon ‘‘Osteolepiformes,’’ now proved to be paraphyletic (Ahlberg and Johanson, 1998), and so are also probably elpistostegalians, that include Panderichthys and Tiktaalik (Figure 1.12) (Daeschler et al., 2006). Onychodontiforms are only known from marine Devonian sediments and display a curious assemblage of sarcopterygian characters (intracranial joint), actinistian characters (unlobed anterior dorsal fin and diphycercal tail, also shared with Dialipina), and actinopterygian‐like characters (large and posteriorly expanded maxilla). They are currently regarded as the sister group of either actinistians or crown‐group sarcopterygians (i.e., actinistians and rhipidistians) (Figure 1.15). In addition to these taxa, the early Devonian of China has yielded a number of sarcopterygians that are referred to monotypic genera, such as

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Meemannia, Psarolepis, Achoania, Styloichthys, and Kenichthys, and currently regarded as either stem sarcopterygians, stem rhipidistians, or stem tetrapodomorphs (Figure 1.15) (Zhu et al., 1999, 2001, 2006; Zhu and Yu, 2002; Zhu and Ahlberg, 2004). Psarolepis, in particular, is a puzzling form that allies classical sarcopterygian characters (e.g., folded dentine in teeth, cosmine, intracranial articulation), some reputedly actinopterygian characters (e.g., structure of the preopercular, median rostral bone), and characters that occur outside osteichthyans, in placoderms, chondrichthyans, and acanthodians (e.g., median fin spines, placoderm‐like shoulder girdle), the latter being probably general gnathostome characters that have been lost in all other osteichthyans. Therefore, it has been suggested that Psarolepis is a stem osteichthyan (Zhu et al., 1999). Phylogenetic analyses would, however, rather place it as a stem sarcopterygian (Zhu and Yu, 2002; Zhu et al., 2006). Meemannia is yet another lower Devonian stem sarcopterygian that is morphologically very close to the currently accepted (or imagined) ancestral morphotype of actinopterygians and sarcopterygians (Zhu et al., 2006). Interestingly, these five taxa all occur in the same locality of South China and range in age from 416 to 400 Myr. This suggests that the minimum age for the divergence between actinopterygians and osteichthyans is about 416 Myr. 7. HOW STABLE IS VERTEBRATE PHYLOGENY? There is a widespread belief that, besides some problematical nodes (see above), the vertebrate tree is relatively stable at the level of the major extant terminal taxa. The current consensus about vertebrate phylogeny is a consequence of an overall agreement between morphology‐based and most molecular sequence‐based phylogenies. There are, however, some discordant views about vertebrate phylogeny, the most striking incongruence with morphology‐based trees being Arnason et al.’s (2001, 2004) consideration of mitochondrial DNA sequence data (Figure 1.4). This gnathostome tree has been the subject of little debate because it was regarded as simply the result of methodological biases, notably the inadequacy of mitochondrial DNA for resolving very deep divergences (Zardoya and Meyer, 2001). Could some paleontological data accommodate Arnason et al.’s (2004) tree? The latter raises two major questions: (1) the monophyly of actinopterygians (cladistians þ actinopterans) and (2) the monophyly of sarcopterygians. Cladistians still trouble molecular phylogenies but are grouped with actinopterans into the actinopterygians on the basis of a few morphological characters. Morphologists classically regard sarcopterygians as a well‐ supported clade. During the last decade, new early Devonian sarcopterygians

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have raised questions about sarcopterygian monophyly, as currently defined. Notably, Psarolepis (see above) displays some classical sarcopterygian characters, along with characters that are only known in non‐osteichthyan gnathostomes. The early Devonian chondrichthyan Pucapampella, one of the earliest known chondrichthyans, also raises the question of the distribution of certain osteichthyan morphological characters. Although it possesses the characteristic prismatic calcified cartilage of chondrichthyans, the braincase of Pucapampella also displays a complete ventral fissure (once regarded as the homologue of the sarcopterygian intracranial joint). In addition, the overall braincase morphology of Pucapampella is strikingly similar to that of the earliest known actinopterygian and sarcopterygian braincases. In a diVerent (nonphylogenetic) context, Jarvik (1981) considered that sarcopterygian skull morphology was an image of the ideal gnathostome skull morphology. At any rate, it now seems that we are coming closer and closer to the ancestral morphotype of the crown‐group gnathostomes, and that endoskeletal cranial characters suggest that it was more similar to the osteichthyan condition than to the modern chondrichthyan one. Although this is not enough to refute the current gnathostome phylogeny, it does show, at least, that its morphological bases are not as stable as once believed. Notably, it addresses the question of the reputed primitiveness of the chondrichthyans, as did Arnason et al.’s (2001) results. The paleontological ‘‘black box’’ of crown‐group gnathostome divergences seems thus situated in time somewhere in the Silurian period between 440 and 415 Myr ago. 8. FOSSILS AND PHYSIOLOGY Fossils have only three unique properties that no other source of biological data can replace: (1) they provide morphological character combinations which no longer exist in living organisms; (2) they provide a minimal age for characters, and thus the taxa that they define; and (3) they may show geographical (and ecological) distributions that are diVerent from the present day. Property (1) is essentially used by phylogeneticists as it may help in resolving (or, at any rate, better support) relationships between extant taxa by answering questions about homologies or homoplasies. To evolutionary morphologists, it provides material evidence for ‘‘transitional forms’’ in such scenarios of evolutionary transition, as the ‘‘agnathan–gnathostome’’ or the ‘‘fish–tetrapod’’ transitions. In rare cases, this property may allow inferences about the distribution of physiological characters, when reflected in hard tissues, such as the presence of bone in a jawless vertebrate, which suggests that the physiological functions involved in bone production did exist before the rise of jaws. Property (2) is now much used by molecular

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phylogeneticists for calibrating molecular clocks (however reliable they may be), but may be of interest to physiologists to explore the stability of a physiological character through time. Finally, property (3), which is mainly useful to historical biogeographers, may have bearings on inferences about the past physiological adaptations of living taxa. For example, physiologists are sometimes surprised to learn that, until about 370 Myr, all lungfishes were marine. In sum, very few fossil data are directly informative to primitive vertebrate physiologists. Generally, they concern the physiology of hard tissues (bone, teeth, calcified cartilage) and are relevant to calcium regulation. Some anatomical data that can be linked to particular functions (e.g., evidence for a pineal foramen that suggests the presence of a photosensory pineal organ, or pelvic claspers that suggest internal fecundation) can provide a minimum age for the latter by inference from one particular phylogenetic tree. In fact, questions about the evolution of physiological functions arise essentially from their distribution in living taxa, and the role of fossils is to provide a basis for the calibration of the divergence times of these taxa. In a sense, evolutionary physiology is in much the same situation as molecular phylogenetics, relative to paleontological data. The literature about fossil fishes probably contains many morphological data on skeletal structures or exceptional soft‐tissue preservations that may be relevant to physiology, but paleontologists are rarely able to properly assess their bearings on this field. Traces of activity left by fossil fishes are very rare and poorly informative as to the mode of living. Fish trails in the sediment, referred to as Undichna, that were left by the fins or fin spines of fishes trapped in shallow pools, can hardly be assigned to a particular taxon. The most interesting of these traces are those clearly left by early Devonian osteostracans, as they represent a unique source of information about the locomotion of an ‘‘ostracoderm’’ and show that the fish moved by short successive ‘‘jumps’’ (Morrissey et al., 2006). Lungfish estivation burrows are known from the Permian and can be readily identified by the articulated lungfish skeletons found inside them (McAllister, 1992). Stomach contents are frequently found in fossil fishes and provide some information about their diet. They also allow to reconstruct the original trophic network (Maisey, 1994). The stomach contents of ‘‘ostracoderms’’ (known in anaspids, euphaneropids, and thelodonts) consist of fine‐grained sediment, show no large food particle, and suggest microphagous particulate feeding. In contrast, all major Devonian gnathostome groups in which the stomach contents are known show evidence for predatory habits. Fossils provide some information about reproduction strategies and sequences of skeletal development in early fishes, for example, fish eggs, egg

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capsules, and growth series from juvenile to adults are known in heterostracans, placoderms, acanthodians, many actinopterygians, and some piscine sarcopterygians (coelacanths, lungfishes, and tristichopterid tetrapodomorphs). A Carboniferous coelacanth (Rhabdoderma) shows that the young had a yolk sac, like Latimeria. Among early fishes, evidence for larval development with metamorphosis is only known in the Devonian lungfish Dipterus (Janvier, 1996a). Finally, by unraveling characters or character combinations that no longer exist in the present day, fossils may challenge functional models based exclusively on living taxa. An interesting example is provided by the ‘‘polybranchic’’ condition in extinct jawless vertebrate taxa, which display an extremely large number of gills, such as some early Devonian galeaspids and the late Devonian euphaneropids. While most studies on fish respiration are based on the living hagfishes, lampreys, and gnathostomes, which have from 15 to 5 pairs of gill pouches or gill arches, the 33 gill pairs of euphaneropids or the 45 gill pairs of certain galeaspids (Figure 1.16) may raise questions relating to gill ventilation (Janvier, 2004a; Janvier et al., 2006). Notably, it suggests that for jawless fishes with lamprey‐like gill pouches and passive inspiration, the ventilatory problems posed by life in dysoxic environments may have been solved by increasing the number of gills, whereas the active inspiration of jawed fishes would not require this specialization (Mallatt, 1996). 9. THE ENVIRONMENT OF EARLY FISHES: MARINE VERSUS FRESHWATER VERTEBRATES Most of the works that allude to the physiology of early fossil fish taxa focus on the question of their freshwater or marine habitat, and sometimes the possible evidence for anadromy (Denison, 1956; White, 1958; GriYth, 1987; Hardisty et al., 1989; Janvier, 1996a). The environment of the earliest vertebrates remains vividly debated and rooted in certain geologic traditions. The first Silurian and Devonian fish remains described in the beginning of the nineteenth century were preserved in sandstones (e.g., the ‘‘Old Red Sandstone’’ of Britain and the Baltic States) and generally associated with plant remains, but rarely with marine invertebrates. In addition, these heavily armored fishes were regarded as ‘‘ganoids,’’ a group which classically included living bichirs, gars, and catfishes, all reputedly freshwater. Progressively, the received wisdom became that all these early fishes lived in freshwater and occasionally passed into the sea, when found in marine sediments. In the mid‐ twentieth century, paleontologists began to raise the question of the evidence for freshwater environment when dealing with sediments and fossils. Marine

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A Incurrent opening

B

Branchial fossae C1

C2

Branchial basket

Fig. 1.16. Polybranchic vertebrates. (A and B) Endoskeletal head shield (braincase) of two early Devonian galeaspids showing the roof of the oralobranchial chamber and the numerous branchial fossae that housed the gills or gill pouches. (A) Duyunolepis, (B) Zhaotongaspis, and (C) specimen (C1) and reconstruction (C2) of the late Devonian euphaneropid Euphanerops, showing the very elongated branchial basket. Scale bar ¼ 10 mm. [Redrawn and modified from Janvier (1996a by permission of Oxford University Press, and 2004a).]

sediments can be readily characterized by the presence of such invertebrates as brachiopods or echinoderms, for which no freshwater representative is known (Figure 1.17), but freshwater has no obvious paleontological ‘‘signature,’’ at any rate for such ancient periods as the Paleozoic. To make a long story short, there are currently two competing trends in the interpretation of these environments: some paleontologists consider the ‘‘Old Red Sandstone’’ as having been deposited in marginal marine environments, whereas others consider that most of these sediments were deposited in either fluvial or

1.

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f e

g

a c a d b a

Fig. 1.17. Reconstruction of a typical marine environment at the end of the middle Devonian (385 Myr), in what is now northern France. The fish fauna is dominated by placoderms (a, arthrodires; b, antiarchs; and c, ptyctodonts) and large sarcopterygians (d, lungfishes; and e, onychodonts), but also includes small primitive actinopterygians (f, ‘‘paleonisciforms’’) and acanthodians (g). Many of these fish taxa, notably arthrodires, antiarchs and acanthodians also occur further north in the contemporary, but reputedly non‐arine, ‘‘Old Red Sandstone’’ of Scotland. [Drawing by P. Janvier, in Lelie`vre et al. (1986).]

lacustrine environments (see review in Janvier, 1996a; Lelie`vre, 2001). The arguments of the former are essentially based on the geologic context (sedimentologic characteristics, geographical position relative to the closest marine deposits), and those of the latter are that the same Silurian and Devonian fishes, sometimes at the species level, may occur in both reputedly freshwater and marine environments. Moreover, many of these early fish taxa found in reputedly freshwater deposits generally display such a broad global distribution that, considering current Devonian paleogeographic reconstructions, they must have dispersed via marine environments. Geochemistry could theoretically settle the question. Notably, stable isotope ratios of certain elements, such as 86Sr/87Sr, significantly diVer between fresh and marine waters, but mainly at higher latitudes. However, attempts at using this criterion on Paleozoic fishes have been scarce (Schmitz et al., 1991), and the results must be taken with great reservation because of the numerous biases induced by diagenesis or percolation, especially for such ancient periods. In addition, the geochemical signals for marginal marine environments (and organisms) situated at low latitudes (tropical waters) or

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submitted to frequent variations of salinity, as was probably the environment of the ‘‘Old Red Sandstone’’ deposits, are often unclear. It is probably the reasons why the signal found for ‘‘Old Red Sandstone ostracoderms’’ (mainly heterostracans) by Schmitz et al. (1991) is ambiguous: freshwater for some samples, marine for others, or undiagnostic. The question is thus not yet settled, but one must keep in mind the fact that the Devonian was a period of extensive peneplanization, during which the continental margins were unusually flat and occupied by vast deltas and tidal flats. The environment of most Silurian and Devonian (and probably earlier) fishes was thus comparable to the present‐day major tropical deltas and mangroves. This question of the marine versus freshwater environment is not restricted to the early Paleozoic. Many late Paleozoic and Mesozoic fish sites also pose the same problem, in connection with the presence of fish taxa whose extant representatives are either essentially or exclusively marine. For example, the Carboniferous and Permian xenacanthiform sharks of the intramontane basins of Europe were almost certainly freshwater, although the same taxa are also known elsewhere from undoubtedly marine sites. Similarly, the earliest known Devonian actinistians were all marine, as is also Latimeria, but several Carboniferous and Mesozoic actinistian taxa are regarded as freshwater, essentially on the basis of the sedimentologic context. A single stable isotope analysis has been performed on a coelacanth from a Cretaceous lacustrine deposit, and eVectively provided a clear freshwater signal, for both the fish and the sediment (Poyato‐Ariza et al., 1998). Anadromy has been frequently invoked to explain the occurrence of the same Paleozoic fish species or genera in marine and reputedly freshwater localities (Halstead, 1973; Trewin and Davidson, 1999). Another argument, albeit weak, in favor of anadromy is the homogeneity in size of the populations of certain fossil fish species and the absence of populations of intermediate sizes (Nielsen, 1949). Only two of the major living fish groups, hagfishes and chimaeriforms, are exclusively marine. Hagfish physiology is supposed to preclude any possibility for the group to have once been freshwater, but the presumed freshwater environment of the Carboniferous hagfish Myxineides is ambiguous and only based on a supposedly intramontane geologic context. Neither the fossil chimaeriforms nor any stem chimaeriform, or fossil euchondrocephalan, has ever been found in a reputedly nonmarine geologic formation. The earliest (Cambrian–Ordovician) vertebrates are all marine, and the freshwater versus marine debate principally begins with the late Silurian and Devonian fishes. However, indisputable freshwater fishes occur first in the latest Devonian or the early Carboniferous. The early phase of vertebrate and even gnathostome evolution thus took place almost certainly in marine waters, though in coastal environments, but the conquest of freshwater

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environments occurred many times independently. Among the major extinct taxa, it may have occurred in placoderms, but certainly in acanthodians. As far as living taxa and their closest fossil relatives are concerned, it occurred once in lungfishes and lampreys, respectively, possibly twice in actinistians, at least four times in elasmobranchs, and many times in actinopterygians. 10. CONCLUSIONS The character combinations that are displayed by the fossil piscine vertebrates rarely overturn phylogenies based on living taxa. However, they provide key information for dating the earliest occurrence of taxa and estimating ‘‘ghost ranges.’’ Therefore, the assessment of a living fish as ‘‘primitive’’ (or ‘‘ancient’’) does not only rest on its vague resemblance to an early fossil form, as in the case of Latimeria, but also on the minimum age of the taxon it belongs to, including when this is based on inferences from the range of its sister group, as in the case of Polypterus. Moreover, regarding the biology of the living primitive fishes, one must keep in mind that their stem‐ group relatives may have lived in environments that were radically diVerent, as exemplified by lungfishes. The geologic context of the early fossil fishes can indeed provide information about their environment, but in such cases as the marine versus freshwater habits the data may be ambiguous, and any large‐ scale conclusions about evolutionary fish physiology should consider them with great reservations. Attempts at inferring the physiology of ancient fishes on the basis of their presumed descendants have generally been made by paleontologists with little factual background. It may be timely for physiologists to consider shared physiological characters as an additional source of data for phylogeny reconstructions and also to take a new look at certain fossil data for hints into the physiology of the past. REFERENCES Ahlberg, P. E., and Johanson, Z. (1998). Osteolepiforms and the ancestry of tetrapods. Nature 395, 792–794. Arnason, U., Gullberg, A., and Janke, A. (2001). Molecular phylogenetics and Gnathostomous (jawed) fishes: Old bones, new cartilage. Zool. Scr. 30, 249–255. Arnason, U., Gullberg, A., Janke, A., Joss, J., and Elmrot, C. (2004). Mitogenomic analyses of deep gnathostome divergences: A fish is a fish. Gene 333, 61–70. Arratia, G. (2004). Mesozoic halecostomes and the early radiation of teleosts. In ‘‘Mesozoic Fishes 3‐ Systematics, Palaeoenvironments and Biodiversity’’ (Arratia, G., and Tintori, A., Eds.), pp. 279–316. Pfeil, Munich. Bardack, D. (1991). First fossil hagfish (Myxinoidea): A record from the Pennsylvanian of Illinois. Science 254, 701–703.

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2 CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES ANTHONY P. FARRELL

1. Introduction 1.1. Scope of the Chapter 1.2. Measurement Systems: Their Benefits and Limitations 2. An Overview of Evolutionary Progressions 2.1. Anatomical Patterns 2.2. Physiological Patterns 3. Details of the Cyclostome Circulatory Systems 3.1. Hagfishes 3.2. Lampreys 4. Details of the Sarcopterygii (Lobe‐Finned Fishes) Circulatory Systems 4.1. Coelacanth 4.2. Dipnoi (Lungfishes) 5. Details of the Circulatory Systems in Polypterids, Gars, and Bowfins 5.1. Polypterids (Bichirs and Reedfish) 5.2. Garfishes 5.3. Amia (Bowfins) 6. Details of the Sturgeon Circulatory Systems 6.1. Cardiac Anatomy 6.2. Circulatory Patterns 6.3. Cardiac Dynamics 6.4. Circulatory Control 7. Conclusions

The cardiovascular system is crucial by virtue of its role in transporting nutrients, respiratory gases, hormones, and waste products. This chapter focuses on circulatory form and function: the anatomy of the cardiovascular system, cardiac dynamics, and cardiovascular control. Studying circulatory control in any fish is particularly diYcult because discrete circulations of specific organs are not easily accessible. Therefore, by necessity, most information on cardiovascular control in primitive fishes is limited largely to the control of cardiac output (Q), as well as control of blood flow through the gills, to air‐breathing organs, and the gastrointestinal tract. Unusual 53 Primitive Fishes: Volume 26 FISH PHYSIOLOGY

Copyright # 2007 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(07)26002-9

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adaptations of primitive fishes that deviate from those piscine features common to elasmobranchs and teleosts are highlighted. The chapter starts with the most primitive fishes, the cyclostomes, and moves through the cardiovascular anatomy of the coelacanth to the cardiovascular anatomy and physiology of dipnoans, the forerunners to tetrapods. It then closes by covering the limited physiological information for Polypterids, gars, bowfins, and sturgeons. By comparing cardiovascular adaptations among these primitive fishes, this chapter examines the evolutionary roots and the evolutionary divergence of the piscine cardiovascular system. 1. INTRODUCTION 1.1. Scope of the Chapter This chapter assumes that the reader has a general knowledge of the circulatory form and function in elasmobranchs and teleosts, as described elsewhere (Randall, 1968; Olson and Farrell, 2006). Additional details can be found in books and reviews, such as Randall (1970), Satchell (1971, 1991), Johansen (1971), Farrell (1984, 1991), Santer (1985), and Butler and Metcalfe (1988), as well as two previous volumes of Fish Physiology (12A and 12B; 1992) that were dedicated to the cardiovascular system. At a finer scale, the cellular structure of fish hearts has been reviewed by Yamauchi (1980). The physiological processes that govern transsarcolemmal and intracellular ion movements associated with cardiac excitation and contraction are being slowly unraveled for teleosts (see reviews by Tibbits et al., 1992; Shiels et al., 2002; Vornanen et al., 2002), but not yet for primitive fishes. Goodrich (1930) is an excellent starting place for readers interested in a description of the fish circulatory system in a broader evolutionary context, with detailed physiological perspectives provided by Johansen (1965), Johansen and Hanson (1968), Johansen and Burggren (1980), and Burggren et al. (1997). The neural and humoral controls of the circulatory system have been reviewed from several evolutionary perspectives (Laurent et al., 1983; Nilsson, 1983; Nilsson and Axelsson, 1987). Linkages can be made between the present chapter and others in this volume, for example, Chapter 3 (nervous controls) and Chapter 5 (respiratory functions), and with the description of cardiorespiratory control in tropical fishes found in an early volume of this series (see Reid et al., 2006 in volume 21). An impressive fact is that many of the older observations of circulatory anatomy, some approaching 200 years old, were remarkably accurate in many respects and thus continue to provide a foundation for our understanding of the piscine circulatory system. Newer techniques, such as vascular casting with synthetic resins and electron microscopy, have added important details

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of the microcirculation, as well as correcting older literature where necessary. This chapter, rather than fully citing the older literature, favors works that are generously referenced and can take the reader back to the roots of important anatomical and physiological discovery in the nineteenth and early twentieth centuries. Among these works are Biology of Myxine (Brodal and Fa¨nge, 1963), Biology of the Lampreys (Hardisty and Potter, 1972), and Biology of the Cyclostomes (Hardisty, 1979). Satchell’s chapter in his book (Satchell, 1991), ‘‘Myxine, a Speculative Conclusion,’’ is fascinating reading. The anatomy of the coelacanth circulatory system is meticulously detailed in the monograph by Millot et al. (1978). Satchell (1976) has provided an extremely insightful chapter on the circulatory system of air‐breathing fish, which has been generously extended by Graham’s (1997) masterful monograph on air‐breathing fishes. Other relevant works appear at the outset of each section. Compared with anatomical studies, physiological studies on primitive fishes are spartan in number. Furthermore, techniques for studying circulatory function are continually emerging and improving. Therefore, older physiological literature may need verification. Concerns include measurement precision, data replication, and animal welfare (the later because of the potential impact of surgical stress and animal restraint on routine physiological variables). Two of these concerns are considered briefly so that the reader can make a more informed decision about the quality of the detailed information presented in this chapter. 1.2. Measurement Systems: Their Benefits and Limitations Early in vivo estimates of blood flow and Q were obtained by either direct measurement with electromagnetic and Doppler flow probes, or indirect measurement using the Fick Principle. Electromagnetic probes report blood flow but require a reliable means to regularly check zero flow. Doppler flow probes have the advantage of being easy to zero in vivo. If either electromagnetic or Doppler flow probes do not fit snuggly to the vessel wall, they become excessively noisy, and if the orientation on the vessel is incorrect, they can underestimate flow. Attention is needed to ensure that the Doppler signal is focused on the center of the vessel. Doppler probes are more diYcult to calibrate and this must be done in situ (with relevant blood flows and blood pressures) after the experiment. Doppler probes measure blood velocity, and a loose fit on a highly elastic cardiac outflow vessel in a fish could underestimate flow if the vessel expands when blood pressure increases. A full strength signal from a Doppler flow probe requires a hematocrit of the order of 10%, which should not be a problem for normocythemic primitive fishes, but this could be an issue during calibration.

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Today’s preferred blood flow measurement technique utilizes a transit time flow probe, which is self‐zeroing, precalibrated, and has a lower tolerance for a snug fit on the vessel. However, transit time flow probes (e.g., Transonic Instruments) are expensive and their temperature sensitivity requires precalibration to the experimental temperature. In addition, probes for small diameter vessels have a limited lead length, which can be a considerable challenge with aquatic animals. Modern Doppler flow probes with their small probe head and fine lead remain the technique of choice for small fish, especially if vessel access is limited. Indirect measurement of flow using the Fick Principle has the advantage of little to no surgical intervention. Nevertheless, it has two shortcomings: (1) it is possible to overestimate Q in fish because oxygen consumed directly by gills and skin is not properly taken into account (Randall et al., 1981) and (2) the possibility of making continuous measurements of gas tensions is limited, which is a problem when fish perform arrhythmic and dynamic behaviors such as air breathing, blood shunting, exercise, and responding to gas tension changes. Hopefully, this latter problem can be resolved using continuous measurements of oxygen tension with fiber optic technologies. Placement of a flow probe around a blood vessel requires surgical intervention. Early studies rarely described how invasive the surgery was and this is a special concern given that early electromagnetic and Doppler flow probes were bulky. Surgical techniques and the size of flow probes have improved considerably over the past 40 years. For example, Q used to be measured by accessing the ventral aorta or bulbus arteriosus via ventral dissection through the pectoral musculature. Beyond tissue and vascular trauma, the pericardium was often cut which alters cardiac pumping (reduces maximum performance—Farrell et al., 1988a; and changes venous blood pressures—Johansen, 1965; Shabetai et al., 1985; Sandblom et al., 2006). In rainbow trout, access to the ventral aorta is now gained through a minor incision in the lateral wall of the isthmus, anterior of the pericardial cavity, and the union of the coronary artery (Axelsson and Farrell, 1993). This approach avoids excessive tissue trauma, opening of the pericardium, and possible occlusion of the coronary artery from a tightly fitting flow probe on the bulbus arteriosus. Routine cardiovascular performance is best assessed after full recovery from anesthesia and surgery. Therefore, measurements made either during anesthesia or shortly after recovery must be treated with caution. Overnight recovery is a generally accepted compromise between recovery and the risk of damage to expensive equipment, even though rainbow trout swim well after just a 2‐h recovery from invasive surgery (Farrell and Clutterham, 2003). Nevertheless, since heart rate typically decreases with recovery time, a protracted recovery may be essential to properly assess certain aspects

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of cardiovascular control. However, even fully recovered fish must be restrained to reduce the risk to expensive recording equipment. Hagfishes, for example, have a remarkable ability to tie multiple knots in any leads attached to them. Therefore, future work should attempt to allow fish to behave more normally. This goes beyond obvious issues of correct lighting, appropriate temperatures, and diurnal rhythms. The use of remote telemetry and video observation will reduce the influence of an investigator’s presence during cardiovascular recordings and allow the animals to behave more normally and with less physical restriction compared to animals physically connected to a recording system. Even simple measurements such as cardiac (cardiosomatic) index need careful inspection for what cardiac chambers (sinus venosus, atrium, ventricle, and conus/bulbus arteriosus) are included in the measurement. While the ventricle is usually the largest of the cardiac chambers contained within the pericardium and atrial mass is about 25% of ventricular mass, this is clearly not the case for either Latimeria or lungfishes (see below). Care is also needed to remove excess blood from the extensive trabecular spaces of the fish heart before weighing (a task that is virtually impossible if blood has coagulated) and to prevent changes in tissue water content through either dehydration in small hearts or fixation techniques. Physiological studies tend to report only relative ventricular mass as part of the calculation of myocardial power output. However, sexual maturation and temperature acclimation influence ventricular mass (Farrell et al., 1988b; Thorarensen et al., 1996). Measurement of blood volume involves marker techniques. Red blood cell markers apparently provide a better estimate of blood volume than plasma markers, which overestimate blood volume to varying degrees and in relation to the equilibration time (Bushnell et al., 1992). In fact, careful consideration of the secondary circulations and blood sinuses is needed to interpret the blood volume estimates because red blood cells do not necessarily move quickly or freely into these fluid compartments. Despite these overarching concerns, the following descriptions take data at face value unless a clear problem is noted. In this regard, it is encouraging to reevaluate Greene’s (1926) arterial blood pressures for chinook salmon and to see that they span the range we now know to be between routine and maximum blood pressure. 2. AN OVERVIEW OF EVOLUTIONARY PROGRESSIONS The purpose of this section is to provide the reader with a general overview of the various patterns of change evident among primitive fishes. This section could have appeared at the end of the chapter as a synthesis

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(i.e., after the detailed information that follows), but I felt it was more useful at the outset to provide a broad framework. For this reason, few citations are found in this section because they appear subsequently. 2.1. Anatomical Patterns 2.1.1. The Heart The number of cardiac chambers contained in the pericardial cavity is three in cyclostomes and four in all other fishes. All fish have a sinus venosus, an atrium, and a ventricle. Cyclostomes lack a ventricular outflow tract within the pericardium. There has been extensive debate on the origins of and the terminology for the ventricular outflow tract (the conus arteriosus, bulbus arteriosus, and truncus cordis: see Smith, 1918; Wright, 1984; Farrell and Jones, 1992; Icardo et al., 2005b). Long ago, Wilder (1876) distinguished a rhythmically contractile ‘‘bulbus arteriosus’’ with several rows of valves in elasmobranchs and ganoids (i.e., Polypterids, gars, and bowfins) from a noncontracting bulbus arteriosus in teleosts. Here, I use the term bulbus arteriosus if cardiac muscle is lacking and conus arteriosus if cardiac muscle is present. All primitive fishes except cyclostomes possess a conus arteriosus proximal to the ventricle, just like elasmobranchs. However, unlike elasmobranchs, the conus in most primitive fish is reduced in size and lacks true valves, and a bulbus arteriosus lies distal to the conus. Furthermore, not all primitive fishes have conal muscle that is rhythmically contractile, at least to the point of altering ventral aortic pressure recordings. The atrium and ventricle of primitive fishes do not diVer from the general piscine arrangement of trabecular muscle (spongy myocardium) with deep lacunae that allow venous blood to almost reach the epicardial surface of the heart. The spongy myocardium derives its nutrition from, and excretes its wastes into, these sinusoids (lacunae, cardiac circulation). The ventricle can have an additional muscle type (compact myocardium) with an arterial (coronary) circulation that surrounds the spongy myocardium. Thus, a variable portion of the ventricle can have a secondary, fully oxygenated blood supply directly from the gills via the hypobranchial artery or dorsal aorta. The most primitive form for the ventricle is an entirely trabecular arrangement found in cyclostomes. Even so, the coronary circulation appeared early in the evolution of the vertebrate heart, being present in elasmobranchs and apparently all other primitive fishes. The need for a more secure, arterial oxygen supply to the heart may reflect both the cardiac evolution toward a higher workload capability, as well as exposure to environmental conditions

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that rendered a venous oxygen supply unreliable (e.g., aquatic hypoxia and exercise). The coronary circulation has appeared, been lost, and reappeared during the evolution of fishes. It has cephalad (hypobranchial artery) and caudal (subclavian and coracoid arteries) origins (Parker and Davis, 1899; Grant and Regnier, 1926; Foxon, 1950; Davie and Farrell, 1991). Rays possess both caudal and cephalad coronaries, and while Latimeria and some Chondrosteans have a caudal supply, other primitive fishes and sharks have a cephalad supply. Most teleosts lack a coronary circulation, but when present it is usually a cephalad supply (a few have both cephalad and caudal supplies, e.g., eels and marlin). While the evolution of the cephalad coronary circulation appears to be tied to evolution of the compact myocardium on both the conus arteriosus (whose cardiac muscle cannot be easily supplied with a vasa vasorum) and the ventricle, the caudal origin of the coronary system in Latimeria is diYcult to reconcile, especially since there is little, if any, compact myocardium on the ventricle (see below). There are indications for some primitive fishes (e.g., Acipenser) that at least a portion of the coronary circulation is not restricted to outer compact myocardium as in most teleosts, but reaches the ventricular trabeculae as in all elasmobranchs. 2.1.2. The Branchial Circulation The number of gill pouches and gill arches is variable among primitive fishes and this results in diVerent numbers of paired branchial arteries. Hagfishes have 5–13 pairs and lampreys have 6. Teleosts and most primitive fish have 4 pairs of branchial arteries, but dipnoans and Amia have 5, although the most anterior branchial artery does not serve a respiratory function. A hyoid arch is present in Acipenser, Lepisosteus (¼Lepidosteus), Lepidosiren, and Protopterus, but absent in Neoceratodus and Polypterus. A pseudobranch is present in Acipenser, Lepisosteus, and Neoceratodus, but absent in Lepidosiren, Protopterus, and Polypterus. A high capillary density is a prerequisite for an eYcient gas exchange surface. The capillaries of mammalian and reptilian lungs, as well as teleost gills, have been described as a vascular sheet, in part because almost 90% of the surface area is occupied by blood vessels (Farrell et al., 1980). The vascular density in the gills of primitive fishes has not been well documented, but appears to be high even in cyclostomes. Thus, the gill surface area of primitive fishes probably has not been overdesigned at the expense of vascular density, except in the gills of air‐breathing fish where the lamellar vessels are ‘‘channelized’’ at the expense of vascular density to provide a more direct routing between the aVerent and eVerent arteries. Protopterus show the most extreme form of these lamellar vascular channels, which even possess an endothelial lining. The lamellar channels in Amia are much less extreme and lack an endothelium, as do teleost secondary lamellar vessels.

60

ANTHONY P. FARRELL

2.1.3. The Systemic Circulation All fish have an anatomically undivided circulation. The air‐breathing lungfishes come closest to having a functionally divided circulation. Also, there appears to be an early evolutionary polarization among vertebrates for the left side of the heart to receive oxygenated blood. Protopterus certainly return blood from the lung to the left side of the atrium and ventricle and keep it largely separated from the systemic venous return. However, recent work (Icardo et al., 2005b) has challenged the idea that the pulmonary vein of Protopterus physically terminates in the atrium proper (see below), although its terminus is such that oxygenated blood is directed to the left side of the atrium. Thus, the veins of all primitive fish anatomically terminate in the sinus venosus. 2.2. Physiological Patterns All primitive fishes have a myogenic heart that often operates at a low heart rate when compared with teleosts at similar temperatures. The potential advantage of these slower heart rates is unclear (possibilities include a lower myocardial oxygen demand or a laminar flow pattern through the heart to prevent mixing of oxygenated and deoxygenated bloodstreams in air‐breathing fish). The pacemaker rate in rainbow trout at 20
C is over 100 min1 and such high heart rates are rarely observed at these temperatures in primitive fishes. Therefore, the low heart rate in primitive fishes is likely a result of a slow pacemaker rate, even though many primitive fishes lack a neural cardiac excitatory mechanism. Ventricular myocytes also show a clear evolutionary progression toward being intrinsically capable of more forceful contractions. The hagfish ventricle has the poorest pressure generating ability and yet it has a mass comparable to that found in teleosts. The lamprey ventricle generates a higher central arterial blood pressure than hagfish but apparently uses a larger ventricle. Ventral aortic blood pressure in other primitive fishes is even higher, often more similar to elasmobranchs and lower than in most teleosts. Thus, although cardiac myocytes superficially share a similar structure among all fishes, the integration of the cellular structures must be diVerent for them to beat with diVerent tensions and at diVerent rates. The cellular basis for these evolutionary shifts in rates and quantities of ion fluxes associated with excitation–contraction coupling needs to be investigated. The progression toward higher arterial blood pressure among primitive fishes has rendered redundant the accessory hearts that assist venous return in hagfishes. Higher blood pressures likely allow regional blood flow to be

2.

CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

61

regulated in a more local and reliable manner with arterial, and perhaps venous, vasoactivity. The evolutionary shift from the 15% blood volume for hagfish to the 3% blood volume for rainbow trout has resulted in circulation time becoming about fivefold shorter because routine Q does not vary greatly among fishes. A faster circulation allows fish to detect variations in blood composition more rapidly. A more rapid detection system is consistent with the other progressive adaptations among primitive fishes of additional sensory systems (e.g., eyes), eVector systems (e.g., neural control of the heart), more complex breathing (e.g., bimodal water and air breathing), and more powerful locomotion. The evolution toward a higher central arterial blood pressure necessitated mechanisms to protect the delicate blood vessels of the branchial circulation. The evolution of a fourth cardiac chamber (the conus or bulbus arteriosus) is likely related to the need for damping the systolic oscillations in blood pressure and flow before they reach the lamellar capillaries. Why the conus has been superseded by the bulbus is unclear, but it corresponds with higher ventral aortic blood pressures. Certainly, the design characteristics of the bulbus arteriosus are such that they allow accurate predictions of ventral aortic systolic and diastolic blood pressures (David Jones, personal communication), but nothing is known about the conus in this regard. Despite this, blood pressures in the secondary lamellae can be higher and more pulsatile than those in mammalian capillaries. Cardiac filling requires energy and this can come in one of the several forms, or in some combination. Vis‐a‐tergo cardiac filling (force from behind) refers to kinetic and potential energy stored in the venous return as well as energy that is generated by a preceding cardiac chamber. Vis‐a‐fronte cardiac filling (force from front) refers to energy from the contraction of the ventricle that is hydraulically coupled to expansion of the atrium and sinus venosus by means of the pericardial cavity acting like a rigid box. Similar to elasmobranchs, a number of primitive fish orders have a stiV pericardium and can utilize vis‐a‐fronte cardiac filling. A complete pericardium is absent in hagfishes, but in lampreys a closed and rigid pericardium assists venous return to the heart. Thus, the evolution of a more powerful ventricle may have been in part to assist venous return and replace the accessory hearts that are not necessarily tightly coupled with the activity of the branchial heart. Alternatively, vis‐a‐fronte filling may have been simply a consequence of the need for a protective structure around the heart. The relationship between the evolution of vis‐a‐fronte filling and vasoactivity in venous vessels is unclear, but both are present in dogfish (Sandblom et al., 2006). Intrinsic modulation of cardiac stroke volume is possible by stretch (the Frank‐Starling mechanism) in hagfishes. Thus, the underlying cellular

62

ANTHONY P. FARRELL

mechanisms, which may be universal among vertebrate hearts, clearly evolved before any neural mechanism to modulate pacemaker rate. Similarly, paracrine excitatory control of cardiac activity (catecholamines stored in cardiac chromaYn tissue) evolved before any neural mechanism to modulate pacemaker rate being present in the cyclostome heart and several other primitive fishes (dipnoans and Lepisosteus). These catecholamine stores provide tonic cardiac stimulation but the stimuli causing their release need further study. The most primitive eVector system is therefore a paracrine adrenergic stimulation of b‐adrenoceptors. This adrenergic transduction mechanism allowed the evolution of humoral control of cardiac tissues by plasma catecholamines well before sympathetic autonomic innervation of the heart first appeared (e.g., in Amia). Therefore, although appropriate cellular transduction mechanisms were present in primitive fish heart, eVerent sympathetic nerve fibers were lacking. Consequently, a clear evolutionary progression exists toward (1) a more rapid eVector system to control cardiac activity and, then, (2) a dual, push–pull (excitatory/inhibitory) control. The neural eVector system to rapidly slow heart rate appeared early in vertebrate evolution. All primitive fishes, with the exception of cyclostomes, have vagal cholinergic (muscarinic) inhibitory cardiac control. A cardiac branch of the vagus nerve is absent in hagfish and first appears in lampreys. However, vagal cholinergic control in lampreys is an obscure nicotinic cardiostimulatory mechanism that predates the muscarinic inhibitory control and, instead, resembles the control of the catecholamine release from stores in the head kidney. Water‐breathing fish respond to environmental hypoxia with bradycardia, but air‐breathing mammals do not, although both possess the same vagal inhibitory control mechanism. Hypoxic bradycardia can reduce Q, if there is no compensatory increase in cardiac stroke volume, which would reduce myocardial oxygen demand. In addition, bradycardia can promote oxygen transfer to the cardiac myocardium by extending diastole which then prolongs blood residence time in the lumen and diastolic blood flow in the coronary arteries. However, in most cases, Q is maintained by an increase in stroke volume, which increases stroke work depending on how Laplace’s law comes into play for trabeculated myocardium with a larger end‐diastolic diameter. Randall (1982) suggested that the bradycardia and increased stroke volume allow stroke volume to be matched with secondary lamellar blood volume, thereby providing for a more even flow of blood through the respiratory lamellae and more eYcient gas exchange. Another advantage might be to prevent an excessive back pressure building up in sensitive gill secondary lamellae if the heart continued to pump blood against a large increase in systemic vascular resistance when strong skeletal muscle contractions compress skeletal muscle capillaries and stop blood flow. Lungfish lack

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CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

63

this hypoxic bradycardia (Fritsche et al., 1993; Sanchez et al., 2001) and so the evolution of lungs may have been associated with the loss of this piscine reflex. Primitive fishes tolerate severely hypoxic environments, which is perhaps not surprising considering that their lineages have survived many environmental changes. This tolerance is expressed in the circulation in at least three important ways: a low cardiac ATP demand, a coronary circulation, and an ability to breathe air. Farrell and Stecyk (2007) have argued that the low myocardial power output of hagfishes has led to a routine cardiac ATP demand that can be completely supplied through their glycolytic ATP capacity during anoxia (rather than by a greatly elevated cardiac glycolytic capacity). How widely this strategy extends among primitive fishes is diYcult to determine given the relatively few measurements of myocardial power output and maximum glycolytic capacity. Nevertheless, given that hagfishes have the lowest ventral aortic blood pressure (one of the main determinants of cardiac power output), the strategy may not be widespread. Instead, it appears that improving oxygen supply to the myocardium through a coronary circulation and air breathing has been a solution to inhabiting hypoxic environments. While the cyclostome heart relies on venous blood for its oxygen supply, all other primitive fishes have a coronary circulation in one form or another. The compact myocardium benefits from the higher arterial oxygen tension during hypoxia and especially during exercise. Farmer (1997) has argued that the evolution of air breathing among fishes was to provide a myocardial oxygen supply during exercise. This has to be subsequent to the evolution of the coronary circulation, since a coronary circulation appeared in elasmobranchs apparently well before air breathing evolved in fishes. Although the apparent loss of coronary support for cardiac muscle for the ventricle (but not the conus) in Lepidosiren (Foxon, 1950) is consistent with Farmer’s suggestion, problematic is the finding that the compact myocardium is more extensive in the facultative air‐breathing Pacific tarpon (Megalops) than in the water‐breathing rainbow trout (Farrell et al., 2007). Air breathing is certainly beneficial to cardiac oxygen supply, but not necessarily at the exclusion of a coronary circulation and probably more so during aquatic hypoxia rather than exercise. A high proportion of primitive fishes are air‐breathers, and this has resulted in numerous cardiovascular modifications that allow varying degrees of functional separation between oxygenated venous return from the air‐ breathing organ and deoxygenated venous return from the rest of the body. (Some of these features are shared by amphibians and reptiles.) Among the three extant genera of lungfishes, there is a clear progression toward a more divided circulation that parallels a greater dependence on air breathing

64

ANTHONY P. FARRELL

(the Australian Neoceratodus only breathes air under hypoxic conditions, while the South American Lepidosiren and the African Protopterus species are obligate air‐breathers). In fact, deoxygenated blood from the body can remain largely separated from the oxygenated blood returning from the lungs during its passage through the lungfish heart and gills. However, no fish possesses an anatomically divided circulation, as is the case in crocodiles, mammals, and birds. Lungfishes show four circulatory changes apparently critical to their successful transition to obligate lung breather: 1. They send pulmonary venous return directly to the atrium and this prevents mixing of pulmonary and systemic venous return in the sinus venosus. 2. They can keep pulmonary and systemic venous return largely separated within the atrium and ventricle (a prelude to a proper anatomically divided, four‐chambered heart). 3. They can direct deoxygenated arterial blood flow from the heart to the lung. 4. Cardiac output can bypass the respiratory surfaces of the gill. 3. DETAILS OF THE CYCLOSTOME CIRCULATORY SYSTEMS The superclass Agnatha contains the two orders Myxiniformes, the hagfishes, and Petromyzontiformes, the lampreys, also known as the cyclostomes. The circulatory system of cyclostomes follows a basic craniate pattern, but commands great interest by informing us on the cardiovascular features likely present in the most primitive fish. 3.1. Hagfishes Excellent summaries of hagfish circulatory systems are found in Cole (1926), Johansen (1963), Hardisty (1979), Forster et al. (1991), and Satchell (1991). A comprehensive historical review of anatomical discoveries (Chapman et al., 1963) suggests that these discoveries date back to Home (1815), Retzius (1824), and Muller (1841). Important histological and cellular studies include those by Augustinsson et al. (1956), Bloom et al. (1963), Leak (1969), Wright (1984), and Fock and Hinssen (2002). Bloom et al. (1961) have summarized the studies on the catecholamine‐containing granules found in cyclostome hearts. 3.1.1. Cardiac Anatomy The hagfishes have a main branchial (systemic) heart and three sets of accessory hearts (see Section 3.1.2). The branchial heart has a sinus venosus, an atrium, and a ventricle (Figure 2.1A), but lacks either a bulbus or a conus.

2.

65

CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

A IJV RACV

LACV VA

PVH Ven

Atr SV

CPV

SupV

PCV

B Internal carotids

Hyoid efferent Efferent branchial

Dorsal aorta Gill pouches

Velar artery External carotid

1

2

5 4 3 Afferent branchials

6

7

Ventral aorta Fig. 2.1. Arrangement of the hagfish circulation. (A) The major inflow and outflow vessels for the branchial heart (Atr, atrium; Ven, ventricle; and SV, sinus venosus) and the portal vein heart (PVH) (arrows indicate direction of flow; VA, ventral aorta; RACV and LACV, right and left anterior cardinal veins; IJV, internal jugular vein; PCV, posterior caudal vein; CPV, common portal vein; and SupV, intestinal portal vein). [Taken from Forster et al. (1991).] (B) A schematic diagram of the major arteries to and from the gill pouches. [Taken from Hardisty (1979).]

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ANTHONY P. FARRELL

The long ventral aorta (that traverses the elongate pharynx to reach the gills) has a swelling just outside of the pericardium that resembles a bulbus arteriosus and has a pair of semilunar valves. The posterior pericardium is connected with the perivisceral coelom and therefore is not completely closed. The hagfish heart is relatively large given its low power‐generating ability (see below). Cardiac index is reported as 0.18% in the Atlantic hagfish Myxine glutinosa (Satchell, 1986). Relative ventricular mass is reported as 0.1% for both Myxine (Johnsson and Axelsson, 1996) and the more active Pacific hagfish Eptatretus cirrhatus, where atrial mass is one‐third of ventricular mass (Forster, 1991). The ventricle is composed of spongy myocardium that lacks a coronary circulation. Hagfish myocytes have a low myofibrillar volume (consistent with a poor pressure‐generating ability), a large sarcoplasmic reticulum, and lack a t‐tubule system, while possessing glycogen and atrionaturetic peptide (ANP) granules in the sarcoplasm, and intercellular desmosome connections (Augustinsson et al., 1956; Bloom et al., 1963; Leak, 1969; Helle and Lonning, 1973; Reinecke et al., 1987). The other main cardiac cell type, found next to the endothelium, is devoid of myofibrils and is filled with densely staining granules associated with Golgi bodies. These granules contain catecholamines (Bloom et al., 1961; Euler and Fa¨nge, 1961; Nilsson, 1983), whose concentration varies considerably among cardiac chambers (Table 2.1). Adrenaline is dominant in the ventricle, while noradrenaline is dominant in the atrium and portal heart. The concentration of adrenaline in the ventricle of the branchial heart of Myxine was approximately the same as in the head kidney of Atlantic cod (Gadus morhua) and 1/100th of that in the chromaYn cells of dogfish (Squalus acanthias). Greene (1902) was the first to suggest that cardiac innervation is lacking in adult hagfish hearts, making them unique among vertebrates. 3.1.2. Circulatory Patterns The circulatory pattern of hagfish shows some important diVerences to the generalized single circulation of fishes. Foremost, the circulatory pattern to the 5–13 pairs of gill pouches is unusual among fishes (Figure 2.1B). Each aVerent branchial artery divides and encircles a water duct, thereby supplying both hemibranchs of a single gill pouch as well as the lamellae that project radially into that gill pouch. Each pair of eVerent branchial vessels unites onto two lateral aortae (an unusual duplication compared with other fishes), which then connect to a single median dorsal aorta. Another unusual feature is that the blood supply to the kidney is entirely arterial, that is the renal portal circulation of other fishes is lacking. In addition, the venous system has unusual asymmetries: the right ductus Cuvier is absent, and the left

2.

67

CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

Table 2.1 Catecholamine (AD, adrenaline; NA, noradrenaline) Concentrations in Tissues (mg g1; Cardiac Chamber and Blood Vessels) and Plasma (nmol liter1) of Primitive Fishes Fish M. glutinosa

M. glutinosa

M. glutinosa

L. fluviatilis L. fluviatilis P. marinus Protopterus

Protopterus

L. platyrhincus

Amia calva

A. naccarii

Tissue

AD

NA

AD (%)

Ventricle Atrium Portal heart Kidney Ventricle Atrium Portal heart Ventricle and atrium Posterior cardinal vein Ventricle Atrium Ventricle Atrium Ventricle Atrium Atrium Prox. intercostal arteries Left cardinal vein Plasma mg/100 ml (stressed fish) Plasma—normoxia Plasma—aquatic hypoxia Plasma—aerial hypoxia Ventricle Atrium Cardinal vein Plasma Plasma Plasma: HCl infusion Plasma: hypoxia

59 8.1 3.1 – 49 13 3.4 21

6.5 18 58 16 6.2 47 53 20

92 31 5 – 89 22 64 53

1.9

49

4

81 127 28 130 9.8 51 4.2 216

12 16 0 6.3 1.3 2.0 71 94

87 89 100 95 88 96 6 70

0.55 11 (27)

0.03 11 (160)

95 50 (17)

5 8

5 8

50 50

18

20

47

0.27 1.8 47.5 54 13.3 730 42.6

0.03 0.23 21.5 4.4 9.0 703 7.8

90 89 69 93 40 49 15

Plasma: normoxia Plasma: hypoxia

4.3 29.9

5.2 45.1

55 60

References Euler and Fa¨nge, 1961

Reported in Bloom et al., 1963 Perry et al., 1993

Reported in Nilsson, 1983 Reported in Bloom et al., 1963 Reported in Bloom et al., 1963 Abrahamsson et al., 1979a

Perry et al., 2005

Nilsson, 1981

McKenzie et al., 1991a McKenzie et al., 1991b Randall et al., 1992

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ANTHONY P. FARRELL

A

B

cbv hvs⬘

scs sca clh

sca⬘ sca⬙

sca⬙ rsc

rdc lcs rdc ls⬘ roc

lac ijv

Heart

Position of

is

pbd ca rac⬘ saa

ph

gut

sv ilt⬘

lac⬘ lac⬙ cysv

gut cpv

pv

ah rpc⬘ gut

lcs ilt

siv

cpc

rs rpc

lpc

Right

cv cv⬙ lcs⬘ scs⬘ cdh Right

cv⬘ mvb

Left lcs⬘ cv⬙ cdh

scs⬘ Left

cv⬙ mvb

Fig. 2.2. Arrangement of major (A) veins and (B) sinuses in hagfish. [ah, anterior hepatic vein; ca, anterior cardinal anastomosis; cbv, branchial constrictor; cdh, caudal heart; clh, cardinal heart; cysv, cystic vein; cpv, common portal vein; cv, caudal vein; hvs, hypophysiovelar sinus; ijv, inferior jugular vein; ilt, intestinal lymphatic trunk; is, intestinal sinus; lac, left anterior cardinal

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CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

69

anterior cardinal vein is larger than the right one and drains into both the branchial and portal hearts (Figure 2.2A). The chronology of the discovery of the accessory portal heart has been described by Fa¨nge et al. (1963) and Johansen (1963). The portal heart has a single chamber that consists of cardiac muscle arranged as thin trabeculae (each containing four to six fibers). It has its own pacemaker activity and looks much like the atrium of the branchial heart. Also like the atrium, the portal heart stores noradrenaline at higher (20 times) concentrations than adrenaline (Table 2.1). The cellular structure of the portal heart has been described for Paramyxine atarii (Endo et al., 1997) and Myxine (Helle and Lonning, 1973) and, much like the branchial heart, its myocytes are characterized by the presence of myofibrils, mitochondria, an extensive sarcoplasmic reticulum, glycogen granules, ANP immunoreactivity, and the absence of a t‐tubular system. The portal heart has two input vessels and one output vessel, each protected by a valve. Its main input comes from the supraintestinal/portal veins that drain the gut circulation. Thus, its main role is to overcome the vascular resistance of the hepatic circulation (Fa¨nge et al., 1963), moving blood through the liver to the sinus venosus via hepatic veins. The portal heart has additional input from the left anterior cardinal vein, but why a portion of the cephalic venous drainage can go directly to the liver via the portal heart is a mystery. Blood volume of hagfishes is around 18% and is the largest of all fishes (Hardisty, 1979). This large volume reflects extensive subcutaneous blood sinuses (Figure 2.2B), whose structure, function, control, and associated accessory hearts have received much attention and debate (Johansen et al., 1962; Satchell, 1984, 1991; Forster, 1997). The original idea that the sinuses were a primitive lymphatic system has been abandoned based on their high plasma protein content (Johansen et al., 1962) and the presence of red blood cells in cannulated hagfish (Forster et al., 1988), albeit at about one‐third of the hematocrit of the primary circulation. There are three large blood sinuses, each connected to arterial vessels via vascular papillae, about the caliber of one red blood cell (Cole, 1926). The secondary circulatory system of teleosts has similar arterial connections that filter red blood cells (SteVensen and Lomholt, 1992), and this has led to the suggestion that hagfish sinuses are the forerunner of the less capacious vein; lcs, lateral chordal sinus; lpc, left posterior cardinal vein; ls, lingual sinus; mvb, median ventral bar of caudal fin; pba, peribranchial anastomosis; ph, portal heart; pv, portal (supra intestinal) vein; rac, right anterior cardinal vein; rdc, right deep anterior cardinal vein; rpc, right posterior cardinal vein; rs, rectal sinus; rsc, right superficial cardinal vein; saa, sinoatrial aperture; sca, subcutaneous anastomosis; scs, subcutaneous sinus; siv, subintestinal (posterior) hepatic vein; sv, sinus venosus]. [Taken from Cole (1926).]

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ANTHONY P. FARRELL

secondary circulatory system (Satchell, 1992). The caudal subcutaneous sinus is the largest sinus, and it drains into an accessory caudal heart. The caudal heart of the hagfish provides venous return from the tail, as do analogous caudal heart structures in sharks and teleosts (see Satchell, 1992 for discussion). The caudal heart arises, just posterior to the last of the mucus glands, as two swellings of the paired caudal veins separated by a medial cartilaginous fin plate (Figure 2.3A and B). The swellings are encased on either side by a fan‐shaped skeletal muscle, each of which is innervated by single motor nerves arising from three spinal roots (Satchell, 1984). In addition to two veins from the subcutaneous sinus and minor inputs from the caudal veins, there is input from the marginal veins that collect blood from radial fin veins. The caudal heart pumps blood into the posterior cardinal vein and contraction of each side of the caudal heart generates a venous pressure pulse (Figure 2.3B). The peribranchial sinus has anastomoses with the anterior cardinal veins (Figure 2.2A). The hypophysiovelar sinus is located in the head region and drains into the inferior jugular vein via the cardinal ‘‘hearts,’’ which Satchell (1991) has considered ‘‘propulsors’’ rather than ‘‘hearts’’ since they are driven by the extrinsic muscles of the velar. 3.1.3. Circulatory Dynamics Various aspects of circulatory physiology in hagfish have been reviewed in Satchell (1984), Forster et al. (1991), and Forster (1997). The electrical properties of hearts from M. glutinosa, Eptatretus stoutii, and E. cirrhatus have been described by Bloom et al. (1963), Chapman et al. (1963), Arlock (1975), Satchell (1986), and Davie et al. (1987). The branchial heart is myogenic, a characteristic of all vertebrate hearts. Desmosomes provide intercellular connections between cardiomyocytes (Leak, 1969). All three cardiac chambers are capable of independent pacemaker activity, although this capability is weakest for the ventricle (Bloom et al., 1963). Pacemaker potentials have been reported in atrial cells from Myxine (Arlock, 1975). Jensen (1965) reported unusually low resting membrane potentialsfor E. stoutii (atrium ¼ –41 mV and ventricle ¼ –48 mV). The branchial heart of both Myxine and E. cirrhatus has a prolonged electrocardiogram (ECG) (Figure 2.4A and B). The electrical conduction times between the cardiac chambers are two to three times longer than other fishes. The prolonged ECG results in atrial depolarization being visible in the trace (a Pr wave; Figure 2.4A). A contributing factor in this electrical delay is likely the long, funnel‐like atrioventricular connection (Figure 2.1). Nevertheless, ventricular dP/dt is about 10 times slower that that found in teleosts

2.

71

CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

A

B CV

SpC Nch

VScS MN

MP CHM

CV MV

CH KMP DCV

LMG VScS

MV RV

C

mmHg 1.25

1.2

Caudal vein pressure

Up

Mechanogram

Down (2.5 s)

Fig. 2.3. Caudal hearts of hagfish. (A) A dorsal view to show the two valved swellings either side of a median plate (hatched, MP) that formed the caudal heart and the direction of blood flow (arrows) in the major inflow and outflow vessels (CV, caudal vein; VScS, vein from subcutaneous sinus; and MV, marginal vein). (B) A lateral view of one side of the caudal heart (CH) illustrating the motor nerves (MN) from the spinal column (SpC) that innervate the caudal heart muscle (CHM) (Nch, notochord; KMP, knob of median plate; LMG, last of the series of mucus glands; RV, radial vein; and DCV, distal caudal vein). (C) A tracing of blood pressure in the caudal vein and a mechanogram from the surface of a caudal heart. Note that the venous pulse pressure is caused by the alternate activity of each side of the caudal heart. [Taken from Satchell (1992).]

and this is probably due to slow muscle activation kinetics (Satchell, 1986; Davie et al., 1987). Routine heart rates in hagfishes are typically reported as 20–30 min1 at
10 C, and an unimpressive maximum of 35 min1 in vivo (but 15–42 min1 at 8–10
C in anesthetized E. stoutii; Chapman et al., 1963). Greene (1900, 1902) and Augustinsson et al. (1956) conclusively showed that neither vagal stimulation nor applied acetylcholine altered branchial heart rate. Therefore, any control of heart rate in hagfish must be aneural. (Curiously acetylcholine has been isolated from both branchial and portal cardiac tissues.) There is very strong evidence that the hagfish heart is under a tonic, paracrine b‐adrenergic stimulation, likely from cardiac catecholamine stores.

72

ANTHONY P. FARRELL

A

R

ECG

T

Pr V

Pressure cm H2O

18 15

Q S

P

Ventral aorta

12 Ventricle

9 6 3

Atrium

0 0 A

1 B

C

2 D EF

3

s

cm H2O

B 8

Dorsal aorta

6

R

T

P Q

ECG

S

Pressure cm H2O

10 Ventral aorta

8 6 4

Ventricle Atrium

2

0 A

B

1 C

D

2 s E F

Fig. 2.4. ECGs and superimposed traces from cardiac chambers and major arteries of lightly anesthetized hagfishes. (A) Pacific hagfish E. cirrhatus [taken from Davie et al. (1987)] and (B) Atlantic hagfish M. glutinosa [taken from Satchell (1986)]. Note the higher pressure development in the Pacific hagfish.

2.

CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

73

Depletion of catecholamine stores revealed positive inotropic and chronotropic eVects of adrenaline and noradrenaline (Fa¨nge and Ostlund, 1954). Likewise, Chapman et al. (1963) found a modest inhibitory inotropic eVect of reserpine, which then allowed a restorative, stimulatory eVect of adrenaline on isolated E. stoutii hearts. Sotalol reduced intrinsic heart rate of the working, perfused Myxine hearts (Figure 2.5A) but did not prevent a modest 3–4 min1 stretch‐induced increase in heart rate (Johnsson and Axelsson, 1996). Indeed, in vivo injections of sotalol in Myxine (Figure 2.5B) and propranolol in E. cirrhatus produced a large reduction in heart rate to around 15 min1 (Axelsson et al., 1990; Forster et al., 1992). Thus, temperature and the slow release of endogenous catecholamines are the two main modulators of heart rate in hagfishes. An unresolved question regarding the control of heart rate is to what degree and under what conditions there is a paracrine regulation of cardiac contractility and heart rate (see Reid et al., 1998 for general discussion of catecholamine release in fishes). Perfused Myxine hearts normally release endogenous catecholamines (Bloom et al., 1963) but at low levels (1 nmol liter1; Perry et al., 1993). In perfused hearts, this release is unaVected by either acidosis or high filling pressure, but is stimulated tenfold by nonphysiological conditions of 60‐mM potassium and carbachol (a cholinergic agonist), and 100‐fold with pituitary extracts. Thus, it is possible that the cardiac tissues have a very high aYnity for catecholamines and paracrine release results in very little overflow into the circulation. Alternatively, there may be a central humoral control (Perry et al., 1993). Potentially, release could even be triggered by a very low threshold to stretch, which could then kick‐start a quiescent heart (Axelsson et al., 1990). But whether heart rate declines under long‐term adverse conditions, thereby slowing the hagfish circulation, is still not known. Another unresolved question regarding the control of heart rate is to what degree increased venous return or cardiac filling can increase heart rate. Results are highly variable among studies, with a quadrupling of heart rate in E. stoutii (up to 30 min1 at 15
C; Jensen, 1961) versus a modest 20% increase (again up to 30 min1 at 10
C; Chapman et al., 1963). These responses could reflect either a stretch‐induced pacemaker eVect (as suggested by Jensen, 1961) or a release of catecholamine stores (as suggested by Johansen, 1963). More recent studies of working, perfused hearts from E. cirrhosus and Myxine point to a more modest stretch‐induced eVect because intrinsic heart rates in hagfish are similar to those recorded in vivo (Table 2.2) and only a modest (<15%), if any, increase in heart rate occurs with cardiac stretch (Forster et al., 1992; Johnsson and Axelsson, 1996). On the basis of in vitro studies, it appears that humoral eVects of catecholamines on heart rate are likely very modest. Heart rate in working,

74

ANTHONY P. FARRELL

Power output (mW g−1 VM)

A

Vs Q (ml min−1 kg−1 BM) (ml beat−1 kg−1 BM)

Control Sotalol treated

P VA (kPa) P DA (kPa)

A

1.5

*

1.0 *

0.5 40 B

*

30 20

*

10 0.8 C 0.6

*

0.4 0.2

* 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 Pin (kPa)

B

f H (beats min−1) Q (ml min−1)

2.0

2

0 2

0 3

0 30

10 Sotalol

1 min

Fig. 2.5. Cardiac performance in Myxine and the inhibitory eVect of b‐adrenergic blockade. (A) Performance of the working, perfused heart illustrating (1) the Frank‐Starling eVect of cardiac filling pressure (Pi) on cardiac stroke volume (Vs), cardiac output (Q), and myocardial

2.

75

CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

Table 2.2 A Comparison of Cardiovascular Performance for Hagfishes Myxine 10
C

Power output (mWg1) Cardiac output (ml min1 kg1) Heart rate (min1) Stroke volume (ml kg1) Ventral aortic pressure (kPa) Dorsal aortic pressure (kPa) Branchial resistance (Pa min kg ml1) Systemic resistance (Pa min kg ml1)

Eptatretus 17
C

In vivo

In vitro— max

In vivo

In vitro— max

0.15 (0.62) 8.7 (25) 22 (25) 0.41 (1.0) 1.0 (1.6) 0.77 (1.0) 31 (24)

0.50 29.4 23 1.3 1.8 NA NA

0.42 (0.88) 15.8 (23) 25 (29) 0.67 (1.0) 1.6 (2.3) 1.3 (1.9) 20 (17)

0.37 22 32 0.71 1.4 NA NA

89 (40)

NA

84 (82)

NA

Values in parentheses are the peak in vivo response to an injection of 10‐nmol kg1 adrenaline. Myxine data taken from Axelsson et al. (1990) and Johnsson and Axelsson (1996). Eptatretus data taken from Forster (1989) and Forster et al. (1992).

perfused Myxine heart was largely insensitive to adrenergic agonists (increases of just 3–5 min1), cholinergic agonists, and atropine (Johnsson and Axelsson, 1996), confirming earlier findings with adrenaline for Myxine and E. stoutii hearts (Fa¨nge and Ostlund, 1954; Chapman et al., 1963). Furthermore, plasma catecholamines are normally in the nanomolar range (Table 2.1) and even severe, acute hypoxia that reduced blood oxygen tension below the P50 for hemoglobin did not significantly increase these plasma catecholamine levels (Perry et al., 1993). Cardiac performance and contractile properties have been studied in unanesthetized hagfish (Forster et al., 1988, 1992; Axelsson et al., 1990), lightly anesthetized hagfish (Johansen, 1960; Chapman et al., 1963; Satchell, 1986; Davie et al., 1987), and working, perfused heart preparations (Forster, 1989, 1991; Johnsson and Axelsson, 1996). Like all vertebrate hearts, the branchial and portal hearts both follow the Frank‐Starling law of the heart: an increase in cardiac filling pressure can increase stroke volume severalfold (Figure 2.5A). Stroke volume can reach 1.3 ml kg1 in Myxine and 0.71 ml kg1 power output and (2) the inhibitory eVect on this response of the b‐adrenergic blocking agent sotalol. [Taken from Johnsson and Axelsson (1996).] (B) In vivo cardiovascular recordings (PVA and PDA, ventral and dorsal aortic blood pressures) to illustrate the marked slowing of heart rate ( fH) after sotalol injection. [Taken from Axelsson et al. (1990).]

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ANTHONY P. FARRELL

in E. cirrhatus (Table 2.2). Venous blood pressures and cardiac filling pressures are above ambient (Figure 2.4A and B), which is consistent with vis‐a‐ tergo cardiac filling (Johansen, 1960; Satchell, 1986). Cardiac output is routinely 9–16 ml min1 kg1 (Table 2.2), a range that is comparable with benthic teleosts at similar temperatures (Forster et al., 1991; Farrell and Jones, 1992). Routine circulation time is estimated as a sluggish 12 min for Myxine and 6 min for E. cirrhatus (based on a blood volume of 150 ml kg1 and ignoring the 30% volume found in sinuses, which has a turnover time of many hours; Forster, 1997). Maximum Q is unimpressive in hagfish (22–30 ml min1 kg1; Table 2.2) but decreases circulation time to 3–5 min. Consistent with the absence of a bulbus arteriosus, flow traces from the ventral aorta show little diastolic run oV and a zero flow (Figure 2.6A). Hagfish hearts stand out from all other vertebrates by their low and slow pressure‐generating ability. Intracardiac pressure recordings in anesthetized animals (Figure 2.4A and B) have been confirmed in vivo and with working, perfused heart preparations (Table 2.2). A peak ventricular pressure of just 1.04 kPa was reported for Myxine (Johansen, 1960) and 2.6 kPa for E. stoutii (Chapman et al., 1963). End‐systolic volume is low for the hagfish ventricle (Hol and Johansen, 1960), as in rainbow trout (Franklin and Davie, 1992). Chapman et al. (1963) found little eVect of temperature on peak ventricular performance of E. stoutii between 11 and 21
C; peak inotropic responsiveness occurred around 14
C. The hagfish heart uses oxygen eYciently, with a myocardial oxygen consumption rate that is not vastly diVerent to teleost values (around 0.4‐ml O2 s1 mW1; Forster et al., 1991). Furthermore, the glycolytic capacity of the heart can support most of the routine myocardial power (Forster, 1991; Farrell and Stecyk, 2007), suggesting that cardiac contractility is unlikely to be severely compromised by a poor oxygen supply. The poor cardiac contractility of the hagfish heart reflects a low myofibril content of cardiomyocytes, but other cellular processes related to excitation–contraction coupling need to be studied in this regard. Hagfishes have a low central arterial blood pressure (1.0–1.6 kPa; Table 2.2). This results in routine and maximum myocardial power output values that are lower than all other vertebrates. E. cirrhatus has a slightly more powerful heart than Myxine (Table 2.2). Maximum arterial pressure is an unremarkable 1.8–2.3 kPa, about one‐fifth of the maximum pressure attained by the rainbow trout heart. Approximately one‐third of arterial blood pressure is lost across the gill circulation (Table 2.2) (Bushnell et al., 1992). Johansen (1960) reported mean dorsal aortic blood pressure as 0.52– 0.78 kPa for Myxine, a range similar to subsequent reports (Table 2.2), but higher than the 0.4 kPa reported for E. stoutii (Chapman et al., 1963).

2.

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CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

B

P (kPa)

3

A

2 1

Ventral aortic pressure Dorsal aortic pressure

PVA (kPa)

2

fH (beats min−1)

0 40 0

PDA (kPa)

2

Q and VS (% change)

30

0

2s 10

2 min

R (% change)

Q (ml min−1)

3

fH (beats min−1)

0

30 20 10 80 =Q = Vs

60 40 20 0 −20 50

= Systemic = Branchial

25 0 −25 −50

0

1

2

3 4 5 6 Time (min)

7

8

9 10

Fig. 2.6. Cardiovascular performance in Myxine. (A) In vivo cardiovascular recordings to illustrate the zero diastolic flow in the ventral aorta (PVA and PDA, ventral and dorsal aortic blood pressures; Q, cardiac output; and fH, heart rate). [Taken from Axelsson et al. (1990).] (B) The stimulatory eVect of an intraarterial injection of adrenaline (P, blood pressure; Vs, cardiac stroke volume; Q, cardiac output; and R, vascular resistance). [Taken from Forster et al. (1992).]

The accessory portal heart has weaker contractions, as well as a lower output, than the branchial heart. Blood pressure was reported as 0.25 kPa (pulsing between 0.39 and 0.19 kPa) for Myxine (Johansen, 1960) and similarly 0.03–0.28 kPa for E. cirrhatus (Davie et al., 1987), that is, mean pressures that are about one‐fifth of those produced by the systemic heart. Flow was 0.1– 0.3 ml min1kg1 in Myxine (Johansen, 1960). Whether performance of the portal heart increases appreciably, say during digestion, has not been tested. Fa¨nge et al. (1963) noted that the portal heart could be distended to the size of the ventricle. Chapman et al. (1963) reported portal heart rates of 43–60 min1 at 8–10
C in anesthetized E. stoutii. For Myxine, beating rates of 47 min1 at 20
C and 24 min1 at 10
C (Fa¨nge et al., 1963) suggest a Q10 around 2. The myogenic portal heart beat is uncoupled from that of the branchial heart beat and the two hearts do not necessarily beat synchronously. Pacemaker activity

78

ANTHONY P. FARRELL

of the portal heart originates upstream in the supraintestinal vein, as demonstrated by the vein continuing to pulse and responded with an increase in pulse frequency with electrical and mechanical stimulation when severed from the heart (Fa¨nge et al., 1963), which is similar to the behavior of the portal heart when filled (Johansen, 1960). The modest inhibitory eVect of sotalol on portal heart rate in vitro and the modest stimulatory eVect of adrenaline following sotalol application suggest a tonic b‐adrenergic control similar to the branchial heart (Johnsson and Axelsson, 1996). The caudal accessory heart regulates venous return blood from the tail and the posterior subcutaneous sinuses. Excellent descriptions of hagfish caudal heart function and comparisons among fishes are provided by Satchell (1984, 1991, 1992). The caudal heart in hagfish, like other fishes, is not myogenic, but is reflexly driven by spinal nerves (Figure 2.3B). It has a rate faster than the branchial and portal hearts, can pause for many minutes, and becomes active shortly after swimming. Greene (1900) provided the first mechanograms of the beating caudal heart, demonstrating an arrhythmic beat in vivo, a steady beat in vitro, and inhibition by touch to the skin (suggesting aVerent neural control). Chapman et al. (1963) reported pulse rates of 60–72 min1 at 8–10
C in anesthetized E. stoutii. The alternate contractions of the paired skeletal muscles, with a slight pause between them, create an intriguing double pressure pulse of about 0.002 kPa superimposed on a diastolic venous blood pressure of about 0.162–0.165 kPa (Figure 2.3C). 3.1.4. Circulatory Control In vivo studies of circulatory control in nonanesthetized hagfish are quite limited (Forster et al., 1988, 1992; Axelsson et al., 1990). In addition, drug injection studies must use small volumes and be interpreted with care because hagfish have a slow circulation and a sluggish regulation of blood pressure (barostatic reflexes are impossible with an aneural heart) (Forster, 1997; see also Chapman et al., 1963). Consequently, increases in blood volume through injections can produce slowly compensated hypertension. A further complication is that blood pressure is also vulnerable to muscular activity. Adrenaline injection in Myxine increases Q and heart rate (Figure 2.6B) to levels close to maximum cardiac performance (see Table 2.2). This result could be interpreted as a consequence of either direct adrenergic stimulation of the heart or venous vasoconstriction mobilizing venous return to trigger Frank‐Starling and pacemaker stretch responses. Gill and systemic vascular resistances in hagfishes are at the low end of the range for fishes (Table 2.2) (Bushnell et al., 1992). In vivo injection of adrenaline decreases systemic vascular resistance without altering branchial vascular resistance (Axelsson et al., 1990). However, adrenaline and acetylcholine constrict perfused gill preparations from Myxine, while isoprenaline, noradrenaline, and adenosine produce vasodilatations (Axelsson et al., 1990;

2.

CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

79

Sundin et al., 1994). Acetylcholine and adrenaline constrict the perfused systemic vasculature of Myxine (Reite, 1969). Spontaneous swimming activity modestly increases ventral aortic blood pressure and heart rate without changing dorsal aorta blood pressure (Forster et al., 1988). Postactivity, heart rate increases further, blood pressure is restored, and hematocrit in the sinuses increases (Forster, 1997). Blood flow into the sinus is regulated by arterial blood pressure (Forster, 1997) and gill vasoactivity (Sundin et al., 1994). Blood flow distribution between the primary circulation and sinuses of hagfish gills is likely regulated by tonic adrenergic mechanisms since adrenaline (more so than isoproterenol) decreases sinus blood flow in perfused gills from 50% to about 10%. Thus, the common neural and humoral vascular smooth muscle control mechanisms (cholinergic and a‐adrenergic vasoconstrictions and b‐adrenergic vasodilation) are found in hagfishes. Likewise, Myxine possess many paracrine vascular smooth muscle control mechanisms. Using an isolated ventral aorta preparation, Evans and Harrie (2001) showed that endothelin‐1 elicited a strong contraction and NO a modest contraction, whereas a strong relaxation was elicited by ANP, a prostaglandin I2 agonist, and prostaglandin E2. Prostacyclin was without eVect. Hagfishes inhabit hypoxic sediments, burrow into prey during feeding, and tolerate extreme hypoxia (Hansen and Sidell, 1983). Hypoxia tolerance is aided by a high blood volume (to buVer anaerobic wastes), high cardiac glycogen stores, a peculiarly thick cardiac glycocalyx (which may be important in protecting the extracellular calcium supply to cardiac myocytes from the eVects of extracellular acidosis; Poupa et al., 1985), and an extremely low myocardial power output. On the basis of Forster’s (1991) estimates of the glycolytic ATP‐generating capacity of the E. cirrhatus heart, maximum glycolytic ATP turnover rate (41 nmol ATPs1 g1) actually lies below that for the trout, but ATP turnover rate normalized to work output (146 nmol J1) is similar to other vertebrates (Farrell and Stecyk, 2007). As a result of this cardiac glycolytic capacity closely matching routine needs, hagfish can likely circumvent the need for a myocardial oxygen supply (Farrell, 1991). Therefore, hagfish may not need to downregulate cardiac activity during anoxia as a protective mechanism, a feature that could be important for the assimilation of food and the distribution of glucose during feeding. Acute (<20 min) hypoxia actually increased rather than decreased myocardial power output (up to 0.4 and 0.7 mW g1 in Myxine and Eptatretus, respectively) and Q (Figure 2.7) possibly because the fish became agitated. However, the eVects of chronic hypoxia in hagfishes are unknown, other than Myxine can maintain the same ‘‘relative cardiac performance’’ (calculated as the product of heart rate and displacement of the pericardium) for 3 h of anoxia at 5
C and, over a 20‐h anoxic period, during which cardiac glycogen stores are depleted from 22 to 0.9 mmol g1 (Hansen and Sidell, 1983).

80 1.0 0.8

Normoxia Severe hypoxia

25 20

0.6 0.4 0.2 0.0

B

Cardiac output (ml min −1 kg −1)

A Cardiac powe routput (mW g −1)

ANTHONY P. FARRELL

15 10 5

Myxine

Eptatretus C

0

Myxine

Eptatretus

2.5

Arterial pressure (kPa)

2.0 1.5 1.0 0.5 0.0

Myxine

Eptatretus

Fig. 2.7. EVect of an acute, severe hypoxia exposure on cardiac performance of M. glutinosa and E. cirrhatus. Note that performance is not depressed with hypoxia. [Taken from Farrell and Stecyk (2007).]

3.2. Lampreys Fa¨nge (1972) has provided an excellent summary of the lamprey circulatory system, which has been studied to a lesser extent than the hagfish. Hardisty (1979) has compared the two cyclostome circulatory systems. 3.2.1. Cardiac Anatomy Descriptions and historical citations on various aspects of cardiac anatomy are found in Augustinsson et al. (1956), Bloom et al. (1961), Bloom (1962), Johansen (1963), Kilarski (1964), and Wright (1984). The branchial heart of the lamprey is unusually large by vertebrate standards. Remarkably, the adult cardiac index [reported as 0.59% in Fa¨nge (1972) and 0.25–0.41% (but only 0.1% in ammocoetes) in Hardisty

2.

CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

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(1979)] exceeds the ventricular index of tunas (but note that relative ¨ stadal and ventricular mass was only 0.07% for Lampetra planeri; O Schiebler, 1971). The ventricle is composed of spongy myocardium and lacks a coronary circulation. The blood volume of lampreys is large (8%; Fa¨nge, 1972) by vertebrate standards, and about half that of hagfishes. The cardiomyocytes contain well‐defined myofibrils, sarcoplasmic reticulum, and desmosomes (Bloom et al., 1961; Kilarski, 1964), similar to the situation in hagfishes. Lampreys are extant representatives of the first vertebrate with cardiac innervation. The vagus nerve travels along the median jugular vein and innervates the sinus venosus. Vagal stimulation accelerates the heart via nicotinic receptors (Augustinsson et al., 1956), a unique feature among vertebrate hearts because cardiac vagal stimulation typically causes bradycardia via muscarinic receptors (Nilsson, 1983). Lampreys, like hagfishes, have specialized cardiac cells with catecholamine‐ containing granules located in a loose subendothelial layer (Augustinsson et al., 1956; Bloom et al., 1961, 1963; Lignon, 1979). The ratio of adrenaline to noradrenaline varies among tissues, with adrenaline being dominant in the ventricle and atrium, and noradrenaline being dominant in the sinus venosus, similar to the portal heart of hagfishes (Table 2.1). The granules are arranged like chains of ganglionic cells throughout the three cardiac chambers (and to a lesser extent in the jugular vein), giving them a resemblance to the varicosities of adrenergic nerve terminals, but without any synaptic structures (Augustinsson et al., 1956; Falck et al., 1966). The sinus, atrium, and ventricle are fully enclosed in a pericardium that is reinforced by cartilage. There is no bulbus or conus arteriosus and a ventral aorta (truncus arteriosus) lies outside the pericardium. Two semilunar valves guard the outflow of the ventricle. The wall of the ventral aorta contains vascular smooth muscle and collagen, but apparently lacks elastin (Wright, 1984, but see Fa¨nge, 1972) and is thicker than that of hagfish, presumably reflecting a higher blood pressure (see below). Near to the heart, and unique among vertebrates, is an ‘‘intra‐arterial cushion,’’ which is a loose arrangement of connective tissue that greatly restricts the lumen of the ventral aorta (Figure 2.8A). The function of this cushion is unknown, but could either help create turbulent blood flow (to limit settling of red blood cells; see Wright, 1984 for discussion) or aid in vasoconstriction (see below for vasoactivity). 3.2.2. Circulatory Patterns The gill vascular pattern in Lampetra japonica has been superbly detailed (Nakao and Uchinomiya, 1978). Seven pairs of aVerent branchial arteries serve the anterior and posterior hemibranchs of adjacent gill pouches

82

ANTHONY P. FARRELL

VA

A VA

CA

D D L CA

V V

B Internal carotids

Hyoid efferent Efferent branchial

Dorsal aorta Gill pouches

Velar artery External carotid

1

2

4 5 3 Afferent branchials

6

7

Ventral aorta Fig. 2.8. Major vessels associated with the gills of the lamprey. (A) A histological section of the ventral aorta (VA and CA) just outside of the pericardium and ventricle (V) to illustrate the vascular cushion that protrudes into the lumen (L) beyond the ostial valves (arrows). [Taken from Wright (1984).] (B) A schematic diagram of the arterial arrangement. [Taken from Hardisty (1979).]

(Figure 2.8B) and, in doing so, they resemble other fish rather than hagfish, where an aVerent branchial artery supplies just one gill pouch. The eVerent branchial arteries mimic this pattern and join the dorsal aorta. AVerent and eVerent lamellar arteries are lacking in lampreys (Figure 2.9A). Blood vessels in the secondary lamellae have a sheetlike arrangement that is bounded by a large diameter marginal vessel (about twice that of the lamellar sheet thickness) and an axial plate that is shared by opposing secondary lamellae (Figure 2.9B). Arteriovenous anastomoses that are protected by microvilli, as in hagfishes, provide extensive connections between the aVerent filament

2.

83

CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

A Anterior

re Ad

afa eba pca

efa

ibp aba

aca

Av hbo

B Inner ridge Efferent filament a.

Nerve Smooth muscle

Marginal channel Respiratory region (Secondary lamella)

Axial blood lacunae Lamellar blood lacunae Canvernous body

Osmoregulatory region l... Chloride cells:

Afferent filament a. Nerve Filament vein Adipose tissue Peribranchial venous sinus

Smooth muscle

Fig. 2.9. Circulatory pattern for the lamprey gill. (A) Major vessels to and from the gill (Av, ventral aorta; aba and eba, aVerent and eVerent branchial arteries; afa and efa, aVerent and eVerent filament arteries; aca and pca, anterior and posterior collecting artery; ibp, internal branchiopore; re, oesophageal branch; Ad, dorsal aorta; and arrows, direction of blood flow) and (B) major vessels to and from a secondary lamella. [Taken from Nakao and Uchinomiya (1978).]

84

ANTHONY P. FARRELL

artery and the gill sinuses. Sinus blood of lampreys has a low hematocrit. There is rich innervation along the eVerent branchial artery (Figure 2.9B). Lampreys have four unusual circulatory features. One feature is the lack of a renal portal system, already noted for hagfishes, such that the kidney only has an arterial supply. Intestinal ‘‘vascular couples’’ are another unusual circulatory arrangement (detailed by Baxter, 1958). These are composite vessels made up of an artery contained within a vein. In this case, each couple involves an intestinal artery vessel from the dorsal aorta and intestinal vein returning to the posterior cardinal vein. The arrangement arises developmentally by an invasion of a vein into the outer connective tissue of the artery. Their functional significance is unknown (Baxter, 1958). The large (600 mm) vascular cushions (sphincters) in the dorsal aorta associated with the origins of the segmental parietal arteries are a third unusual feature. Again, they may induce turbulent blood flow to ensure that red blood cells leave the dorsal aorta during sluggish aortic blood flow. Baxter (1958) emphatically rejected the presence in lampreys of either valves in the wall of the dorsal aorta or lymphatics. The fourth unusual feature is the developmental loss of the left ductus Cuvier in adult lampreys (Fa¨nge, 1972). 3.2.3. Circulatory Dynamics In vivo heart rate is 33–50 min1 at 16
C in adult Lampetra fluviatilis (Hardisty, 1979) and 23–37 min1 at 15
C in Geotria australis (Macey et al., 1991). Similar heart rates at 18–20
C are apparent from traces presented for isolated L. fluviatilis hearts (Augustinsson et al., 1956; Falck et al., 1966). A heart rate of 8 min1 at 11
C for L. planeri ammocoetes (Lignon, 1979) seems unusually low. For Geotria, the Q10 is 1.2 between 5 and 15
C and 2.2 between 15 and 25
C (Macey et al., 1991). For Entosphenus tridentatus, heart rate increased from 25 to 45 min1 between 4 and 20
C, and then jumped to between 80 and 130 min1 from 20 to 25
C (Johansen et al., 1973), perhaps signifying arrhythmias at these unusually high temperatures for lampreys. Lampreys possess two cardioaccelerator mechanisms and therefore are unusual among vertebrates. Neural regulation of the myogenic lamprey heart has been detailed by Augustinsson et al. (1956) and Falck et al. (1966), who conclusively confirmed Carlson’s (1906) unusual observation that stimulation of the vagal nerve accelerated heart rate. The findings that acetylcholine, nicotine, and even cigarette smoke cause cardioacceleration led to the conclusion that the cardiac receptors were nicotinic. These researchers (see also Lignon, 1979) also characterized a positive inotropic and chronotropic eVect through the stimulation of b‐adrenergic receptors. Adrenergic stimulation likely involves the paracrine action of the cardiac chromaYn tissue. In isolated lamprey hearts, b‐adrenergic antagonists

2.

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85

and reserpine treatment (to deplete catecholamine stores) have negative inotropic and chronotropic eVects and subsequently enhance the stimulatory eVects of b‐adrenergic agonists. Extracellular calcium, especially with acidosis, also enhances the positive inotropic eVect of catecholamines, suggesting b‐adrenergic modulation of intracellular calcium availability, similar to the situation in teleosts (Shiels et al., 2002; Vornanen et al., 2002). Perfused ammocoete hearts show an overflow of adrenaline into the perfusate (Lignon, 1979). While the mechanism behind catecholamine release from the lamprey heart has not been adequately studied, it is not a result of vagal stimulation (Augustinsson et al., 1956). Humoral chronotropic responses are also possible in lampreys because circulating catecholamine levels rise during stress (Dashow et al., 1982), and both adrenaline and noradrenaline increase in vivo heart rate to 47 min1 in Geotria (Macey et al., 1984). In the absence of cardiac vagal inhibitory innervation, slowing of the lamprey heart must come about by the withdrawal of either the excitatory vagal tone or the excitatory paracrine/humoral adrenergic tone. The physiological importance of stretch‐induced tachycardia in lampreys needs to be revisited. Although Jensen (1969) reported tachycardia (22–44 min1) in Petromyzon marinus, an excessive increase in ventricular filling pressure (up to 2.1 kPa) makes the physiological significance of this finding questionable. Johansen et al. (1973) reported subambient central venous pressures (0.4 to 0.1 kPa) for E. tridentatus. These data indicate vis‐a‐fronte suction filling is important as a result of the rigid pericardium in lampreys (Satchell, 1991). Information on cardiac performance and blood pressures in lampreys is conspicuously absent from general reviews (Bushnell et al., 1992; Farrell and Jones, 1992). Instead, indirect information points to a more powerful heart than hagfishes. The Fick estimate of routine Q was 32 ml min1 kg1 for E. tridentatus (Johansen et al., 1973). Geotria has a similar routine oxygen uptake as E. tridentatus (Macey et al., 1991) and so Q may be similar. Routine Q in lampreys is potentially about the same as maximum Q in hagfishes. Mean dorsal aortic blood pressure is between 2.5 and 4.4 kPa in E. tridentatus, with pulse pressure usually less than 1.2 kPa (Johansen et al., 1973). This means that the lamprey heart can generate a central arterial blood pressure two to four times greater than the Myxine heart, but it uses apparently two to six times more cardiac mass. As a result, performance of the lamprey heart is superior to hagfishes likely because of a larger ventricular mass. Potentially, the mass‐specific myocardial power output is much lower in lampreys than in teleosts, but accurate measurements are needed to test this prediction.

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ANTHONY P. FARRELL

3.2.4. Circulatory Control Recovery from stress in Geotria decreases heart rate from 64 to 20 min1 over 4 h. Since this decline in heart rate is slower and smaller than the fivefold decrease in oxygen uptake (145‐ to 25‐ml O2 h1 kg1; Macey et al., 1991), cardiac stroke volume and venous oxygen content are restored faster than heart rate. Seasonal variation in heart rate in Geotria is in proportion with oxygen uptake, but independent of temperature (Macey et al., 1991). The basis for this regulation is unknown. Using isolated ventral aortic tissue, Evans and Harrie (2001) showed that P. marinus has many of the paracrine vascular control mechanisms found in Myxine. DiVerences for the lamprey included very strong contractions with endothelin, strong relaxations with NO and prostacyclin, and acetylcholine having no eVect. Studies with isolated dorsal aortic tissue have led to the suggestion that a primordial feature of the earliest vertebrates is the hypoxic response of large vessels being mediated by the gas H2S (Olson et al., 2006). Both H2S and hypoxia produce similar responses (these are vessel‐specific vasodilations and vasoconstrictions) in at least one representative species of each vertebrate class and show essentially identical depolarizing eVects on the transmembrane potential. Other criteria used in reaching this conclusion include: H2S being vasoactive at relevant physiological concentrations; blood vessels producing H2S enzymatically; the competitive eVects of H2S and hypoxia; inhibitors of H2S synthesis partially or completely blocking hypoxic responses; and the addition of cysteine, the precursor for H2S production, enhancing the hypoxic response (Olson et al., 2006). 4. DETAILS OF THE SARCOPTERYGII (LOBE‐FINNED FISHES) CIRCULATORY SYSTEMS 4.1. Coelacanth Nothing is known of the circulatory physiology of coelocanths except what can be deduced from anatomical studies. Circulatory anatomy for Latimeria chalumnae is extensively detailed by Millot et al. (1978) and the following account is taken from this treatise. The ventricle is composed largely of muscular trabeculae (Figure 2.10A) without any remarkable features. The atrium is larger than the ventricle and has ostial valves protecting the inflow and outflow. The sinus venous also has trabecular structures (Figure 2.10A), which are unusual among fishes. The heart is contained in a tough, fibrous pericardium and there are vestiges of

2.

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CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES

B

A a.br.aff. 1

2 cm

a.br.aff. 3 a.br.aff. 4 per. trc.a.

2 cm

a.br.aff. 2

valv.c.a

par.vt. c.a. cav.vt. o.at.vt. vt. at.

cav.at. v.s.oes.

valv.sin.at. can.C.g.

o.sin.at. can.C.dr.

v.jug.i.g. cav.sin.v. per. sirr.v. v.s.int

can.C.g. per.

v.hep.g. V.C. v.pulm.

v.pulm.

Fig. 2.10. The coelacanth heart and its major inflow and outflow vessels. [a.br.aV., aVerent branchial arteries; at, atrium; c.a., conus arteriosus and its valves (valv.c.a.); can.C.g., left ductus Cuvier; can.C.dr., right ductus Cuvier; cav.sin.v., sinus venosus lumen; cav.vt, ventricular lumen; o.at.vt., atrioventricular ostium; o.sin.at., sinoatrial ostium; par.vt., retracted ventricle; per., pericardium; sin.v., sinus venosus; trc.a., truncus arteriosus; valv.sin.at., sinoatrial valve; V.C., vena cava; v.hep.g., left hepatic vein; v.jug.i., internal jugular vein; v.pulm., pulmonary vein; v.s.int., subintestinal vein; v.s.oes., oesophageal vein; and vt., ventricle]. [Taken from Millot et al. (1978).]

the pericardioperitoneal canal found in elasmobranchs. Both features raise the possibility of vis‐a‐fronte cardiac filling. A 35‐kg coelacanth is reported to have a 20‐g heart, which converts to a cardiac index of 0.57%. What proportion of this large relative cardiac mass is ventricle is unclear. The cardiac outflow tract is unusually long (Figure 2.10B), consisting of a posterior conus arteriosus and an anterior bulbus arteriosus, each of similar length. Two sagittal valves protect the opening of the ventricle and the conus contains four longitudinal rows of five to six valves that increase in size

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anteriorly. The short ventral aorta fans out into three pairs of aVerent branchial arteries, with the most posterior pair branching further to supply the third and fourth branchial arches. In addition, there is a vestige of an aVerent hyoidean artery. The eVerent branchial arteries unite onto the anterior dorsal aorta and its first major posterior branch is the coeliomesenteric artery. Two lateral coronary arteries supply the ventricle and conus arteriosus. They have a caudal origin from the subclavian arteries and reach the heart laterally in association with the ductus Cuvier (Figure 2.11A). This caudal coronary arrangement is similar to that found in skates, rays, and some Chondrosteans. Large coronary veins drain into the sinus venosus. Despite a well‐developed coronary circulation, a thick compact myocardium is not evident in any cardiac illustration, which raises the possibility that coronary vessels of Latimeria largely invest ventricular trabeculae. The venous arrangement in Latimeria is illustrated in Figure 2.11B. Notably, a vestigial pulmonary vein can be traced from the fat‐filled lung, but there is no vestigial pulmonary artery from the fourth eVerent branchial artery, as in the dipnoans. The vestigial lung (air bladder) is instead supplied by several small arterial branches from the dorsal aorta. 4.2. Dipnoi (Lungfishes) The lungfish circulatory system has commanded the most attention of all primitive fishes because lungfish are the oldest of extant air‐breathing fish and are at the root of tetrapod evolution. In fact, many evolutionary parallels have been drawn between the lungfish and urodele circulatory systems. However, despite a steady interest in the macrocirculation after the early anatomical studies (Muller, 1842; Gunther, 1871) and the pioneer physiological studies (summarized in Johansen, 1970), few physiological studies have followed the excellent review of the dipnoan circulatory system by Burggren and Johansen (1986). The circulatory adaptations of dipnoans are placed in the broader context of all air‐breathing fishes by Graham (1997) and Olson (1994). The hearts and circulatory patterns of lungfishes diVer from the typical piscine arrangements in a number of important respects. Here, the emphasis is on how the basic fish circulatory system was modified for air breathing. As noted already, Neoceradatus breathes air only under hypoxic conditions, while Lepidosiren and Protopterus species are obligate air‐breathers. Also, Lepidosiren and Protopterus can live for long periods out of water (estivation). Along with the greater dependence on air breathing, Lepidosiren and Protopterus show greater deviation from the normal piscine circulatory design and a progression toward a more divided circulatory system than

A

2 cm

c.a.

can.C.dr. v.jug.i.dr. at.

sin.v.

vt. a.cor.dr. v.cor.dr.

B v.caud.

v.s.ch.g. v.s.ch.dr.

v.card.pg.

v.gén.dr.

v.cut.lat. v.s.cl. a.abd.l.

can.C.dr. can.C.g. v.pulm.

v.ng.an. v.s.cd. v.ren.aff.

v.interrén. v.s.int. v.hep.g. sin.v. v.jug.i. v.ng.pelv.

V.C.

v.hep.dr.

v.intr.int. v.gastr.int. v.int.d. v.hep.g. v.bd.gastr.int.dr. v.bd.gastr.int.g. v.gastr. trc.p.hep.

Fig. 2.11. (A) Lateral view of the right caudal coronary supply to the coelacanth heart. (B) The major posterior veins of the coelacanth. [a.br.aV., aVerent branchial arteries; a.cor.dr., right coronary artery; at., atrium; c.a., conus arteriosus and its valves (valv.c.a.); can.C.g., left ductus Cuvier; can. C.dr., right ductus Cuvier; cav.sin.v., sinus venosus lumen; cav.vt., ventricular lumen; o.at.vt., atrioventricular ostium; o.sin.at., sinoatrial ostium; par. vt., retracted ventricle; per., pericardium; sin.v., sinus venosus; trc.a., truncus arteriosus; valv.sin.at., sinoatrial valve; v.caud., caudal vein; v. interren., interregnal vein; V.C., vena cava; v.cor.dr., right coronary vein; v.gast., gastric vein; v.hep.g., left hepatic vein; v.jug.i., internal jugular vein; v.pulm., pulmonary vein; v.s.int., subintestinal vein; v.s.oes., oesophageal vein; and vt., ventricle]. [Taken from Millot et al. (1978).]

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ANTHONY P. FARRELL

B

1 2 1 1 C

A

A

A P

SV

L R R

L

V

C

1 23 4 5

d.ch v.ch sw sp.v Vn Dr

Fig. 2.12. The lungfish heart. (A) A scanning electron microscopic image of the inside of the sinus venosus (SV) of the Protopterus heart to illustrate the pulmonary vessel is embedded in its wall (arrow) and the left (L) atrium is larger than right (R) atrium (A). [Taken from Icardo et al. (2005a).] (B) A scanning electron microscopic image of a sagittal section through the ventricle (V) of the Protopterus heart to illustrate the left (L) and right (R) sides to the ventricle that are

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Neoceratodus. To reiterate and as detailed below, lungfishes show four circulatory changes critical for the transition to obligate air breathing with a lung: They do not mix pulmonary and systemic venous return in the sinus venosus because of a separate return of pulmonary blood to the atrium; pulmonary and systemic flows remain largely separated during their passage through the atrium and ventricle; deoxygenated arterial blood flow can flow directly from the heart to the lung; and Q can bypass the gill respiratory surface. Radiographic imaging of blood flow streams (Johansen and Hol, 1968; Szidon et al., 1969) and measurements of gas tensions (Johansen et al., 1968) have provided the key evidence for this functional separation between the pulmonary and systemic circulations. 4.2.1. Cardiac Anatomy In Neoceratodus, the single pulmonary vein joins the sinus venosus (Foxon, 1950). This union is unlike other air‐breathing fishes, where the venous drainage from the air‐breathing organ is less central. Protopterus and Lepidosiren have an even more unusual cardiac modification since they are the only fishes known to separate the pulmonary and systemic venous return to the heart. After the paired pulmonary veins unite, the single pulmonary vein becomes embedded in the wall of the sinus venosus of Protopterus and Lepidosiren (Figure 2.12A). The vein then runs longitudinally toward the atrioventricular plug and fuses with the membranous pulmonalis fold of the atrium. Meticulous examination of the inflow tract of Protopterus led Icardo et al. (2005a) to conclude that the pulmonary vein terminates in the sinus venosus so close to the sinoatrial junction that oxygenated blood from the pulmonary vein empties directly into the left lobe of the atrium. Thus, they rejected the previously held idea that the pulmonary vein actually terminated in the atrium (unlike all other fishes where veins do not terminate beyond the sinus venosus). Instead, the pulmonary vein in Protopterus has a functional termination in the atrium and an anatomical termination in the sinus venosus. All three genera have a single atrium that is partially divided externally and internally into a larger right and smaller left side, and that is heavily trabeculated. This appearance caused Gunther (1871) to incorrectly refer to the heart as trilocular (Figure 2.12B). Lepidosiren shows the greatest degree separated by the vertical septum (VS) and the atrioventricular plug (P), and through the outflow tract of the heart (C) with its intralumenal spiral and bulbar folds (1), which fuse at the asterisk. [Taken from Icardo et al. (2005b).] (C) A schematic diagram of the outflow tract in Lepidosiren, which has a reduced spiral valve (sp.v) compared with Protopterus. (The aVerent brachial arteries are numbered; d.ch., dorsal channel; v.ch., ventral channel; sw, swelling in the spiral valve; pr.v, proximal semilunar valves; Vn, ventral; and Dr, dorsal). [Taken from Satchell (1976).]

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ANTHONY P. FARRELL

of atrial subdivision and Neoceratodus the least. The left lobe of the atrium represents the pulmonary channel into which the pulmonary vein empties and is formed in part by the pulmonary fold in the wall of the atrium and the atrioventricular plug. A sinoatrial valve guards the entrance into the right atrium, but the pulmonary vein is apparently not valved at its termination. The atrioventricular channel has an atrioventricular plug (cushion), formed from a cartilaginous core surrounded by connective tissue (see Icardo et al., 2005a for details). The atrioventricular plug moves into and out of the atrioventricular orifice during cardiac contraction and relaxation, and while serving as a one‐way valve during ventricular systole, it allows the right and left bloodstreams to remain separated as they leave the atrium. This type of separation would be diYcult with the typical ostial valve found in other fishes. The rounded lungfish ventricle is highly trabeculated and compact myocardium is not evident (Figure 2.12B). Again, in all three genera, the ventricle is divided into right and left sides by a large muscular ridge, the vertical septum, which extends from the apex between the dorsal and ventral walls (Figure 2.12B). The vertical septum is developed to the greatest extent in Lepidosiren and least in Neoceratodus. Collectively, the arrangement of the pulmonary vein, the pulmonary fold, the atrioventricular plug, and the vertical septum results in oxygenated blood returning from the lung being preferentially directed through the left side of the atrium to the left side of the ventricle. Deoxygenated systemic venous return is preferentially directed through the larger right side of the atrium to the right side of the ventricle. To what degree the highly trabeculated structure of the cardiac chambers restricts intracardiac mixing by immobilizing blood during diastole [as suggested by Shelton (1976) for amphibian hearts] is unclear. The ability of lungfish to direct deoxygenated blood flow from the heart to the lung depends on specializations in the cardiac outflow tract and in the circulation (see Section 4.2.2). The long outflow tract from the lungfish heart, described by Icardo et al. (2005b), is perhaps the most complex of all fishes (Figure 2.12B and C). In Protopterus and Lepidosiren, it has three sections, a 270
rotation, conal valves, and incomplete partitioning of its lumen with a spiral valve. The net eVect of these anatomical adaptations is that the separation of oxygenated and deoxygenated blood is maintained to a large degree within the outflow tract. As a result, deoxygenated systemic blood is preferentially directed to the two posterior gill arches, while the oxygenated pulmonary blood is preferentially directed to the two anterior gill arches (Johansen and Hol, 1968; Johansen et al., 1968; Szidon et al., 1969). The proximal portion of the outflow tract displays several rows of valves on the dorsal and ventral walls. It possesses a thick layer of well‐vascularized, circumferentially arranged, and compact myocardium and should be termed

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conus arteriosus (Icardo et al., 2005b). A large fold of loose connective tissue, the spiral fold, arises on the inside left wall and runs most of the length of the outflow tract in Protopterus and Lepidosiren. The spiral fold is greatly reduced in Neoceratodus, especially in the distal region (Figure 2.12C). In Protopterus and Lepidosiren, a smaller bulbar fold arises in the middle portion of the outflow tract, but it does not fuse with the spiral fold until the distal portion of the outflow tract. The medial and distal portions of the outflow vessel contain predominantly elastic and connective tissue and should be termed bulbus arteriosus (Icardo et al., 2005b). A cephalad coronary artery supply reaches to the compact cardiac muscle of the conus arteriosus and, to a limited extent, the ventricle. The possibility of coronary vessels reaching the large ventricular trabeculae has not been explored. Innervation of the lungfish heart has been described in Abrahamsson et al. (1979a,b), Nilsson (1983), and Axelsson et al. (1989). Vagal innervation reaches the sinus venosus and probably the atrium (see Section 4.2.3). Spinal autonomic innervation is absent. Instead, a putative adrenergic control is provided by the large masses of chromaYn cells that line the inner wall of the atrium (but not the ventricle), the left cardinal vein, and curiously the walls of the intercostal arteries near the dorsal aorta (Abrahamsson et al., 1979a). Noradrenaline predominates in the atrium, while adrenaline predominates in the other locations, with the exception of the plasma (Table 2.1). 4.2.2. Circulatory Patterns The circulatory patterns of the gills and lungs are summarized in Burggren and Johansen (1986). Early macroscopic studies include Boas (1880), Spencer (1892), Lankester (1878), Robertson (1913), and Bugge (1961). The functional arrangement of the gill arteries is summarized in Johansen et al. (1968), Szidon et al. (1969), Gannon et al., (1983), and Fishman et al. (1985). The circulatory pattern of the gill diVers considerably among the three genera, and Protopterus shows the greatest divergence from the basic piscine pattern (Burggren and Johansen, 1986). The gill microcirculation of Protopterus is detailed in Laurent et al. (1978). The ventral aorta is almost nonexistent because four aVerent branchial arteries originate immediately outside the pericardium, similar to elasmobranchs. In Neoceratodus, the first and second (anterior) gill arches are filament‐bearing holobranchs and their eVerent branchial arteries directly join the dorsal aorta. However, the two anterior gill arches in Protopterus and Lepidosiren are not true holobranchs because the arches lack gill filaments and the aVerent branchial artery passes directly to the dorsal aorta (Figure 2.13A). Thus, oxygenated blood from the ventral root of the bulbus arteriosus reaches to the dorsal aorta and the systemic circulation

94

ANTHONY P. FARRELL

A

C

A P Right Pulmonary Artery

da

B

Ductus

Aorta

p.a

I

p.v

2a 3a

4a

c 6a

Fig. 2.13. The circulation pattern in the obligate air‐breathing lungfish Protopterus. (A) A vascular cast of the gills showing that the anterior two gills arches are devoid of gill arches and each contains a single vessel that connects directly to the dorsal aorta (A), while the posterior two gill arches possess a coarse arrangement of gill filaments and their eVerent flow is directed more to the pulmonary artery (P). [Taken from Jesse et al. (1967).] (B) A schematic diagram of the vascular arrangement of the gills and the lung. (Aortic arches are numbered; c, conus; da, dorsal aorta; and p.a and p.v, pulmonary artery and vein.) [Taken from Satchell (1976).] (C) A close of the connecting vessel between the third and fourth gill arch and the origin of the pulmonary artery. [Taken from Szidon et al. (1969).]

without going through a gill respiratory surface, which is not the case for Neoceratodus. The gill circulation of Neoceratodus is distinguished by the absence of a hemibranch on the hyoid arch. However, in Protopterus and Lepidosiren, the aVerent branch from the first gill arch supplies an additional hemibranch located on the hyoid arch (Figure 2.13B). This is analogous to the pseudobranch because the eVerent vessels of the hyoid arch go on to form the main vessel supplying the brain, the internal carotid artery. All three dipnoan genera have primary gill filaments and secondary lamellae on their third and fourth (posterior) gill arches. The gill filaments

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in Lepidosiren are sparse and coarse (Laurent et al., 1978). The aVerent branchial artery of the fourth gill arch also supplies blood to a hemibranch located on a fifth gill arch (Figure 2.13B). The posterior gill arches are supplied predominantly with deoxygenated systemic venous blood from the dorsal channel of the bulbus arteriosus. Thus, combined with a complex arrangement of the posterior eVerent branchial arteries (Figure 2.13B), dipnoans can preferentially direct deoxygenated blood to the lung and be highly successful air‐breathers. The fourth eVerent branchial artery is contiguous with the pulmonary artery (Szidon et al., 1969), while the third eVerent branchial artery goes more directly to the dorsal aorta. However, the third and fourth eVerent branchial arteries have a short connecting vessel, the ductus (ductus arteriosus; Figure 2.13C). The ductus is highly invested with vascular smooth muscle (up to 30 layers as compared with just 5–7 in the dorsal aorta) and clearly plays a regulatory role in blood flow distribution between the systemic and pulmonary circuits (see below). The lung is perfused by two pulmonary arteries that originate at the base of the fourth eVerent branchial arteries close to the dorsal aorta. The left pulmonary artery perfuses the lung from a ventral aspect and becomes branched partway down the lung, while the right pulmonary artery perfuses the lungs from the dorsal side. Neoceratodus has one lung, and Protopterus and Lepidosiren have two lungs. The vascular pathways in the posterior gill filaments of Protopterus are highly modified. Three arterio‐arterial vascular shunt pathways allow (Laurent et al., 1978) blood to bypass the normal lamellar respiratory area. These vascular adaptations would be important either in severely hypoxic water, when oxygen could be lost across to the water from blood across the gill secondary lamellae, or in estivating lungfish when secondary lamellae might collapse during air exposure. A short shunt vessel connecting the base of the aVerent filament artery to the base of the eVerent filament artery provides one bypass route (Figure 2.14A). This shunt has not been reported for other fishes. The aVerent filament artery that continues around the tip of the filament provides a second bypass route, although it does taper and lose vascular smooth muscle, (Figure 2.14A). The vessels of the secondary lamellae resemble second order arterioles, designed more for throughput rather than for exchange, as is the high‐density vascular sheet typically found in teleost secondary lamellae (Farrell et al., 1980). They also have distinct origins and terminations on the aVerent and eVerent filament arteries, possess vascular smooth muscle, are lined with endothelium, and are covered by multiple layers of epithelium (Figure 2.14B and C) (Laurent et al., 1978). All these features are normally absent in secondary lamellae. Angiogenesis in the gill lamellae of Protopterus must follow a unique trajectory among fishes and further work is needed to ascertain the role that pillar cells might play

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ANTHONY P. FARRELL

a-a a-v

A

aa2 aa1 bv ea2 S

aba eba n v m cg

na

B

ea1

C

ea1

pa eba

IV V

2 mm

VI

aa1

100

Fig. 2.14. Details of the vascular arrangement in the posterior gill arches of Protopterus. (A) A schematic of vascular arrangement of a gill filament illustrating the shunt vessels (S) between the aVerent (aba) and eVerent (eba) branchial arteries, as well as the primary aVerent and eVerent primary (aa1, ea1) and the secondary (aa2, ea2) aVerent and eVerent arteries (bv, branchial vein; cg, cartilage; m, muscle; na, nutritive artery; v, vein; and arrows, direction of blood flow) [inset shows detail of the tip of the filament with the connection between the aVerent (aa) and eVerent (ea) filament arteries (a‐a, arterioarterial capillaries and a‐v, arteriovenous system).

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in lamellar vessel development. Like other fishes, Protopterus gills possess a well‐developed arteriovenous system, with vessels originating from the eVerent filament artery (Laurent et al., 1978). Gannon et al., (1983) concluded that the arterial system of the gift filaments of Neoceratodus was similar to that of the elasmobranch pattern and lacked the specializations and shunt vessels evident in Protopterus. In addition they observed that both the lung and the secondary lamellar contained highly vascularized capillary sheets. 4.2.3. Circulatory Dynamics Dipnoans are prime candidates for high‐quality studies of cardiovascular physiology. Studies of circulatory physiology in lungfish are very limited, unlike studies of the anatomy and respiratory properties of the circulatory system. Also, the classical studies by Johansen et al. (1968) on unanesthetized lungfishes and by Szidon et al. (1969) on anesthetized Protopterus have inconsistencies. For Protopterus at 18
C, heart rate, stroke volume, and Q are reported as 15 min1, 1.3 ml kg1, and 20 ml min1 kg1, respectively (Johansen et al., 1968). For Protopterus at 25
C, dorsal aortic flow is reported as 34 ml min1 (range 20–48 ml min1; body mass was not given and varied from 0.3 to 4 kg) (Szidon et al., 1969). Cardiac output increased with air breathing but values were not given. Using the Fick Principle, Q in Neoceratodus apparently increased sevenfold following 45 min of hypoxia (Johansen et al., 1967), which suggests Q had been extremely low during hypoxia. In contrast, direct measurement of Q with a Doppler flow probe failed to find such large changes in Q during hypoxia with Neoceratodus (Fritsche et al., 1993). Compared with teleosts, mean arterial blood pressures seem to be lower for all three genera of dipnoans. Johansen et al. (1968) suggested, without convincing data and great variability in the individual blood pressure tracings (1.9–2.3 kPa), that arterial blood pressures in Neoceratodus were higher than in the other two genera. While diastolic and systolic blood pressures in the ventral aorta were reported as 2.6 and 4.2 kPa, respectively (Fig. 2 in Johansen et al., 1968), peak ventricular pressure was only 2.3 kPa in another figure (their Fig. 3). Also, in a later figure (their Fig. 19), the highest ventral aortic blood pressure was clearly associated with the heart being unable to pump blood through the gills. A mean ventral aortic blood pressure of 2.7 kPa (pulse pressure ¼ 1.1 kPa) may be normal for Neoceratodus. (B) A vascular cast of the coarse arrangement of the filaments and the eVerent branchial arteries (eba) on the IV, V, and VI aortic arches. A close‐up of a vascular cast near the tip of a gill filament illustrating the large diameter and direct vascular connections between the aVerent (aa1) and eVerent (ea1) filament arteries. [Taken from Laurent et al. (1978).]

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ANTHONY P. FARRELL

For anesthetized Protopterus, Szidon et al. (1969) showed a representative peak ventricular pressure of 2.0 kPa (Figure 2.15) and suggested that peak ventricular pressure can reach 2.5 kPa, but then stated that the postbranchial blood pressure was 3.0 kPa in an unanesthetized specimen. Assuming Q is 20–35 ml min1 kg1, mean ventral aortic pressure is 3.0 kPa and relative ventricular mass is 1g kg1 body mass, routine myocardial power output of lungfish can be estimated as 1.0 to 1.8 mW g1 ventricular mass. Heart rates are comparatively low in dipnoans: 29 min1 at 27
C in Lepidosiren (Axelsson et al., 1989), 38–41 min1 at 25
C in Lepidosiren (Sanchez et al., 2001), 47 min1 in Protopterus at 25
C (Perry et al., 2005), 32 min1 in Protopterus (from Fig. 15 in Johansen et al., 1968), 20–30 min1 and 30 min1, respectively, in anesthetized and unanesthetized Protopterus at 25
C (Szidon et al., 1969), 22 min1 at 20
C in Neoceratodus (Fritsche et al., 1993), and 24 min1 in Neoceratodus (from Fig. 14 in Johansen et al., 1968). It may be that a low heart rate and a gentle cardiac contraction are needed to ensure laminar, rather than turbulent, flow in the cardiac chambers, and thereby aid the separation of oxygenated and deoxygenated bloodstreams. The thick pericardium suggests that vis‐a‐fronte cardiac filling is important in dipnoans. In fact, subambient cardiac filling pressures were reported for all three genera by Johansen et al. (1968), who concluded that suctional attraction by the heart was important for the systemic veins; however, the pulmonary veins had above ambient blood pressures. Szidon et al. (1969) similarly concluded that vis‐a‐fronte filling was important based primarily on subambient pressure measurements within the pericardial space of Protopterus, but in their figures, venous pressures were above ambient (Figure 2.15; these experiments referenced the pressure transducer to the center of the lungfish out of water, leaving a large margin for a referencing error). Beyond a passive capacity to separate bloodstreams, the conus arteriosus may play an active role in the circulation of dipnoans (Satchell, 1991). Distinct rhythmic conal contractions were present in blood pressure traces for Neoceratodus (Johansen et al., 1968), but not for either anesthetized or unanesthetized Protopterus (Johansen et al., 1968; Szidon et al., 1969). Perhaps the contractile function was lost as dipnoans evolved a greater dependency on air breathing. Even so, secondary pressure oscillations became apparent when blood pressure in the aVerent branchial artery of Protopterus was excessively elevated following acetylcholine injection (Johansen et al., 1968). Measurements of dorsal aortic blood pressures are variable. EVerent branchial blood pressures were reported as 1.7–2.0 kPa in Protopterus (Perry et al., 2005), 2.6 kPa in Lepidosiren (Axelsson et al., 1989), and 2.9 (Szidon et al., 1969) and 2.1–2.3 kPa in Neoceratodus (Johansen et al., 1968; Fritsche et al., 1993). Collectively, these data point to the latter authors’ suggestion that about one‐third of the arterial blood pressure can be lost across the dipnoan

2.

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CARDIOVASCULAR SYSTEMS IN PRIMITIVE FISHES O

P

QRS

O

P

A ECG

mmHg 20

Ventricular ejection

Isovolumic ventricular contraction

Bulbus

10

Ventricle

0 mmHg 5 Vena cava Sinus venosus B Atrium Atrial systole Sinus venosus systole

Pericardium

0

−2 1s

Fig. 2.15. ECG and superimposed traces from the cardiac chambers and the pericardial cavity of lightly anesthetized Protopterus. [Taken from Szidon et al. (1969).]

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gills, as in other fishes. However, when nearly simultaneous measurements of blood pressure were made in the aVerent branchial artery and the coeliac artery of Protopterus, there was a very small transbranchial vascular resistance (i.e., a similar diastolic blood pressure and a systolic diVerence of just 0.23 kPa), which then disappeared when Protopterus was exposed to air (Johansen et al., 1968). This result was taken to indicate that the vascular resistance of the proximal gill arches is very small, as expected given the lack of primary filaments. The implication of this finding is profound because the eVerent branchial blood pressure of the anterior gill arches must be greater than that in the posterior gill arches, and yet both are connected to the dorsal aorta. Thus, blood cannot flow from the posterior gill arches into the dorsal aorta unless there is a means to either increase vascular resistance in the anterior arches (vasoconstriction) or decrease vascular resistance in the posterior arches (perhaps opening of gill shunt vessels). Conversely, blood from the posterior arches will tend to flow into the pulmonary circulation rather than the dorsal aorta. In addition, unless the ductus is closed, blood will easily flow from the dorsal aorta into the pulmonary arteries retrograde via the posterior eVerent branchial arteries. Clearly, vasoactive control systems are important for eVective perfusion of the lung since the anatomical arrangement in the gills is not entirely adequate by itself. As shown in the next section, some aspects of this control have been elucidated. 4.2.4. Circulatory Control The Lepidosiren heart has a routine cholinergic inhibitory tone (Axelsson et al., 1989). Also, resting heart rate decreased from 32 to 25 min1 when propranolol was injected, suggesting a b‐adrenergic tonus (Axelsson et al., 1989). In contrast, Fritsche et al. (1993) found neither vagal nor adrenergic tone on resting heart rate in Neoceratodus. An abrupt, fright‐induced bradycardia was abolished with atropine treatment in both Lepidosiren and Neoceratodus, suggesting a common vagal cardioinhibitory mechanism in dipnoans (Axelsson et al., 1989; Fritsche et al., 1993). Adrenergic cardiac control is possible through humoral and paracrine mechanisms, but adrenergic innervation is lacking (Abrahamsson et al., 1979a). Paced ventricular strips from Protopterus show a positive inotropic response to adrenaline but no response to cholinergic stimulation with carbachol (Abrahamsson et al., 1979b). Conversely, paced atrial strips show a strong negative inotropic eVect with cholinergic stimulation and no eVect with adrenaline. However, injections of adrenaline into Protopterus and Neoceratodus have failed to change heart rate (Johansen and Reite, 1967; Fritsche et al., 1993), and so experiments with adrenergic antagonists are needed to properly describe catecholamine control of cardiac function in vivo.

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Despite their functional vagal cardioinhibitory control system, Protopterus lack the typical piscine bradycardia response to aquatic hypoxia; heart rate remains at 46–48 min1 at a water oxygen tension of 2.6 kPa (Perry et al., 2005). Absence of bradycardia in response to aquatic hypoxia (3–4 kPa) was noted earlier for Lepidosiren (Sanchez et al., 2001) and Neoceratodus (Fritsche et al., 1993). Also, aquatic hypoxia did not alter oxygen uptake (Perry et al., 2005) and plasma catecholamine levels (Table 2.1). Likewise, aerial hypoxia (4.6 kPa) had no eVect on heart and ventilation rates, but depressed oxygen uptake by fivefold (Perry et al., 2005), suggesting some combination of a decrease in cardiac stroke volume and tissue oxygen extraction. Lungfish increase pulmonary blood flow after an air breath to ensure that lung ventilation and perfusion are closely matched. However, quantifying such changes in vivo has proven diYcult because of limited vascular access to implant flow probes. Johansen et al. (1968) reported that spontaneous air breathing was consistently associated with increased pulmonary flow and, sometimes, increased heart rate and Q. Unfortunately, no numerical information was reported for this response, or for the stated tachycardia and pressor eVects associated with artificial lung inflation, or for the stated bradycardia and depressor eVects associated with lung deflation. Instead, pulmonary flow was estimated to vary between 20% and 70% of Q. Other reports suggest that the change in pulmonary blood flow with air breathing ranges from as little as 50% to over fourfold. Szidon et al. (1969) reported a mean flow in the left pulmonary artery of Protopterus of 18 ml min1 that varied more than fourfold from 7 to 33 ml min1 (body mass was not given). However, if we assume similar blood flow rates in the right and left pulmonary arteries, such a response would mean that total pulmonary blood flow could exceed their measurement of Q. When Lepidosiren were taking air breaths every 4–12 min, pulmonary blood flow increased 50% (from 13 to 20 ml min1 kg1) and heart rate increased 10% (Axelsson et al., 1989), with some of the tachycardia being preemptive. This increase in pulmonary flow was blocked by atropine pretreatment, suggesting that a cholinergically mediated vasodilation (possibly of the ductus) can regulate pulmonary flow. In Neoceratodus, pulmonary flow increased with air breathing (without aVecting heart rate) through a decrease in pulmonary vascular resistance and a slight increase in resistance of, and decrease in flow in, the coeliacomesenteric circulation (Fritsche et al., 1993). Also, pulmonary blood flow was estimated to increase 25% after 25 min of progressive hypoxia in Neoceratodus (Johansen et al., 1967). In Protopterus, pulmonary blood flow decreased with swimming and aerial exposure to 5% carbon dioxide (again numerical values were not presented; Johansen et al., 1968). Pulmonary blood flow must be under active control because the ratio of systemic and pulmonary blood flow can change, that is a change in

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pulmonary blood flow goes beyond that associated with the change in Q. In this regard, vasoactivity in the pulmonary artery and the well‐innervated ductus becomes important. Fishman et al. (1985) proposed the following general mechanism for cyclic control of pulmonary flow in Protopterus. During submersion when the posterior gills are engaged in gas exchange with the water, the gill shunt vessels are closed, the pulmonary artery is constricted, and the ductus is relaxed. When Protopterus breathe air, the pulmonary artery opens, the gill shunts open, and the ductus closes. This hypothesis is supported by vascular casts from estivating Protopterus showing a clear constriction site at the base of pulmonary artery and dilated gill aVerent to eVerent shunt vessels (Laurent et al., 1978), as well as by studies of putative vasoactivity control mechanisms with isolated and perfused vessels of Protopterus (Table 2.3). Even so some of these in vitro results are contradictory. For example, while Johansen and Reite (1967) found that both the ductus and pulmonary artery constricted with acetylcholine, Fishman et al. (1985) found opposite responses of the two vessels to acetylcholine. In vivo studies in Neoceratodus revealed cholinergic vasoconstriction of the pulmonary artery and cholinergic relaxation of the ductus (perhaps through NO release) (Fritsche et al., 1993). Acetylcholine increased pulmonary blood flow, while atropine injection decreased pulmonary vascular resistance. In the same study with Neoceratodus, adrenaline injection increased pulmonary blood flow, dorsal aortic pressure, and coeliacomesenteric vascular resistance, and decreased coeliacomesenteric blood flow. Because sotalol injection increased vascular resistance in both the pulmonary and coeliacomesenteric circulations, and phentolamine decreased dorsal aortic blood pressure, at least three tonic mechanisms appear to influence pulmonary blood flow in Neoceratodus: cholinergic vasodilation, a‐adrenergic vasoconstriction, and b‐adrenergic vasodilation. Despite these studies, a complete understanding of vasoactive control for the ductus and pulmonary artery in dipnoans is lacking. Even less is known concerning the control of the gill shunt vessels (e.g., how is blood flow distributed among gill arches? What determines inter‐ and intrabranchial shunt patterns?). A cholinergic control mechanism may be important because injection of acetylcholine in Protopterus increased branchial vascular resistance to such a degree that Q and pulmonary blood flow stopped (Johansen et al., 1968). The decrease in gill resistance with air exposure in Protopterus (Johansen et al., 1968) contrasts with the large increases in gill vascular resistance seen for water‐breathing teleosts during air exposure. Holmgren et al. (1994) examined the in vivo responses of Neoceratodus to several neuropeptides (neurotensin, galanin, bombesin, substance P, and cholecystokinin 8) and concluded that they followed the general

Table 2.3 Vasoactivity of the Isolated Ductus and the Proximal Pulmonary Artery of the African Lungfish Protopterus Ductus Fishman et al., 1985 Hypoxia Hyperoxia Hypercapnia Dopamine Noradrenaline Adrenaline Isoproterenol Acetylcholine PGE2 Histamine Serotonin

Relaxes Contracts NA Contracts (strong) Contracts (strong) NA Relaxes (weak) Relaxes (weak) atropine blocks Relaxes (strong) NA NA

Pulmonary artery

Johansen and Reite, 1967 No response NA No response NA Relaxes (slow) Relaxes (slowly) Relaxes (weak) Contracts (strong) atropine blocks NA Contracts (strong) Contracts (strong)

Fishman et al., 1985 No response NA NA No response No response NA No response Contracts (strong) atropine blocks NA NA NA

Johansen and Reite, 1967 No response NA No response NA Relaxes (slowly) Relaxes (slowly) Relaxes (weak) Contracts (strong) atropine blocks NA Contracts (strong) Contracts (strong)

A

p.a

B a.bl

p.v

c

6a D

C

Fig. 2.16. Unusual features in ganoid circulatory systems. (A) A schematic diagram of the circulatory arrangement of the gills and lung (abl) of the air‐ breathing Polypterus. (Aortic arches are numbered; c, conus; p.a and p.v, pulmonary artery and vein). [Taken from Satchell (1976).] (B) A vascular cast

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vertebrate pattern of cardiovascular control by these peptides. In addition, Abrahamsson et al. (1979b) found muscarinic and adrenergic constrictions of the spleen in Protopterus. 5. DETAILS OF THE CIRCULATORY SYSTEMS IN POLYPTERIDS, GARS, AND BOWFINS The fish orders comprising the Polypterids, gars, and bowfins have been grouped together in early literature as the ganoids. Collectively, their primitive status is reflected in the possession of a conus arteriosus [termed contractile bulbus arteriosus by Wilder (1876)] that contains serial rows of valves. Here, they are grouped because so little is known of their individual circulatory physiology and because all three orders are facultative air‐ breathers (Smatresk and Cameron, 1982; Graham, 1997). Amia rarely breathe air at 24
C, but at 30
C, and when exposed to aquatic hypoxia, they take regular air breaths every 10 min or so (Randall et al., 1981). While the gill structure of garfish is reduced (Potter, 1927), Amia have well‐ developed gills (Daxboeck et al., 1981; Olson, 1981). 5.1. Polypterids (Bichirs and Reedfish) Polypterus possess a number of anatomical features that have drawn attention. They have four gill arches, and the most posterior arch bears a single hemibranch without a gill slit behind it (Figure 2.16A). While much debate has been given to whether this last gill arch is a fifth gill arch with an accompanying loss of the fourth gill arch or a reduced fourth gill arch, or (see Britz and Johnson, 2003), recent literature favors the latter view, in part because the fourth gill arch possesses its own aVerent and eVerent branchial arteries. A cephalad coronary artery from the subclavian artery supplies the compact myocardium of the conus arteriosus and the ventricle. A thick‐ walled coronary vein drains into the ductus Cuvier (Budgett, 1901). Budgett (1901) has shown Polypterus with a similar eVerent branchial artery arrangement as Amia (and lungfish). The fourth eVerent branchial artery of a gill filament from the air‐breathing garfish Lepisosteus with most lamellae removed to reveal channelized vessels in the lamella vessels. [Taken from Smatresk and Cameron (1982).] (C) A vascular cast of a secondary lamella from the air‐breathing Amia to show the limited degree of channelized lamellar vessels with the exception of the larger inner (IM) and outer (OM) marginal vessels. [Taken from Olson (1981).] (D) A histological cross‐section through the secondary lamellae of Amia to show the fusion of adjacent lamellae (S), which results in lamellar vessels being buried in epithelium, as they are in the basal region of the lamellae near to the filament (F). [Taken from Olson (1981).]

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passes directly to the pulmonary artery and is connected via a ductus to the third eVerent branchial artery. However, the third eVerent branchial artery joins the coeliac artery directly, rather than joining the dorsal aorta (Figure 2.16A). The lungs are supplied by a pair of pulmonary arteries, with the left pulmonary artery passing over the left ductus Cuvier en route to the lung. Left and right posterior pulmonary veins drain the posterior two‐third of the lungs. These vessels unite to form a common pulmonary vein, which then empties into the large posterior vena cava (Kerr, 1910) that runs ventral to the right lung between the posterior caudal vein and ductus Cuvier. The two anterior pulmonary veins unite before entering the vena cava at the same point (Magid, 1967). While beautifully describing the pattern of venous system, Magid (1967) termed this large collecting vessel the hepatopulmonary vein because it received numerous collateral venous branches from the lung before receiving the hepatic veins caudal to the union of the common pulmonary vein. The caudal vein also connects to an unusual pairing of posterior cardinal veins, which have lateral connections, a dominant right side, and empty into the ductus Cuvier. Additional examples of venous asymmetry in Polypterus, first noted by Muller (1845; as quoted in Magid, 1967), are the relatively small and short left ductus Cuvier and small left jugular vein. Surprisingly, physiological studies are lacking for the circulatory system of Polypterids, despite the fact that they are extant fish with paired lungs. 5.2. Garfishes Although the gill structure of garfish is reduced, remarkable modifications are lacking (Landolt and Hill, 1975). Smatresk and Cameron (1982) examined the gill and pulmonary circulations in Lepisosteus oculatus using corrosion vascular casts, radiolabeled microspheres, and gas tensions. They concluded that the arterial system of the gill filaments was similar to the typical teleost pattern. Three paired aVerent branchial arteries originated on the ventral aorta, with the third and fourth gill arches having a common aVerent branchial artery. Similarly, the eVerent branchial arteries united into a common vessel before directly entering the dorsal aorta. No major nonrespiratory shunt vessels were evident. The vascular pathways in the secondary lamellae of garfish are unusual in that the pathways are clearly ‘‘channelized’’ (Figure 2.16B), but not to the extremes seen in protopterus. Lamellar vascular channels provide a more direct flow of blood across the respiratory surface and reduce the vascular density of the exchange area. Nevertheless, oxygen is lost as blood passes through the lamellae, as indicated by oxygen tensions in the ventral aortic blood exceeding those in the dorsal aortic blood when the garfish are breathing air

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(Smatresk and Cameron, 1982). A few vascular anastomoses between the aVerent filament artery and the central venous sinus have been observed. Blood flow to the lung is derived caudally from the dorsal aorta as numerous paired branches (Potter, 1927). Venous drainage from the lung is also by means of numerous paired vessels that join only to the right side of the posterior cardinal vein (Potter, 1927). L. oculatus had a routine heart rate of 33 min1 at 20
C, a ventral aortic blood pressure of 2.5 kPa, and a dorsal aortic blood pressure of 1.9 kPa (Smatresk and Cameron, 1982). These arterial blood pressures are somewhat lower than those of teleosts. However, similar to teleost fish, gill vascular resistance was about 25% of the total vascular resistance. Air breathing was found to have no eVect on heart rate and arterial blood pressures. In the absence of direct measurements, Smatresk and Cameron (1982) used Fick calculations for carbon dioxide to estimate Q. During hypoxia, Q increased from 31 to 40 ml min1 kg1, with a modest hypertension (ventral aortic blood pressure ¼ 3.0 kPa and dorsal aortic blood pressure ¼ 1.9 kPa), tachycardia (up to 36 min1), and increased air‐breathing frequency (from 1 h1 to 7 h1). Using these measurements and assuming a ventricular mass of 1 g kg1 body mass, routine myocardial power output can be estimated as 0.8 mW g1, increasing to 2.0 mW g1 during hypoxia. Blood flow to the lung was estimated as 19% of Q during normoxia. Pulmonary flow increased from 5.9 to 12.1 ml min1 kg1 during hypoxia. Consequently, most of the increase in Q during hypoxia was diverted directly to the lung (Smatresk and Cameron, 1982). During normoxia, the proportion of blood flow was similar for each gill arch and remained unchanged during hypoxia. There is no evidence for sympathetic innervation of the garfish heart since postganglionic fibers are absent in the atrium and ventricle (Nilsson, 1981). The atrium of Lepisosteus platyrhinchus receives vagal innervation. Catecholamine stores are found in the sinus venosus, atrium, and the small coronary vessels in the conus arteriosus, but not in the ventricle (Table 2.1). Both the atrium and ventricle may have adrenergic control via either humoral or paracrine mechanisms because adrenaline had a positive inotropic eVect on paced atrial and ventricular muscle strips (Nilsson (1981). Carbachol had a negative inotropic eVect on the atrium, but not the ventricle (Nilsson, 1981). The garfish spleen contracted with both adrenaline and noradrenaline, as in teleosts (Nilsson, 1981). 5.3. Amia (Bowfins) The heart of Amia has no unusual features. A cephalad coronary artery, which originates in the first and second gill arches (Randall et al., 1981), supplies the compact myocardium of the ventricle. The conus arteriosus is

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greatly reduced and gives way to a longer bulbus arteriosus. Three aVerent branchial arteries arise from the ventral aorta, with the posterior one dividing to supply the third and fourth gill arches, as in garfish. The gill respiratory dimensions in Amia are similar for all four gill arches (Olson, 1981). The central vessels of the secondary lamellae lack an endothelial lining (Olson, 1981) and are not channelized to the extent seen in garfish (Figure 2.16D). The secondary lamellae on apposing gill filaments are fused by epithelium (Figure2.16C) (Bevelander, 1934), presumably to prevent lamellar collapse in air, and this means that the most marginal lamellar blood channels ( just like basal lamellar channels in most fish) are buried in epithelium which restricts gas transfer (Olson, 1981). However, these potential intralamellar nonrespiratory shunts have a limited benefit when Amia breathe air during aquatic hypoxia because oxygen can be lost from the gills and the transfer factor for this oxygen loss was calculated to be the same as that for oxygen uptake during water breathing (Randall et al., 1981). The few connections between the aVerent filament artery and the secondary circulation of the gills are outnumbered by those from the eVerent filament artery (Olson, 1981; but see Daxboeck et al., 1981). Thus, no major nonrespiratory shunt vessels have been identified from vascular corrosion casts of Amia gills (Olson, 1981; Randall et al., 1981). The first and second eVerent branchial arteries supply the pseudobranch, the cranial vessels, and the dorsal aorta. The third and fourth eVerent branchial arteries are modified to supply the air bladder (Olson, 1981; Randall et al., 1981). The fourth eVerent branchial artery is directly confluent with the air bladder artery. The third and fourth eVerent branchial arteries are connected by a short ductus near their dorsal emergence from the gill arches. The ductus is orientated to favor flow from the third into the fourth eVerent branchial artery, and thus blood flow from the fourth gill arch may reach the dorsal aorta only via the ductus and the third eVerent branchial artery. A single coeliacomesenteric artery arises from the dorsal aorta just caudal to the junction of the third eVerent branchial artery (Olson, 1981). A more direct connection between the coeliacomesenteric artery and the third eVerent branchial artery has been suggested in the schematic diagram of Randall et al. (1981) and in some of the specimens examined by Olson (1981). Studies of cardiovascular physiology are limited. The Fick estimate for Q was 66–69 ml min1 kg1 during regular air breathing at 30
C. The air bladder received 39% (25.9 ml min1 kg1) of Q (Randall et al., 1981), a percentage that would represent almost the entire flow from the third and fourth gill arches. Wilder (1876) had earlier suggested that blood flow in the posterior dorsal aorta and the air bladder should be similar given their similar vessel diameters. McKenzie et al. (1991b) measured routine heart

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rate (30 min1) and dorsal aortic blood pressure (2.9 kPa) in Amia at 20
C. A single tracing in Amia at 27
C of ventral aortic flow after an air breath showed Q increasing and heart rate remaining unchanged (32 min1; Johansen et al., 1970). By combining these measurements routine Q in Amia has an impressive cardiac stroke volume of 2 ml kg1. During hypoxia at 24
C, Amia increased Q to 96.3 ml min1 kg1 (stroke volume ¼ 3 ml kg1; but note these estimates were based on some 30
C data), which is an impressive Q among fishes. At the same time, blood flow to the air bladder increased to 25.9 ml min1 kg1, but not at the expense of the systemic circulation, which remained unchanged at 44 ml min1 kg1, that is, all of the increase in Q was directed to the air bladder. Aquatic hypoxia (6.3 kPa) and NaCN injection into the buccal cavity both decreased heart rate by about 15%. Aquatic hypoxia increased and NaCN decreased dorsal aortic blood pressure. Adrenaline and noradrenaline infusions both increased heart rate by 30–40% and dorsal aortic blood pressure by 60–70% (McKenzie et al., 1991b). 6. DETAILS OF THE STURGEON CIRCULATORY SYSTEMS 6.1. Cardiac Anatomy The anatomy of the sturgeon heart has a few notable peculiarities. The ventricle has nodular tissue located in the subendocardium (Hertwig, 1873; Khloponin, 1979; Fa¨nge, 1986). This nodular tissue can be several millimeters thick in a 30‐kg fish and has outer and inner layers resembling, respectively, the cortical and medullar regions of the thymus. It apparently serves as a lymphohemopoietic organ, is well vascularized, and contains lymphocytes and young forms of white blood cells (Fa¨nge, 1986; Icardo et al., 2002b). Such a function for the ‘‘heart’’ may be unique among fishes. Myklebust and Kryvi (1979), who examined the cardiac ultrastructure of Acipenser stellatus, showed that the myofibrils and much of the sarcoplasmic reticulum have peripheral cellular locations, while the mitochondria and the nucleus have central locations. T‐tubules were not observed and large (þ300 nm) glycogen granules were observed only in the atrium. Nerves and nerve endings were apparent among the myocytes but their locations were not given. The outflow tract of the heart comprises a proximal conus arteriosus and distal bulbus arteriosus within the pericardium [see descriptions for Acipenser naccarii in Icardo et al. (2002a,b) and Guerroero et al. (2004). The conus is rich in collagen but not elastin, is surrounded by well‐vascularized, compact myocardial muscle, and contains rows of conal valves (cushions).

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In contrast, the bulbus is rich in elastin, lacks cardiac muscle, and carries the coronary artery on its surface. Embryonic development of the heart has been carefully documented for A. naccarii (Icardo et al., 2004). Notably, the conus and its endocardial cushions appear 4 days posthatch and the coronary artery appears 10 days posthatch. The coronary circulation has diVerent origins among sturgeons: a cephalad origin in the paddlefish Polyodon spathula (from the most posterior eVerent filament artery) and a caudal origin in the shovelnose sturgeon Scaphirhynchus platorynchus (from the subclavian artery) (Danforth, 1916; Foxon, 1950). In addition to supplying the compact myocardium of the conus and ventricle, the coronary circulation apparently serves the inner spongy myocardium of the ventricle in the beluga sturgeon (Huso huso), stellate sturgeon (A. stellatus), and Acipenser gu¨ldensta¨dti, much like the situation in skates and sharks (Romensky, 1978). 6.2. Circulatory Patterns For A. stellatus, the circulatory pattern in the gills (Byczkowska‐Smyk, 1962) and the swim bladder (supplied by a branch of the coeliacomesenteric artery and drained to the hepatic portal system; Jasinski, 1965) are not unusual. 6.3. Cardiac Dynamics For A. naccarii at 23
C, heart rate and dorsal aortic blood pressure are reported as 62 min1 and 2.8 kPa (McKenzie et al., 1995), and 60 min1 and 2.5–3.0 kPa (Agnisola et al., 1996). For Acipenser transmontanus at 19
C, heart rate and dorsal aortic blood pressure are reported as 48 min1 and 2.9 kPa (Crocker et al., 2000). Routine heart rate is under modest b‐adrenergic and cholinergic tonus, as suggested by the findings of tachycardia (up to 67 min1) after atropine injection, bradycardia (down to 52 min1) after injection of propranolol (McKenzie et al., 1995), and a 9% increase in heart rate after isoproterenol injection (Crocker et al., 2000). Kisch (1950) reported heart rate as 70 min1 (temperature was not given) and a fright‐induced bradycardia reflex for Acipenser sturio. In A. transmontanus at 19
C, Q was 36 ml min1 kg1 and stroke volume was 0.83 ml kg1 (Crocker et al., 2000). In an anesthetized A. naccarii at 23
C, Q was 13 ml min1 kg1 and stroke volume 0.2 ml kg1 (Agnisola et al., 1996). Systemic vascular resistance was 88 Pa min kg ml1. Following a struggle, Q increased by 29%. Hypercarbia (2 h at 2.6 kPa) decreased systemic resistance, which produced systemic hypotension despite an increase in Q to 68 ml min1 kg1 (occasionally up to 82 ml min1 kg1). Combining these

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in vivo data, and assuming a blood pressure loss of one‐third across the branchial circulation and a relative ventricular mass of 1 g kg1 body mass, routine myocardial power output is estimated at 2.3–5.3 mW g1. By comparison, isolated, perfused ventricles from A. naccarii with a low spontaneous heart rate (20 min1 at 20
C) had a very low basal power output (0.3 mW g1; Agnisola et al., 1996). Heart rate was independent of filling pressure and output pressure. However, power output was doubled at maximum stroke volume and increased a further 50% when diastolic output pressure was increased from 2.0 kPa to a maximum level, such that the maximum power output was 1.5 mW g1. Subambient filling pressures were reported for the perfused heart (Agnisola et al., 1996). Thus, vis‐a‐fronte cardiac filling seems likely especially since the heart is surrounded by a fibrous pericardium and there is pericardioperitoneal connection, as in sharks. 6.4. Circulatory Control Hypoxia causes bradycardia. In A. naccarii at 23
C, acute hypoxia (2.5 kPa for 20 min) and externally applied NaCN slowed heart rate (cholinergically mediated) without a pressor eVect on dorsal aortic blood pressure (McKenzie et al., 1995). Agnisola et al. (1996) found that moderate hypoxia (6.6 kPa for 30 min1) had no cardiovascular eVects, but deep hypoxia (4.6 kPa for 15 min) produced bradycardia (4–50) and, in contrast, systemic hypotension (dorsal aortic blood pressure ¼ 2.0–2.5 kPa). Adrenaline injections cause tachycardia and systemic hypertension, while propranolol causes bradycardia and systemic hypotension in A. naccarii (McKenzie et al., 1995). Blood flow in the coeliacomesenteric artery was 20% of routine Q (Crocker et al., 2000). Struggling increased splanchnic vascular resistance, which rapidly reduced splanchnic blood flow by 72%. An a‐adrenergic vasoconstriction of the systemic and splanchnic circulations was demonstrated by increases in vascular resistance after phenylepherine injection, but splanchnic vascular resistance was unaVected by isoproterenol injection, even though it increased Q and decreased systemic vascular resistance. 7. CONCLUSIONS There is certainly still much to be discovered about the circulatory physiology of primitive fishes. For many orders, other than cyclostomes and lungfishes, there are just a few physiological studies of cardiovascular control, and none for Polypterids. More studies are certainly needed to either validate or refute the speculation presented here in the synthesis and the

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detailed descriptions. In a much earlier evolutionary synthesis based on brain, lobe fin, and cardiac anatomy, Wilder (1876) pleaded ‘‘guilty’’ to not including any ‘‘osteological characters by which fossil forms may be collocated with the living.’’ Today we can be more confident that the study of extant species can reveal important information about the evolution of the vertebrate circulatory system. Encouragement is provided by the derived circulatory diversity among lungfishes (Burggren and Johansen, 1986), as well as the relics of circulatory design (e.g., the vestigial pulmonary vessel in Latimeria). Likewise, the ontogenetic loss of a ductus Cuvier in lampreys and the developmental studies of the sturgeon cardiac outflow tract indicate that much remains to be discovered in terms of evolutionary trends from studies of cardiovascular development. Therefore, this chapter closes with two remarks and no apology. The evidence presented here for very diVerent circulatory anatomy and physiology in hagfishes and lampreys must surely place them further apart from each other than their current phylogeny indicates. Many more circulatory similarities can be described for other, more phylogenetically divergent primitive fishes. Finally, primitive fishes are clearly survivors and this is reflected well in their cardiac hypoxia tolerance. Hagfishes and perhaps lampreys apparently took the path of ‘‘low cardiac ATP demand.’’ Other primitive fishes have a higher cardiac ATP demand, but their hearts benefit from an additional oxygen supply in their coronary circulation and from air breathing. A notable exception is Latimeria, which appears to have adopted a sedentary lifestyle in stable and remote marine environments. Added to this is the remarkable tolerance of hypercarbia by sturgeon, that is, levels that will anesthetize salmon. Therefore, exciting discoveries remain ahead for researchers who tackle why the hearts of primitive fishes are so tolerant. ACKNOWLEDGEMENTS The assistance of Christopher Wilson, Joanna Bernhardt, and Linda Hansen in locating and organizing reference material and figures is gratefully appreciated.

REFERENCES Abrahamsson, T., Holmgren, S., Nilsson, S., and Pettersson, K. (1979a). On the chromaYn system of the African lungfish, Protopterus aethiopicus. Acta Physiol. Scand. 107, 135–139. Abrahamsson, T., Holmgren, S., Nilsson, S., and Pettersson, K. (1979b). Adrenergic and cholinergic eVects on the heart, the lung and the spleen of the African lungfish, Protopterus aethiopicus. Acta Physiol. Scand. 107, 141–147. Agnisola, C., McKenzie, D. J., Taylor, E. W., Bolis, C. L., and Tota, B. (1996). Cardiac performance in relation to oxygen supply varies with dietary lipid composition in sturgeon. Am. J. Physiol. 271, R417–R425.

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Randall, D. J. (1982). The control of respiration and circulation in fish during exercise and hypoxia. J. Exp. Biol. 100, 275–288. Randall, D. J., Cameron, J. N., Daxboek, C., and Smatresk, N. (1981). Aspects of bimodal gas exchange in the bowfin, Amia calva L. (Actinopterygii: Amiiformes). Respir. Physiol. 43, 339–348. Randall, D. J., McKenzie, D. J., Abrami, G., Bondiolotti, G. P., Natiello, F., Bronzi, P., Bolis, L., and Agradi, E. (1992). EVects of diet on responses to hypoxia in the sturgeon (Acipenser naccarii). J. Exp. Biol. 170, 113–125. Reid, S. G., Bernier, N. J., and Perry, S. F. (1998). The adrenergic stress response in fish: Control of catecholamine storage and release. Comp. Biochem. Physiol. 120C, 1–27. Reid, S. G., Sundin, L., and Milson, W. K. (2006). The cardiorespiratory system in tropical fishes: Structure, function, and control. In ‘‘The Physiology of Tropical Fishes’’ (Val, A. L., Almeida‐Val, V. M. F., and Randall, D. J., Eds.), pp. 225–275. Academic Press, San Diego. Reinecke, M., Betzler, D., and Forssmann, W. G. (1987). Immunohistochemistry of cardiac polypeptide hormones (cardiodilatin/atrial natriuretic polypeptide) in brain and hearts of Myxine glutinosa. Histochemistry 86, 233–239. Reite, O. B. (1969). The evolution of vascular smooth muscle responses to histamine and 5‐hydroxytryptamine. I. Occurrence of stimulatory actions in fish. Acta Physiol. Scand. 75, 221–239. Retzius, A. (1824). Yeterligare Bigrag till Anatomien ab Myxine glutinosa. Kongl. Vetenskapsacademiens Handligar (Stockholm) 408–431. Robertson, J. I. (1913). The development of the heart and vascular system of Lepidosiren paradoxa. Q. J. Micro. Sci. 59, 53–132. Romensky, O. Y. (1978). Blood supply for compact and spongy myocardium in fish, amphibians and reptiles. Arkh. Anat. Gistol. Embriol. 75, 91–95. Sanchez, A., Soncini, R., Wang, T., Koldkjaer, P., Taylor, E. W., and Glass, M. L. (2001). The diVerential cardio‐respiratory responces to ambient hypoxia and systemic hypoxaemia in the South American lungfish, Lepidosiren paradoxa. Comp. Biochem. Physiol. 130A, 677–687. Sandblom, E., Axelsson, M., and Farrell, A. P. (2006). Central venous pressure and mean circulatory filling pressure in the dogfish Squalus acanthias: The roles of adrenergic controls and the pericardium. Am. J. Physiol. 291, R1465–R1473. Santer, R. (1985). Morphology and innervation of the fish heart. Adv. Anat. Embryol. Cell Biol. 89, 1–102. Satchell, G. H. (1971).‘‘Circulation in Fishes,’’ 131 London, Cambridge University Press. Satchell, G. H. (1976). The circulatory system of air breathing fish. In ‘‘Respiration of Amphibious Vertebrates’’ (Hughes, G. M., Ed.), pp. 105–123. Academic Press, London. Satchell, G. H. (1984). On the caudal heart of Myxine (Myxinoidea: Cyclostomata). Acta Zool. 65, 125–133. Satchell, G. H. (1986). Cardiac function in the hagfish, Myxine (Myxinoidea: Cyclostomata). Acta Zool. 67, 115–122. Satchell, G. H. (1991). ‘‘Physiology and Form of Fish Circulation,’’ p. 235. Cambridge University Press, Cambridge. Satchell, G. H. (1992). The venous system. In ‘‘Fish Physiology’’ (Hoar, W. S., Randall, D. J., and Farrell, A. P., Eds.), Vol. 12A, pp. 141–183. Academic Press, New York. Shabetai, R. D., Abel, C., Graham, J. B., Bhargave, V., Keyes, R. S., and Witzum, K. (1985). Function of the pericardium and pericarioperitoneal canal in elasmobranch fishes. Am. J. Physiol. 248, H198–H207. Shelton, G. (1976). Gas exchange, pulmonary blood supply, and the partially divided amphibian heart. Perspect. Exp. Biol. 1, 247–259.

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Shiels, H. A., Vornanen, M., and Farrell, A. P. (2002). Force‐frequency relationship in fish hearts—a review. Comp. Biochem. Physiol. 132A, 811–826. Smatresk, N., and Cameron, J. (1982). Respiration and acid‐base physiology of the spotted gar, a bimodal breather. I. Normal values and the response to severe hypoxia. J. Exp. Biol. 96, 263–280. Smith, W. C. (1918). On the process of the disappearance of the conus arteriosus in teleosts. Anat. Rec. 15, 65–72. Spencer, W. B. (1892). Note on the habits of Ceratodus forsteri. Proc. R. Soc. Vict. 4, 81–84. SteVensen, J. F., and Lomholt, J. P. (1992). The secondary vascular system. In ‘‘Fish Physiology’’ (Hoar, W. S., Randall, D. J., and Farrell, A. P., Eds.), Vol. 12A, pp. 185–217. Academic Press, New York. Sundin, L., Axelsson, M., Nilsson, S., Davison, W., and Forster, M. (1994). Evidence of regulatory mechanisms for the distribution of blood between the arterial and the venous compartments in the hagfish fill pouch.. J. Exp. Biol. 190, 281–286. Szidon, J. P., Lahiri, S., Lev, M., and Fishman, A. P. (1969). Heart and circulation of the African lungfish. Circ. Res. 25, 23–38. Thorarensen, H., Young, G., and Davie, P. (1996). 11‐Ketoestosterone stimulates growth of heart and red muscle in rainbow trout. Can. J. Zool. 74, 912–917. Tibbits, G. F., Moyes, C. D., and Hove‐Madsen, L. (1992). Excitation‐contraction coupling in the teleost heart. In ‘‘Fish Physiology’’ (Hoar, W. S., Randall, D. J., and Farrell, A. P., Eds.), Vol. 12A, pp. 267–304. New York, Academic Press. Vornanen, M., Shiels, H. E., and Farrell, A. P. (2002). Plasticity of excitation‐contraction coupling in fish cardiac myocytes. Comp. Biochem. Physiol. 132A, 827–846. Wilder, B. G. (1876). Notes on the North American Ganoids, Amia, Lepidosteus, Acipenser and Polyodon. (Taken from: 1875, Proc. Am. Assoc. Adv. Sci. 24 ). Salem Press, Salem, Massachussetts. 149–197. Wright, G. M. (1984). Structure of the conus arteriosus and ventral aorta in the Sea Lamprey, Petromyzon marinus, and the Atlantic hagfish, Myxine glutinosa: Microfibrils, a major component. Can. J. Zool. 62, 2445–2456. Yamauchi, A. (1980). Fine structure of the fish heart. In ‘‘Hearts and Heart‐like Organs’’ (Bourne, G. H., Ed.), pp. 119–148. Academic Press, New York.

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1. Introduction 2. Development of the CNS 3. The Brains of Primitive Fishes 3.1. Agnathans (Hagfishes and Lampreys) 3.2. Sarcopterygians (Lobe‐Finned Fishes) 3.3. Actinopterygians (Early Ray‐Finned Fishes) 4. Functional Classification of Cranial Nerves in Fishes 5. The Visual System 5.1. The Optical Apparatus 5.2. Retina and Visual Function 5.3. Spectral Filters 5.4. Visual Sensitivity 5.5. Visual Resolution 5.6. Visual Input to the CNS 5.7. Nonvisual Photoreception 6. Chemoreceptive Systems 6.1. Olfaction 6.2. Gustation 6.3. Solitary Chemoreceptor Systems 6.4. Common Chemical Sense 7. Octavolateralis System 7.1. Audition 7.2. Vestibular Control 7.3. Lateral Line 8. Electroreception 8.1. Structure, Function, and Evolution of Ampullary Receptors 8.2. Role in Passive Electrolocation 9. Concluding Remarks

The nervous and sensory systems of lampreys, hagfishes, the coelacanth, lungfishes, and the basal ray‐finned fishes, such as bichirs and reedfishes, paddlefishes and sturgeons, garfishes and bowfins, are reviewed and compared 121 Primitive Fishes: Volume 26 FISH PHYSIOLOGY

Copyright # 2007 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(07)26003-0

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with closely related groups of ‘‘primitive’’ fishes. Anatomical, physiological, molecular, and behavioral data are discussed in relation to both ecological and phylogenetic relationships. In addition to overviews of their embryology, development, and gross anatomy, a functional classification of the cranial nerves is also presented. The peripheral and central components of the visual (including nonvisual photoreception), chemoreceptive (olfaction and gustation), octavolateralis (audition and lateral line), and electroreceptive systems are examined in some detail, highlighting the physiological basis for behavior wherever possible. Although a number of neuroanatomical, neurochemical, physiological, and molecular studies have greatly enhanced our understanding of brain evolution in these important groups, physiological studies are remarkably still scarce and need to be undertaken in order to trace the origins of craniate brains and the evolutionary constraints placed on neural plasticity and function. 1. INTRODUCTION The central nervous system (CNS) and sensory systems of ‘‘primitive’’ fishes have received considerable attention, and these studies provide crucial insights into the evolution and origins of the structure and function of the nervous systems of vertebrates. As in more advanced fishes, the peripheral sense organs and their central input are under intense selection pressure according to their ecological niche and the evolutionary constraints on their sensory and motor lifestyles. The aquatic environment is diVerent to the terrestrial environment in its ability to convey sensory information, and the physical environment has been shown to play a major role in the propagation and reception of signals. This chapter will concentrate on the nervous and sensory systems of the Agnatha (Myxiniformes, hagfishes and Petromyzontiformes, lampreys), the Sarcopterygii or lobe‐finned fishes (Coelacanthiformes, coelacanth and the Dipnoi, lungfishes), and the basal Actinopterygii (Polypteriformes, bichirs and reedfishes; Acipensiformes, paddlefishes and sturgeons; Semionotiformes, garfishes; and Amiiformes, bowfins), although reference will be made to other closely related groups in order to reveal phylogenetic trends in the evolution of specific neural characters. Knowledge of these ‘‘living fossils’’ is really our only window into the physiology and behavior of the ancestral vertebrates and the processes of brain evolution that have shaped the diversity of the nervous systems in extant species. In reviewing the research done on the nervous and sensory systems in primitive fishes, it became apparent that an appreciable wealth of anatomical knowledge exists, with little or no physiological studies for many species.

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Therefore, anatomy forms the basis of some of the sections, although structure–function relationships are discussed wherever possible. Special emphasis is placed on the few physiological studies and the physiological mechanisms influencing behavior. 2. DEVELOPMENT OF THE CNS In anamniotes, the embryo is dependent on nutrition from either a large yolk sac within the egg (oviparous fishes) or via a direct placenta‐like connection to the mother (viviparous fishes). Cleavage divisions in the animal pole exceed those in the vegetal pole but eventually the blastomeres migrate and surround the yolk. The dorsal margin of the embryo thickens and gastrulation takes place, where mesodermal cells move inside the ectodermal layer and migrate back toward the animal pole. At this stage, the ectoderm begins to fold onto itself to form the neural tube, which gives rise to nearly all the neurons and glia. Invading ectoderm then forms a continuous layer over the neural tube, which is sealed oV at both ends. The lumen forms the ventricles of the brain (rostrally) and the central canal of the spinal cord (caudally). The undiVerentiated nerve cells within the neural crest grow processes, which either grow out to the periphery (aVerents) or invade the neural tube (eVerents). The evolution of these neural crest cells and the subsequent development of complex head structures are thought to be a major reason for the success of vertebrates (Gans and Northcutt, 1983). The development of oligodendrocytes (centrally) and Schwann cells (peripherally) at this time is also thought to underlie the success of jawed vertebrates. These cells produce myelin to ensheath the neural processes (axons) and increase the conduction velocity of neural impulses, a distinct advantage over the jawless protochordates (lancelets) and agnathans (hagfishes and lampreys) (Zalc and Colman, 2000). The rostral portion of the neural tube enlarges and diVerentiates to form three primary vesicles; the prosencephalon, the mesencephalon, and the rhombencephalon. The prosencephalon subdivides to form the telencephalon and the diencephalon (thalamus and hypothalamus), and after flexion, the rhombencephalon or hindbrain further subdivides to form the metencephalon (pons and cerebellum) and the myelencephalon (medulla oblongata). The mesencephalon becomes the optic tectum and tegmentum. The identity and boundaries of segmental units or compartments of the vertebrate brain (neuromeres) depend on cell surface molecules such as Eph and a number of transcription factor‐encoding genes such as Hox and Krox20 (see review by Murakami et al., 2005). A comparison of the expression patterns of the Pax6 gene in the dogfish, Scyliorhinus canicula, and the lamprey, Lampetra

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fluviatilis during neurulation, reveals that this gene plays an important role in brain regionalization in primitive fishes and that this has been conserved during vertebrate evolution (Derobert et al., 2002). 3. THE BRAINS OF PRIMITIVE FISHES 3.1. Agnathans (Hagfishes and Lampreys) A number of studies have concentrated on establishing the morphotype of the craniate brain based on the brains of both hagfishes and lampreys (Wicht and Northcutt, 1992, 1994; Fritzsch and Northcutt, 1993; Northcutt, 1996; Wicht, 1996). The neuroanatomy of these jawless fishes has also received much attention (Bone, 1963; Braun, 1996; Northcutt, 1996; Niewenhuys and Nicholson, 1998; Wicht and Niewenhuys, 1998). The hagfish brain comprises telencephalic, diencephalic, mesencephalic, and rhombencephalic divisions, although the presence of a metencephalic/cerebellar division is questionable in lampreys (Wicht, 1996) (Figure 3.1A–D). There is no cerebellum in hagfishes

Fig. 3.1. (A and B) The brain of the Atlantic hagfish, M. glutinosa in dorsal (A) and lateral (B) views. (C and D) The brain of the river lamprey, L. fluviatilis in dorsal (C) and lateral (D) views. The tela choroidea are removed on the left‐hand side. [(A) and (B) adapted from Wicht and Niewenhuys (1998).] [(C) and (D) adapted from Niewenhuys and Nicholson (1998).] Ac, auricula cerebelli; Cc, corpus cerebelli; D, diencephalon; Ht, hypothalamus; M, mesencephalon; OB, olfactory bulbs; OT, optic tectum; Po, pedunculus olfactorius; R, rhombencephalon; S, spinal cord; and Tel, telencephalon. Abbreviations apply to Figures 3.1–3.3. Reproduced with kind permission of Springer Science and Business Media.

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(Figure 3.1A and B). The rhombencephalon is divided into dorsal (visceral and somatosensory) and ventral (branchiomotor and somatomotor) zones. The spinal cord has both ascending and descending projections between the mesencephalic and rhombencephalic areas. In myxinoids, the brain is large and approaches the high encephalization quotients of teleosts and amphibians (Platel and Delfini, 1981). The cerebellum and choroid plexus are lacking, and in hagfishes, sulci do not always demarcate the boundaries of brain divisions. The narrow optic nerves form a chiasm and decussate within the brain rather than occur externally. The ventricular system is almost obliterated by the compression of the brain in the rostrocaudal axis (Wicht and Niewenhuys, 1998). The brains of lampreys are slender and possess a well‐developed ventricular system and a choroid plexus in the roof of the mesencephalon, a unique feature not found in other craniates. The ratio of brain to body weight is also the lowest among craniates (Platel and Delfini, 1986). The spinal cord is hypertrophied in lampreys, is ribbonlike, and extends throughout the length of the vertebral canal (Figure 3.1C and D). The forebrain of hagfishes and lampreys diVers appreciably. In lampreys, the telencephalic hemispheres are formed from the paired lateral evaginations of the rostral neural tube, while in hagfishes, the forebrain is composed of paired anterior and posterior evaginations from the prosencephalic vesicle, which include the olfactory bulb and the pallial and subpallial grisea (Wicht, 1996). 3.2. Sarcopterygians (Lobe‐Finned Fishes) The lobe‐finned fishes comprise the lungfishes and the coelacanth. The three main groups of lungfishes are the African, for example, Protopterus, the South American Lepidosiren, and the Australian Neoceratodus. All lungfish brains show little histological diVerentiation and are considered ‘‘simple’’ compared to other primitive fishes and teleosts (Striedter, 2005). The spinal cord is cylindrical and the rhombencephalon resembles that of urodele amphibians. The cranial nerves reveal a pattern similar to that of other gnathostomatous fishes, although the anterior lateral line nerve is divided into two branches; pars dorsalis and pars ventralis (Niewenhuys, 1998a). The mesencephalic optic tectum has retained its embryonic, tubelike appearance. The telencephalon consists of three parts: the telencephalon impar, the cerebral hemispheres, and the olfactory bulbs. The brains of Protopterus and Lepidosiren (Figure 3.2A and B) are very similar and diVer to that of Neoceratodus. In Neoceratodus forsteri, the olfactory bulbs are connected to the cerebral hemispheres by short hollow peduncles instead of being sessile as in the other two groups (Niewenhuys, 1998a). Neural development and a range of neural characters have been useful in clarifying phylogenetic

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Fig. 3.2. A, B. The brain of the South American lungfish, Lepidosiren paradoxa in dorsal (A) and lateral (B) views. The tela choroideae are removed on the left‐hand side. (C and D) The brain of the coelacanth, L. chalumnae in dorsal (C) and lateral (D) views. [(A) and (B) are adapted from Niewenhuys (1998a) and (C) and (D) are adapted from Niewenhuys (1998b).] For abbreviations see Figure 3.1. Reproduced with kind permission of Springer Science and Business Media.

relationships between this group and tetrapods (Bemis and Burggren, 1986; Northcutt, 1986b; Kemp, 2000). The coelacanth, Latimeria chalumnae, has a small brain occupying less than 1% of the total endocranial volume and appears superficially to comprise a mixture of neural features from both teleosts and cartilaginous fishes (Millot and Anthony, 1965; Striedter, 2005). Sitting within the otico‐ occipital division of the braincase and covered with adipose tissue, the brain is long and slender and gives rise to very elongate cranial nerves and ‘‘stretched’’ olfactory peduncles (Niewenhuys, 1977, 1998b; Northcutt et al., 1978) (Figure 3.2C and D). The spinal cord has slight pelvic and

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pectoral thickenings associated with the species’ extraordinarily mobile, paired fins. All the major brain divisions can be identified, especially the cerebellum, which is well developed (diVerentiated into a corpus cerebelli and a lobus vestibulolateralis). The mesencephalon is small and not well diVerentiated. The olfactory bulbs are greatly separated, apposed to the olfactory mucosa, and connected to the telencephalon by slender stalks (Niewenhuys, 1998b) (Figure 3.2C and D). An elegant reconstruction of the cranial nerves of the prenatal coelacanth reveals that (1) the optic nerves are interdigitated and therefore provide a partial decussation of retinal input to the brain; (2) the profundal ganglion and ramus are separate from the trigeminal system in contrast to hagfishes, lampreys, lungfishes, and tetrapods; and (3) there are three postotic lateral line nerves (Northcutt and Bemis, 1993). 3.3. Actinopterygians (Early Ray‐Finned Fishes) The early ray‐finned fishes are a collection of bony fishes: the Polypteriformes or bichirs and reedfishes (Polypterus and Erpetoichthys formerly Calamoichthys), the Acipenseriformes or sturgeons and paddlefishes (Acipenser, Polyodon, and Psephurus), the Semionotiformes or garfishes (Lepisosteus), and Amiiformes or bowfins (Amia). According to Northcutt and Braford (1980), Polypterus possesses a higher brain weight to body weight ratio than the other basal actinopterygians. In this genus, the rhombencephalon is well developed and the cerebellum is not fused as in most other vertebrates but is paired, connected only by a thin lamella (Niewenhuys, 1998c). The habenular ganglia are asymmetrical with the right ganglion larger than the left. The telencephalon is large and the two elongated hemispheres are connected by the anterior commissure. Bichirs appear to retain all six pairs of lateral line nerves that characterize the earliest gnathostomes and, unlike most non‐teleost bony fishes, have lost the spiracular organ (Piotrowski and Northcutt, 1996). The hypobranchial nerve of bichirs is formed by only two spinal nerves, a pattern seen also in gars. The rhombencephalon, hypothalamus, and cerebellum are all well developed in the Acipenseriformes. The rhombencephalon contains the termination of fibers from the viscerosensory, somatosensory, electroreceptive, lateral line, auditory, and vestibular systems. The telencephalon is large and of the everted type, that is the walls have recurved laterally during ontogenesis (Niewenhuys, 1998d; Striedter and Northcutt, 2006). The relatively minimal development of the mesencephalic tectum in Acipenser naccarii (Figure 3.3) is a derived characteristic that is shared with other species of sturgeon, suggesting that the visual system is not well developed in this group (Va´zquez et al., 2002). In the developing paddlefish, Polyodon spathula, the brain fills the braincase in larval forms but not in larger fish, and as

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Fig. 3.3. (A and B) The brain of the reedfish, E. calabaricus in dorsal (A) and lateral (B) views. The tela choroideae are removed on the left‐hand side. (C and D) The brain of the shovelnose sturgeon, Scaphirhynchus in dorsal (C) and lateral (D) views. The tela choroideae are removed on the left‐hand side. [(A) and (B) adapted from Niewenhuys (1998c) and (C) and (D) adapted from Niewenhuys (1998d).] For abbreviations see Figure 3.1. Reproduced with kind permission of Springer Science and Business Media.

the rostrum protrudes forward, the position of the brain, barbels, and eyes all change (Larimore, 1949). The garfishes and bowfins possess similar brains with relatively small cerebella and optic tecta, when compared to the brain of teleosts, that is Salmo, although gars have a higher brain weight to body weight ratio than most other primitive fishes (Striedter, 2005). A number of studies tracing the

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innervation of the brain stem (Niewenhuys and Pouwels, 1983), lateral line (Song and Northcutt, 1991a), retina (Northcutt and Butler, 1976; Butler and Northcutt, 1992; Collin and Northcutt, 1995), and tectum (Northcutt, 1982) have previously been published and are reviewed in Meek and Niewenhuys (1998). 4. FUNCTIONAL CLASSIFICATION OF CRANIAL NERVES IN FISHES Cranial nerves are components of the peripheral nervous system that connect directly to the brain rather than the spinal cord (Figure 3.4A and B). The numbers assigned to a cranial nerve normally indicate both its location on the brain stem in the rostro‐caudal direction and its function. The cranial nerves of fishes are classified as primarily sensory (carrying somatic sensory information including touch, pressure, vibration, temperature, or pain), special sensory (carrying the sensations of smell, sight, hearing, or balance), motor (carrying information to somatic muscle), or mixed (comprising sensory and motor axons). The size and development of the cranial nerves are directly related to their importance and organizational complexity. A total of 20 cranial nerves have now been identified in vertebrates, although some of these are not found in fishes, i.e. vomeronasal (Butler and Hodos, 1996) (Figure 3.4A and B). The olfactory nerve (nI) carries sensory information from the olfactory epithelium situated at the base of the nares into the olfactory bulbs where they aggregate with axons sensitive to the same water‐soluble stimuli within glomeruli. Olfactory information is then carried to the telencephalon via the olfactory tract. The optic nerve (nII) contains the axons of ganglion cells located within the inner retina, which terminate in the hypothalamus, thalamus, pretectum, and optic tectum. The optic nerve may be pleated in some species (Collin, 1989). The epiphyseal nerve carries aVerent and eVerent axons from the epithalamus (epiphysis and habenular nucleus) to the diencephalon and midbrain. The terminal nerve is closely associated, but separate to, the olfactory nerve input and projects from a ganglia, situated generally between the olfactory bulbs and the telencephalon, to a large range of sites throughout the CNS including the retina. Terminal nerve fibers may be luteinizing hormone‐releasing hormone (LHRH) immunoreactive and may be involved in reproductive behaviors. Six extraocular muscles are innervated by the oculomotor (nIII), the trochlear (nIV), and the abducens (nVI) motor nerves, which appear to be found in all primitive fishes except hagfishes (with no extraocular eye muscles). Interestingly, lampreys possess only five extraocular eye muscles. The complement of

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A

1 mm B

d dr vr

MLLN

Vlll

STL

PLLN

pd

vX

pv

Pr buc max mV

mVll mAV

2 mm Fig. 3.4. (A) Photograph of the cleared head of a juvenile Senegal bichir, P. senegalus stained with Sudan black to reveal the peripheral course of the cranial nerves. (B) Camera lucida drawing of the course of the cranial nerves seen in (A). buc, buccal ramus of anterodorsal lateral line nerve; d, dorsal ramus of posterior lateral line nerve; dr, dorsal root of anterodorsal and anteroventral lateral line nerves; gAD, sensory ganglion of anterodorsal lateral line nerve; gV, sensory ganglion of trigeminal nerve; hy‐op, hyo‐opercular ramus of anteroventral lateral line and facial nerves; hym, hyomandibular trunk of anteroventral lateral line and facial nerves; mAV, mandibular ramus of anteroventral lateral line nerve; max, maxillary ramus of trigeminal nerve; MLLN, middle lateral line nerve; mV, mandibular ramus of trigeminal nerve; mVII, mandibular ramus of facial nerve; pd, pars dorsalis of lateral ramus of posterior lateral line nerve; PLLN, posterior lateral line nerve; Pr, profundal nerve; pv, pars ventralis of lateral ramus of posterior lateral line nerve; so, superficial ophthalmic ramus of anterodorsal lateral line nerve; ST, supratemporal sensory canal; STL, supratemporal lateral line nerve; VIII, octaval nerve; vr, ventral root of preoptic lateral line nerve; vx, visceral trunk of vagal nerve; X, vagal nerve. [Adapted from Piotrowski and Northcutt (1996) with kind permission of S. Karger AG, Basel.]

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extraocular eye muscles in gnathostomes is four recti (inferior, superior, lateral, and medial) and two oblique (inferior and superior) muscles, which control eye movements and the retraction of the nictitating membrane where present, that is, in cartilaginous fishes. The sensations of pain, temperature, touch, and proprioception from the skin and muscles of the head and jaws are conveyed to the CNS via the trigeminal (nV) nerve. Three major branches (ophthalmic, maxillary, and mandibular) of the trigeminal nerve terminate within either the descending or principal nuclei. In the coelacanth, the mucosal walls are innervated by the profundus nerve (a homologue of the ophthalmic branch of the trigeminal). Taste buds are often distributed within the oral cavity, pharynx, gills, and over the barbels and skin of the body. The responses of gustatory fibers conveyed from taste cells are carried by three diVerent nerves (the ventrolateral regions of the facial nVIIVL, glossopharyngeal nIXVL, and vagus nXVL) and terminate within the two divisions of the nucleus solitarius, that is, rostral (also called the gustatory nucleus) and caudal regions. The input to these subdivisions is topographic. The dorsal component of the facial nerve (nVD) contains motor axons and innervates the superficial muscles of the head, including muscles of the cheeks, lips, and nares. Ingestion of food into the mouth is controlled by the motor component of nV and the dorsal component of nVII. Swallowing is controlled by inputs from the dorsal glossopharyngeal (NIXD), dorsal vagus (nXD), and accessory nerves (nXI), which convey eVerent axons to the pharynx and palate from the nucleus ambiguous in the medulla. The octaval or vestibulocochlear nerve (nVIII) comprises two components (auditory and vestibular), which are found in all primitive fishes and terminate primarily in the octaval column but also the cerebellum and the reticular formation (but not in lampreys). In fishes, the octaval column comprises at least four nuclei: anterior, magnocellular, descending, and posterior, and receives information from both the auditory and vestibular systems. In hagfishes, the eighth cranial nerve projects primarily to the ventral nucleus of the area acousticolateralis. Unique to aquatic vertebrates and all primitive fishes is the lateral line, which detects either water movements (mechanosensory) or electrical fields (electrosensory) using neuromasts and/or pit organs and either ampullary and/or tuberous organs, respectively. Input from these two systems is mediated by up to six nerves: anterodorsal, anteroventral, otic, middle, supratemporal, and posterior lateral line nerves. The mechanosensory fibers of the lateral line terminate within two regions (nucleus medialis and nucleus caudalis), although in electroreceptive non‐teleost gnathostomes, a third region also receives input (nucleus dorsalis). In electroreceptive teleost fishes, the input terminates in a long electrosensory lateral line lobe within the dorsal zone of the medulla.

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5. THE VISUAL SYSTEM 5.1. The Optical Apparatus All primitive fishes possess single‐chambered eyes, which scale positively with body size, although primitive fishes with long bodies, that is lampreys (Petromyzon marinus) and reedfishes (Erpetoichthys calabaricus), do not obey the same allometric relationships as other fishes (Howland et al., 2004). With the exception of some species of hagfishes (i.e., Paramyxine and Myxine sp.), which possess degenerate eyes and lack a cornea, iris, and lens (Holmberg, 1977), the eyes of primitive fishes all possess a cornea and lens and focus light onto a diVerentiated retina. The cornea is the first optical interface and acts as a protective goggle and is composed predominantly of collagen fibers (stroma) interposed between an epithelium and an endothelium. The fish cornea confers little, if any, refractive power due to the comparable refractive indices of the cornea and the surrounding aquatic media. In some primitive fishes, that is lampreys (van Horn et al., 1969), the cornea is split into dermal (continuous with the skin) and scleral (continuous with the eyecup) components, which allows the underlying globe to move freely. The evolution of the dermal cornea or secondary spectacle is thought to streamline the head and, in benthic species, protects the eye from abrasion. Microprojections (microridges, microplicae, and microvilli) extend from the surface of the superficial corneal epithelial cells and stabilize the tear film to provide an optically smooth interface (Collin and Collin, 2006). In lampreys and lungfishes, microholes in the cornea constitute the surface openings of large mucus‐secreting cells. The mucus provides a protective coating during burrowing and estivation (Collin and Collin, 2001). Colored pigments within the cornea and lens act as short wavelength‐ absorbing filters in some primitive fishes (the sea lamprey, P. marinus; the Australian lungfish, N. forsteri; and the bowfin, Amia calva) to minimize chromatic aberration and to tune the light reaching the retinal photoreceptors. In agnathans and elasmobranchs, sutural fibers inhibit the corneal stroma from swelling in response to low temperatures and changes in osmotic pressure associated with moving between saltwater and freshwater. Multilayered stacks of materials (such as connective tissue, modified rough endoplasmic reticulum, collagen fibrils, and cytoplasmic plates) with diVerent refractive indices are common inclusions in some teleost and non‐teleost fishes, that is the bowfin A. calva (Munk, 1968), and produce iridescence (Collin and Collin, 2001). In the sturgeon Acipenser sp. and the garfish Lepisosteus platyrhinchus, the cornea is supported by a loose meshwork of cellulofibrous tissue (or annular ligament) to support the iridocorneal angle (Collin and Collin, 1993). In water, and in the absence of any corneal refraction, the refractive power of the fish eye lies solely with the spherical lens. Although it is assumed

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that all primitive fishes adhere to this general mechanism, the lenses of downstream transformers of the sea lamprey, P. marinus, exhibit positive spherical aberration, while adults are corrected for spherical aberration (Bantseev et al., 2005). In biological lenses, short wavelengths of light are refracted more than long wavelengths of light (chromatic aberration). This results in blue light focusing closer to the lens than red light. However, interruptions to the otherwise smooth refractive index gradients of the lens in lampreys, lungfishes, and teleosts bring the focal length of the light spectrum back to a single focus on the retina. These multifocal lenses allow the incident light to be focused on a single layer of retinal photoreceptors, thereby optimizing the capture of light by multiple cone types involved in mediating color vision (Kro¨ger et al., 1999). The size of the pupillary aperture can improve the level of spherical aberration by reducing the number of rays passing through the lens periphery. The shape and size of the pupil varies across fishes from a circular to a U‐ or W‐shaped with multiple apertures to a slit. Lampreys possess little, if any, active movement of the iris, while most elasmobranchs elicit rapid changes in pupil shape in response to ambient light levels. Accommodatory mechanisms have been subject to appreciable selection pressure in primitive fishes and vary across many of the groups. Many hagfishes do not possess a lens and in both hagfishes and lampreys, the eyes do not possess intraocular eye muscles, a ciliary ganglion, and an Edinger‐Westphal nucleus. However, in other agnathans (lampreys), a cornealis muscle lying in the head next to the eye is thought to retract the cornea, and thereby the closely apposed lens, toward the retina during accommodation. However, stimulation of the cornealis muscle does not elicit lens movement (Sivak and Woo, 1975), and other static forms of accommodation may provide a focused image on the retina. In lampreys, some batoid elasmobranchs, and developing teleosts, the eyes are not symmetrical, where the dorsal retina sits closer to the lens than the ventral retina. This ‘‘ramped retina’’ allows both near and far objects to be focused on the retina simultaneously. Both the garfish, Lepisosteus osseus oxyurus, and the bowfin, A. calva, possess a retractor lentis muscle, which is responsible for accommodatory lens movement in the same direction (toward the retina) as in teleosts (Sivak and Woo, 1975). Cartilaginous fishes possess a protractor lentis muscle within the ventral papillae of the ciliary body, which moves the spherical lens anteriorly (toward the cornea) accommodating for near objects. 5.2. Retina and Visual Function With the exception of the hagfish retina, which is undiVerentiated, the retina of both primitive and advanced fishes possesses all of the major retinal neurons found in other vertebrates. These include photoreceptors, horizontal,

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bipolar, amacrine, and ganglion cells, which are arranged into three nuclear layers and two plexiform (synaptic) layers. In lampreys, most (75%) of the ganglion cells lie within the inner nuclear layer. Axons of these ‘‘ectopic’’ ganglion cells join those lying within the ganglion cell layer to exit the retina at the boundary of the inner nuclear and inner plexiform layers at the optic nerve head, eVectively negating the blind spot (Fritzsch and Collin, 1990; Fritzsch, 1991). In all other primitive fishes, most of the ganglion cells lie within the ganglion cell layer, with a small proportion ‘‘displaced’’ to the inner nuclear layer, and their axons traverse the retina within the nerve fiber layer abutting the inner limiting membrane. A great deal of work still remains to be done on the evolution of the inner retina in primitive fishes, but it appears that lampreys possess a population of biplexiform ganglion cells that make direct connections with the photoreceptors and inner nuclear layer cells (Fritzsch and Collin, 1990). The photoreceptors within the outer retina of a range of extant primitive fishes have received more attention. Their visual pigments phototransduce light energy into electrical impulses that trigger a cascade of enzymatic reactions that amplify the signal and ultimately change the rate of neurotransmitter release from their synaptic terminals. The signals are conveyed to the ganglion cells via bipolar interneurons. The visual pigments comprise a chromophore based on either vitamin A1 (rhodopsin) or A2 (porphyropsin) covalently bonded to an opsin protein, composed of seven transmembrane a‐helices embedded within the outer segment discs. The amino acid sequence of the opsin protein and the type of chromophore used determines the spectral sensitivity/tuning of the visual pigment and therefore the range of wavelengths to which an animal is sensitive. The possession of an A2‐based visual pigment by the pouched lamprey, Geotria australis, at the time this species enters the sea, contrasts with the situation in the comparable stage of P. marinus and in marine teleosts and elasmobranchs, which generally have vitamin A1‐based visual pigments (rhodopsins). Interestingly, during the upstream migration of P. marinus, the chromophore becomes A2‐based (Harosi and Kleinschmidt, 1993), as is typically the case in freshwater teleosts. The photoreceptors of both downstream and upstream migrants of the Pacific lamprey, Entosphenous tridentatus, possess a vitamin A1‐based photopigment, typical of rhodopsin (Crescitelli, 1972). When considered together with the finding of the entire complement of visual pigments incorporating a chromophore based on vitamin A2 in the anadromous white sturgeon Acipenser transmontanus, it appears that migration between freshwater and saltwater may not be suYcient to induce a paired A1/A2 visual pigment system (Whitmore and Bowmaker, 1989; Sillman et al., 1995). Given the interspecific variability, it is premature to predict whether rhodopsin or

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porphyropsin is the ancestral photopigment. However, it is interesting to note that only vitamin A1, and not A2, could be isolated from the liver in Myxine glutinosa (Vigh‐Teichmann et al., 1984). Rods mediate dim light (scotopic) vision and cones mediate bright light (photopic) vision. In jawed vertebrates, rod visual pigments are classified as Rh1, while cone visual pigments fall into four classes (long wavelength‐sensitive or LWS, ultraviolet (UV)‐sensitive or SWS1, blue wavelength‐sensitive or SWS2, and medium wavelength sensitive or Rh2). The presence of more than one cone type each with a diVerent spectral sensitivity provides the basis for color vision. The eyes of present day hagfishes, such as M. glutinosa and Eptatretus stoutii, are poorly developed and lie beneath muscle (M. glutinosa) or unpigmented skin (E. stoutii) (Locket and Jorgensen, 1998). On morphological criteria, only a single rodlike photoreceptor has been identified in M. glutinosa (Locket and Jorgensen, 1998), although Vigh‐Teichmann et al. (1984) found two unidentified classes of outer segments that could be distinguished on immunocytochemical, but not ultrastructural, criteria. Despite the degenerative state of the retina in hagfishes, M. glutinosa and Eptatretus burgeri both respond to changes in illumination by active locomotory movements, possibly ensuring that it remains buried during the day or maintains its circadian rhythm of activity (Kabasawa and Ooka‐Souda, 1989). However, the relative contributions of the retinal photoreceptors in the eye and those situated in the cloacal region (Myxine sp.) or the tail (Lampetra sp., Young, 1935; Ronan and Bodznick, 1991) still need to be assessed. The retinae of a number of northern hemisphere (or holarctic) lampreys have been examined morphologically and the consensus is that two photoreceptor types exist, a short and a long, and are putatively a rod and a cone receptor, although the classification of these receptors has been a subject of contention for many years (reviewed in Crescitelli, 1972; Collin et al., 1999) (Figure 3.5A). On the basis of the continuity of the outer segment discs with the extracellular matrix and a pattern of protein labeling throughout the outer segment (following incorporation of radioactively labeled amino acids to indicate the process of outer segment disc renewal), both receptor types were considered cones in the sea lamprey, P. marinus, by Dickson and Graves (1979). In contrast, the southern hemisphere lamprey, G. australis, possesses five morphologically distinct photoreceptor types, all of which possess cone‐like characteristics and contain a diVerent visual pigment (LWS, SWS1, SWS2, RhA, and RhB), providing the basis for pentachromatic vision (Figure 3.5B). Three of these opsin genes are orthologous to the visual pigment classes of gnathostomatous (jawed) vertebrates, but the other two (RhA and RhB) have evolved by an independent gene duplication event,

136 — 507 nm — 552 nm — 614 nm

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Catfish Rh1 Dogfish Rh1 36 Skate Rh1 Chameleon Rh1 99 Coelacanth Rh1 Goldfish Rh1 Dunnart Rh1 92 Mouse Rh1 83 100 Human Rh1 Coelacanth Rh2 Goldfish Rh1-1 100 100 100 Goldfish Rh1-2 79 Chameleon Rh2 Geotria RhB 96 Geotria RhA 86 Lamprey Petromyzon RhA 100 Lamprey Lampetra RhA 100 Geotria SWS2 68 Goldfish SWS2 100 Newt SWS2 93 81 Chameleon SWS2 Geotria SWS1 95 Frog SWS1 Chameleon SWS1 89 Wallaby SWS1 96 Mouse SWS1 100 97 Human SWS1 Geotria LWS Goldfish LWS 84 100 Chameleon LWS Wallaby LWS 97 Mouse LWS 65 Human LWS-R 100 100 Human LWS-G Fruitfly Rh4 99 100

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Fig. 3.5. Spectral sensitivity of lamprey photoreceptors. (A) Upper two panels: summary of the calculated photoreceptor quantal spectral sensitivity curves for three of the five types of photoreceptors in downstream (left) and upstream (right) migrants of G. australis. Middle two panels:

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which occurred within the agnathan lineage (Collin et al., 2003b) (Figure 3.5B). Given the absence of the Rh1 opsin gene within the agnathan lineage, scotopic vision may have evolved exclusively within the gnathostomatous lineage prior to the evolution of the cartilaginous fishes. The functional characterization of rods and cones requires further examination in the early vertebrates at both the physiological (photokinetics) and the biochemical (phototransduction genes) levels. Interestingly, the lamprey visual pigments react with hydroxylamine in a similar manner to cone pigments (Hisatomi et al., 1988), they do not saturate at high light intensities (Govardovskii and Lychakov, 1984) and appear to be designed for photopic vision. Multiple cone types have been retained within the cartilaginous (three cone types in the shovelnose rays, Hart et al., 2004), dipnoan (four cone types in the Australian lungfish, Bailes et al., 2006), early ray‐finned (up to three cone types, that is, in the sturgeon and paddlefish, Munk, 1964, 1968, 1969; Sillman et al., 1999), and teleostean (up to seven cone types in cichlids, Parry et al., 2005) fishes. Color‐coding mechanisms and various subtypes of color opponent horizontal cells are also present in primitive fishes, that is in the bowfin, A. calva, the shortnose garfish, Lepisosteus platostomus, and the Siberian sturgeon, Acipenser baeri (Burkhardt et al., 1983; Gottesman and Burkhardt, 1987). Therefore, it appears that all vertebrate classes possess the potential for color vision, but this has not yet been confirmed behaviorally in the non‐actinopterygian fishes. The comoran coelacanth appears to have lost the SWS1, SWS2, and LWS opsin genes and retained the Rh1 and Rh2 opsin genes that are tuned to detect the full spectrum of light available at the depth it inhabits (200 m) (Dartnall, 1972; Yokoyama, 2000).

spectral sensitivity curves for the visual pigments in the short (SR) and long (LR) receptor types in the river lamprey, L. fluviatilis, giving lmax values of 517 and 555 nm, respectively. Lower two panels: normalized absorbance spectra from the outer segments of the short (SR) and long (LR) receptors in the sea lamprey, P. marinus, giving lmax values of 525 and 600 nm, respectively (left), and the relative quantal spectral sensitivity of the whole photoreceptor in M. mordax (lmax 514 nm), based on both the visual pigment and ellipsosome spectra and the dimensions of the inner and outer segment (right). PB, postbleaching and BL, baseline. [Figure from Collin and Trezise (2006).] (B) Phylogenetic tree (based on codon‐matched nucleotide sequences comparisons) showing the relationships between the opsin genes of G. australis, the northern hemisphere lampreys, Lampetra japonica and P. marinus, representative jawed vertebrates, and an invertebrate out‐group. See text for explanations of the diVerent opsin groups. The dashed line indicates the predicted genetic complement of opsins present in the most recent common ancestor of the jawed and jawless vertebrates, 540 mya. The number at each branch point reflects its robustness (maximum 100). The scale bar is calibrated in nucleotide substitutions per site. [Reproduced from Collin et al. (2003b) with kind permission from Science Publishers Inc.]

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5.3. Spectral Filters Filtering mechanisms in fishes are common and typically comprise the accumulation of short wavelength‐absorbing pigment within the ocular media (cornea, lens, and vitreous humor) (Siebeck and Marshall, 2001) and within the inner regions of some photoreceptor types (myoidal pigment and oil droplets) (Collin et al., 1999; Vorobyev, 2003). These spectral filters narrow the absorption spectrum of the visual pigment housed in the outer segment of the photoreceptors, shift the peak absorption of the visual pigment toward longer wavelengths, and decrease the absorption eYciency. Accumulations of yellow myoidal pigment exist in lampreys (Collin et al., 1999), Australian lungfishes (Bailes et al., 2006), and reptiles (Barbour et al., 2002). Oil droplets exist in the lobe‐finned (lungfish, Robinson, 1994; Bailes et al., 2006) and early ray‐finned (sturgeon and paddlefish, Sillman, et al., 1999) fishes and may be red or colorless. Oil droplets allow the discrimination of more colors under bright light conditions. Intracellular structures resembling oil droplets lie within the photoreceptor inner segments in two species of lamprey (Collin and Potter, 2000; Collin and Tresize, 2006) and some teleostean cyprinids (Nag and Bhattacharjee, 1995). Termed ellipsosomes, on the basis of their elliptical shape, these structures are of mitochondrial origin and may either contain a heme pigment, thereby acting as a spectral filter (cyprinids), or lack any light‐absorbing pigment and may act as an intracellular focusing device (lampreys, Collin and Potter, 2000). 5.4. Visual Sensitivity Increased visual sensitivity is often mediated by the adaptive advantage of a mirror or tapetum located behind the retina. Sensitivity is increased by reflecting light back onto the photoreceptors for enhanced photon capture, an early invention in vertebrate evolution. Of the 33 species of lampreys described, a single species (Mordacia mordax) possesses a mixture of reflective needles and pigment granules within the retinal pigment epithelium, which elicits a yellow eyeshine (Collin and Potter, 2000). Retinal tapeta are present in the garfishes and several species of teleosts. These comprise spheres containing astaxanthin, phenolic compounds, or lipids packed into a hexagonal array within the confines of the retinal pigment epithelial cell membrane. All these spheres are reflective and elicit a colored reflex produced by diVuse scattering. Choroidal tapeta typically with guanine as the reflector are found in elasmobranchs (sharks, skates, rays, and ratfishes), Polypteriformes (bichirs), Semionotiformes (gars), Acipenseriformes (sturgeons), Dipnoi (lungfishes), the coelacanth, and a few nocturnal ray‐finned fishes (Nicol and Arnott, 1973). In cartilaginous fishes, the choroidal tapetum is occlusable, masking the mirrored surface with pigment granules in bright light.

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Another way of increasing sensitivity to a range of ambient light conditions is to diVerentially place the rod photoreceptors adapted for dim light vision closer to the incident light (at the outer limiting membrane) during times of low light. Under these conditions, the cone photoreceptors migrate toward the back of the retina. The opposite occurs in bright light, where the cones adopt a position at the outer limiting membrane and the rods are masked by the migration of the melanosomes within the retinal pigment epithelium. Retinomotor movements are not present in lampreys (Walls, 1942), reedfishes, Calamoichthys calabaricus and Polypterus delhezi, and in the African lungfishes Protopterus dolloi (PfeiVer, 1968a). In teleosts, not all photoreceptor types undergo photomechanical movements. There appears to be a trade‐oV between the level of photomechanical retinomotor movements (inherently slow) and the evolution of more rapid pupillary movements. In evolutionary terms, pupillary and retinomotor movements appear to be most developed within the elasmobranchs and teleosts, respectively. 5.5. Visual Resolution In contrast to optimizing sensitivity with either retinomotor movements or a tapetum, many fishes are specialized for acute vision, sampling a particular part of their visual field with high spatial resolving power. The lamprey retina is specialized for acute vision, where ganglion cell densities increase to form an area centralis in central retina (Fritzsch and Collin, 1990). All of the fishes examined thus far, irrespective of their phylogenetic origins, possess some form of retinal specialization, which may be in the form of a concentric increase in cell density (area centralis) or an elongated increase in cell density across the retinal meridian (horizontal streak). The silver lamprey, Ichthyomyzon unicuspis, possesses an area centralis located in central retina (peak of over 4000 ganglion cells per mm2), which relocates to the dorsal peripheral retina (peak of over 3600 ganglion cells per mm2) during growth. In the Florida garfish, L. platyrhinchus, a pronounced horizontal streak lies across the ventral meridian of the eye. This specialization subtends the surface of the water, where this predator preys on live fish with its long snout armed with needlelike teeth (Collin and Northcutt, 1993). Together with a temporal area centralis, which receives input from the frontal visual field, much of its visual field is monitored with increased spatial resolving power without the need for scanning eye movements. Environmental cues and the symmetry of each species’ perceived world play a large role in the topography of retinal cells rather than any phylogenetic relationships. The first appearance of a retinal invagination or foveal pit in predatory fishes appears to be in the euteleosts, that is in the seahorse and the sandlance (see reviews by Collin, 1997; Collin, 1999; Collin and Shand, 2003). Although there is little known about the resolution of primitive vertebrate eyes, there

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does appear to be a large variation in the type and total number of retinal ganglion cells in lampreys (12,000–35,000, Vesselkin et al., 1989; Fritzsch and Collin, 1990), elasmobranchs (100,000, Collin, 1988), and garfishes (80,000 cells, Collin and Northcutt, 1993), reflecting functional diVerences in acuity and function. The homogeneous distribution of large a‐like ganglion cells across the retina in L. platyrhinchus suggests that they possess large receptive fields and are motion sensitive as found in various other species of vertebrates (Cook et al., 1992; Collin and Northcutt, 1993). The topographic distribution of rods and cones in the paddlefish, P. spathula, and the Australian lungfish, N. forsteri (Bailes et al., 2006), reveals that these primitive species lack a retinal specialization for acute vision, but have increased receptor size to increase sensitivity, while maintaining chromatic sampling of their visual environment. 5.6. Visual Input to the CNS The optical image formed by the visual apparatus is transformed into an electrical image by the retina, which is conveyed to the visual centers of the brain via the optic nerve. The optic nerve comprises the axons of the retinal ganglion cells and eVerent fibers. In lampreys, the optic nerve is avascular and contains an ependymal core and unmyelinated axons. However, typically the axons of gnathostomatous (jawed) fishes are myelinated and form fascicles or bundles subdivided by astroglia. The optic nerves from the left and right eyes cross (decussate) at the optic chiasm crossing as separate nerves (most teleosts) or interlacing with each other (cartilaginous fishes and a few teleosts). Three major fiber tracts are present in virtually all vertebrates, that is the basal optic tract, the axial or medial optic tract, and the marginal optic tract (Fritzsch, 1991). In lampreys, 75% of the ganglion cells are ‘‘displaced’’ to the inner nuclear layer, while less than 1% of these ganglion cells exist in jawed vertebrates. In gnathostomatous vertebrates, the displaced ganglion cells project to the basal optic root and the basal optic nucleus, but this pathway does not exist in hagfishes (Wicht and Northcutt, 1990) and receives little input in lampreys (Fritzsch and Collin, 1990; Fritzsch, 1991). The axial optic tract occurs in lampreys and conveys retinopetal (eVerent) fibers rather than retinofugal (aVerent) fibers (Rubinson, 1990). In most fishes, contralateral and ipsilateral projections from the eyes terminate in the suprachiasmatic nucleus, the posterior parvocellular preoptic nucleus, the lateral geniculate nucleus, the dorsolateral thalamic nucleus, the pretectal nuclei, and the optic tectum. In all species examined, retinal input to the optic tectum is retinotopic and is restricted to the stratum opticum (SO), the stratum fibrosum et griseum superficiale (SFGS), the

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stratum griseum centrale (SGC), and the junction between the stratum album centrale (SAC) and the stratum periventriculare (SPV), although some phylogenetic variation exists. Ipsilateral (non‐decussating) input to the visual centers of the brain is thought to be an inherent component of the visual pathway in all vertebrates, including hagfishes (15%) and lampreys. However, the relationship between ipsilaterally projecting ganglion cells and the extent of the binocular visual field is not well understood in early fishes. The optic tracts in both hagfishes and lampreys project bilaterally to the preoptic, thalamic, and pretectal nuclei and terminate in the mesencephalic optic tectum (Wicht and Northcutt, 1990) and may represent the ancestral condition, which has been retained in hagfish (Fritzsch, 1991). In garfishes, the mediorostral and ventrolateral regions of the optic tectum receive ipsilateral input from the retina and subtend the dorsal and ventral binocular fields, respectively (Collin and Northcutt, 1995). Discrete ipsilateral input to the entire optic tectum is found in Australian lungfishes (Northcutt, 1980) and juvenile teleosts (Collin et al., 2001), suggesting that there may be phylogenetic diVerences in binocular partitioning (when compared to most modern teleosts). Ipsilateral input via the intertectal and posterior commissures does not appear to occur in all non‐actinopterygian fishes and appears to have evolved independently many times (Northcutt and Butler, 1992). Retinal projections to non‐teleost actinopterygians, that is sturgeon, and to chondrichthyans (sharks) terminate bilaterally in the preoptic area, thalamus, area pretectalis, nucleus of the posterior commissure, optic tectum, and the nuclei of the accessory optic tract (Ito et al., 1999). In both lampreys and hagfishes, two tegmental cell groups (the reticular mesencephalic area and the nucleus M5 of Schober) give rise to centrifugal fibers and, as in teleosts, also make contact with bipolar, horizontal, and ganglion cells. In the river lamprey, L. fluviatilis, it is thought that postsynaptic ganglion cell responses are the synaptic potentials from amacrine cell contacts and that the amacrine cells are also directly stimulated by retinopetal fibers (Vesselkin et al., 1996). The centrifugal system responds to chemical cues such as sex pheromones and regulates visually mediated sexual and reproductive behavior in addition to altering ganglion cell responses to color contrast. The increased proportion of eVerents in agnathans (2% in lampreys and 5% in hagfishes) in contrast to jawed vertebrates (0.5%) suggests that this is the ancestral condition. 5.7. Nonvisual Photoreception Almost all organisms have evolved some form of endogenous time‐ keeping mechanism or biological clock to respond to changes in environmental conditions, for example seasons, tides, light cycles, and temperature. The

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receptors that sense these regular, environmental perturbations are diverse in their structure, function, location, and role. The synchronization or entrainment between the external environment and internal rhythms regulates the period and phase of the rhythm, and, for the majority of animals, this biological clock is entrained by changes in both the intensity and the spectral composition of light. Photoentrainment, or using the transition between light and dark (dawn and dusk) to adjust circadian phase to local time, is considered primarily to be mediated by nonvisual photoreceptors (Foster and Provencio, 1999). These photoreceptors do not contribute to image‐forming vision or object detection (rods and cones in the eye) but respond only to brightness information. However, as both detector systems occur in all vertebrate classes, the selection pressures underlying their coevolution and maintenance are crucial to survival. 5.7.1. Pineal and Parapineal Organs The pineal makes the hormone melatonin, which is produced only in the dark portion of the light/dark cycle and provides a slow signal that is important in regulating circadian and/or photoperiodic behaviors. The pineal arises from an evagination from the roof of the diencephalon and sits beneath a translucent area or ‘‘window’’ in the skull. Pineal photoreceptors appear to be highly specialized for detecting gradual changes in environmental light rather than transient light stimuli (Shand and Foster, 1999). Light responses from the pineal may be color‐coded with sensitivity extending into the UV range (Koyanagi et al., 2004). There is no pineal body described in hagfishes, but in holarctic lampreys and G. australis, pinealocyte outer segments are immunolabeled with both anticone and antirod opsin antisera (Garcia‐Fernandez and Foster, 1994; Garcı´a‐Ferna´ndez et al., 1997). For G. australis and M. mordax, metamorphosis and reproduction are both accompanied by migration between freshwater rivers and the sea, occurring at very precise times of the year and are most likely controlled by seasonal changes in photoperiod. Pinealectomy has been shown to aVect all the phases of the life cycle (Joss, 1973). In M. mordax, the parapineal is absent (Eddy and Strahan, 1968). Elasmobranchs possess UV or blue sensitive photoreceptors, which serve to monitor circadian and circannual variations in light intensities (Vigh‐Teichmann et al., 1990). The outer segments of photoreceptors in the pineal have also been discovered in the Dipnoi (lungfishes, P. dolloi, Ueck, 1969) and the coelacanth L. chalumnae (Hafeez and Merhige, 1977). The parapineal arises from a dorsal evagination from the diencephalon (Vollrath, 1981). In lampreys, the parapineal organ consists of a vesicle that sits beneath the pineal organ and possesses a few photoreceptors. Parapinealocytes are not labeled by anticone opsin antisera in the sea lamprey P. marinus (Garcia‐Fernandez and Foster, 1994), but are densely labeled in

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the river lamprey L. fluviatilis (Garcı´a‐Ferna´ndez et al., 1997) with potentially two types of parapinealocytes based on opsin immunoreactivity (Vigh‐ Teichmann et al., 1984). The parapineal has not been found in adult lungfishes (African lungfish, P. dolloi, Ueck, 1969) or elasmobranchs (Bertolucci and Foa´, 2004). 5.7.2. Deep‐Brain Photoreceptors As early as 1935, J. Z. Young revealed that deep‐brain photoreceptors were responsible for body movements in blinded and pinealectomized lampreys following illumination of the head (Young, 1935) and since then all nonmammalian vertebrates have been found to possess deep‐brain photoreceptors (Foster et al., 1994). Although photons are scattered and selectively filtered by neural tissue, large amounts of light penetrate deep into the brain, thereby producing a measure of environmental irradiance and hence time of day. There are two classes of encephalic photoreceptors: cerebrospinal fluid (CSF)‐contacting neurons in the hypothalamus of lampreys, reptiles, and birds (Garcia‐Fernandez and Foster, 1994) and cells within the nucleus magnocellu´ lvarez‐Viejo et al., 2004). The hypolaris preopticus in fish and amphibians (A thalamic and septal nuclei of the periventricular CSF‐contacting neuronal system are already present in the lancelets and hagfishes (Vigh et al., 2002; David et al., 2003). 5.7.3. Dermal Photoreceptors Dermal cells are regulated by photoreceptor cells in the CNS via pituitary melanocyte‐stimulating hormone, pineal melatonin, or directly by photopigments localized within the dermal cells themselves. Dermal chromatophores or iridophores of many fishes and amphibians are directly sensitive to light and will aggregate or disperse pigment granules on light exposure. Dermal photoreceptors are able to initiate behavioral responses in lampreys and teleosts, where, following illumination, an ‘‘eyespot’’ on the tail of both ammocoete (Ronan and Bodznick, 1991) and adult (Ulle´n et al., 1993) lampreys mediates tail movement and avoidance behavior, respectively. Hagfishes have also been reported to possess light‐sensitive pigment in both the tail and a line of unpigmented skin running down the back in E. burgeri (Patzner, 1978). Iridophores and chromatophores also occur in the cornea and act either as a yellow filter, to reduce chromatic aberration and/or enhance contrast perception (chromatophores), or reflect bright downwelling light (iridophores, Muntz, 1976). Yellow/orange pigment granules within the corneal stroma can migrate across the teleost fish cornea in response to environmental lighting conditions (Siebeck et al., 2003). Recent ultrastructural studies of the eyes of the river lamprey L. fluviatilis reveal corneal pigment in the primary spectacle (S.P.C., unpublished data).

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6. CHEMORECEPTIVE SYSTEMS Anamniotic gnathostomes possess five types of chemosensory systems: (1) olfaction or smell for social interactions, homing, alerting to the presence of food and avoiding predators; (2) gustation or taste used in feeding and the localization, acceptance, or rejection of food; (3) isolated chemosensory receptors for feeding and predator avoidance; (4) a common chemical sense (free nerve endings) for irritant detection (Finger, 1997); and (5) chemoreceptors involved in cardiovascular responses to changes in oxygen and carbon dioxide levels. The olfactory epithelium lies within a water‐filled chamber at the base of the nares and comprises a series of folded lamellae adorned with sensory receptors to detect water‐soluble odors. The gustatory system comprises taste buds or aggregations of sensory receptors situated often on raised papillae within the oral cavity, pharynx, or over the head and body. Solitary chemoreceptors exist as small, encapsulated nerve endings over the epidermis or, more commonly, on the pectoral fin rays. The common chemical sense is closely associated with the somatosensory system. Branchial and extrabranchial chemoreceptors play a role in controlling cardiorespiratory responses to changes in oxygen and carbon dioxide levels in addition to pH. These receptors, sensitive to dissolved gases and innervated by cranial nerves V, VII, IX, and X, will not be discussed further in this chapter, but see Chapters 4 and 5 for a comprehensive review. 6.1. Olfaction 6.1.1. Olfactory Epithelium Olfaction constitutes a vital role in the lives of primitive fishes, especially hagfishes and lampreys. In hagfishes, water enters a single median nasohypophysial duct and then passes ventrally to pass over the olfactory organ (Figure 3.6A). The olfactory organ is housed within a single cartilaginous capsule but comprises left and right convoluted epithelial surfaces divided by a medial septum (Northcutt, 1989a). Caudal to the brain, water is then pumped from another chamber with the help of a velum, which forces water backward to the gills and out through the branchiopores (Zeiske et al., 1992; Braun, 1996) (Figure 3.6A). A similar arrangement is found in lampreys, where water passes over an olfactory median organ before being pumped toward the gills. The pumping mechanism is an enlarged sac dorsal to the branchial pharynx, which contracts and subsequently recoils to force water in and out of the nasal cavity (Kleerekoper and van Erkel, 1960; Gradwell, 1972). Agnathan water sampling may be described as cyclosmate (reliant on pumping). This is in contrast to the isosmate (reliant on cilia)

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A v

nd n

ol

ve np

ol np 2 mm

B

C

Sensory m

Nonsensory

10 µm

2 µm

Fig. 3.6. (A) The olfactory organ of the hagfish, M. glutinosa, showing the head in sagittal section and in transverse section (inset). The broken line indicates the level of section of the inset. The arrows show the direction of the water current. n, nostril; nd, nasal duct; np, nasopharyngeal duct; ol, olfactory lamellae; v, valve; and ve, velum. (B and C) Scanning electron micrographs of the olfactory epithelium of the gray reef shark, Carcharhinus amblyrhincos, in low (B) and high (C) power. Note the clear diVerentiation between the sensory and nonsensory epithelium. The sensory epithelium is characterized by both microvillous (m) and ciliated epithelial cells. [(A) is adapted from Zeiske et al. (1992). (B and C) are courtesy of Vera Schluessel.]

arrangement in the garfish, L. osseus, where ciliary action moves an odorant wave front over the olfactory lamellae only once (Bashor et al., 1974). In both hagfishes and lampreys, the presence of a prenasal sinus and an adenohypophysis, respectively, has received much attention along with the relationship between the olfactory and endocrine systems (Gorbman, 1995). In bony fishes, the olfactory epithelium is located within specialized nares, one on each side of the dorsal surface of the head, often with an inhalent and an exhalent opening. The cartilaginous ratfish, Chimaera monstrosa, possesses internal nares. Bipolar neurons of the olfactory epithelium are

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generally characterized by ciliated apical surfaces and a basal axon that forms the first cranial nerve (olfactory nerve, nI, Figure 3.6B and C). The olfactory epthelium of lampreys is composed of only ciliated receptors, whereas hagfish olfactory epithelial cells possess both cilia and microvilli (Thornhill, 1967; Theisen, 1976). A similar dichotomy is revealed in lungfishes, where the African lungfish (Protopterus annectens) possesses both microvillous and ciliated receptor cells, but the Australian lungfish (N. forsteri) has only microvillous receptor cells as adults but both as juveniles (Hansen et al., 1994). The olfactory organs of dipnoans (Derivot et al., 1979) and the coelacanth (Millot and Anthony, 1965; PfeiVer, 1969) have also been described. 6.1.2. Sensitivity and Odor Discrimination The olfactory epithelium is sensitive to a range of odors, including amino acids, bile acids, steroid pheromones, and aromatic compounds. For teleosts, sensitivity thresholds vary for amino acids (10–9 M in Ictalurus catus, Caprio, 1980), steroids (10–13 in Carassius auratus, Sorensen et al., 1987), and prostaglandins (10–13 M in Misggurnus anguillicaudatus, Kitamura and Ogata, 1980). In the hagfish M. glutinosa, the olfactory system reveals a sensitivity threshold of 10–6 for both amino acids and steroids (Doving and Holmberg, 1974). Acids (such as taurocholic acid) are produced by the bile to solubilize ingested fat and are then released into the environment, where they can be used as a signal by conspecifics, especially in the context of homing and sex. In anadromous lampreys, individuals return to their natal stream to spawn and eventually die based on an acute sensitivity to specific amino acids and bile acids produced by ammocoetes and other fishes (Teeter, 1980; Li et al., 1995). Similarly, hagfish are thought to rely heavily on olfaction to find prey and, with their limited visual and hydrodynamic abilities, chemosensory sensitivity may be their most important sensory modality. DC recordings [analogous to an electro‐olfactogram (EOG)] from the olfactory epithelium of hagfishes reveal that L‐alanine is detected at lower concentrations than L‐glutamine and that g‐aminobutyric acid, glutathione, and 4‐hydroxy‐l‐proline are also potent stimuli (Doving and Holmberg, 1974). Sea lampreys (P. marinus) undergo three major phases during their life cycle (a larval phase, where they travel down freshwater streams after metamorphosis; a marine phase, where they become parasitic on large teleosts; and an upstream phase, returning to their natal stream to spawn and die) (Hardisty and Potter, 1971). In the upper regions of the streams, spermiating male bile acids are thought to function as a mating pheromone, while larval bile acids act as a migratory pheromone. The bile acid 3kPZS acts as a potent male pheromone to adult female lampreys and allows discrimination between ovulating females and ever present larvae in the rapids, where the released bile acids would be rapidly diluted (Siefkes and

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Li, 2004). Suzuki (1978) suggests that calcium ions play an important role in the generation of olfactory receptor potentials (resting membrane potentials of the olfactory neurons in the lamprey Entosphenus japonicus range from 36.7 to 60.0 mV, Suzuki, 1977). In general, olfactory acuity varies among species. In studies utilizing a ‘‘natural’’ stimulus of beef heart extract, rather than pure compounds, PfeiVer (1969) revealed that sensitivity thresholds varied between 10–11 g liter 1 in C. calabaricus and 10–14 g liter 1 in the bichir Polypterus bichir, which was about 10 times more sensitive (macrosmatic) than the teleost Phoxinus. Olfactory receptor proteins are encoded by a large multigene family, which may consist of up to 1000 genes in mammals (Buck and Axel, 1991) but in the order of 50–100 genes in teleost fishes (Weth et al., 1996). Individual olfactory receptor genes are selectively expressed in a small subpopulation of olfactory neurons, each of which expresses only one or a few receptor types (Freitag et al., 1998). Comparison of the deduced amino acid sequences of the olfactory receptor gene family reveals that there are two basic types (Types I and II) that are thought to be specialized for detecting water‐soluble odorants and volatile odorants, respectively. Analysis of the receptors in L. chalumnae shows that although both Types I and II are both present, the Type II receptor genes represent nonfunctional pseudogenes (Freitag et al., 1998). It is interesting to speculate whether the diminishing importance of the Type II receptors in these lobe‐finned ‘‘living fossils,’’ which hold such vital clues to the evolution of tetrapods, has resulted either from selection pressures associated with its origin or a new function for the recognition of volatile odorants. 6.1.3. Primary and Secondary Olfactory Input to the CNS Hagfishes and lampreys both send large numbers of olfactory axons into the olfactory bulbs (primary input) and telencephalon (secondary input). The secondary projections in hagfishes are so pronounced that a large, laminated olfactory cortex has been identified within the telencephalon (Wicht and Northcutt, 1992). In lampreys, the receptor cells within the left and right epithelia project to the ipsilateral olfactory bulbs and form spatially and functionally distinct arrangements. The number of types of olfactory receptors in lampreys is relatively small and only a few odorants stimulate olfactory activity (Li et al., 1995; Frontini et al., 2003). From the olfactory bulbs, axons of the mitral cells leave the glomerular zones and form fiber bundles (medial and lateral) collectively called the olfactory tract, which terminate within various regions of the telencephalon. In contrast to the olfactory nerve axons, which are all unmyelinated, the olfactory tract is composed of a mixture of myelinated and unmyelinated axons. In all primitive fishes studied thus far (Lampetra planeri, I. unicuspis, Polypterus palmas, C. calabaricus, A. calva, and P. dolloi), a subset of primary olfactory projections bypass the olfactory bulbs and terminate in both the

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telencephalon and the diencephalon, with the exception of Acipenser ruthenus, that does not possess extrabulbar projections to the diencephalon (see review by Hofmann and Meyer, 1995). The function of these extrabulbar olfactory projections is speculative but is thought to be involved in the detection of waterborne substances that are either in high enough concentrations or are suYciently distinct to elicit a response without the processing necessary for bulbar projections. Despite their putative function, there is little evidence to suggest that extrabulbar projections are of olfactory origin, indicating that their inclusion within the terminal nerve complex may be more appropriate (Demski, 1993; von Bartheld, 2004). 6.1.4. The Terminal Nerve Originally discovered in the African lungfish, P. annectens, by Pinkus (1894), the terminal nerve (or nervus terminalis) is considered to be a group of ganglion cells, which possess fibers projecting from the nasal epithelium, that bypass the olfactory bulb and terminate in various regions of the CNS. The nervus terminalis is considered to be a separate cranial nerve (n0), but its projection patterns and characterization are variable in the range of species examined, making it diYcult to trace its origins or function. In lungfishes, an anterior root enters the brain among the olfactory nerve and bulb, while the posterior root enters the CNS at the level of the optic nerve (as the nervus praeopticus, SewertzoV, 1902). The two components of the terminal nerve in dipnoans possess diVerent neurochemical signatures (anterior root is GnRH immunoreactive and the posterior root is acetylcholinesterase immunoreactive, von Bartheld et al., 1988, 1990). The situation appears to be diVerent for lampreys that have lost the GnRH or FMRF‐amide terminal nerve components but retained other features thought to be characteristic of this system (Eisthen and Northcutt, 1996). Bichirs (Polypterus) possess similar projection patterns to lampreys and lungfishes (von Bartheld and Meyer, 1986). The terminal nerve within the brain of both the bichir, Polypterus senegalus (Chiba, 1997), and the gar, Lepisosteus oculatus (Chiba, 2005), is immunoreactive to neuropeptide Y and GnRH, suggesting that it acts as a neuromodulator, especially in reproductive behaviors via the hypothalamic pituitary system. Von Bartheld (2004) has recently suggested that the terminal nerve in primitive fishes is extremely variable and is probably a component of all primitive species, albeit defined using diVerent anatomical, neurochemical, and developmental criteria. 6.2. Gustation Taste or the central perception of both mechanical and chemical stimulation by food is mediated by taste buds. Taste buds are pear‐shaped organs, which consist of up to 100 specialized epithelial cells concentrated into

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aggregations over the oropharynx and often also covering the body surface or branchial cavity. Unlike olfactory neurons, taste receptors lack an axon, where their bases are synaptically connected to aVerent nerve fibers via chemical synapses. From developmental studies, taste buds are considered secondary sensory cells (Reutter and Witt, 1993) and are always stimulated by nutritionally important substances. In most anamniotes, the diVerent sized villi at the apical ending protrude above the surface of the epithelium. In teleost fish, taste buds are able to discriminate between palatable substances before ingestion and thereby act over very small distances (Kasumyan, 1997). The gustatory system comprises peripheral receptors or taste buds, which are innervated by the facial (nVII), glossopharyngeal (nIX), and vagal (nX) cranial nerves (Finger, 1983). Oropharangeal taste buds appear to be the primitive condition with external taste buds over the head and even trunk regions evolving independently a number of times (Northcutt, 2004). Lampreys (P. marinus and I. unicuspis) do not appear to possess external taste buds (Baatrup, 1985), but have terminal (ciliated) buds over the internal surfaces of the pharyngobranchial ducts (Mallat and Ridgeway, 1984), which respond preferentially to amino acids. Given the similar distribution, innervation, and central projections of the internal buds in lampreys and the taste buds of teleosts, Braun and Northcutt (1997) have considered them to be homologous. External taste buds are frequently observed over the head of gars, that is L. platyrhinchus (Norris, 1925; Song and Northcutt, 1991b), the bowfin, A. calva (Allis, 1889), and sturgeons (Norris, 1925; Figure 3.7A and B), but not in Polypterus (PfeiVer, 1968b) and Polyodon (Norris, 1925). This distribution suggests that the earliest ray‐finned fishes possessed only internal taste buds and that external taste buds evolved independently in chondrosteans and neopterygians. Although there is considerable variation in the morphology of taste buds, four types have been characterized: dark cells (Type I), light cells (Type II), basal cells, and stem cells (Reutter and Witt, 1993; Finger, 1997). In Lepisosteus, taste buds are composed of two types of elongated light cells and one type of dark cell (Reutter and Hansen, 2005) (Figure 3.7C and D). In contrast, Amia taste buds contain two types of light cells and two types of dark elongated cells. On the basis of the height of the microvillar projections above the epidermis, both the Australian lungfish, N. forsteri, and the sturgeon, Scaphirhynchus platorynchus, possess three types of light cells and one type of dark cell (Reutter and Hansen, 2005). AVerent synapses are common in the buds of both species, while eVerent synapses occur only in Lepisosteus taste buds (Reutter et al., 2000). The internalized taste buds within the oropharynx in the plesiomorphic representatives of the sarcopterygian group of lobe‐finned fishes (Latimeria and Neoceratodus) appear to confirm that this is the primitive pattern and that protopterid

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B

A

TB

200 µm C

20 µm D

Cd CI1

Cd

1 µm

Cd

CI2

Cd

1 µm

Fig. 3.7. (A) Scanning electron micrograph of the tip of the barbel of the sturgeon, A. baeri, showing a series of epidermal hillocks each containing several taste buds. (B) High power of a hillock in A. baeri showing three taste bud (TB) receptor regions. (C and D) Transmission electron micrographs of the apical region of two types of light (Cl1 and Cl2) and dark (Cd) taste receptors in the garfish, L. oculatus. Note one type of light cell has a single large villus. [Reproduced with permission from Reutter and Hansen (2005).]

lungfishes (that possess external taste buds, PfeiVer, 1968b) have independently evolved external taste buds as has occurred in teleosts (especially over barbels, fin rays, and around the mouth, Hansen and Reutter, 2004). Taste buds are used to assess palatability of food and may be ‘‘tuned’’ to specific prey items or nutritional needs or both. At least teleost fish taste buds are sensitive to diVerent amino acids such as arginine, proline, and alanine

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(Caprio et al., 1993). Presumably, taste buds in lampreys may be used to monitor respiratory currents for the presence of potential food particles (Finger, 1997). Little is known about the taste preferences of other primitive fishes. However, using the teleost systems as a guide, one may expect that taste preferences are highly species specific, that there is a strong similarity in the taste preferences between geographically isolated fish populations, and that environmental factors, such as water temperature and pollutants (heavy metals and low pH water), may shift taste preferences and fish‐feeding motivation (Kasumyan and Doving, 2003). 6.3. Solitary Chemoreceptor Systems Solitary chemosensory cells are present in lampreys, elasmobranchs, teleost fishes, and some amphibians (Whitear, 1992). The hagfishes, Eptatretus stouti and Myxine sp., do not possess taste buds but possess solitary chemosensory receptors over the oral tentacles, at the opening of the prenasal duct and in the epidermis surrounding the mouth and oropharynx (Retzius, 1892; Patzner et al., 1977; Georgieva et al., 1979). These solitary receptors are innervated by either cranial (trigeminal) or spinal nerves, whose central projections form a continuous dorsolateral tract, which runs the length of the neuraxis (Whitear, 1992; Braun, 1996; Finger, 1997). Hagfishes also possess 180,000 Schreiner organs (Schreiner, 1919), which are similar to the solitary receptor organs, within the oropharynx and over the entire external surface of the body (Northcutt, 2004). Fibers from the Schreiner organs over the barbels, snout, oral epidermis, and velum terminate within the medullary trigeminal complex, while those on the body project to the dorsal spinal cord. Those organs over the pharynx terminate within the visceral column (vagal lobe and fasciculus communis) (Matsuda et al., 1991; Finger, 1997). There is no information pertaining to the stimuli that elicit responses in the sensory bud system of hagfishes. However, the large representation of the terminal field in the medulla of the CNS (occupying 10%) suggests an important role. In both ammocoetes and adult Lampetra planeri, solitary chemosensory receptors occur over the head and trunk that are characterized by apical microvilli and a basal synapse (Northcutt, 1989a). The oligovillous solitary chemosensory cells of lampreys physiologically respond to epithelial secretions (sialic acid), fish mucous, and water tainted by the odor of other fish. Sialic acid is stimulatory in concentrations as low as 10–9 and could be a meaningful stimulus to parasitic lampreys in addition to being important in social behavior (Baatrup and Doving, 1985). The identification of sialic acid in the epithelial cells of the pouched lamprey, G. australis (Lethbridge and Potter, 1979), could be also detected by the numerous solitary oligovillous cells on the male genital papilla (Whitear and Lane, 1983).

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6.4. Common Chemical Sense The common chemical sense is mediated by epithelial free nerve endings that are innervated by cranial and spinal nerves, many associated with the somatosensory system. Stimulation of these nerve endings (nociceptors and thermoreceptors) by various chemicals produces sensations including ‘‘pain,’’ warmth, and cold; however, others may elicit irritation and subsequently aversive behavior (Rovainen and Yan, 1985; Andres and von Du¨ring, 1993). 7. OCTAVOLATERALIS SYSTEM The octavolateralis system comprises end organs responsible for audition, vestibular control, and the lateral line, which all utilize hair cells as receptors and branches of the eighth (octaval) and lateral line nerves (Northcutt, 1981; McCormick, 1982; Platt et al., 1989). In contrast to earlier theories that suggested that the ear was derived from the lateral line, the term ‘‘octavolateralis system’’ should not inherently suggest acoustic function, nor imply evolutionary or ontogenetic origins (see review by Popper et al., 1992). It was Wever (1974) who first suggested that the auditory system and the lateral line may have evolved concurrently, but there is still much to learn about the role each system plays in the detection of propagated (sound) energy at low frequencies. The lateral line appears to respond to particle motion below 1 Hz, whereas the low limit of the inner ear in most fishes is about 35–50 Hz (Popper, 1983). Therefore, the two systems are complementary with the auditory system operating as a high‐frequency sound detector and the lateral line acting as a low frequency detector. The lateral line and auditory innervation also maintain their separate integrity to at least the level of the midbrain or the torus semicircularis in A. calva (McCormick, 1981; Northcutt, 1981). 7.1. Audition Three major evolutionary patterns exist with respect to the vertebrate ear. In hagfishes, a single torus with two rings of sensory epithelia and a single macula comprise the ear. The lamprey ear comprises two semicircular canals with trifid cristae and a partially divided macula, while the gnathostomatous ear has three distinct semicircular canals and at least two or more maculae with otoconia (Fritzsch, 1998). In general, the angular acceleration‐sensing semicircular canals and the linear accelerating‐sensing systems of the otoconia‐ bearing maculae have remained relatively constant (Lewis et al., 1985).

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Classically, the utricle has been considered as the primary vestibular organ for postural control and the sacculus as the primary auditory end organ (with the lagena used for both). However, the utricle has undergone large evolutionary change where it lies either in a recess of its own (elasmobranchs and lungfishes) or in the common crus of the anterior and horizontal canal (bony fish and tetrapods, Fritzsch, 1998), and all three maculae may be involved in both vestibular and auditory functions (Popper and Fay, 1993; Popper and Platt, 1993). The eighth cranial nerve is divided into (1) the anterior ramus, which innervates the anterior horizontal semicircular cristae, the utricular macula, and part of the saccular macula and (2) the posterior ramus, which innervates most of the saccular macula, the lagena macula, the macula neglecta, and the posterior semicircular crista (Meredith and Butler, 1983; Popper and Northcutt, 1983). A single macula communis is present in the hagfish, Myxine, and two ampullae appear as dilations on either end. Subdivisions of this torus are thought to be homologous to the utricular and lagenar maculae of gnathostomes (Lowenstein and Thornhill, 1970). The macula communis in lampreys can be divided into anterior, vertical, and posterior subdivisions, which are thought to be homologous to the utricular, saccular, and lagenar maculae in gnathostomes (Lowenstein et al., 1968; Popper and Hoxter, 1987). 7.1.1. The Inner Ear and Hair Cells The mechanism of transduction from a mechanical signal to neurotransmitter release is still not well understood but is mediated by bundles of hair cells, composed of a single long kinocilium and a number of stereocilia. On movement of the surrounding endolymph, and thereby the cupula, the hair cells are deflected possibly changing the ionic permeability and/or resistance of their membranes to produce a voltage diVerential. The inner ear in six species of hagfishes, all possess a labyrinth containing a single macula and two cristae in a single semicircular canal. The macula consists of a horizontal, a middle vertical, and a posterior horizontal component, each of which is covered by numerous round statoconia (otoconia) (Retzius, 1881; Jorgensen et al., 1998). The hair cell bundles contain long kinocilia (up to 35 mm in length) and lack a cupula. The ear of the coelacanth, L. chalumnae, possesses fewer otoliths than the three found in bony fishes, where the upper part of the inner ear adheres to the gnathostomatous pattern of a moderately sized utricle and three perpendicularly oriented semicircular canals (Millot and Anthony, 1965; Bernstein, 2003). The lower part comprises the sacculus with a large otolith and separated lagenar recess. Analyses of larval and adult specimens of Latimeria have revealed that both ears are linked by a canalis communicans, which is enclosed by cartilage (Bernstein, 2003; Fritzsch, 2003). Where the

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communicans enters the saccular/lagena region, an end organ lies innervated by the lagenar branch of the statoacoustic nerve VIII. The end organ is proported to represent a basilar papilla, a potential precursor to the cochlea of tetrapods (Fritzsch, 1987, 2003), although Bernstein (2003) considers this structure more suited to sensing pressure changes during movements involving the intracranial joint rather than for audition. Many species of elasmobranchs also have one or two accessory maculae, that is the macula neglecta, which contain patches of sensory epithelium covered by a gelatinous membrane. In sharks and rays, the macula neglecta possesses high numbers of hair cells (267,000 in the gray reef shark, Carcharhinus menisorrah, and 6,000 in the thornback ray Raja clavata), which are reported to detect forces directed posteroventrolaterally in the posterior canal duct, suggestive of an auditory function (Corwin, 1977, 1983). Physiological recordings confirm auditory sensitivities of between 40 and 200 Hz for the ray, R. clavata (Corwin, 1983), and between 31 and 375 Hz for the lemon shark, Negaprion brevirostris (Corwin, 1981), where both nonotolithic (macula neglecta) and otolithic (sacculus) organs may mediate sound source localization (Corwin, 1981). Popper (1977) has reported that sharks can behaviorally detect sound at frequencies of up to 1000 Hz and that certain low frequency signals attract sharks from large distances. The sensory cells in the sacculus are innervated by both aVerent and eVerent fibers, and although the precise number of neurons innervating the otolithic organs is unknown for most primitive fishes, the number of sensory cells varies (216,000 cells in the lagena macula; 56,400 cells in the utricular macula; and 8,600 cells in the saccular macula) in the bowfin, A. calva. These high receptor numbers indicate a high convergence of information in the order of 90 sensory cells to ganglion cells (Popper and Northcutt, 1983), which is appreciably higher than the hair cell to myelinated axon ratio of 57:1 found in the macula neglecta in the shark C. menisorrah (Corwin, 1977). In the sturgeon and the bichir, the saccular sensory hair cells are divided into two groups as opposed to four in non‐osteriophysans (Popper, 1983). The sacculus and lagena comprise one continuous chamber in the sturgeon, S. platorynchus, and are connected through a wide canal in the bowfin, A. calva (Figure 3.8A and B). Unlike the condition in teleosts, early ray‐ finned fishes have horizontally oriented cells rostrally, and vertically oriented cells caudally in the saccular macula. The horizontally oriented cells also appear to be derived from vertically oriented cells through ontogenetic shifts in macula curvature (Popper, 1978). Therefore, the most primitive ray‐finned fishes may have originally been equipped with vertically oriented hair cells, where the subsequent evolution of horizontally oriented hair cells enabled them to do a vectorial analysis of acoustic signals associated with sound localization (Schuijf and Buwalds, 1980; Platt and Popper, 1981). The

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A

B K

de ac

u

hc

cn

pc

S cn

um

Im

cn ab

pb sm s In

I

Fig. 3.8. (A) Illustration of the medial side of the left ear of the bowfin, A. calva, based on an early depiction by Retzius (1881). The macula neglecta is not shown. ac, crista of the anterior semicircular canal; ar, anterior ramus of the otic nerve; cn, branches of otic nerve to the various cristae; de, ductus endolymphaticus; h, crista of the horizontal semicircular canal; l, lagena; lm, lagenar macula; ln, lagenar branch of the eighth nerve; pb, posterior branch of the otic nerve; pc, crista of the posterior semicircular canal; s, sacculus; sm, saccular macula; u, utricle; and um, utricular macula. (B) Ciliary bundles from the saccular macula. Note the long kinocilia (K) and shorter stereocilia (S). [Reproduced from Popper and Northcutt (1983) with kind permission of John Wiley & Sons, Inc.]

saccular macula in lungfishes is diVerent to that of teleosts, and there is no discrete boundary between vertical and horizontal groups of hair cells, which comprise up to 80 cilia each (Platt et al., 2004). DiVerences in the size of the ciliary bundles located in the striolar and extrastriolar regions of the lagenar and saccular maculae may indicate that they respond to diVerent types of stimuli and have diVerent frequency response characteristics. 7.1.2. Sound Source Localization, Sensitivity, and Frequency Tuning The perpendicular orientation of the hair cells in the saccular and lagena in both the bichir, P. bichir, and the sturgeon, S. platorynchus, suggests that these primitive species, like teleosts, are able to detect signals at right angles from one another and therefore localize a sound source (Popper, 1978). Whether the direct displacement of the otolith is mediated by bone conduction (appreciable in these two species) or pressure displacement is unknown. The hearing abilities of the paddlefish, P. spathula, and the lake sturgeon, Acipenser fulvescens, have revealed that both fish are responsive to sounds ranging in frequency from 100 to 500 Hz and their lowest hearing thresholds are acquired from frequencies in a bandwidth of between 200 and

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300 Hz (Lovell et al., 2005). Both species are not sensitive to sound pressure but to particle motion and will be able to detect the knocks and moans produced during their breeding season, thereby serving as some form of intraspecific communication. Both P. spathula and A. fulvescens do not possess an internal division of their saccule and lagena, a feature shared with African lungfishes, Protopterus (Platt et al., 2004). The saccule bears hair cells divided into two oppositely oriented groups allowing these species to locate the source of a sound in both the horizontal and vertical planes, relying on the stimulation of ciliary bundles oriented specifically along the sound propagation axis (Lu and Popper, 1998). 7.2. Vestibular Control In vertebrates, a membranous labyrinth contains specialized organs that mediate the reception of gravity, linear, and angular accelerations. This mechanosensory complex is innervated by the eighth cranial nerve. Fishes lack a cochlea but otherwise the semicircular canal system has not changed much during vertebrate evolution (Platt, 1983a,b). The semicircular canal system monitors rapid head movements and provides spatial orientation. This is achieved by the displacement of endolymph within the canals, which stimulate hair cells located in a dome‐like enlargement (ampulla) and which are covered by a gelatinous cupula. Most fishes are considered to possess Type II vestibular hair cells but diVerent hair cell bundle forms have been diVerentiated on the diameter of the stereocilia, bundle size, and the length of the kinocilium. Three otolithic organs (utriculus, sacculus, and lagena) extend from the bases of the semicircular canals and are covered by either crystalline structures of aragonite (bony fishes) or aggregations of calcite crystals (cartilaginous fishes). The utriculus is thought to be the superior part of the vestibular system responding primarily to movement and postural changes. The inferior part of the ear (encompassing the sacculus and lagena) responds primarily to vibration and acoustic stimuli. The hair cells of non‐teleosts such as the bichir (P. bichir) and the sturgeon (S. platorynchus) have been characterized (Popper, 1978), and hair cell bundles with long kinocilia located near the margins of the otolithic organs have been described in chondrosteans (Popper, 1978), elasmobranchs (Lowenstein et al., 1964), and agnathans (Lowenstein et al., 1968). The vestibular maculae in the lampreys, L. fluviatilis and E. japonicus, possess only two ciliary bundle forms, where the forms with the shortest stereocilia are localized in areas with vibration sensitivity (Lowenstein et al., 1968; Lowenstein, 1970). The large size of the kinocilium dictates that the hair cell bundle has an orientation (Figure 3.8B), where displacement toward the kinocilium produces

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excitation and displacement away from the kinocilium produces inhibition. Although the parts of the labyrinth concerned with balance and postural control seem to show less variability than the parts related to acoustic reception, the demarcation between hair cells of diVerent polarities lies near the curving anterior edge of the utricular macula in both the shovelnose sturgeon, S. platorynchus, and the bichir, P. bichir, which is wide and disc shaped, respectively (Popper, 1978). The otoconia‐bearing organs can also detect linear accelerations. Surgical lesions and examination of the resultant behavioral deficits have established that the utricle has a gravistatic function and the saccule has a specialized acoustic function. The lagena is thought to participate in both (Schoen and von Holst, 1950). 7.2.1. Semicircular Canals and Balance Hagfishes possess a single canal (simple torus), which is oriented about 55 to the vertical plane (Lowenstein and Thornhill, 1970). In the hagfish, Myxine a large internal radius of the single semicircular canal is the result of the need to increase the sensitivity within a canal that must signal rotation in three planes, while the two cristae within the canal are known to respond to angular accelerations despite the absence of a cupula (McVean, 1991). Lampreys possess two separated semicircular canals. An additional horizontal canal evolved with the jawed vertebrates. Lampreys and hagfishes also appear to possess a more primitive pattern of oculomotor innervation (Fritzsch, 1991) than in jawed vertebrates. This reorganization may be related to the evolution of the inner ear. The innervation pattern of four extraocular muscles supplied by the oculomotor nucleus (two contralateral and two ipsilateral), one muscle by the trochlear, and one muscle by the abducens nucleus in elasmobranchs and lungfishes appears to have evolved into the tetrapod pattern of four extraocular muscles innervated by the oculomotor nucleus (one contralateral and three ipsilateral) and one muscle by the trochlear, and one muscle by the abducens nucleus after the acquisition of the horizontal canal (Fritzsch, 1998). The large Mu¨ller and Mauthner cells in the lampreys, I. unicuspis and P. marinus, also respond to vestibular stimulation as part of a general arousal response (Rovainen, 1980), where in addition to other motor functions, reticulospinal neurons participate in polysynaptic vestibular reflexes (Rovainen, 1979). 7.2.2. Vestibulo‐Ocular Control Visual input is important for controlling body orientation in the three dimensionalities of the water column. Hagfish canal aVerents are about half as sensitive as those of other vertebrates due to their lack of image formation and not having to stabilize their eyes (McVean, 1991). However, the neural

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mechanisms controlling spatial orientation (roll and pitch angles in the vertical plane and the yaw angle in the horizontal plane) with respect to the eyes have received a great deal of attention in lampreys. Vestibular information is crucial for the roll control and vestibular reflexes are tuned to stabilize a dorsal‐side‐up orientation (Ulle´n et al., 1995a,b). Lampreys rely on extratectal inputs to evoke the dorsal light response (a roll tilt toward the light) and negative phototaxis (lateral turn away from the light) (Ulle´n et al., 1995b) and use the eyes to compensate for any vestibular impairment (Degliagina, 1997). Lampreys have also been used to highlight the striking capacity of the CNS to compensate for unilateral loss of the vestibular system by restoring the symmetry of reticulospinal commands that control rotation around the horizontal axis (Degliagina et al., 1993; Pavlova et al., 2004). Behavioral responses to vestibular stimuli depend on modulation by the CNS, where the state of arousal, adaptive compensation, and eVerent innervation can play a large role (Platt, 1983a). Responses to the dorsal light reflex depend on the excitability of the fish, and restoration of normal responses following vestibular lesions may take over a month in teleosts. EVerent neural pathways can modulate postural behavior and ‘‘reaVerence’’ would allow receptors to retain sensitivity to other external stimuli. 7.3. Lateral Line Unlike the auditory portion of the ear and the closely related electrosensory system, the mechanosensory lateral line is thought to have arisen from a single common ancestor. Lateral line organs are mechanoreceptors, which are direction selective neuromasts containing hair cells (with a relatively immobile kinocilium and up to 100 stereocilia) over the surface of a sensory macula. Embedded within a gelatinous cupula, the hair cells are connected by fine extracellular threads, where the displacement of the stereocilia is proportional to the water flow (Dijkgraaf, 1963). Movement of the sterocilia relative to the kinocilium provides a directional indicator (deflection toward the kinocilium causes a depolarization and an increase in the aVerent firing rate; deflection away from the kinocilium causes a hyperpolarization and a decrease in aVerent nerve firing) (Blaxter, 1987). The ability to detect both particle motion and pressure may have evolved independently for both the ear and the lateral line. Significant changes in the relative excitation of diVerent lateral line canal neuromasts can result from mixing the pressure‐ induced and motion‐induced signals (Coombs et al., 1988). These changes could encode information about the distance of a source, about sudden changes in acceleration of the source, and about small changes in its relative position (Gray, 1984).

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7.3.1. Structure and Function of Neuromasts Neuromasts may be located superficially or in canals. Superficial neuromasts lie in shallow dermal pits or grooves and function by projecting their cupula into open water with slow‐moving fishes possessing more superficial neuromasts than fast moving ones (Dijkgraaf, 1962). On the other hand, canal neuromasts are stimulated by local displacements of the canal fluid perpendicular to the surface of the skin. Pores to the external environment allow water to pass into the semicompartmentalized canals, where the hair cells act as displacement sensors allowing the animal to detect something moving toward it. Occluded canals produce an acceleration of the water past the neuromast, thereby amplifying the signal (Coombs et al., 1988). Seven lateral line canals represent the basic pattern in fishes: supraorbital, infraorbital, mandibular, otic, temporal, supratemporal, and trunk canals (Song and Northcutt, 1991b). The hagfish lateral line is composed of rudimentary clusters of a single type of ciliated cell sitting in a series of two or three shallow epidermal grooves (Braun and Northcutt, 1997). Although there is a central kinocilium surrounded by stereocilia, there is no apparent orientation and no cupula. The development (Wicht and Northcutt, 1995), innervation (Braun and Northcutt, 1997), and central projections (Kishida et al., 1987) indicate that the lateral line of hagfishes is homologous to other craniates but its absence in the genus Myxine (Braun and Northcutt, 1997), the lack of eVerent innervation, and large interindividual variation suggest that the role and importance of this sense is still to be elucidated. It is interesting to note that instead of lateral line receptors, mechanoreceptive‐lamellated receptors are present in the hyperdermal cutaneous layer (Andres and von Du¨ring, 1993). These spindle‐shaped or cylindrical corpuscles are innervated by unmyelinated spinal nerve axons and are thought to be encapsulated stretch receptors, which appear to have evolved in this group independently in addition to species of anurans and reptiles (von Du¨ring and Seiler, 1974; von Du¨ring and Miller, 1978). Lampreys possess three classes of lateral line receptors, and the appearance and polarized orientation of their hair cells are similar to those in gnathostomes, except that they do not receive eVerent innervation and lack a cupula (Lane and Whitear, 1982; Fritzsch et al., 1989). The lateral line in hagfishes comprises only a few shallow epidermal grooves containing a single class of sensory hair cell (Braun, 1996), which could respond to spatial disturbances in the hydrodynamic field surrounding the animal. All the electroreceptors are innervated by the anterior lateral line nerve (Ronan and Northcutt, 1983). While cartilaginous fishes possess canal neuromasts, in addition to pit lines, lateral line receptors in lungfishes either lie in grooves

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or are superficial. It appears that neuromasts in lampreys, lepidosirenid lungfishes, and some teleosts have independently evolved from being housed in grooves and/or canals to occurring as lines of superficial neuromasts (Northcutt, 1989b). This tendency toward more superficially placed neuromasts may be correlated with a progression into more still water environments and/or sedentary behavior, where these types of neuromasts may function as proprioceptors of swimming velocity (Blaxter, 1987). The reduction of the lateral line canals in the bichir, Polypterus, and the lungfishes, Protopterus, Neoceratodus, and Lepidosiren, could also be the result of selection for changes in neuromast function, the nonadaptive reduction of dermal bone around the lateral line canals and their neuromasts (Webb and Northcutt, 1997), or a by‐product of the need to truncate their development in an environment prone to desiccation (Northcutt, 1989b). 7.3.2. Frequency Sensitivity and Object Localization The apical surfaces of canal neuromasts are 2–4 times larger and more oval shaped than those of superficial neuromasts, and canal neuromasts possess 4–15 times more hair cells than do superficial neuromasts in the Florida garfish, L. platyrhinchus (Song and Northcutt, 1991b) (Figure 3.9). Similar diVerences have been noted in the cladistian, Polypterus, by Webb and Northcutt (1988). These morphological diVerences may reflect physiological diVerences in the transduction properties and the sensitivity of the neuromasts (van Netten and Kroese, 1989). It appears that canal neuromasts respond better to high frequencies than do superficial neuromasts and that superficial neuromasts may respond preferentially to water velocity in contrast to acceleration (Kroese and Schellart, 1987). 8. ELECTRORECEPTION Electroreceptors are found in most primitive fishes, including lampreys, elasmobranchs, non‐teleost ray‐finned fishes (such as polypterids and chondrosteans), some teleosts (siluriforms, gymnotids, mormyrids, and gymnarchids), dipnoans, crossopterygians, and aquatic amphibians (urodeles and apodans) (Bodznick and Northcutt, 1981; Bullock et al., 1982; Jorgensen, 1982; Bullock and Heiligenberg, 1986; Northcutt, 1986a; Blaxter, 1987; Zakon, 1988; Jorgensen, 2005). Although both hagfishes and lampreys possess a lateral line, only the lampreys possess an electrosense (Ronan and Bodznick, 1986). All bony fish with electroreception possess cathodally stimulated receptors with the exception of some members of the neopterygians (four groups of teleosts mentioned above), which are stimulated by anodal signals

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al OT

SO

sp

IO MD

ml ST PT T

dl ll

TL

PO hl

mdl

vl gl

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Fig. 3.9. Drawing of the lateral view of the head of the Florida gar, L. platyrhinchus, illustrating the relative positions and extent of the cephalic superficial (pit) lines and canals of the lateral line. al, anterior pit line; dl, dorsal trunk pit line; gl, gular pit line; hl, horizontal pit line; IO, infraorbital canal; ll, lateral trunk pit line; MD, mandibular canal; mdl, first mandibular motor ramus of trigeminal nerve; ml, middle pit line; OT, otic canal; PO, preopercular canal; PT, posttemporal canal; SO, supraorbital canal; sp, spiracular diverticulum; ST, supratemporal commissure (or canal); T, temporal canal; TL, trunk canal; and vl, vertical pit line. [Reproduced from Song and Northcutt (1991b) with kind permission of S. Karger AG, Basel.]

(Northcutt, 1986a). The electroreceptors occur in two types (ampullary and tuberous organs) but only the ampullary receptors are present in primitive fishes. Evolved for the detection of weak electromagnetic fields produced by living organisms (and inanimate sources), electroreception was thought to be present in the ancestor of the earliest vertebrates and was reinvented after the ancestors of the teleost fishes lost the sense (New, 1997). Electrical fields provide a rich source of information about prey and mate locations (Tricas et al., 1995), local electrogenic landmarks (Peters and Bretschneider, 1972; Pals et al., 1982), and the animal’s orientation with respect to currents induced by the earth’s magnetic field (Kalmijn, 1982; Paulin, 1995). Some teleosts are able to generate electrogenic signals or electric organ discharges for interspecific communication (mediated by tuberous organs) but this will not be discussed further. 8.1. Structure, Function, and Evolution of Ampullary Receptors Ampullary receptor organs are superficial structures embedded within the epidermis and connected to the surface by a canal or ampullary pore filled with a mucopolysaccharide gel. At the base of the canal is an ovoid capsule containing aggregations of receptor cells, each of which supplies an aVerent axon that projects to the medulla. The length of the canal changes according to the position of the receptors over the head and, at least in elasmobranchs, to environmental factors such as osmoregulatory constraints and concomitant changes in skin resistance (Kalmijn, 1982; Raschi and Mackanos, 1989).

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Lamprey electroreceptors were once considered to be taste buds (Johnston, 1902) and were subsequently termed end buds. However, they are considered to be homologous to electroreceptors (Bodznick and Northcutt, 1981; Bullock et al., 1982, 1983; Fritzsch et al., 1984; Ronan and Bodznick, 1986). Scattered over the head and body trunk, each organ comprises between 3 and 25 sensory cells surrounded by support cells. Each ampullary organ usually comprises a single kinocilium surrounded by numerous stereocilia/microvilli (as found in cladistians and dipnoans, Jorgensen, 1984), although in lampreys, elasmobranchs, and chondrosteans, a kinocilium is lacking (Waltman, 1966; Ronan and Bodznick, 1986). There is a wide variation in the number and distribution of electroreceptor organs (see review by Collin and Whitehead, 2004 for elasmobranchs), ranging from 6 tubules in the rostral organ of the coelacanth (Millot and Anthony, 1956) to 75,000 in the paddlefish, Polyodon (Jorgensen et al., 1972; Pettigrew and Wilkens, 2003) (Figure 3.10). The ciliated receptors in the coelacanth lie within a chamber at the base of three tubules within the ethmoid chondrocranium on each side of the head (Bemis and Hetherington, 1982; Jorgensen, 1991). The octavolateralis nucleus in the brain of L. chalumnae is also hypertrophied, indicative of a well‐developed electroreceptive capacity (Northcutt, 1980). Ampullary electroreceptors are broadly tuned to low‐frequency electric fields from less than 0.1 to 25 Hz. This low‐frequency range of sensitivities corresponds well to the frequency ranges of standing or modulated fields produced by aquatic environments (New, 1997). Ampullary receptors are tonic receptors with a long‐lasting response to low‐frequency stimulation and possess threshold sensitivities of less than 20 nV cm 1 in elasmobranchs to 100 mV cm 1 in other taxa (New, 1997; Tricas and New, 1998). At least in elasmobranchs, the ampullae of Lorenzini are also thought to detect changes in ambient water temperature using the glycoprotein‐based (semiconductor‐ like) gel within the epidermal canal to elicit a thermoelectric signal (Brown, 2003). Ampullary organs appear to be the ancestral condition and are restricted to the head, except in lungfishes, which also possess trunk and tail ampullary organs (Gibbs, 2004). The loss of electroreception in the neopterygians is thought to be pleiotropically linked to the reduction of the cranial dermal armor (Moy‐Thomas and Miles, 1971; Lauder and Liem, 1983), a significant reorganization of the bones of the cranium and a reduction in size and thickness of the scales (Northcutt and Gans, 1983; Northcutt, 1986a). The evolutionary mechanism underlying the reinvention of electroreception (based on the ampullary system of receptors) in the teleosts, that is in the mormyrid, gymnotiform, gymnarchid, and siluriform lineages, is not well understood. Specialization of the superficial neuromasts of the lateral line, which underwent subsequent changes to give rise to tuberous organs, has been hypothesized (New, 1997), although electroreceptors do not exhibit a

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A

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c

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PA1 PA2

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Hz 80 60 40 20

D Skin Canal Hair cells in epithelium Excitatory synapses

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ALLn ganglion Brain

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Receptive Head field Rostrum Paddlefish

Fig. 3.10. (A) A paddlefish, P. spathula, attacking dipole wires (asterisk) that are mimicking the electrical discharges of live prey. (B) A schematic diagram of the ampullary electroreceptors found in the rostrum of the paddlefish. Note that primary aVerent nerve endings PA1 and PA2 innervate ciliated sensory epithelial cells, which mediate signal transduction. C, cilia; M, microvilli; NE, nerve endings; RC, receptor cell; and SC, supporting cell. (C) Diagram of an electroreceptor in the paddlefish rostrum. e, photograph of pipette electrodes in two canals of one large cluster, which is the receptive field of the aVerent in (D) and (E). SIZ, presumed spike initiating zone. (D) Raw recording of the spontaneous firing of an electroreceptor aVerent with its spontaneous firing frequency (top trace) and simultaneous pipette recordings from two canals. (E) Expanded segment showing canal oscillations at 26.7 Hz (middle trace) and 53 Hz (bottom trace, left). [(A) and (B) are from Pettigrew and Wilkins (2003) and are reproduced with kind permission of Springer Science and Business Media. (C), (D), and (E) are reproduced from Neiman and Russell (2004).]

neuromast‐like developmental stage and it may be that developing ampullary electroreceptors have arisen from epithelial cells overlying a new class of aVerent nerve fiber. 8.2. Role in Passive Electrolocation The ampullary electroreceptors are used to detect animate and inanimate electric fields, by measuring minute changes in potential between the water at the skin surface and the basal surface of the receptor cells. Epidermal pores

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and the jelly‐filled canals comprising each electroreceptor organ ensure that the potential within the ampullary lumen is the same as that at the surface. The hair cells of each receptor act as voltage detectors and release neurotransmitter onto the primary aVerent neurons according to the diVerence between the basal and apical potentials (Tricas, 2001). The primary aVerent neurons encode stimulus amplitude and frequency data that is sent to the brain (Montgomery, 1984; Tricas and New, 1998), where a sophisticated set of filter mechanisms are used for extracting the weak electrosensory signals from a much stronger background noise, predominantly created by the animal’s own movements (see review by Bodznick et al., 2003). Therefore, the distribution of the ampullary organs may provide information about the electric field’s intensity, its spatial configuration, and possibly the direction of its source (Tricas, 2001). The behavioral relevance of this level of sensitivity was uncovered in a series of experiments involving both experimental and free‐living elasmobranchs, which induced feeding responses toward either buried fish or a pair of buried electrodes, that could not otherwise be detected using other sensory modalities. Feeding was subsequently terminated when the bioelectric field of either source was masked by thin plastic film (Kalmijn, 1982). Therefore, the high sensitivity of electroreceptors enables elasmobranchs to localize prey by detecting the very faint potentials associated with the ionic leakage of the gills (modulated by ventilatory movements) of buried teleosts. Wounded crustaceans produce higher bioelectric fields (1000 mV cm 1). Bodznick and Northcutt (1981) revealed that lampreys (Lampetra tridentata) possess sensitivity thresholds (0.1 mV cm 1) comparable to electrosensory freshwater teleosts. Watt et al. (1999) have also shown that the ampullary organs in the Australian lungfish, N. forsteri, use passive electroreception to perceive weak electric fields emanating from hidden prey in much the same way as elasmobranchs. The rostrum of the paddlefish, P. spathula, possesses a rostrum (not unlike that of the platypus, Pettigrew and Wilkens, 2003) that is adorned with ampullary electroreceptors, which act as an antenna (Figure 3.10B). The rostrum is considered a sensory device with suYcient sensitivity to detect the electric fields of planktonic prey with a sensitivity threshold of 10 mV cm 1, a considerably higher sensitivity than the sensitivity of individual electroreceptors (Wilkens et al., 1997; Russell et al., 1999). Paddlefishes use this rostrum to laterally strike at planktonic prey using its electric sense passively without the use of visual, chemical, and hydrodynamic senses at distances of 8–9 cm (Wilkens et al., 2001) (Figure 3.10A). Higher concentrations of receptors along the edges of the rostrum and its saccade‐like motion through the water may serve to enhance prey detection by increasing the width of the electrical scan field (Pettigrew and Wilkens, 2003). It appears that a single receptor traversing an electrical field receives an electrical signal over time that

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contains suYcient information necessary for the dorsal octavolateral nucleus to extract the location, size, and orientation of a source, rather than relying on topographic input (Hofmann et al., 2005). Recent findings of two noise oscillators in the electroreceptors in paddlefish, one in the sensory epithelia and the other in the aVerent terminals, reveal that there are mechanisms for (1) driving spontaneous firing, conferring the advantage that both inhibitory and excitatory stimuli can be detected (aVerent oscillator); (2) dealing with high levels of synaptic convergence (45,000 hair cells onto 1 aVerent axon) (aVerent oscillator); (3) increasing receptor sensitivity near threshold by mediating stochastic resonance (epithelial oscillator); and (4) encoding water temperature by frequency modulation of aVerent firing (epithelial oscillator) (Neiman and Russell, 2004, 2005) (Figure 3.10C–E). Behavioral response to weak cathodal stimulation has been confirmed in the sea lamprey, P. marinus, with a threshold sensitivity of 30 mV cm 1 (Chung‐Davidson et al., 2004). Using neuronal activity markers such as Fos, FosB, and Jun, it has also been established that, in addition to the involvement of the octavolateralis region of the medulla and the torus semicircularis (Gonza´lez et al., 1999), the habenula‐fasciculus retroflexus‐interpeduncular nucleus system also receives input in this species. 9. CONCLUDING REMARKS In this chapter, an attempt has been made to reveal the evolution and complexity of the nervous and sensory systems in primitive fishes. Drawing on detailed information available on the neuroanatomy of specific representatives, structure–function relationships have been developed wherever possible in order to gain insights into the physiology. Unfortunately, this has not always been possible for each of the major groups of fishes. Inevitably, and due in part to the accessibility of both the animals and the neural system(s) under consideration, detailed analyses of some species have not been undertaken. However, in some neural systems, a wealth of information at the neuroanatomical, neurochemical, physiological, and molecular levels has greatly enhanced our understanding of brain evolution and the mechanisms regulating the genesis of new neural tissues as opposed to the modification of existing neural organization. However, physiological studies are remarkably still scarce and need to be undertaken in order to trace the origins of craniate brains and the evolutionary constraints placed on neural plasticity. In combination with molecular and embryological investigation, a multidisciplinary approach will allow a more comprehensive understanding of brain complexity, function, and therefore behavior. A clear understanding of their behavior in the natural environment can also help protect these unique animals that hold so many keys to brain evolution.

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Webb, J. F., and Northcutt, R. G. (1997). Morphology and distribution of pit organs and canal neuromasts in non‐teleost bony fishes. Brain Behav. Evol. 50, 139–151. Weth, F., Nadler, W., and Korsching, S. (1996). Nested expression domains for odorant receptors in zebrafish olfactory epithelium. Proc. Natl. Acad. Sci. USA 93, 13321–13326. Wever, E. G. (1974). The evolution of vertebrate hearing. In ‘‘Handbook of Sensory Physiology, Vol. 1, Auditory System’’ (Keidel, W. D., and NeV, W. D., Eds.), pp. 423–454. Springer, Berlin. Whitear, M. (1992). Solitary chemosensory cells. In ‘‘Fish Chemoreception’’ (Hara, T. J., Ed.), pp. 103–125. Chapman and Hall, London. Whitear, M., and Lane, E. B. (1983). Multivillous cells: Epidermal sensory cells of unknown function in lamprey, Entosphenous japonicus. J. Ultrastruct. Res. 43, 1–17. Whitmore, A. V., and Bowmaker, J. K. (1989). Seasonal variation in cone sensitivity and shortwave absorbing visual pigments in the rudd, Scardinius erythophthalmus. J. Comp. Physiol. A 166, 103–115. Wicht, H. (1996). The brains of lampreys and hagfishes: Characteristics, characters, and comparisons. Brain Behav. Evol. 48, 248–261. Wicht, H., and Niewenhuys, R. (1998). Hagfishes, Myxinoidea. In ‘‘The Central Nervous System of Vertebrates’’ (Niewenhuys, H., Ten Donkelaar, J., and Nicholson, C., Eds.), Vol. 1, pp. 497–550. Springer‐Verlag, Berlin. Wicht, H., and Northcutt, R. G. (1990). Retinofugal and retinopetal projections in the Pacific hagfish, Eptatretus stouti. Brain Behav. Evol. 36, 315–328. Wicht, H., and Northcutt, R. G. (1992). The forebrain of the pacific hagfish: A cladistic reconstruction of the ancestral craniate forebrain. Brain Behav. Evol. 40, 25–64. Wicht, H., and Northcutt, R. G. (1994). An immunohistochemical study of the telecephalon and the diencephalon in a myxinoid jawless fish, the Pacific hagfish Eptatretus stouti. Brain Behav. Evol. 43, 140–161. Wicht, H., and Northcutt, R. G. (1995). Ontogeny of the head of the Pacific hagfish (Eptatretus stouti, Myxinoidea): Development of the lateral line system. Philos. Trans. R. Soc. Lond. B. 349, 119–134. Wilkens, L. A., Russell, D. F., Pei, X., and Gurgens, C. (1997). The paddlefish rostrum functions as an electrosensory antenna in plankton feeding. Proc. R. Soc. Lond. B 264, 1723–1729. Wilkens, L. A., Wettring, B., Wagner, E., Wojtenek, W., and Russell, D. (2001). Prey detection in selective plankton feeding by the paddlefish: Is the electric sense sufficient? J. Exp. Biol. 204, 1381–1389. Yokoyama, S. (2000). Color vision of the coelacanth (Latimeria chalumnae) and adaptive evolution of rhodopsin (RH1) and rhodopsin‐like (RH2) pigments. J. Hered. 91, 215–220. Young, J. Z. (1935). The photoreceptors of lampreys. I. Light sensitive fibres in the lateral line nerves. J. Exp. Biol. 12, 229–238. Zakon, H. H. (1988). The electroreceptors: Diversity in structure and function. In ‘‘Sensory Biology of Aquatic Animals’’ (Atema, J., Fay, R. R., Popper, A. N., and Tavolga, W. N., Eds.), pp. 813–850. Zalc, B., and Colman, D. R. (2000). Origins of vertebrate success. Science 288, 271–272. Zeiske, E., Theissen, B., and Breucker, H. (1992). Structure, development, and evolutionary aspects of the peripheral olfactory system. In ‘‘Fish Cemoreception’’ (Hara, T. J., Ed.), pp. 13–39. Chapman and Hall, London.

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4 VENTILATORY SYSTEMS EMILY COOLIDGE MICHAEL S. HEDRICK WILLIAM K. MILSOM

1. Introduction 2. Respiratory Strategies 3. Respiratory Organs 3.1. Water Breathing 3.2. Air Breathing 4. Ventilatory Mechanisms 4.1. Cutaneous Gas Exchange 4.2. Ventilation of External Gills 4.3. Ventilation of Internal Gills 4.4. Ventilation of ABOs 5. Respiratory Control 5.1. Hypoxic and Hypercarbic Ventilatory Responses and Reflex Pathways 5.2. Receptors Involved in Reflex Ventilatory Control 6. Conclusions

Primitive fishes are widespread in geographical distribution, and the diverse nature of their aquatic environments has given rise to a tremendous adaptive radiation in respiratory physiologies. The group of primitive fishes comprises water‐breathing, bimodal breathing, and obligate air‐breathing species, which possess a variety of respiratory strategies, respiratory organs, pumping mechanisms, and control systems that integrate multiple exchange sites and receptors into their overall ventilatory response. Many studies have described the reflex responses of this group to environmental perturbations such as hypoxia and hypercarbia, but one area that clearly needs more study is our understanding of the mechanisms by which peripheral receptors and neural pathways are centrally integrated to produce the complex ventilatory behaviors seen in these fishes. Although these species are highly evolved and adapted to their particular ecological niches, and represent significant 181 Primitive Fishes: Volume 26 FISH PHYSIOLOGY

Copyright # 2007 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(07)26004-2

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departures in morphology, ecology, and behavior from the stem groups that gave rise to them, further examination of the mechanisms underlying their respiratory behavior may lead to greater insight into the evolutionary forces that have shaped the transition from living in water to living on land among vertebrates.

1. INTRODUCTION The first chapter in this volume has clearly defined those groups of living fishes that are considered ‘‘primitive fishes.’’ In this chapter, we will examine what is known of the respiratory systems of these species. While there is an extensive literature describing respiratory processes in fish, much of this literature comes from studies of elasmobranchs and teleosts. These are considered ‘‘modern fishes’’ and previous volumes in this series have focused exclusively on these groups (Volumes 5, 10, 12, and 17; Randall et al., 1981a; Graham, 1997; Maina, 2003). The challenge of this chapter is to summarize data specific to the primitive fishes and build on previous reviews on this topic (Randall et al., 1981a, Burggren et al., 1985; Shelton et al., 1986). Several things are of note in this regard. Ventilatory systems regulate the diVusion of respiratory gases in (O2) or out (CO2 and NH4) of the body, and hence are strictly linked to the regulation of all metabolic processes. They are also highly modified as a function of the environments within which these fish live. The various groups of primitive fishes have a widespread geographical range of distribution, and the diverse nature of their aquatic environments has given rise to a tremendous adaptive radiation in respiratory strategies. Thus, the group of primitive fish defined in this volume consists of water‐breathing, bimodal breathing, and obligate air‐breathing fish, possessing a variety of respiratory strategies, respiratory organs, pumping mechanisms, and control systems. Furthermore, all extant species of fish (primitive fish included) are highly derived and represent significant departures in morphology, ecology, and behavior from the stem groups that gave rise to them. Given this background, it is extremely difficult to distinguish ‘‘primitive’’ from ‘‘derived’’ physiological characteristics, particularly given how plastic physiological processes can be at the organismal level. In summarizing studies of these processes, trends will be shown that are suggestive of evolutionary progression. All attempts to describe evolutionary trends based on the physiology of present‐day fishes, however, must be viewed within this context.

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2. RESPIRATORY STRATEGIES Because environmental hypoxia is such a pervasive problem for aquatic organisms, the ventilatory adaptations to hypoxia have been investigated in fishes for many years. As with all fish, primitive fish have developed strategies (behavioral, morphological, anatomical, physiological, and biochemical) either to avoid low O2 conditions, increase O2 transfer from the environment to the tissues, reduce O2 demands, or some combination of these. Many species constantly sense and monitor environmental conditions and migrate to better areas (Junk et al., 1983; Wootton, 1990). Other species increase O2 extraction and/or reduce O2 demands through a host of physiological and biochemical adjustments including regulation of diVerent Hb fractions, adjustment of intraerythrocytic levels of organophosphates, changes in hematocrit and Hb concentration, and metabolic suppression— almost all under catecholaminergic control. These are described in detail in Chapter 5 of this volume (Milligan and Wood, 1987; Perry and Kinkead, 1989; Nikinmaa, 1990; Randall, 1990; Val et al., 1992; Almeida‐Val and Val, 1993). They also include increased O2 uptake via the respiratory system. In general terms, nearly all aquatic organisms exhibit increases in ventilation either as increases in ventilatory frequency, tidal volume, or both, in order to improve O2 extraction at the gills. A surprisingly large number of the ‘‘primitive fishes’’ are also air‐breathing fishes. Indeed, of the bony fishes, only two Chondrosteans, the sturgeon and the paddlefish, have retained total dependency on aquatic gas exchange. For some species, air breathing is a facultative event that occurs only when water O2 levels are low. These species tend to have functional gills that are used in conjunction with air breathing (e.g., gar, bowfin). For other species, air breathing is an obligatory behavior and these species rely primarily, if not exclusively, on O2 taken from the air. These species tend to have greatly reduced gill structure (e.g., lungfish). There are many species that utilize both strategies either as a function of developmental age or environmental conditions. Thus, the gar, Lepisosteus, is a facultative air‐breather at low temperatures but becomes an obligate air‐breather when O2 uptake increases at higher temperatures (Rahn et al., 1971). Obligate air‐breathing fish, such as Protopterus, do not respond to aquatic hypoxia but, instead, respond to aerial hypoxia. In facultative air‐breathers such as Lepisosteus or Amia, there is a reduction in gill ventilation and a behavioral switch to air breathing as the major source of O2 acquisition under hypoxic conditions. These are generalizations, however, and more specific examples of the ventilatory response to hypoxia are detailed below.

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Environmental hypercarbia, although a prevalent and important ventilatory stimulus in freshwater, has not been studied to the same extent as environmental hypoxia. Because increased ventilation cannot ameliorate the eVects of environmental hypercarbia, alternative respiratory strategies must be employed when fish encounter such a stimulus. Moreover, owing to the Root eVect in fish Hb, environmental hypercarbia may produce secondary eVects on ventilation through reductions in O2 content. 3. RESPIRATORY ORGANS 3.1. Water Breathing 3.1.1. Gills From the Agnatha to the elasmobranchs to the bony fish, there is a shift from pouched gills to septal gills to opercular gills (Figure 4.1). The fundamental structure of the gill remains the same but the eYciency with which the actual gas exchange surface, the secondary lamellae, is exposed to the water flowing through the gills increases progressively (see below). While most of these gills are internal and situated within the respiratory cavity, external gills

A

B

Juvenile

C

Adult

Lamprey

Shark

Teleost fish

Fig. 4.1. Schematic diagrams of (above) the arrangement and coverings of the pharyngeal slits and (below) the individual pharyngeal arches of a (A) lamprey (ammocoetes larva on left and adult on right), (B) shark, and (C) teleost fish. Black arrows indicate the direction of water flow. [Modified from Kardong (2002).]

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are common in the larval stages of primitive jawed fishes and are also found in the adults of some Polypterids and Dipnoans (Burggren et al., 1985). The diVusing capacity of any species can be increased by alterations in the number of gill arches, the length and number of gill filaments on each arch, the spacing of the lamellae along the filament, the surface areas of individual gill lamellae, the thickness of the water–blood interface, and the resistance to water flow through the gill sieve (Hughes, 1984). Changes in any or all of these variables occur as a function of lifestyle and habitat throughout all taxonomic groups of fishes. The diVusing capacity of any individual fish can also be changed in a number of ways. These include increasing the number of lamellae perfused at any one time (and hence the functional area available for gas transfer) (Booth, 1978), redirecting blood through sections of lamellae exposed to gill water flow, and reducing lymphatic space (Randall et al., 1981a). All result in a reduction in diVusion distance between blood and water and an increase in the surface area across which gas exchange occurs. These are the general trends. Specific details are available for many species. Thus, as mentioned above, the gills of agnathans are located in saclike pouches. Lampreys have seven pairs of gill pouches while hagfish have from 5 to 14 pairs, depending on the species. The anterior and posterior walls of each pouch bear a hemibranch on which the gill filaments are diVerentiated into secondary lamellae. The anatomy of the secondary lamellae, their epithelial thickness, and their total surface area in lampreys are similar to those in teleost fish (Hughes and Morgan, 1973; Lewis, 1976; Lewis and Potter, 1976; Hughes, 1984). In elasmobranchs each gill possesses a central partition, the interbranchial septum, which is covered on both sides by primary lamellae (or gill filaments). In the bony fishes, the interbranchial septum is reduced or absent and the gill filaments often arise directly from the base of the branchial arch. This frees the individual filaments in such a way that water flow over the filaments is enhanced (Figure 4.1). In the Chondrosteans (sturgeon and paddlefish), the gill septum is reduced and only about 50% of the gill filaments attach to the septum. The general organization of the secondary lamellae is similar to that of other bony fishes although the total surface area is greatly reduced (Byczkowska‐Smyk, 1962; Hughes, 1984). In Neopterygii (bowfin and gar), the interbranchial septum is well developed proximally but the distal one‐third of the gill filaments are unsupported (Randall et al., 1981b; Smatresk and Cameron, 1982). While the gills are well developed in general, in Amia, the first gill arch bears no hemibranch, whereas in Lepisosteus, it possesses only a posterior hemibranch. Despite this, the total surface area of the gills of Amia is similar to that of a teleost with similar activity levels (Daxboeck et al., 1981). Furthermore, in Amia, the secondary lamellae are fused into a lattice of rectangular pores, which

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is believed to prevent their collapse in air (Daxboeck et al., 1981). The reduced first gill arch and strengthened secondary lamellae are believed to be adaptations associated with air breathing. In the Dipnoi, there are considerable diVerences in gill structure between the three extant species. The Australian lungfish, Neoceratodus forsteri, is an obligate water‐breather with well‐developed gills. The African lungfish, Protopterus sp., and the South American lungfish, Lepidosiren paradoxa, on the other hand are all obligate air‐breathers with diVerent degrees of reduction in their gill development and surface area (Johansen and Lenfant, 1967; Brauner et al., 2004) (Figure 4.2). For species possessing well‐developed air‐breathing organs (ABOs), there are conflicting functional requirements placed on the design of their gills. This arises because the more O2‐rich blood draining the ABO returns to the heart and must then pass through the gills before entering the systemic circulation. In the process, the potential exists for significant loss of O2 to the hypoxic water during transit through the gills (Randall et al., 1981a). As a result, many of these fish exhibit reductions in functional gill surface area. This may be in the form of reductions in the number of gill arches, the number of secondary lamellae, secondary lamellar thickening, or presence of gill vascular shunts. The extent to which any or all of these occur is generally a function of the dependence of the species on air breathing. 3.1.2. Skin Although the gills are the primary gas exchange organs in water, the skin also plays a significant role in many species of fish. Agnathans lack dermal bone and the skin is smooth and without scales. At least 80% of O2 uptake in the hagfish, Myxine glutinosa, can occur across the skin (SteVensen et al., 1984) while the respiratory role of the skin of adult lampreys has A

B

Fig. 4.2. Scanning electron micrographs (SEM) of the gills from (A) a typical bony fish and (B) the obligate air‐breathing teleost Arapaima gigas. Scale bar, 500 mm. [Figure courtesy of Colin Brauner.]

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been estimated to be only 20% of total O2 uptake (Korolewa, 1964). The remaining primitive fishes, including both the actinopterygians and sarcopterygians, are bony fishes. While bone is pervasive throughout the endoskeleton in this class, the trend is reversed in groups such as the sturgeons, paddlefishes, and some lungfishes in which the endoskeleton is primarily cartilaginous. Despite this, all have scales but cutaneous gas exchange is still very significant in many, particularly in their larval stages. In most air‐ breathing fish, while other specialized exchange surfaces become the major site of O2 uptake from air, the gills and/or skin remain one of the major sites of CO2 excretion (into water). In Amia, the gas exchange ratio (CO2 elimination/O2 uptake) is 0.21 for the ABO and 1.61 for the gills (Randall et al., 1981b). CO2 excretion is exclusively aquatic in Neoceratodus, whereas in Protopterus and Lepidosiren air breathing can account for up to 30% of CO2 elimination (Johansen et al., 1970). 3.2. Air Breathing 3.2.1. Lungs and Respiratory Gas Bladders An accessory or alternative strategy employed by a large number of phyletically primitive fishes is air breathing. The diversity of sites and surfaces that are utilized for gas transfer from air to blood in fish is remarkable. While a few species do utilize their gills for gas exchange in air, this is a rare occurrence and most air‐breathing fish utilize other surfaces. Graham (1997) has put forward a simplified classification scheme for structures utilized by fish for aerial gas exchange (ABO). He suggests that ‘‘even though air breathing has evolved numerous times and independently, the location of aerial exchange sites has remained largely under the conservative influence of structures ‘‘predisposed’’ for air gulping and sites in the body where gas storage and the requisite vascularization could be developed.’’ This scheme divides structures into three groups: (1) those associated with the skin; (2) structures associated with organs in the head region (buccal and opercular cavities, pharynx) or along the digestive tract; and (3) the lungs and respiratory gas bladders (Carter and Beadle, 1931; Johansen, 1970; Randall et al., 1981a). In the primitive fishes, however, structures associated with air breathing fall into only the last category (lungs or respiratory gas bladders). At least 47 species from 24 genera of bony fish are known to breathe air using a lung or a respiratory gas bladder. By the scheme put forward by Graham (1997), gas bladders have an embryonic origin from the side or dorsal aspect of the alimentary canal, are not paired, do not always have a glottis (and may or may not retain an open pneumatic duct), and in most cases receive blood in parallel with the systemic circulation and lack a specialized pulmonary circulation. Lungs, on the other hand, have an embryonic origin from the ventral wall of the alimentary canal, are paired, possess a valvular glottis in

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the floor of the alimentary canal, and have a proper pulmonary circulation in which eVerent vessels return blood directly to the heart (not the vena cava) (Figure 4.3). By this scheme, lungs are possessed only by the lungfishes (Neoceratodus, Lepidosiren, and Protopterus) and the polypterids (Polypterus and Erpetoichthys). Gas bladders are found in both Amia and the garfishes and are scattered throughout the teleosts. Among these fishes, respiratory gas bladders diVer greatly in complexity (Jarvik, 1980; Graham, 1997). Note that this scheme implies that both lungs and gas bladders must have evolved independently multiple times. The relative roles of the various gas exchange surfaces vary tremendously between species. In the reedfish, Calamoichthys calabaricus, when in oxygenated water, total O2 uptake is 28% from the gills, 32% across the skin, and 40% across the lungs (Sacca and Burggren, 1982). In Amia and Lepisosteus, while the gills are the major site of gas exchange at low temperatures (10
C), at warmer temperatures (20–25
C), the ABO accounts for 40–75% of O2 uptake (Johansen et al., 1970; Rahn et al., 1971; Randall et al., 1981a; Smatresk and Cameron, 1982b). The relative contributions of aquatic and aerial O2 uptake in the three genera of lungfish range from nearly 100% from

A

B Pulmonary artery

Swim bladder artery Dorsal aorta

Dorsal aorta

Lung

Swim bladder Gill Postcava Swim bladder vein

Gill Postcava Pulmonary vein

Heart

C

Heart

D

Fig. 4.3. Schematic diagrams illustrating the generalized circulation to (A) the ABO of a typical bony fish and (B) a lungfish (see text for details). Panels (C) and (D) illustrate the relation of the ABO and lung to the oesophagus as seen from the side and in cross section. [Modified from Kardong (2002).]

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water in Neoceratodus to 80–90% from air in Protopterus and Lepidosiren (Johansen et al., 1970). 3.2.2. Skin Many fish that spend time out of water (amphibious fish) do use their skin for aerial gas transfer and, although subject to uncontrolled water loss and limited as an organ for O2 uptake, the surface is adequate for CO2 excretion (Graham, 1997). 4. VENTILATORY MECHANISMS No matter what the exchange organ, water or air must move actively across the respiratory surfaces to increase the rate of diVusion. Invariably, this requires muscular action. 4.1. Cutaneous Gas Exchange While cutaneous gas exchange in aquatic organisms is often viewed as purely a passive phenomenon, active movement of water over the skin surface is common (Feder and Burggren, 1985). This may involve general body movements or more specialized movements. Larvae of the lungfish, Neoceratodus, possess cilia which develop respiratory currents across the general body surface and inside the opercula (Whiting and Bone, 1980). Larvae of Monopterus use movements of the pectoral fins to produce water currents that run over the body surface counter to the direction of skin blood flow (Liem, 1982). 4.2. Ventilation of External Gills While external gills are rare in general, they are common in the larval stages of primitive jawed fishes (Burggren et al., 1985). The external gills of larval Protopterus are ciliated and produce convective currents prior to the development of a muscular apparatus for active movement of the gills. Intrinsic musculature does develop and the external gills of lungfishes are capable of sweeping movements that irrigate the gills and break up stagnant boundary layers surrounding the gills (Greenwood, 1958). 4.3. Ventilation of Internal Gills 4.3.1. Phylogenetic Perspectives One of the characteristics that define all chordates is the possession of pharyngeal slits, at least at some point during their development (Cameron et al., 2000). It is believed that these pharyngeal slits likely first evolved

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from the corners of the mouth to aid in suspension feeding in primitive chordates and protochordates (Gutmann, 1981). This allowed a one‐way flow of water—in at the mouth and out through the pharyngeal slits. Initially, this form of suspension feeding depended solely on ciliary pumps to create the flow of water (Gilmour, 1979). As the primitive chordates enlarged, the flanks of the body weakened favoring the evolution of supporting structures between successive slits. These ultimately gave rise to cartilaginous pharyngeal arches. Initially, the walls of the slits were associated with mucus‐bearing cilia that served to trap suspended particles; respiration was primarily cutaneous. Only secondarily did the walls defining the slits become associated with gills and begin to participate in respiratory gas exchange. At this point, water entering the mouth could bring suspended food and O2 to the animal. This increase in feeding eYciency gave rise to more active lifestyles. With this came the evolution of a muscular buccal pump that helped to produce the food bearing current. This allowed animals to attain larger mass and led to the loss of ciliary mechanisms for moving water (Sanderson and Wassersug, 1990) (Figure 4.1A). Transitional species probably became raptorial feeders, plucking individual particles selectively from suspensions or oV surfaces. The supporting structures of the first pharyngeal slit moved forward and evolved into jaws, further increasing feeding eYciency and giving rise to the origins of active predation and a shift away from sessile suspension feeding (Mallatt, 1996). With this, the pharyngeal slits were no longer required for feeding but the active lifestyle demanded greater gas exchange than was provided by cutaneous exchange alone. With removal of the constraints placed on the pharyngeal slits for feeding, true gills evolved, and the pharyngeal slits and buccal pump that originally evolved for feeding gave rise to gills for breathing with water flow being driven by a buccal pump involving muscles primarily innervated by the trigeminal and facial nerves. Muscles in the walls of the pharyngeal arches, innervated by the glossopharyngeal and vagus nerves acted as accessory muscles to stabilize and maintain the gill curtain (Mallatt, 1996; Kardong, 2002). This is the situation found today in the cartilaginous and bony fishes, and to some extent in the agnathans (hagfish and lampreys). 4.3.2. Agnatha Ventilation in Agnatha is powered by muscular velar folds and by compression and expansion of the branchial apparatus. In the larvae of the lamprey as well as in adult hagfish, the primary respiratory pump is the velum. This is a muscular structure attached to the roof of the pharynx in the midline. It consists of two leaves that are tightly furled at rest. Scrolling and unscrolling of the velum on each side of the buccal cavity, together with

4.

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synchronized contraction and relaxation of the branchial pouches, produce a flow of water in at the nostril (singular for hagfish) and out across the gills via the branchial pouches. This is an amazingly eVective mechanism that can generate water flows that are not dissimilar from those produced by many teleost fishes (Figure 4.4) (Shelton, 1970; Rovainen and Schieber, 1975).

A

Vel.

Bucc.

Ph.

Vel.ch. B

C

1

2

3

4

Fig. 4.4. (A) Schematic sagittal section through the anterior trunk of a hagfish (from Johansen and Strahan, 1963). (B) Schematic diagram of the velar chamber with the left pharyngeal wall removed. 1, resting; 2, velar scroll beginning to unroll; 3, velar scroll unrolled to the full extent; and 4, velar scroll beginning to roll again. Arrow shows direction of water flow. [From Johansen and Strahan (1963).] (C) Schematic of a lateral view of the velum scrolling and unscrolling to move water through the pharynx. [From Kardong (2002).] Bucc., buccal cavity; Ph., pharynx; Vel., velum and Vel. ch., velar chamber.

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In adult lampreys, the velum is reduced and ventilation is primarily generated by compression and expansion of the branchial apparatus alone. Closure of the velum and active compression of the branchial apparatus drive water out through the branchial pouches. Relaxation of these muscles allows the elastic branchial apparatus to recoil into its expanded position passively drawing water back in. Although unidirectional water flow (in at the mouth and out over the gills) is possible, in most adult lampreys the pattern of water flow is tidal with water flowing out and back in through the openings of the branchial pouches. A partition dividing the pharynx thus allows the lamprey to attach to its prey by suction and use its tongue to scrape flesh into the esophagus while continuing to ventilate tidally through the branchial pouches (Johansen and Strahan, 1963; Jensen, 1966; Randall, 1972). 4.3.3. Bony Fishes In most fishes, the buccal and opercular cavities form dual pumps on either side of the gill curtain. Both cavities are expanded simultaneously by muscular action creating a suction that closes the operculae and draws water in through the mouth. Both cavities are then compressed by muscular action while the mouth closes, forcing water over the gill curtain and out through the operculae. Because of a slight diVerence in pressure between buccal and opercular cavities, water flows almost continuously across the gills in one direction (Figure 4.5A) (Hughes, 1984). This basic mechanism powers gill ventilation in all primitive and modern jawed fishes (McMahon, 1969; Shelton, 1970; Hughes, 1984). 4.3.4. Acipenseriformes (Sturgeon) and Polypteriformes (Birchirs) Many species of these two groups are bottom feeders eating buried invertebrates and carrion. Their mouths are modified for sucking mud. In most, the first gill arch is reduced to a spiracle. When these fish are in open water they ventilate the gills just as other fish do. In sturgeon (Acipenser transmontanus), when the mouth is buried, the fish ventilate only via the opercular opening (the spiracle plays little role) but in a unique way that retains a unidirectional flow of water over the gills. During buccal and opercular expansion, water enters the opercular cavity through a permanent aperture in the dorsal margin of the operculum. This water then continues into the buccal cavity flowing dorsally over the gill sieve, not passing over the gill lamellae. During buccal compression, the water then passes over the gill sieve in a normal fashion and exits via the operculum (Figure 4.5B) (Burggren, 1978). It has been postulated that a similar pattern is produced using the spiracle in Polypterus (Magid, 1966).

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Dual pump A

Buccal cavity

Gill curtain

Oral valve

Opercular cavity Opercular valve

Force phase Suction phase

B

Buccal cavity

Spiracle

Dorsal opercular channel

Buccal cavity Spiracle

Dorsal opercular channel

Fig. 4.5. Schematic diagrams illustrating the dual pump found in most bony fishes (A), and the modified pumping mechanism found in sturgeon (B). See text for details on all pumping mechanisms. [From Burggren (1978) and Kardong (2002).]

4.4. Ventilation of ABOs 4.4.1. Sarcopterygian Fishes In air‐breathing fish, the buccal pump usually exclusively produces ventilation. In sarcopterygian fishes, an initial buccal expansion phase draws both air from the ABO and fresh air from the environment into the buccal cavity

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A

Exhalation

Water pressure

Compression (2) Expansion (1) Buccal cavity Sphincter Lung

Inhalation

Expansion (3) Compression (4)

B Expansion

Compression

Fig. 4.6. Schematic diagrams illustrating the two‐stroke buccal pump found in sarcopterygian fishes (A), the four‐stroke pump found in most actinopterygian fishes (B). See text for details on all pumping mechanisms. [From Randall et al. (1981a) and Kardong (2002).]

simultaneously. Lung emptying is due to a combination of elastic recoil, contraction of muscles within the lung wall, hydrostatic forces, and the negative pressure created by buccal expansion. In the next step, buccal compression in series with jaw closure and sealing of the opercula forces mixed air into the lungs with any excess being expelled through the mouth, operculae, or nares (Figure 4.6B) (McMahon, 1969; Brainerd, 1994). 4.4.2. Actinopterygian Fishes While in sarcopterygian fishes, air breathing occurs in two phases, in actinopterygian fishes this occurs in four phases. In the former case, initial buccal expansion occurs with the mouth closed and draws air from the ABO into the buccal cavity. This may be assisted by elastic recoil of the ABO as well as compression of muscles in the wall of the ABO. Hydrostatic pressure gradients in submerged fish may also assist in this air movement. This air is then expelled during buccal compression through the mouth or operculae. A second buccal expansion now draws in fresh air through the open mouth and the subsequent buccal compression, which takes place with the operculae and mouth closed, forces this air into the ABO (Figure 4.6A) (Liem, 1988; Brainerd, 1994).

4.

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4.4.3. Polypterids A notable exception to this general trend is found in the polypterids (Polypterus and Erpetoichthys) in which elastic recoil from emptying of the lungs leads to aspiration breathing. Exhalation in these fishes is driven by contraction of the lung wall, which also deforms the body wall. When the muscles subsequently relax, a negative, recoil pressure is created within the lungs, enhanced by the ganoid scale‐reinforced skin and body wall, which serves to reinflate the lungs (Figure 4.7) (Purser, 1926; Brainerd et al., 1989).

Stiff dermal armor

Contraction of lung Escaping spent air

Fresh air sucked in by aspiration

Inward buckling of ventral wall

Elastic recoil

Fig. 4.7. Schematic diagrams illustrating the modified dual pump found in polypterids. See text for details on all pumping mechanisms. [From Liem et al. (2001).]

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Claims of suction filling of lungs by estivating Protopterus (Lomholt et al., 1975) and of ABOs by Arapaima (Farrell and Randall, 1978) have not been substantiated (DeLaney and Fishman, 1977; Greenwood and Liem, 1984). 5. RESPIRATORY CONTROL The ability to assess respiratory control in fishes requires experiments that distinguish between the diVerent sites where respiratory stimulation might occur. For example, hypoxia and/or hypercapnia may stimulate externally oriented (water) or internally oriented (blood or brain tissue) chemoreceptors. Respiratory control in purely water‐breathing fishes supports the hypothesis that there are two separate populations of O2‐sensitive chemoreceptors: in general, one population monitors the internal environment (blood) and elicits ventilatory reflexes while those that monitor the external environment (water) elicit both ventilatory and cardiovascular reflexes. Mechanical deformation of the gills or ABO is sensed by mechanoreceptors that may be integrated into the overall ventilatory response. Thus, a full understanding of respiratory control, and the mechanisms involved in these responses, needs to distinguish between stimuli arising from water, blood, and, if present, the ABO. 5.1. Hypoxic and Hypercarbic Ventilatory Responses and Reflex Pathways 5.1.1. Agnathans There has been little work on the ventilatory responses to hypoxia and/or hypercarbia in hagfishes or lampreys. Hagfishes are known to be very hypoxia tolerant with a low metabolic rate (Munz and Morris, 1965) and high anaerobic potential (Sidell et al., 1984). Measurements of ventilation of hagfishes are rare, probably owing to the diYculty of making direct measurements of ventilation on an animal that produces copious amounts of slime when disturbed (Lim et al., 2006). Branchial ventilation has been measured in animals at rest in 7
C water and the frequency was 18 beats min1 generating a water flow of 0.019 ml min1 g1 (SteVensen et al., 1984). Given that hagfish are hypoxia, and perhaps, anoxia tolerant, it is possible that ventilation in these animals is oxyconforming; that is, ventilation may be depressed on hypoxic exposure and hagfish rely primarily on anaerobic metabolism. On the other hand, hypoxic exposure results in the release of catecholamines in hagfish (Bernier et al., 1996), and catecholamines have been shown to increase ventilation in fish. The ventilatory response to hypoxia in hagfish awaits experimentation in these animals and a solution to the ‘‘slime problem.’’

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Compared with hagfishes, there has been considerably more work on the ventilatory responses to hypoxia/hypercarbia in lampreys. Larval lampreys (ammocoetes) are not particularly hypoxia tolerant, although hypoxia tolerance varies inversely with temperature (Potter et al., 1970). Ventilation frequency in Ichthyomyzon hubbsi ammocoetes increases with hypoxia, but eventually decreases over time when PO2 < 10 mmHg at 15.5
C (Potter et al., 1970). Direct measurements of ventilation in larval sea lampreys (Petromyzon marinus) indicate that severe hypoxia (4% O2) increases ventilation by nearly tenfold with increases in both respiratory frequency and stroke volume that account for the large increase in minute ventilation (Rovainen and Schieber, 1975). In the same study, aquatic hypercarbia (3% CO2) also significantly increased minute ventilation by about fivefold with increases in both ventilatory frequency and stroke volume (Rovainen and Schieber, 1975); however, the authors were unsure whether the increased ventilation in response to hypercarbia resulted from the reduction in aquatic pH or a direct eVect of CO2 as a ventilatory stimulus. Adult lampreys (Lampetra fluviatilis) increase ventilation frequency in response to reductions in O2 saturation (Claridge and Potter, 1975). At a temperature of 9.5
C, frequency increased from 60 breaths min1 with 100% air saturation to 180 breaths min1 at 15% air saturation. As a consequence, they can maintain or even increase their O2 uptake down to a PO2 as low as 10 mmHg even in comparatively warm water (Claridge and Potter, 1975). Further reductions to 7.5% saturation, however, were lethal. Although ventilation volume was not measured, it is likely that volume increases also contributed to the overall increase in ventilation. 5.1.2. Dipnoans Members of the three extant lungfish genera, Protopterus, Lepidosiren, and Neoceratodus, occupy an important position in the evolution of tetrapods. In addition, these groups have provided a substantial amount of information about the transition from aquatic to aerial ventilation. The African and South American lungfishes, Protopterus and Lepidosiren, respectively, are primarily obligate air‐breathers and exhibit little or no response to aquatic hypoxia (Johansen and Lenfant, 1967, 1968), whereas the Australian lungfish, Neoceratodus, is a facultative air‐breather that uses its gills as the primary gas exchange organ (Johansen et al., 1967a; Fritsche et al., 1993). African lungfish ventilate the gills, but gill ventilation has been found both to be unresponsive to aquatic hypoxia (Johansen and Lenfant, 1968) and to exhibit increases in response to hypoxia (Jesse et al., 1968). The lungfish in the latter study were smaller than in the study by Johansen and Lenfant (1968), thus developmental stage might be responsible for the diVerences between the two studies. Branchial denervation in Protopterus abolished the

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increase in gill ventilation and enhanced air‐breathing behavior during hypoxia (Lahiri et al., 1970), suggesting that branchial O2 chemoreceptors are a vital component of aerial respiratory control in these animals. Experiments with the African lungfish, Protopterus amphibius, indicate that juvenile members of this species use the gills to obtain about 70% of total O2 uptake, whereas adults use aquatic respiration to obtain about 10–15% of the total O2 uptake (Johansen et al., 1967b). However, a study with Protopterus aethiopicus indicates that in hypoxic water, both juvenile and adult animals obtain more than 90% of O2 uptake via aerial respiration (Seifert and Chapman, 2006). Although African lungfish are largely unresponsive to hypoxic water, lungfish forced to breathe hypoxic gas from the aerial environment or when N2 is injected into the lung significantly increase air‐ breathing frequency (Burggren and Johansen, 1968; Johansen and Lenfant, 1968; Perry et al., 2005), suggesting that lungfish have internally oriented O2‐sensitive chemoreceptors. The Australian lungfish, as a facultative air‐ breather, inhibits gill ventilation and increases air‐breathing frequency in response to aquatic hypoxia (Johansen et al., 1967a; Fritsche et al., 1993), similar to the responses of other facultative air‐breathing fish such as gar (Lepisosteus), bowfin (Amia) and reedfish (Erpetoichthys) (Pettit and Beitinger, 1981; Smatresk et al., 1986; McKenzie et al., 1991a). Hypercarbia in the African lungfish reduces gill ventilation rate while increasing air‐breathing frequency (Johansen and Lenfant, 1968). In the South American lungfish, prolonged aquatic or combined aquatic/aerial hypercarbia elicits an initial large increase in air‐breathing frequency that steadily declines to preexposure levels after 8 h (Sanchez et al., 2005). 5.1.3. Polypterids The African reedfish (Erpetoichthys calabaricus), a bimodally breathing fish, is capable of supporting metabolism exclusively by air breathing in aquatic hypoxia (Pettit and Beitinger, 1981), although in normoxic conditions it obtains most of its O2 requirements through gill ventilation (Pettit and Beitinger, 1985). Reedfish are also amphibious, making terrestrial excursions that are supported entirely by air breathing (Sacca and Burggren, 1982). This fish also does not avoid low dissolved O2 concentrations (0.5 mg liter1) in comparison to an obligate water‐breathing fish (Percina caprodes), suggesting that habitat selection and distribution of E. calabaricus is not limited by aquatic hypoxia (Beitinger and Pettit, 1984). Gill ventilation in E. calabaricus is reduced in hyperoxia (100% O2), and is completely inhibited in aquatic hypoxia when the fish have access to aerial normoxia (Pettit and Beitinger, 1981); however, exposure to combined aquatic and aerial hypoxia does not alter gill ventilation from control levels (Pettit and Beitinger, 1985), suggesting a complex interaction of O2 partitioning between the aquatic and aerial environments. In the same study, gill ventilation

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increased significantly in response to low (0.5%) or high (5.0%) levels of CO2 (Pettit and Beitinger, 1985). Air breathing in E. calabaricus is strongly stimulated by aquatic hypoxia (3% O2), but also increases in response to combined hypoxia/hypercarbia (8% O2/5% CO2), or by hypercarbia alone (5% CO2) (Pettit and Beitinger, 1985). The birchir (Polypterus senegalus), the only other extant polypterid, also increases air breathing in response to aquatic hypoxia (Magid, 1966) and is an obligate air‐breather even under normoxic conditions (Magid and Babiker, 1975). Overall, air breathing is an important feature of the respiratory physiology of polypterid fishes and allows them to invade hypoxic/hypercarbic habitats. 5.1.4. Chondrostei The ventilatory responses of chondrosteans to aquatic hypoxia have been the subject of several studies, but have been limited to sturgeons with no apparent examination of the ventilatory responses to hypoxia in paddlefishes. The majority of the studies with sturgeon report typical ventilatory responses to hypoxia including increases in ventilatory frequency and volume. One study, however, reported that the sturgeon A. transmontanus was an oxyconformer, lowering its metabolic rate and ventilation in response to progressive hypoxia (Burggren and Randall, 1978). This view was challenged by a study in a congener (Acipenser baeri) that demonstrated oxyregulation and increases in respiratory frequency and gill amplitude in response to progressive hypoxia (Nonnotte et al., 1993). The discrepancies between the two studies appear to result from technical diVerences in the measurements of ventilation: the study by Burggren and Randall (1978) used a flexible tube sewn directly to the mouth of the sturgeon which may have imposed a resistance to gill water flow (see Nonnotte et al., 1993 for discussion). Other studies support the view that sturgeon respond to hypoxia with increased ventilatory frequency and increased opercular pressure amplitude (Maxime et al., 1995; McKenzie et al., 1995) and are, until a critical PO2 is reached, oxyregulators. Stimulation of external chemoreceptors in the sturgeon by addition of NaCN to the inspired water elicited a transient bradycardia and stimulated ventilation, whereas intra‐arterial injections of NaCN stimulated ventilation and had no eVect on heart rate (McKenzie et al., 1995), supporting the hypothesis that two separate populations of chemoreceptors monitor the external and internal environments and are responsible for the cardioventilatory responses to hypoxia (Randall, 1982). 5.1.5. NEOPTERYGII Garfishes (Lepisosteus spp.) and bowfin (Amia calva) are bimodally breathing fish with functional gills and a well‐vascularized gas bladder that is used for air breathing. Several studies have examined gill ventilation and air breathing in these species in response to hypoxic and hypercarbic

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challenges. These fishes shift the emphasis of ventilation from gill ventilation to air breathing as temperature increases or in response to aquatic hypoxia. In the spotted gar, Lepisosteus oculatus, severe hypoxia (12 mmHg) results in a significant increase in air‐breathing frequency from about 1 breath h1 to 8–9 breaths h1, which supports 100% of the metabolic requirements of the animal (Smatresk and Cameron, 1982a). Gill ventilation was significantly depressed in hypoxia, decreasing from 35 breaths min1 to 21 breaths min1 (Smatresk and Cameron, 1982a). The depression of gill ventilation is thought to limit the diVusional loss of O2 from blood to water during severe hypoxia. A similar study in conscious longnose gar (Lepisosteus osseus) revealed that progressive hypoxia significantly stimulated air breathing, but had little eVect on gill ventilation frequency (Smatresk, 1986). Opercular pressure amplitude, used as an index of gill ventilation volume, initially increased with moderate hypoxia, but was inhibited with more severe hypoxia, as shown previously. Smatresk (1986) also used NaCN applied to the inflow water and injected intra‐arterially in an attempt to distinguish between externally oriented and internally oriented O2 chemoreceptors that mediate the ventilatory responses to hypoxia. NaCN applied externally or internally stimulated air‐breathing frequency, indicating that O2‐sensitive chemoreceptors monitor both the external and internal environments (Smatresk, 1986). Gill ventilation, however, was inhibited by NaCN applied externally, similar to the hypoxic response, but stimulated gill ventilation when injected intra‐arterially, suggesting that internal hypoxia exerts the dominant control over gill breathing in gar (Smatresk, 1986). Studies with anesthetized, spontaneously breathing gar where internal and external O2 levels were precisely regulated show that hypoxia, whether internal or external, consistently stimulated air breathing whereas the responses to hypoxia by gill breathing were more complex (Smatresk et al., 1986). Hypoxia appeared to consistently inhibit gill ventilation, regardless of internal O2 levels, but there was also an interaction between internal and external O2 chemoreceptors for establishing gill ventilation levels. Hypercarbia significantly stimulated gill ventilation in spotted gar, but had no consistent eVect on air breathing (Smatresk and Cameron, 1982b). Bowfin (A. calva) is also a facultative air‐breather that responds to hypoxia by increasing air‐breathing frequency (Johansen et al., 1970; Randall et al., 1981a; McKenzie et al., 1991a). There are conflicting reports on the response of gill ventilation to aquatic hypoxia. Some studies have shown that severe hypoxia inhibits gill ventilation (Johansen et al., 1970), as seen in gar, but studies using both hypoxia and NaCN show that bowfin does not inhibit gill ventilation (McKenzie et al., 1991a). In Amia, denervation of the cranial nerves serving the gill arches (glossopharyngeal and vagus) combined with pseudobranch ablation (innervated by the facial nerve) eliminated

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the air‐breathing responses to brief (15 min) aquatic hypoxia (McKenzie et al., 1991a). Although denervation experiments can produce side eVects such as altered behavior (see below), metabolic depression, and stress, cranial nerve denervation has been used successfully to elucidate chemoreceptor pathways in a number of fish species (Burleson et al., 1992). Additionally, Amia uses two types of air breaths that appear to be linked to gas exchange and buoyancy functions (Figure 4.8) (Hedrick and Jones, 1993; Hedrick and Jones, 1999). Type I breaths (exhalation followed by inhalation) are gas exchange breaths and are predominantly stimulated by aquatic hypoxia, or by internal hypoxia created by forcing bowfin to breathe hypoxic aerial gas mixtures (Figure 4.8A). Type I breaths also exhibit a periodicity with a mean interbreath interval of about 30 min in normoxia and 16 min in hypoxia (Hedrick et al., 1994). The periodic air‐breathing pattern is thought to arise from periodic fluctuations in blood PO2 that stimulate Type I (gas exchange) breaths. Type II breaths (inhalation only) are stimulated by aerial hyperoxia (Hedrick and Jones, 1993) or by gas bladder deflation (Hedrick and Jones, 1999), supporting the hypothesis that Type II breaths regulate gas bladder volume and have a buoyancy‐ related function (Figure 4.8B). These studies point to the importance of buoyancy as a proximate factor in the air‐breathing behavior of primitive air‐breathing fishes. It is unknown if gar, or other air‐breathing fishes that

A

B Inhale

Inhale Type II

→

Type I

→

B 60

0

T

−10

TE →

→

0

Exhale

100 ms Inhale

Inhale →

→

Air flow (ml s−1)

30

80 40 TE

T

Exhale

→

→

0 0

−20

Fig. 4.8. Records of air flow (ml s1) for two A. calva in control conditions illustrating (A) Type I and (B) Type II air‐breaths. For Type I breaths, the transfer phase (T) and expiratory time interval (TE) is shown. [From Hedrick and Jones (1993).]

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use the gas bladder as a buoyancy organ, also exhibits air‐breathing mechanisms similar to the Type II breaths described in bowfin. Complete branchial denervation in Amia, without pseudobranch ablation, did not abolish air‐ breathing responses to aquatic hypoxia, but did interfere with the ability to capture inhaled air by Type I air‐breaths (Hedrick and Jones, 1999). Air‐breathing frequency actually increased due to the inability to transfer inhaled gas to the gas bladder; the increase in frequency was due to a large increase in the number of Type II air‐breaths further supporting the hypothesis that Type II breaths monitor gas bladder volume and have a buoyancy‐ related function (Hedrick and Jones, 1999). These experiments also suggest that some components of branchial innervation, perhaps arising from gill mechanoreceptors, play an important role in the coordination of ventilatory muscles important for air breathing. 5.2. Receptors Involved in Reflex Ventilatory Control 5.2.1. O 2‐Sensitive Chemoreceptors Physiological data provides indirect evidence for the location of O2‐sensitive chemoreceptors. However, in combination with whole‐animal responses, putative locations for chemoreceptors may be distinguished using electrophysiology and histology. Chemoreceptive cells are characterized by neurotransmitter storage and possess irregular discharge at rest, which increases frequency and irregularity exponentially with decreasing levels of O2 (PO2 or CaO2). In all classes of vertebrates, O2‐sensitive chemoreceptors are composed of glomus cells (Type I), companion cells (Type II), and nerve tissue. Emphasis has been placed on the role of the glomus cells in aVerent sensing, and these cells are characterized by cytoplasmic vesicles, ribosomes, endoplasmic reticulum, and mitochondria, similar to endocrine tissue. Putative chemoreceptive cells, neuroepithelial cells (NEC), in the gills of water‐breathing fish share many similar characteristics with the glomus cells of the mammalian carotid body. Examination of the NECs have shown serotonin (5‐HT) to be the major monoamine present in the dense‐cored vesicles and 5‐HT has been used as a marker for these cells (Dunel‐Erb et al., 1982; Zaccone et al., 1992; Goniakowska‐Witalinska et al., 1995; Jonz and Nurse, 2003). Neuroepithelial cells have been identified in a few primitive fish, including the gills and lungs of Protopterus (Figure 4.9), the swimbladder of Polypterus, and the gills of bowfin (Zaccone et al., 1989; Adriaensen et al., 1990; Goniakowska‐ Witalinska et al., 1995). Therefore, it appears that putative O2 chemoreceptors carrying aVerent information to a respiratory center are present in both the respiratory structures of the gills and ABOs.

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A

B

A

NE

g Fig. 4.9. (A) Serotonin‐like immunoreactive cells (brown) within the epithelium of the alveoli of Protopterus annectens (100) (PAP method) [(from Zaccone et al. (1989)] and (B) low‐power electron micrograph of a solitary neuroendocrine cell in the pneumatic duct region of the lung of P. aethiopicus. Dense cored vesicles are observed in the basal cytoplasm (6800). [From Adriaensen et al. (1990).] NE, neuroendocrine cell.

5.2.2. Peripheral CO 2‐Sensitive Chemoreceptors Ventilation, aquatic, aerial or both, increased in fish exposed to environmental acidosis, hypercapnia, and arterial acidosis. Acid–base disturbances come hand‐in‐hand with changes in CO2 dissociation, so it is diYcult to isolate the stimulus for potential chemoreceptors as solely CO2 or concomitant fluctuations in pH. CO2 sensitivity may have evolved with primitive bimodal breathing fish, as these animals continually face the challenge of both O2 and CO2 regulation. Although the O2 ventilatory drive is supported by the lower content of O2 than CO2 in water, obligate air‐breathing fish, such as the African lungfish, increased both air‐breathing frequency and branchial ventilation in mildly hypercarbic water (Jesse et al., 1968; Johansen and Lenfant, 1967; Johansen et al., 1967a). However, higher levels

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of CO2 in the water decreased aquatic ventilation. The isolated brainstem of L. osseus is responsive to changes in superfused CO2 as well as changes in O2 (Wilson et al., 2000); however, Amia is not responsive to any changes in arterial CO2 levels (McKenzie et al., 1991b). There is no histological identification of specific CO2‐sensitive chemoreceptors in primitive fish; however, the proposed location is again the gills. A separate population of CO2/Hþ chemoreceptors, separate from the O2‐sensitive NECs, projecting aVerent sensory input may exist. Additionally, CO2 sensitivity may be associated with central chemosensitivity, and research using hypercarbic and acidotic superfusates on isolated brains is discussed briefly in the next section. 5.2.3. Central CO 2 Chemosensitivity The central site for respiratory integration remains unknown for both water‐breathing and air‐breathing fish. Numerous studies have suggested two separate central pattern generators (CPGs) for gill ventilation and air breathing, presumably located in the medulla (Rovainen, 1977; Ballintijn, 1987; Taylor et al., 1999). [For more detailed reviews on the location and function of CPG in fish, see Ballintijn (1987) and Taylor et al. (1999).] The isolated brainstem prep of longnose gar (L. osseus) showed a motor pattern for lung ventilation that was responsive to CO2, and the authors correlated this proposed central chemoreceptor to a CPG (Remmers et al., 2001). However, this is not true of all air‐breathing fish. Amia do not appear to have central chemoreceptors mediating cardioventilatory responses, as altering gas tensions and pH on the extradural fluid has no eVect on any cardioventilatory variables in normoxic water (Hedrick et al., 1991). 5.2.4. Mechanoreceptors Physical displacement of respiratory passages and gas exchange surfaces is detected by mechanoreceptors, which are a broad group of receptors sensitive to changes in flow, volume, transpulmonary pressure, and tension. Mechanoreceptors are characterized by simple free nerve endings located in connective tissue or muscle and it is the deformation of these nerve endings that results in changes in membrane and channel geometry, ion flux, and membrane potential, and therefore a change in discharge frequency. Typically, the sensory modalities and response characteristics of these mechanoreceptors are determined by the location of receptors. In fish, mechanoreceptors have been identified in the buccal and opercular cavities, pharynx, gill arches, gill rakers and filaments, and ABOs. In general, there are two divisions of mechanoreceptors based on their response to the degree or rate of change during inspiration and expiration. Slowly adapting receptors (SAR) demonstrate a static change in discharge

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that is proportional to the degree of change in flow, volume, pressure, or tension. The second group, rapidly adapting receptors (RAR), responds to the rate of change, showing a dynamic change in discharge with an initial burst on inspiration and a second burst corresponding to deflation of the respiratory surfaces. Mechanoreceptors that are sensitive to both the degree and rate of change are characterized as SAR with a dynamic aspect that gradually levels oV to a steady state in proportion to the change in volume, pressure, and tension. a. Gill Mechanoreceptors. Although proprioceptors do exist in the buccal and opercular cavities of fish, the gills are the primary site of mechanoreceptors aVecting ventilatory control in water‐breathing fish. Within the gills, mechanoreceptors have been identified along the gill filaments, gill rakers, and in the cartilaginous strip between the gill arches (De Graaf and Ballintijn, 1987). The receptors of the gill filaments are of the slowly adapting variety with a dynamic response to displacement. Gill raker mechanoreceptors are phasic and predominantly detect damaging material and work to maintain the gill sieve by producing the ‘‘cough’’ reflex of fish and a reflect bradycardia (De Graaf and Ballintijn, 1987). However, there is no evidence that they contribute to respiratory control (Burleson et al., 1992). b. ABO Mechanoreceptors. With the addition of air breathing in fish, mechanoreceptors are found in both the gills and ABOs. Studies documented both SAR and RAR of their ABOs in lungfish and gar (DeLaney et al., 1983; Smatresk and Azizi, 1987), while bowfin only appeared to have SAR of its swimbladder (Milsom and Jones, 1985). The SAR of the lungfish increased firing with progressive inflation, but also displayed a dynamic component as a function of the inflation rate (Fishman et al., 1989). Receptors sensitive to the degree of physical change in the lung predominated, while RAR fired briefly only during inflation and deflation. AVerent information from both of the degree and rate of inflation in the lungfish was transmitted to the brainstem where it likely plays a role in ventilatory control (Fishman et al., 1989). Lungfish stretch receptors responded strongest to transpulmonary pressure yet they also had a unique inhibition of receptor discharge in intrapulmonary hypercapnia (DeLaney et al., 1983). Receptor inhibition by intrapulmonary CO2 concentrations also occurred in the spotted gar, L. oculatus (Smatresk and Azizi, 1987); however in A. calva, the receptors were insensitive to CO2 concentrations (Milsom and Jones, 1985). The ABOs of these primitive fish appeared to function both in buoyancy control as well as gas exchange, thus adding to the importance of aVerent information of stretch receptors (Johansen et al., 1967b; Smatresk and Azizi, 1987). In support of this hypothesis, rapid deflation of the ABO in Amia elicited a Type II breath while inflation causes fish to stop an air‐breath attempt (Hedrick and Jones, 1999).

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6. CONCLUSIONS The ‘‘primitive fishes’’ described in this volume display a wide variety of ventilatory patterns and control mechanisms. As this chapter has illustrated, many of these fishes are facultative or obligate air‐breathers, thus increasing the complexity of control mechanisms when several sites and receptors are integrated into the overall ventilatory response. Because many of these fishes use both water and air breathing in their respiratory behavior, they are important for understanding the physiological mechanisms in the transition from water to land. Many studies have described the reflex responses to environmental perturbations such as hypoxia and hypercarbia, but one area that clearly needs more study is how peripheral receptors and neural pathways are centrally integrated to produce the complex ventilatory behaviors seen in these fishes. Although these fishes are highly evolved and adapted to their particular ecological niches, further examination of mechanisms underlying respiratory behavior may lead to greater insight into the evolutionary forces that have shaped the transition from water to land among vertebrates. REFERENCES Adriaensen, D., Scheuermann, D. W., Timmermans, J.‐P., and De Groodt‐Lassel, M. H. A. (1990). Neuroepithelial endocrine cells in the lung of the lungfish Protopterus aethiopicus. An electron‐ and fluorescence‐microscopial investigation. Acta Anat. 139, 70–77. Almeida‐Val, V. H. M., and Val, A. L. (1993). Evolutionary trends of LDH isozymes in fishes. Comp. Biochem. Physiol. 105B, 21–28. Ballintijn, C. M. (1987). Evaluation of central nervous control of ventilation in vertebrates. In ‘‘The Neurobiology of the Cardiovascular System’’ (Taylor, E. W., Ed.), pp. 3–27. Manchester University, Manchester, UK. Beitinger, T. L., and Pettit, M. J. (1984). Comparison of low oxygen avoidance in a bimodal breather, Erpetoichthys calabaricus and an obligate water breather, Percina caprodes. Environ. Biol. Fish 11, 235–240. Bernier, N. J., Harris, J., Lessard, J., and Randall, D. J. (1996). Adenosine receptor blockade and hypoxia‐tolerance in rainbow trout and Pacific hagfish. I. EVects on anaerobic metabolism. J. Exp. Biol. 199, 485–495. Booth, J. H. (1978). The distribution of blood flow in the gills of fish: Application of a new technique to rainbow trout (Salmo gairdneri). J. Exp. Biol. 73, 119–129. Brainerd, E. L. (1994). The evolution of lung‐gill bimodal breathing and the homology of vertebrate respiratory pumps. Am. Zool. 34, 289–299. Brainerd, E. L., Liem, K. F., and Samper, C. T. (1989). Air ventilation by recoil aspiration in polypterid fishes. Science 246, 1593–1595. Brauner, C. J., Matey, V., Wilson, J. M., Bernier, N. J., and Val, A. L. (2004). Transition in organ function during the evolution of air‐breathing; Insights from Arapaima gigas, an obligate air‐breathing teleost from the Amazon. J. Exp. Biol. 207, 1433–1438.

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5 GAS TRANSPORT AND EXCHANGE C. J. BRAUNER M. BERENBRINK

1. Introduction 2. Partitioning of O2 and CO2 Exchange Across the Respiratory Surfaces 2.1. Primitive Ray‐Finned Fishes (Actinopterygii) 2.2. Lobe‐Finned Fishes (Sarcopterygii) 2.3. Jawless Fishes (Agnatha) 3. Blood O2 Transport 3.1. General Principles of Hb Function 3.2. Factors AVecting the ArterioVenous O2 DiVerence 3.3. Survey of Extant Primitive Fishes 4. Transport and Elimination of CO2 4.1. General Model of CO2 Transport and Excretion 5. Synthesis 5.1. How Do Primitive Fishes Compete with Other Fishes? 5.2. Primitive Fishes and the Evolution of Vertebrate Blood O2 and CO2 Transport Characteristics

Gas exchange is a prerequisite of vertebrate life. In terms of structural and functional diversity and habitats occupied, extant teleosts clearly outcompete extant primitive fishes; however, there are a few aspects related to gas exchange that may have contributed to the survival of these primitive fishes. Most of the primitive fishes either have the ability to breath air, have the ability to tolerate aerial exposure (and in some cases estivate), or are tolerant to aquatic hypoxia. Many of the bimodal breathers retain fully functional gills, which at times allow strictly aquatic breathing over prolonged periods which may be important for aerial predator avoidance or surviving ice cover in temperate climates. While air breathing is important for surviving aquatic hypoxia, it is also important in enhancing O2 uptake during exercise. Living primitive fishes occupy strategic positions in the evolutionary tree of 213 Primitive Fishes: Volume 26 FISH PHYSIOLOGY

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vertebrates and may shed light on the evolution of blood O2 and CO2 transport characteristics. Evolutionary reconstruction indicates that the increase in the Bohr–Haldane eVect in primitive ray‐finned fishes was followed first by a gradual increase in the magnitude of the Root eVect and then a gradual reduction in specific Hb buVer value. This was followed by the evolution of a choroid rete mirabile and ocular O2 secretion in the last common ancestor of Amia calva and teleosts. Finally, the adrenergic red blood cell Naþ/Hþ exchanger was never present in primitive ray‐finned fishes or primitive teleosts and only evolved in advanced teleosts. No such evolutionary trends are observed in primitive lobe‐finned fishes. 1. INTRODUCTION The uptake of O2 from the environment and elimination of metabolically produced CO2 are prerequisites of vertebrate life. A great deal is known about the diVerences in O2 and CO2 transport and exchange between water and air‐ breathing vertebrates; however, this stems largely from studies on teleost fishes in the former, and mammals in particular in the latter. Fishes possess great diversity in gas exchange strategy, ranging from completely water breathing to obligate air breathing, and thus occupy a crucial phylogenetic position in the transition of life from water to land which has large implications for gas exchange (Dejours, 1988; Graham, 1997). Relatively little in relation to O2 and CO2 transport and exchange is preserved within the fossil record, and consequently, reconstruction of the evolution of gas exchange is limited largely to studies on extant species. In the following sections, primitive fishes will be discussed going backward in time from the closest living relatives of teleosts to successively more distantly related groups of primitive fishes. We first discuss the relative roles of the respective gas‐exchange surfaces [gills, skin, and air‐ breathing organs (ABOs)] to O2 and CO2 exchange in each primitive fish group. We then discuss general aspects of O2 and CO2 exchange, largely on the basis of what is known in teleosts and then what is known for primitive fishes. Finally, we discuss how this information on primitive fishes helps to identify some general trends in the evolution of vertebrate blood gas transport characteristics. 2. PARTITIONING OF O2 AND CO2 EXCHANGE ACROSS THE RESPIRATORY SURFACES In typical water‐breathing teleosts, the gills are the predominant surface for both O2 and CO2 exchange; but in some cases, there can be appreciable O2 uptake across the skin. Many of the primitive fish groups discussed in this chapter contain species that are facultative or obligate air‐breathers. Thus, the gills, skin, and ABOs are all potential sites for gas exchange in many primitive

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fishes. There has been considerable interest and research conducted on the morphologies of the respective gas exchange structures (see Chapter 4, this volume) and a great number of direct measurements of the relative role and eYciency of each structure to both O2 and CO2 exchange, which are briefly summarized below. In most air‐breathing fishes studied to date, there appears to be a spatial separation of O2 and CO2 exchange. That is, the majority of O2 uptake may occur across the ABO, and the majority of CO2 excreted across the gills and/or skin. This is largely related to the fact that the capacitance coeYcient for CO2 does not change much between water and air, while that for O2 is 20‐ to 30‐fold higher (depending on the temperature) in air than water (Dejours, 1988). Because ventilation‐rate volume (ventilation frequency
volume) of gas exchangers in fish is largely regulated to secure adequate O2 uptake, ventilation‐ rate volume of the ABO in an air‐breather is greatly reduced relative to that of the gills in a water‐breather. The reduced ventilation‐rate volume is suYcient for O2 uptake, but insuYcient for CO2 elimination across the ABO, and consequently CO2 diVuses out across the gills and/or skin (Dejours, 1988; Graham, 1997). The spatial uncoupling of O2 and CO2 transport has interesting implications for gas exchange in fish, given that at least in most teleost fishes there is a tight interaction between O2 and CO2 exchange that resides at the level of Hb in the red blood cell (RBC) (Jensen, 1989; Brauner and Randall, 1996, 1998; Brauner and Val, 1996; Nikinmaa, 2001). Air breathing not only permits fishes to survive exposure to aquatic hypoxia but also allows them to maintain normal levels of metabolism and activity in aquatic hypoxia, provided O2 taken up in the ABO is not subsequently lost across the gills. Consequently, many air‐breathing fishes possess circulatory adaptations in the gills. Of the four gill arches, eVerent vessels from the third and fourth arches give rise to the pulmonary artery leading to the ABO, and venous return from the ABO is direct to the heart. The first and second gill arches lead exclusively to the dorsal aorta. The creation of a double circulatory loop is most developed in the obligate air‐breathing lungfishes but present to some degree in some of the other groups described below (reviewed in Graham, 1997). Those primitive fishes that are not air‐breathers tend to be tolerant of aerial exposure and/or aquatic hypoxia and thus are very tolerant of adverse environments. The following sections review the limited information that exists for primitive fishes. 2.1. Primitive Ray‐Finned Fishes (Actinopterygii) 2.1.1. Bowfin (Amiiformes) Like many basal teleosts, the bowfin Amia calva uses a swim bladder for accessory air breathing, with air‐breathing frequency and the fraction of aerial _ O2 increasing with rising temperatures, hypoxia, and exercise (Johansen et al., M 1970; Farmer and Jackson, 1998).

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The gills in A. calva are well developed and similar to those of teleost fishes with a few minor modifications (Daxboeck et al., 1981; Olson, 1981) (Figure 5.1). At temperatures below 10–15  C in normoxia, A. calva is an exclusive water‐breather (Johansen et al., 1970; Horn and Riggs, 1973). Under these conditions, a high arterial PO2 (PaO2) of about 110 mmHg (measured in a branch of the celiac artery) approaches the PO2 of inspired water (Table 5.1). This was taken to indicate countercurrent O2 exchange between water and blood in the gills (Johansen et al., 1970). Air‐breathing frequency increases almost linearly with an increase in temperature above 10  C, to an upper thermal tolerance of 35  C (Horn and Riggs, 1973). At 20  C, the ABO accounts for about 35% of the total O2 consumed in normoxia, and at 30  C contribution by ABO increases to 75% at which time A. calva is an obligate air‐breather (Johansen et al., 1970). The role of the ABO in CO2 excretion is not proportional to the O2 uptake, accounting for 25% and 40% of total CO2 removal at 20 and 30  C, respectively. Thus, at 30  C, the majority of CO2 is excreted aquatically, presumably across the gills, although transcutaneous excretion has not been measured (Johansen et al., 1970). During exposure to hypoxia, the dependence on the ABO remains temperature dependent. At 10  C, there is no change in air‐breathing frequency down to a water PO2 (PwO2) of 70–80 mmHg, and in many specimens, no changes down to 40 mmHg (Johansen et al., 1970). There is a large increase in branchial ventilation over this PwO2 range. At a PwO2 of 70–80 mmHg, the ABO is responsible for about 50% of the total O2 uptake at 20  C, and almost 100% at 30  C. Johansen et al. (1970) postulated that A. calva possess shunts in the gills to bypass the lamellae and prevent O2 loss from the blood to hypoxic water during air breathing. However, after this study, there has neither been physiological (Randall et al., 1981) nor anatomical evidence (Daxboeck et al., 1981; Olson, 1981) for shunts of this nature in the gill. The creation of a double circulatory loop is present to some degree based on anatomy, but is not as developed as in the obligate air‐breathing lungfish. Activity level also influences the role of the ABO in gas exchange (Johansen et al., 1970). At a temperature of 24  C in fish at rest, O2 uptake from the air was 10% of whole animal metabolic rate, the remainder secured from the water across the gills and/or skin. When fishes were forced to swim at a low but sustained swimming velocity, there was an increase in air‐ breathing frequency and O2 uptake across the ABO was elevated to 66% of whole‐animal metabolic rate. Despite the large increase in aerial O2 uptake, there was also a ca 50% increase in O2 uptake from the water (Farmer and Jackson, 1998), indicating that there is still some capacity to elevate aquatic gas exchange in resting fish at this temperature.

5.

217

GAS TRANSPORT AND EXCHANGE

106

m

105 Amia calva

Gill surface area (cm2)

104 Lampetra fluviatilis

tei

os

le Te

Acipenser transmontanus Scyliorhinus stellaris Latimeria chalumnae

103 au

st

102

is

wo

su

t Ka

s nu

la pe

u an

Neoceratodus forsteri

ps

O

Lepisosteus oculatus 101

Lepidosiren paradoxa

100

10 −1 0.01

0.1

1 Body mass (kg)

10

100

Fig. 5.1. Comparison of gill surface area in relation to body weight in primitive fishes. Bilogarithmic plot. The shaded area indicates the typical range for teleosts as obtained by extrapolation of the regression lines for the sluggish toadfish Opsanus tau and the highly active skipjack tuna Katsuwonus pelamis (Muir and Hughes, 1969; Hughes and Gray, 1972). For primitive fishes, actual data points and, if applicable, regression lines are given, except for the river lamprey L. fluviatilis, where minimal and maximal values from Lewis (1980) are shown. The single value for the Australian lungfish N. forsteri was estimated from Figure 6 in Hughes (1976). Further references: A. transmontanus (white sturgeon; Burggren et al., 1979), A. calva (bowfin; Daxboeck et al., 1981), Latimeria chalumnae (coelacanth; Hughes, 1995), Lepidosiren paradoxa (South American lungfish; de Moraes et al., 2005), and Lepisosteus oculatus (spotted gar; Landolt and Hill, 1975). Values for the elasmobranch nursehound Scyliorhinus stellaris from Hughes et al. (1986) are included for comparison. Note the teleost‐like gill surface area of bowfin and river lamprey, the reduced gill surface area in the coelacanth, and the diVerence between the obligate and facultative air‐breathing South American and Australian lungfishes, respectively.

Table 5.1 Whole‐Blood and Hemoglobin Characteristics of Primitive Fishes Species

PaO2

PvO2

[Hb] (g/dl)

Hct (%)

MCHC (g/dl)

Actinopterygii Amia calva

P50

F

nH

bHb

4 11 15 110

218

43.7

5.8

31.1*

22.8

254

9

1.3

0.43

24

2.6

0.51 0.497

4.5

35–59

47 7.5

26.4

284

9.1

29

314

1.0 6.4

Atractosteus tristoechus Lepisosteus oculatus

24

20

7.0–8.0

24

0. 5

Comments

Whole blood, 15  C, PCO2 0–1 Whole blood, 15  C, PCO2 12 Whole blood, 15  C, PCO2 15 Whole blood, pH 7.6, 15  C Whole blood, pH 7.6, 27  C Whole blood, pH 7.5, 30  C, *ventral aortic blood Whole blood, pH 7.60–7.67, 20  C Whole blood, pH 7.72, 20  C Washed RBCs 15  C, no CO2 25  C

Whole blood in vitro, 1% CO2, pH 7.8, 20  C

References

Black, 1940 Black, 1940 Black, 1940 Johansen et al., 1970 Johansen et al., 1970 Randall et al., 1981

McKenzie et al., 1991a

McKenzie et al., 1991b Weber et al., 1976b Berenbrink et al., 2005 Siret et al., 1976 Smatresk and Cameron, 1982a

30

0. 5

9*

Lepisosteus osseus

16*

Whole blood in vitro, 2% CO2, pH 7.4, 20  C *Whole blood buVer value in mmol HCO 3 per liter and pH unit, winter fish, 10  C *Whole blood buVer value in mmol HCO 3 per liter and pH unit, summer fish, 15  C

219

9.0 Lepisosteus platyrhincus Acipenser transmontanus

0.525 90

5.0þ

115

5.5

21.5

21

262

0.55

0.40

0.50

Acipenser baeri

80* 70* 6.9

*

8.6

25  C, *Haldane coeYcient Whole blood in vitro, PO2 3.5, pH 7.81, 15  C Whole blood in vitro, pH 8.23–7.27, 15  C Whole blood in vitro, pH 8.14–7.20, 20  C *pH 7.85, 18  C *pH 7.85, 15  C

Smatresk and Cameron, 1982a

Rahn et al., 1971

Rahn et al., 1971

Lenfant and Johansen, 1972 Berenbrink et al., 2005 Burggren and Randall, 1978 Crocker and Cech, 1998

Crocker and Cech, 1998 Maxime et al., 1995 Nonnotte et al., 1993 O. Kepp and M. Berenbrink, unpublished data

(continued)

Table 5.1 (continued ) Species Acipenser naccarii Acipenser ruthenus Polypterus senegalus

220

Erpetoichthys calabaricus

PaO2 72

PvO2

[Hb] (g/dl)

Hct (%)

MCHC (g/dl)

nH

P50

F

7.4þ

23.4 30

316

4.3–14

17–43

253–334

23.5

2.68

0.43

7.5

22

341

17.9

2.0

0.247

bHb

Comments pH 7.91, 23  C

9.4 15.4*

11.6

15  C Whole blood in vitro, PCO2 6, pH 7.7, 30  C, F calculated from pH 7.6–7.3, *whole blood buVer value in mmol HCO 3 per liter and pH unit Whole blood in vitro, PCO2 7, pH 7.56, 25  C, F calculated from pH 7.56– 7.00  25 C

11.9 Sarcopterygii Latimeria chalumnae

3.4

20

170

3.3

1

0.51

9.0*

Whole blood, pH 7.8, 20 ºC, F calculated from pH 8.8–6.6, *whole blood buVer value in mmol HCO 3 per liter and pH unit

References McKenzie et al., 1997 Clementi et al., 1999 Berenbrink et al., 2005 Vokac et al., 1972

Beitinger et al., 1985

Berenbrink et al., 2005 Kepp and Berenbrink, unpublished data Wood et al., 1972

3.7þ

2.1

1

11.9

Neoceratodus fosteri

38.9

20

221 Lepidosiren paradoxa

30

81

43

76

49

5.5þ

31

177

11

6.0

30

200

22

7.0 4.1þ

35 15.7

200 261

10.5

0.234

6.5 7.0

28 39.8 21

232 176 18.6

2.00

0.295 0.31 0.66

29.8

1.97

0.44

6.2

33.5

185

0.62

2.27

13.3

0.48

Whole blood in vitro, pH 7.3, 15  C Calculated based on number and location of histidines Whole blood, pH 7.5, PCO2 3.5, 18  C, *whole blood buVer value in mmol HCO 3 per liter and pH unit Whole blood, pH 7.5, PCO2 16, 20  C Whole blood, PCO2 6, 23  C Whole blood, 25  C, F calculated from pH 7.62– 7.38 Whole blood, 35  C, F calculated from pH 7.71– 7.39

0.33

Whole blood

20 35

16.5

25  C

Hughes and Itazawa, 1972 Berenbrink, 2006

Lenfant et al., 1966

Kind et al., 2002

Johansen, 1970 Johansen and Lenfant, 1967 Johansen, 1970 Johansen et al., 1978 Bassi et al., 2005

Bassi et al., 2005

Isaacks et al., 1978 Powers et al., 1979 Amin‐Naves et al., 2004 Sanchez et al., 2001 Berenbrink et al., 2005

(continued)

Table 5.1 (continued ) Species Protopterus aethiopicus

Protopterus amphibius

222 Protopterus annectens Protopterus dolloi Protopterus sp. Lampreys Lampetra fluviatilis

PaO2

PvO2

[Hb] (g/dl)

Hct (%)

MCHC (g/dl)

P50

nH

F

bHb

0.28

36

Comments Whole blood, 25  C Whole blood, 23  C Whole blood, 25  C

References Lahiri et al., 1968

7.0

25.3

277

0.35

6.2

25

248

0.47

7.4 7.1

270 245

5.8þ

27.4 29 32.7 26.5

219

0.20 0.29

8.7þ

38.3

227

0.41 0.68

6.9

32.1

215

Babiker, 1979

7.8

15.3 30.1

259

Perry et al., 2005 Johansen, 1970 11.8

Whole blood from nonestivating fish > 2 years Whole blood from estivating fish

1.21

Adult fish whole blood, 10  C, pH 7.75 Larvae whole blood, 10  C, pH 7.75 2C

1.8

6.1

31.4þþ

194

9.5

33.5þþ

284

1.03 0.9*

3.0

15  C Intracellular F *Haldane coeYcient

Swan and Hall, 1966 Lenfant and Johansen, 1968 DeLaney et al., 1977 Jensen et al., 2003 Bartlett, 1978b Johansen et al., 1976b

Johansen et al., 1976b

Bird et al., 1976

Bird et al., 1976

Nikinmaa and Weber, 1984 Nikinmaa et al., 1995 Jensen, 1999

Lampetra (Entosphenus) tridentata

58 77

Petromyzon marinus

120

24 40

6.7

21.3

315

6.5þþ

24.6

263

18

1.88

23.6

1.89

40.8

1.52

0.41

0.63

3.5*

4.78*

223 Hagfishes Eptatretus cirrhatus

Eptatretus stoutii

90–110

17.2 3.0 2.4þ

12.6

12.3

1.38

0.43

Whole blood in vitro

2–4

1.0

0.0

Whole blood, in vitro

242

0.35*

Myxine glutinosa 4.1

19.1

Whole blood 14  C, pH 7.6 *Whole blood buVer value in mmol HCO 3 per liter and pH unit Whole blood in vivo, 10  C Whole blood, 10  C, PCO2 1.5 Whole blood, 10  C, PCO2 35 Intracellular F *Whole blood buVer value in mmol HCO 3 per liter and pH unit, deoxygenated

215

8.2

*Haldane coeYcient

Johansen and Lenfant, 1972; Johansen et al., 1973

Tufts, 1991 Ferguson et al., 1992 Ferguson et al., 1992 Ferguson et al., 1992 Ferguson et al., 1992

Wells et al., 1986 Wells and Forster, 1989 Manwell, 1958; Johansen and Lenfant, 1972 Jensen, 1999 Larsson et al., 1976

PaO2 and PvO2 refer to the in vivo partial pressure (mmHg) of O2 in arterial and venous blood, respectively, P50 refers to the PO2 (mmHg) at which whole blood is 50% saturated, nH refers to the Hill number at 50% hemoglobin saturation, F refers to the Bohr coeYcient, bHb refers to the hemoglobin buVer value in organic phosphate‐free, deoxygenated hemolysates at physiological pH and Cl and is given in mmol per mmol Hb4 and pH, unless otherwise indicated. Comments refer to conditions under which P50, nH, F, and bHb were made unless otherwise indicated by * in that row. þHb concentration ([Hb4]) estimated from O2 capacity, þþhematocrit (Hct) or [Hb4] calculated from mean cellular Hb concentration (MCHC) and [Hb4] or Hct, respectively.

224

C. J. BRAUNER AND M. BERENBRINK

Immediately after exhaustive exercise, fish that had access to air experienced less of a metabolic acidosis than those denied air access, presumably due to the increased ability to secure O2 aerially (Gonzalez et al., 2001). Interestingly, the rate of recovery from acidosis following exhaustive exercise was slower in fish with than without access to air, which the authors attribute to a reduced ability to excrete CO2 and Hþ during air breathing due to inadequate gill ventilation (Gonzalez et al., 2001). In general, A. calva utilizes aerial respiration to deal with aquatic hypoxia at higher temperatures (20–30  C) and to augment O2 uptake during periods of temperature‐ or activity‐induced elevations in metabolism. The fraction of total CO2 excreted across the ABO is less than the fraction of total O2 uptake across this organ, consistent with many other air‐breathing fishes. 2.1.2. Gars (Lepisosteiformes) Of the seven species of extant gars found in two genera, most of the research conducted on gas exchange to date is on Lepisosteus. Like A. calva and many basal teleosts, gars use a swim bladder for accessory air breathing (Graham, 1997). Under resting conditions, the spotted gar Lepisosteus oculatus is generally considered to be a facultative air‐breather. It possesses reasonably well‐developed gills, although with a slightly reduced mass specific total gill surface area relative to that observed in studied teleosts (Landolt and Hill, 1975) (Figure 5.1). An elevation in temperature (Smatresk and Cameron, 1982b), or a reduction in aquatic O2 levels (Smatresk and Cameron, 1982a), results in an elevation in air‐breathing frequency. At 20–25  C, 70–80% of _ O2 was obtained from the air and air breathing became obligatory total M (Rahn et al., 1971). The gills remained the principal site for CO2 excretion at all temperatures, with the ABO contributing 0% and 8% to the total CO2 excretion at 13 and 25  C, respectively (Rahn et al., 1971). During exposure to severe aquatic hypoxia (PwO2 ¼ 12 mmHg), 100% of the O2 uptake was across the ABO; however, the majority of CO2 was excreted to the water (Smatresk and Cameron, 1982a). L. oculatus does not appear to actively avoid O2‐depleted water, provided it has access to air (Graham, 1997). An exercise‐induced elevation in metabolic rate also stimulates air breathing in L. oculatus. During a low‐level exercise‐induced elevation in metabolic rate, aerial O2 uptake accounted for 53% of total uptake (Farmer and Jackson, 1998). During forced exhaustive exercise, the animals continuously breathed air during the exercise bout (30–40 min) and during recovery (Burleson et al., 1998). Consequently, exposure to water with PwO2 < 19 mmHg had a minimal eVect relative to aquatic normoxia on the duration over which the fish could sustain the swimming bout, or the pattern of recovery following exhaustive exercise, provided the animals had unlimited access to air.

5.

GAS TRANSPORT AND EXCHANGE

225

Recovery from exercise was associated with relatively minor changes in gill ventilation, but a large elevation in air‐breathing frequency relative to preswim levels further emphasizing the reliance on air‐breathing in this species during periods of elevated metabolism. 2.1.3. Sturgeons and Paddlefishes (Acipenseriformes) Sturgeons and paddlefishes are strictly water‐breathers, unlike many of the other primitive fishes, and the gills possess filaments with relatively well‐ developed lamellae that are similar to those of teleost fishes. Paddlefishes are filter feeders, and juvenile Polyodon spathula are obligate ram‐ventilators in normoxia and swim continuously shortly following hatch and throughout life (Burggren and Bemis, 1992). While they have the ability to ventilate their gills at low swimming speeds (<0.6–0.8 body lengths s1), juveniles are not tolerant of hypoxia and die at a PwO2 < 90 mmHg (Burggren and Bemis, 1992). The paddlefishes have reduced scales, and thus transcutaneous gas transfer may occur; however, this has not been measured directly. The open swim bladder in Acipenseriformes is not used as an accessory ABO (Graham, 1997) and probably only participates in buoyancy regulation. In the white sturgeon, Acipenser transmontanus, the total gill surface area relative to body weight is at the lower margin of the values in typical teleosts (Figure 5.1), which is thought to be related to a low activity level and metabolic rate (Burggren et al., 1979). The eYciency of the gills for gas exchange in the genus Acipenser is demonstrated by high‐average normoxic PaO2 values ranging between 70 and 110 mmHg at temperatures between 23 and 15  C, depending on species (Table 5.1) (Burggren, 1978; Nonnotte et al., 1993; Maxime et al., 1995; McKenzie et al., 1997; Crocker and Cech, 1998). Average dorsal aortic blood pH was between 7.73 and 7.91 in these studies. At these PO2 and pH levels, dorsal aortic hemoglobin‐O2 (HbO2) saturations above 90% can be calculated from in vitro O2 equilibrium curves of whole blood from A. transmontanus (Burggren and Randall, 1978; Crocker and Cech, 1998). A unique feature in this species, and probably also in other members of this genus, is the ability to eYciently ventilate the gills with water drawn in through the upper part of the opercular slits when the retractable, tubelike mouth is blocked, as may occur during feeding (Burggren, 1978). In sturgeons, up to 10% of O2 uptake may occur across the skin (Burggren et al., 1979). Although the sturgeons are water‐breathers, anecdotally they have been reported to be extremely tolerant of aerial exposure. Whether this is associated with transcutaneous gas exchange or the possession of lamellae that do not collapse and are still functional in air is not known. In addition to aerial exposure, white sturgeons are very tolerant of high aquatic CO2 levels (Crocker and Cech, 1998; D. Baker and C. J. Brauner,

226

C. J. BRAUNER AND M. BERENBRINK

unpublished data), and sturgeons in general are very tolerant of aquatic hypoxia [Acipenser baeri (Nonnotte et al., 1993; Maxime et al., 1995, 1998), Acipenser oxyrinchus and Acipenser brevirostrum (Baker et al., 2005), and A. transmontanus (Burggren and Randall, 1978)]. Burggren and Randall (1978) reported that white sturgeons were oxyconformers during exposure _ O2 during to short‐term hypoxia, exhibiting 50%, 15%, and 5% of control M exposure to a PwO2 of 100, 60, and 30 mmHg, respectively. However, in studies on the Siberian (Acipenser baeri) and the Adriatic sturgeon (Acipenser naccarii), no oxyconforming behavior was observed, indicating that these species were oxyregulators (Randall et al., 1992; Nonnotte et al., 1993; McKenzie et al., 1997), like most other fishes studied to date. 2.1.4. Reedfish and Bichirs (Polypteriformes) In the gray bichir, Polypterus senegalus, and the reedfish, Erpetoichthys calabaricus, the ABO is quite well developed (Magid, 1967; Lechleuthner et al., 1989). However, under normoxia all O2 uptake occurs from the water (Babiker, 1984b; Pettit and Beitinger, 1985). In E. calabaricus, transcutaneous O2 uptake accounts for up to 32% of whole‐animal metabolic rate (Sacca and Burggren, 1982). During elevated activity levels or exposure to hypoxia, aerial respiration increases in both species indicating that they are facultative air‐breathers (Babiker, 1984b; Pettit and Beitinger, 1985). In P. senegalus smaller than 25 g, the lungs are not well developed and aquatic hypoxia does not result in air breathing. At this stage of development, a water O2 level of 1.8 mg liter1 results in 100% mortality (Babiker, 1984b). With an increase in body mass up to about 100 g, there is a progressive, almost linear increase in percentage of total O2 consumed that is secured from the ABO (up to 80%) during exposure to aquatic hypoxia (2.8 mg liter1 O2; calculated PwO2 of 57 mmHg). At 100 g, survival was 100% in animals with free access to air during exposure to low aquatic O2 (1.8 mg liter1; calculated PwO2 of 37 mmHg). In adults, air‐breathing frequency increases linearly with a reduction in water O2 levels down to 2.4 mg liter1 (calculated PwO2 of _ O2 is secured across the 49 mmHg) or less, at which time 95% of total M ABO (Babiker, 1984b). Both P. senegalus and E. calabaricus survive low aquatic PO2 values through air breathing, and do not strongly avoid O2 poor waters. E. calabaricus makes voluntary excursions onto land and is tolerant of aerial exposure for several hours without negative eVects (Sacca and Burggren, 1982; Pettit and Beitinger, 1985). However, there are contradictory reports on whether this is associated with an increase or decrease _ O2 (Sacca and Burggren, 1982; Pettit and Beitinger, 1985). in M The creation of a double circulatory loop is present to some degree based on anatomy, but is not as developed as in the obligate air‐breathing lungfish.

5.

GAS TRANSPORT AND EXCHANGE

227

2.2. Lobe‐Finned Fishes (Sarcopterygii) 2.2.1. Coelacanths (Coelacanthiformes) Superficially, the general gill structure in Latimeria chalumnae is similar to that of teleosts. However, it has been hypothesized that their O2 extraction eYciency may be quite low (Hughes, 1976). The lamellae are less well developed, and mass specific total gill surface area is reduced relative to other strictly water‐breathing fishes (Figure 5.1). However, this is a general characteristic of deep dwelling fishes and may not reflect the ancestral condition of lobe‐finned fishes. Metabolic rate has never been measured directly; but on the basis of the low mass specific total gill surface area, metabolic rate has been calculated to be relatively low (i.e., 10 ml kg1 h1) (Hughes, 1976) in comparison with teleost fishes. It is likely that the gills are the predominant sites for O2 and CO2 exchange. However, nothing is known of the potential for transcutaneous gas exchange. 2.2.2. Lungfishes (Dipnoi) The role of the lungs, gills, and skin in O2 and CO2 exchange in lungfishes has been studied extensively in the past century, largely with the goal to gain insight into how aerial respiration may have arisen in ancestral tetrapods. There are a number of excellent reviews on the topic to which the reader is referred (see Johansen, 1970; Burggren and Johansen, 1986; Graham, 1997). Of the three extant lungfish genera, the Australian lungfish (Neoceratodus forsteri) is a facultative air‐breather and its physiology diVers markedly from the obligate air‐breathing lungfishes of South America (Lepidosiren paradoxa) and Africa (Protopterus species). In the two most extensively studied species of obligate air‐ breathing lungfishes, L. paradoxa and P. aethiopicus, the lung has long been thought to be the dominant site for O2 uptake and the gills and/or skin has been the dominant site for CO2 excretion (Johansen and Lenfant, 1967; Lenfant and Johansen, 1967, 1968; McMahon, 1970; Burggren and Johansen, 1986). In adults of these species, aerial O2 uptake satisfies 90–97% of the animal’s O2 requirements, and CO2 excretion to the aquatic environment comprises 40–77% of total CO2 excretion. The circulatory system of these obligate air‐breathing lungfishes has received a great deal of attention due to the degree of separation between pulmonary and systemic blood flow. The eVerent blood vessels from gill arches 3 and 4 give rise to the pulmonary artery leading to the lung, and the pulmonary venous return is delivered directly to a functionally divided heart (see Chapter 2, this volume). Oxygenated blood returning from the lung is then directed toward gill arches 1 and 2, which are largely devoid of respiratory lamellae, and this prevents O2 loss to the water (see Graham, 1997 for a review). While it has long been assumed that the lamellae on

228

C. J. BRAUNER AND M. BERENBRINK

arches 3 and 4 are retained for CO2 excretion, there have been few studies that have tried to delineate the relative role of the gills and skin in CO2 excretion. In L. paradoxa, a detailed morphological analysis revealed that 99.15% of the physiological diVusing capacity lies in the lungs, 0.85% in the skin, and only 0.0013% in the gills (de Moraes et al., 2005). This is reflected in the extremely reduced gill surface area shown in Figure 5.1. From these data, the authors conclude that gills are virtually without importance as a gas exchange organ. Despite the relatively small diVusing capacity of the skin, this route could still account for a large proportion of CO2 excretion (de Moraes et al., 2005). Clearly, physiological studies are required to delineate the relative roles of the gills and skin to CO2 elimination in these obligate air‐breathing lungfishes. In L. paradoxa, temperature has a large eVect on the role of the lung in CO2 excretion. A 20  C increase in temperature (from 15 to 35  C), resulted in a 15‐fold elevation in metabolic rate, and CO2 excretion across the lung increased from less than 15% to about 70% of total CO2 excreted (Amin‐Naves et al., 2004). Thus, with an elevation in metabolic rate, the lung may take on a greater role in CO2 excretion; however, this was associated with a respiratory acidosis where PaCO2 increased from 12 to 35 mmHg, and pHa decreased from 7.6 to 7.35 (Amin‐Naves et al., 2004). In a study on an obligate air‐breathing species of African lungfish, P. dolloi, the lung plays the dominant role in both O2 and CO2 exchange (91% and 76%, respectively; Perry et al., 2005). During exposure to aerial and aquatic hypercapnia, only the former induced a respiratory acidosis. The apparent lack of CO2 uptake across the gills during hypercapnia could be due to a limited role of the gills for CO2 exchange, or, alternatively, CO2 excretion across the lung could be suYcient to excrete the CO2 taken up across the gills in this species. Thus, the textbook description of the relative role of the lungs and gills to obligate air‐breathing lungfishes in terms of O2 and CO2 exchange is not as clear as once thought, and is clearly in need of further detailed investigation. The Australian lungfish, N. forsteri, is a facultative air‐breather and obligate water‐breather incapable of surviving prolonged aerial exposure, unlike P. aethiopicus and L. paradoxa. Gill surface area is slightly reduced compared to teleosts (Figure 5.1), but aerial respiration can augment O2 uptake during elevated metabolic rate associated with activity or during exposure to low water PO2 . Interestingly, during exposure to aquatic hypoxia, N. forsteri responds with an immediate, large, and sustained increase in gill ventilation, which diVers markedly from the obligate air‐breathing lungfishes P. aethiopicus and L. paradoxa, which exhibit no change in branchial ventilation, consistent with the limited role of gills for O2 uptake in the latter (Burggren and Johansen, 1986; Sanchez et al., 2001). The increase in gill ventilation during hypoxia in N. forsteri is maintained during hypoxia even

5.

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following the onset of air breathing (Fritsche et al., 1993; Kind et al., 2002). In P. aethiopicus, P. amphibius, and P. annectens, juvenile stages are more dependent on aquatic O2 uptake than adults (Johansen et al., 1976a; Babiker, 1979; Burggren and Johansen, 1986; Seifert and Chapman, 2006), and juveniles respond to hypoxic water by elevating both gill ventilation and air‐breathing frequency. The greater dependence on aquatic O2 uptake in juvenile animals is likely a reflection of a less‐developed lung (Babiker, 1979). Clearly, there are interesting developmental changes that occur within these obligate air‐breathing lungfishes that remain to be investigated. All lungfishes are able to tolerate aquatic hypoxia through the use of air breathing, as is the case for many other primitive fishes. However, both L. paradoxa and the species of Protopterus possess yet another line of defense against unfavorable environments, in that they can estivate. In estivating animals, the metabolic rate is greatly reduced (to 1–20% of resting rate in water) and the animals become ureotelic (Fishman et al., 1986; reviewed by Graham, 1997). Thus, in addition to being virtually unaVected by poorly oxygenated waters through breathing air, they tolerate desiccation of the environment for long periods of time. 2.3. Jawless Fishes (Agnatha) 2.3.1. Lampreys (Petromyzontiformes) Both larval and adult stages of the lamprey Geotria australis are capable of surviving out of water in humidified air for up to at least 4 days (Potter et al., 1996b) without apparent negative eVects. During this time, metabolic rate is similar to that measured in G. australis held in water, and most of the O2 uptake during air exposure is cutaneous (Potter et al., 1996b). However, in air‐exposed adult G. australis, the gills are responsible for 87% of O2 uptake and 80% of CO2 excretion, indicating that the gills in adults retain their integrity in air (Potter et al., 1997). Lampreys, such as G. australis, are exposed to air when they leave the water to overcome barriers during their upstream migration (Potter et al., 1996a), and thus aerial exposure may be common. Total gill surface area to body weight in adult river lamprey, Lampetra fluviatilis, is similar to that of active teleosts (Figure 5.1) (Lewis and Potter, 1976), and metabolic rate in resting and exercising Petromyzon marinus appear to be in the order of that of teleosts (Beamish, 1973). The gills are the predominant site of O2 uptake, and presumably CO2 excretion, in water. 2.3.2. Hagfishes (Myxiniformes) Hagfishes (Eptatretus cirrhatus and E. stoutii) have a metabolic rate that is considerably lower than in teleost fishes of similar mass and temperature, and is among the lowest recorded among vertebrates (Munz and Morris, 1965; Forster, 1990). The skin in hagfishes may play a significant role in O2 uptake,

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and, on the basis of calculations by SteVensen et al. (1984) for the Atlantic hagfish (Myxine glutinosa), may be responsible for up to 80% of whole‐animal metabolic rate! Dermal capillaries have been identified in several species of hagfishes (Potter et al., 1995). During feeding, this may be important when the head is buried in the prey, preventing ventilation through the nostril.

3. BLOOD O2 TRANSPORT The general blood O2 transport characteristics of fishes have been reviewed in detail in a previous volume of the Fish Physiology series, including comprehensive chapters on Hb structure and function (Jensen et al., 1998a), general O2 transport (Nikinmaa and Salama, 1998), hematocrit and blood O2‐carrying capacity (Gallaugher and Farrell, 1998), the physiology of the Root eVect (Pelster and Randall, 1998), and the linkage between O2 and CO2 transport (Brauner and Randall, 1998). These chapters extensively dealt with teleost fishes and also covered elasmobranchs and agnathans. However, the basal members of the ray‐finned fishes and lobe‐finned fishes were usually only treated in passing, if at all. Here we first recapitulate general aspects of vertebrate blood O2 transport and then review the fragmentary information on primitive fishes. 3.1. General Principles of Hb Function Reversible binding of O2 to Hb inside RBCs is crucial to the uptake, transport, and delivery of O2 in whole blood of nearly all vertebrates. Briefly, O2 reversibly binds to the Fe atom in a heme group, which itself is attached to a globin polypeptide chain. Hbs of jawed vertebrates consist of a‐type and b‐type globins, which align to form tetramers consisting of two ab‐dimers and thus contain four O2‐binding sites. In most cases, O2 binding by the tetrameric Hb of jawed vertebrates can be satisfactorily explained by the two‐state allosteric model, whereby Hb is in equilibrium between a low‐O2 aYnity, T(ense)‐state conformation, and a high‐O2 aYnity, R(elaxed)‐state conformation. Changes in the O2‐binding properties of Hb can be explained by a number of factors interacting diVerentially with the two conformations, thereby stabilizing one form over the other. Hb in the jawless Agnatha contains only one general type of globin chain, which is in monomer–oligomer equilibrium and has a higher O2 aYnity in the monomeric than in the dimeric or tetrameric aggregation state. Again, factors aVecting the equilibrium between aggregation states determine the whole‐blood O2‐binding properties.

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The Hb‐O2‐binding characteristics are generally believed to be fine‐tuned to meet the variable O2 requirements of an organism. Increased metabolic demand for O2 due to, for example, exercise, digestion, or an increased body temperature, can principally be met by an increased cardiac output and/or an increased arteriovenous O2 diVerence. In the following sections, the latter will be discussed in this respect. With the exception of Hb‐less icefishes, changes in the amount of physically dissolved O2 between arterial and venous blood of vertebrates are usually negligible when compared to the changes in Hb‐bound O2. Therefore, the arteriovenous O2 diVerence is mainly a function of the diVerence in Hb‐O2 saturation between arterial and venous blood and blood Hb concentration.

3.2. Factors AVecting the ArterioVenous O2 DiVerence 3.2.1. Blood Hb CONCENTRATION The amount of O2 that can be maximally taken up by the blood in addition to physically dissolved O2 is a direct function of the functional blood Hb concentration and is termed the O2 capacity. Several early studies did not correct their O2 capacity measurement for physically dissolved O2, which is insignificant at high Hb concentrations, but can make up a large fraction of total O2 at low Hb concentration, high PO2 , or low temperature. There is generally a strong positive correlation between blood Hb concentration and hematocrit, both within and across species (Graham, 1997; Gallaugher and Farrell, 1998). This supports the early finding of a relatively constant Hb concentration inside RBCs across vertebrates (Wintrobe, 1934). Table 5.1 gives values for blood Hb concentration, hematocrit, and mean cellular Hb concentration in a number of primitive fishes. As can be seen, blood Hb concentration can be extremely variable even within the same species, making it diYcult to identify adaptive trends in this parameter when comparing species in diVerent environments or with diVerent lifestyles (Gallaugher and Farrell, 1998). During exercise or hypoxia, a short‐term response of several teleosts is to release additional RBCs form their spleen and thereby increase blood Hb concentration; a more long‐term response involves increased production of new RBCs. With few exceptions, hypoxia induces erythropoiesis also in teleost fishes as in other vertebrates (Gallaugher and Farrell, 1998). 3.2.2. The O 2 Equilibrium Curve and Its Modulators Outside extremely low and high O2 saturations, the shape of the O2 equilibrium curve can be satisfactorily characterized by the overall aYnity and cooperativity of Hb‐O2 binding. A useful, inverse measure for Hb‐O2

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aYnity is the P50, the PO2 required for half‐maximal saturation of Hb with O2. The cooperativity of Hb‐O2 binding determines the steepness of the O2 equilibrium curve in the vicinity of the P50. Cooperativity is expressed as the Hill‐coeYcient, nH, which is usually determined at 50% O2 saturation and is obtained as the slope of the line that results when the O2 equilibrium curve data are transformed such that the log of the oxyHb to deoxyHb ratio is plotted against log PO2 . Values for nH around unity signify noncooperative, hyperbolic O2 equilibrium curves, whereas nH values increasing above unity indicate increasing degrees of cooperativity. In fish Hbs that display a Root eVect, nH values below unity are frequently observed at low pH and are thought to reflect heterogeneity in subunit O2 aYnity. a. Hb Multiplicity. In the simplest case, Hb‐O2 saturation at any one PO2 can be calculated from the prevailing P50 and nH value. However, many fish species, primitive and modern, contain multiple Hbs in their blood, which often diVer significantly in aYnity and cooperativity. This can result in undulatory O2 equilibrium curves because at increasing PO2 values, the Hb fraction with the highest O2 aYnity is saturated significantly before the others, resulting in a flattening of the overall curve at intermediate PO2 values (Maginniss et al., 1980). Whenever O2‐binding sites diVer in O2 aYnity, either as a result of diVerent Hb isoforms or because of subunit heterogeneity, the apparent nH values are reduced as a mathematical consequence, sometimes below unity. b. Evolutionary and Ontogenetic Changes. Even with only one major Hb component, the shape of the O2 equilibrium curve is quite plastic and influenced by several factors. Over evolutionary timescales, genetic changes in the amino acid sequence of the globin chains can aVect the intrinsic O2 aYnity and cooperativity. Further, over the life span of individuals, ontogenetic changes in the expression of genetically diVerent Hbs have been described in virtually all major vertebrate groups such that embryos, juveniles, and adults often express diVerent, life stage‐specific Hbs (Nikinmaa, 1990). c. Organic Phosphates. Over even shorter timescales, phenotypic changes in the shape of the O2 equilibrium curve of jawed vertebrates may be brought about by species‐specific organic phosphate molecules, which bind allosterically to the b‐ chains of deoxygenated Hb and thereby reduce Hb‐O2 aYnity. In contrast to mammalian and avian RBCs, which contain high concentrations of 2,3‐diphosphoglycerate (2,3‐DPG or 2,3‐BPG) and inositol‐pentaphosphate (IP5), respectively, the principal organic phosphates of most fishes are ATP and GTP, in varying amounts. The RBC concentrations of ATP and GTP are reduced during hypoxic acclimation or

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on RBC swelling, leading to a higher Hb‐O2 aYnity under these conditions (Val, 2000). d. Temperature. Due to the exothermic nature of Hb‐O2 binding, increased temperatures usually decrease Hb‐O2 aYnity. This physicochemical property of Hb will necessarily cause a shift in O2 aYnity of blood between metabolically active, heat‐producing tissues and relatively cooler, water‐exposed respiratory organs. O2 aYnity will also vary with daily and seasonal temperature changes and with the evolution of regional or complete endothermy, unless compensated by phenotypic or genetic mechanisms. e. pH. Hb‐O2 aYnity is also profoundly aVected by pH and this eVect dynamically changes O2‐binding properties in the time frame of seconds when blood circulates between arterial and venous capillaries because of the metabolic acids produced and released by the tissues. In the physiological pH range, a decrease in pH lowers O2 aYnity and consequently increases P50, causing a right‐shift of the O2 equilibrium curve. This is the so‐called Bohr eVect, various aspects of which have been reviewed, including a series of papers celebrating the centennial of the first description of the phenomenon by Bohr et al. (1904; Giardina et al., 2004; Jensen, 2004; Berenbrink, 2006). The magnitude of the Bohr eVect is often quantified by the Bohr coeYcient F, which gives the change in log P50 on a unit change in pH: f¼

D log P50 1 ¼  DzHþ 4 D log pH

ð1Þ

Alternatively, F can be estimated from acid–base titrations of Hb as one‐ fourth of the number of protons that are released from tetrameric Hb when it changes from being completely deoxygenated to being fully oxygenated at constant pH (Haldane eVect), as also shown in Eq. (1). Strictly, the two expressions for the Bohr eVect are only equivalent when the shape of the O2 equilibrium curve is symmetrical (otherwise the log of the median PO2 has to be used), and in the absence of other eVectors that may interact diVerentially with oxygenated and deoxygenated Hb (including CO2, and the anion A of the acid AH that is used to change pH) (Wyman, 1964). In whole blood in vivo, in the presence of multiple Hbs or organic phosphate modulators, these conditions are rarely achieved. The source of the so‐called Bohr protons [DzHþ in equation (1)], and thereby also the molecular mechanism of the Bohr eVect, is quite diVerent in Hbs of jawed vertebrates and agnathans, and increasing evidence suggests that the mechanism also diVers within the jawed vertebrates (Berenbrink, 2006).

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The Bohr eVect is advantageous in increasing O2 unloading by a right‐ shift of the O2 equilibrium curve in acidic tissues and enhancing O2 loading by a left‐shift at the respiratory organs, where CO2 is released and pH increases. Its ultimate benefit is more accurately described by the extent to which it increases the arteriovenous O2 diVerence, which does not only depend on the shift of logP50 with pH (i.e., F) but also on the respective arterial and venous PO2 values and nH (Bartels, 1972). But even when these parameters are identical, the extent to which a given acid load in the tissues aVects the arteriovenous O2 diVerence in two species may be quite diVerent. This is due to species‐specific diVerences in (1) the transfer mechanisms of extracellular acid–base loads to the interior of the RBCs, where protons ultimately act on Hb‐O2 aYnity; (2) the net charge of Hb, which aVects RBC pH at a given extracellular pH; and (3) the degree of intracellular proton buVering, mainly by Hb itself (Sections 3.2.2.g and 3.2.2.h). Many fishes show an extreme reduction of Hb‐O2 aYnity at low‐pH values, which results in whole blood not being fully saturated with O2 even when equilibrated with air (Root, 1931). In some cases, the blood at low pH cannot even be fully saturated when equilibrated with pure O2 at pressures of several atmospheres. This has been ascribed to a decrease in Hb‐O2 capacity and is known as the Root eVect (Scholander and Van Dam, 1954). However, it is somewhat arbitrary to define a fixed PO2 above which failure to become fully saturated constitutes a Root eVect and below which that failure constitutes a Bohr eVect. Clearly, on the basis of the saturation criterion at fixed PO2 alone, the distinction between a strong Bohr eVect and a small Root eVect is diYcult (Farmer et al., 1979), as shown by the debate about whether the amphibian Xenopus laevis possesses a Root eVect (Perutz and Brunori, 1982; Bridges et al., 1985; Kister et al., 1989; Berenbrink et al., 2005). Dilute hemolysates usually have a much higher O2 aYnity than whole blood because of the diminished interaction between organic phosphates and Hb. Thus, the failure of air‐equilibrated hemolysates to become completely saturated at low pH and physiological temperature is often accepted as indicating the presence of a Root eVect, and the percentage of Hb remaining deoxygenated under these conditions is used to quantify the eVect (Farmer et al., 1979; Berenbrink et al., 2005). In contrast to the Bohr eVect, many Root eVect Hbs show a conspicuous decrease in nH at low pH, and this property has been proposed to diVerentiate between the two eVects (Perutz and Brunori, 1982). In teleosts and the bowfin, the presence of a Root eVect is associated with the presence of specialized vascular countercurrent exchangers in the eye or swim bladder (choroid and swim bladder retia mirabilia, singular: rete mirabile; Farmer et al., 1979; Berenbrink et al., 2005). The Root eVect allows

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the acid‐induced oZoading of Hb‐bound O2 even at high PO2 values. Together with countercurrent multiplication by the retia mirabilia, this is used to fill the swim bladder with O2 against high hydrostatic pressures for buoyancy regulation or to sustain the high metabolic demand of the poorly vascularized retina of many fishes (Pelster and Randall, 1998; Waser and Heisler, 2005). Theoretically, a Root eVect may be used to increase the PO2 in other tissues. However, as there are no reports of gas‐exchanging vascular retia mirabilia outside the eye and swim bladder, which could act as countercurrent multipliers, the degree of PO2 elevation is necessarily limited. With a blood O2 solubility coeYcient of 2 mmol liter1 mmHg1, a typical tetrameric blood Hb concentration of 1 mmol liter1, and a maximal Root eVect of 50%, fully oxygenated blood that is acidified in a closed system will show a theoretical PO2 increase of 1000 mmHg. Although in vivo the assumption of a closed system is practically never fulfilled, especially so in the absence of the barrier function of a rete mirabile, a Root eVect may be the cause behind PO2 values slightly above air saturation in postbranchial blood of exercised striped bass (Nikinmaa et al., 1984) and higher than expected PO2 values in red muscle tissues of rainbow trout (McKenzie et al., 2004). However, it is currently unclear whether blood in red muscle is suYciently acidotic, even transiently, for a significant Root eVect to occur. Clearly more research is needed before a physiological function of the Root eVect outside the eye and swim bladder can be ascertained. f. CO2. A large part of the eVect of elevated blood PCO2 on Hb‐O2 transport is caused by the pH change generated by the hydration of CO2 to carbonic acid and its subsequent dissociation to bicarbonate and protons. However, as in mammals, a specific eVect of CO2 has also been reported for the Hb‐O2 aYnity of some fishes (Farmer, 1979). Again as in mammals, CO2 is thought to bind preferentially to the N‐terminal amino groups of deoxygenated Hb as a carbamino compound, thereby decreasing Hb‐O2 aYnity. The eVect is stronger at high pH values because CO2 reacts with the unprotonated N‐terminal amino groups, whereby the Bohr eVect may be partially oVset in the presence of CO2. However, the eVect of CO2 is not as strong in teleosts as in mammals, most likely because of the blockage of some of the N‐terminal amino groups in teleosts (Jensen et al., 1998a). It is unclear whether CO2 competes with organic phosphates in binding to deoxygenated fish Hbs, as shown for human Hb (Kilmartin and Rossi‐Bernardi, 1973), and whether CO2 will have an appreciable eVect in the presence of physiological ATP and GTP concentrations. Purely water‐breathing fishes are generally characterized by lower blood PCO2 values than air‐breathers (Rahn, 1966), questioning the physiological role of CO2 in modulating Hb‐O2 aYnity in these fishes (but see hagfishes below).

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g. RBC pH Regulation. Possession of even the strongest Bohr eVect Hb would be physiologically meaningless if the changes in plasma acidity that occur between tissues and respiratory surfaces were not transferred from the plasma to the RBC interior. In all jawed vertebrates studied so far, a powerful anion exchange protein (AE1, band 3) rapidly equilibrates acid–base equivalents in form of the bicarbonate anion across the RBC membrane (Nikinmaa and Salama, 1998; Jensen, 2004). Whether blood CO2 increases by respiratory acidosis or whether it is generated during a metabolic acidosis when increased proton concentrations in the plasma lead to the formation of CO2 from plasma bicarbonate, CO2 readily diVuses across the RBC membrane into the RBC. Here the equilibrium of the CO2 hydration reaction is shifted toward the formation of bicarbonate and protons. RBC bicarbonate is exchanged for plasma chloride and leaves the cell, whereas the proton acidifies the RBC interior. The net eVect is an entry of Hþ and Cl (the dissociation products of HCl) by which extracellular pH changes are directly transmitted to the RBC cytosol. Sodium and potassium, the main intra‐ and extracellular cations, are regulated at a rather constant level, and in the absence of acid–base relevant ion transporters other than AE1, the passive equilibrium distribution of protons across the RBC membrane largely depends on the net charge of membrane impermeable anions, mainly Hb and plasma proteins (Nikinmaa, 1990; Nikinmaa and Salama, 1998). The much higher concentration of intracellular Hb compared to plasma proteins together with its negative charge causes a higher intracellular than extracellular proton concentration. Across the physiological plasma pH range, intracellular pH is therefore distinctly lower than plasma pH. The transmembrane pH diVerence is directly influenced by changes in Hb charge, such that it decreases on deoxygenation when the binding of Bohr protons neutralizes negative charges on Hb. Titration of negative charges on intracellular Hb at low pH also causes the transmembrane pH diVerence to diminish. Therefore, a unit decrease in plasma pH causes a somewhat smaller decrease in RBC pH so that the Bohr coeYcient F in studies on whole blood or isolated RBCs needs to be related to the intracellular pH when it is compared with studies on Hb in solution. Due to their negatively charged phosphate groups, organic phosphates contribute to the number of impermeable intracellular negative charges, and their concentration therefore also aVects the transmembrane Hþ equilibrium of RBCs. Thus, a decrease in intracellular organic phosphates will decrease the transmembrane pH diVerence (Nikinmaa and Salama, 1998). In many teleost species and lampreys, secondarily active transport of protons elevates intracellular pH above its equilibrium value (Nikinmaa and Huestis, 1984; Nikinmaa, 1986; Berenbrink and Bridges, 1994a,b).

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In lampreys, RBC pH is continually elevated above the Hþ equilibrium distribution by a Naþ/Hþ exchanger (NHE), increasing Hb‐O2 aYnity because of the strong Bohr eVect of lamprey Hb (described below). In teleosts, a similar mechanism is activated by catecholamines, whose plasma levels increase during exercise stress or hypoxia. As in lampreys, the ensuing increase of RBC pH via the adrenergic NHE increases Hb‐O2 aYnity in teleosts, which often have a very large Bohr eVect in addition to a Root eVect. The method of blood sampling in many early studies very likely caused elevated plasma catecholamine levels. If the species under investigation possessed an adrenergic NHE, the reported whole‐blood O2‐binding properties clearly would not represent resting conditions. In Atlantic cod RBCs, hypercapnic acidosis activates another secondarily active, pH regulatory transporter, which has been characterized as sodium‐ dependent chloride/bicarbonate exchange. This transport mechanism elevates intracellular pH and will thereby also aVect Hb‐O2 binding (Berenbrink and Bridges, 1994a). h. Specific Hb BuVer Capacity. The extent to which a given acid load entering the RBC aVects intracellular pH and thereby the Bohr and Root eVect critically depends on the specific buVering capacity of Hb (bHb). Hb is the main nonbicarbonate blood buVer and bHb is the molar concentration of acid–base equivalents that is required to change the pH of an Hb solution of a given molar concentration (in the absence of other buVers) by one unit. It is determined by acid–base titration at constant oxygenation status and yields negative values because addition of acid decreases the pH. In the following sections, values for bHb refer to measurements on deoxygenated, tetrameric Hb at physiological temperature, chloride concentration, and RBC pH. 3.2.3. Arterial andVenous PO2 Hb‐O2 saturation is principally determined by the shape of the prevailing Hb‐O2 equilibrium curve and PO2 . High PaO2 values ensure full blood O2 saturation in respiratory organs. Generally, PaO2 in gills and lungs is a function of the PO2 in inspired water or air and therefore environmental PO2 . It is aVected by the extent and type of ventilatory movements and the general anatomy of the gas exchange organs, such as countercurrent arrangement of gill blood and water flow (Chapter 4, this volume). As O2 transfer _ O2 is from water or air to blood ultimately occurs by diVusion, maximal M limited by the respiratory surface area of gills and lungs and the length of the water to blood diVusion path. Strictly water‐breathing teleosts fall into two broad groups regarding the level of resting PaO2. Contrary to the suggestion of generally low PaO2

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values in water‐breathing fishes (Lenfant et al., 1970), available data suggest that salmonids and other teleosts that require well‐oxygenated water, such as Atlantic cod or striped bass, have a high normoxic resting PaO2 around 100 mmHg (Nikinmaa and Salama, 1998), similar to or sometimes even exceeding values in the air‐breathing birds and mammals. On the other hand, cyprinids show normoxic resting PaO2 values of 20–40 mmHg. This is not due to a low diVusion capacity of the gills in these animals but is regulated by intermittent ventilation as shown by the increased PaO2 values under exercise‐induced hyperventilation (Jensen et al., 1983; Koldkjær and Jensen, 1998; see Nikinmaa and Salama, 1998). Species with low resting PaO2 values commonly have a high blood O2 aYnity, such that nearly complete Hb‐O2 saturation can nevertheless be achieved. These species are often also hypoxia tolerant, like the common carp, but so far the diVerences in the regulation of PaO2 values in teleosts have not been systematically addressed and it is unclear to what extent phylogenetic position plays a role. PO2 at the principle site of O2 consumption in the mitochondria can be considered to be close to zero, and therefore the diVusive O2 transfer from venous blood to tissue mitochondria is directly related to venous PO2 (PvO2). Tissue O2 supply is therefore enhanced by Hbs, which oZoad O2 at relatively high venous PvO2 values. The other major factor determining the rate of diVusive O2 transfer from blood to mitochondria in a given tissue is the diVusive O2 conductance. The latter depends on factors such as the geometry of the structures through which diVusion occurs (e.g., capillary density and length), the O2 diVusion coeYcients of the various tissue components, their O2 capacitance coeYcients, and whether myoglobin‐facilitated diVusion occurs (Dejours, 1975). Except under special circumstances like in O2 secretory structures, PvO2 must be lower than PaO2 for tissue oxygenation, and Hbs requiring only a small decrement from PaO2 for oZoading significant amounts of O2 (i.e., Hbs with PaO2 and PvO2 in the steepest part of the O2 equilibrium curve) are most eYcient for tissue O2 supply. On the other hand, to ensure near complete saturation of Hb in arterial blood, PaO2 should ideally be regulated in the flat upper part of the O2 equilibrium curve, that is, away from the steep portion. Perhaps it is for this reason that PaO2 usually exceeds PvO2 by a safety margin of several mmHg. In primitive air‐breathing fishes, the accessory ABOs are arranged in parallel to the systemic tissues and even if PO2 in blood leaving these organs is high, the admixture of systemic mixed venous blood in an undivided heart will subsequently reduce it. If the gills are reduced, collapsed, or contain shunt pathways, the PO2 of mixed blood will be little changed and constitute the systemic arterial PO2 . If the gills are well developed, not collapsed, and contain no shunts, PO2 of mixed blood can be increased or decreased, depending on whether the PO2 gradient to inspired water is positive or negative.

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3.3. Survey of Extant Primitive Fishes 3.3.1. Ray‐Finned Fishes (Actinopterygii) a. Bowfin (Amiiformes). Relative to many teleost fishes, the P50 in whole blood of A. calva is quite low, ranging from 4 to 24 mmHg depending on the temperature, pH, and CO2 level (Table 5.1). The Bohr coeYcient (F) in whole blood is largely temperature independent and ranges from –0.43 to 0.51 (Table 5.1). The Bohr eVect in washed RBCs at 15  C in the absence of CO2 is large, with F ¼ 1.0 (Table 5.1) (Weber et al., 1976b). It is unclear whether the somewhat reduced Bohr eVect in whole blood is due to a specific CO2 eVect since arterial PCO2 is low when the fish is water breathing (>4 mmHg, Randall et al., 1981). A. calva blood contains at least five diVerent Hb components with broadly similar functional properties and amino acid compositions (Weber et al., 1976b). At a PO2 of 150 mmHg, a PCO2 of 26 mmHg depressed Hb‐O2 saturation in A. calva whole blood by only 8% (Black and Irving, 1938). Johansen et al. (1970) similarly found that a pH change from 8.0 to 7.2 only caused a 10% reduction in O2 capacity and judged the Root eVect in A. calva whole blood as insignificant. The apparent lack of a Root eVect in whole blood is surprising given the importance of the Root eVect for O2 secretion (Waser and Heisler, 2005), the presence of a well‐developed choroid rete mirabile in the eye of A. calva and PO2 values considerably above air saturation close to the retina (Wittenberg and Wittenberg, 1974). In contrast, the hemolysate shows a strong Root eVect of about 60% (Weber et al., 1976b; Berenbrink et al., 2005). Plasma pH changes during and after exhaustive exercises are transferred to the RBC interior (Gonzalez et al., 2001). The changes in RBC chloride concentration and water content with changes in extra‐ and intracellular pH in vitro and the inhibition of proton equilibration across the RBC membrane by the anion exchange inhibitor DIDS further all point to a passive equilibration of protons across the RBC membrane (Tufts et al., 1994). Using the formula obtained for washed A. calva RBCs by the latter authors, the extracellular pH of 7.2 in Johansen et al. (1970) corresponds to an RBC pH of about 6.8. This value is suYcient for 50% Hb deoxygenation in the hemolysate at saturating ATP concentrations (Figure 4 in Weber et al., 1976b). A. calva RBCs do not possess an adrenergic NHE (Tufts et al., 1994; Berenbrink et al., 2005), which could otherwise have elevated RBC pH and thereby shifted the onset of the Root eVect to lower plasma pH values in the study by Johansen et al. (1970). Perhaps reduction of O2 capacity by only 10% at pH 7.2 in the latter study was due to less than saturating organic phosphate concentrations in the RBCs. Saturating levels of organic phosphates are known to shift the onset of the Root eVect to higher pH values (Weber et al., 1976b). From this it follows that blood pH in the choroid rete mirabile of A. calva must be below 7.2 for maximal exploitation of the Root eVect.

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b. Gars (Lepisosteiformes). At 20  C, whole blood of the spotted gar, L. oculatus, had a P50 of 24.1 mmHg at 1% CO2 in vitro, which increased to 30.4 at 2% CO2. The shape of the O2 equilibrium curves was distinctly sigmoidal, and possessed F ¼ 0.5 (Table 5.1) (Smatresk and Cameron, 1982a), which was similar to the value measured above in whole blood from A. calva. RBCs of L. oculatus and the Florida gar Lepisosteus platyrhincus lack significant adrenergic NHE activity (Berenbrink et al., 2005). In L. platyrhincus, the Bohr eVect in organic phosphate‐free hemolysates at physiological temperature, pH, and chloride concentrations was F ¼ 0.525 and bHb was 8.6 per mmol Hb4 and pH. This magnitude of the Bohr eVect is distinctly lower and the value for bHb distinctly higher than in isolated Hbs of A. calva and most teleosts under the same conditions (Table 5.1) (Berenbrink et al., 2005). Changing the PCO2 of air‐equilibrated whole blood of L. oculatus from 1% to 2% CO2 caused pH to drop from 7.8 to 7.4 and was associated with a statistically significant decrease in O2 capacity by about 13%, which was taken to indicate a Root eVect (Smatresk and Cameron, 1982a). However, it is not clear to what extent the decrease in Hb‐O2 binding was caused by oxidation of Hb, which readily occurs at low pH values. Hemolysates of L. oculatus and L. platyrhincus show a Root eVect of about 43% and 40%, respectively (Berenbrink et al., 2005). The presence of strong Root eVect Hbs in Lepisosteiformes may be surprising in view of the general absence of a choroid or swim bladder rete mirabile outside teleosts and A. calva (Berenbrink et al., 2005). If the reduction of whole‐blood O2 capacity at pH 7.4 was indeed caused by a Root eVect (Smatresk and Cameron, 1982a), the low blood pH values under severe exercise (Burleson et al., 1998) indicate that the Root eVect may be quite important at physiological blood pH values. However, in the absence of gas exchanging retia mirabilia in gars, which will concentrate CO2 as well as O2, it is unclear whether low enough RBC pH values can be achieved to elicit the full magnitude of the Root eVect in vivo. Despite the absence of a choroid rete mirabile, maximal ocular PO2 values ranged between 72 and 145 mmHg in L. oculatus and alligator gar [Atractosteus (formerly Lepisosteus) spatula], with an average of 90 mmHg (Wittenberg and Wittenberg, 1974). Even the lowest recorded individual ocular PO2 value is about twice as high as the highest recorded average dorsal aortic PO2 value of 37 , which was observed after recovery from exhaustive exercise in normoxic water at 23–24  C (Burleson et al., 1998). Ocular PO2 measurements were performed on restrained animals submerged in surface water between 22 and 33  C immediately after they had been caught from bottom temperatures of 23  C. However, even when it is assumed that the eye is a closed system and does not consume O2 (neither

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of which is true), the calculated temperature‐induced increase in PO2 by the decreased O2 solubility is only 20% (Wittenberg and Wittenberg, 1974). This indicates that ocular PO2 is elevated above PaO2 in the absence of a choroid rete mirabile and suggests that the strong Root eVect of Lepisosteus Hbs may be involved in O2 delivery to the eye. Even in the absence of a distinct choroid rete mirabile, a certain degree of countercurrent gas exchange may contribute to the elevated ocular PO2 given the general blood supply characteristics of vertebrate eyes. The ophthalmic artery and vein often share a single entry/ exit point in the sclera. From here, arterial and venous networks fan out in close vicinity to supply and drain the choroid capillaries. This arrangement by necessity already creates favorable conditions for countercurrent gas exchange. Clearly more studies are necessary to establish whether the eyes of gars resemble an intermediate stage in the evolution of O2 secretion, which is so powerfully developed in A. calva and teleosts (Wittenberg and Wittenberg, 1974; Berenbrink et al., 2005). c. Sturgeons and Paddlefishes (Acipenseriformes). No published information appears to exist on the blood O2 transport characteristics of the two paddlefish genera and all information for Acipenseriformes stems from studies on just one out of four sturgeon genera, namely Acipenser. Whole blood P50 is 21.5 mmHg in A. transmontanus (Table 5.1). The Bohr coeYcient in whole‐blood ranges from 0.4 to 0.55 (Table 5.1). Intracellular RBC pH in A. naccarii is much lower than plasma pH (McKenzie et al., 1997). Studies on washed RBCs of A. baeri suspended in CO2/bicarbonate buVered salines show that extracellular pH changes are transferred to the RBC interior to a similar extent as in rainbow trout RBCs under the same conditions (A. J. da Silva and M. Berenbrink, unpublished data). The latter study provided evidence for a small Root eVect as RBC acidification by CO2 at a PO2 of 150 mmHg caused 15% and 20% deoxygenation at pH 7.1 and 6.6, respectively. Plasma catecholamine levels sharply increase during mild and severe acute hypoxia in A. naccarii and A. transmontanus (Randall et al., 1992; Maxime et al., 1995), an adrenergic NHE, however, appears to be missing in washed RBCs of A. baeri and sterlet (Acipenser ruthenus; A. J. da Silva and M. Berenbrink, unpublished data; Berenbrink et al., 2005), as well as in A. transmontanus (Baker, Rummer, and Brauner, unpublished data). The RBC GTP concentration in A. naccarii is equal to, and in A. ruthenus is twice as high as the RBC ATP concentration (Clementi et al., 1999; O. Kepp and M. Berenbrink, unpublished data). The Bohr eVect and P50 of purified hemolysates of A. naccarii in the presence of physiological chloride concentrations and excess GTP are similar to the values in washed RBCs in phosphate buVer (Clementi et al., 1999).

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The Hb system of A. naccarii consists of two Hb components in equal amounts composed of three a‐ and two b‐chain types, with the individual Hb components apparently containing diVerent a‐ and b‐chains in each component (Clementi et al., 2001a,b). The small Root eVect in RBCs of A. baeri (see above) is also seen in hemolysates, which show Root eVect magnitudes of 15–26% in A. ruthenus, A. naccarii, and A. baeri (Clementi et al., 1999; Berenbrink et al., 2005). No swim bladder rete mirabile is present in the genus Acipenser (Mu¨ller, 1839; Bridge, 1904), and swim bladder O2 secretion also appears to be absent (Fa¨nge, 1966). Despite the generally more benthic lifestyle, the swim bladder is regressed only in a few sturgeon species (Rauther, 1922). Presumably a normal‐sized swim bladder is useful during the long migrations undertaken by some sturgeon species. No choroid rete mirabile has been found in the genera Acipenser, Scaphirhynchus, and Polyodon (Mu¨ller, 1839; Rauther, 1937; Wittenberg and Haedrich, 1974). However, Rodrı´guez and Gisbert (2001) have mentioned a choroid gland, an old name for the choroid rete mirabile, in 5‐ to 6‐day‐old larvae of A. baeri. The structure is described as conjunctive tissue with two pigmented layers and hence may not constitute the vascular countercurrent exchange system usually described by this name. TretjakoV (1926) has described a suprachorioidea in the starry sturgeon, A. stellatus, consisting of closely arranged sinusoid veins, of which the larger veins regularly alternate with larger arteries. He suggested that the choroid rete mirabile of teleosts may have evolved from a similar arrangement (TretjakoV, 1926). PO2 and pH in choroid capillaries in the eyes of sturgeons are not known, so it is unclear whether the small Root eVect is used in ocular O2 secretion. Arterial pH values of 7.1 or below have been measured during external hypercapnia or deep hypoxia (Burggren and Randall, 1978; Crocker and Cech, 1998), and the very much lower RBC pH compared to plasma pH (McKenzie et al., 1997) all suggest that the Root eVect may be occurring in vivo in the general circulation, let alone under the more acidic conditions in tissues. d. Reedfish and Bichirs (Polypteriformes). The P50 of in vitro whole blood of the gray bichir (P. senegalus) at 30  C is 23.5 mmHg (Table 5.1) (Vokac et al., 1972). Hb‐O2 binding is sigmoid with nH ¼ 2.68 (calculated from Fig. 2B in Vokac et al., 1972) and F ¼ 0.43 (Table 5.1). Using this value and assuming that the normoxic, resting arterial blood pH at 30  C is about 7.55, like in other facultative air‐breathing ray‐finned fishes such as A. calva and Lepisosteus species (Ultsch and Jackson, 1996), the P50 in arterial blood is calculated to be 27.3 mmHg. RBCs of P. senegalus contain high levels of ATP and GTP, which change during ontogeny as well as hypoxia acclimation (Babiker, 1984a). The sum of

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these nucleotides changes little during hypoxia acclimation of juveniles that have not yet developed the ability to breathe air, or during maturation from juveniles to bimodally breathing adults. However, in both cases the relative contribution of GTP increases, from 20% to 35% during hypoxia acclimation and 5–30% during maturation (Babiker, 1984a). Unfortunately, whole blood Hb‐O2 aYnity under these conditions is not known. Because GTP is a more eVective organic phosphate than ATP, a decrease in Hb‐O2 aYnity may be expected (Val, 2000). In E. calabaricus, whole blood in vitro has a P50 of 17.9 mmHg and an nH value of 2.0 at 25  C, and F is 0.247 (Table 5.1). Air exposure for 4 h does not change P50 or the Bohr eVect but significantly increases nH (Beitinger et al., 1985). Assuming an in vivo normoxic resting arterial pH at 25  C of 7.65 (close to values in A. calva and Lepisosteus species), a P50 of 17.0 mmHg can be calculated. E. calabaricus RBCs contain ATP and GTP in equal amounts and relatively high 2,3‐DPG levels of about 40% of the sum of ATP and GTP. These levels show no major changes after 4‐h air exposure (Beitinger et al., 1985). Referring to the latter work, Graham (1997) has erroneously stated that reedfish RBCs also contain IP5, which is the major organic phosphate in birds but also occurs in lungfishes (see below). Whole blood of P. senegalus shows a small Root eVect. Hb‐O2 saturation at 30  C and a PO2 of 200–220 mmHg is close to 100% between pH 7.74 and 7.47 and decreases to about 93% between pH 7.16 and 7.00 (Vokac et al., 1972). However, the maximal Root eVect in hemolysates of the ornate bichir (Polypterus ornatipinnis) and E. calabaricus are somewhat higher with 14% and 15%, respectively, at 25  C (Berenbrink et al., 2005). Retinal PO2 in Polypteriformes is not known, but choroid and swim bladder retia mirabilia are missing in all members of this group that have been studied (Bridge, 1904; Wittenberg and Haedrich, 1974). Berenbrink et al. (2005) found no evidence of an adrenergic NHE in washed RBCs of P. ornatipinnis. 3.3.2. Lobe‐Finned Fishes (Sarcopterygii) a. Coelacanths. At 20  C and a pH of 7.8, Wood et al. (1972) determined the rather low P50 of 3.3 mmHg in the blood of L. chalumnae. In the same study, whole blood F was 0.51 (Table 5.1). A low P50 and the presence of a Bohr eVect were confirmed by Hughes and Itazawa (1972), who measured a P50 value of 2.1 mmHg and showed that P50 in whole blood increases with increasing temperature, as in most other vertebrates. Both groups found that blood‐O2 binding was essentially noncooperative at high pH values (nH ca 1.0), although Wood et al. (1972) showed an unusual increase in cooperativity at low pH (nH ca 1.6 at pH 6.7).

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Studies on purified Hb at 20  C revealed even lower P50 values than in whole blood but similar values for the Bohr coeYcient. A change from hyperbolic to cooperative Hb‐O2 binding below pH 7.5 was observed and an increase in P50 and nH in the presence of phosphate buVer was demonstrated (Wood et al., 1972; Bonaventura et al., 1974). Increasing temperature decreased Hb‐O2 aYnity to a similar extent as in other fish Hbs (DH ¼ 10.4 kcal per mol Hb; Wood et al., 1972). Unlike mammalian Hbs, but similar to elasmobranchs, coelacanth Hb is insensitive to high urea concentrations, which correlates with high urea concentrations in the blood of elasmobranchs and the coelacanth (Mangum, 1991). Wood et al. (1972) also found a 12% reduction in O2 capacity when blood was acidified with 3% CO2 in air and attributed this to a moderate Root eVect. In contrast, Hughes and Itazawa (1972) did not find any evidence of a Root eVect because blood O2 capacity at pH 7.30 and 6.88 at 15  C and pH 6.55 at 28  C was the same. However, pH 7.3 is 0.5 pH units below the estimated normal arterial pH value in L. chalumnae (about 7.8, see below), and it is possible that the maximum decrease in O2 capacity via the Root eVect already occurs at higher pH values than this. Unfortunately, Hb concentration and methemoglobin (metHb) fraction were not reported, precluding the calculation of functional Hb‐O2 saturation under these experimental conditions. Similarly, the pH values before and after equilibration to 3% CO2 in air in the study by Wood et al. (1972) are not known, and it is unclear whether the 10% decrease in O2 capacity represents a maximal reduction or whether further acidification would have resulted in even higher decreases. In addition, since 10% metHb had already been formed in their sample and lower pH values generally increase the rate of metHb formation (Wallace et al., 1982), it is possible that this was the cause of reduced Hb‐O2 binding at low pH and that L. chalumnae blood does not show a Root eVect. Gorr et al. (1991a) have determined the amino acid sequence of L. chalumnae Hb, and on the basis of the molecular mechanism for the Root eVect proposed by Perutz and Brunori (1982), they predicted in a subsequent paper the absence of a Root eVect in L. chalumnae Hb (Gorr et al., 1991b). However, the model of Perutz and Brunori for the Root eVect mechanism has been subsequently challenged (Ito et al., 1995; Yokoyama et al., 2004; see Berenbrink, 2006). Thus, the prediction by Gorr et al. (1991b) about the absence of a Root eVect may be invalid. The question whether L. chalumnae possesses a large Root eVect Hb is still open. The high‐O2 aYnity of L. chalumnae blood deserves special comment. However, before discussing its physiological relevance, it is worthwhile to briefly mention three possible reasons for measuring erroneously high blood O2 aYnities. First, long storage times until analysis may reduce RBC organic phosphates and thereby increase Hb‐O2 aYnity. The natural organic

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phosphate, if any, that modulates Hb‐O2 aYnity in L. chalumnae RBCs is not known. At pH 7.47, which may be close to physiological RBC pH at a plasma pH of 7.8, ATP has only a small eVect on P50 of isolated Hb in Tris buVer (Wood et al., 1972), but the eVects of other organic phosphates, such as GTP, 2,3‐DPG, IP5, or IP2, have not been tested. Another reason for the measurement of high‐O2 aYnities may be metHb formation. 10% of the sample was oxidized to methemoglobin in the study by Wood et al. (1972). Although metHb itself is unable to bind O2, it is known to increase the average O2 aYnity of the remaining functional heme groups (Falcioni et al., 1977; Kwiatkowski et al., 1994). Mangum (1991) showed that procedures normally removing metHb did not reduce the high‐O2 aYnity in L. chalumnae Hb preparations, although the initial metHb concentration was not reported. Finally, the animals live at much higher hydrostatic pressures than those under which O2 equilibrium curves are normally established, and it is not known how this diVerence aVects blood O2 aYnity. Bearing the above caveats in mind, a high blood O2 aYnity in L. chalumnae may signify tolerance to a low‐O2 environment (Wood et al., 1972). Ever since the pioneering work of Krogh and Leitch (1919), a correlation between high blood O2 aYnities and hypoxic habitats has been noted in water‐breathing vertebrates. Thus, common carp Cyprinus carpio, which frequently encounters hypoxic environments, has a whole blood P50 of ca 7 mmHg at pH 7.9 and 20  C (Weber and Lykkeboe, 1978). This contrasts with a P50 of ca 23 mmHg at pH 7.93 and 15  C in rainbow trout Oncorhynchus mykiss (Soivio et al., 1980). Thus, rainbow trout, which live in well‐oxygenated waters and are relatively hypoxia intolerant, have more than a threefold higher P50 value, despite the lower measurement temperature that tends to decrease P50. Using their submersibles GEO and JAGO, Fricke and Hissmann (2000) established the depth profile of physically dissolved O2 in the habitat of L. chalumnae. They found that the caves, which are used as resting places during the day, were located at depths corresponding to an O2 minimum layer at about 200 m. Dissolved O2 concentration decreased from about 7.6 mg liter1 in 27  C surface water to about 5.1 mg liter1 in 20  C water at the level of the caves, as estimated from Fig. 1 in Fricke and Hissmann (2000). However, to assess the gradient for diVusive O2 uptake under these conditions, the actual PO2 of the water, which was not given, is more important than the absolute O2 concentration. Using the O2 solubility coeYcient for seawater at 20  C (tabulated in Dejours, 1975), a PO2 of about 100 mmHg or 2/3 of air‐equilibrated seawater can be calculated. During the night, L. chalumnae uses energy‐saving drift‐hunting at depths below the level of the caves, where dissolved O2 concentrations gradually increase again (ca 6.0 mg liter1 at 400 m and 12.5  C; Fricke and Hissmann, 2000). However, the increased concentration of dissolved O2 is accompanied by an

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increased solubility at the lower temperature prevailing at 400 m such that the calculated PO2 remains unchanged at about 100 mmHg. Thus, a high blood O2 aYnity in L. chalumnae may indeed be related to the low‐O2 availability in its habitat and ensures full Hb‐O2 saturation in the gills. Another reason for a high blood O2 aYnity may be the low gill surface area and relatively thick water–blood diVusion barrier of L. chalumnae gills measured by Hughes (1972, 1995), which results in a very low diVusing capacity of the water–blood barrier and may result in a large PO2 drop between inspired water and arterial blood. As the PO2 of inspired water is already low due to environmental hypoxia, a high‐O2 aYnity may be necessary for adequate oxygenation of Hb at low arterial blood PO2 . On the basis of the low body mass specific gill area (Figure 5.1), Hughes _ O2 and a sluggish lifestyle (1972, 1976, 1995) has predicted a low resting M for L. chalumnae, similar to some deepwater teleosts. The latter has been confirmed by observations from submersibles (Fricke and Hissmann, 2000). From a high blood O2 aYnity it follows that PvO2 must be comparatively low in order to allow significant O2 unloading in tissues. The P50 values measured by Wood et al. (1972) and Hughes and Itazawa (1972) suggest that PvO2 values as low as 2–3 mmHg are required for 50% Hb‐O2 unloading. Unloading may be facilitated by slightly lower venous than arterial pH values and the Bohr eVect of L. chalumnae blood, but the noncooperative, hyperbolic shape of the blood O2 equilibrium curve appears disadvantageous, as a given arteriovenous PO2 diVerence at a given P50 causes smaller changes in Hb saturation when compared to the cooperative, sigmoid O2 equilibrium curves found in many other vertebrate groups. L. chalumnae is live bearing (Smith et al., 1975) with a 98‐kg female containing as many as 26 fetuses with a near‐term body mass up to about 500 g (Heemstra and Greenwood, 1992). The extent of maternal trophic input in L. chalumnae has been a matter of intense debate, and suggestions of prenatal oophagy, uterine cannibalism, and placentotrophy in an earlier volume of the Fish Physiology series (Wourms et al., 1988) have been strongly criticized (Heemstra and Greenwood, 1992). Nevertheless, Wourms et al. (1991) demonstrated an extensive plexus of blood vessels in a specialized area in each of the uterine compartments closely surrounding individual fetuses. These blood plexuses lie in close apposition to the heavily vascularized yolk sac and are discussed as yolk sac placenta specialized for molecular transport and gas exchange by Wourms et al. (1991). Live bearing has independently evolved in many elasmobranch and teleostean lineages (Reynolds et al., 2002). In several cases, maternal‐fetal O2 transfer appears to be facilitated by a shift toward a distinctly higher blood O2 aYnity in fetal blood compared to maternal blood (Ingermann et al., 1984; Weber and Hartvig, 1984; King, 1994). It will be most interesting to see whether this

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is also true for fetal L. chalumnae blood because of the already very high blood O2 aYnity of the adult described above. Early studies showed that blood of adult L. chalumnae contains a single tetrameric Hb composed of two diVerent subunit types conforming to the general a2b2 subunit structure of other jawed vertebrates (Weber et al., 1973; Bonaventura et al., 1974). The amino acid sequences of the a‐ and b‐chains were determined by Gorr et al. (1991a), who found that these chains share 51 and 48 identical residues with the respective chains in human HbA. The molecular mechanism behind the low cooperativity and low intrinsic O2 aYnity of L. chalumnae Hb was explored by molecular modeling and comparison with human HbA. These studies are exceedingly diYcult when dealing with a large number of amino acid diVerences. Nonetheless, Gorr et al. (1991b) were able to predict a distinct diVerence in the mechanism of the Bohr eVect between Latimeria Hb and human HbA, despite the similar Bohr coeYcient displayed by these two Hbs. They also predicted the absence of a Root eVect in L. chalumnae Hb (see above). In contrast to the a‐chains of most teleost Hbs, the N‐terminal amino groups of the globin chains in L. chalumnae are not acetylated (Gorr et al., 1991b). b. Lungfishes (Dipnoi). Blood respiratory characteristics in the facultative air‐breathing, nonestivating N. forsteri were investigated by Lenfant et al. (1966). At 18  C during normoxia at rest, this species is predominantly water breathing and it is assumed that blood in the pulmonary artery represents systemic arterial blood, that is that no central blood shunting occurs. Under these conditions, a PaCO2 of 3.5 mmHg is as low as in typical water‐breathing fishes (Table 5.1). The PaO2 of 40 mmHg is rather low. Nevertheless an Hb‐O2 saturation of 95% can be calculated for these conditions from in vitro whole‐blood O2 equilibrium data. This is due to a low P50 of 11 mmHg and sigmoid Hb‐O2 binding (Lenfant et al., 1966). The Bohr eVect was rather high with F ¼ 0.620. In a more recent study on N. forsteri at 20  C, P50 was 22.0 mmHg and nH was 2.27 at pH 7.5 and PCO2 of 16 mmHg. The Bohr eVect was considerably lower with F ¼ 0.48 (Kind et al., 2002). These authors found a significant left‐shift of the O2 equilibrium curve with hypoxia acclimation, although ATP levels did not change. However, GTP, which makes up 30% of the NTPs in N. forsteri (Isaacks and Kim, 1984), was not measured. In contrast to N. forsteri, the other two genera of lungfishes are obligatory air‐breathers grouped in their own separate family. Working on juvenile L. paradoxa, Johansen and Lenfant (1967) clearly demonstrated a selective passage of oxygenated pulmonary blood and systemic venous blood through the only partially divided heart because PO2 in dorsal aortic blood exceeded pulmonary arterial PO2 (10–43 mmHg versus 5–16 mmHg, respectively).

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Higher dorsal aortic PO2 values of 80–100 mmHg have been reported from somewhat larger specimens (Amin‐Naves et al., 2004). In vivo whole blood P50 at 23  C and PCO2 of 6 mmHg was 10.5 mmHg in the study by Johansen and Lenfant (1967), showing a rather high Hb‐O2 aYnity. The Bohr coeYcient was F ¼ 0.234. In later studies on adult Lepidosiren, slightly higher values of F ¼ 0.293 and 0.31 have been measured (Johansen, 1970; Johansen et al., 1978). All three foregoing values are less than half the value of F ¼ 0.66 determined at 25  C by Bassi et al. (2005). The latter study found a smaller Bohr eVect at 35  C, where F was 0.44. In contrast, Powers et al. (1979) found F ¼ 0.33 at 20  C and 30  C (Table 5.1). The values for the Bohr coeYcient reported in P. aethiopicus are equally variable, ranging from F ¼ 0.28 to 0.35 and 0.47 at 23–25  C (Lahiri et al., 1968; Swan and Hall, 1966; Lenfant and Johansen, 1968) (Table 5.1). Some of the above authors have oVered an adaptive explanation for the particular size of the Bohr eVect measured in their study. These interpretations are tenuous because it is not known to what extent the variations are due to experimental technique, phenotypic plasticity, or inheritable, genetic diVerences between individuals, populations, and species. Several of the whole‐blood O2 equilibrium curves in earlier studies by Johansen and coworkers were obtained by the mixing method, which was previously successfully used on invertebrate blood (Lenfant and Johansen, 1965) but, as later admitted by the authors, may not work well with fish blood (Johansen et al., 1976b). Problems include accurate PO2 measurements in bloods with high rates of O2 consumption and depletion of organic phosphates in anoxically incubated subsamples (Scheid and Meyer, 1978; Tetens and Lykkeboe, 1981). The latter may also be a problem in the study by Johansen and Lenfant (1967), where whole blood was stored for more than 48 h before O2 equilibrium curves were determined. Evidence for some individual and phenotypic plasticity of the Bohr eVect comes from studies on estivating African lungfishes (Johansen et al., 1976b). At 26  C, in vitro whole blood F in two active P. amphibius was 0.29 and 0.20 relative to 0.41 and 0.68 in two individuals that had been estivating for 28–30 months. The elevated whole blood F in estivating animals was associated with a large reduction in RBC GTP levels, whereas ATP levels remained unchanged. In a companion study, it was shown that estivation did not change the functional properties or relative proportion of the three Hb isoforms in organic phosphate‐free solutions. Addition of ATP increased the Bohr eVect, but the eVects of GTP on the Bohr eVect have not been reported. At constant pH, GTP had a stronger eVect on P50 than ATP (Weber et al., 1976a). The dynamics of the Bohr eVect changes between estivating and active animals in vivo are not known, but it is possible that

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part of the variable Bohr eVect in active animals can be explained by the time that has elapsed since the last estivation. Although F in whole blood is correlated with RBC GTP levels, the mechanism for the increased Bohr eVect during estivation remains to be investigated. Babiker (1984a) confirmed decreased GTP levels in RBCs of estivating P. annectens and further showed an increase in relative and absolute GTP levels during the ontogenetic change from almost completely water‐breathing juveniles to obligatory air‐breathing adults. Babiker (1984a) further showed that acclimation to aquatic hypoxia in maturating juveniles led to an increase in GTP levels while ATP levels decreased slightly. Unfortunately, there are no data on P50 and the Bohr eVect under these conditions, but it is possible that ontogenetic changes and levels of previous hypoxic exposure contribute to the variation of the Bohr eVect within species. In contrast to P. amphibius, P. annectens and L. paradoxa contain only one major Hb isoform, and studies on their functional properties in solution suggest distinct interspecific diVerences in their intrinsic O2 aYnity and interaction with allosteric modifiers (Weber et al., 1976a; Phelps et al., 1979). Unlike N. forsteri, Protopterus species and L. paradoxa are unusual in containing significant amounts of inositol‐diphosphate (IP2) and uridine phosphates (UTP and UDP) in their RBCs (Bartlett, 1978b; Isaacks et al., 1978). The eVects of these organic phosphates on Hb‐O2 binding are unknown. High urea concentrations accumulate in the blood of African lungfishes during estivation (Smith, 1930). A plasma osmolality increase from 235 to 650 mOsm over 13 months of estivation has been reported (DeLaney et al., 1977). Urea concentrations in estivating L. paradoxa are unknown, but the RBCs of the species have unusually high urea permeability (Kim and Isaacks, 1978). In contrast to humans, high urea does not decrease P50 in P. amphibius hemolysates (Weber et al., 1976a). RBCs of P. aethiopicus possess an anion exchanger and extracellular pH changes are transferred to the interior, suggesting passive proton equilibration across the RBC membrane (Jensen et al., 2003). RBCs of this species express b‐adrenergic receptors, and aerial (but not aquatic) hypoxia significantly increases plasma catecholamine levels in the related P. dolloi (Koldkjær et al., 2002; Perry et al., 2005). However, in neither of these two species, nor in L. paradoxa, could an adrenergic NHE be demonstrated using diVerent methods (Koldkjær et al., 2002; Berenbrink et al., 2005; Perry et al., 2005). A Root eVect is absent in whole blood of all three lungfish genera (Johansen, 1970), which has been confirmed for the hemolysates of an unidentified Protopterus species and L. paradoxa (Farmer et al., 1979; Berenbrink et al., 2005). A choroid rete mirabile is also absent (Wittenberg and Haedrich, 1974).

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3.3.3. Jawless Fishes (Agnatha) The biology of hagfishes has been reviewed in two compilations of articles by Brodal and Fa¨nge (1963) and, more recently, by Jørgensen et al. (1998). A similar compilation about the biology of lampreys has been edited by Hardisty and Potter (1971, 1972). All three compilations contain chapters about the blood O2 transport characteristics (Manwell, 1963; Riggs, 1972; Fago and Weber, 1998). Comparative aspects of blood O2 transport in agnathans relative to teleosts, elasmobranchs, and terrestrial vertebrates are discussed by Nikinmaa (1990, 1992, 1997, 2001). As mentioned earlier, agnathan Hbs consist of high O2 aYnity monomers, which aggregate to low O2 aYnity dimers and tetramers at low pH and PO2 . a. Lampreys (Petromyzontiformes). Despite the general anatomical diVerences between the gills in agnathans and jawed fishes, gill O2 diVusion capacity is high in the Pacific lamprey Lampetra tridentata and the sea lamprey P. marinus as indicated by high normoxic PaO2 values of up to 77 and 120 mmHg at 14 and 10  C, respectively, which is associated with an Hb‐O2 saturation of about 95% (Table 5.1) (Johansen et al., 1973; Tufts, 1991). At 10  C and pH of 7.75, whole blood P50 in L. fluviatilis is 11.8 mmHg in the adults but only 1.8 mmHg in the larvae (Bird et al., 1976). The higher O2 aYnity of the larvae has been attributed to their buried lifestyle in presumably hypoxic sediments, whereas the parasitic adults have an active lifestyle in more oxygenated waters, actively attaching themselves to teleost prey, and undertaking long spawning migrations (Bird et al., 1976). When the Bohr coeYcient is related to extracellular pH values, lamprey blood shows low F values between 0.1 and 0.3. However, since a functional anion exchanger is lacking and extracellular pH changes are not readily transferred to the RBC interior in lampreys, this underestimates the pH sensitivity of Hb‐O2 binding and values of F ¼ 0.63 to 1.03 have been measured for the intracellular Bohr coeYcient (Table 5.1) (Ferguson et al., 1992; Nikinmaa et al., 1995). At high pH, cooperativity of Hb‐O2 binding is saturation dependent, nH at 35%, 50%, and 80% saturation increases from 1.00 to 1.21 and 2.32, respectively (Bird et al., 1976). At lower pH values, Hb‐O2 binding becomes hyperbolic and nH is close to 1.00 regardless of saturation (Nikinmaa, 1993). After exhaustive exercise, normoxic dorsal aortic pH in P. marinus at 10  C fell from 7.9 to ca 7.55. However, as this change was not transferred to the intracellular RBC compartment, pHi stayed constant (Tufts, 1991). Nevertheless, arterial Hb‐O2 saturation fell transiently to about 80% because PaO2 decreased from 120 to about 80 mmHg. Acute hypoxia (PwO2 ¼ 40–50 mmHg) in L. fluviatilis resulted in a large _ O2 , increase in ventilation frequency, accompanied by a 50% increase in M

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which after 1 week was reduced to 125% of normoxic control values (Nikinmaa and Weber, 1984). This was paralleled by an acclimatory increase in Hb‐O2 aYnity, brought about by an increase in RBC pH and the Bohr eVect, and also by a decrease in intracellular Hb concentration, which is known to shift the monomer–oligomer equilibrium toward the higher aYnity monomeric Hb form (Nikinmaa and Weber, 1984). RBC pH in lampreys is elevated above the proton equilibrium distribution by the activity of an NHE mechanism as first shown by Nikinmaa (1986). The high RBC pH together with the strong Bohr eVect shifts the P50 of Hb to the region of active teleosts. If RBC pH was as low as in a rainbow trout at the same temperature, P50 in L. fluviatilis would be as high as 73 mmHg (Nikinmaa, 1993). The same study showed that at low RBC pH (6.8), brought about by very high PCO2 values (152 mmHg), L. fluviatilis Hb failed to become saturated even at PO2 values of 530 mmHg and that Hb‐O2 saturation at PO2 152 mmHg was below 40%, indicating an eVect analogous to the Root eVect in ray‐finned fishes. Swim bladders are absent in lampreys and no choroid rete mirabile has been found (Wittenberg and Haedrich, 1974). Under physiological conditions, catecholamines do not significantly activate the NHE in RBCs of P. marinus and L. fluviatilis (Tufts, 1991; Virkki and Nikinmaa, 1994). RBCs of the Pacific lamprey, L. (formerly Entosphenus) tridentatus, contain high levels of ATP and 2,3‐DPG and a small amount of GTP (Johansen et al., 1973; Bartlett, 1982). However, Hb‐O2 binding in this species is not aVected by 2,3‐DPG (Johansen et al., 1973), which is in line with the absence of the typical binding site in the crevice of deoxygenated tetramers, where organic phosphates bind in jawed vertebrate Hbs. Lamprey hemolysates often contain multiple Hbs, several of which have been sequenced. The mechanism of the strong Bohr eVect has been elucidated in a major Hb component of P. marinus. Structural X‐ray crystallography and functional studies on mutant Hbs obtained by site‐directed mutagenesis have revealed that, unlike in Hbs of all other vertebrate classes, the Bohr eVect is brought about by preferential proton binding in the deoxygenated Hb to a cluster of glutamic acid residues in the interface of the dimeric Hb and to the distal histidine of the heme group (Heaslet and Royer, 1999; Qiu et al., 2000). b. Hagfishes (Myxiniformes). Blood O2 transport in the hagfish E. cirrhatus was studied by Wells et al. (1986; Table 5.1). At 16  C, they found a high normoxic resting PaO2 of about 90–110 mmHg, which was not reduced during 10–15 min of swimming at 0.4 body lengths per second. PvO2 was rather high at 17.2 mmHg, falling to 3.5 mmHg during swimming. PCO2 was low in arterial and venous blood, as typical for water‐breathers, and pH was 7.92 in arterial and 7.77 in venous blood. Arteriovenous pH diVerence

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slightly increased to 0.25 pH units during swimming. Hematocrit value and Hb concentration were comparatively low relative to lampreys and jawed vertebrate fishes (Table 5.1), and physically dissolved O2 composed 15% of normoxic arterial O2 content. In vitro, whole‐blood O2 equilibrium curves were slightly sigmoid with nH ¼ 1.38. The latter did not change with saturation or pH. At pH 7.8, P50 was 12.3 mmHg and F ¼ 0.43 (Wells et al., 1986) (Table 5.1). These data are quite diVerent from observations on the congeneric Pacific hagfish Eptatretus (formerly Polistotrema or Bdellostoma) stoutii (Manwell, 1958, 1963; Johansen and Lenfant, 1972). Whole blood of the latter species did not show a Bohr eVect at physiological blood pH. Above pH 7.0, O2 binding was strictly hyperbolic (nH ¼ 1.0) and P50 was 2–4 mmHg between 5 and 18  C and was unaVected by CO2 at constant pH (Manwell, 1958; Johansen and Lenfant, 1972) (Table 5.1). Similarly, Manwell (1963) reported a high blood O2 aYnity in Atlantic hagfish (M. glutinosa) RBCs, very low cooperativity, and no Bohr eVect. From a crude visual estimation of arterial and venous Hb‐O2 saturation in exposed dorsal aortic and liver vessels in restrained animals, Manwell (1958, 1963) concluded that hagfish Hb essentially functions over a restricted PO2 range of maximally 10 mmHg, with very low venous PO2 values. It is possible that these diVerences are related to diVerences in the lifestyle and environmental PO2 of the species, with some preferring rocky substrates and others living in muddy burrows (Wells et al., 1986). However, methodological diVerences also need to be taken into account since Manwell (1958, 1963) used RBCs in phosphate buVers, whereas Wells et al. (1986) used whole blood, pH‐adjusted with CO2. The above diVerence is important since E. stoutii RBCs appear to lack the anion exchanger (Ellory et al., 1987). Thus, extracellular pH changes with the membrane permeable CO2 are more likely to change RBC pH than those with phosphate buVers. In M. glutinosa whole blood in vitro, extracellular pH changes caused by changes in PCO2 are transferred to the RBC interior, but intracellular pH is much lower than in lampreys (Tufts and Boutilier, 1990). In further contrast to the river lamprey, no RBC pH regulation could be observed after intracellular acidification (Nikinmaa et al., 1993). Studies on the isolated multiple Hbs of M. glutinosa have further shown that the very small Bohr eVect of F ¼ 0.07 can be increased to F ¼ 0.17 in the presence of CO2 (Bauer et al., 1975). These authors showed a specific CO2 eVect, which decreased Hb‐O2 aYnity at constant pH. However, these experiments were performed at low chloride concentrations, which have been shown to facilitate the Bohr eVect of M. glutinosa Hb (Fago and Weber, 1998). The CO2 eVect described by Bauer et al. (1975) has later been ascribed to an interaction between bicarbonate ions and Hb (Fago et al., 1998).

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RBCs of E. stoutii contain high ATP and ADP levels, together with small amounts of GTP (Bartlett, 1982). However, in the presence of chloride at least ATP, 2,3‐DPG, and inositol hexaphosphate do not aVect O2 aYnity or cooperativity in isolated M. glutinosa Hb (Bauer et al., 1975; Fago and Weber, 1995). 4. TRANSPORT AND ELIMINATION OF CO2 4.1. General Model of CO2 Transport and Excretion There have been a number of reviews on CO2 excretion in vertebrates in general (Swenson and Maren, 1978; Klocke, 1987, 1988; Swenson, 1990) and fish in particular (Perry, 1986; Tufts and Perry, 1998) to which the reader is referred; however, the basic teleost model is briefly discussed here. As in all vertebrates, the majority of total CO2 is transported in the blood as HCO 3 in fish. At the tissues, CO2 diVuses down its partial pressure gradient into the þ blood, and once within the RBC is converted to HCO 3 and H in the presence of high levels of carbonic anhydrase (CA). There is no plasma accessible CA in the gill of teleosts, and thus CO2 hydration/dehydration in the blood is restricted to within the RBC. In general, HCO 3 is transported across the RBC anion exchanger (AE1) in exchange for Cl (with the exception of hagfishes and lampreys discussed below), and the majority of HCO 3 is transported in the plasma compartment. A varying proportion of the protons resulting from CO2 dehydration are then bound to Hb, either through the ability of Hb to act as a buVer or through the binding of protons associated with deoxygenation (Haldane eVect), minimizing the pH changes that occur within the RBC during CO2 loading. In general, nonoxygenation‐dependent Hþ buVer sites in Hb under physiological conditions consist predominantly of histidine residues (Jensen, 1989), and in some cases the terminal amines of the a‐ and b‐chains. In general, animals appear to have Hbs with relatively high buVer values at fixed oxygenation status and small Haldane coeYcients, as in mammals and elasmobranchs, or low buVer values and large Haldane coeYcients, as in teleost fishes (Jensen, 1989). These two patterns represent two diVerent, but equally eVective strategies for CO2 transport and excretion (Jensen, 1989). Despite the binding potential of Hb for Hþ, there is often a reduction in pH during blood tissue transit due to metabolic CO2 production, that in conjunction with the Bohr eVect (see above) facilitates O2 delivery to the tissues. This has been well described in the blood of vertebrates (Bartels, 1972; Lapennas, 1983; Riggs, 1988). Lapennas (1983) has conducted a theoretical analysis which concludes that the optimal Bohr coeYcient for O2 delivery at the tissues is one‐half of the respiratory quotient (rate of CO2 production/rate of O2

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consumption by the tissues) which in most animals is between 0.35 and 0.5. This value represents a compromise between the sensitivity of the Hb to changes in pH (Bohr eVect) and the net acidification in the blood associated with metabolic CO2 production and the binding of protons to Hb during deoxygenation (Haldane eVect). The reverse process occurs at the gas exchange site responsible for CO2 elimination (gills or ABO), where the majority of CO2 excreted consists of HCO 3 transported from the plasma into the RBC (Perry et al., 1982; Brauner et al., 2000) (via AE1) where it combines with Hþ to form CO2 and diVuses down its concentration gradient to the environment. Again, protons are generally either titrated from Hb or released from Hb during oxygenation (Haldane eVect), and HCO 3 dehydration occurs at a rapid rate in the presence of high RBC levels of CA. In fish which possess a low‐Hb buVer value and large Haldane eVect (i.e., teleosts), there is a tight interaction between O2 uptake and CO2 removal at the gills (Jensen, 1986, 1989; Brauner et al., 1996; Brauner and Jensen, 1998; Brauner and Randall, 1998). The rate‐limiting step in CO2 excretion is thought to exist at the RBC AE1 (Perry, 1986), and when all steps associated with CO2 excretion are considered, the gills of fish are thought to be diVusion limited for CO2 (Perry and Gilmour, 2002). A number of aspects that influence the pattern of CO2 transport and elimination in primitive fish groups diVer from teleosts and air‐breathing vertebrates. These include aspects related to (1) Hb and RBC function, in particular Hb buVer values and the magnitude of the Haldane eVect which influence the degree of interaction between O2 and CO2 exchange (Brauner and Randall, 1996; Brauner and Randall, 1998), as well as RBC permeability to HCO 3 via AE1, and (2) the catalytic activity and location of CA. The limited research conducted to date on these two aspects in primitive fishes is discussed below. 4.1.1. Hemoglobin and RBC Function a. Ray‐finned fishes (Actinopterygii) i. Bowfin (Amiiformes). Blood bicarbonate concentration at physiological pH and PCO2 is 6–7 mM at 20  C and corresponds to the typical values for water‐breathers (Johansen et al., 1970). The latter authors noted a nonbicarbonate buVering capacity similar to the blood of other primarily water‐ breathing fishes. The specific buVer capacity of the composite Hb system bHb is 6.4 mol Hþ per mol Hb4 and pH, a value distinctly lower than in other primitive ray‐finned fishes such as L. platyrhincus, A. ruthenus, and E. calabaricus, but very similar to many teleosts such as dolphin fish, rainbow trout, and European eel (Coryphaena hippurus, O. mykiss, and Anguilla anguilla; Berenbrink et al., 2005) (Table 5.1; Figure 5.2F). A. calva RBCs appear to possess a high rate of Cl/HCO 3 exchange, and in conjunction with the

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presence of intermediate levels of RBC CA activity (relative to agnathans and teleosts), and the presence of a plasma CA inhibitor, it is thought that blood CO2 transport is similar to that of teleost fishes (Gervais and Tufts, 1999). ii. Gars (Lepisosteiformes). In longnose gar (L. osseus) exposed to 10  C, PCO2 and bicarbonate concentration of ventricular blood were low as expected for strict water‐breathers and average pH was 7.831 (Rahn et al., 1971). In animals at 25  C, pH was 7.440, which is lower than predicted by the general temperature–pH relationship of ectotherms (0.016/ C, Ultsch and Jackson, 1996). The low pH at 25  C was primarily due to an elevation in PCO2 associated with air breathing (Rahn et al., 1971). In L. platyrhincus, bHb was 8.6 mmol Hþ/mmol Hb4 and pH. While the Bohr eVect in these Hbs has not been measured directly, the Haldane coeYcient in organic phosphate‐free hemolysates at physiological temperature, pH, and chloride concentrations was F ¼ 0.525. Assuming numerical identity between the Bohr and Haldane coeYcients, the magnitude of the Haldane eVect is lower, and bHb higher than in Hbs of A. calva and most teleosts under the same conditions (Table 5.1; Figure 5.2) (Berenbrink et al., 2005). iii. Sturgeons and Paddlefishes (Acipenseriformes). The Adriatic sturgeon A. naccarii is the only primitive ray‐finned fish whose globins have at least been partially sequenced (Clementi et al., 1999). These partial sequences are maximally 30 amino acids long but already contain some very interesting information that sets them apart from teleosts. In contrast to virtually all teleosts, the N‐terminal amino groups of the a‐globins are not acetylated and hence are available for binding of CO2 as carbamino compound or for buVering of protons. The number of physiological buVer groups is further increased by the presence of histidines in position 9 of both the a‐ and b‐chains and in position 2 of the b‐chains. In teleosts, these positions are usually occupied by amino acids that do not buVer at physiological pH. This is consistent with a distinctly higher bHb of 9.4 mol Hþ per mol Hb4 and pH in A. ruthenus as compared to L. platyrhincus, A. calva, and most teleosts (Table 5.1; Figure 5.2F) (Berenbrink et al., 2005). iv. Reedfish and Bichirs (Polypteriformes). No measurements of in vivo blood CO2 transport characteristics exist for this group. However, in vitro characterization of the blood of P. senegalus reveals a typical CO2‐combining curve, where the majority of HCO 3 is held within the plasma compartment (Vokac et al., 1972). The whole blood buVering capacity was calculated to be 15.4 mmol HCO 3 per liter and per pH unit (Vokac et al., 1972). Hemolysates of the reedfish E. calabaricus show a bHb value of 11.6 mmol/mmol Hb4 and pH, which is similar to values in many tetrapods and elasmobranchs, but higher than the values in more advanced ray‐finned fishes, especially those in many teleosts (Table 5.1; Figure 5.2F) (Berenbrink

256 A

RBC pH regulatory NHE

Primitive fishes

B

Choroid rete mirabile

60

40

0

4

3

2

1

C

Root effect (%) 20

Fixed acid Haldane effect (mol H+ per mol Hb4)

0 0

−5

−10

−15

Agnatha (84)

Sarcopterygii (23,558)

Tetrapoda (23,550)

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C. J. BRAUNER AND M. BERENBRINK

Gnathostomata (48,085)

(8)

?

?

Osteichthyes (47,239)

(44)

Actinopterygii (23,681)

Teleostei (23,637)

Chondrichthyes (846)

(84)

?

Fig. 5.2. (continued)

Myxine glutinosa Lampetra fluviatilis Scyliorhinus stellaris Mustelus asterias Squalus acanthias Coryphaena hippurus Oncorhynchus mykiss Cyprinus carpio Scleropages jardinii Amia calva Lepisosteus platyrhincus Acipenser ruthenus Erpetoichthys calabaricus Latimeria chalumnae Lepidosiren paradoxa Xenopus laevis Alligator mississippiensis Gallus gallus Sus scrofa Homo sapiens

D

E

F bHb (mol H+ per mol Hb4 per pH)

257

a

b c e

e

Homo

Sus

Gallus

Alligator

Xenopus

Lepidosiren

Latimeria

Erpetoichthys

Acipenser

Lepisosteus

Amia

Scleropages

Cyprinus

Oncorhynchus

Coryphaena

Squalus

Lampetra

Myxine

G

Mustelus

GAS TRANSPORT AND EXCHANGE

Scyliorhinus

5.

a c d b

a a b c d e

- Fixed-acid Haldane effect increases - Root effect increases - Hb buffer value decreases - Choroid rete mirabile evolves - RBC pH regulation evolves

Fig. 5.2. Selected blood O2 and CO2 transport characteristics and other relevant factors in representative primitive fishes and other vertebrates. (A) Number of primitive fish species compared to other vertebrate groups. (B) Presence (solid circles) of RBC pH regulatory Naþ/Hþ exchange. (C) Presence (solid circles) of a choroid rete mirabile. (D) Root eVect in RBCs or Hb solutions, expressed as maximal percentage deoxygenation induced by decreased pH at PO2 ¼ 150 mmHg compared to pH 8 or higher. (E) Fixed‐acid Haldane eVect, expressed as maximal number of protons taken up per Hb tetramer on changing the oxygenation status of hemolysates at constant pH from fully oxygenated to fully deoxygenated in the absence of organic phosphates and at physiological chloride concentrations. (F) Specific Hb buVer value, bHb, expressed as mol protons released per mol Hb tetramer on a unit increase in pH, obtained in deoxygenated hemolysates in the absence of organic phosphates and at physiological chloride concentrations and pH. (G) Reconstruction of the evolution of blood respiratory gas transport characteristics on a vertebrate phylogenetic tree (for detailed methods see Berenbrink et al., 2005). Dashes through tree branches indicate the level at which certain characteristics (a–e) were gained. References: (A) Nelson (1994); (B) Berenbrink et al. (2005) and Wittenberg and Haedrich (1974); (C) Berenbrink et al. (2005), Nikinmaa (1986), and Nikinmaa et al. (1993); (D) Berenbrink et al. (2005) and Nikinmaa (1993); (E and F) Berenbrink et al. (2005), Jensen (1999), Jensen et al. (1998b), and Siggaard‐Andersen (1975); and (G) phylogenetic tree assembled from information given in Berenbrink et al. (2005) and Takezaki et al. (2003). ‘‘?’’ indicates no data. See text for further details.

et al., 2005). The Haldane coeYcient in P. senegalus calculated from in vitro CO2 dissociation curves was determined to be 0.10 vol% CO2 per vol% O2 capacity (Vokac et al., 1972) and the Bohr coeYcient measured in the same study was F ¼ 0.43. The latter value is low compared with values observed in most teleosts, and this occurs in conjunction with relatively high buVer values.

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b. Lobe‐Finned Fishes (Sarcopterygii) i. Coelacanths (Coelacanthiformes). The physiological pH range of L. chalumnae blood is not known. Assuming that the general relationship between resting arterial pH and body temperature in elasmobranchs and marine teleosts (Ultsch and Jackson, 1996) holds, values of 7.85 to 7.75 can be expected at the habitat temperatures of 13–25  C, respectively, that were measured by Fricke and Hissmann (2000). The limited work conducted on aspects related to CO2 transport in L. chalumnae indicates that it has a small to moderate Haldane eVect (F ¼ 0.5; Bonaventura et al., 1974) and a fairly high‐Hb buVer value based on the total number of histidine residues (36 per tetramer) (Gorr et al., 1991b). Wood et al. (1972) determined a whole blood nonbicarbonate buVer value of 9.0 mmol HCO 3 per liter and pH unit. The primary sequence of Latimeria globin chains allows an estimation of the number of physiological buVer groups per tetrameric Hb and this can be used to predict bHb (Berenbrink et al., 2005). Using the number of predicted physiological buVer groups per Hb tetramer and the regression line obtained by phylogenetically independent contrast from Berenbrink (2006), bHb is calculated to be 11.9 mmol Hþ per mmol Hb4 and pH. This value is twice as high as in the bowfin A. calva and typical teleosts, somewhat lower than the value measured for the South American lungfish, but in the same range as the values measured for various tetrapods, elasmobranchs, and E. calabaricus as a member of the most basal living actinopterygian lineage (Table 5.1; Figure 5.2F) (Berenbrink et al., 2005). Thus, in terms of CO2 transport and excretion, L. chalumnae possesses characteristics that are more similar to that of elasmobranchs than teleosts (Jensen, 1989), namely a high Hb buVer value and moderate Haldane eVect. ii. Lungfishes (Dipnoi). In L. paradoxa, the Haldane eVect is small and the buVer value of oxygenated blood was high in relation to typical water‐ breathers (Johansen and Lenfant, 1967). This has also been found in P. aethiopicus (Jensen et al., 2003). Rodewald et al. (1984) sequenced the globin chains of the single major Hb of L. paradoxa. The N‐terminal amino groups are free and thus available for proton buVering and CO2 binding, unlike in most fish investigated to date. The Hb contains 50 histidines per tetramer (Rodewald et al., 1984) and the total number of physiological buVer groups has been estimated to be 40 per tetramer, which corresponds to one of the highest bHb values measured so far of 16.5 mmol Hþ per mmol Hb4 and pH (Berenbrink et al., 2005). In the African lungfish P. aethiopicus, the RBC volume approaches 7000 mm3 (Koldkjær et al., 2002; Jensen et al., 2003), more than 30‐fold larger than that commonly observed in teleost fishes (200 mm3; Jensen and Brahm, 1995). The plasma compartment in the blood of P. aethiopicus contains the highest concentration of total CO2 in the blood. Thus, CO2

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excretion is likely to be very dependent on RBC Cl/HCO 3 exchange, the rate of which may be limited by the large size of lungfish RBCs. The rate coeYcient for unidirectional RBC Cl eZux is much slower than that measured in rainbow trout (O. mykiss) (Jensen et al., 2003). No studies have been conducted to determine the degree to which the large cells may aVect CO2 excretion in vivo. c. Jawless Fishes (Agnatha). Both lampreys and hagfishes appear to virtually lack RBC anion exchange (Ellory et al., 1987; Nikinmaa and Railo, 1987), which greatly alters the general pattern of CO2 excretion from most other vertebrates. River lampreys (L. fluviatilis) have Hbs with relatively low buVer values (1–1.5 titratable groups per monomer due to a low number of histidines in the Hb) but a relatively large Haldane eVect (0.9 Hþ per monomer on deoxygenation). In contrast, the Atlantic hagfish (M. glutinosa) exhibits the reverse pattern, with relatively high‐Hb buVer values (four to five titratable groups per monomer) and a relatively small Haldane eVect (0.35 Hþ per monomer on deoxygenation) (Jensen, 1999) (Table 5.1; Figure 5.1E). In the river lamprey, these Hb–Hþ equilibrium characteristics result in a very interesting system that helps to partially compensate for the lack of an RBC AE1 that would otherwise be assumed to limit the rate of CO2 excretion in vivo. It must be remembered that the amount of HCO 3 formed for a given PCO2 increases with increasing intracellular pH. Because of the large Hþ binding associated with deoxygenation in conjunction with a low Hb buVer value, deoxygenation results in an alkalinization of the RBC by 0.3–0.4 pH units, and venous pHi is greater than arterial pHi in vivo (Tufts et al., 1992). Furthermore, RBC membrane Naþ/Hþ exchange maintains the RBC pHi for a given pHe at a more alkaline level than in most other fishes (Tufts et al., 1992; Nikinmaa, 1997). Thus, the relatively alkaline RBC results in a greatly elevated HCO 3 concentration within the erythrocyte (Tufts and Boutilier, 1989), and the majority of CO2 excreted at the gills will consist of HCO 3 carried within the RBC that combines with Hþ released from Hb during oxygenation. Thus, in terms of CO2 transport and excretion, there is a tight coupling of CO2 excretion with O2 uptake in the river lamprey, that is accomplished in the absence of AE1. The pHi in the Atlantic hagfish M. glutinosa is not elevated as it is in river lampreys, and the Hb–Hþ characteristics appear less eVective in facilitating CO2 removal in the absence of AE1. However, the Hb of the Atlantic hagfish exhibits oxygenation‐linked binding of HCO 3 (Fago and Weber, 1998; Fago et al., 1998) analogous to that observed in crocodiles, where deoxygenated  Hb binds HCO 3 . Thus, during tissue capillary transit, HCO3 could be bound to Hb as O2 is delivered, and transported within the RBC until blood reaches the gills. Oxygenation‐dependent HCO 3 binding does not exist in lamprey

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Hb, and thus there appear to be two very diVerent strategies to compensate for the lack of RBC AE1 in Atlantic hagfish and river lamprey, which likely reflects the early evolutionary divergence of the two groups. The lack of RBC AE1 in both lampreys and hagfishes indicates that the RBC may have originally evolved to transport both O2 and CO2 (Tufts and Boutilier, 1989). In the absence of appreciable RBC HCO 3 permeability, extracellular acid loads cannot be buVered by Hb within the RBC (Nikinmaa, 1997), and the intra‐ and extracellular compartments within the blood functionally operate as two separate compartments. With the incorporation of AE1 into vertebrate RBCs, extracellular protons can be buVered by Hb with the export of HCO 3 into the plasma, and the two‐compartmental system results in the majority of HCO 3 being transported in the plasma, and the total CO2 content of blood for a given PCO2 , to be elevated. 4.1.2. CA Activity and Location CA is a ubiquitous enzyme that catalyzes the hydration/dehydration of CO2 and is involved in processes related to CO2 transport and excretion (as described above), but also ion regulation, acid‐base balance, and fluid secretion among others. CA is found in very high concentrations within the erythrocyte of all vertebrates, second only in concentration to Hb, and plays a crucial role in CO2 transport and excretion. In teleost fishes, there is no plasma accessible CA in the gill, and thus HCO 3 dehydration at the gills is restricted to within the RBC. In the elasmobranchs (Gilmour et al., 2001), CA accessible to plasma is present in the gills, and there is significant non‐ RBC HCO 3 dehydration associated with CO2 excretion. CA is also found in the endothelium of the ABO of many fishes, and this is also thought to play an important role in CO2 excretion from the ABO. The following sections describe what is known about CA characteristics in the RBCs, gills, and ABOs of primitive fishes. a. RBC CA. It has been proposed that there may be a trend from agnathans to teleosts toward a faster RBC CA isozyme (Tufts et al., 2003). However, it was cautioned that this trend was based on comparison of a few broad phylogenetic groups from diVerent studies and using diVerent methodologies. In a later study, the single cytoplasmic CA isozyme expressed in numerous tissues in the sea lamprey (P. marinus) exhibited a turn‐over rate that was similar to that of teleosts such as the rainbow trout (Esbaugh and Tufts, 2006). Furthermore, the amino acid sequence of the active site pocket of the lamprey RBC CA isozyme is very similar to that of rainbow trout, which is known to be a high‐turnover isozyme. These data indicate that the lamprey RBC CA isozyme is in fact a high‐turnover isozyme, and thus a high‐activity CA isozyme was present in RBCs early in vertebrate evolution.

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On the basis of the amino acid sequences of the putative active site, as well as the hydrophobic core of RBC CA, the emerging consensus is that RBC CA is highly conserved during vertebrate evolution. For example, relative to mammalian CA VII, the active site of lamprey (Esbaugh and Tufts, 2006), gar (Lund et al., 2002), and rainbow trout (see Esbaugh and Tufts, 2006) all only diVered by 3 of 36 amino acids. Biochemical measurements on RBC CA activity in lamprey yield a value, an order of magnitude lower than that of rainbow trout; however, this could be accounted for solely by the lower CA concentration within lamprey RBCs (Esbaugh et al., 2004). The fact that large diVerences in RBC CA concentration exists among the few primitive fish groups investigated to date [i.e., sea lamprey (P. marinus) < A. calva < O. mykiss (Gervais and Tufts, 1999; Esbaugh et al., 2004)] is interesting given that CA levels appear to be far in excess of that thought to be required in vivo. b. Gill CA. High levels of CA are found in the gills of most fishes studied to date. However, for the most part, it appears to be restricted to the cytoplasm where it plays an important role in ion and acid–base regulation þ by catalyzing the conversion of CO2 to HCO 3 and H ; crucial counterions  þ for Cl and Na exchange in particular (Perry and Laurent, 1990; Randall and Brauner, 1998). In elasmobranchs, membrane‐bound CA that is plasma accessible in the gills has been identified, and there is significant non‐RBC HCO 3 dehydration associated with CO2 excretion (Gilmour et al., 2001). Plasma accessible CA in the gills of the dogfish Squalus acanthias is also thought to be important in equilibrating postbranchial CO2 and Hþ, for chemoreceptors regulating ventilation (Gilmour et al., 1997; Henry et al., 1997). In general, very little is known about the subcellular localization of CA within the gills of primitive fishes. In the sea lamprey, P. marinus, there does not appear to be any plasma accessible membrane‐bound CA in the gill (Henry et al., 1993). In A. calva, subcellular fractionation indicates that the majority (>97%) of CA exists within the cytosol, and the small amount associated with membranes was not likely membrane bound (Gervais and Tufts, 1998). Clearly a great deal of more research is required to determine whether elasmobranchs represent the only fish group to have plasma accessible CA in the branchial circulation. c. CA in the ABO. There have been a number of studies investigating the possible role of CA in aerial CO2 excretion in the ABO of fishes. In the lungs of air‐breathing vertebrates, plasma accessible CA is available through a membrane‐bound CA (CA IV), which is anchored to the extracellular luminal surface of capillary lung endothelium (Heming et al., 1993), and this is thought to be the case in all air‐breathing animals (Stabenau and Heming, 2003). While extracellular HCO 3 dehydration is thought to play a role in

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CO2 excretion during lung capillary transport, the low plasma buVer value, and therefore lack of Hþ availability for HCO 3 dehydration, limits the degree to which this facilitates CO2 excretion (Bidani and Heming, 1991). Current estimates in the lungs of mammals indicate that less than 10% of total CO2 excretion may be associated with plasma accessible CA. Membrane‐bound CA has been identified in the ABO of bowfin. Interestingly, the membrane‐bound CA in the bowfin ABO was about three times less sensitive to the plasma CA inhibitor than RBC CA (Gervais and Tufts, 1998), and thus plasma CA inhibitors may be more eVective at scavenging and inhibiting CA released from lysed RBCs while minimally aVecting membrane‐bound CA as has been observed in mammals (Heming et al., 1993). In the lungfish P. dolloi, injection of an impermeant CA inhibitor that was hypothesized to inhibit any plasma accessible CA did not significantly aVect CO2 excretion rate into water or air, or alter arterial PCO2 or pH. Although it is not known whether plasma accessible CA exists in this species, if it does, it does not appear to contribute to CO2 excretion (Perry et al., 2005), and in general, the degree to which ABO CA influences aerial CO2 excretion remains controversial (Graham, 1997). Membrane‐bound CA in the lungs of vertebrates has also been proposed to ensure complete pH/PCO2 equilibration during blood capillary transit (Henry and Swenson, 2000), which may be important for the control of ventilation; however, this remains to be investigated in primitive fishes. 5. SYNTHESIS 5.1. How Do Primitive Fishes Compete with Other Fishes? In terms of species numbers, living primitive fishes are dwarfed by the enormous species richness of their extant relatives, which had the same or even less time available for speciation. Thus, the 84 agnathan, 8 primitive lobed‐finned, and 44 primitive ray‐finned species are opposed by the at least 500‐fold more speciose gnathostomes, tetrapods, and teleosts, respectively (Figure 5.2A). A priori species number is not necessarily a measure of the superiority of some structure or function in one group over that in another group since highly asymmetric species numbers in two sister groups can also evolve in simulations under a model of random speciation and extinction (Slowinski and Guyer, 1993). However, in terms of structural and functional diversity and habitats occupied, extant teleosts and tetrapods clearly outcompete living primitive ray‐finned and lobe‐finned fishes, respectively, and the same is true comparing

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all these jawed vertebrates with the living agnathans. After emerging onto land, the new environment presumably allowed adaptive radiation and the explosion of species numbers in tetrapods, compared to aquatic lobefins. Another adaptive radiation (or several successive ones) occurred in the aquatic habitat, giving rise to the current diversity of teleosts fishes, perhaps related to an additional whole‐genome duplication event in teleosts as compared to all other vertebrates (see below). The question arises: to what extent does the respiratory physiology of living primitive fishes contribute to their survival, given the competition by teleosts and by terrestrial, aerial, and secondarily aquatic lobe‐fins, such as man, birds, and seals, which prey on them? 5.1.1. Primitive Ray‐Finned Fishes (Actinopterygii) In contrast to many bimodally breathing members of teleosts, air‐ breathing primitive fishes such as the Polypteriformes, A. calva, and the genus Lepisosteus retain fully functional gills, which at times allow strictly aquatic breathing over prolonged periods. This may be important to avoid aerial or terrestrial predators lurking at the water surface (Graham, 1997; Farmer, 1999). It is of additional importance in temperate zone fishes like A. calva and L. osseus, where ice cover in winter may prevent air breathing for months (Rahn et al., 1971). Alternatively, these two genera are also able _ O2 almost completely aerially even under exercise in hot, to support their M severely O2‐depleted water. Thus, Burleson et al. (1998) concluded that using their ABO, ‘‘gar can maintain activity under . . . conditions that incapacitate virtually every other fish in their environment.’’ Sturgeons and paddlefishes are unusual among primitive ray‐finned fishes in that they do not breathe air. Both groups have evolved a highly specialized feeding mode, together with unusual ventilatory adjustments. Sturgeons are able to ventilate their gills via water intake through the dorsal part of the gill slit while their protrusible mouth is involved in suctional feeding, whereas paddlefishes are continually moving filter feeders, which use ram ventilation (see above). These specializations may contribute to their success despite competition by teleosts. 5.1.2. Primitive Lobe‐Finned Fishes (Sarcopterygii) The extraordinary capacity of African lungfishes to estivate in cocoons for months or even years in hard‐baked mud until favorable conditions are encountered again allows them to inhabit desiccation‐prone swamp areas and is unparalleled by teleosts. The South American lungfish L. paradoxa is not known to produce a cocoon but also estivates in damp burrows when water levels fall. Functional separation of deoxygenated systemic venous blood and oxygenated pulmonary blood in the heart of lungfishes provides

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their blood–gas transport system with an eYciency that is usually only found in the single‐loop arrangement of pure water‐breathers or the double‐loop arrangement found in birds and mammals. Interestingly, the swamp eel Synbranchus marmoratus, perhaps the only teleost able to compete with Lepidosiren in its natural habitat, has also evolved a way to avoid central mixing of oxygenated ABO blood and deoxygenated systemic blood, namely by using its gills as an ABO (Graham, 1997). For the coelacanth, Fricke and Hissmann (2000) have suggested that a low metabolic rate, together with a low‐energy drift‐hunting strategy and the peculiar electroceptive rostral organ for prey detection, has enabled L. chalumnae to successfully compete with modern ray‐finned fishes in a hypoxic and low biomass deepwater environment. The extremely low P50 would also be beneficial during exposure to hypoxia, but these low values need to be reconfirmed. 5.1.3. Jawless Fishes (Agnatha) Surprisingly, at least in one way both groups of living agnathans have turned the emergence of teleosts and tetrapods to their advantage. Thus, adult pacific lamprey L. tridentatus parasitize on teleosts and sperm whales (Hart, 1973), and, when given the opportunity, the Atlantic hagfish M. glutinosa feeds on teleostean, avian, and mammalian carcasses, often penetrating with head and anterior body into the carcass (Strahan, 1963). In both cases, respiratory water intake via the mouth or nostril is impeded. In lampreys, this can be compensated by unique tidal ventilation of gill pouches via their external openings (Randall, 1972). Hagfishes with their heads immersed in a carcass may rely on O2 uptake via the skin of their elongated bodies (Strahan, 1958; SteVensen et al., 1984). Feeding on the evolutionary younger groups of teleosts and tetrapods is clearly a secondary specialization, and fossils demonstrate that today’s jawless fishes are at least structurally quite diVerent from their early agnathan ancestors (Benton, 2000; Chapter 1, this volume) and thereby probably also from the last common ancestor with jawed vertebrates. 5.2. Primitive Fishes and the Evolution of Vertebrate Blood O2 and CO2 Transport Characteristics Living primitive fishes occupy strategic positions in the evolutionary tree of vertebrates and may shed light on the respiratory physiology of the first vertebrates and the first Osteichthyes (Figure 5.2A). Reconstructing the evolution of blood O2 and CO2 transport characteristics and their condition in ancestral species from the condition in living species is a fascinating task that may unravel general trends in physiological evolution but has its pitfalls.

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5.2.1. A Note of Caution In most cases, it is not clear to what extent phenotypic similarities and diVerences between species are genetically based and therefore subject to Darwinian evolution. Genetic components are diYcult to clarify because of the enormous phenotypic plasticity of blood O2 and CO2 transport characteristics and because diVerent experimental methods and approaches sometimes give significantly diVerent values for the same variable (see above discussion of the Bohr eVect in lungfishes). Because the primary structure of Hb is undoubtedly genetically determined, one useful approach to unravel evolutionary trends in blood O2 and CO2 transport is to relate Hb‐O2‐binding characteristics to its molecular structure. Although the already large number of nucleotide and amino acid sequences for fish globins seems to be ever increasing, there is a conspicuous lack in globin sequences and associated structure–function analyses in primitive fishes. No Hb sequences are available for primitive ray‐finned fishes such as Polypteriformes, gars, bowfin, and even basal teleosts such as the Osteoglossiformes. Even the Elopomorpha are only covered by globins from the highly modified Anguilliformes. Similarly among primitive lobe‐finned fishes, no sequences are available for the Australian and African lungfish species. Another diYculty is that, whether due to insuYcient data or an attempt to simplify, discussions of evolutionary trends in terms of vertebrate blood O2 and CO2 transport characteristics are often limited to the larger subgroups, which are treated sequentially, such as from agnathans, elasmobranchs, teleosts to one or more groups of tetrapods (Lenfant et al., 1970; Wood and Lenfant, 1987; Nikinmaa, 1990, 1997, 2001). A hidden danger of such studies is that, even if unintended by the authors, they can easily be interpreted in a teleological and anthropocentric way such that living members of increasingly closer relatives to tetrapods (or mammals or man) are interpreted to have changed little since they split from the last common ancestor with tetrapods and are taken to represent a linear succession of stages that the ancestors of tetrapods underwent before reaching their present form. In strong contrast to such a view, increasing evidence points to an additional, whole‐genome duplication event in the ray‐finned fish lineage as compared to lobe‐finned fishes (including tetrapods) that may have provided the genetic basis for the adaptive radiation and evolutionary novelties in modern teleosts (Meyer and Schartl, 1999; Hoegg et al., 2004; Crow et al., 2006; but see Donoghue and Purnell, 2005). Yet, although not explicitly stated, an inherent assumption in the older literature appears to be that the fishlike ancestors of tetrapods went through a teleost‐like state before they acquired the current blood O2 and CO2

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transport characteristics. For example, it was held that to venture onto land, the tetrapod ancestors had to reduce the strong CO2 sensitivity of blood O2 binding (i.e., the Bohr and Root eVects) typical of modern teleosts and had to acquire a higher blood‐buVering capacity than found in teleosts (Carter, 1931; Root, 1931). Studies of primitive sarcopterygians (lungfishes and coelacanth) and actinopterygians (Polypteriformes, sturgeons, gars, and bowfin) now suggest that the last common ancestor of these groups had a high blood buVer value and that the low‐Hb buVer capacity, the Root eVect, and the exceptionally large Bohr eVect evolved only within the actinopterygians after they split form the ancestor of tetrapods (see below; Berenbrink et al., 2005; Berenbrink, 2006). Thus, the jawed vertebrate ancestors of air‐breathing tetrapods never went through a teleost‐like state and were more likely to show elasmobranch‐like blood O2 transport properties. Similarly, modern lungfishes are often seen as resembling the ancestors of today’s tetrapods at a time when they first emerged onto land. Most scientists now agree that lungfishes are the closest living relatives of tetrapods; however, that does not mean that lungfishes have survived unchanged since their split from the lineage that gave rise to tetrapods. Thus, the first lungfishes may have been non‐air‐breathing fishes of open marine habitats (Marshall and Schultze, 1992; Cloutier and Ahlberg, 1996; Chapter 1, this volume). Among living lungfishes, the Australian N. forsteri is the least dependent on air breathing and does not estivate. In this regard, it may more closely resemble ancestral forms than the obligate air‐breathing and estivating African and South American lungfish genera Protopterus and Lepidosiren, respectively. However, fossils of estivating lungfishes in cocoons can be dated back to 286 mya (Graham, 1997), whereas the fossil record of the group containing N. forsteri is dated back to only 245 mya (Schultze, 1993). Thus, given the current uncertainty regarding the relationships of fossil lungfishes, it is also possible that N. forsteri secondarily lost the ability to estivate and changed from an obligate to a facultative air‐breather. This is supported by their lung anatomy, whereby the dorsal unpaired lung in N. forsteri may be derived from the more primitive arrangement of paired ventral lungs in African and South American lungfishes (Schultze, 2003). Moreover, despite the ventral location of the primitive lungfish lung and amphibian lungs, anatomical diVerences suggest that the amphibian lung is not directly derived from the organ in lungfishes (Perry and Sander, 2004). Thus, it appears possible that air breathing evolved independently in modern lungfishes and tetrapods and that consequently the partially divided heart with functional separation of pulmonary and systemic blood pathways was also acquired independently. Another pitfall of this kind in the reconstruction of ancestral respiratory physiology is illustrated by the use of modern amphibians to infer the mode

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of CO2 elimination in the first vertebrates emerging onto land in the Devonian. It was proposed that much like present‐day amphibians these protoamphibians eliminated CO2 via their moist skin and thereby avoided the general respiratory acidosis otherwise occurring when due to a presumed general decrease in ventilation rates the first terrestrial air‐breathers could no longer readily eliminate CO2 (Howell, 1970; see also Ultsch, 1996). However, fossils show that in contrast to modern amphibians, several protoamphibians were heavily scaled and rather large, with a low surface area to volume ratio, creating unfavorable conditions for CO2 elimination via the skin. Thus, Ultsch (1996) proposed that protoamphibians living in hypercapnic waters had already coped with elevated blood CO2 levels before they ventured onto land by evolving ion‐regulatory mechanisms for an equivalent rise in plasma bicarbonate. In the following discussion, it may be helpful to remind oneself of the provocative but well‐supported statement that lungfishes share more derived characters with—and therefore are more closely related to—a cow than to a salmon (Gardiner et al., 1979). 5.2.2. Trends in the Evolution of Vertebrate Blood O 2 and CO 2 Transport Characteristics Bearing these points in mind, we tentatively identify some general trends in the evolution of vertebrate blood respiratory properties. It is generally accepted that vertebrate life began in water and the blood respiratory properties of the earliest vertebrates were presumably shaped by the physicochemical consequences of water breathing such as the relative ease of CO2 release into the water and the relatively low availability of O2. This is reflected in the unanimously low CO2/bicarbonate levels and the high intrinsic Hb‐O2 aYnity in virtually all present‐day water‐breathers, primitive or modern. Lampreys are a curious exception because of their low intrinsic Hb‐O2 aYnity. This may therefore be a secondary specialization. As hypothesized by Wood and Lenfant (1987), the generally high intrinsic O2 aYnity of vertebrate Hbs may be related to the lower atmospheric PO2 values prevalent in the Devonian, where several of the main vertebrate lineages evolved. It is often presumed that the lack of stable tetrameric Hbs consisting of two ab‐heterodimers in living agnathans represents the ancestral condition of vertebrates (Wood and Lenfant, 1987). This seems plausible since a‐ and b‐globins appear to have arisen by duplication of an ancient globin gene (Goodman et al., 1975). However, if molecular evidence, which suggests that living lampreys and hagfishes are not consecutive sister groups of extant jawed vertebrates but rather together form the sister group of gnathostomes

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(Takezaki et al., 2003; Chapter 1, this volume), is accepted, this is not the single most parsimonious explanation. Under this scenario, a last common ancestor of agnathans and gnathostomes with a2b2 tetramers is equally parsimonious. Similarly, lack of significant RBC AE in agnathans is considered the primitive, ancestral state in the ancestor of agnathans and jawed vertebrates. However, it should be remembered that, again accepting that lampreys and hagfishes together are the sister group to gnathostomes, it is conceivable that RBC AE was present in all early vertebrates and only secondarily lost in an ancestor of living agnathans. In previous reviews, the first organophosphate that was used to modulate vertebrate Hb function was considered to be ATP, which was found to be the predominant organophosphate in some shark RBCs (Wood and Lenfant, 1987). However, GTP, which is a stronger eVector than ATP, is found in higher concentrations than ATP in some other sharks and in a member of the Holocephali, the sister group of elasmobranchs (Bartlett, 1978a). Evolutionary analysis of RBC organic phosphate concentrations in a large number of vertebrates confirms that both ATP and GTP were likely present in significant amounts in the first jawed vertebrates (Val, 2000). Even some agnathans contain traces of GTP next to large amounts of ATP, ADP, and, surprisingly, 2,3‐DPG (Johansen et al., 1973; Bartlett, 1982). Although we do not know about ancestral vertebrates, organic phosphates in living agnathans have only an unspecific anion eVect on the monomer–oligomer equilibrium in hagfish Hb, and no eVect at all on lamprey Hb (Nikinmaa, 2001). Leaving speculations about the presence or absence of stable tetrameric Hbs, AE1, or organophosphate eVects in RBCs of ancestral vertebrates aside, Figure 5.2G illustrates the most parsimonious reconstructions of some other blood gas transport properties, which are better substantiated. These reconstructions are not aVected by the alternative positionings of lampreys as the sister group to hagfishes or jawed vertebrates. They use linear parsimony as the optimality criterion for reconstruction (Berenbrink et al., 2005; Berenbrink, 2006). The fixed‐acid (i.e., CO2‐independent) Haldane eVect, DzHþ, is mechanistically linked to the fixed‐acid Bohr eVect and numerical similar to 4F in the absence of other interacting allosteric eVectors (Eq. 1). Its value is reconstructed to be below 1.5 mol Hþ per mol Hb tetramer in the last common ancestor of vertebrates (Figure 5.2G). This is still seen in M. glutinosa, living sharks, and the non‐amniotic sarcopterygians L. paradoxa and X. laevis (Figure 5.2E). Increased fixed‐acid Haldane eVects above 1.5 mol Hþ per mol Hb tetramer evolved three times independently, namely in L. fluviatilis, ray‐finned fishes, and to a lesser extent in amniotes (Figure 5.2E,G). In primitive ray‐finned fishes, the evolutionary increase in the fixed‐acid

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Haldane eVect is followed first by a gradual increase in the Root eVect and then a gradual reduction in specific Hb buVer value. This was followed by the evolution of a choroid rete mirabile and ocular O2 secretion in the last common ancestor of A. calva and teleosts. Finally, the adrenergic NHE was never present in primitive ray‐finned fishes or primitive teleosts and only evolved in RBCs of advanced teleosts (Figure 5.2G) (Berenbrink et al., 2005). No such evolutionary trends are observed in primitive lobe‐finned fishes, which only very rarely possess Hbs that are more than 10% deoxygenated at low pH and air equilibration as seen in X. laevis, have usually high Hb buVer values, no choroid rete mirabile, and no pH regulatory NHE in their RBCs (Figure 5.2G) (Berenbrink et al., 2005). However, in some advanced tetrapods Hb amino acid sequence data suggest similar reductions in Hb buVer values as seen in teleosts (Berenbrink, 2006). Interestingly, the elevated fixed‐acid Haldane eVect in lampreys is associated with exactly the same changes in RBC physiology as seen in primitive ray‐finned fishes. Thus, lamprey RBCs show a strong Root eVect, the lowest ever measured specific Hb buVer value, and RBC pH regulation by an NHE, although a choroid rete mirabile never evolved (Figure 5.2B–G) (Nikinmaa, 1986, 1993; Jensen, 1999). The sequence in which these diVerences evolved from the condition in the ancestral vertebrates is not resolved. It is not known why this trend is not seen in advanced lobe‐fins (amniotes, i.e., reptiles, birds, and mammals), which present the third case that shows an (albeit modest) evolutionary increase in the fixed‐acid Haldane eVect. Berenbrink et al. (2005) and Berenbrink (2006) have discussed diVerences in the mechanism of the fixed‐acid Bohr eVect for the evolution of a Root eVect in ray‐finned and lobe‐finned fishes and the implications of terrestrial air breathing and the associated elevation in blood CO2/bicarbonate buVer capacity for the evolution of specific Hb buVer values and RBC pH regulation. Lampreys and teleosts are among the most active aquatic vertebrates, with lampreys in some rivers undertaking the same exhausting spawning migrations, overcoming the same rapids and other obstacles as salmonids. Little is known about the exercise physiology of sharks, but at least in comparison with hagfishes and primitive lobe‐finned fishes, it appears that the suite of respiratory blood–gas transport characteristics, which independently evolved in lampreys and ray‐finned fishes, ideally poised them for exercising in water. ACKNOWLEDGEMENTS CJB was supported by an NSERC Discovery grant and MB’s research was supported by funds from BBSRC, United Kingdom. We would like to thank Pia Koldkjær and Kim Suvajdzic

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for editorial assistance, Pia Koldkjær and Mathew Regan for commenting on an earlier version of this chapter, and the reviewers for valuable criticisms and suggestions.

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6 IONIC, OSMOTIC, AND NITROGENOUS WASTE REGULATION PATRICIA A. WRIGHT

1. Introduction 1.1. Origins in Seawater 1.2. ‘‘Parting of the Ways’’: The Move to Freshwater 1.3. Key Sites of Osmoregulation and Nitrogen Excretion in Fishes 2. Ionic and Osmotic Regulation 2.1. In Seawater 2.2. In Freshwater 2.3. Moving Between the River and Sea 3. Nitrogen Excretion 3.1. Toxic Ammonia 3.2. Synthesis of Nitrogen End‐Products 3.3. Excretion 3.4. The Challenges of Estivation 4. Concluding Remarks

Among primitive fishes, there is a diversity of strategies that have evolved to cope with ion, water, and nitrogen balance. The whole physiological spectrum is found from ionic and osmotic conformation to the regulation of body fluids distinct from the environment. The most primitive of vertebrates, the marine hagfish iono‐ and osmoconforms to its seawater environment, whereas their euryhaline relatives, the lampreys, iono‐ and osmoregulate. The gills of Agnathans contain both pavement and mitochondrial rich cells, but the arrangement of cells and structural features are unique relative to euryhaline teleosts. Coelacanths are osmoconformers but ionoregulators, maintaining high internal urea levels like the elasmobranchs. In many primitive species, ammonia is the dominant excretory product as it is in most teleost fishes. The exception is the coelacanth and estivating lungfish that synthesize urea via the urea cycle and excrete urea. Membrane transporters have been isolated in fish that regulate urea and possibly ammonia movements between tissue 283 Primitive Fishes: Volume 26 FISH PHYSIOLOGY

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compartments and to the environment. Nitrogen excretion during early life stages presents a particular challenge in encapsulated embryos dependent on yolk protein catabolism. As yet, little is known about how primitive fish embryos face these challenges. Research on primitive fish species will broaden our knowledge of the evolution of osmoregulation and excretion in fish and terrestrial vertebrates. 1. INTRODUCTION The focus of this chapter is on the Agnathans (lampreys and hagfishes), the Sarcopterygians (the coelacanth and the lungfishes), and the primitive Actinopterygians such as the Polypteriformes (bichirs and reedfish), the Chondrostean Acipenseriformes (paddlefishes and sturgeons), and the Neopterygians (gars and the bowfin). The systematics and phylogeny of these fishes is outlined by P. Janvier in Chapter 1. This chapter concerns the regulation of ions, water, and nitrogen end‐products. Although the endocrine control of these processes is important in their regulation, the current chapter focuses on the sites, structures, and mechanisms involved in iono‐ and osmoregulation. Readers are referred to two comprehensive chapters on ‘‘Peripheral Endocrine Glands’’ by J. Youson in this volume (Chapters 8 and 9) for a more detailed review of endocrine control. 1.1. Origins in Seawater Over the last 100 years or so, scientists have debated whether the first fishes evolved in freshwater or seawater (Smith, 1932, 1961; Munz and McFarland, 1964; Fa¨nge, 1998). These arguments are based on osmoregulatory structures present in extant fishes, including the most primitive jawless fishes, the Agnathans. Collective opinion weighs in on the seawater side (GriYth, 1994; Holland and Chen, 2001; Chang et al., 2006); early Agnathans are thought to have inhabited shallow seas or estuaries (Helfman et al., 1997). The living jawless hagfishes are entirely marine. They are unique among vertebrates in having plasma ion concentrations and osmolarity roughly the same as seawater, similar to marine invertebrates. 1.2. ‘‘Parting of the Ways’’: The Move to Freshwater Smith (1932) first noticed the interesting contrast between the blood osmotic concentration of the hagfish Myxine glutinosa with that of the lamprey Petromyzon fluviatilis. Hagfish iono‐ and osmoconform to their seawater habitat, whereas lamprey iono‐ and osmoregulate in either seawater or freshwater. It has taken decades to form a more complete understanding of vertebrate osmoregulation, but the following prescient statement by Smith

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(1932) captures so much. It is thus possible that these two groups lead back to a ‘‘parting of the ways’’ in the evolution of body fluid regulation. (The two groups he refers to are the hagfish and the lamprey.) After early beginnings in seawater, lamprey ancestors moved back to freshwater, no later than the early Cretaceous (Chang et al., 2006). Osmotic control that evolved first in lamprey and teleost ancestors has been an adaptive trait with a selective advantage, in both freshwater and marine environments (Robertson, 1963). Although the hagfish lineage has survived for millions of years, their unique form of iono‐ and osmoconformation has not appeared in any other group of aquatic vertebrate. 1.3. Key Sites of Osmoregulation and Nitrogen Excretion in Fishes The gills, kidney, intestine, urinary bladder, and integument are the key sites of ion exchange, nitrogen elimination, and osmoregulation in most fishes (for a review see Marshall and Grosell, 2006). In many of the primitive fish groups, there is limited information to verify the involvement of these sites or the importance of one tissue over another. However, it is probably safe to say that the gill is the dominant site of exchange of ions, water, and nitrogenous waste products in most of the primitive fishes discussed below. Among primitive fishes, with the exception of lungfish, the gill has a large surface area in contact with flowing water and with the aid of specialized branchial epithelium, materials are transported between the blood and water. The kidney is also an important structure, particularly in freshwater fishes where passive water gain is countered by a high urine output with reabsorption of key monovalent ions (see Section 2.2); however, renal nitrogen excretion is typically low. The intestine and urinary bladder are important for absorption of ions and water, but will not be considered further due to the lack of research on primitive fishes. The integument in most fish presents a barrier to the exchange between the internal and external environments, and may only play a critical role in a few unusual species (Wood, 1993; see Section 2.2). It should also be noted that a postanal gland, similar in structure to the elasmobranch rectal gland, has been described in the coelacanth Latimeria chalumnae (reviewed by Locket, 1980). This gland probably represents an additional site of NaCl secretion. 2. IONIC AND OSMOTIC REGULATION 2.1. In Seawater 2.1.1. Ionoconform, Osmoconform: The Hagfish Strategy Hagfish are the only aquatic vertebrate known that ionoconform as well as osmoconform to their seawater environment (Figure 6.1A) (Robertson, 1963; Evans, 1993; Karnaky, 1998). They are not found in water of low salinity,

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Fig. 6.1. Schematic representation of ionic and osmotic balance in (A) hagfish, (B) coelacanth, (C) seawater lamprey, and (D) freshwater lamprey. Total plasma osmolality (mosmol kg
1), plasma osmolality attributed to NaCl (mosmol kg
1), and plasma urea concentrations (mmol kg
1) are given inside each diagram, were appropriate. Passive fluxes are represented by a dashed line, whereas active mechanisms are shown as a solid line. [A, values from McDonald and Milligan (1992), B, GriYth (1991), and C and D modified from Bartels and Potter (2004).]

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contrasting sharply with their Agnathan relatives, the lampreys (see Section 2.1.3, below). They gain weight in hyposmotic water, slowly recovering after 7 days, whereas in hyperosmotic water they lose weight and fail to recover (McFarland and Munz, 1965). Smith (1932) established that the ionic composition of hagfish blood approximated the inorganic ion concentrations of seawater (Figure 6.1A), a very diVerent osmoregulatory strategy compared to osmoconforming coelacanth and elasmobranch fishes (Figure 6.1B; see Section 3.3.3). Similar to elasmobranchs, the intracellular osmotic composition of marine hagfish has a large organic component, with trimethylamine oxide (TMAO) concentrations exceeding 200 mM and one‐third lower inorganic ion levels relative to serum levels (Bellamy and Jones, 1961). There has been very little attention to the mechanisms of iono‐ and osmoregulation in hagfish, possibly because it was assumed that iono‐ and osmoconforming with the environment requires minimal eVort. Large mitochondrial rich cells (MRCs) are numerous on hagfish gill lamellae (Mallat and Paulsen, 1986; for a review see Bartels, 1998; Choe et al., 1999), but unlike seawater‐acclimated lampreys (see Section 2.1.3) the MRCs appear singly, sandwiched between gill pavement cells. A leaky paracellular junction that is so clearly observed between MRCs in marine teleosts and lampreys and allows for the passive leak of Naþ is not apparent in hagfish gills. Hagfish gill MRCs stain for Naþ/Kþ ATPase (Mallat et al., 1987; Choe et al., 1999) and Naþ/Hþ exchanger isoforms (NHE) are also expressed in the M. glutinosa gill but cell localization has yet to be determined (Edwards et al., 2001; Choe et al., 2002). Evans (1984) proposed that NHE and Cl
/HCO
3 exchangers were operating in parallel in hagfish gill epithelium for acid or base excretion, and provided a ‘‘preadaptation’’ for ion regulation in species that later inhabited freshwaters. This proposition was later supported by McDonald et al. (1991) who showed that acid–base disturbances in M. glutinosa were fully corrected by gill mechanisms, probably involving NHEs. Indeed, gill NHE mRNA is upregulated in hagfish gill tissue following an acid infusion (Edwards et al., 2001). It might be expected of the iono‐ and osmoconforming hagfish that passive water influx (found in freshwater teleosts) or passive water eZux (found in seawater teleosts) would be minimal, raising the question of kidney structure and function (GriYth, 1994; Fels et al., 1998). The hagfish kidney is unusual in having large glomeruli (500–1500 mm) and two archinephric ducts or ureters (Riegel, 1998). There have been only a few studies on kidney function (Karnaky, 1998). Overall, the hagfish kidney functions in the reabsorption of glucose and amino acids and secretion of some ions (Munz and McFarland, 1964; Riegel, 1998); however, there are some discrepancies in the literature whether reabsorption of Naþ or Cl
occurs (McInerney, 1974; Alt et al., 1981). Little progress has been made over the last 40 years or so on understanding of iono‐ and osmoregulation in hagfish. More information is required on

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the stability of plasma NaCl concentrations under diVerent physiological conditions (e.g., feeding, exercise, and acid–base disturbances) to determine if NaCl in the blood is regulated under some circumstances. This would go a long way in understanding the potential roles of gill MRCs and specific ion transporters. Osmotic disturbances may be primarily due to the composition of the diet in this stenohaline fish. Using traditional methods to study ion fluxes and water balance, it would be valuable to know how hagfish compensate for a meal with a high water content (e.g., teleost tissues) versus an isosmotic meal (e.g., invertebrate, elasmobranch tissues). 2.1.2. Ionoregulate, Osmoconform: The Coelacanth Strategy An overall understanding of ionic and osmotic regulation is lacking in Latimeria, with only two surviving species of Coelacanths, L. chalumnae (Comoro Islands and vicinity) and L. menadoensis (discovered in 1999 oV the coast of Indonesia) (Holder et al., 1999). It is well accepted that L. chalumnae plasma and tissues contain elevated concentrations of urea similar to marine elasmobranchs and they osmoconform to their seawater environment (Figure 6.1B). What is less clear is whether plasma osmolality is somewhat greater (as in elasmobranchs); equal to or slightly less than (GriYth, 1991) the local marine environment. The confusion over this issue no doubt relates to a limited number of samples that have been collected from either frozen (Pickford and Grant, 1967) or moribund specimens (GriYth et al., 1976). On the basis of available evidence, it does appear that plasma NaCl concentrations are 25% lower than values reported in marine elasmobranchs (McDonald and Milligan, 1992). If this is indeed the case, then there are several implications. First, other organic osmolytes besides urea and TMAO (GriYth et al., 1974) must be present in the plasma to add up to 1000 mosmol kg
1. Second, to maintain a combined osmolality of plasma NaCl at 350 mosmol kg
1 (Figure 6.1B), a value almost identical to stenohaline marine teleosts (346 mosmol kg
1; McDonald and Milligan, 1992), L. chalumnae, must have powerful mechanisms to secrete NaCl. Are these mechanisms partly in the gill (i.e., chloride‐type cells) or solely in the postanal gland? The structure of the kidney resembles other osteichthyes (Locket, 1980), but measurements of a single urine sample are insuYcient to understand renal function (GriYth et al., 1976). There is much to learn and physiologists await the opportunity to study multiple live Latimeria. 2.1.3. Ionoregulate, Osmoregulate: The Alternative Strategy In seawater, marine teleosts as well as lampreys and sturgeons maintain body fluid osmolality and NaCl concentrations at about one‐third of their environment (Morris, 1972; Potts and Rudy, 1972; Beamish, 1980a). The seawater origins of Agnathans [with lampreys later entering freshwater

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(Chang et al., 2006)], but freshwater origins of Teleostei (with subsequent forays to seawater) suggest that convergent evolution may explain the remarkable similarity of gill and renal osmoregulatory mechanisms in these two groups of fish (Bartels and Potter, 2004). Fish ion and osmoregulation have been well reviewed (Marshall, 2002; Bartels and Potter, 2004; Marshall and Grosell, 2006). Ionic and osmotic gradients result in the constant influx of NaCl and loss of body water that are counterbalanced by active excretion of NaCl across the gills and replenishment of water by drinking (Figure 6.1C). The branchial epithelium, therefore, plays an important role in ionoregulation. The structure and cellular composition of the lamprey gill have been extensively described (reviewed by Bartels and Potter, 2004). Chloride cells in lamprey gills in seawater form long rows and lack accessory cells that are associated with chloride cells in teleosts. The apical crypts so distinctive in teleost gills in seawater are absent in lampreys. Despite these small structural diVerences, the lamprey chloride cells share most of the other characteristics typical of teleost fishes and other salt‐secreting epithelia, such as a high density of mitochondria, basolateral membrane elaboration, and a leaky paracellular pathway (Laurent, 1984; Karnaky, 1986; Bartels and Potter, 2004). Furthermore, the mechanism of active NaCl secretion, involving Naþ/Kþ ATPase, Naþ/Kþ/2Cl
cotransporter, and a chloride channel (Marshall and Grosell, 2006), is assumed to be present in seawater lamprey gills (Bartels and Potter, 2004), but this has not been verified. The kidney plays a small role in lamprey osmoregulation in seawater, producing low volumes of urine as would be expected of marine osmoregulators (Logan et al., 1980). Similar to teleosts, lamprey and sturgeon kidneys preferentially secrete divalent ions in the urine (Pickering and Morris, 1970; Logan et al., 1980; Krayushkina et al., 1996). Following transfer from freshwater to brackish water, glomeruli size declines and the tubule cells and brush border were reduced in two sturgeon species, Acipenser naccarii (Cataldi et al., 1995) and Huso huso (Krayushkina et al., 1996), implying reduced function in hypersaline water. 2.2. In Freshwater In freshwater, iono‐ and osmoregulation in primitive fishes is accomplished as outlined in Figure 6.1D for freshwater lamprey, similar to freshwater teleosts. Plasma is hyperosmotic to the surrounding media and therefore passive water gain and NaCl loss must be compensated by the elimination of copious volumes of urine and the active uptake of ions via the branchial epithelium. The majority of studies on primitive fish osmoregulation have focused on lampreys and sturgeon, with far fewer on bowfin,

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gar, paddlefishes, and birchirs. Elegant studies by Bull and Morris (1967) and Morris and Bull (1968, 1970) established that freshwater ammocoete larva of Lampetra planeri carefully regulate serum and tissue water and ion content, that external calcium aVects gill permeability to ions, and that sodium influx is dependent on both internal and external sodium concentrations. The life cycle of all species of lamprey consists of a larval phase (ammocoetes) in freshwater, metamorphosis into young adults that may migrate downstream to the ocean, if anadromous, a marine trophic phase, followed by a return migration upstream to freshwater streams where they spawn and die (Beamish, 1980b). The gill cell composition of lampreys changes with life cycle and external salinity. Larval gills have both ammocoete MRCs and intercalated MRCs, as well as pavement cells. Downstream migrants (freshwater) retain the intercalated MRCs and pavement cells, and new chloride cells develop (Youson and Freeman, 1976; Bartels and Potter, 2004). Choe et al. (2004) further identified two subtypes of MRCs based on immunohistochemical staining of the gills of freshwater adult lampreys. They proposed a model of ion transport where MRC‐A that express Naþ/Kþ ATPase are responsible for Naþ uptake, whereas MRC‐B that stain for carbonic anhydrase and V type Hþ ATPase transport Cl
. Verification of this model will require sophisticated separation of MRC‐A and MRC‐B type cells, similar to isolated cell studies in freshwater teleosts (see review by Marshall and Grosell, 2006). Kidney function in freshwater primitive fishes is probably comparable to teleosts. In bowfin and lampreys, the kidney reabsorbs Naþ and Cl
, and has a relatively high glomerular filtration rate that is correlated with urine flow rate (Logan et al., 1980; Butler and Youson, 1988). The renin‐angiotensin system (RAS) has been identified in the river lamprey Lampetra fluviatilis (Cobb et al., 2002; Brown et al., 2005). In vertebrates, the RAS plays an important role in blood volume and pressure regulation, and studies in L. fluviatilis indicate that the RAS responds to external water salinity changes (Rankin et al., 2001; Brown et al., 2005). Ionic and osmotic balance in submerged African lungfish has not been well studied, but presents an interesting challenge given the reduction of gill surface area (Laurent et al., 1978) and reliance on lung respiration (Perry et al., 2005a,b). Moreover, the absence of gill convection during terrestrial episodes may further exacerbate osmoregulatory control. Wilkie et al. (2007) have discovered that Protopterus dolloi remains in ionic and osmotic balance after a 6‐month episode on moist land, partly because they exchange water and ions across their ventral body surface. In fact, their data indicates that the majority of water exchange in submerged lungfish also occurs across the ventral skin, which may play a similar role as the pelvic region in amphibians. This is a fascinating avenue for future study.

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2.3. Moving Between the River and Sea The life history of anadromous lampreys and sturgeon involves an initial freshwater phase, followed by migration to the sea and a later return to freshwater streams to spawn. In lamprey (Petromyzon marinus), anadromous populations are far better at maintaining plasma osmolality with rising external salinity relative to landlocked populations (Beamish et al., 1978). Plasma osmolality increases on exposure to saline water in sturgeon (McEnroe and Cech, 1985; Cataldi et al., 1995; Krayushkina et al., 1996; Altinok et al., 1998; McKenzie et al., 2001; Martı´nez‐Al´varez et al., 2002; Rodrı´guez et al., 2002) and lamprey (Beamish, 1980a). Gill chloride cell size and number increase when sturgeon are transferred to hypersaline waters (Altinok et al., 1998) accompanied by an upregulation of gill Naþ/Kþ ATPase activity in some species (McKenzie et al., 1999; Rodrı´guez et al., 2002), but not all (Jarvis and Ballantyne, 2003). These responses are comparable to euryhaline teleosts moving from fresh to seawater environments, although far more details of molecular and cellular changes have been uncovered in teleosts (for a review see Marshall and Grosell, 2006). Gill cell composition in euryhaline lamprey is distinct from teleosts. Prior to migration down river to the sea, the surface of the gill MRC of young adult lampreys is covered by the flanges of adjacent pavement cells, with only a relatively small circular area exposed covered with microvilli (Peek and Youson, 1979; Mallat et al., 1995; Bartels and Potter, 2004). After young lampreys enter seawater, the pavement cells retract revealing a larger rectangular microvilli‐free chloride cell surface area, as well the paracellular channel between adjacent cells widens. These excellent structural studies now need to be linked to functional investigations to understand the role of ion transport proteins in specific cell types. 3. NITROGEN EXCRETION 3.1. Toxic Ammonia In aqueous solution, ammonia exists both as NH3 and NHþ 4 , according to the equation: NH3 þ H3 Oþ $ NHþ 4 þ H2 O The term ammonia represents the sum of the NH3 and NHþ 4 concentrations. The pK of the reaction is about 9.5 so that at fish blood pH values (pH  8) þ  about 96% of ammonia will be in the NHþ 4 form at 25 C. NH4 is charged and larger than NH3 and therefore has a lower diVusivity compared to NH3.

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Ammonia may accumulate in fish under a variety of conditions and, if severe, can result in convulsions, coma, and eventually death (for reviews see Ip et al., 2001, 2004; Randall and Tsui, 2002). Most of the fish discussed in this chapter are primarily ammoniotelic, that is, they excrete primarily ammonia. If environmental conditions preclude normal rates of ammonia elimination (e.g., elevated water pH, air exposure, or limited access to water), then endogenously produced ammonia may accumulate in the fish. Elevated environmental ammonia occurs in natural and hatchery freshwaters. The reversal of the branchial blood‐to‐water ammonia gradient results in ammonia uptake and elevated plasma and tissue ammonia concentrations in a variety of fish species, including lungfish (Chew et al., 2005). The toxicity of elevated environmental ammonia varies with water pH, temperature, salinity, and oxygen levels (for review see Ip et al., 2001). As well, intraspecific variation, developmental eVects, nutritional status, and acute versus chronic exposure all impact ammonia toxicity. There is a paucity of data on primitive fishes. In one study by Fontenot et al. (1998), the 96‐h median‐lethal concentration (96‐h LC50) for NH3 for fingerling shortnose sturgeon Acipenser brevirostrum was 0.58  0.21 mg liter
1 (mean  SD, 18  C). As a comparison, the 96‐h LC50 value for NH3 in both rainbow trout (Oncorhynchus mykiss) and fathead minnow (Pimephales promelas) was 0.37 mg liter
1 (14  C) (Thurston et al., 1981). With very little solid data on ammonia tolerance between diVerent orders of fishes, it is not easy predicting which fish might demonstrate a higher tolerance to environmental ammonia. The strongest guidelines may be environmental or ecological considerations. For example, there is evidence that freshwater fishes are less susceptible to ammonia toxicity compared to their seawater counterparts (Ip et al., 2001). The tolerance to elevated water ammonia levels may be high in fish that form aggregations, burrow into confined spaces, or encounter low water volumes. Thus, there is an apparent correlation between ammonia and hypoxia tolerance in fish (for review see Walsh et al., 2006). For example, high densities of hagfish have been observed feeding on whale carcasses in deep ocean environments (Martini, 1998). Rotting flesh combined with hundreds of relatively large (0.5 m) ammonia‐excreting hagfish may create a local environment high in ammonia. As well, hagfish normally reside in mud burrows on the ocean floor, such a confined space may also result in elevated external ammonia. Due to these circumstances, it is possible that hagfish may have evolved a high tolerance to ammonia, but this has not been tested. Another Agnathan, the lamprey burrows into soft mud for several years in the larval stage (ammocoete) and feeds on detritus (Moore and Mallatt, 1980). Depending on the rate of water exchange near the ammocoete surface, endogenous ammonia may accumulate in the local environment. In fact, the 96‐h LC50 value for NH3 was 1.7 mg liter
1 in P. marinus

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ammocoetes (Wilkie et al., 1999), fivefold higher relative to the values reported for teleosts (see above). Lungfish encounter limited water availability or a complete absence of water during estivation (see Section 3.4), and therefore may frequently encounter ammonia loads and have a correspondingly higher tolerance for ammonia. LC50 values have not been reported in the African lungfish, but this group of fish certainly appears to be ammonia tolerant. P. dolloi survives 6 days in water containing 100 mmol liter
1 NH4Cl and barely accumulates ammonia in the extracellular compartment (Chew et al., 2005). It has recently been discovered that their remarkable insensitivity to elevated external ammonia may be partly linked to the excretion of acid. Wood et al. (2005a) found that when P. dolloi are exposed to 309 mmol liter
1 NH4Cl for 7 days they excrete both CO2 and titratable acid (e.g., Hþ) into their external environment, lowering water pH to as low as pH 3.7 in one case (even with aeration). Environmental acidification ensures that the highly diVusible NH3 remains low in the external water, thereby lowering the overall uptake of ammonia by the lungfish. P. dolloi also detoxifies excessive ammonia by conversion to urea via the urea cycle (see Section 3.2.2) when confronted with exceptionally high external ammonia concentrations (Chew et al., 2005). Hence, the African lungfish may have multiple strategies of coping with this toxic compound. It is likely that African lungfish rank up there with other highly ammonia‐tolerant species, such as the mudskipper (Periophthalmodon schlosseri), that can survive also in 100‐mmol liter
1 NH4Cl and has a 96‐h LC50 for NH3 of 7.6 mg liter
1 (Ip et al., 2004)! The brain of fish, as well as all vertebrates, is the most vulnerable organ to elevated plasma ammonia levels (Felipo and Butterworth, 2002; Walsh et al., 2006). This topic has been extensively reviewed elsewhere and will only be briefly described here. In the case of high extracellular ammonia, if NH3 is the primary permeant species, cytosol pH will increase. If mostly NHþ 4 enters the cell, intracellular pH will decrease. Any pH change will influence the function of intracellular processes. Ammonia has numerous other eVects that appear to be due to the unique properties of the NH3 or NHþ 4 molecules þ þ themselves. NHþ can directly substitute for K or H in ion exchangers, 4 disrupting ion balance and nerve propagation (Cooper and Plum, 1987). Elevated brain ammonia in fish interferes with normal cell metabolism and the synthesis of the neurotransmitter, glutamate (Wicks and Randall, 2002). The most important detoxification enzyme, glutamine synthetase (GSase), catalyzes the conversion of glutamate and NHþ 4 to glutamine and is induced in the brain of some ammonia‐exposed teleost fishes (Wicks and Randall, 2002; Wright et al., submitted for publication). As well, tolerance to elevated environmental ammonia levels is correlated with constitutive activities of GSase in the brain of closely related Batrachoididae fishes (Wang and

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Walsh, 2000). Comparable data on brain metabolism in ammonia‐exposed primitive fishes would be a step toward understanding the evolution of ammonia detoxification mechanisms. 3.2. Synthesis of Nitrogen End‐Products Nitrogen end‐products are the result of protein catabolism. Ingested or cellular proteins are hydrolyzed to the component amino acids by proteolytic enzymes. Excess amino acids are catabolyzed forming ammonia, the primarily nitrogen end‐product in fishes. Ammonia may be further ‘‘repackaged’’ as urea, glutamate, or glutamine. Several reviews have been written on nitrogen metabolism and excretion in fishes (Wood, 1993, 2001; Walsh, 1998; Anderson, 2001; Ip et al., 2001; Walsh and Mommsen, 2001; Wilkie, 2002). 3.2.1. Ammonia Figure 6.2 describes the three pathways for ammonia synthesis. Amino acid transferase enzymes transfer an amino group from the L‐amino acid to a‐ketoglutarate (a‐KG) forming glutamate and a‐keto acid (Figure 6.2A). Mitochondrial glutamate dehydrogenase (GDH) catalyzes the conversion of glutamate to a‐KG and NHþ 4 . Hepatic and red muscle mitochondrial GDH activities are comparable between bowfin (Amia calva), a Holostean fish and various teleost species (Chamberlin et al., 1991; Felskie et al., 1998). In lamprey (P. marinus) GDH activities in liver, intestine, and muscle vary with developmental stage. In the parasitic phase, liver GDH activity and protein abundance were six times higher relative to the lamprey ammocoete or upstream migrant (Wilkie et al., 2006). The authors proposed that high levels of GDH in the liver of parasitic lamprey allow for rapid catabolism of amino acids when feeding opportunities arise. This supposition is further strengthened by parallel high activities of two liver transferase enzymes, alanine, and aspartate aminotransferase in the parasitic lampreys (Wilkie et al., 2006). Ammonia is also created when AMP is degraded to IMP, catalyzed by AMP deaminase (Figure 6.2B). Although present in fish liver (Casey and Anderson, 1983), it probably only makes a significant contribution to ammonia synthesis in skeletal muscle tissue after exhaustive exercise (Mommsen and Hochachka, 1988; Wright et al., 1988). Finally, the breakdown of glutamine also results in the generation of ammonia and glutamate, catalyzed by glutaminase (GLN) (Figure 6.2C). The reverse reaction, catalyzed by GSase, consumes ammonia (Figure 6.2D). Thus, the balance between the two enzymes will determine the net ammonia synthesis in fish tissues (Chamberlin et al., 1991). In bowfin, liver GLN activity is higher than GSase activities (Chamberlin et al., 1991), suggesting a net production

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Fig. 6.2. Pathways for ammonia synthesis in and out of the mitochondrion. (A) L‐Amino acids are transaminated forming glutamate and an a‐Keto acid. Glutamate enters the mitochondrion where the enzyme GDH deaminates glutamate forming a‐ketoglutarate (a‐KG) and NHþ 4 . (B) In the purine nucleotide cycle, the adenylate AMP is degraded to IMP and NH3, catalyzed by AMP deaminase. (C) Glutamine may enter the mitochondrion where the enzyme GLN catalyzes the reaction forming glutamate and NH3. (D) Glutamate and NHþ 4 are combined by the enzyme GSase to form glutamine. GS is typically cytosolic in ammoniotelic fish, but mitochondrial in ureotelic fish.

of ammonia by the liver typical of other ammonotelic fishes. Ureotelic species such as elasmobranchs and most likely the coelacanth (not measured) have much higher liver GSase activities because available ammonia is scavenged to form glutamine, the nitrogen donating substrate for the urea cycle (see below). 3.2.2. Urea Urea is formed by three known pathways in fish: uricolysis, arginolysis, and the urea cycle (Figure 6.3). Uric acid arises from a purine ring that is formed by a series of complex reactions involving glutamine, aspartate, glycine, HCO
3 , and phosphoribosyl pyrophosphate (PRPP) (Figure 6.3A). Uric acid degradation to urea occurs in the peroxisomes in fish (Noguchi

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Fig. 6.3. Pathways for urea synthesis in the peroxisome (A) and mitochondrion (B), (C). Uricolysis is depicted in (A), where a purine ring is formed by a series of complex reactions involving 2 glutamine, aspartate, glycine, HCO
3 , and PRPP. Uric acid is degraded by uricase (URC) to allantoin which is further degraded by allantoinase (ALN) and allantoicase (ALC) to urea. Ariginolysis (B) is the simple conversion of arginine to ornithine and urea catalyzed by arginase. The urea cycle in fish is initiated by the enzyme carbamoyl phosphate synthetase III (CPS III) that combines glutamine and HCO
3 to form carbamoyl phosphate, which in turn is converted to citrulline catalyzed by ornithine carbamoyl transferase (OTC). Citrulline is converted to arginine by two enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL), the final step is arginolysis (B) forming urea.

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et al., 1979) and involves three enzymes, uricase (URC), allatoinase (ALN), and allatoicase (ALC). Activities for all three uricolytic enzymes were first reported in lungfish in an aquatic habitat (Brown et al., 1966). Later studies revealed that urea synthesis via the urea cycle in estivating lungfish far surpassed the capacity of uricolysis (Forster and Goldstein, 1966). It is likely that all fish have the capacity to degrade uric acid because the pathway plays an important role in nucleic acid metabolism. It is surprising therefore that in hagfish (Bdellostoma cirrhatum renamed Eptatretus cirrhatus), no uricolytic enzymes were detected (Read, 1975), a finding that has not been confirmed in this or other hagfish species. In contrast, the full suite of uricolytic enzymes were found in liver of lamprey ammocoetes (P. marinus), and the level of activities were similar to teleost values (Wilkie et al., 1999). The first uricolytic gene, uricase (or urate oxidase), has been cloned in African lungfish (P. annectens), as well as in several teleost species (Andersen et al., 2006). Although the allantoicase gene has been identified in the puVer fish (Fugu rubripes) genome database (Vigetti et al., 2003), cloning of allantoicase and allantoinase genes in fish has not been successful to my knowledge. Given that several enzymes in the uricolytic pathway have been lost during the evolution of higher vertebrates, much would be gained from understanding the evolution of the genes coding for the uricolytic enzymes in primitive as well as other fishes. Urea can also be formed from arginine degradation in fish mitochondria catalyzed by arginase (ARG) (Figure 6.3B), independent of a complete urea cycle. ARG is widespread in fish tissues, including hagfish (Read, 1975), lamprey (Read, 1968; Wilkie et al., 1999, 2004, 2006), lungfish (Janssens and Cohen, 1966), coelacanth (Brown and Brown, 1967), sturgeon and gar (Cvancara, 1969), as well as bowfin (Felskie et al., 1998). Two mitochondrial isoforms of ARG, ARG type I and II, are coded by two genes in puVer fish (Takifugu rubripes) and four genes in rainbow trout (O. mykiss) (Wright et al., 2004), whereas ARG type I in terrestrial ureotelic vertebrates is cytosolic (Ikemoto et al., 1990). At what stage during the transition to a terrestrial habitat did ARG type I lose its mitochondrial leader sequence? Mommsen and Walsh (1989) proposed that this shift in the intracellular location of liver ARG first appeared in the lungfish. Although enzyme activity data support this notion (Mommsen and Walsh, 1989), the sequencing of lungfish ARG genes (type I and II) would provide valuable information for a more complete phylogenetic analysis and lead to a better view of the evolution of the urea cycle in vertebrates. The urea cycle is the main pathway for urea synthesis in terrestrial vertebrates (amphibians, mammals), as well as elasmobranchs (Anderson, 2001), coelacanths, lungfish (GriYth, 1991), and a few teleosts (Walsh and

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Mommsen, 2001). Five enzymes form the backbone of the pathway: carbamoyl phosphate synthetase III (CPS III), ornithine carbamoyl transferase (OTC), argininosuccinate synthetase (ASS) and lyase (ASL), and ARG (Figure 6.3B and C). CPS III requires glutamine as the nitrogen donating substrate and, therefore, mitochondrial GS is considered an important accessory enzyme. The properties of CPS III and the subcellular location of ARG distinguish the fish urea cycle from that of amphibians and mammals (Anderson, 2001). In terrestrial vertebrates, CPS I utilizes ammonia as the N‐donating substrate and ARG is cytosolic. A third CPS cousin, CPS II, is prevalent in all vertebrate tissues, is part of the pyrimidine pathway, requires glutamine as a substrate, but does not require the eVector N‐acetyl glutamate (required by both CPS I and III). Confusion in the literature arose a few decades ago when it was first reported that CPS activity was fairly common in fish (Cvancara, 1974), but subsequent work clarified the importance of careful assay technique to provide the correct conditions for the urea cycle‐related CPS III and separate it from CPS II (Felskie et al., 1998). Thus, it is now clear that hagfish (Read, 1975; Mommsen and Walsh, 1989), lamprey (Wilkie et al., 1999, 2004), and bowfin (Felskie et al., 1998) have extremely low or nondetectable levels of CPS III and lack a functional hepatic urea cycle. There is limited information on urea cycle enzymes in sturgeon, paddlefish, and bichir; however, the absence of the pathway is reported in Mommsen and Walsh (1989). Despite this, the bichir was assigned a CPS type III enzyme by Mommsen and Walsh (1989) and would be interesting to investigate further given this fish’s predilection for river margins, flood plains, and swamps, similar to lungfishes. The coelacanth, L. chalumnae, first came to the attention of urea cycle enthusiasts with the reports of Pickford and Grant (1967) and Brown and Brown (1967) that blood and tissue urea levels as well as OTC and ARG activities were similar to the ureosomotic elasmobranchs (GriYth, 1991). A number of follow‐up studies expanded on these initial findings but fascinating questions remain. For example, during early development, coelacanth pups are carried within the oviducts of the mother (Wourms et al., 1991) and obtain nutrition from large attached yolk sacs (Locket, 1980). Are these developing young ureosmotically independent as have been found in little skate (Raja erinacea) embryos (Steele et al., 2004) or is urea cycle capacity only apparent after release from internal maternal support? African lungfish express all urea cycle enzymes (Janssens and Cohen, 1966) and detoxify ammonia to urea when water supplies become limited (Janssens and Cohen, 1968; see also Section 3.4). Mommsen and Walsh (1989) classified CPS in the lungfish P. aethiopicus as type I, with a preference for ammonia over glutamine as the N‐donating substrate. This designation was later challenged by Loong et al. (2005) who claimed that hepatic CPS activities in P. aethiopicus and P. annectens were 30‐ to 60‐fold higher if

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glutamine was the available substrate rather than ammonia. It is possible, however, that both subtypes of CPS exist, but that the developmental stage or previous environmental conditions of the lungfish play a role in determining whether CPS I or III dominates (P. Walsh, personal communication). Clearly, sequence information on lungfish CPS genes would help to illuminate the controversy. 3.3. Excretion Aquatic animals tend to be ammoniotelic, whereas terrestrial animals excrete mostly urea (ureotelic) or uric acid (uricotelic) (reviewed by Wright, 1995). The key nitrogen excretory product in fish is ammonia, but elasmobranchs (and probably the coelacanth) release primarily urea (ureotelic) as a by‐product of their ureosmotic strategy (Table 6.1). Other nitrogenous end‐ products including amino acids, proteins, creatine, creatinine, and unknown substances, which may account for 20–40% of the total nitrogen excreted in teleost fishes, are less well understood (Kajimura et al., 2004). 3.3.1. Ammonia Fish excrete ammonia mostly as NH3 down the blood‐to‐water partial pressure gradient (Wilkie, 2002; Evans et al., 2005). To determine if NH3 diVusion dominates at the gill, researchers have manipulated external water pH (Wright and Wood, 1985). The diVusion trapping model predicts that NH3 excretion will increase in acid environments and decrease in alkaline environments (Wright and Wood, 1985; Wilkie and Wood, 1991). This phenomena is nicely illustrated in sturgeon (Acipenser ruthenus) fingerlings acclimated gradually for 1 week to water pH values ranging from pH 4.0‐ to 9.4 (fish died at pH 9.6) (Figure 6.4). If one takes 500 mmol kg
1h
1 as the control excretion rate (pH 7–8.4), then there is an 80% increase in Jamm at pH 4.0 and a 34% decrease in Jamm at pH 9.4, with virtually no change in Jurea. The very high rate of Jamm under acid conditions would be hard to maintain and it is not surprising that some mortalities were noted after 2 days at pH 4.0. þ There is evidence for some branchial NHþ 4 eZux possibly linked to Na influx in fish (Wilkie, 2002; Evans et al., 2005). Interestingly, in the hagfish M. glutinosa, Evans (1984) proposed that branchial ammonia excretion was dominated by NH3 diVusion, with no evidence for Naþ‐dependent NHþ 4 transport. In general, gill NHþ 4 diVusion may be more important in seawater compared to freshwater fish. In seawater lamprey and sturgeon, leaky paracellular junctions adjacent to gill MRCs (see Section 2.1.3) may provide a passageway for NHþ 4 . In contrast, in the hagfish gill MRCs are tightly associated with pavement cells and this arrangement probably prevents NHþ 4 diVusion between cells.

Table 6.1 Ammonia (Jamm) and Urea (Jurea) Excretion Rates and the Percentage of Nitrogen Wastes Excreted as Urea (% urea) in Hagfish, Lamprey, Elasmobranch, Lungfish, Sturgeon, and Bowfin Species Species

300

Hagfish Eptatretus stoutii Myxine glutinosa

Lamprey Petromyzon marinus

Entosphenus tridentatus Elasmobranch Squalus acanthias Raja erinacea Lungfish Protopterus dolloi

Jamm (mmol N kg
1h
1)

Jurea (mmol N kg
1h
1)

Urea (%)

Comments

References

73 218 200

5 – –

6 – –

SW SW SW

Walsh et al., 2001 Evans, 1984 McDonald et al., 1991

50 100

9 15

18 13

Wilkie et al., 1999 Wilkie et al., 2004

2500

200

7

119

0

0

FW (ammocoetes) FW (adults) fasted FW (adults, fed trout blood) FW

11 150 124

547 411 111

98 73 47

SW SW (adult) SW (embryo)

Wood et al., 1995 Steele et al., 2005 Steele et al., 2004

170

21

11

2‰

Wood et al., 2005

Wilkie et al., 2004 Read, 1968

Protopterus aethiopicus Protopterus annectens Protopterus dolloi

63 96 133 265

66 50 42 221

51 34 24 46

FW FW FW (fasted) FW (fed)

Loong et al., 2005 Loong et al., 2005 Lim et al., 2004 Lim et al., 2004

469

–

–

FW (juvenile)

208

16

7

FW

142

15

10

9‰ SW

Acipenser oxyrhynchus

438

–

–

FW (juvenile)

Acipenser brevirostrum

381

–

–

FW (juvenile)

Acipenser gueldenstaedti

724

95

12

FW (juvenile)

594

130

18

10‰ SW (juvenile)

Dabrowski et al., 1987 Altinok and Grizzle, 2004 Altinok and Grizzle, 2004 KieVer et al., 2001 KieVer et al., 2001 Gershanovich and Pototskij, 1995 Gershanovich and Pototskij, 1995

607

60

9

FW

Sturgeon Acipenser baeri Acipenser oxyrinchus desotoi

301

Bowfin Amia calva

McKenzie and Randall, 1990

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PATRICIA A. WRIGHT

Fig. 6.4. Ammonia (Jamm) and urea (Jurea) excretion rates (mmol kg
1 h
1) in Sturgeon fingerlings A. ruthenus in response to 1 week of exposure to water of varying pH. [Reprinted from Gershanovich and Pototskij (1995) with permission from Elsevier.]

Gill ammonia transporters have been isolated in two teleost species, Rivulus marmoratus (Hung et al., 2007) and O. mykiss (Nawata, et al., submitted manuscript), which share sequence similarities to the Rhesus‐associated glycoproteins (RhG) (Huang and Peng, 2005). There is an ongoing controversy whether RhG proteins transport NH3 (Ripoche et al., 2004), NHþ 4 (Nakhoul þ et al., 2005), and/or mediate NHþ /H exchange (Handlogten et al., 2004) in 4 mammals. In crab gills, an Rh‐like protein (RhCM) is thought to actively transport NHþ 4 across the cuticle (Weihrauch et al., 2004). Huang and Peng (2005) have reported the presence of ‘‘genuine Rh genes in hagfish’’ and suggest further characterization of these genes may shed light on CO2 conductance across red blood cell membranes. Thus, across the spectrum of organisms and tissues many putative roles have been associated with RhG genes and the path forward in fish will be clearly very interesting. The major site of Jamm is typically the gills in fish (Smith, 1929). Divided chamber experiments performed by Read (1968) on the lamprey Entosphenus tridentatus demonstrated that 87% of the ammonia was eliminated across the gills, 8% across the skin, and 4% via the kidneys. Little attention has been paid to cutaneous ammonia excretion, but premetamorphic lamprey larvae have a thinner integument (Youson, 1980) and possibly rely less on branchial excretion while in burrows compared to adults; however, this has not been tested. In the submerged lungfish P. dolloi, ammonia and urea excretion are almost equally partitioned between the anterior (internal and external gills) and posterior end (most of the skin and urinary opening) (Wood et al., 2005).

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What percentage of the posterior excretion is attributable to the skin versus urine is unknown; however, the skin may be the key site of urea elimination following prolonged terrestrialization in lungfish (see Section 3.4). Feeding has a profound eVect on nitrogen excretion rates in fish, but most Jamm values in the literature are for starved fish (Wood, 2001). One of the most dramatic examples of the influence of nutrition is found in the parasitic lamprey (P. marinus) (Wilkie et al., 2004), where postprandial Jamm was 25 times higher after a blood meal (Table 6.1). Parasitic lampreys may consume up to 30% of their body weight/day and are very eYcient at assimilating the energy in a blood meal (Farmer et al., 1975). The African lungfish (P. dolloi) appears to take a diVerent approach to a surfeit of amino acids following feeding. Although Jamm does increase significantly after a meal, urea synthesis is stimulated to a greater extent resulting in higher tissue urea contents and a greater proportion of nitrogen excreted as urea (Lim et al., 2004). 3.3.2. Urea In jawless and ray‐finned primitive fishes, Jurea constitutes 6–18% of total ammonia and urea excretion (Table 6.1). Jurea depends on both the simple diVusion of urea as well as facilitated transport (Walsh and Smith, 2001). The first fish urea transporter (UT) was isolated and characterized in the dogfish shark (Squalus acanthias) kidney (ShUT; Smith and Wright, 1999). ShUT, homologous to the mammalian facilitated transporter UT‐A2 family, shares sequence similarity with UTs cloned from the kidneys of other elasmobranch species (Janech et al., 2003, 2006; Morgan et al., 2003; Hyodo et al., 2004). A survey of several marine fish indicates that UT gill expression may be fairly widespread, although no signal was detected in hagfish (Eptatretus stoutii) (Walsh et al., 2001). This negative result requires further investigation because a teleostean UT probe was used. Other UT isoforms may be present in hagfish or branchial urea transport may be solely dependent on simple diVusion. Due to the unique position of hagfish in the fish evolutionary tree, a more complete picture of Agnathan urea transport would be valuable. There have been only a few studies in the primitive fishes that report fluctuations in Jurea with changing physiological conditions. Jurea is not particularly sensitive to changes in the external water pH (Figure 6.4) or salinity (Altinok and Grizzle, 2004). Feeding enhances Jurea, just as it does Jamm (see above). In parasitic lamprey feeding on rainbow trout, Jurea was elevated by 15‐fold initially and remained elevated for 8 h after the meal (Wilkie et al., 2004). More remarkably, two lampreys (P. marinus) were caught parasitizing basking sharks and Jurea was as high as 9000 mmolNkg
1h
1 after the meal (Wilkie et al., 2004). This surge in Jurea is presumably necessary to clear the lamprey body fluids of the excessive

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PATRICIA A. WRIGHT

urea concentrations taken in with the shark blood. The rapid eZux of urea under these conditions may be dependent, in part, on the upregulation of gill UTs (Wilkie et al., 2004). 3.3.3. Retention of Urea in Coelacanths and Elasmobranchs The coelacanth, holocephalans (chimaeras), and marine elasmobranchs evolved a strategy of osmoregulation that sets them apart from all other known fish species, namely the retention of urea to counterbalance the osmotic strength of seawater. Like many other evolutionary experiments, it is not apparent why an excretory waste product (urea) would be retained at relatively high concentrations as an osmolyte (for a discussion see Walsh and Mommsen, 2001). Kirschner (1993) compared the energetic costs of hyposmotic regulation by marine teleosts with ureosmotic regulation by marine elasmobranchs and concluded that they were similar. Urea is less toxic than ammonia at the same concentration, but urea at high concentrations denatures proteins (Yancey, 2001). In elasmobranchs, the destabilizing eVects of urea are counterbalanced by methylamines or other organic osmolytes (Yancey, 2001). In some marine elasmobranchs, the ratio of urea to TMAO concentrations is 2:1, whereas in other species a variety of counteracting osmolytes along with TMAO have an additive eVect (Steele et al., 2004, 2005). Embryos of the oviparous little skate, R. erinacea, have a urea: TMAOþ other stabilizing osmolytes ratio of 2.3:1 (4 months) and 2.7:1 (8 months) (Steele et al., 2004), whereas the ratio was 1.68:1 in the skeletal muscle of adult skates (Steele et al., 2005). These findings imply developmental changes in osmolyte regulation. Coelacanth hemoglobin is unaVected by urea concentrations >3 M, similar to elasmobranchs (Mangum, 1991). Are other coelacanth proteins sensitive, insensitive, or urea‐requiring, as has been found in some elasmobranchs? In the coelacanth, skeletal muscle TMAO was 300 mmol liter
1 (Lutz and Robertson, 1971) considerably higher than the little skate value of 50 mmol liter
1 (Steele et al., 2005) or 180 mM in the shark Scyliorhinus canicula muscle (Robertson, 1989). These diVerences raise many interesting questions about the coelacanth counterbalancing osmolyte strategy. For example, do coelacanth pups within the mother (Wourms et al., 1991) retain the same organic osmolyte ratios as adults? Are these osmolyte concentrations sensitive to external salinity changes, as reported in elasmobranch embryos (Steele et al., 2004) and adults (Steele et al., 2005)? Maintaining elevated urea concentrations in body fluids is a considerable challenge for coelacanths and elasmobranchs, given the large blood (400 mmol liter
1)‐to‐water (0 mmol liter
1) gradient across the gills and the obligatory release of more or less isosmotic urine. In marine elasmobranchs, renal reabsorption of urea has been long established, but the

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mechanisms involved have not been resolved. The passive countercurrent reabsorption of urea by the kidney (Boylan, 1972; Lacy et al., 1985) is probably dependent, in part, on UT proteins (Smith and Wright, 1999) with diVerent functional characteristics and regional heterogeneity (Walsh and Smith, 2001; Janech et al., 2003, 2006; Morgan et al., 2003; Hyodo et al., 2004). Although sequence and tissue distribution information on UTs in elasmobranchs is valuable, what is now needed is a more complete understanding of the functional role of various tubule segments. This work has been impeded by the complexity of the elasmobranch nephron structure. On the other hand, the coelacanth kidney is thought to be more similar to the Osteichthyes (see Section 2.1.2); a more detailed structural analysis needs to be performed for comparisons with the elasmobranch nephron. GriYth (1991) postulated that coelacanths do not reabsorb urea from the renal filtrate based on samples collected from the urinary bladder of a moribund specimen (GriYth et al., 1976). Urea is energetically expensive to produce and, therefore, it is hard to imagine how the coelacanth could manage such a high rate of urea loss via the urine unless urine flow is remarkably low. A secondary active Naþ/urea antiporter in the gills of the dogfish shark is thought to transport urea out of the gill epithelial cells back into the blood against the urea concentration gradient (Fines et al., 2001). The gene coding for this Naþ/urea antiporter has not been isolated in elasmobranchs, nor in the mammalian kidney where active Naþ‐coupled urea transport is thought to play an important role (Kato and Sands, 1998). Dogfish gill basolateral membranes have a very high cholesterol content (Fines et al., 2001) that decreases the permeability to urea (Pugh et al., 1989). Hill et al. (2004) reported that apical and basolateral gill membrane vesicles in dogfish and marine flounder share relatively low permeabilities to water and urea. A picture of branchial urea retention in elasmobranchs is starting to emerge, but no comparable data is available for coelacanth. Even though it is unlikely that physiologists will obtain live coelacanth for whole‐animal experiments, fresh gill and kidney tissue could provide a wealth of information on ultrastructure and molecular composition. 3.3.4. The Developmental Perspective An understanding of nitrogen excretion during early development in teleost fishes (Korsgaard et al., 1995; Wright and Fyhn, 2001) may shed some light on unstudied ammoniotelic primitive fishes (hagfish, lungfish, and Actinopterygii fishes) or provide comparisons to the one group that has been examined, the lampreys. Ammonia excretion (Jamm) can be detected in many teleost embryos very early after fertilization and depending on the species, Jurea can account for a significant fraction of total nitrogen excretion (Wright and Fyhn, 2001). GriYth (1991) proposed that urea synthesis via

306

PATRICIA A. WRIGHT

the urea cycle arose in early gnathostome fishes as a protective mechanism to ensure low tissue ammonia levels during a long embryonic development phase solely dependent on yolk proteins and amino acids for energy. Indeed, urea cycle enzymes, including the key enzyme CPS III, are expressed in freshwater and marine teleosts embryos encompassing a variety of early life histories (Wright et al., 1995; Chadwick and Wright, 1999; Terjesen et al., 2001; Barimo et al., 2004). Interestingly, in most of these species the urea cycle is not operational in the adult stage. The exception is the embryos of the gulf toadfish, Opsanus beta, which develop into ureagenic adults, turning on urea synthesis under stressful conditions (for a review see Walsh, 1997). An alternative route for ammonia detoxification during early development is glutamine synthesis, which may or may not feed into the urea cycle. Early induction of GSase genes in rainbow trout embryos and subsequent formation of the active enzyme before hatching may be necessary to prevent excessive accumulation of ammonia (Essex‐Fraser et al., 2005). Given this background on teleost early development, it would be fascinating to know more about the ontogeny of nitrogen excretion in the oldest living vertebrate, the hagfish. Female hagfish are thought to produce 20–30 yolky encapsulated embryos varying in size between 20 and 70 mm (Martini, 1998). In large embryos such as these, ammonia diVusion to the surrounding seawater would be comparatively slow, and therefore detoxification pathways such as the urea cycle and/or glutamine synthesis might be imperative. Embryonic hagfish have rarely been found over the last 100 years (Martini, 1998). The picture is brighter for the lampreys. Larval ammocoetes synthesize low levels of urea via uricolysis, not the urea cycle (Wilkie et al., 1999). When exposed to elevated external ammonia, plasma and tissue ammonia levels increased in ammocoetes without changes in urea or glutamine (Wilkie et al., 1999). Premetamorphic lampreys have a depressed metabolic rate compared to postmetamorphic stages (Wilkie et al., 2001), partly explaining the low rates of nitrogen excretion relative to postmetamorphic stages (Table 6.1). After metamorphosis, parasitic and upstream‐migrant lampreys express only very low or nondetectable levels of the urea cycle enzymes, CPS III and OTC (Wilkie et al., 2006). Is the urea cycle expressed in embryonic lamprey? Embryos of P. marinus are about 1–2 mm in diameter (Richardson and Wright, 2003), have a relatively large yolk sac, and hatch after 20 days (22  C) in the nest (Applegate, 1950). One might predict that lamprey embryos consuming yolk proteins may induce urea cycle enzymes prior to hatching, but later repress the activity of urea cycle enzymes when metabolic rate and protein intake is low when larvae inhabit mud burrows and feed on detritus.

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3.4. The Challenges of Estivation The three families of lungfish tolerate terrestrial conditions to varying degrees. Some species of African lungfish Protopterus estivate (i.e., survive the dry season by forming a cocoon or a protective layer of dried mucus and reduce metabolic rate), whereas the South American Lepidosiren will partially estivate in a moist environment, and the Australian Neoceratodus is completely aquatic and does not estivate. GriYth (1991) and Graham (1997) presented excellent reviews of the older literature on nitrogen metabolism and excretion in the three families of lungfish. There have been several new studies on the African lungfish Protopterus, mostly due to the ease of shipping these animals to laboratories far away from their native habitat. P. dolloi form dry, brown mucus cocoons in the laboratory when water is removed and animals are kept slightly moist for 30 or 40 days (Chew et al., 2004; Wood et al., 2005). During estivation, Protopterus and Lepidosiren maintain low tissue ammonia levels, but accumulate urea in order to avoid ammonia toxicity (Janssens, 1964; Carlisky and Barrio, 1972; Chew et al., 2004; Wood et al., 2005). Urea synthesis in these air‐exposed lungfish occurs via the hepatic urea cycle (see Section, 3.2.2). The activities of urea cycle enzymes were enhanced by approximately twofold in P. dolloi, although the increase is remarkably modest given that these fish were without water for 40 days (Chew et al., 2004). Moreover, the data suggest that P. dolloi maintains a high reserve capacity for urea synthesis under control or immersed conditions, even when they are ammoniotelic (Chew et al., 2003). This may be beneficial if low water or impending drought is not readily anticipated by the fish. In the nonestivating Neoceratodus forsteri, the rate of urea synthesis is 100 times lower than that in Protopterus (Goldstein et al., 1967). A ‘‘washout’’ of urea was observed in Protopterus when estivating lungfish were returned to water (Smith, 1930; Janssens, 1964). An initial pulse of Jurea (peak 0–1 h) was followed by a second pulse (peak 12 h) of greater magnitude (Wood et al., 2005). When lungfish were placed in divided chambers to separate the anterior (gills) from the posterior (most of the skin and urine/feces) end of the fish, more Jurea occurred from the posterior end in the second phase of the urea ‘‘washout’’ (Figure 6.5). Further characterization of the second pulse of Jurea suggested that facilitated type UTs may have been mobilized to accommodate the enormous flux at this time (Wood et al., 2005). Isolation of lungfish UTs and their tissue distribution will be an important next step. The environmental or endogenous signal(s) that stimulates estivation has not been identified in lungfish. Ip et al. (2005) hypothesized that a subtle increase in external salinity as the river water evaporates prior to a drought

308

PATRICIA A. WRIGHT

Fig. 6.5. Nitrogen excretion in the African lungfish (Protopterus dolloi) submerged (A) and after 21 days of terrestrial conditions (skin remained moist), (B) 0–3 h and (C) 12–13 h. Nitrogen

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may be one instigator of estivation in P. dolloi. Multiple factors are probably involved and identification of changes in hormone‐signaling pathways would be valuable information toward a more thorough understanding of the control of estivation. Bowfin is tolerant of air‐exposure and there were suggestions in the literature that Amia estivates. McKenzie and Randall (1990) attempted to induce estivation in A. calva in the laboratory by gradually air‐exposing fish over a 10‐day period, or elevating external water ammonia or decreasing water oxygen levels. None of these treatments induced estivation, and A. calva died following 3–5 days in air. 4. CONCLUDING REMARKS In the area of ionic, osmotic, and nitrogenous waste regulation in primitive fishes, there are as many gaps in our knowledge as there is detailed information. For example, considerable research has focused on gill morphology, cell type, and ultrastructure of the Agnathans, but limited work has been directed toward the functional role of gill subtypes and the expression of ion transporters. Likewise, an explosion of papers on lungfish nitrogen excretion has unveiled fascinating responses to environmental perturbations but much less is known of ionic and osmotic regulation. As outlined in specific sections above, molecular approaches may provide the missing links in a number of cases (e.g., the urea cycle enzyme CPS in lungfish, expression of UTs and Rh‐factor ammonia transporters in gills). Finally, we have negligible data on any aspect of osmoregulation and nitrogen excretion in birchir, gar, bowfin, and paddlefish (not to mention coelacanth!). Studies of extant primitive fishes may provide more than just data on another fish species, but lead to a broader understanding of how early vertebrates evolved under changing external conditions (e.g., ions, salinity, water availability).

excretion was partitioned between the anterior (i.e., head) and posterior (i.e., body) compartments by placing lungfish in divided chambers under (A) control aquatic conditions (N ¼ 10), (B) after return (0–3 h) to aquatic conditions following 21 days of terrestrial conditions (N ¼ 5), and (C) 12–13 h after return to aquatic conditions following 21 days of terrestrial conditions (N ¼ 9). Asterisks indicate significant diVerence ( p  0.05) from the aquatic rates in panel A, whereas crosses indicate significant diVerence ( p  0.05) from the corresponding rates in the anterior compartment. [After Wood et al. (2005b) with permission from University of Chicago Press.]

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ACKNOWLEDGEMENTS The author wishes to thank Drs. Wilkie, Wood and Terjesen for access to unpublished material, Tom Binder for helpful discussions on lamprey development, Ian Smith for graphics, Kim Ong for digging up references and Lori Ferguson for skilled clerical help. The comments of two anonymous reviewers are very much appreciated. Financial support was provided by the Natural Sciences and Engineering Research Council.

REFERENCES Alt, J. M., Stolte, H., Eisenbach, G. M., and Walvig, F. (1981). Renal electrolyte and fluid excretion in the Atlantic hagfish Myxine glutinosa. J. Exp. Biol. 91, 323–330. Altinok, I., and Grizzle, J. M. (2004). Excretion of ammonia and urea by phylogenetically diverse fish species in low salinities. Aquaculture 238, 499–507. Altinok, I., Galli, S. M., and Chapman, F. A. (1998). Ionic and osmotic regulation capabilities of juvenile Gulf of Mexico sturgeon, Acipenser oxyrinchus de sotoi. Comp. Biochem. Physiol. 120, 609–616. Andersen, Ø., Aas, T. S., Skugor, S., Takle, H., van Nes, S., Grisdale‐Helland, B., Helland, S. J., and Terjesen, B. F. (2006). Purine‐induced expression of urate oxidase and enzyme activity in Atlantic salmon (Salmo salar). Cloning of urate oxidase liver cDNA from three teleost species and the African lungfish Protopterus annectens. FEBS J. 273, 2839–2850. Anderson, P. M. (2001). Urea and glutamine synthesis: Environmental influences on nitrogen excretion. In ‘‘Fish Physiology Vol. 20, Nitrogen Excretion’’ (Wright, P., and Anderson, P., Eds.), pp. 239–278. Academic Press, San Diego. Applegate, V. C. (1950). Natural history of the sea lamprey Petromyzon marinus, in Michigan. US Department of Interior, Fish and Wildlife Service, Special Scientific Report: Fisheries. no. 55. Barimo, J. F., Steele, S. L., Wright, P. A., and Walsh, P. J. (2004). Ureotely and ammonia tolerance in early life stages of the gulf toadfish. Opsanus beta. J. Exp. Biol. 207, 2011–2020. Bartels, H. (1998). The gills of hagfishes. In ‘‘The Biology of Hagfishes’’ (Jørgensen, J. M., Lomholt, J. P., Weber, R. E., and Malte, H., Eds.), pp. 205–222. Chapman and Hall, London. Bartels, H., and Potter, I. C. (2004). Cellular composition and ultrastructure of the gill epithelium of larval adult lampreys. J. Exp. Biol. 207, 3447–3462. Beamish, F. W. H. (1980a). Osmoregulation in juvenile and adult lampreys. Can. J. Fish. Aquat. Sci. 37, 1739–1750. Beamish, F. W. H. (1980b). Biology of the North American anadromous sea lamprey, Petromyzon marinus. Can. J. Fish. Aquat. Sci. 37, 1924–1943. Beamish, F. W. H., Strachan, P. D., and Thomas, E. (1978). Osmotic and ionic performance of the anadromous sea lamprey, Petromyzon marinus. Comp. Biochem. Physiol. 60A, 435–443. Bellamy, D., and Jones, I. C. (1961). Studies on Myxine glutinosa. I. The chemical composition of the tissues. Comp. Biochem. Physiol. 3, 175–183. Brown, G. W., Jr., and Brown, S. G. (1967). Urea and its formation in coelacanth liver. Science 155, 570–573. Brown, G. W., Jr., James, J., Henderson, R. J., Thomas, W. N., Robinson, R. O., Thompson, A. L., Brown, E., and Brown, S. G. (1966). Uricolytic enzymes in liver of the Dipnoan Propterus aethiopicus. Science 153, 1653–1654. Brown, J. A., Cobb, C. S., Frankling, S. C., and Rankin, J. C. (2005). Activation of the newly discovered cyclostome rennin‐angiotensis system in the river lamprey Lamprey fluviatilis. J. Exp. Biol. 208, 223–232.

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Smith, H. W. (1961). ‘‘From Fish to Philosopher.’’ American Museum of Natural History, New York. Steele, S. L., Yancey, P. H., and Wright, P. A. (2004). Osmoregulation during early embryonic development in the marine little skate Raja erinacea; response to changes in external salinity. J. Exp. Biol. 207, 2021–2031. Steele, S. L., Yancey, P. H., and Wright, P. A. (2005). The little skate Raja erinacea exhibits an extrahepatic ornithine urea cycle in the muscle and modulates nitrogen metabolism during low‐salinity challenge. Physiol. Biochem. Zool. 78, 216–226. Terjesen, B. F., Chadwick, T. D., Verreth, J. A. J., Rønnestad, I., and Wright, P. A. (2001). Pathways for urea production during early life of an air‐breathing teleost, the African catfish Clarias gariepinus Burchell. J. Exp. Biol. 204, 2155–2165. Thurston, R. V., Russo, R. C., and Vinogradov, G. A. (1981). Ammonia toxicity to fishes. EVects of pH on the toxicity of the un‐ionized ammonia species. Environ. Sci. Technol. 15, 837–840. Vigetti, D., Binelli, G., Monetti, C., Prati, M., Bernardini, G., and Gornati, R. (2003). Selective pressure on the allantoicase gene during vertebrate evolution. J. Mol. Evol. 57, 650–658. Walsh, P. J. (1997). Evolution and regulation of urea synthesis and ureotely in (Batrachoidid) fishes. Annu. Rev. Physiol. 59, 299–323. Walsh, P. J. (1998). Nitrogen excretion and metabolism. In ‘‘The Physiology of Fishes’’ (Evans, D. H., Ed.), 2nd edn., pp. 199–214. CRC Press, Boca Raton. Walsh, P. J., and Mommsen, T. J. (2001). Evolutionary considerations of nitrogen metabolism and excretion. In ‘‘Fish Physiology Vol. 20, Nitrogen Excretion’’ (Wright, P., and Anderson, P., Eds.), pp. 1–30. Academic Press, San Diego. Walsh, P. J., and Smith, C. P. (2001). Urea transport. In ‘‘Fish Physiology, Vol. 20, Nitrogen Excretion’’ (Wright, P. A., and Anderson, P. M., Eds.), pp. 279–307. Academic Press, San Diego. Walsh, P. J., Wang, Y., Campbell, C. E., DeBoeck, G., and Wood, C. M. (2001). Patterns of nitrogenous waste excretion and gill urea transporter mRNA expression in several species of marine fish. Mar. Biol. 139, 839–844. Walsh, P. J., Veauvy, C. M., McDonald, M. D., Pamenter, M. E., Buck, L. T., and Wilkie, M. P. (2006). Piscine insights into comparisons of anoxia tolerance, ammonia toxicity, stroke and hepatic encephalopathy. Comp. Biochem. Physiol. Part A (in press). Wang, Y., and Walsh, P. J. (2000). High ammonia tolerance in fishes of the family Batrachoididae (toadfish and midshipman). Aquat. Toxicol. 50, 205–219. Weihrauch, D., Morris, S., and Towle, D. W. (2004). Ammonia excretion in aquatic and terrestrial crabs. J. Exp. Biol. 207, 4491–4504. Wicks, B. J., and Randall, D. J. (2002). The eVect of sub‐lethal ammonia exposure on fed and unfed rainbow trout: The role of glutamine in regulation of ammonia. Comp. Biochem. Physiol. 132A, 275–285. Wilkie, M. P. (2002). Ammonia excretion and urea handling by fish gills: Present understanding and future research challenges. J. Exp. Zool. 293, 284–301. Wilkie, M. P., and Wood, C. M. (1991). Nitrogenous waste excretion acid‐base regulation, and ionoregulation in rainbow trout (Oncorhynchus mykiss) exposed to extremely alkaline water. Physiol. Zool. 64, 1069–1086. Wilkie, M. P., Wang, Y., Walsh, P. J., and Youson, J. H. (1999). Nitrogenous waste excretion by the larvae of a phylogenetically ancient vertebrate: The sea lamprey (Petromyzon marinus). Can. J. Zool. 77, 707–715. Wilkie, M. P., Bradshaw, P. G., Joanis, V., Claude, J. F., and Swindell, S. L. (2001). Rapid metabolic recovery following vigorous exercise in burrow‐dwelling larval sea lampreys (Petromyzon marinus). Physiol. Biochem. Zool. 74, 261–272.

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Wilkie, M. P., Turnbull, S., Bird, J., Wang, Y. S., Claude, J. F., and Youson, J. H. (2004). Lamprey parasitism of sharks and teleosts: High capacity urea excretion in an extant vertebrate relic. Comp. Biochem. Physiol. 138A, 485–492. Wilkie, M. P., Claude, J. F., Cockshutt, A., Holmes, J. A., Wang, Y. S., Youson, J. H., and Walsh, P. J. (2006). Shifting patterns of nitrogen excretion and amino acid catabolism capacity during life cycle of the sea lamprey (Petromyzon marinus). Physiol. Biochem. Zool. 79, 885–898. Wilkie, M. P., Morgan, T. P., Galvez, F., Smith, R. W., Kajimura, M., Ip, Y. K., and Wood, C. M. (2007). The African lungfish (Protopterus dolloi): Ionoregulation and osmoregulation in a fish out of water. Physiol. Biochem. Zool. 80, 99–112. Wood, C. M. (1993). Ammonia and urea metabolism and excretion. In ‘‘The Physiology of Fishes’’ (Evans, D. H., Ed.), pp. 379–425. CRC Press, Boca Raton. Wood, C. M. (2001). Influence of feeding exercise, and temperature on nitrogen metabolism and excretion. In ‘‘Nitrogen Excretion’’ (Wright, P. A., and Anderson, P. M., Eds.), pp. 201–218. Academic Press, San Diego. Wood, C. M., Walsh, P. J., Chew, S. F., and Ip, Y. K. (2005a). Ammonia tolerance in the slender lungfish (Protopterus dolloi): The importance of environmental acidification. Can. J. Zool. 83, 507–517. Wood, C. M., Walsh, P. J., Chew, S. F., and Ip, Y. (2005b). Greatly elevated urea excretion after air exposure appears to be carrier mediated in the slender lungfish (Protopterus dolloi). Physiol. Biochem. Zool. 78, 893–907. Wourms, J. P., Atz, J. W., and Stribling, M. D. (1991). Viviparity and the maternal‐embryonic relationship in the coelacanth Latimeria chalumnae. Environ. Biol. Fishes 32, 225–248. Wright, P. A. (1995). Nitrogen excretion: Three end products, many physiological roles. J. Exp. Biol. 198, 273–281. Wright, P. A., and Fyhn, J. H. (2001). Ontogeny of nitrogen metabolism and excretion. In ‘‘Nitrogen Excretion’’ (Wright, P. A., and Anderson, P. M., Eds.), pp. 149–200. Academic Press, San Diego. Wright, P. A., and Wood, C. M. (1985). An analysis of branchial ammonia excretion in the freshwater rainbow trout eVects of environmental pH change and sodium uptake blockade. J. Exp. Biol. 114, 329–353. Wright, P. A., Randall, D. J., and Wood, C. M. (1988). The distribution of ammonia and Hþ between tissue compartments in lemon sole (Parophrys vetulus) at rest, during hypercapnia and following exercise. J. Exp. Biol. 136, 149–175. Wright, P. A., Felskie, A. K., and Anderson, P. M. (1995). Induction of ornithine‐urea cycle enzymes and nitrogen metabolism and excretion in rainbow trout (Oncorhynchus mykiss) during early life stages. J. Exp. Biol. 198, 127–135. Wright, P. A., Campbell, A., Morgan, R. L., Rosenberger, A. G., and Murray, B. W. (2004). Expression of arginase type I and II genes in rainbow trout: Influence of fasting on liver enzyme activity and mRNA levels in juveniles. J. Exp. Biol. 207, 2033–2042. Yancey, P. H. (2001). Nitrogen compounds as osmolytes. In ‘‘Fish Physiology Vol. 20, Nitrogen Excretion’’ (Wright, P. A., and Anderson, P. M., Eds.), pp. 309–341. Academic Press, San Diego. Youson, J. H. (1980). Morphology and physiology of lamprey metamorphosis. Can. J. Fish Aquat. Sci. 37, 1687–1710. Youson, J. H., and Freeman, P. A. (1976). Morphology of the gills of larval and parasitic adult sea lamprey Petromyzon marinus L. J. Morphol. 149, 73–104.

7 LOCOMOTION IN PRIMITIVE FISHES D. J. MCKENZIE M. E. HALE P. DOMENICI

1. Introduction 2. Swimming Modes and Associated Morphological Adaptations 2.1. BCF Swimming 2.2. MPF Swimming 2.3. Modes of Fast‐Start Behavior 3. Locomotor Muscles 4. Neuromotor Coordination 4.1. Axial Rhythm Generation Circuits 4.2. Mauthner Neurons and the Evolution of the Startle Neural Circuit 5. Locomotor Performance and Physiology 5.1. Continuous Swimming Performance 5.2. Fast‐Start Performance 6. Conclusions

Swimming is critical to the ecology of many fishes as it determines, for example, their ability to forage, to escape predators, and to migrate. The information about locomotion in primitive fishes is, however, sparse and unevenly distributed. For example, sustained exercise performance has been studied in the primitive groups with a migratory lifestyle such as lampreys and sturgeons, whereas fast‐start performance has been investigated in sit‐and‐wait predators such as the bichirs and gars. Very little is known about locomotion in lungfishes, typically considered rather sedentary species. The lamprey has been a model for studies of the neural circuits that drive axial locomotion, but this information has yet to be put into a clear evolutionary context relative to other fishes. Indeed, there have been relatively few studies aimed explicitly at investigating the evolution of locomotor performance in primitive fishes, aside from some studies on startle behaviors. Great caution should be exercised when making literature‐based comparisons between ‘‘the teleosts’’ and single 319 Primitive Fishes: Volume 26 FISH PHYSIOLOGY

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primitive species, or discrete phylogenetic groups with particular life histories and morphologies (e.g., sturgeons). The evidence does indicate, however, that primitive fishes perform exercise less well than teleosts with similar lifestyles. This is true for swimming endurance, critical speed swimming, and fast starts. Thus, the evolution of fishes to the teleosts has, in fact, been associated with evolution of ‘‘better’’ exercise performance. It is tempting to speculate that improved locomotor ability might have contributed to the dominance of the teleosts today. 1. INTRODUCTION Swimming is critical to the ecological performance of many fishes as it determines, for example, their ability to forage, to escape predators, and to migrate. Some of the features common to all fishes are in fact locomotor in nature: the myotomal muscle anatomy that oscillates the body and tail to power swimming, the giant Mauthner cells (M‐cells) that receive input from the sensory arm of the startle reflex and control the motor output, and the neuronal patterns of the spinal cord that control swimming (Bone et al., 1995). The evolution of fishes to the dominant modern Euteleosts has, however, involved much modification to the basic chordate locomotor plan that is found in amphioxus and the agnathans. Modifications to the anatomy of the skeleton, muscles, and fins, and also improvements in neuromuscular control, allow the Euteleosts a wider spectrum of more accurate and more rapid locomotor movements than are found in any of the primitive fishes, including the modern elasmobranchs (Bone, 1978; Bone et al., 1995; Helfman et al., 1997; Chapter 10, this issue). Thus, as far as locomotion is concerned, some features of the plesiomorphic fish species can, without risk of misrepresentation, be defined as truly ‘‘primitive.’’ The information about locomotion in primitive fishes is, however, sparse and unevenly distributed. This is because locomotor performance and physiology will, to some degree, reflect the ecology and life history strategy of fishes, and this has tended to attract researchers to investigate particular aspects of locomotion in particular species. For example, sustained exercise performance has been studied in the primitive fish groups with a migratory lifestyle such as lampreys and sturgeons, whereas fast‐start performance has been investigated in sit‐and‐wait predators such as the bichirs and gars. For the lungfishes, typically considered rather sedentary species, very little is known about locomotor performance. The lamprey has been a model for studies of the neural circuits that drive axial locomotion, but this information has yet to be put into a clear evolutionary context relative to other fishes. Indeed, there have been relatively few studies aimed explicitly at investigating

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the evolution of aspects of locomotor performance in primitive fishes, aside from some studies on startle behaviors. This chapter will begin with a review and summary of what is known about swimming modes, locomotor muscles, and neuromotor coordination. This will be followed by a review of locomotor performance and physiology, with comparisons of swimming endurance, prolonged exercise performance, anaerobic burst exercise, and fast starts. 2. SWIMMING MODES AND ASSOCIATED MORPHOLOGICAL ADAPTATIONS Primitive fishes have evolved a wide range of swimming modes associated with their varied morphologies and ecologies. This diversity is most clear when comparing slow routine swimming among species. Lampreys and hagfish rely on axial locomotion whereas, in addition to retaining axial locomotion in many species, chondrichthyans have also evolved an array of fin functions and morphological adaptations of these fins for locomotion. The extant sarcopterygians fishes, lungfishes and the coelacanth, have dextrous fins. The many paired and median propulsive fins of the coelacanth are particularly striking (Fricke and Hissmann, 1992; Hissmann and Fricke, 1996). Like the chondrichthyans, the primitive non‐teleost actinopterygian fishes demonstrate a diversity of swimming modes. While chondrosteans rely heavily on axial locomotion at all speeds, other species use axial movement primarily during bursts of high‐speed swimming. For slow swimming and hovering, the bowfin, Amia calva, has evolved dorsal fin propulsion, amiiform locomotion (Breder, 1926) and lepisosteid and polypterid species use paired pectoral fins (Grubich and Westneat, 2004). The lepisosteids and polypterids also have dermal armor which is thought to be important for axial movements, particularly in the former group (Long et al., 1996). Traditionally, aquatic locomotion has been divided into swimming with the median and paired fins (MPF) and swimming with the body and caudal fin (BCF) (Breder, 1926; Lindsey, 1978). All fishes, including the primitive species, tend to be classified according to the mode they use during routine swimming, but are often capable of swimming with more than one mode, transitioning between them in association with changes in swimming speed. In general, the activities are independent such that fishes swim either with the median and/or paired fins or with the axis and caudal fin. Although there are exceptions in several teleosts (Batty, 1981; Arreola and Westneat, 1996; Hove et al., 2001; Mu¨ller and van Leeuwen, 2004; Thorsen et al., 2004), to our knowledge no such MPF–BCF coordination pattern has been reported in primitive fishes. Startle responses have been classified within the BCF

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category (as ‘‘transient’’ BCF locomotion; Webb, 1994) because, in general, the axis powers propulsion. The movement and motor control diVer, however, from that of axial swimming and so here startle responses will be considered separately, after addressing the routine swimming modes. 2.1. BCF Swimming Axial locomotion is classified into various swimming modes that are based on the portion of the body that oscillates during movement (Breder, 1926; Lindsey, 1978). The rostrocaudal extent of axial bending has implications for force generation, axial muscle function, and motor control (Wardle et al., 1995), and so examination and comparison of axial swimming modes can provide a useful source of hypotheses about the functional morphology of swimming. For many of the primitive fishes, there is too little information available to allow their BCF swimming to be definitively classified according to previously defined modes which are, in any case, often described for specific highly specialized taxa. There is, however, some information available about the extent of rostrocaudal bending in various primitive fishes. At the same time, information on BCF swimming throughout the complete speed range is not available for many species, and so these may have a broader range of swimming modes than is currently described. Similarly, for a number of species in which primary swimming modes are fin based, such as polypterids and Amia, axial swimming may only be used for short bursts at the highest swimming speeds (Breder, 1926; Westneat et al., 1998; Hale et al., 2002). 2.1.1. Axial Undulation in Hagfish and Lampreys: Body Bending in Elongate Fishes Both hagfishes and lampreys are elongate species that swim by propagating waves of bending along the full length of the body. This type of locomotion is exhibited by elongate fishes that have a relatively uniform shape along the length of the trunk and tail, and is often called anguilliform after such propulsion in the Anguilla eel genus. In the hagfish, approximately one propulsive wavelength is transmitted along the body (estimated from Long et al., 2002), while more than one propulsive wave is propagated at a given time in lamprey (Williams et al., 1989). The hagfish and lampreys are unusual in that they exhibit relatively high‐amplitude body bending in rostral regions. This characteristic, and the fact that one or more wavelengths are propagated within a body length, is believed to generate propulsive force along the whole body. This diVers from many teleosts, which use axial bending primarily to wag the caudal fin (Wardle et al., 1995). In fact, work on the American eel

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(Anguilla rostrata) indicates that there is relatively little rostral bending during aquatic locomotion (Gillis, 1998). Thus, Agnathans may in fact exhibit more characteristically anguilliform locomotion than members of the genus Anguilla. Despite their similarities to elongate teleosts, Long et al. (2002) discovered some interesting and unexpected characteristics in the swimming kinematics of hagfish. In particular that, at low speeds, tail beat amplitude decreased with increasing length‐specific swimming speed. This is in contrast to elongate salamanders and eels where amplitude remains relatively constant or increases (Gillis, 1997, 1998). Tail beat frequency does, nonetheless, increase with increasing speed in hagfish, and Long et al. (2002) hypothesized that the decreased amplitude occurs because notochord stiVness increases with tail beat frequency. It is surprising that there has been relatively little research on Agnathan swimming modes, considering the position of lampreys and hagfish at the base of vertebrate phylogeny, and the extensive literature on the neurobiology of motor control in lampreys. The recent work on hagfish suggests that there are likely to be interesting and fundamental principles of swimming that have yet to be described in these unusual fishes. 2.1.2. The Role of The HeterocercalTail in Axial Swimming of Sharks and Sturgeons In general, sharks and sturgeons swim by oscillation of the body axis and heterocercal tail. The heterocercal shape, an asymmetric profile with greater caudal extension of the dorsal margin, is unusual in fishes which, for the most part, have dorsoventrally symmetrical homocercal caudal fins. There has been considerable interest in heterocercal tail function (Grove and Newell, 1936; Alexander, 1965; Webb, 1986; Ferry and Lauder, 1996; Liao and Lauder, 2000; Wilga and Lauder, 2004). In particular, the relationship of the heterocercal tail to lift because both sharks and sturgeons are negatively buoyant and so must generate upwardly directed lift as well as thrust during swimming. In recent years, flow visualization has provided new insights into the role of the heterocercal tail in such force generation (Ferry and Lauder, 1996; Liao and Lauder, 2000; Wilga and Lauder, 2004). Vortices shed from the tail are directed ventrally and posterior to the body during swimming, indicating the generation of forces in forward and dorsal directions (Liao and Lauder, 2000; Wilga and Lauder, 2002). In the Pacific spiny dogfish (Squalus acanthias), the heterocercal tail generates an unusual ring within a ring vortex wake, which is probably due to a combination of the asymmetry of the tail itself and the asymmetry of its movement through the water, with dorsal edge leading ventral edge (Wilga and Lauder, 2004). The implications of this vortex structure for locomotion are, as yet, unclear.

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In addition to lift generation by the tail, both sharks (bamboo Chiloscyllium punctatum and leopard Triakis semifasciatus) and the white sturgeon (Acipenser transmontanus) generate lift by orienting their bodies at a positive angle of attack relative to the horizontal (Wilga and Lauder, 1999, 2002). As swimming speed increases more lift is generated and, in sturgeon, this is associated with the adoption of shallower body angles (Wilga and Lauder, 1999). The pectoral fins had been thought to play a significant role in generating lift, but flow visualization indicates that this may not be the case (Wilga and Lauder, 1999, 2002; Liao and Lauder, 2000). The relationship between caudal fin morphology and orientation of force generation is not, however, as simple as it may seem. Shape diVerences in the heterocercal tails of leopard and bamboo sharks did not make an appreciable diVerence to their vortex shedding (Wilga and Lauder, 2002). There do, however, appear to be diVerences in how the heterocercal tail is controlled, whereby sturgeon have a greater degree of control over the angle of vortex shedding than do sharks, which would allow sturgeons greater maneuverability (Liao and Lauder, 2000). In addition, Lauder (2000) has demonstrated that a homocercal tail can also generate vortices directed ventrolaterally, in some species. 2.1.3. Skin and Axial Undulation in Primitive Fishes Skin may play many roles in axial locomotion. In addition to streamlining the body and providing support for internal organs, the skin can be integrally bound to muscle by connective tissues (Westneat et al., 1993) and has been shown to transmit force in sharks and eels (Wainwright et al., 1978; Hebrank, 1980). Several basal actinopterygian groups have unusual skin that is plated with extensive dermal armor. Although there is a clear evolutionary trend toward a loss of plate in favor of lighter and thinner scales in fishes (Moy‐Thomas and Miles, 1971; Lauder and Liem, 1983; Webb et al., 1992), extant polypterids and lepisosteids have retained heavily armored heads and chevron‐shaped plate rows ringing the body along their lengths. While it is unclear whether polypterids use axial bending for swimming at anything but high burst speeds, the longnose gar (Lepisosteus osseus) and presumably other gar species use axial undulation over an extensive range of swimming speeds. Like many teleosts, tail beat frequency in L. osseus increased with swimming speed, as did tail beat amplitude and wave speed (Webb et al., 1992; Long et al., 1996). Several studies on longnose gar have examined the role of skin in swimming. Webb et al. (1992) compared steady swimming between gar and the tiger musky, a similarly shaped but unarmored teleost (a hybrid of two species in the genus Esox). The results indicated that body armor did not

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have a large eVect on performance because the gar and musky exhibited similar patterns of body bending, similar propulsive wavelengths (of near one body length), and comparable mechanical power requirements (Webb et al., 1992). Long et al. (1996) addressed directly the function of dermal plating in the swimming mechanics of the gar by investigating how the removal of plate rows or the sectioning of connective tissue between adjacent rows aVected swimming kinematics. Decoupling of the rostral and caudal scale rows, by cutting the connective tissue between rows in the caudal region of high lateral bending, decreased tail beat frequency and propulsive wave speed (Long et al., 1996). Modeling of the morphology and body bending indicated that the body plates provided a mechanical advantage to the myomeres in generating body bends. Taken together, these studies indicate that armored skin plays an integral role in locomotion of L. osseus but, despite its body armor, the gar shows relationships between basic kinematics and swimming speed that are comparable with most other nonarmored fish that have been studied. 2.2. MPF Swimming MPF swimming modes are based on discrete diVerences in the fins used during locomotion and the pattern of actuation or coordination of those fins. For example, batoids, the skates and rays, are pectoral fin swimmers but some species oscillate their fins while others flap them (Breder, 1926; Webb, 1994) and others still can alternate between the two modes (Rosenberger, 2001). The movements of paired fins can also be coordinated into diVerent patterns. For example, juveniles of some reef fish species will alternate their pectoral fins at low speeds but actuate them synchronously at high speeds (Hale et al., 2006). The evolution and diversity of paired fins and elaboration of median fins among primitive fish groups is striking and a rich area for research on swimming biomechanics. 2.2.1. Locomotion in Rays and Skates: Pectoral Fin Specialists Skates, rays, and their relatives have greatly expanded pectoral fins that serve as their primary propulsors. Two pectoral fin swimming modes have been identified within the group. Rajiform locomotion, named for the Rajidae family of skates, passes a rostral to caudal wave of oscillation along the pectoral fin (Breder, 1926). In contrast, mobuliform locomotion involves dorsoventral flapping of the fins. This type of fin actuation is named for the ray family Mobulidae that includes large pelagic rays such as the manta rays. These named behaviors represent the opposing ends of a spectrum of pectoral fin activity and some ray species can perform both oscillation and undulation. The fin movement patterns can be quantified with regard to the number of bending waves propagated along the fin during locomotion, the mean wave

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number (Rosenberger, 2001; Rosenberger and Westneat 1999). In a study of eight batoid species, Rosenberger (2001) found that mean wave number varied from 0.4 to 1.4, with a graded continuum across species within that range. Several species changed wave number with swimming speed, decreasing the number of waves propagated along the fin with increasing swimming speed and thereby generating a more oscillatory pattern of fin movement. 2.2.2. Fin Use and Coordination in the Highly Maneuverable Swimming of the Coelacanth The coelacanth, Latimeria chalumnae, is remarkable for its fin structures and functions. As in other sarcopterygians, the paired fins have evolved mobility and a range of actuation patterns. In the coelacanth, the posterior dorsal fin and the anal fin are highly mobile and appear to have complex patterns of neural control and movement. The internal structures of the posterior dorsal fin (second dorsal fin) and the anal fin are similar to those of the paired fins (Millot and Anthony, 1958), and it has been proposed that this is due to changes in gene expression from a typical sarcopterygian median fin gene expression pattern to a pelvic or pectoral fin expression pattern (Ahlberg, 1992). In contrast, the anterior dorsal fin (first dorsal fin) can be erected or depressed and is thought to stabilize the body and to function in social signaling but not to generate thrust for locomotion (Fricke and Hissmann, 1992). Because of this species’ relationship to the terrestrial tetrapods, there has been great interest in exploring potential locomotor similarities. In field‐ recorded video of coelacanths, Fricke et al. (1987) found that the fins were only used in swimming and not for walking on the sea floor. When at the bottom, coelacanths either tucked the fins against the body or rested on the fin tips (Fricke et al., 1987). During swimming, it is likely that the fin movements not only provide thrust but also stabilize the body. Pectoral fin rowing is used during slow forward swimming, along with sculling of the pelvic fins, posterior dorsal fin, and anal fin. All of the paired and median fins, except the anterior dorsal fin, can also be used to push water forward and so swim backward (Fricke and Hissmann, 1992). When swimming, coelacanths use many coordination patterns of fins and axis, as well as body postures that can include head‐down and upside down orientations (Fricke and Hissmann, 1992). These patterns are thought to help coelacanths maneuver in drift currents (Fricke et al., 1987). In general, the paired pectoral fins and pelvic fins alternated nearly 180
out of phase between the left and right sides of each pair. The second dorsal fin and anal fin were actuated synchronously 100
out of phase from the pectoral fins and pelvic fins. Coelacanths also possess a small epicaudal fin at the tip of their tail (Hissmann and Fricke, 1996). Epicaudal fin beats were observed in the field, which were not coordinated with the paired or unpaired fins

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(Hissmann and Fricke, 1996). The epicaudal fin beats occurred in three specific situations: in curved, forward swimming; during narrow turns on a point; and during station holding. The epicaudal fin appeared to have a locomotor function in turns and curves but Hissmann and Fricke (1996) suggested that the fin served a sensory function during station holding. 2.2.3. Fin Function in Basal Actinopterygian Fishes Little work has been performed on the paired and median fin movements of basal actinopterygian fishes. Grubich and Westneat (2004) found diVerences in pectoral fin use during swimming by the shortfin bichir (Polypterus palmas), the longnose gar, and Amia. Although all three used the pectoral fins for slow swimming, Polypterus and Amia exhibited synchronous fin coordination whereas Lepisosteus alternated the fins for both slow forward and slow backward swimming. Amia is better known for amiiform locomotion, swimming by passing waves of bending down the length of the dorsal fin (Breder, 1926). Similar patterns of fin movement can be observed in the dorsal fins of seahorses and pipefishes (Ashley‐Ross, 2002) and the anal fins of gymnotids (gymnotiform locomotion) (Lighthill and Blake, 1990). 2.3. Modes of Fast‐Start Behavior Although this discussion of locomotor modes has focused on steady swimming, startles also fall into several distinct categories (reviewed by Domenici and Blake, 1997). The typical startle reflex of bony fishes is the C‐start in which the initial movement is a bend into a C‐shape away from the direction of the stimulus. This reflex is driven by one of a pair of large reticulospinal M‐cells and several other related neurons in the hindbrain (discussed below). If the fish is elongate, like many larvae, the head and tail may cross in the high‐amplitude bend and look more like an ‘‘O’’ than a ‘‘C.’’ This may simply reflect high flexibility in the larvae, there is no information available to indicate whether either the hydrodynamics or the neural control of these extreme bends is fundamentally diVerent from those of C‐starts. The S‐start occurs in some teleost species when escapes are elicited from the tail and, in the muskellunge (Esox masquinongy), it is actively controlled and fundamentally diVerent from the C‐start in patterns of muscle activity (Hale, 2002). The withdrawal or retraction response involves the fish bending at existing bends in the body so that, in general, where there was a bend at rest before the startling stimulus, there is a tighter bend after the withdrawal (Currie and Carlsen, 1985, 1987; Meyers et al., 1998; Bierman et al., 2004; Ward and Azizi, 2004). In response to head stimulation, this results in the head being withdrawn away from the stimulus. This response has been observed in elongate fish, many of which are substrate dependent, such as

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eels (Meyers et al., 1998; Ward and Azizi, 2004), which often live in rock formations or reefs and larval lampreys (Currie and Carlsen, 1985) which live in sandy substrates but extend their heads into the water column to feed. The primitive fishes exhibit both C‐start and withdrawal responses. The lamprey uses a withdrawal response (Currie and Carlsen, 1985). The coelacanth and West African lungfish (Protopterus annectens) use a C‐start (Fricke et al., 1987; Meyers et al., 1998). Several Polypterus species use a C‐start (Westneat et al., 1998; Tytell and Lauder, 2002), while their sister genus the ropefish (Erpetoichthys calabaricus) uses a withdrawal (Bierman et al., 2004). L. osseus and A. calva both perform C‐starts (Figure 7.1) (Westneat et al., 1998; Hale et al., 2002). It has been suggested, therefore, that the withdrawal reflex is the primitive condition with C‐start evolving and then being secondarily lost in the ropefish (Figure 7.1) and other elongate species like the eel (Meyers et al., 1998; Ward and Azizi, 2004). The Pacific spiny dogfish shows a C‐start response much like that of teleost fishes (Domenici et al., 2004). They also show variability in the rate of turning, from escape responses with relatively low turning rates to fast responses with relatively high turning rates. Similar patterns have been observed in teleosts (Domenici and Batty, 1997). The chimera, the only chondrichthyan species known to have M‐cells as an adult, also performs C‐starts in which pectoral fins are used, possibly for orientation (Domenici and Jordan, unpublished observations). Although S‐start behavior has only been studied in teleost fishes, it has been observed in some lepisosteid species that commonly perform C‐starts (Bierman and Hale, personal communication). 3. LOCOMOTOR MUSCLES Previous volumes of this series have provided exhaustive reviews about fish locomotor muscle, in particular in teleosts and elasmobranchs but also in primitive fishes (Bone, 1978; Sa¨nger and Stoiber, 2001; Shadwick and Gemballa, 2006; Syme, 2006), so the reader is referred to these and this section shall be limited to a brief summary. A prominent feature of all fish groups is their large myotomal muscle mass composed of relatively discrete groups of metabolically diVerent fiber types, which is responsible for BCF swimming. Locomotion results from contraction of this segmental axial musculature, which is divided into myotome blocks by myoseptal connective tissue partitions, on which the muscle fibers insert. Muscle blocks on opposing sides of the incompressible notochord or vertebral column work antagonistically, causing the body to bend laterally (Bone, 1978; Webb, 1998; Shadwick and Gemballa, 2006). The notochord is a plesiomorphic character of adult hagfish, lamprey, sturgeon, and latimeria, but is more or less reduced

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Fig. 7.1. Startle behaviors of several basal actinopterygian fishes [images from Westneat et al. (1998) and Bierman et al. (2004)]. A. calva and P. palmas perform C‐starts in response to stimulation near the head (Westneat et al., 1998). E. calabaricus, the sister genus to Polypterus, performs retraction to both head (not shown) and tail stimulation (Bierman et al., 2004). (Reproduced with permission of the company of Biologists).

in most bony fishes by development of vertebral elements around it (Bone et al., 1995). All fishes, even lancelets and the Agnatha, have myotomes with complex three‐dimensional shapes and, in the gnathostomes, these have evolved anterior and posterior projecting cones (reviewed by Shadwick and Gemballa, 2006). The notochord/vertebral column is positioned dorsally so the greater bulk of the myotome lies below the notochord. To avoid dorsonventral inflection when myotomes contract, ancestral forms were V‐shaped

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(amphioxus) and this shape is also found in many larvae. All other adult fishes, however, have more complex shapes, with myotomes folded into W’s (reviewed by Bone, 1978; Shadwick and Gemballa, 2006). The arrangement of muscle fibers within the myotomes increases in complexity with the evolution of fishes, and this is an adaptation to allow all of the fibers, whatever their position in the myotome, to contract to a similar extent when the body flexes, and in an isometric manner so that the muscles operate at the most advantageous point of the force–velocity curve (Alexander, 1969; Shadwick and Gemballa, 2006). It is the insertion of the muscle fibers into the myosepta that transmits muscular forces to the notochord/backbone along tensile collagenous fibers. In gnathostomes, there is also a collagenous horizontal septum that divides the myomeres into epaxial and hypaxial elements and contributes to this force transmission. Each myoseptum can span more than one vertebral segment, and the extent of this elongation is related to swimming mode, being greatest in carangiform and thunniform swimmers. Indeed, Shadwick and Gemballa (2006) demonstrated that it is the swimming mode rather than phylogenetic position that defined the degree of elongation, with lamnid sharks, scombrids, and tunas having similar and much longer myoseptal lengths than dogfish, bichirs, sturgeons, gar, and teleosts that exhibit noncarangiform locomotion. The skin also plays a role in force transduction, in part, by generating high intramuscular pressures that contribute to fast‐start performance in some primitive species (Westneat et al., 1998). It is well‐established that all fishes have a distinct anatomical and metabolic division of their muscle fibers. ‘‘Slow oxidative’’ (SO) fibers have, as the name implies, a slow twitch frequency that relies almost exclusively on oxidative phosphorylation to provide ATP. The SO fibers typically represent a minor proportion of the muscle mass, distributed as a thin layer just below the skin. They are well‐vascularized to provide oxygen and nutrients and contain myoglobin, hence the common name ‘‘red muscle.’’ These fibers are used for steady aerobic swimming at relatively slow speeds. Fast glycolytic (FG) fibers have a fast twitch frequency and rely almost exclusively on endogenous anaerobic fuels, initially phosphagen hydrolysis and then anaerobic glycolysis. These fibers comprise the main part of the myotome and are used for short bursts of high‐speed swimming (Bone, 1978; Webb, 1998; Sa¨nger and Stoiber, 2001). Most fish species also possess intermediate fibers, which are intermediate between SO and FG in terms of their distribution, energy metabolism, and role in steady versus burst swimming. The reader is referred to Bone (1978), Sa¨nger and Stoiber (2001), Shadwick and Gemballa (2006), and Syme (2006) for extensive reviews of muscle structure, innervation, and function, with the vast majority of information confined to the teleosts. Bone (1978) has argued that the large muscle mass reflects the density of the medium in which fishes live, which exerts great friction drag, while the

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anatomically discrete muscle types reflect the conflicting demands of allowing both low‐speed cruising economy and also short bursts of high speed. He reviewed the comparative ultrastructure of muscle fibers in Agnatha, elasmobranchs, and teleosts. One particular feature of note is that Agnathans possess well‐defined SO, FG, and intermediate fibers but these are distributed as a regular mosaic arrangement of the fiber types within compartmental muscle units, where SO fibers form a sheath around FG fibers and intermediate fibers. Since then, Kryvi et al. (1980) and Radaelli et al. (1999) have demonstrated that sturgeon possess all three muscle types, distributed in a pattern that is very similar to that of elasmobranchs and teleosts. Both authors, however, reported that FG fibers are typically smaller and of more variable diameter in sturgeons than in either elasmobranchs or teleosts. A major anatomical change during evolution of the fishes has been in the innervation of white muscle. All fish groups except the more advanced members of the teleosts have focal polyneural innervation of FG fibers, whereas the advanced teleosts (and also salmonidae) have evolved diVuse multiple innervation along the fiber length. This may reflect some advantage, perhaps in the relative control of the use of FG fibers during gait transitions (Bone, 1978; Syme, 2006). The literature has tended to focus on the myotomal muscle, with less attention paid to muscles used for MPF locomotion. In the teleosts, these tend to comprise largely SO muscle fibers, reflecting the fact that MPF locomotion is almost exclusively an aerobic activity (Webb, 1998), although there is no information about muscle composition in fin muscles of the primitive fishes. 4. NEUROMOTOR COORDINATION Research on primitive fishes has provided important information about the neural circuits that drive axial locomotion and, more broadly, the basic organization of simple motor networks such as central pattern generators, neural circuits that generate rhythmic activity, and startle circuits. The relative simplicity of serially reiterated myomeres and longitudinally distributed neuron populations has made axial locomotion amenable for study. Of particular focus have been two common axial locomotor behaviors: rhythmic axial swimming and startle responses. Axial undulatory rhythms are used for locomotion by the large majority of fishes. In many, they are the primary mode of locomotion. In others, they supplement fin swimming at the highest burst swimming speeds. Neural control of steady axial swimming has been investigated in depth in the lamprey (reviewed by Grillner et al., 1998; Grillner, 2003). The lamprey is a

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useful model for this research because of the viability of in vitro preparations and the relatively small size of the spinal cord (reviewed by Parker, 2006). Preparations of adult lamprey have been used to identify the spinal cord neurons involved in rhythmic swimming, their physiology, and their connections within neural circuits. This, together with work on frog tadpoles (Roberts et al., 1998) and in zebra fish (Hale et al., 2001; Ritter et al., 2001; Saint‐Amant and Drapeau, 2001), has provided the current understanding of basic circuit organization and diversity. The startle response of fishes has become a model for a simple motor circuit. The large paired Mauthner neurons, important cells for driving the startle, have been widely studied to examine basic principles of nerve cell function (reviewed by Zottoli and Faber, 2000). The M‐cells have their cell bodies in the hindbrain and an axon that extends the length of the spinal cord, and have been examined across a wide array of fish and aquatic amphibian taxa. The spinal cord neurons with which they synapse have been studied to the greatest extent in zebra fish and goldfish. Nonetheless, behavioral and electromyographical studies performed in a wide range of primitive fishes, including lampreys, bichirs, and Amia, have provided additional insight into the diversity of this behavior and its motor control. 4.1. Axial Rhythm Generation Circuits Undulatory axial rhythms involve neural and muscular activity that alternates between the left and right sides of the body within each segment, and that propagates in waves rostrocaudally along the trunk and tail. The muscle activation pattern emanates from rhythm‐generating centers that are organized segmentally along the length of the spinal cord. Each center is composed of two half‐centers, with each half‐center controlling a myomere hemisegment. The basic model for organization of an axial rhythm‐generating center in the lamprey has three primary components: ipsilateral excitatory interneurons, commissural inhibitory interneurons, and motor neurons (reviewed by Grillner et al., 1998; Grillner, 2003) (Figure 7.2). The ipsilateral interneurons excite the other excitatory neurons, the inhibitory interneurons and the motor neurons on the same side of the body. The commissural inhibitory interneurons inhibit the contralateral half‐center to prevent conflicting muscle activity on the opposite side of the body. The motor neurons take the signal from the spinal cord circuits to the axial muscle to initiate and sustain muscle contraction. Several mechanisms are thought to be important in producing alternating activity. First, as excitation levels decrease to the inhibitory interneurons on, for example, the right side, the inhibition of the excitatory interneurons on

7.

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Left axial muscle

M E

Left margin of spinal Cord

I

M I Spinal cord midline

E

Right axial muscle

Right margin of spinal cord

Fig. 7.2. Model for the axial rhythm generation center in the lamprey [based on Grillner et al. (1998)]. Excitatory ipsilateral interneurons (E) excite all cells within each half‐center (dark‐ outlined box) including motoneurons (M) that excite collateral muscle and commissural inhibitory interneurons (I) that inhibit the contralateral half‐center. Left–right rhythms of muscle activity and body bending can be produced by alternating activity between two half‐centers. By coordinating a series of centers in the spinal cord, that activity may be propagated along the trunk and tail.

the contralateral left side decreases. Eventually, the right side inhibitory interneurons no longer exert any eVects on the left side excitatory interneurons (reviewed by Grillner et al., 1998; Grillner, 2003). At this stage, these left excitatory interneurons can excite the commissural inhibitory interneurons on their own left side, and so immediately inhibit activity of the right side. In addition, stretch receptor neurons provide sensory feedback during axial bending (Di Prisco et al., 1990), firing when the side of the body in which they are located is stretched. That is, if the fish bends to the left, it will activate stretch receptors on the right. Two types of stretch receptor have been identified in lampreys. One excites the half‐center on the same side of the cord to contract and the other inhibits the contralateral half‐center. This activity causes the body to bend to the right, decreasing activation of the stretch receptors on the right side but, instead, activating those on the left. Although the coordination of the half‐centers into a rhythm‐generating center in the lamprey is now understood, a few issues remain unclear. For example, whether half‐centers alone can generate rhythmic bursting and the organization into centers coordinates left and right sides, or whether the rhythmic activity is a consequence of the interaction of half‐centers within a center. Several studies have found that hemisection of a region of the spinal cord results in the loss of rhythm generation in that region (Buchanan, 1999; Jackson et al., 2005). Others have found that stimulation of hemisected regions of cord did result in rhythmic bursting (Cangiano and Grillner, 2003). Complementary research on rhythm‐generating centers involved in axial locomotion has been conducted in the Xenopus tadpole. While it is outside the scope of this chapter to review detailed morphological and physiological diVerences between these species, there are many similarities in the basic

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organization of the rhythm‐generating circuit with primary components that include excitatory ipsilateral neurons, commissural inhibitory cells, and motor neurons (reviewed by Roberts et al., 1998). An interesting finding that has yet to be confirmed in the lamprey is that motor neurons take an active part in modulating neural activity, with connections between the motor neurons and from motor neurons to interneurons within a half‐center (reviewed by Roberts et al., 1998). The circuit model above is for simple alternating activity. In axial locomotion, however, this activity has to be propagated down the body for eVective thrust generation in many species. Intersegmental coordination has received less attention than the interaction of center components. In lampreys, intersegmental coupling is not necessary for rhythm generation of a particular center but is important for the generation of rhythmic movement (Buchanan, 1986, 1999; Miller and Sigvardt, 2000). Intersegmental coordination may be mediated by the previously identified excitatory interneurons which send axonal processes to nearby segments rostral and caudal to the segment containing the cell body (reviewed by Grillner, 2003). The diversity of axial locomotor patterns across fishes varies from the whole‐body undulations of anguilliform locomotion, discussed above, to thunniform locomotion in which the body is held rigid and the axial muscle is used to actuate the caudal fin at the caudal peduncle. Knower et al. (1999) suggested that there may be considerable diversity in spinal circuit organization across fishes. Lampreys, Xenopus tadpoles, and zebra fish all undulate across a large portion of the trunk and tail and, therefore, much of the diversity in axial movement patterns remains to be explored. Such comparative work may provide important information about the evolution of rhythmic circuits and the coordination of rhythm‐generating centers. 4.2. Mauthner Neurons and the Evolution of the Startle Neural Circuit Work on startle circuits has focused on the large, paired reticulospinal M‐cells located medial to the otic vesicles, one cell body on each side of the hindbrain with an axon that crosses the midline of the hindbrain and extends the full length of the spinal cord contralateral to the cell body (reviewed by Zottoli and Faber, 2000). M‐cells are some of the largest vertebrate neurons and have been identified in the great majority of fish families examined (reviewed by Zottoli, 1978), including many primitive fishes (Table 7.1). The M‐cells appeared early in vertebrate evolution (Figure 7.3). Hagfish do not possess them but they occur in most major extant groups: lampreys, cartilaginous fishes, lobe fin fishes, basal actinopterygian fishes, and most families of teleosts. The M‐cells form the sensory arm in the various forms of startle reflexes among diVerent species. As mentioned above, this is the

Table 7.1 Presence or Absence of Mauthner Cell (M‐Cell) Neurons in Primitive Fishes and Approximate Fish Length and M‐Cell Diameter (Calculated from Stefanelli, 1980) Superclass

Class

Subclass/ infraclass

Agnatha

Gnathostomata

Order Myxinoformes Petromyzontiformes

Elasmobranchii

Sarcopterygii

Coelacanthiformes Dipnoi

Actinopterygii

Polypteriformes

Chondrostei Neopterygii Teleostei

Acipenseriformes Semionotiformes Amiiformes Anguilliformes Esociformes Pleuronectiformes Cypriniformes Perciformes

Species Lampetra planeri Petromyzon fluviatilis P. marinus S. acanthias Mustelus mustelus Scyliorhinus stellaris Raja asterias L. chalumnae P. annectens Protopterus dolloy Polypterus bichir Calamoichthys calabaricus Acipenser sturio Lepidosteus osseus A. calva A. anguilla Esox lucius Solea vulgaris Carassius auratus Uranoscopus faber

M‐cell Noa Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No

a From Zottoli (1978). A few modern elasmobranchs and teleost representatives are also listed for comparison [(calculated from Stefanelli (1980)].

Fish length, M‐cell diameter 12 cm, 100 mm 22 cm, 130 mm 40 cm, 160 mm 10 cm, 80 mm 30 cm, 100 mm 32 cm, 50 mm 32 cm 20 cm, 500 mm 30 cm, 400 mm 32 cm, 200 mm 62 cm, 250 mm 60 cm, 100 mm 90 cm, 120 mm 20 cm, 100 mm 15 cm, 120 mm 15 cm

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Lepomis

Oncorhynchus

Carassius

Anguilla

Amia

Lepisosteus

Polypterus

Erpetoichthyes

Dipnoi

Anura

Chimaeriformes

Petromyzontiformes

Myxiniformes

Amphioxiformes

A

Startle behavior NA: M-cell absent Axial retraction Fast start Fast start/escape jump Pectoral fin Equivocal B

Stage 1 motor pattern NA: M-cell absent Bilateral EMG Unilateral EMG Equivocal/no data

Fig. 7.3. Motor control characteristics of the Mauthner‐associated startle response mapped onto a phylogeny of chordates [modified from Hale et al. (2002)]. (A) Startle behavior varies among the species studied. Although most perform a fast start, other movement patterns have also been observed. Axial retraction appears to have evolved independently multiple times and may be the primitive condition. (B) During the startle response axial muscle may be active unilaterally or bilaterally. This character varies across the phylogeny suggesting that the startle circuit may not be as conservative as previously believed.

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C‐start bend in most but, in a number of elongate species, M‐cells drive a withdrawal response (see above, Meyers et al., 1998; Bierman et al., 2004; Ward and Azizi, 2004). It is unclear whether M‐cells function in the S‐start response. Electrophysiological and morphological studies in goldfish have identified primary components of the hindbrain and spinal cord circuits associated with the M‐cell and startle response, and these represent the current models of hindbrain and spinal cord circuit organization (Fetcho and Faber, 1988; Scott et al., 1994; Zottoli and Faber, 2000). Although it has not been feasible to test these models at the cellular level in a range of species, they do predict patterns of muscle activity that can be more readily examined. Specifically, they suggest that there should be strong muscle activity along the length of the body on one side and no activity on the opposite side. It might be presumed that the startle neural circuit is evolutionarily conserved because of the presence of the M‐cells and a C‐start response in many fishes and larval amphibians. When, however, the EMG patterns indicative of reticulospinal input to spinal cord circuits (Foreman and Eaton, 1993), and their activity, are mapped onto a phylogeny (Figure 7.3), it becomes clear that the startle circuit has evolved considerably within vertebrates. Even among the basal actinopterygian fishes, EMG data reveals striking diversity in motor control variables (reviewed by Hale et al., 2002). While the goldfish model appears to hold for some species such as longnose gar and rainbow trout (Oncorhynchus mykiss) (Hale et al., 2002), other species exhibit EMG patterns that are not consistent with it (reviewed by Hale et al., 2002). In several basal actinopterygians, such as Polypterus (Westneat et al., 1998; Tytell and Lauder, 2002) and Amia (Westneat et al., 1998), there is extensive bilateral activity during stage 1 (the first muscular contraction) that is slightly higher in the direction of bending. Furthermore, patterns of muscle activity in goldfish include a low level of bilateral activity during the initial startle bend (Foreman and Eaton, 1993). In addition to the various muscle activity patterns comprising C‐start responses, M‐cells also appear to drive the withdrawal startle response in a number of species (Figure 7.1), including those of E. calabaricus and eels (Meyers et al., 1998; Bierman, et al., 2004; Ward and Azizi, 2004). Phylogenetic mapping indicates that neural control of the withdrawal response has evolved multiple times in fishes. Together the interspecific diVerences in startle responses (C‐start versus withdrawal) and the diVerences in C‐start behaviors indicate that there is considerable diversity in neural control among the startle reflexes driven by the M‐cells. The diversity in motor pattern and behavior may be correlated with morphology of the M‐cell itself. Stefanelli (1980) reviewed the neuroanatomy of M‐cells in various species of fishes, including the primitive fishes. He suggested that cell size and shape follow two main evolutionary lines, that

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is, the Batracomorph line (Chondrostei, Sarcopterygii, and Amphibians) and the Telesteomorph line (including all teleost fishes), departing from the Petromyzontiformes line. This classification is based on cell shape and size, which is much larger in the Batracomorph line (see Table 7.1), with most values greater than 200 mm in species of Chondrostei and Sarcopterygii (and also amphibians, Stefanelli, 1980), while cell size in teleosts is typically around 100 mm (in individuals of diVerent species ranging from about 5 to 10 cm in length, Stefanelli, 1980). E. calabaricus have also been found to have relatively large M‐cells (200 mm) (Bierman et al., 2004), suggesting that the Telesteomorph line may have evolved from the Batracomorph line. The classification of M‐cells by size is, however, complicated by the diVering sizes of the species examined. While cell size increases with fish size in certain species, this is not always the case (Stefanelli, 1980). Indeed, the relationship between cell and fish size is further complicated by the fact that cell size regresses in certain species in the adult stage (Stefanelli, 1980). Zottoli (1978) has suggested an alternative but complementary evolutionary scheme for M‐cell diversification, based on the shape of the cell body and pattern of dendritic processes. The tree starts out with Agnatha, and splits into the Chondrichthian and Osteichthian lines. This latter line gives rise to the Amphibia. Clearly, there is considerable diversity in shape and size of the M‐cells that needs to be addressed further in terms of its evolutionary and functional implications. The diversity in motor pattern and behavior may also be correlated with morphology of the M‐cell’s axon cap, a structure of neuronal fibers and glial cells that surrounds the initial segment of the axon. A well‐ developed axon cap was proposed to be associated with the presence of hindbrain circuits that drive the C‐start whereas a reduced axon cap in eels to be associated with withdrawal behavior (Meyers et al., 1998). E. calabaricus, however, performs withdrawals and has a more complete axon cap (Bierman et al., 2004), which is similar to that of the lungfish which performs a C‐start (Meyers et al., 1998). Thus, any relationship between M‐cell structure and form of startle responses would appear to be more complicated than previously thought. 5. LOCOMOTOR PERFORMANCE AND PHYSIOLOGY 5.1. Continuous Swimming Performance In the current context, ‘‘continuous swimming’’ is taken to imply all rhythmic BCF activity that is of much greater duration than startle responses. Continuous swimming activities are typically categorized according to their duration and the muscle types and energetic substrates that they

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rely on (Beamish, 1978; Webb, 1993, 1998). Two extremes are recognized, the first being sustained aerobic swimming that can be maintained for extended periods (hours or days) without any muscular fatigue and is, therefore, a purely aerobic activity dependent on SO muscle function (Webb, 1998). Thus, sustained swimming will be at relatively low tail beat frequencies and speeds during, for example, foraging, maintaining position against currents, and migrating against little or no opposing water currents. The other extreme is burst swimming, which can only be sustained for short periods (seconds) prior to fatigue because it is an anaerobic activity dependent on FG muscle function (Webb, 1998). This comprises the much higher tail beat frequencies and swimming speeds that are achieved by recruiting the large FG muscle mass, for example during predator–prey chases or when negotiating velocity barriers during upriver migration. Intermediate between these extremes are those speeds at which fishes can swim for prolonged periods but which can eventually lead to fatigue. Fatigue might occur when the aerobic metabolic demands of the SO muscle (and heart) exceed the capacity for O2 and nutrient supply by the respiratory and cardiovascular systems (Brett, 1964; Jones and Randall, 1978; Claireaux et al., 2005) and/or if FG muscle recruitment is too frequent and/or intense (Peake and Farrell, 2004). These prolonged speeds might be used for particular periods of upriver migration (Standen et al., 2004) or perhaps portions of predator–prey interactions when the danger is not imminent. The information on continuous swimming performance in primitive fishes is patchy. Measurements of continuous swimming activity in the wild have been made with telemetry and video. Researchers have also quantified aspects of continuous swimming performance in laboratory studies, using flumes and swimming respirometers to measure swimming endurance, performance in the critical swimming speed (Ucrit) protocol, and also burst swimming ability. There is also some information about the physiology of recovery from exercise to exhaustion in various primitive fishes. 5.1.1. Field Measurements of Performance Swimming activity in the wild has been investigated by telemetry [including electromyogram (EMG) telemetry] in the sea lamprey (Stier and Kynard, 1986; Almeida et al., 2002; Quintella et al., 2004) and two species of sturgeon (McKinley and Power, 1992; Geist et al., 2005), and by sonic telemetry and underwater filming in the coelocanth (Fricke et al., 1987; Fricke and Hissmann, 1994; Hissmann et al., 2000). Patterns of habitat use have been studied by telemetry in a few other species, from which some idea of activity levels can be inferred (Paukert and Fisher, 2000; Stancill et al., 2002; Mlewa et al., 2005).

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a. The Migratory Lampreys. Lampreys can swim for hundreds of kilometers in their anadromous spawning migrations and so there is some information available about their performance in the field. Skidmore (1959) and Wigley (1959) reported that rates of upstream movement (ground speeds) were slow, less than 5 cm s1 (Beamish, 1974). Direct observations report that the fishes hug the banks and avoid areas of rapid flow, and use their oral sucker to attach to rocks and recover from bursts of activity when negotiating velocity barriers (Hardisty and Potter, 1971). Telemetry has been used to explore patterns of activity during such migrations by the anadromous sea lamprey, Petromyzon marinus (Stier and Kynard, 1986; Almeida et al., 2002; Quintella et al., 2004). The migrating lampreys are most active at dusk and during the night, and typical ground speeds during upriver migration are shown in Table 7.2. These speeds are less than those measured in acipenserids or teleosts (Table 7.2). Quintella et al. (2004) used EMG telemetry to investigate patterns of swimming activity in migrating P. marinus as it negotiated areas with diVerent flow rates in the river Mondego (Portugal). The fish swam with a sustained pattern typical of aerobic exercise in areas of slow flow, but utilized short periods of intense burst swimming to cross areas of rapid current. The periods of intense activity lasted an average of 30 s and, when successive bouts of such activity were required to overcome areas of rapid flow, the lampreys would attach to convenient structures using their oral sucker and so rest immobile to recover prior to another bout of activity (Quintella et al., 2004). b. The Coelocanth: A Drift‐Hunter. Field observations of Latimeria oV Grande Comoro Island in the Indian Ocean revealed that they are nocturnal, occupying caves during the day but making foraging forays after sunset with restricted home ranges that occupy only a few kilometers of the coastline (Fricke and Hissmann, 1992; Hissmann et al., 2000). Fricke et al. (1987) and Fricke and Hissmann (1992) filmed coelocanths from a submersible and reported that the fish could travel as much as 8 km, either drifting passively with the current or swimming slowly within 1–3 m of the bottom. Hissmann et al. (2000) used sonic tags to follow nocturnal foraging activities and confirmed that they are extremely slow‐swimming drift hunters, which forage close to the bottom. After sunset, they typically left their cave and foraged for linear distances of up to 7 km, often drifting down the bottom contour into deeper water, presumably to depths where prey was more abundant. Estimated ground speeds for various types of spontaneous swimming activities are shown in Table 7.2. Fricke and Hissmann (1992) do, however, report that coelocanths could achieve swimming speeds of 310 cm s1, equivalent to 1.9 body lengths s1, in escape responses.

Table 7.2 Measures of Swimming Performance in the Field for Various Primitive Fishes, Modern Elasmobranchs, and Teleosts Range of speeds Length (cm)

Species

N

cm s1

BL s1

341

Superclass Agnatha P. marinusa

63–80

39

P. marinusa

83–91

10

0.18–1.07

Class Sarcopterygii L. chalumnaeb

130–170

6

0.03–0.09a

L. chalumnaeb

100–170

2

3–19

Subclass Chondrostei Acipenser sp.a Acipenser sp.a Acipenser sp.a A. nudiventrisa A. gueldenstaedtii a

ca. 110 ca. 110 ca. 110 80–130 10–120

6

130–500

1.2–4.5

25–58 5–70

0.2–0.5 0.05–0.9

A. fulvescensb,c

80–95

3

29–78

71

15

12–25

34–43

32

1–68

A. transmontanus

P. spathulab

b,c

Mean speed

3–97

cm s1

BL s1

Time

T (
C)

42  15

0.6

24 d

17–21

42  2

0.49

15–24 h

11–15

5

165 15 33 15 33

0.17–0.25

10–24

1.5 0.14 0.3 0.1–0.2 0.3–0.5

16 d

15 –, 19

1h 1d 1d many h many h

20 20 20

24 h

10

3x6d

12–22

8 months

2–22

Location

References

Connecticut River, United States River Mondego, Portugal

Stier and Kynard, 1986

Grand Comoro, Indian Ocean Grand Comoro, Indian Ocean

Fricke and Hissmann, 1992 Hissmann et al., 2000

Volga river, Russia Volga river, Russia Volga river, Russia Volga river, Russia Kuban river, Russia Mattagami River, Ontario, Canada Snake River, Idaho, United States Missouri River, United States

Malinin et al., 1971 Malinin et al., 1971 Malinin et al., 1971 Malinin, 1973 Malinin, 1973

Almeida et al., 2002

McKinley and Power, 1992 Geist et al., 2005

Roush et al., 2003

(continued)

Table 7.2 (continued ) Range of speeds Species P. spathulab

Length (cm)

N

cm s

84–100

6

0–111

1

Sphyrna lewini

342

Infraclass Teleostei Oncorhynchus gorbuschkaa,c O. gorbuschkaa O. nerkaa

a

1

BL s

Class Elasmobranchii Isurus oxyrinchusb 80–145 b

Mean speed

1

cm s

64 5

47–57

93

51–183

114

47–57

9

21–114

57

200

71–292

64

1.1–4.6

BL s1

Time

T (
C)

Location

3 months

24–29

Keystone Reservoir, Oklahoma, United States

Paukert and Fisher, 2000

0.55

7–45 h

24

Sepulveda et al., 2004

0.81  0.1

20–57 h

22–29

Southern California Bight Kane’ohe Bay, HI, United States

many days

14–17

Fraser River, Canada Fraser River, Canada Fraser River, Canada

Standen et al., 2002d

2–8 h

Measured during spawning migration. Routine activity. c Swimming speeds derived from EMG telemetry of tail beat frequency. d Direct comparison of swimming versus ground speeds. Where stated, mean values are SE; BL s1, body lengths s1. All swimming speeds are ground speeds, except where stated. b

References

Lowe, 2002

Standen et al., 2002d Standen et al., 2004

7.

LOCOMOTION

343

c. The Lungfishes: Not as Sedentary as Might be Thought?. All lungfishes inhabit slow‐moving or stagnant waters and are considered relatively sedentary; there are few field measures of activity and no reports of ground speeds for movements. Grigg (1965) reported that adult Queensland lungfish, Neoceratodus forsteri, were most active at night in a large outdoor tank, and appeared to use air breathing to meet the oxygen demands of activity. In the wild, the same species migrated actively upstream when they had been displaced downstream by flooding (Stuart and Berghuis, 2002). Mlewa et al. (2005) used radiotelemetry to reveal that African marbled lungfish, Protopterus aethiopicus, in Lake Baringo, Kenya, are actually quite active swimmers that occupy relatively large home ranges, up to almost, 20 km2. The lungfishes could change their position by up to 5 km day1, although Mlewa et al. (2005) were not able to estimate actual swimming speeds. d. The Sturgeons: Migratory Inhabitants of Large River Systems. All species of sturgeon inhabit large river systems, and many perform long anadromous reproductive migrations as adults (Rochard et al., 1991). Table 7.2 provides some measurements of performance in the field. Sonic tracking observations of migrating Russian sturgeon (Acipenser gueldenstaedtii) and stellate sturgeon (Acipenser stellatus) in the Volga river reported complex activity patterns, with much variation in swimming speeds and the alternation of periods of activity with periods of rest (Gayduck et al., 1971; Shubina, 1971; Lindsey, 1978). (Malinin et al., 1971; Malinin, 1973) also reported very variable swimming speeds in various species of sturgeons migrating in the Volga and other Russian rivers draining into the Caspian Sea (Table 7.2). McKinley and Power (1992) used EMG telemetry to investigate patterns of routine swimming activity by lake sturgeon (Acipenser fulvescens), inhabiting a tract of the Mattagami river (Ontario, Canada). The sturgeon showed highly variable tail beat frequencies and swimming speeds (Table 7.2), with no evidence of diurnal patterns. The authors had calibrated the tail beat frequency provided by the EMG signals against measures of oxygen consumption during forced exercise in a tunnel respirometer, at diVerent temperatures (see below). They calculated that the spontaneous swimming activity might account for less than 50% of the aerobic metabolic scope for activity at warm summer temperatures (19
C), for slightly more than 50% of scope in autumn (at 10
C) and for more than the scope for activity in winter (at 5
C), possibly indicating increased reliance on anaerobic swimming at low temperatures (McKinley and Power, 1992). Geist et al. (2005) also used EMG telemetry to investigate movements and swimming speeds of juvenile white sturgeon as a function of diVerences in water flow in the Snake River below the Hells Canyon Dam (Idaho, United States). They

344

D. J. MCKENZIE ET AL.

found that the total distance moved and area occupied were greatest when river flow was low and stable, although they do not report ground speeds for the fish. The tail beat frequency derived from the EMG signal indicated that median swimming speed was also lowest when flow was low (Table 7.2). The total energy used (as derived from previous calibration of the EMG signals against oxygen consumption during forced exercise in a tunnel respirometer, see below) was similar under all flow regimes because the fishes selected refuges when river flow was high. Chandler et al. (2003) modeled the same sturgeon’s habitat in the river as a function of the energy required to inhabit areas of diVering flows, and found that the distribution of the sturgeon as revealed by the EMG telemetry coincided with the areas that were energetically the least expensive. Thus, the lower the flow in the river, the larger the habitat the sturgeon utilized. e. The Paddlefish: A Migratory Filter‐Feeder. The paddlefishes are the most derived forms of the Acipenseriformes and the North American paddlefish, P. spathula, is a highly mobile filter‐feeder inhabiting large midwestern rivers of the central United States (Bemis and Grande, 1992). It also performs extensive spawning migrations (Rosen et al., 1982; Paukert and Fisher, 2000; Stancill et al., 2002; Roush et al., 2003). Sonic telemetry revealed that routine movements of adult males in Keystone reservoir (created by impoundment of the Arkansas river, Oklahoma) occurred predominantly at night and during periods of low water flow, with speeds up to 4 km h1 (Table 7.2) (Paukert and Fisher, 2000). Juvenile paddlefish also swam at similar speeds in a main stem Missouri river reservoir (Table 7.2), with complex seasonal changes in diurnal activity patterns (Roush et al., 2003). In both of these studies, movements were both up and down river, so actual swimming eVort is not known. Upriver spawning migrations are stimulated by high water flows in spring and, although ground speeds and movements are not available, adult paddlefish are reported to migrate up to 45 km day1 (Paukert and Fisher, 2000). f. Other Primitive Fishes. It is not clear to what extent locomotion is important in the parasitic life history of the hagfishes. Little is also known about movement patterns of primitive actinopterygians other than the acipenserids. Bichirs and the reedfish inhabit marginal swamps, freshwater (FW) lagoons, and rivers in western Africa, favoring sheltered inshore habitats. They are piscivorous, foraging largely at night, and lying quiet by day (Worthington, 1929). There are, however, no measurements of their patterns of habitat use or swimming performance in the wild. The gars also occupy slow‐moving rivers or stagnant waters (swamps and lakes). They are reported to migrate up rivers during their spawning season (Jones et al., 1978) but

7.

LOCOMOTION

345

there are no field measurements of their patterns of habitat use or swimming performance. Amia are found in swampy, vegetated lakes, and rivers and, although they are also reported to be somewhat migratory during their spawning season (Jones et al., 1978), their locomotor behavior and activity in the wild has not been specifically investigated. g. A Comparison with Modern Elasmobranchs and Teleosts. Table 7.2 also shows some spontaneous swimming speeds for modern elasmobranchs and teleosts. Spontaneous routine swimming speeds of sharks (Lowe, 2002; Sepulveda et al., 2004) are quite similar to those of the sturgeons and paddlefish. Underwater video has revealed that migratory teleosts such as sockeye salmon (Oncorhynchus nerka) show alternation between periods of sustained low‐intensity exercise and periods of high‐speed burst swimming as they negotiate velocity barriers in rivers (Standen et al., 2004). This pattern of swimming is similar, therefore, to that observed by Quintella et al. (2004) in migrating lampreys (although these attached to stones to recover from burst exercise), and may explain the highly variable swimming speeds observed by Malinin et al. (1971; Malinin, 1973) in migrating sturgeon. The migratory salmonids, however, swim at much higher ground speeds than migratory lampreys or sturgeons, with the exception of the data reported by Malinin et al. (1971) for an unidentified sturgeon species (Table 7.2). 5.1.2. Laboratory Studies: Endurance Curves Endurance is the time for which a fish can swim at a given speed prior to fatigue. It comprises measures of all swimming speeds from sustained through prolonged to burst. Such data therefore provide an excellent picture of swimming capacity in fishes, revealing the speeds at which fishes transit from sustained to prolonged and then to burst swimming. As would be expected, endurance is inversely related to swimming speed. Data are typically presented as fatigue curves, where endurance is plotted against a range of swimming speeds on a semilog graph, revealing characteristic inflections in the relationship as fish move from sustained, through prolonged, and into burst swimming speeds (Videler, 1993) (Figure 7.4). The first section is vertical and is located at maximum sustained speed: all speeds to the left can, in theory, be sustained indefinitely. The subsequent section, showing an exponential decline as swimming speed increases, covers prolonged speeds (Figure 7.4). The second inflection occurs when the fishes transition to pure FG muscle activity for burst speeds, which endure less than 20 s and where there is a much shallower relation of the brief endurance times with speed (Figure 7.4). Such curves are very time‐ consuming to construct and endurance has only been studied in one species of lamprey (Beamish, 1974; Hanson, 1980) and two species of sturgeons (Peake et al., 1997; Adams et al., 1999), all species which perform extensive migrations.

346

D. J. MCKENZIE ET AL.

10,000

Endurance (min)

1000

100

10

1

0.1

0.01 0

50

100

150

200

250

300

Swimming speed (cm s−1) Fig. 7.4. Swimming endurance curves for various primitive fish species and two representative teleosts. Triangles, P. marinus [data replotted from Beamish (1974) with permission from the American Fisheries Society; plus supplementary data plotted from Hanson (1980)]; gray circles and dotted line, A. fulvescens [data replotted from Peake et al. (1997) with permission from NRC Research Press]; white circles and solid line, S. platorynchus [data replotted from Adams et al. (1999) with permission from the ASIH]; dashed line, O. mykiss [data replotted from Bainbridge (1960) with permission from the Company of Biologists]; dotted and dashed line, Salvelinus alpinus [data replotted from Beamish (1980) with permission from Elsevier].

Beamish (1974, 1978) reported that endurance at a range of prolonged swimming speeds was positively correlated with water temperature, fish mass, and fish length in wild‐caught sea lamprey (P. marinus) acclimated in the laboratory to temperatures of 5, 10, or 15
C. There was an exponential decline in endurance as swimming speed was increased (Figure 7.4). Hanson (1980) found that endurance at high (burst) speeds was relatively independent of speed in spawning run adults of the same species seasonally acclimatized to temperatures of about 15
C (Figure 7.4). Peake et al. (1997) found that endurance at sustained and prolonged speeds increased with temperature and length in lake sturgeon (A. fulvescens) acclimated in the laboratory to temperatures of 7, 14, or 21
C. This sturgeon species, however, appeared unable to generate any burst swimming speeds (Figure 7.4). In juvenile hatchery‐reared pallid sturgeon (Scaphirhynchus albus) at 12
C, endurance was positively related to fish length (Adams et al., 1999), and this species was able to swim at burst speeds (Figure 7.4). Adams et al. (1999)

7.

LOCOMOTION

347

also reported that at high swimming speeds the pallid sturgeon often appressed themselves to the swim tunnel bottom where they could maintain station without body or caudal fin motion. Despite this limited database, some clear similarities and diVerences emerge by comparison with teleosts. Endurance typically increases with length in all fishes (Videler, 1993) and temperature also has a positive eVect on sustained and prolonged performance in teleosts, similar to that observed in the sea lamprey (Beamish, 1974, 1978) and lake sturgeon (Peake et al., 1995), although beyond an optimum temperature, performance tends to decline in teleosts (Brett, 1964; Beamish, 1978). This optimal temperature was clearly not exceeded in the studies on the two primitive fishes (Beamish, 1974, 1978; Peake et al., 1997). The overall shape of the endurance curve is similar in the lamprey and sturgeons to that of teleosts (Figure 7.4), with the exception of the lack of burst speeds in lake sturgeon (Peake et al., 1997). The endurance of lampreys for ‘‘prolonged’’ speeds is similar to that of sturgeons (Peake et al., 1997; Adams et al., 1999) but all of these fishes perform less well than two representative migratory teleosts (Bainbridge, 1960; Beamish, 1980). On the other hand, endurance at burst speeds appears to be similar in lampreys and teleosts (Figure 7.4). 5.1.3. Laboratory Studies: Critical Speed Swimming The physiology and performance of sustained and prolonged swimming has been studied quite extensively in teleosts, although most work has been on salmonids (Beamish, 1978; Jones and Randall, 1978; Videler, 1993; Webb, 1993, 1998). Many authors have investigated traits such as maximum sustained swimming speed and maximum prolonged speed in teleosts (reviewed by Videler, 1993) but these traits have not been investigated in primitive fishes. The Ucrit protocol developed by Brett (1964) is one of the most widely applied techniques for quantifying prolonged swimming performance in the laboratory. It involves incremental stepwise increases in swimming speed until the fish fatigues, which allows the calculation of Ucrit as a single performance trait (Brett, 1964; Beamish, 1978). During the protocol, the fish will initially utilize SO muscle but, at a certain speed, FG muscle will be recruited and this leads relatively rapidly to fatigue. Thus, Ucrit measures aspects of prolonged swimming performance (Beamish, 1978). Although the exact physiological and ecological significance of Ucrit are open to question (Plaut, 2001; Nelson et al., 2002; McKenzie et al., 2004; Peake and Farrell, 2006), individual performance has been shown to be consistent and repeatable in a diversity of fish species (Jain et al., 1998; McKenzie et al., 2007), indicating that the protocol does provide a useful means of gauging and comparing performance between species. Furthermore, given that the initial stages of the protocol rely on SO muscle work, there is an exponential increase in oxygen uptake as swimming speed increases (Brett, 1964;

348

D. J. MCKENZIE ET AL.

Beamish, 1978; Webb, 1993). Thus, if rates of oxygen uptake are measured, the protocol can provide values for cardiorespiratory performance variables such as the maximum metabolic rate of aerobic activity (active metabolic rate, AMR) and the scope to increase metabolic rate above standard or routine metabolism (aerobic metabolic scope, MS) (Brett, 1964; Fry, 1971; Beamish, 1978). The Ucrit respirometry protocol has been used extensively in teleosts to investigate the impact on performance of variables such as fish size (Brett and Glass, 1973) and various environmental factors (Beamish, 1978; Randall and Brauner, 1991) such as water temperature (Brett, 1967; Claireaux et al., 2006; McKenzie et al., 2007), salinity (Brauner et al., 1992, 1994), dissolved gases (Randall and Brauner, 1991), and pollutants (Waiwood and Beamish, 1978; Ye and Randall, 1991; MacKinnon and Farrell, 1992; McKenzie et al., 2007a). The literature on all other fish groups is very poor by comparison. As detailed below, measurements of performance and metabolism in primitive fishes are restricted to some Agnathans, some acipenseriformes, one species of gar, and Amia. a. Hagfishes and Lampreys. The hagfishes have a low‐pressure circulation in a blood system containing large venous sinuses, and no neural control of the heart, which might not be considered as adaptations for high‐ performance sustained exercise (Farrell, 1991). There are no reports of Ucrit in hagfishes but Wells et al. (1986) and Forster et al. (1988) reported that Eptatretus cirrhatus swam spontaneously when exposed to a water current of 20 cm s1 equivalent to 0.4 body lengths s1 for 10–15 min in an annular flume. They exhibited increases in heart rate and a decline in venous PO2 , in other fishes these responses are considered to reflect increased cardiac output and increased oxygen consumption by working SO muscle (Holeton and Randall, 1967; Stevens and Randall, 1967; Farrell and Clutterham, 2003). Table 7.3 shows Ucrit values for two species of lamprey. Beamish (1974, 1978) reported that Ucrit of wild‐caught adult P. marinus increased with fish length (Figure 7.5) and also progressively with acclimation temperature up to 15
C (Table 7.3). Beamish (1973) reported measures of the maximum metabolic rates of P. marinus when swimming in a respirometer (AMR; Fry, 1971) at 10
C (Table 7.4). It is interesting that the Ucrit reported by Beamish (1974) is not dissimilar from the actual ground speeds covered by migrating P. marinus (Quintella et al., 2004). Mesa et al. (2003) and Dauble et al. (2006) measured Ucrit in wild‐caught adults and juveniles, respectively, of the Pacific lamprey (Lampetra tridentata), and this was similar to P. marinus (Table 7.3). b. Sturgeons and Paddlefish. These are the primitive fishes for which there is the most information about critical speed swimming. Table 7.3 carries Ucrit values for six species of sturgeons and a paddlefish, using both

Table 7.3 Critical Swimming Speeds in Various Primitive Fishes, Modern Elasmobranchs and Teleosts Ucrit Species

N

Length (cm)

Superclass Agnatha P. marinus

53

14.5–39.0

5–100

L. tridentata L. tridentata

24 30

63  3 14  1

383  45

Subclass Chondrostei A. transmontanus

349

A. transmontanus A. transmontanus

4 4 14

34  2

8 8 8 63 63 4

68  2 71  2 68  2 15 120 30–52

U interval

T (
C)

U increment

5 10 15 15 12

Gradually to highest

10 min at max

10 cm 15 cm

30 min 5 min

800–1100 800–1100 288  34

10 10 12

10 cm 10 cm 5 cm

2400  670 2600  440 2200  240

Mass (g)

cm s1

BL s1

16.6–33.6 16.8–34.7 24.2–41.3 86  7 36  10

0.9–1.2 0.9–1.2 1.1–1.7 1.4 2.4  0.6

30 min 30 min 15 min

56

1.2 0.8 1.65  0.05

10 cm 10 cm 10 cm 5 cm 5 cm 5 cm 5 cm 5 cm 10 cm

variable variable variable 10 min 10 min 10 min 10 min 10 min 20 min

75 65 65 26 97 39 45 47 68  12

1.1 0.9 0.9 1.7 0.8 0.9 1.0 1.0

A. medirostris

19

1132  424

14 19 24 14 14 7 14 21 11

A. medirostris

11 8 11

68  1

1132  424 1132  424 700–1600

19 24 19

10 cm 10 cm 5 cm

20 min 20 min 60 min

80  16 56  21 53

0.8

10

65  1

700–1600

19

5 cm

60 min

54

0.8

A. fulvescens

Comments

Prolonged speeds; acute temperature changes

References

Beamish, 1974; 1978 Mesa et al., 2003 Dauble et al., 2006 Burggren, 1978

Lips sewed shut

Values derived by modeling data for fishes of diVerent sizes

Counihan and Frost, 1999 Geist et al., 2005 Geist et al., 2005 Geist et al., 2005 Peake et al., 1995 Peake et al., 1995

Mayfield and Cech, 2004

Lankford et al., 2005 28‐day exposure to various acute stressors

(continued)

Table 7.3 (continued ) Ucrit Length (cm)

U interval

cm s1

BL s1

350

T (
C)

U increment

197  18 204  19 156  12

19 24 23

10 cm 10 cm 0.25 BL

20 min 20 min 30 min

45  2 52  2 83

1.3 1.4 3.2  0.2

27  1

158  16

23

0.25 BL

30 min

76

2.8  0.2

4

21  1

34  8

10

5 cm

30 min

19  4

0.9

S. pallidus

6 6

19  1 20  1

25  4 30  7

20 10

5 cm 5 cm

30 min 30 min

37  3 15  3

1.9 0.7

P. spathula

8 5

21  2 82

36  3 2.5  0.2

20 22

5 cm 0.5 BL

30 min 15 min

36  3 14

1.7 1.7  0.1

10 6

27  2 30

55  16 1000

25 15

5 cm variable

1 min 20 min

51.4  7 75

1.9 2.5

Species

N

A. medirostris

11 11 8

35  1 36  1 26  1

9 S. platorynchus

A. naccarii

Subclass Neopterygii L. osseus A. calva

Mass (g)

Comments

References Allen et al., 2006

Fishes reared in FW

McKenzie et al., 2001a

Fishes reared in salinity of 11 g liter1 Adams et al., 2003 Adams et al., 2003 10 min rest period between velocity increments

Burggren and Bemis, 1992 Webb et al., 1992 Farmer and Jackson, 1998

Class Elasmobranchii Negaprion brevirostris T. semifasciata

Sphyrna lewini Infraclass Teleostei O. nerka

O. nerka O. mykiss

351

Salvelinus alpinus Micropterus salmoides Esox sp. A. anguilla D. labrax

1

70

1650

24

9 cm

30 min

77

1.1

6

37  1

173  11

19–23

9 cm

30 min

53  4

1.4  0.1

3 11

111  4 57  3

4660  285 614  22

18–23 21–28

9 cm 10 cm

30 min 30 min

77  5 65  11

0.7  0.1 1.2  0.2

9–37

9–17

5

0.5 BL

60 min

41 – 54

4.4–3.3

6 20 20 6

3–1432 1 – 1962 64  1 34  1

8–54 6–61 2690  100 534  66

15 20 18 15

0.5 BL 0.5 BL 0.15 BL 0.25 BL

60 min 60 min 20 min 30 min

51 – 178 37 – 136 133  2 76

6.7–3.3 6.4–2.1 2.1  0.1 2.2  0.1

11 10

35 10

12 25

10 cm 10 cm

10 min 60 min

100 35.1

2.8 3.5

10 8

19  1 31  3

25  5 106  4

25 23

5 cm 0.5 BL

1 min 30 min

64  11 44  3

3.4 1.4  0.1

7

37  2

514  21

15

10 cm

30 min

79  2

2.1  0.1

4

U, speed; Ucrit, critical swimming speed; BL s1, body lengths s1.

Graham et al., 1990 Graham et al., 1990 Lowe, 1996 Brett and Glass, 1973

Lee et al., 2003 Shingles et al., 2001 Jones et al., 1974 Farlinger and Beamish, 1977 Webb et al., 1992 McKenzie et al., 2003 Chatelier et al., 2005

352

D. J. MCKENZIE ET AL.

160 140

Ucrit (cm s−1)

120 100 80 60 40 20 0 0

20

40

60 80 Length (cm)

100

120

140

Fig. 7.5. The relationship of body length to Ucrit for various primitive fishes, and elasmobranch and a representative teleost. Bold black line, P. marinus [data replotted from Beamish (1974) with permission from the American Fisheries Society]; white triangles and dotted line, A. fulvescens [data replotted from Figure 4b in Peake et al. (1995)]; white circles and solid black line, A. medirostris [data replotted from Allen et al. (2006) with permission of NRC Research Press]; white squares and dash/dot line, Triakis semifasciata [data replotted from Graham et al. (1990), with permission from the Company of Biologists]; dashed line and single white diamond, O. nerka [from data in Table 4 of Brett and Glass (1973) and from Tables 2 and 3 of Lee et al. (2003)].

wild‐caught and hatchery‐reared specimens maintained under a variety of diVerent environmental conditions. Table 7.4 carries data for respiratory metabolism of prolonged exercise in some of these same species. Interspecific comparisons are complicated somewhat by the diVerent animal sizes and environmental conditions but most species have relatively similar performance, rarely achieving Ucrit higher than 2 body lengths s1, the highest Ucrit being in juvenile hatchery‐reared Adriatic sturgeon, Acipenser naccarii (McKenzie et al., 2001a). When exposed to a current in a swim tunnel, many sturgeon species will respond by appressing themselves against the bottom and using their pectoral fins to maintain position without swimming. This behavior is particularly evident in the lake sturgeon (McKinley and Power, 1992), the pallid sturgeon and the shovelnose sturgeon (Adams et al., 2003), and the white sturgeon (Geist et al., 2005), less so in the Adriatic sturgeon (McKenzie et al., 2001a) and the green sturgeon, Acipenser medirostris

Table 7.4 Elements of Exercise‐Related Respiratory Metabolism in Various Primitive Fishes, Modern Elasmobranchs and Teleosts

Species

N

Mass (g)

T (
C)

Superclass Agnatha P. marinus 9

34–72

10

Subclass Chondrostei A. transmontanus 4

800–1100

10

2400  670 2600  440 2200  240 4000–6000

14 19 24 5

A. transmontanus

SMR (mmol O2 kg1 h)

RMR (mmol O2 kg1 h)

2.00

AMR (mmol O2 kg1 h)

Net scope

Factorial scope (AMR/SMR)

353

14.84

12.84

7.43

1.56

4.46

2.90

2.86

10.51  1.22 14.85  2.30 13.34  1.50 0.38

21.06  1.31 23.00  1.34 21.47  0.97 2.73

10.54 8.15 8.12 2.34

2.00 1.55 1.61 7.09

1.34 1.71 6.75  0.78

4.16 4.99 17.12  1.90

2.81 3.28 10.37  1.47

3.09 2.92 2.53

A. fulvescens

8 8 8 10

A. naccarii

10 10 8

156  12

10 19 23

9

158  16

23

8.78  0.34

19.84  0.69

11.06  0.62

2.26

11

700–1600

19

5.8  0.4

18.6  1.3

12.3  1.1

3.1

10

700–1600

19

8.5  0.4

17.7  0.8

9.0  1.1

2.1

5

2.5  0.2

22

7.77

4.27

2.2

690  129

26

15.65

9.75

2.65

A. medirostris

P. spathula

Class Elasmobranchii Sphyrna lewini 17

3.50

5.91  0.47

Comments

References

Beamish, 1973 RMR for fish ‘‘at rest’’ RMR for fish swimming at 0.5 BL s1

Burggren, 1978 Geist et al., 2005

McKinley and Power, 1992

Fishes reared in FW

McKenzie et al., 2001a

Fishes reared in salinity of 11 g liter1 Lankford et al., 2005 28‐day exposure to various acute stressors RMR for fish swimming at 0.5 BL s1

Burggren and Bemis, 1992 Lowe, 2001

(continued)

Table 7.4 (continued )

Species

N

Infraclass Teleostei O. nerka

354

O. nerka

Mass (g)

T (
C)

SMR (mmol O2 kg1 h)

RMR (mmol O2 kg1 h)

AMR (mmol O2 kg1 h)

Net scope

Factorial scope (AMR/SMR)

50

15

2.7

25.7

23.0

9.6

500 1000 3000  110 2690  100 2260  190 534  66

15 15 12 18 8 15

1.9 1.7

3.04  0.86

23.6 23.0 28.25  0.30 18.58  0.81 16.12  0.84 19.39  1.68

21.7 21.3 20.10  0.26 13.33  0.81 11.06  0.75 16.35

12.6 13.7 3.46 3.54 3.18 6.38  2.58

8.16  0.17 5.25  0.19 5.06  0.36

O. kisutch O. mykiss

12 20 13 6

A. anguilla

8

106  4

23

1.55  0.27

7.35  1.26

5.80

4.80  0.37

D. labrax

7

514  21

15

1.92  0.47

9.93  1.0

7.83  1.22

5.17

SMR, standard metabolic rate; RMR, routine metabolic rate; AMR, active metabolic rate.

Comments

Values derived by modeling data for fishes of diVerent sizes

References

Brett and Glass, 1973

Lee et al., 2003

Shingles et al., 2001 McKenzie et al., 2003 Chatelier et al., 2005

7.

LOCOMOTION

355

(Lankford et al., 2005; Allen et al., 2006). This behavior is presumably used in their natural environment as a rheotactic response to velocity barriers (Geist et al., 2005). Nonetheless, the critical speeds measured in the laboratory are all higher than the swimming speeds observed during routine activity of sturgeons in the field (McKinley and Power, 1992; Geist et al., 2005), and also compare very favorably with the speeds observed in migrating sturgeons (Table 7.2; cf Table 7.3). During the Ucrit protocol, oxygen uptake has been reported to increase either as a linear function of swimming speed (McKinley and Power, 1992), as an exponential function (Geist et al., 2005), or in a manner that is described almost equally well by either function (McKenzie et al., 2001a). The eVects of body length on Ucrit have been reported for the lake sturgeon (Peake et al., 1995) and the green sturgeon (Allen et al., 2006). As has been described in teleosts (Beamish, 1978), absolute Ucrit performance (cm s1) increased with fish size (Figure 7.5) whereas relative performance (body lengths s1) declined (Table 7.3). A number of studies have investigated the eVects of temperature on Ucrit and associated respiratory metabolism (Tables 7.3 and 7.4). Overall, the sturgeons appear to be quite tolerant of relatively high water temperatures, performing exercise well at temperatures above 20
C (Table 7.3). The data indicate that the optimal temperature for exercise and cardiorespiratory performance in the white sturgeon lies between 14 and 19
C (Geist et al., 2005), while the green, lake, pallid, and shovelnose (Scaphyrhincus platorynchus) sturgeons are clearly adapted to higher maximum seasonal temperatures in their habitats (McKinley and Power, 1992; Peake et al., 1995; Adams et al., 2003; Mayfield and Cech, 2004; Allen et al., 2006). Some species, therefore, must have a rather wide zone of thermal tolerance, for example the lake sturgeon inhabits areas where waters are covered in ice for long periods of the year. McKenzie et al. (2001a) investigated the eVects of water salinity on Ucrit performance in Adriatic sturgeon. In this species, breeding occurs in FW, but it is believed that both juveniles and adults predominantly inhabit estuarine brackish water (BW) environments and also venture into coastal waters for brief foraging trips (Tortonese, 1989; Rochard et al., 1991; Rossi et al., 1992). There was no significant diVerence in Ucrit between fish raised in either FW or in BW at a salinity of 11 g liter1 (Table 7.3), nor in aerobic scope for activity (Table 7.4). This despite the fact that fish in BW exhibited significantly reduced growth rates and elevated standard metabolic rate (Table 7.4). McKenzie et al. (2001b) exposed sturgeon from the FW and BW groups to salinity challenge (an abrupt increase in salinity to 28 g liter1) and found that, at 24 h, this significantly impaired swimming performance. Swimming was energetically less eYcient and this was presumably a consequence of the measured increases in tissue ion concentrations and reductions in tissue

356

D. J. MCKENZIE ET AL.

Ucrit (body length s−1)

4

3

2

1 80

120

160

Plasma ion concentration (mequiv

200

240

liter −1)

Fig. 7.6. Least‐squares linear regression analysis describing the relationship of mean (SE) critical swimming speed (Ucrit) versus mean (SE) Naþ (open circles) and Cl– (solid circles) measured in the plasma of Adriatic sturgeon (A. naccarii) following exercise to fatigue. When reading from left to right down the regression lines, the circles represent a group raised in FW, a group raised in BW at 11 g liter1, the BW group at 24 h following abrupt transfer to seawater at 28 g liter1, and FW group following the same abrupt transfer (see text for further details). [From McKenzie et al. (2001b) with permission from the NRC Research Press.]

moisture (McKenzie et al., 2001b). The changes in tissue ions and water content were, however, significantly less severe in the animals from BW and, as a result, they exhibited better exercise performance after the challenge than the FW fish did (McKenzie et al., 2001b). Indeed, there was a direct linear relationship between Ucrit and plasma osmolarity or the concentrations of sodium or chloride (Figure 7.6). The results of the seawater challenge experiments indicate that exposure to BW in estuarine environments promotes osmoregulatory adaptations that could then improve the ability of the sturgeon to forage in marine waters (McKenzie et al., 2001b). Lankford et al. (2005) investigated the impact of chronic stress on swimming performance in green sturgeon. Daily exposure of the sturgeon to a random series of acute stressors (5‐min chasing, 5‐min confinement, and 10‐min water reduction) for 28 days had no eVect on the rather low‐Ucrit achieved by this species (Table 7.3) despite the fact that aerobic scope for activity was reduced consequent to a stress‐related increase in routine metabolic rate (Table 7.4). The consistent increase in metabolic rate after 28 days exposure to the acute stressors was taken as an indication that this species was not able to habituate to chronic stress (Lankford et al., 2005).

7.

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6 Buccal water intake

Oxygen uptake (mmol kg−1 h−1)

Opercular water intake 4

2

0 0.0

0.4

0.8

1.2

Swimming speed (body lengths s−1) Fig. 7.7. Oxygen consumption (10
C) at diVerent swimming speeds in a representative experiment from Burggren (1978) on the white sturgeon, A. transmontanus (forklength 62 cm). The relationship during both buccal water intake (solid lines) and strictly opercular intake (dotted lines) is shown. Figure reproduced with permission from Elsevier.

A number of studies have investigated interactions between ventilation and the performance of sustained swimming in Acipenseriformes. Burggren (1978) investigated the potential role of the spiracle and incomplete opercular flap (this does not completely cover the gills in most acipenserids) in maintaining gill ventilation in the white sturgeon, as an adaptation for bottom dwelling. Burggren (1978) reported some intriguing results whereby sewing the lips closed did not stop the sturgeon from swimming at low speeds but did significantly curtail aerobic scope (Figure 7.7) and, therefore, Ucrit (Table 7.3). This indicates that this species is able to maintain gill ventilation while foraging at low speeds across silty substrates that eVectively block inhalation through the mouth (Burggren, 1978). Indeed, this semitidal ventilatory mode (Burggren, 1978) would be suYcient to meet the metabolic demands of the spontaneous swimming speeds measured on this species in the wild (Geist et al., 2005).

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D. J. MCKENZIE ET AL.

The filter‐feeding strategy in P. spathula requires extensive foraging activity. Burggren and Bemis (1992) demonstrated that the constant spontaneous swimming observed in Polyodon reflects an adaptation for ram ventilation of the gills that, presumably, coevolved with the filter‐feeding lifestyle. In juvenile paddlefish at 25
C, the transition to complete ram ventilation occurred at about 50% of Ucrit (Table 7.3) and the speed of spontaneous swimming activity was 70–80% of Ucrit (Burggren and Bemis, 1992). Aerobic scope was similar to sturgeon (Table 7.4) but metabolic rate rose very rapidly as swimming speed increased and, given that the juvenile paddlefish swam spontaneously at speeds close to their Ucrit, they appeared to operate routinely in the upper portion of their aerobic scope for activity (Burggren and Bemis, 1992; Peake, 2004). Burggren and Bemis (1992) suggested that, although this might indicate an energetically expensive lifestyle, it would allow some energy to be conserved by ram ventilation. It would be interesting to investigate energetics of spontaneous swimming and ram ventilation in larger animals because telemetry indicates that they do swim at relatively high speeds in their natural environment (Paukert and Fisher, 2000; Roush et al., 2003). McKenzie et al. (2007b) found evidence that swimming also contributes to gill ventilation in the Adriatic sturgeon. Like Polyodon, this sturgeon species also exhibits constant spontaneous swimming activity although at only about 15% of its Ucrit (McKenzie et al., 1995, 2001a). When, however, the sturgeon is allowed to swim it is more tolerant of aquatic hypoxia (Figure 7.8). That is, it can regulate aerobic metabolism and blood oxygen content down to levels of hypoxia that cause significant declines in these variables in static animals (McKenzie et al., 2007b). Paradoxically, McKenzie et al. (2001a) found that the species never exhibits a complete transition to ram ventilation as it swims to Ucrit, but the results of McKenzie et al. (2007b) indicate that forward motion may nonetheless contribute to irrigation of the gills. c. The Gars and Amia: A Role for Air Breathing. The only measure of Ucrit in this group was reported by Webb et al. (1992) for longnose gar (Table 7.3). The neopterygians are air‐breathing fishes and Farmer and Jackson (1998) investigated the interactions between ventilation and activity in Amia and the spotted gar, Lepisosteus oculatus. During incremental increases in sustained swimming speeds, most of the Amia exhibited an exponential increase in their air‐breathing frequency (Figure 7.9). Although the authors do not report a Ucrit for the fish, Figure 7.9 reveals that the ‘‘30‐cm long’’ Amia were all able to achieve swimming speeds in excess of 2 body length s1 (Table 7.3). Somewhat surprisingly, during recovery from this exercise protocol, the Amia did not breathe air any more frequently than they had done prior to exercise (Farmer and Jackson, 1998). If the air breathing during incremental exercise reflected an exponential increase in oxygen demand, then the absence

7.

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LOCOMOTION

6

MO2 (mmol kg−1 h−1)

5

4

3

2

1 0

5

10

15

20

P O2 (kPa) Fig. 7.8. EVects of progressive hypoxia on mean (SEM) rates of oxygen uptake (MO2 ) in the Adriatic sturgeon, A. naccarii, under static conditions (gray symbols) or when swimming at a speed equivalent to 0.5 body lengths s1 (white symbols). The hatched line is a least‐squares linear regression through the mean data points obtained under static conditions, showing a linear decline in MO2 with PO2 . The regression is described by the equation MO2 ¼ 0.182 PO2 þ 1.850, R2 ¼ 0.940. The horizontal solid line shows mean normoxic MO2 in swimming fish, the sloped solid line is a linear regression through those data points for swimming animals where there was a progressive decline in MO2 below normoxic values. The intersection of the two solid lines indicates the critical water PCO2 below which the swimming sturgeon could no longer regulate aerobic metabolism (see text for further details). [Figure reproduced from McKenzie et al. (2007b) with permission from the The Fisheries Society of the British Isles.]

of such behavior during recovery presumably indicates that there was no elevated postexercise increase in oxygen consumption (EPOC). This, in turn, implies that the exercise was entirely aerobic, at least in part thanks to the air breathing. Indeed, Farmer and Jackson (1998) found that both the Amia and the spotted gar increased the proportion of their aerobic metabolism obtained from air versus water when performing low‐intensity sustained swimming. Thus, it would appear that these fish will continually be breaking the surface when, for example, performing spawning migrations or chasing prey. There are, in fact, other reports of interactions between activity and respiration in primitive fishes. Grigg (1965) reported field observations whereby the Queensland lungfish breathed air most frequently when active, for example, at night and during breeding behaviors or predator–prey interactions. Magid (1965) reported that increased spontaneous activity in the gray bichir, Polypterus senegalus, was associated with a marked increase in

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D. J. MCKENZIE ET AL.

1.25

1.25

Amia 4

Amia 1 1.00

1.00

0.75

0.75

0.50

0.50

0.25

0.25 0.00

0.00 0

0.5

1.0

0.5

1.0

Amia 5

Amia 2 Surface events (min−1)

0 1.25

1.25 1.00

1.00

0.75

0.75

0.50

0.50

0.25

0.25

0.00

0.00 0

0.5

0

1.0

0.5

1.0

0.5

1.0

1.25

1.25

Amia 6

Amia 3 1.00

1.00

0.75

0.75

0.50

0.50

0.25

0.25 0.00

0.00 0

0.5

1.0 0 Flume speed (m s−1)

Fig. 7.9. The frequency of surfacing events, presumed to indicate air‐breaths, as a function of flume speed for A. calva swimming in an open flume at 15
C. The fish were observed at rest for 1 h, during which time none of them surfaced. The flume was then increased in speed every 20 min until either the fishes showed signs of exhaustion or the highest flume speed was reached. [Figure reproduced from Farmer and Jackson (1998) with permission from the Company of Biologists.]

air‐breathing frequency. Both Grigg (1965) and Magid (1965) suggested that air breathing may be at least as important for sustaining activity metabolism as it is for tolerating hypoxia. d. A Comparison of Performance with Elasmobranchs and Teleosts. As is visible from Table 7.3, primitive fishes generally exhibit similar Ucrit to modern elasmobranchs but lower than most teleosts, when compared against

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361

species with similar lifestyles. One exception is the European eel, Anguilla anguilla, which has similar Ucrit to the lampreys, both being anguilliform swimmers (Table 7.3) (Beamish, 1974; McKenzie et al., 2003; Mesa et al., 2003; Dauble et al., 2006). Figure 7.5 confirms that, for a given body length, the lampreys, sturgeons, and sharks generally exhibit a lower Ucrit than migratory teleosts such as salmonids. For example, at a body length of 40 cm, the sockeye salmon has a higher Ucrit than any of the primitive fishes (Figure 7.5). The swimming performance of the tunas exceeds that of any primitive fish of a similar body length, and this includes the lamnid sharks which exhibit homologous morphophysiological adaptations for sustained high swimming performance (Bernal et al., 2001). Webb et al. (1992) demonstrated with a direct comparison that the gar has a lower Ucrit than a teleost predator that lives in a similar environment and with similar predatory habits, the tiger musky, Esox sp. (Table 7.3). The maximum metabolic rate of activity, and aerobic scope, of lampreys and sturgeon are similar to those of elasmobranchs but generally somewhat lower than those of teleosts (Table 7.4). In particular, teleosts appear to have a higher factorial scope than the other fish groups. In many teleosts, there is a positive eVect of water temperature on Ucrit, up to an ‘‘optimum’’ temperature beyond which performance declines (Brett and Glass, 1973; Claireaux et al., 2006). The same would, presumably be true in the lampreys and sturgeon but evidence for this was obtained only for the white sturgeon by Geist et al. (2005). Brauner et al. (1992) found that salinity challenge impaired the ability of coho salmon (Oncorhynchus kisutch) to perform a Ucrit test, with a relationship between increased plasma ions and reduced Ucrit performance that was consistent with the results subsequently obtained by McKenzie et al. (2001b) on the Adriatic sturgeon. In salmonids, however, prior acclimation to BW had no eVect on their subsequent tolerance of salinity challenge (Morgan and Iwama, 1991), quite unlike the beneficial eVect observed in A. naccarii (McKenzie et al., 2001b). Other euryhaline teleosts such as seabass, Dicentrarchus labrax, can tolerate acute exposure from FW to SW, or vice versa, with no eVects on ion‐osmotic homeostasis or Ucrit (Chatelier et al., 2005). 5.1.4. Laboratory Studies: Burst Swimming and Recovery from Exhaustion Burst swimming is considered to rely almost exclusively on FG muscle function and, therefore, to be an anaerobic activity (Wood, 1991; Webb, 1998). Hanson (1980) found that adult P. marinus could achieve burst speeds of up to 10 body lengths s1, which is comparable to the maximum burst speeds of many teleosts (Videler, 1993). Dauble et al. (2006) found, however, that juvenile Pacific lamprey could only achieve burst speeds of about 5 body

362

D. J. MCKENZIE ET AL.

lengths s1. Sturgeons appear to perform burst swimming much less eVectively than either lampreys or teleosts (Peake et al., 1997; Adams et al., 1999) (Figure 7.4). There is no information about burst swimming performance of other primitive fishes. The metabolic imbalances associated with exhaustion from burst exercise, and processes of recovery from these, have been studied extensively in teleosts, particularly salmonids (Wood, 1991; Wang et al., 1994; Richards et al., 2002). Phosphocreatine (PCr) and ATP are the fuels for the initial stages of burst exercise and then carbohydrates, glucose, and glycogen provide fuels when the high‐energy phosphagens have been depleted. The glycolysis leads to a large accumulation of tissue lactate (Wood, 1991; Schulte et al., 1992; Wang et al., 1994). Recovery is an aerobic process, associated with a large EPOC, with rapid repletion of the phosphagens and slower recovery of glycogen and metabolism of lactate, with fatty acid oxidation making a significant contribution to fuel repletion (Wood, 1991; Wang et al., 1994; Richards et al., 2002). The protocol can include measuring recovery from fatigue in a Ucrit test, but typically involves manual chasing of the animal until it becomes refractory to further stimulation. This latter protocol of exhaustion causes much more severe imbalances than Ucrit fatigue (Wood, 1991) (Table 7.5). This area of exercise physiology has also been studied in a number of primitive fishes. a. The Agnathans: Rapid Recovery in the Lamprey. The hagfish exhibits a typical acid–base response to exhaustive exercise. Davison et al. (1990) subjected E. cirrhatus to repeated bouts of burst swimming until they became refractory in a swim flume and measured a severe decline in blood pH, an increase in plasma lactate, and a very considerable accumulation of lactate in their myotomal muscle (Table 7.5). The animals had reduced muscle lactate by about 40% by 4‐h following exhaustion but there was no evidence that the hagfish had used their large subcutaneous sinuses preferentially to store lactate during recovery. The lampreys appear able to recover quite rapidly from the metabolic imbalances incurred following exhaustive exercise. Tufts (1991) exercised P. marinus to exhaustion by manual chasing, and reported an immediate significant decline in blood pH (Table 7.5). This acidosis had both a respiratory and metabolic component, with a significant increase in plasma lactate (Table 7.5). Blood pH and CO2 partial pressures (PCO2 ) recovered rapidly, within 1 h following exercise, although lactate levels did not recover until 4 h following exhaustion. Boutilier et al. (1993) also found that plasma pH dropped immediately but then recovered within 1 h in sea lampreys chased manually to exhaustion (Table 7.5). They found that exhaustion was associated with a significant depletion of PCr, ATP, and glycogen in the

Table 7.5 Metabolic Responses to Exhaustive Exercise in Various Primitive Fishes, a Modern Elasmobranch and Teleosts Plasma Species

Protocol

Superclass Agnatha E. cirrhatus 100 min repeated bursts in a flume P. marinus Manual chasing Manual chasing Larval Manual chasing P. marinus L. tridentata Fatigue from Ucrit

T (
C)

Lactate (mM)

18

7.7–7.0

1–5

10 10 15

7.9–7.6 (0) 7.9–7.6 (0)

1–6 (0.5)

11

7.5–6.9 (0)

1–9 (0)

Subclass Chondrostei A. medirostris Manual chasing

16

A. oxyrhynchus A. naccarii

15 23

Manual chasing Fatigue from Ucrit

pH

FG muscle pH

7.2–6.7 (0)

ATP (mmol g1)

5–4 (0) 4–3 (0)

0.5–1.5 (0) 0.1–1.0 (0)b 0.8–1.7 (0) 0.6–1.4 (0)

PCr (mmol g1)

22–6 (0) 27–12 (0)

11–6 (0)

Glycogen (mmol g1)

Lactate (mmol g1)

References

8–48

Davison et al., 1990

14–5 (0) 17–5 (0)

1–25 (0) 1–8 (0)

Tufts, 1991 Boutilier et al., 1993 Wilkie et al., 2001

34–18 (0)

3–15 (0)

Mesa et al., 2003

14–10 (0)

1–6 (0)

KieVer et al., 2001; Baker et al., 2005b Baker et al., 2005 McKenzie et al., 2001a

Subclass Neopterygii L. osseus Fatigue from burst swimming in flume A. calva Manual chasing

23

7.8–7.3 (0)

1–12 (2)

Burleson et al., 1998

15

7.9–7.7 (0)

0.3–5 (0.5)

Gonzalez et al., 1998

Class Elasmobranchii S. acanthias Manual chasing

11

7.8–7.4 (1)

1–9 (4)

Infraclass Teleostei O. mykiss Manual chasing

15

7.8–7.5 (0)

1–20 (2)c

S. trutta

10

8.0–7.7 (0)

0.7–2.4 (0)

Fatigue from Ucrit

3–1 (0.2)

20–6 (0)

15–1 (0.2)

9–39 (0)

Richards et al., 2003

7.2–6.7 (0.2)

8–5 (0)

38––24 (0)

14–1 (0)

2–38 (0)

7.2–6.8 (0)

13–10 (0)

17–5 (0)

31–27 (0)

8–19 (0)

Wang et al., 1994; Milligan and Wood, 1986c Beaumont et al., 2000

For plasma and tissue variables, data are for control and maximum exhausted values, separated by a dash. The value in parentheses indicates the time (h) when the maximum exhausted value occurred.

364

D. J. MCKENZIE ET AL.

locomotor muscle. There was a significant muscle acidosis that had both a respiratory and a metabolic component, involving a significant increase in both PCO2 and lactate concentrations (Table 7.5). The recovery of muscle PCr and adenylates was rapid, and completed within 1‐h postexhaustion, whereas complete lactate clearance and glycogen restoration required up to 4 h. Muscle PCO2 had not recovered at 4‐h postexhaustion and the authors speculated that this was due to a limited capacity for CO2 transport from muscle to blood, and then also within the blood itself (Boutilier et al., 1993). Lampreys lack a functional chloride–bicarbonate exchanger on the erythrocyte membrane (Tufts and Boutilier, 1989; Tufts, 1991; Tufts et al. 1996) hypothesized that CO2 transport and excretion in exercised lampreys might be limited by access to erythrocytic carbonic anhydrase (CA) for bicarbonate hydration/dehydration reactions. The authors did not, however, find that infusion of CA into the plasma had any eVects on CO2 transport or acid–base status. Wilkie et al. (1998) demonstrated that the excretion of protons across the body surface accounted for the rapid recovery of extracellular pH in sea lampreys following exercise (Tufts, 1991; Boutilier et al., 1993), although the majority of the excess protons that caused the intracellular muscle acidosis were metabolized in situ. Wilkie et al. (2001) went on to demonstrate that larval sea lampreys (ammocoetes) are also adept at recovering from exhaustive exercise, despite the fact that they are considered to lead a sessile lifestyle, burrowed in the sediment. Exhaustion led to a significant depletion of whole‐ body PCr and glycogen, large accumulations of lactate, and a profound metabolic acidosis (Table 7.5). There was a large EPOC with over fivefold increases in oxygen uptake during recovery (Wilkie et al., 2001). Nonetheless, rapid rates of proton excretion, lactate elimination, and glycogen replenishment were observed, indicating rapid recovery of metabolic homeostasis. Mesa et al. (2003) found that the Pacific lamprey showed a plasma acidosis when fatigued in a Ucrit protocol (Table 7.5), but recovered this within 1 h. Plasma and muscle lactate peaked at fatigue (Table 7.5) but had recovered within 4 h. b. Sturgeons: A Reduced Response to Exhaustive Exercise? There is some evidence that sturgeon may exhibit a reduced physiological response to exhaustive exercise. In the shortnose sturgeon, Acipenser brevirostrum, chasing manually to exhaustion caused a slight depletion of muscle PCr that was recovered within 1 h. There was no significant eVect on glycogen stores and only a modest increase in muscle lactate concentrations (KieVer et al., 2001) (Table 7.5), although lactate did not return to control levels until 6‐h postexercise. Plasma lactate increased very little (Table 7.5) and had recovered within 2‐h postexhaustion. This sturgeon species and the Atlantic sturgeon, Acipenser oxyrhynchus, both showed a rather modest EPOC, only increasing

7.

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metabolic rate by about twofold following fatigue (KieVer et al., 2001). Baker et al. (2005) confirmed that these two species show little increase in plasma lactate following manual chasing to exhaustion (Table 7.5). The Adriatic sturgeon also shows a relatively modest increase in plasma lactate following exercise to fatigue in a Ucrit protocol (McKenzie et al., 2001a) (Table 7.5). c. Gars and Amia: A Role for Air Breathing. In the neopterygians, there is some evidence that air‐breathing behaviors influence recovery from exhaustive exercise (Burleson et al., 1998; Gonzalez et al., 1998). Burleson et al. (1998) demonstrated that air breathing was stimulated in longnose gar exposed to burst exercise in a flume, and that this allowed the fish to perform equally well in either normoxic or hypoxic water. Both normoxic and hypoxic gar showed an immediate acidosis on exhaustion, with both a respiratory and metabolic component. Lactate accumulated in the blood rather slowly, peaking between 2‐ and 4‐h postexercise (Table 7.5). The restoration of acid– base balance in hypoxic water was, however, slower than in normoxic water because the hypoxia caused a reflex inhibition of gill ventilation and, therefore, inhibited excretion of CO2 and protons into the water (Burleson et al., 1998). Gonzalez et al. (1998) showed that air‐breathing behavior was also stimulated in Amia when they were chased manually to exhaustion. This caused the expected acidosis, with both respiratory and metabolic components, and accumulation of lactate (Table 7.5). If, however, the Amia were obliged to rely exclusively on water breathing during recovery (by being denied access to air), they were able to recover acid–base homeostasis more rapidly. Gonzalez et al. (1998) suggested that this is because, when allowed to breathe air, gill water flow is reduced and therefore this compromises excretion of CO2 and acid equivalents into the water. It must be presumed that the increased air breathing in exhausted gar and Amia (Burleson et al., 1998; Gonzalez et al., 1998) would contribute significantly to EPOC, even if there is a trade‐oV against recovery of acid–base balance. This remains to be demonstrated. d. A Comparison with Elasmobranchs and Teleosts. The metabolism of anaerobic burst exercise is clearly an ancestral feature in fishes, showing a common pattern from hagfish to teleosts (Table 7.5). The work on lampreys (Tufts, 1991; Boutilier et al., 1993; Wilkie et al., 1998, 2001; Mesa, 2003) indicates that they recover plasma pH and lactate after exhaustive exercise much more quickly than do neopterygians (Burleson et al., 1998; Gonzalez et al., 1998), teleosts (Wang et al., 1994), or elasmobranchs (Richards et al., 2003), for which these variables remain significantly aVected for over 4 h following exhaustion. In muscle, while PCr recovers rapidly (within 1 h) in all

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D. J. MCKENZIE ET AL.

fish groups studied to date, the dynamics of muscle ATP and lactate recovery are also much slower in teleosts and elasmobranchs than in lampreys, taking up to 6 h rather than just 1 h (Wood, 1991; Boutilier et al., 1993; Wang et al., 1994; Richards et al., 2003). Sturgeons appear to exhibit a reduced physiological response to exhaustive exercise by comparison with other fish species (Table 7.5). They also exhibit very reduced EPOC by comparison with exhausted Agnathans or teleosts, exhibiting a twofold increase in oxygen uptake (KieVer et al., 2001) rather than over fivefold (Scarabello et al., 1991; Wilkie et al., 2001). The Pacific spiny dogfish is unusual because it exhibited no postexhaustion increase in plasma PCO2 (Richards et al., 2003) such that the exercise‐ related acidosis was purely metabolic, unlike lampreys (Tufts, 1991; Boutilier et al., 1993), neopterygians (Burleson et al., 1998) and teleosts (Wood and Perry, 1985). Richards et al. (2003) suggest that this may be because elasmobranchs have extracellular CA in the plasma (Gilmour et al., 2001), which may facilitate CO2 transport and excretion following exercise. Recovery from exhaustive exercise in elasmobranchs is also interesting because they cannot metabolize lipids as a metabolic fuel in extrahepatic tissues (Moyes et al., 1990; Richards et al., 2003). Thus, while teleosts use lipid oxidation to meet the aerobic metabolic demands of recovery (Wang et al., 1994; Richards et al., 2002), elasmobranchs appear to use ketone bodies (Richards et al., 2003). 5.2. Fast‐Start Performance As mentioned above, the C‐start startle reflex is an ancestral feature of fishes that is exclusively dependent, for its performance, on the function of FG muscle. Fast‐start performance, the motor output of the reflex, has only been studied in a few sarcopterygians, primitive actinoperygians, an elasmobranch, and, indeed, relatively few teleosts. As a result, few firm conclusions can be drawn about the comparative performance of primitive fishes, although some tendencies are visible at a qualitative level. Turning rate (i.e., the average angular velocity of the anterior portion of the body during the stage 1 of an escape response) gives an indication of how fast fishes can change direction. It is the outcome of various components such as the speed of muscle contraction, the amount of muscle power generated, the flexibility of the fish, and the resistance to bending due to hydrodynamic forces. As shown in Figure 7.10, primitive fishes such as the West African lungfish, the coelacanth, two species of bichir, the longnose gar, and Amia appear to have comparable turning rates to those of a modern elasmobranch (S. acanthias) and various teleosts (Fricke and Hissmann, 1992; Meyers et al., 1998; Domenici, 2001; Hale et al., 2002; Tytell and Lauder, 2002; Domenici et al., 2004).

7.

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LOCOMOTION

Turning rate (degrees s−1)

10,000 Pa Ac Pa Ps

1000

Lo

100 0.01

0.1

Lc

1

10

Length (m) Fig. 7.10. The relationship between fast‐start average turning rates and length in teleosts (squares), primitive fishes (filled triangles), and a modern elasmobranch (the spiny dogfish S. acanthias, open triangle). Data on nonprimitive fishes are from Domenici (2001). Data on spiny dogfish are from Domenici et al. (2004) (average value for fast escape responses). Data on primitive fishes are from the African lungfish (P. annectens (Pa); from Meyers et al. (1998); data estimated from a single fast start), L. chalumnae (Lc), average value of two fast starts, estimated from figure 14 in Fricke and Hissmann (1992), Polypterus palmas (Pp); L. osseus (Lo) and A. calva (Ac) from Hale et al. (2002), and P. senegalus (Ps) from Tytell and Lauder (2002). The slope and elevations of the relationships between turning rate (TR) and length in nonprimitive (log TR ¼ 1.19 log Length þ 2.3) and primitive fishes (log TR ¼ 0.82 log Length þ 2.8) are not significantly diVerent from each other (p > 0.1 and p > 0.05, respectively).

There are also some data on typical measures of ‘‘distance‐time’’ performance such as speed and acceleration. In terms of speed, primitive fishes appear to be somewhat slower than the teleosts (Figure 7.11). In particular, two primitive actinopterygians, P. palmas and L. osseus, are much slower than those of all other fish tested to date (Domenici, 2001; Hale et al., 2002) except for the dogfish, S. acanthias (Domenici et al., 2004). This finding requires further investigation. Given that turning rates are similar between primitive fishes and the modern elasmobranches and teleosts (Figure 7.10), the diVerences in speed may be due to body design and the kinematics of the tail motion. On the other hand, acceleration in primitive fish seems to be within the range of teleosts (Figure 7.12), albeit at the lower end and similar to acceleration in the dogfish (Domenici et al., 2004). Any diVerences in acceleration may also be obscured, however, by the higher errors and variability inherent to measurements of acceleration as compared to speed. In their comparison of the fast‐start performance of tiger musky (Esox sp.) with that of the longnose gar, Webb et al. (1992) found that the gar traveled only 65% of the distance covered by musky during their escape response. Although turning rates, speed, and acceleration were not provided, Webb

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10

Speed (m s −1)

Lc

1

Ps

Ac Lo Pp

0.1 0.01

0.1

1

Length (m) Fig. 7.11. The relationship between fast‐start speed and length in teleosts (squares), primitive fishes (filled triangles), and a modern elasmobranch (the spiny dogfish S. acanthias, open triangle). The regression line is based on nonprimitive fishes only (log Speed ¼ 0.49 log Length þ 0.59; r2 ¼ 0.66; p < 0.0001; N ¼ 36). Data on nonprimitive species are from a review on fish performance by Domenici (2001). Data on spiny dogfish are from Domenici et al. (2004) (average value for fast escape responses). Data on primitive fish are based on five species, that is L. chalumnae (Lc), average value of two data, that is from Fricke et al. (1987) and estimated from figure 14 (left panel) in Fricke and Hissmann (1992), P. palmas (Pp); L. osseus (Lo) and A. calva (Ac) from Hale et al. (2002) and P. senegalus (Ps) from Tytell and Lauder (2002).

et al. (1992) suggested that the reduced performance in gar was due to a lower proportion of muscle mass (40% of the total mass in gar versus 51% in musky) together with higher inertia of the skin (20% of the total mass in gar versus 6% in pike). Overall, the results indicate that certain primitive fishes exhibit poorer fast‐start performance than that of the teleosts in terms of speed. Further work is needed to elucidate the basis for these diVerences, and also to investigate other performance variables that comprise the response. For example turning radius, which is an important element of maneuverability (Domenici and Blake, 1997), has not been studied at all in primitive fishes. 6. CONCLUSIONS It is clear that the primitive fishes exhibit quite considerable diversity in their swimming modes and physiology, despite the relatively small number of species when compared to modern teleosts. Although primitive species such as the lamprey have been developed as models for studies of neuromotor

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1000

100

Ac Pp Ps

10

1 0.01

0.1 Length (m)

1

Fig. 7.12. The relationship between fast‐start acceleration and length in teleosts (squares), primitive fishes (filled triangles), and a modern elasmobranch [the spiny dogfish S. acanthias, open triangle, data from Domenici et al. (2004)]. Data for teleosts fishes are from Domenici (2001). Data on primitive fishes are from Westneat et al. (1998) [P. palmas (Pp) and A. calva (Ac)] and Tytell and Lauder (2002) [P. senegalus (Ps)].

coordination, it is a shame that so few studies have directly investigated the evolution of aspects of locomotor performance and physiology (Hale et al., 2002). Although various aspects of locomotion are common to almost all fishes, such as the myotome muscle blocks and the BCF locomotion they power; the M‐cells and the startle response, or the physiology of sustained exercise and recovery from exhaustion, the evidence does indicate, overall, that the primitive fishes perform exercise less well than teleosts with similar lifestyles. Great caution should, of course, be exercised when making comparisons between ‘‘the teleosts’’ and single primitive species, or discrete phylogenetic groups with particular life histories and morphologies (e.g., sturgeons). Nonetheless, the primitive fishes perform less well in terms of their swimming endurance (Figure 7.4), their critical speed swimming (Figure 7.5) (Table 7.3), and their fast starts (Figures 7.10 and 7.11). Thus, the evolution of the fishes to the modern teleosts has, in fact, been associated with the evolution of ‘‘better’’ exercise performance, as suggested by Bone et al. (1995). It is tempting to speculate, therefore, that improved locomotor ability might have contributed to some extent to the dominance of the teleosts today (see Chapter 10, this volume). For species such as the parasitic hagfishes and the drift‐hunting coelocanths, their lifestyle may not place a great deal of selective pressure on traits of locomotor performance, although the coelocanths are able to perform

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rapid escape responses (Fricke and Hissmann, 1992). For the lungfishes, too little is known of their locomotor performance, and the role of locomotion in their natural histories, to draw any significant conclusions, although there is some information to indicate that they are more active than might be commonly assumed (Grigg, 1965; Stuart and Berghuis, 2002; Mlewa et al., 2005). Wilkie et al. (2000) suggested that the ability of lampreys rapidly to recover from exhaustive exercise would be important for periods of intensive swimming and burrowing as larvae, for example, to escape predators. It may also, however, reflect adaptations for their subsequent migratory behavior as adults. Indeed, Quintella et al. (2004) reported a swimming pattern during upriver migration that comprised periods of intense exercise interspersed with periods where they recovered while attached by their sucker to a stone. It is intriguing that the poor performance of the sturgeons at burst swimming speeds (Peake et al., 1997; Adams et al., 1999) is coupled with a reduced physiological response to exhaustive exercise (KieVer et al., 2001; Baker et al., 2005). It is tempting to speculate that the sturgeon might avoid burst swimming in their natural environment by appressing themselves to the substrate (McKinley and Power, 1992; Adams et al., 2003; Geist et al., 2005), and can thereby overcome velocity barriers in rivers by proceeding slowly along the bottom (Chandler et al., 2003; Geist et al., 2005). Air breathing is important to the swimming performance of the lungfishes (Grigg, 1965) and the primitive actinopterygians (Magid, 1965; Burleson et al., 1998; Farmer and Jackson, 1998). It is interesting that the armored skin of some primitive actinopterygians may not, in fact, represent any hindrance to their locomotor performance in steady swimming (Long et al., 1996) although it may impair their performance of escape responses (Webb et al., 1992). There is clearly plenty of work that remains to be performed on swimming in the primitive fishes, and such studies would provide invaluable insights into how aquatic locomotion has evolved in the vertebrates. REFERENCES Adams, S. R., Hoover, J. J., and Killgore, K. J. (1999). Swimming endurance of juvenile pallid sturgeon, Scaphirhynchus albus. Copeia 1999, 802–807. Adams, S. R., Adams, G. L., and Parsons, G. R. (2003). Critical swimming speed and behavior of juvenile shovelnose sturgeon and pallid sturgeon. Trans. Am. Fish Soc. 132, 392–397. Ahlberg, P. E. (1992). Coelacanth fins and evolution. Nature 358, 459. Alexander, R. McN. (1965). The lift produced by the heterocercal tails of Selachii. J. Exp. Biol. 43, 131–138. Alexander, R. McN. (1969). The orientation of muscle fibres in the myomeres of fishes. J. Mar. Biol. Assoc. UK 49, 263–290. Allen, P. J., Hodge, B., Werner, I., and Cech, J. J. (2006). EVects of ontogeny, season, and temperature on the swimming performance of juvenile green sturgeon (Acipenser medirostris). Can. J. Fish Aquat. Sci. 63, 1360–1369.

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8 PERIPHERAL ENDOCRINE GLANDS. I. THE GASTROENTEROPANCREATIC ENDOCRINE SYSTEM AND THE THYROID GLAND JOHN H. YOUSON

1. Introduction 2. Endocrine Pancreas and Related Gastrointestinal Endocrine System 2.1. Background and Definitions 2.2. Agnatha 2.3. Gnathostomes 2.4. Phylogenetic Considerations 3. Thyroid Gland 3.1. Background 3.2. Agnatha 3.3. Gnathostomes 3.4. Phylogenetic Considerations 4. Summary and Conclusions

This chapter focuses on the phylogenetic development of the gastroenteropancreatic (GEP) system and the thyroid gland as supported by descriptions of their changing distribution and structure in agnathans (hagfishes and lampreys) and ancient bony fishes. The inclusion of the systems in a chapter together is because they have a mesoderm derivation and their products have some involvement in regulating intermediary metabolism. The GEP system shows clear phylogenetic patterns from protochordates to the ancient agnathans through more generalized teleosts. These patterns are viewed in the structure and distribution of the pancreatic islet organs and in the molecular structure of the generated peptides (insulin, somatostatin, and both the glucagon‐ and neuropeptide Y‐family of peptides). Several cases are provided where distinct diVerences in anatomical or molecular structures in the GEP system support existing views on inter‐ and/or intra‐group relationships among fish species. The follicular thyroid glands and their generated thyroid hormones in 381 Primitive Fishes: Volume 26 FISH PHYSIOLOGY

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adult agnathans and the ancient bony fishes are of similar basic structure, but in the former group their glands are poorly vascularized. The lamprey follicular thyroid develops from larval, nonfollicular thyroid tissue, the endostyle, during metamorphosis. Anatomical and molecular techniques show the homology between the endostyles of larval lampreys and those in protochordates. Phylogenetic patterns in thyroidology are not pronounced in ancient bony fishes, but there are some distinct, species‐specific features. A future focus in generating data on the structure and function of the thyroid gland in ancient bony fishes is important to help remove many from their threatened status. 1. INTRODUCTION This is the first of two chapters on peripheral endocrine systems of the extant fishes with an ancient lineage. The term peripheral is not new to the endocrine system and here it refers to those glands outside of the nervous system. This term would exclude discussion of the urophysis, an aggregation of nerve cell bodies in the posterior region of the spinal cord in teleosts and elasmobranchs (Bentley, 1998). This gland is equated with the neurohypophysis where axons terminate as neurohemal junctions with blood vessels. Although the adenohypophysis is derived from stomodeal endoderm, it is best considered in conjunction with the neurohypophysis as part of the pituitary and is a subject matter of a magnitude that requires a separate chapter. Peripheral endocrine glands do include the parathyroid and the ultimobranchial glands, both derived from pharyngeal endoderm and the source of calcitonin (CT), but these glands are not present or they have not been described in detail in any of the groups of fishes under consideration in this volume. There is an evidence of immunoreactive CT, even in the pharyngeal epithelium of protochordates (Robertson, 1986), and many of the species under study in the chapter have either the gene for CT (eel, lungfish, bichir, sturgeon, and gar) or like the lamprey and hagfish have a CT‐like substance in their blood (Suzuki et al., 1999; Suzuki, 2001). However, there is no definitive gland in any of these subjects. This will be the only reference to parathyroids, ultimobranchial bodies, and/or CT in either of the two chapters. The rationale for bringing up the subject of parathyroid and ultimobranchial glands here is that the two endocrine systems, the thyroid gland and the gastroenteropancreatic (GEP) system, to be discussed in the present chapter also are derived from embryonic endoderm. The thyroid tissue arises from the floor of the pharynx and the pancreas as diverticula of the embryonic gut. These two endocrine systems are also of fundamental importance in generating hormones involved in the regulation of intermediary metabolism. The subjects of the second chapter, the adrenal gland and the corpuscles of Stannius, are at least partially derived

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from embryonic mesoderm and have an intimate association with the kidney. A common regulatory function of products of these two glands is not obvious but at least one feature is their involvement in creating a homeostatic internal environment for the organism. The thyroid gland and the GEP system of the ancient fishes have long been a major focus of research in comparative endocrinology. The hagfishes and lampreys have received considerable attention, and there is a vast literature because of the passionate interest in these organisms by the late Aubrey Gorbman on the thyroid and by Sture Falkmer on the GEP. Both Gorbman and Falkmer were pioneers of comparative endocrinology and sought to find the bridge between vertebrates and invertebrates by studying their respective systems in protochordates and even lower invertebrate groups. As will be seen in the pages to follow, molecular biology has taken us in entirely new and exciting directions to show thyroid tissue homology, but still following in the footsteps of the pioneers. The products of principal islet tissue of the fish pancreas were of major interest to coworkers of Frederick Banting, the most high‐profile discoverer of insulin (MacLeod, 1922; McCormick, 1925). Today, medical science is exploring ways of using fish principal islets in xenotransplantation (Alexander et al., 2006). This chapter provides an update on the extensive literature of thyroid gland and the endocrine pancreas that was provided in an earlier volume of this Fish Physiology series (Epple, 1969; Gorbman, 1969). As I had extensive, personal encounters with both August Epple and Aubrey Gorbman, and was greatly influenced by their contributions and devotion to the comparative approach, I honor these pioneers with this chapter. 2. ENDOCRINE PANCREAS AND RELATED GASTROINTESTINAL ENDOCRINE SYSTEM 2.1. Background and Definitions The endocrine pancreas of fishes was a chapter in Volume 2 (Epple, 1969), and the molecular aspects of endocrine pancreatic peptides (Duguay and Mommsen, 1994) can be found in Volume 13 of this book series. There have been many excellent reviews on the subject of the phylogenetic and ontogenetic development of the vertebrate GEP endocrine system by August Epple and coworkers (Brinn, 1973; Epple and Lewis, 1973; Epple and Brinn, 1975, 1986, 1987; Epple, 1987) and by Sture Falkmer and his colleagues (Falkmer and Patent, 1972; Falkmer and Van Noorden, 1983; Falkmer, 1985a,b, 1995). Also the reader is referred to Gapp (1987) and Plisetskaya (1990) and to recent reviews by the author on the phylogenetic and

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ontogenetic development of the fish GEP and endocrine pancreas (Youson and Al‐Mahrouki, 1999; Youson et al., 2006). Unlike any of the references above, this section of the chapter will deal specifically with the literature on the endocrine pancreatic islet cells and related cell types of the stomach and intestine, and their principal products, in the agnathans and the bony fishes of ancient lineage which have been the focus in other chapters of this volume. There has been a lengthy historical ‘‘lead‐up’’ to the present views and terminology associated with the GEP system in fishes. Epple (1969) reviewed the early literature and described four types of pancreas in fishes (cyclostome type, primitive gnathostome type, tetrapod‐like type, and actinopterygian type) that reflect two phylogenetic trends in gnathostomes. These pancreatic types and phylogenetic trends are placed in the context of physiology in a later review of the vertebrate endocrine pancreas (Epple and Brinn, 1987). Falkmer (1995) believed that the endocrine pancreas is an important member of the larger neuroendocrine system and that both its ontogeny and phylogeny are important for understanding the pathogenesis of the tissue. To this end, he was interested in knowing the phylogenetic origin of cells of the islets from unicellular organisms, invertebrates, and protochordates through to the vertebrates. Once again, the readers are encouraged to read this history in the articles listed in this, and the previous, paragraph. The term GEP is used in the present article because it quite nicely sums up the general theme of this section that there is a relationship between certain cell types of the endocrine cells of the pancreas, the stomach, and intestine, that is they together are part of a system. This GEP system in euteleosts is a consequence of ontogenetic and phylogenetic patterns, both of which are reflected in the GEP system of fishes that are considered more ancient or more basal. It is the goal of this section to provide a description of the GEP system and the peptides generated from the system in the more ancient extant fishes to show how their study has provided insight into the evolution of the system in higher organisms. The space does not permit the writer to do an adequate analysis of metabolic aspects of GEP‐generated peptides; therefore, this important feature of this system will not be covered in any great detail. For this aspect of the GEP system, the reader is referred to Nelson and Sheridan (2006) who have provided an excellent summary of past and recent literature and suggestions for the future research directions. Given that there has been some confusion in terminology in studies of development and evolution of the GEP system (Youson and Al‐Mahrouki, 1999; Youson et al., 2006), it is critical to begin the section with some definitions. Ontogeny refers to the development of the GEP of a single species, whereas phylogeny is the evolutionary trend in the GEP in the fishes as a group. The islet organ is the endocrine pancreatic homologue of fishes, irrespective of its form; the term is suggested for adoption because this

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homologue in fishes is present in many forms and names for the homologue are ubiquitous because of this polymorphism. Principal islet is a single focal accumulation of endocrine pancreatic tissue usually, but not always, surrounded by a thin rim of exocrine tissue. A Brockmann body is a major concentration of endocrine pancreatic tissue that may contain principal islets; principal islet and Brockmann body may be used as synonyms if the latter contains a single principal islet. These definitions may seem rather pedestrian, but when one considers that fish islet organs are being considered in xenotransplantation as a source of ‘‘close‐to‐pure’’ endocrine pancreatic tissue (Alexander et al., 2006), the description of the islet organ in fish species should be well defined. Rather than providing some descriptive details of islet organs of the extant, ancient fishes, this section will use diagrammatic presentations to provide an overview of a proposed ontogenetic and phylogenetic development of the GEP in fishes. Although there is a variety of endocrine cells in the gastrointestinal system (Norris, 1997), as mentioned above this section will focus on endocrine cell types that are common throughout the GEP. These cell types are those that elaborate at least one of the following hormones: insulin, somatostatin (SST), and members of the glucagon‐and neuropeptide Y (NPY)‐families of peptides.

2.2. Agnatha There has been a relatively recent review that specifically deals with the enteropancreatic (EP) endocrine system and its generated peptides in hagfishes and lampreys (Youson, 2000). EP is used in this case because there is no stomach in extant agnathans. The hagfishes, and larval and most adult lampreys, have endocrine cells in the oesophagus and have both exocrine (zymogen‐secreting) and endocrine cells in the intestine (Figure 8.1). The exception is Mordacia mordax, a lamprey of the Southern Hemisphere, that has an enlarged left diverticulum of the intestine, a so‐called protopancreas, that houses the exocrine cells (Gillett et al., 1996). This divergent feature in M. mordax has some interesting implications in the view of evolution of the diVerent families of lampreys (Potter and Gill, 2003). The islet organ of hagfishes is positioned at the junction of the extrahepatic bile duct and the intestine and consists of islets, and sometimes follicles, of mostly insulin‐ containing (B) cells; SST‐containing (D) cells make up only
1% of the cell population (Figures 8.1 and 8.2). B and D cells are seen in the intestinal and bile duct epithelium with glucagon‐ and NPY‐family peptides also present in the intestine. The bile duct epithelium is the source of the islets and follicles of the hagfish islet organ (Figure 8.2).

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A Protochordate C

B

LG

LG

SH larval lamprey

NH larval lamprey

F L

E

D

G

SH adult lamprey

L G

Basal actinopterygian

H

Elasmobranch S

L G

J L

Derived euteleost

S

L L G G

Holocephalan S

I S

Hagfish

G

L

NH adult lamprey

Islets with exocrine cells in gut Subepithelial islet Islets intermingled with exocrine tissue Principal islets (with little or no exocrine tissue)

Exocrine pancreas Endocrine pancreas (islet organ) Dark cell : enteroendocrine Hatched cell : exocrine (zymogen)

Fig. 8.1. Diagrammatic representation of the distribution of the endocrine tissue (islet organ) in fishes relative to the exocrine pancreatic tissue, Oesophagus (O), stomach (S), intestine (unlabeled portion of the gut), gall bladder (G), and the bile duct (tube leading from the gall bladder). The epithelial cells of the digestive tube are enlarged relative to the cells of the other structure in order to show endocrine (dark) and exocrine (hatched) cells; the latter are seen in (B)–(F), inclusive. The protochordate has endocrine cells restricted to the gut with the islet tissue and exocrine tissue relationships indicated by the key and the accompanying arrows for the various fish groups. See the text for further detail for each fish group. [Modified from Youson and Al‐Mahrouki (1999).]

The distribution of components of the islet organ of larval lampreys shows some variability between Northern and Southern Hemisphere species (Figure 8.1). Whereas in Holarctic species, the groups of islets are found in submucosal sites at the junction of the oesophagus, anterior intestine, and point of entrance of the extrahepatic common bile duct (EHCB), the EHCB in Southern Hemisphere species enters an enlarged left intestinal diverticulum. Consequently, although there are similar intestinal and oesophageal islets, there are few, if any, associated with the EHCB. The islets of all species contain only B cells, and some islets remain contiguous with the intestinal epithelium from which they arise (Figure 8.2). Intraepithelial clusters of cells are immunoreactive to antisera against NPY‐family peptides and there are also isolated SST‐ and NPY‐immunoreactive cells in most species. It is still questionable whether the intestine of larval lampreys has cells containing glucagon (Youson and Al‐Mahrouki, 1999; Youson, 2000). There is some interspecific variability

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Glucagon/GLP

Insulin cell of open and closed type, respectively

PP cell Both Glucagon and PP immunoreactive cell

Somatostatin cell of open and closed type, respectively A

B

Gut lumen

Gut lumen

Larval lamprey

Bile duct lumen

Protochordate C

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Adult lamprey Gut lumen

Bile duct lumen

Cranial Principal islet Caudal

Hagfish

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Gut lumen

F

Gut lumen

Bile duct

Principal islet

Pancreas Pancreatic duct lumen Pancreatic duct

Single islet cells

Elasmobranch

Islet bud

Large islet with ductlike lumen

Teleost

Fig. 8.2. Diagrammatic representation of the endocrine cell types in the epithelium of the gut, bile duct, pancreatic ducts, and islet tissue of a protochordate (A) and various fishes (B–F). The arrows indicate the possible lines of direction of the phylogenetic development of the islet tissue from the protochordate confinement of endocrine cells in the gut to the single‐cell (insulin) islets of larval lampreys or adult hagfishes. The larval lamprey islets give rise to an adult cranial principal islet, whereas the caudal principal islet can arise from the larval bile duct, like the situation in the hagfish. The islets of teleosts arise from the gut like those of larval lampreys, while those of elasmobranchs originate in the pancreatic ducts. [Modified from Youson and Al‐Mahrouki (1999); a portion of the original is from Falkmer (1985a) and was reproduced with permission).]

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in immunostaining of intestinal islet EP cells, particularly in the species of the Southern Hemisphere. For example, M. mordax larvae have intestinal insulin cells but no staining for SST, and Geotria australis larvae have weak staining for insulin compared to the intense staining for this peptide in the more compacted islet organ in M. mordax (Youson and Potter, 1993). The reader is referred to Youson (2000) for a detailed discussion of the phylogenetic and ontogenetic implications of the islet/intestinal staining of EP endocrine cells in larval lampreys. Metamorphosis in lampreys is marked by a dramatic change in the islet organ between larva and adults (Youson, 1985). The islet organs of adults of Northern and Southern Hemisphere species vary because of diVerences in ontogenetic patterns during their metamorphoses (Figure 8.1). In adult Holarctic species, there is a cranial and a caudal principal islet having diVerent ontogenies (Youson, 1981, 2000; Youson and Elliott, 1989; Youson and Cheung, 1990; Youson and Al‐Mahrouki, 1999). The cranial principal islet, which forms a cap of islet tissue over a small, right diverticulum of the anterior intestine in the cardiac region (Figure 8.1), arises from the larval islet organ and intestinal epithelium at the time of transformation of the larval alimentary canal (Figure 8.2). The caudal principal islet, which is positioned alongside the hepatic portal vein within connective tissue connecting the liver and anterior intestine (Figure 8.1), arises from a transformation of the epithelium of the EHCB (Figure 8.2). There are no components of a biliary tree in adult lampreys (Youson, 1985, 1993). In contrast, there is only a large, cranial principal islet in adult lampreys of the Southern Hemisphere; this islet arises, like in Holarctic species, from the larval islet organ (Figure 8.1). There is no caudal principal islet in Southern Hemisphere species because the larval EHCB is not involved in islet organ ontogeny during metamorphosis; this absence is likely due to the position of the EHCB in larvae, relative to its position in larvae of Holarctic species (Hilliard et al., 1985). Irrespective of the origin or position of the adult lamprey principal islets, they all contain B and D cells in almost equal proportions; a few cells immunoreactive for NPY‐family peptides (F cells) are only present in Petromyzon marinus. No A cells, showing immunoreactivity for glucagon‐family peptides, are present in the principal islets of any species. Immunostaining for SST and insulin shows some species variation in the islet organs that likely reflect the nature of the peptides they contain. B cells stain weakly in G. australis with both antimammalian and antilamprey insulin antisera but intensely with these antisera in M. mordax and P. marinus. D cells of P. marinus immunostain with sera against synthetic human SST‐14 or against lamprey SST‐34 but only anti‐SST‐14 stains these cells in G. australis and M. mordacia. In contrast, SST‐14 and SST‐34 are colocalized in intestinal EP cells, but sometimes only SST‐34 in cells, of both Holarctic and Southern

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Hemisphere lampreys. Antisera of salmon glucagon and salmon glucagon‐ like‐peptide (GLP) immunostain the same cells (i.e., co‐localize) in the intestines of all species of adult lamprey, either in the Northern or the Southern Hemisphere. The EP cells of the intestine of adult lampreys also include NPY‐ family peptides and M. mordax has some insulin‐positive cells (Youson and Potter, 1993). There has been a long history of interest in the bioactivity of EP peptides from hagfishes and lampreys (Epple and Brinn, 1987; Youson, 2000), but it is only recently that we have got to know something of their primary structure (for reviews see Conlon, 2000; Sheridan et al., 2000; Youson, 2000; Conlon and Larhammar, 2005; Nelson and Sheridan, 2005). This interest in primary structure has been driven mainly by the desire to know about the evolution of insulin, SST, and both the NPY‐ and glucagon‐families of peptides, rather than to explain their biological activity. However, the comparisons in the primary structure of insulin and SST between P. marinus, Lampetra fluviatilis, and G. australis have helped to explain the interspecific diVerences in immunostaining for these peptides described above (Conlon, 2000; Youson, 2000). Also, the sequence data, such as the common 5 amino‐acid extension of the N‐terminal of the insulin B‐chain (Conlon et al., 2001), the multiple forms of glucagons in G. australis (Wang et al., 1999a), and a distinction between peptide‐tyrosine‐tyrosine (PYY; Wang et al., 1999b) in species of the Northern and Southern Hemisphere, have been useful adjuncts to support claims of both the monophyly of order Petromyzontiformes and the early divergence of the two Southern Hemisphere families (Geotriidae and Mordaciidae) from the single family, Petromyzontidae, in the Northern Hemisphere (Potter and Gill, 2003). The two forms of PYY in Northern Hemisphere lampreys, PYY and PMY, are a result of accelerated evolution (Conlon and Larhammar, 2005), and their near similarity in Petromyzon and Ichthyomyzon genera (Montpetit et al., 2005) is support for a view that the two genera are close to the ancestral lamprey stock. There is some evidence from expression studies of prepro‐NPY, ‐PYY, and ‐PMY in parasitic (P. marinus) and nonparasitic (Ichthyomyzon gagei) lamprey species that PMY might have a diVering role in the timing of reproductive maturity in these two adult life history types (Figure 8.3). Two proglucagon cDNAs cloned from the intestine of P. marinus, preproglucagon‐ I and ‐II (PPG‐I and ‐II), have diVerent coding potentials and it is possible that lampreys might use diVerential gene expression as a means of regulating production of PPG‐derived peptides (Irwin et al., 1999). PPG‐I encodes glucagon and GLP‐ 1 while PPG‐II encodes GLP‐II and likely glucagons; these data indicate that all three glucagon‐like sequences originated prior to divergence of jawless and jawed vertebrates, likely 1 billion years ago for glucagon and 700 mya for the GLPs (Irwin et al., 1999).

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Ichthyomyzon gagei

Petromyzon marinus 0.5

Gut NPY

2.0

Gut NPY

0.4 1.5

Relative neuropeptide Y- family peptide expression (relative to b-actin)

0.3 1.0

0.2 0.1

0.5

0.0

0.0 Gut PYY

2.0

2.0

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1.5

1.0

1.0

0.5

0.5

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0.0 Gut PMY

Gut PMY

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1.5

1.5

1.0

1.0

0.5

0.5

0.0

0.0 Larva

T

Gut PYY

J

SP

Larva

SP

Fig. 8.3. Gut expression of preproneuropeptide tyrosine (NPY), ‐peptide tyrosine tyrosine (PYY), and ‐peptide methionine tyrosine (PMY) during the life cycle of parasitic lamprey, P. marinus, and the nonparasitic lamprey, I. gagei. The expression levels are normalized to b‐actin and the values are mean  SEM; electrophoretic images appear above the bar. Abbreviations: T, late metamorphosis; J, juveniles; and SP, prespawning adults. [From Montpetit et al. (2005).]

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2.3. Gnathostomes 2.3.1. Sarcopterygii The review by Youson and Al‐Mahrouki (1999) seems to be the most recent account of the GEP endocrine system of the lobe‐finned fishes. There has been no extensive histological study of the GEP of the coelacanth, Latimeria chalumnae, but Epple (1969) described A, B, and D islet cells along pancreatic ducts within a rather compact, extraintestinal structure. The insinuation is that the coelacanth islet organ more closely resembles that in selachian cartilaginous fishes (Epple, 1969). On the other hand, Millot and Anthony (1972) have described a pancreas in L. chalumnae as densely scattered in the connective‐adipose tissue of the pyloro‐duodenal regions; they propose that the coelacanth pancreas is intermediate in morphology to the ‘‘majority of fishes’’ and the compact organs of Selaciae and Dipnoi. The islet organ of Protopterus spp., however, seems to be more similar to that seen in more derived teleosts rather than to the amphibian islet organ (Tagliafierro et al., 1996). That is, the endocrine pancreatic tissue is mainly in a Brockmann body of concentrated islet tissue with a few accessory islets (Scheuermann et al., 1991). In Protopterus annectens, large islets are described (Tagliafierro et al., 1996) and they would conform to our definition of principal islets and the islet organ would be more like that in derived teleosts (Figure 8.1). Epple and Brinn (1975) thought that the lungfish islet organ had a unique status among bony fishes. In contrast to Protopterus spp., the Australian lungfish, Neoceratodus forsteri, has an islet organ with the size and distribution of islets that is more tetrapod‐like (Rafn and Wingstrand, 1981), that is, closer to that seen in more basal actinopterygians (Figure 8.1). Immunohistochemistry of islets from Protopterus spp. shows an euteleost‐ like distribution of A, B, D, and F cells, that is, central B cells with peripheral A, D, and F cells, with some variation between large (primary) and small islets (Hansen et al., 1987; Scheuermann et al., 1991; Tagliafierro et al., 1996). Observations of GEP cells of the alimentary canal in lungfishes seem to be restricted to the intestine of P. annectens (Tagliafierro et al., 1996). The intestine of P. annectens contains cells that are immunoreactive to antisera against mammalian SSTs, glucagon, pancreatic polypeptide (PP), and insulin. The presence of insulin‐ and SST‐immunoreactivity is not novel but is not the norm for the fish intestine (Tagliafierro et al., 1996). The primary structures of insulin from P. annectens (Conlon et al., 1997) and N. forsteri (Conlon et al., 1999) show a common N‐terminal extension of alanine in the B‐chain. Conlon (2000) has implied a close relationship between lungfish and amphibian insulin. Trabucchi et al. (1999) have deduced from cDNAs that P. annectens has two preprosomatostatins (PPSS) with PPSS‐I coding for invariant SST‐14 and SST‐27 and PPPSS‐III for variant

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[Pro2]‐SST‐14 and SST‐24. Fragments of proglucagon are reported in databases for both N. forsteri (Q6RYB5) and Protopterus dolloi (Q6RYB6). The close similarity between NPY in the brain of P. annectens and amphibians has been emphasized and the lungfish NPY seems to have some similarity to PYY normally seen in the GEP of other vertebrates (Vallarino et al., 1995). There has been some recent molecular data on NPY receptors Y5 and Y6 in L. chalumnae that suggest a long functionally important history of the Y5 receptor in sarcopterygian and tetrapod, but not the teleost, lines (Larsson et al., 2007). 2.3.2. Acipenseriformes and Polypteriformes It was reported by Epple and Brinn (1975) and even more recently by Youson and Al‐Mahrouki (1999) that a detailed description of the islet organ and cells of the GEP system in general is not available in Polypterus spp. Epple and Brinn (1975) have described the distribution of the islet organ in the bichir as disseminated islets among the exocrine acini extending along the gastrointestinal tract and into the liver along with the hepatic portal vein. The greatest concentration of islets is near the EHCB. Since the void of information on cell types has not been corrected, we have to rely on data from another polypterid, the reedfish (Calamichthys calabaricus). In this latter species, the islet organ, which consists of widely dispersed islets and an islet of conspicuous dimension (principal islet?) visible by the naked eye near or in the liver, contains B cells and three types of acidophilic cells (Mazzi, 1976). We do know, however, the primary structures of Polypterus insulin and glucagons (Conlon et al., 1998). The insulin A‐chain has some unusual amino acid substitutions not seen in any other insulins, and other substitutions in the B‐chain are only present in divergent mammalian insulin or in the hagfish, Myxine glutinosa. When compared to other fishes, Polypterus insulin most closely resembles insulins of paddlefish (Acipenseriformes) and the gar (Semionotiformes). The Polypterus glucagon sequence most closely resembles this molecule in bowfin (Amiiformes) and gar with only a 4 amino acid diVerence, but unlike insulin, it is quite diVerent (13 and 14 amino acids) from the two paddlefish glucagons. What these data tend to support is what we already know from morphological and fossil‐ record evidence, that is, that the polypterids are likely the most basal living ray‐finned fishes and they have connections (sister group?) with both the Acipenseriformes and the neopterygians. As with the Polypteriformes, our information on the structure and distribution of the islet organ in the paddlefish and sturgeon (Acipenseriformes) is scanty and needs attention (Epple and Brinn, 1987). In the sturgeon, the islet organs have no principal islets or Brockmann bodies but instead a

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mammalian‐like distribution of various‐sized islets among the acinar tissue (Epple and Brinn, 1975) alongside the intestine and at its junction with the stomach and EHCB. The brief description of the islet organ of the paddlefish (Weisel, 1972; Epple and Brinn, 1975) also gives us the impression of a widely dispersed system of small islets which one would consider a more basal distribution than that seen in more derived ray‐finned fishes which have principal islets or Brockmann bodies (Figure 8.1). Two molecular forms of insulin, diVering by one amino acid at position 5 of the A‐chain, are present in the North American paddlefish, Polyodon spathula (Nguyen et al., 1994). These insulins are relatively conserved in structure, have the closest resemblance to insulin from the gar, Lepisosteus spp., and they are more similar to mammalian insulin than are the insulins from teleosts. This similarity to gar insulin is interpreted as supporting the view that the chondrosteans, the ‘‘lower actinopterygians,’’ are more closely related to the Lepisosteiformes than they are to the Amiiformes (Nguyen et al., 1994). P. spathula also has two glucagon molecules, diVering by one amino acid, and a GLP but they tend to support the view that proglucagon‐derived peptides in fish are less well conserved than the insulins (Plisetskaya and Mommsen, 1996). Despite the fact that the two glucagons show some key diVerences in amino acid structure and 10–11 amino acid substitutions from mammalian glucagons, they are equally as eVective as bovine glucagon in stimulating glycogenolysis in cultured hepatocytes (Nguyen et al., 1994). The one amino acid diVerence in the two glucagons translates into a sixfold diVerence in potency of the hormones. In contrast to the duplicate forms of peptides in the paddlefish, in both the Russian and pallid sturgeons there is only one form of insulin (Rusakov et al., 1998; Kim et al., 2000). In addition, there is one form of glucagon, two forms of SST‐14 (an invariant and a variant form), and three forms of PYY in the pallid sturgeon, Scaphirhynchus albus (Kim et al., 2000) GEP system; the GEP does not contain the [Pro2]‐SST‐14 present in the pituitary of the Russian sturgeon (Nishii et al., 1995). The duplication, or the absence of duplication, of GEP peptide genes in these two ancient groups cannot be passed over without some comment on their evolutionary significance. There have been some proposals of how specific gene duplication events influenced the evolution of GEP peptides in fishes (Irwin, 2004; Zhou and Irwin, 2004; Conlon and Larhammar, 2005; Irwin and Wong, 2005; Nelson and Sheridan, 2005). Conlon (2000) has proposed that the appearance of the two insulins, and perhaps the same can be said for the two glucagons, in P. spathula, occurred as a result of a relatively recent gene duplication event. This gene duplication in fishes has been a subject of wide interest in recent literature (Taylor et al., 2003; Conlon and Larhammar, 2005) and, in the case of two insulin genes, has likely resulted in two diVerent functions for insulins within a species

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(Irwin, 2004), rather than a subfunctionalization of the ancestral function (Force et al., 1999). This same view of two fish proglucagon genes with diVerent functions arising through duplication of single‐function, ancestral gene has also been addressed (Irwin, 2001; Zhou and Irwin, 2004). However, diVerences in function of proglucagon‐derived peptides in fish and mammals can be explained through the evolution of glucagon receptor genes (Irwin and Wong, 2005). The receptors for glucagon and the GLPs had their origin before the divergence of fish and mammals, but fish lost the gene encoding for the GLP‐1 class of receptors, hence, the quite common eVects of glucagon and GLP‐1 in fish (Plisetskaya and Mommsen, 1996). Evidence from fish genomes and phylogenetic analysis suggests that duplication of the retained glucagon receptor gene in fish completes the explanation for the shared action of glucagon and GLP‐1 in this vertebrate group. It is diYcult to explain why the GEP peptides of paddlefish and sturgeon followed what seemed to be separate evolutionary paths with respect to duplication. Kim et al. (2001) have described two forms of vasoactive intestinal polypeptide from the pallid sturgeon, S. albus, and have provided data from both sides of the argument of either a tetraploidization event or a diploid status to explain the multiple forms of the various GEP peptides. The number of chromosomes (120–500, depending on the sturgeon species and type of ploidy) suggests that genome duplication events took place during sturgeon evolution (Ludwig et al., 2001), but it is likely that multiple forms of GEP peptides are a consequence of local gene duplication within the Scaphirhynchus lineage rather than from whole‐genome or whole‐chromosome events (Kim et al., 2001). Although the same type of analysis has not been performed on the paddlefish, it is likely that local gene duplication in the Polyodon spp. lineage, independent of whole‐genome or whole‐chromosome duplication, took place to produce the two insulins and glucagons that have similar functions. Present evidence from fish genomes suggests that fish‐specific genome duplications likely result in two genes of each insulin and proglucagon that, if both are maintained, has diVerent coding potential and yield products having potentially diVerent functions (Irwin, 2004; Zhou and Irwin, 2004). The Acipenseriformes are one group that has excellent potential for providing some meaningful evolutionary analysis of GEP gene families (Conlon and Larhammar, 2005). 2.3.3. Amiiformes and Semionotiformes In the initial description of the fish endocrine pancreas in this series on Fish Physiology (Epple, 1969), only very cursory information was available on the nature of the islet organ in ‘‘Holostei.’’ Even nearly 20 years later, the descriptions of the gar and bowfin islet organs were based primarily on light microscopy and some gross anatomical observations (Epple and Brinn, 1986).

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The diVuse nature of the islet organ in the bowfin that was described by McCormick (1925), working in the same department at the University of Toronto as Frederick Banting, was not definitively confirmed until 2001 (Youson et al., 2001). There are no principal islets or large concentrations of islets (Brockmann bodies), but instead the islet organ is diVusely distributed, along with the pancreatic acini, within the mesentery that connects the liver, anterior intestine, stomach, gall bladder, and the EHCB. For this reason, Epple (1969) described the bowfin pancreas as resembling that of higher vertebrates. Immunohistochemistry at the light and electron microscope levels show that the bowfin islets are composed of central B cells and one type of a more peripheral D cell (Figure 8.4). The same antisera against synthetic SST‐14 and salmon SST‐25 that reveals two cell types in diVerent locations in islets of salmon, sea bass and sea bream (Nozaki et al., 1988; Lozano et al., 1991; Agulleiro et al., 1993), colocalize in the same granules of bowfin D cells (Youson et al., 2001). Although some isolated A cells are present, for the most part antisera to glucagon‐ and NPY‐family peptides colocalize in A/F cells located at the outer rim of the islets. Some peripheral cells have rod‐shaped inclusions; on the basis of immunocytochemistry of granules in the cells, it is believed that the inclusions are in A (glucagon‐ containing) cells or in A/F (colocalizing with NPY) cells. It is noteworthy that the inclusions are close to being identical in structure and position (within the cisternae of endoplasmic reticulum) to those described in B or D cells of the hagfish islet organ. Although bowfin and hagfish followed diVerent evolutionary pathways, they are both considered as living fossils. In hagfish, it is suggested that the inclusions represent an early state of hormone processing (Raska et al., 1982). The absence of immunocytochemical labeling in the inclusions of bowfin, but instead in the granules of the same cell, seems to support the view that the inclusions had content that had not proceeded suYciently through posttranslational processing to a step where antigenic determinants were similar to, or as exposed as (due to molecule conformation), those of the proteins in the granule matrices. Extra‐islet immunostaining for A, B, D, and F cells in pancreatic ducts and among the exocrine acini is quite extensive but particularly for cells colocalizing glucagon‐ and NPY‐ family peptides. Immunohistochemistry for GEP cells of the alimentary canal shows only small numbers of cells containing either SST or glucagon‐ and NPY‐family peptides in the anterior and posterior intestine and in the stomach (Figure 8.4). The primary structures of bowfin insulin, SST‐14 and SST‐26, NPY, and both glucagon and GLP‐1 are available (Conlon et al., 1991b,c, 1993; Wang et al., 1993) and were discussed in an earlier volume of this series (Duguay and Mommsen, 1994) and in other subsequent chapters (Conlon, 1995, 2000; Larhammar, 1996; Lin et al., 2000; Sheridan et al., 2000; Irwin, 2001). These sequences have all been very important for

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Fig. 8.4. Immunohistochemistry of cells of the GEP system of the bowfin (A. calva). Red is positive staining for the various antisera with hematoxylin as the counterstain; EP, indicates the exocrine pancreatic tissue. (A) Centrally located B cells (b) stain with antimammalian insulin and

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discussions of the molecular evolution of the gene families of which they are apart and will be discussed together with those from the gar. As with the bowfin, the earliest descriptions of the gar islet organ indicate a diVusely distributed islet tissue in gar (Epple and Brinn, 1975). However, as with this earlier report, a more recent study used routine light and electron microscopy and immunohistochemistry to show the highest concentrations of islets near the EHCB as it enters the intestinal‐stomach junction and beneath a pyloric caeca (GroV and Youson, 1997). This focal accumulation of islets is referred to as a ‘‘Brockmann body‐like accumulation,’’ coined by Epple and Brinn (1975), to distinguish it from the true Brockmann body seen in euteleosts. Similar to the bowfin, B cells are abundant in the center of each gar islet but in contrast to the bowfin, D cells are scattered among the B cells and toward the periphery of the islet (Figure 8.5). A and F cells are present at the outer edge of the islets and colocalization of glucagon‐ and NPY‐family peptides likely occurs. SST is present in cells of the exocrine pancreatic ducts and a few interstitial or acinar cells stain with antisera for members of the other GEP peptide families. Electron microscopy and immunocytochemistry indicate that there are only three cell types in the gar islet, namely B, D, and A/F cells (GroV and Youson, 1998). Other notable features are the wide range of sizes and morphologies of B‐cell granules, relative to those in other ray‐finned fishes and the existence of only one morphological and immunohistochemically distinct D cell. This feature is shared with the bowfin, as most ‘‘more advanced’’ ray‐finned fishes have two types of D cells, D1 and D2, which immunoreact to diVerent antisera and usually have a specific location (Nozaki et al., 1988; Lozano et al., 1991). Another pronounced feature of the gar islet organ is the large number of nerve terminals among the islet cells. Although the dense‐cored vesicles within the nerve terminals do not react with an antiserum to NPY, both anti‐PYY and anti‐VIP immunoreact with nerves and their terminals in teleosts (Jo¨nsson, 1991; Agulleiro et al., 1993). The question arises whether these are the adrenergic postganglionic terminals that have been suggested to be unique to teleosts (Epple and Brinn, 1975)? At any rate, the presence of these terminals in such abundance among the islet

are surrounded by a rim (arrow) of unstained cells. (B) D cells (d) are immunoreactive with anti‐ SST‐25 serum and make up the majority, but not all (arrow), of the peripheral rim of unstained cells seen in (A). (C) F cells (f) immunostain with anti‐anglerfish peptide tyrosine and are present at the islet periphery and among the exocrine acini (arrow). (D) As in (B), anti‐SST‐14 immunostains mostly peripheral islet cells but some (arrowhead) central cells. (E) A few peripheral cells in the islet (a) immunostain with anti‐glucagon. (F) Cell of the stomach gastric gland immunostains with anti‐glucagon. (G) A cell of the anterior intestine immunostains with anti‐ glucagon‐like peptide. Bars: A–E ¼ 28 mm; F and G ¼ 11 mm. [From Youson et al. (2001).]

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Fig. 8.5. Diagrammatic representation of a transverse section of a pancreatic islet from the gar, Lepisosteeus osseus, showing the distribution of insulin‐immunoreactive cells (small dots), cells immunoreactive for glucagon‐and PYY‐family peptides (large dots), and SST‐immunoreactive cells (plus signs). [From GroV and Youson (1997).]

cells suggests some neuromodulation of the islets other than a typical neurotransmitter control. GEP cells are more prominent in the stomach and intestine in the gar, compared to these regions of the alimentary canal of bowfin. In gar alimentary canal, there is some regional diVerentiation of GEP cells as indicated by how they immunoreact to the following antisera: mammalian and lamprey insulins; salmon glucagon and GLP; synthetic SST‐14, salmon SST‐25, and lamprey SST‐34; NPY, PP, and anglerfish PY (GroV and Youson, 1997). All cells immunoreactive to SST antisera are present in the stomach, whereas the NPY‐related peptides and most of the glucagon‐related peptides are concentrated in cells in the intestine. Although both the stomach and the intestine have insulin‐containing cells, the two regions immunoreact to diVerent antisera. It is not common to have insulin‐containing cells in adult fish but even less common to have regional immunoreactivity for this peptide with diVerent antisera. GroV and Youson (1997) have provided a detailed discussion of this phenomenon. In general, the most plausible interpretation for the diVerent insulin immunoreactivity in the two regions of the gar alimentary canal is that it is reflecting variable processing of insulin, as in

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the case of the ratfish (Conlon et al., 1989). Also, one of the two antisera could be detecting insulin‐like growth factor, also seen in ratfish islet organ (Reinecke et al., 1994). The immunoreactivity for SSTs and NPY‐related peptides in the gar is similar to that in the alimentary canal of other fishes, but the absence of immunostaining for glucagon is surprising. This result may be interpreted that it is only the GLP portion of the PPG gene that is expressed in the alimentary canal of the gar. The primary structure of most of the gar GEP peptides was provided long before we know much about the distribution and nature of the peptide immunoreactivity (Pollock et al., 1987, 1988). As with the bowfin, these primary structures have been important in phylogenetic analysis and molecular evolution of the peptides in chordates and are discussed below. The conclusions of studies of the GEP cells of both the islet organs and alimentary canals of the gar and bowfin are that they have a distribution and an immunoreactivity that reflect the antiquity of the organisms, for they share only some features of these tissues in the euteleosts (GroV and Youson, 1997, 1998; Youson and Al‐Mahrouki, 1999; Youson et al., 2001, 2006). Some of the variation in immunohistochemistry for bowfin and gar peptides has been interpreted as being a consequence of diVerences in primary structure of the peptides (GroV and Youson, 1997, 1998; Youson et al., 2001). As mentioned previously, the gar insulin shows much more similarity to that of the paddlefish insulin‐2 rather than to the bowfin insulin, despite the fact that the gar and the bowfin were formerly grouped as holosteans. Furthermore, bowfin insulin is about 14‐fold less potent than porcine insulin in a receptor‐binding assay (Conlon et al., 1991b). This reduced potency is explained by key amino acid substitutions that are not found in the gar; the gar insulin is more similar to teleost insulin (Conlon, 2000). Does this suggest that the Semionotiformes and Amiiformes are not descended from a common ancestor and followed a parallel evolutionary pathway? In contrast, phylogenetic analysis of the NPY‐family peptide, PYY, places the gar and bowfin in a similar clade (Larhammar, 1996) and their sequence identity is more similar to some cartilaginous fishes than it is to teleost PYY (Conlon et al., 1991b). To the knowledge of the author, a gar SST cDNA has not been cloned or a gar SST isolated. This knowledge void for gar SST or PPSS is unfortunate for the bowfin has preprosomatostatin‐I (PPSS‐1) posttranslational processing that is diVerent from that in teleosts or higher vertebrates. SSTs isolated from the bowfin islet organ indicate that an SST‐26 is the dominant mature peptide over invariant SST‐14 (Wang et al., 1993). It is interesting that the dominant forms of SST are also ‘‘large somatostatins,’’ SST‐34 in both hagfishes and lampreys, the agnathans of ancient heritage (Andrews et al., 1988; Conlon et al., 1988a). The immunostaining of D cells of bowfin islet tissue with anti‐SST‐14 and anti‐SST‐25 (Figure 8.4)

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is consistent with their PPSS‐I processing of SST‐14 and SST‐26. To date, there is no evidence of a PPSS‐II in bowfin. Bowfin and gar glucagons diVer by only one amino acid but bowfin GLP contains a deletion and seven residue substitutions compared to gar GLP (Pollock et al., 1988; Conlon et al., 1993). The similarity of bowfin glucagon to human glucagon makes them equally potent in activating glycogenolysis in cultured hepatocytes but, given the large diVerences in human and bowfin GLP‐1 (3 deletions and 15 substitutions), it is not surprising that the bowfin GLP is 23‐fold less potent than human in this glycogenolysis assay (Conlon et al., 1993). It is not known how the gar glucagon and GLP would operate in the glycogenolysis assay, but with only one residue substitution in glucagon compared to bowfin, it is likely that it would have some close‐to‐equivalent potency. 2.3.4. BasalTeleosts It was expected that the gar and bowfin islet organ and related cells in the alimentary canal, and the peptides that the cells generate, might reveal some transitional phase in the phylogenetic development of the GEP system that is present in the euteleosts. This expectation was because the ancestors of at least the Amiiformes or the Semionotiformes led to the teleost line (Gardiner et al., 1996). For the most part, these organisms did not fail to provide features that were considered both non‐teleost and teleost‐like and for this reason they have transitional GEP systems. However, there seems to be a large gap in general topography of the islet organ, in distribution and immunoreactivity of cell types, and in the primary structure of the peptides between the Amiiformes and Semionotiformes and at least the more derived teleosts. For this reason, some consideration is given to the GEP system of basal teleosts. Among what might be called basal teleosts are three subdivisions of division Teleostei: Osteoglossomorpha, Elopomorpha, and Clupeomorpha. The eel is in the order Anguilliformes, and it is the only member of the Elopomorpha in which we have some, but not definitive information, on the islet organ. There is still some controversy whether adult eels have a Brockmann body (Youson and Al‐Mahrouki, 1999), and the general view is that the islet tissue is diVuse (Epple and Brinn, 1975). However, the pancreas itself is compact enough to be removed, in experiments of isletectomy, without disruption to the biliary system (Epple and Brinn, 1986); previously, Epple (1969) referred to this procedure as pancreatectomy in reference to the classical experiments on eels by one of the unrecognized founders of insulin (MacLeod, 1922). According to L’Hermite et al. (1985), the pancreas in leptocephali (larva prior to metamorphosis) extends on the left side from the liver to the posterior part of the stomach and is adherent to this latter organ. The controversy lies with the histological description by L’Hermite

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et al. (1985) that two voluminous islets are present in the middle of the pancreas along with scattered islets in the head and tail of the pancreas of leptocephali. In glass eel and adults, a single islet (called a Brockmann body, but in the definition of this chapter, a principal islet) is present in the mid‐ pancreas with small islets in the head and tail region of a pancreas that is now more dorsally located on top of the stomach and the duodenal bulbous. The principal islet in adult eel may come from the fusion of the two larval islets during metamorphosis. August Epple (personal communication), who performed many experiments with eel pancreas (for review see Epple, 1987; Epple and Brinn, 1986), indicates that ‘‘large islets’’ become more conspicuous in adult eel during lipopexia. Thus, although there may be large islets (principal islets?) in eel pancreas, it is of general view that Brockmann bodies are not present in the eel islet organ. The islet tissue of adult and glass eels contains B, A, and D cells but immunohistochemistry for NPY‐family peptides has not been performed; glucagon immunoreactivity is also seen in cells of the pancreatic ducts (L’Hermite et al., 1985). Surprisingly, the islet tissue of leptocephali contains D cells and weak staining for glucagon but no B cells. The intestine of adults and glass‐eels contains abundant glucagon‐ but few SST‐containing cells, whereas the stomach contains numerous SST‐ but no glucagon‐containing cells. In the leptocephali, the fore‐ and midgut immunostain weakly for only glucagons. Insulin is absent from the epithelium of the alimentary canal at all stages of development. The primary structures of eel insulin, glucagon, GLP, SSTs, and PYY are available (Conlon et al., 1988b, 1991a,b) and have been more than adequately compared with these pancreatic peptides from other fishes in an earlier volume of this series (Duguay and Mommsen, 1994). One noteworthy feature is the diVerence in primary structure of insulin‐primary structure, but conservation of glucagon‐ primary structure, between the American (Anguilla rostrata) and European (Anguilla anguilla) eels (Conlon et al., 1991a). The order Osteoglossiformes (bonytongues) of subdivision Osteoglossomorpha are considered among the most ancient of extant teleosts and old enough that their present distribution was influenced by the movement of the earth’s tectonic plates (Li and Wilson, 1996). Thus, any discussion of the phylogeny of the GEP system in fishes of ancient lineage should include data from this interesting group of fishes. Some earlier investigators (McCormick, 1925; Epple and Brinn, 1975) recognized the value of information on the osteoglossomorph islet organ, but provided only cursory observations. Several studies have given us a clear picture of the structure and distribution of the islet organ and the GEP cells of the alimentary canal of five species of the order Osteoglossiformes (Al‐Mahrouki and Youson, 1998, 1999; Al‐Mahrouki, 2001) and a sixth species classified as a member of a sister group (Al‐Mahrouki, 2001; Youson et al., 2006). The distribution of the islet organ varies with the species but in general, it is distributed along

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with the exocrine acini within the mesentery that connects the outer surface of the gatrointestinal tract, the pyloric caeca, the gall bladder, and the liver. In some species, namely the two arawana species, there are some large islets near where the pyloric caeca connects with the anterior intestine, but for most cases, the islets are of various sizes and spread throughout the area penetrated by the exocrine acini. The conclusion is that the osteoglossomorphs have an ‘‘actinopterygian type’’ (Epple and Brinn, 1975) of islet organ but not one containing aggregations of islets (i.e., a Brockman body) seen in some generalized and more derived teleosts. Only the arawana species contain islets the size of which could be considered a principal islet. Therefore, the osteoglossomorphs have an islet organ that is more similar to the non‐teleost or basal actinopterygian fishes (e.g., the bowfin and gar) than to other teleosts (Figure 8.1). There are A, B, D, and F cells in the islet tissue of five species so far examined but there is some species variability in distribution and immunoreactivity (Al‐Mahrouki and Youson, 1998, 1999) that may reflect some diVerences in evolutionary history of the organisms, in ontogenies of their GEP cells or variations in posttranslational processing of prohormones that influence the immunostaining. The presence of four distinct cell types is a derived feature of the teleost islet organ. In general, however, the distribution of B and D cells is more similar to the distribution of cells in bowfin and gar than to the teleost pattern. Immunocytochemistry in the electron microscope shows two types of D cells, like the situation in more advanced teleosts, even if one of these, a DX cell, shows novel immunoreactivity. The conclusion from histological and immunohistochemical observations of the osteoglossomorph islet organ is that it is transitional in sharing features with other teleosts and with the Amiiformes and the Semionotiformes, the more basal actinopterygians. The primary structures of insulin and SST have been deduced from cloned cDNAs of preproinsulin and preprosomatostatins (PPSS‐I and ‐II) of several species of Osteoglossiformes (Al‐Mahrouki et al., 2001; Youson et al., 2006). For the most part, relative to the known conformational and functional domains seen in insulins of other vertebrate species, the insulin molecules are conserved among all osteoglossomorph species (Al‐Mahrouki et al., 2001). However, there are suYcient amino acid substitutions that when the preproinsulin amino acid sequences are used in a phylogenetic analysis they show the osteoglossomorph species as a monophyletic group. The preproinsulins of the species also have utility in showing intergroup relationships in that phylogenetic analysis placed the species in the same taxonomic relationships that are based on more classical, morphological parameters. PPSS‐I codes for invariant SST‐14 and the extended form, SST‐26, while PPSS‐II codes for variant [Tyr7, Gly10] SST‐14 and the extended form, SST‐27 (Youson et al., 2006). Phylogenetic analysis using the amino acid sequences of the osteoglossomorph PPSS‐I and ‐II supports the monophyly of the group

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Ictalurus punctatus I 54 90

84

Megalobrama pellegrini Danio rerio I Carassius auratus I Gnathonemus petersii I

80

Chitala chitala I Carassius auratus II 100

Danio rerio II Catostomus commersoni II

82

Epinephelus coioides II

100

Lophius americanus II 60 100

63 86

Osteoglossum bicirrhosum II Gnathonemus petersii II Chitala chitala II Pantodon buchholzi II Oncorhynchus mykiss II Chicken

Fig. 8.6. Phylogeny of PPSS sequences I and II in several teleost species analyzed by parsimony with the maximum parsimony shown. Numbers on each lineage indicate the percentage of 500 bootstrap replications that support each lineage; lineages without a number were supported by less than 50% of the bootstrap replication. Of interest are the grouping of bonytongue species (Osteoglossum bicirrhosum, Gnathonemus petersii, Chitala chitala, and Pantodon buchholzi) in both PPSS‐I and ‐II providing support for the monophyly of members of the order Osteoglossiformes. [From Youson et al. (2006).]

but is not as useful as preproinsulin in resolving intergroup relationship among these ancient teleosts (Figure 8.6). 2.4. Phylogenetic Considerations It was emphasized in the opening remarks to this section of the chapter that there has been an extensive discussion on the phylogeny of islet organ in fishes. Although this discussion began long before the contribution to this theme by Epple (1969) in this Fish Physiology series, he likely laid the

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foundation for the contributions on the subject for almost the past 40 years. The writer has provided some relatively recent articles on the subject of phylogeny of the fish islet organ (Youson and Al‐Mahrouki, 1999; Youson et al., 2006), so here I will only summarize the contributions provided in the present section from observations of ‘‘primitive fishes.’’ This section has not dealt with the observations of GEP cells in protochordates, but it is most probable that the story of phylogeny of the islet organ in vertebrates begins in this group (for review see Youson and Al‐Mahrouki, 1999). The protochordates have their GEP cells confined to their gut (Reinecke, 1981; Falkmer, 1985a,b, 1995) and the next step in a putative evolution is the budding of GEP cells to form islets of cells from the epithelium of either the EHCB (hagfish) or the intestine (larval lamprey). Although the epithelium of the gut in protochordates and larval lampreys has several types of GEP cells, the islets that produce the islet organs in larval lampreys are exclusively composed of B cells and those of hagfishes are primarily B cells (Figure 8.2). The adult lamprey islet organ consists of principal islets that are composed of close to equal numbers of B and D cells. A cranial principal islet develops from the intestinal epithelium and, thus, follows the larval lamprey route. A caudal principal islet, as evident in adults of Holarctic lampreys, appears during metamorphosis from the epithelium of the EHCB and, thus, follows the hagfish route of development. In agnathans, the glucagon‐containing cells remain confined to the gut. Where the phylogeny goes from this point depends on how one looks at the evolution of the lobe‐finned fishes and more basal ray‐finned fishes relative to the agnathans and the derived teleosts. The evidence above indicates that the coelacanth and dipnoan islet organ are either more like cartilaginous fishes, because of their compact nature, or even more tetrapod‐like in character. In terms of islet cell types, they have the four cell types like those in euteleosts, and at least in this parameter they are well advanced over Agnatha. The polypterids and Acipenseriformes contribute little to a discussion of phylogeny of the bony fish islet organ, for knowledge is very scanty on its distribution and structure in these two groups. The general view, however, is that their islet organs are very similar to those in both Semionotiformes and Amiiformes, that is, very much basal actinopterygian in character with diVusely distributed islets of small to moderate size (i.e., no principal islets or Brockmann bodies). The gars and the bowfin islet organs do seem to represent a step in the phylogeny of the islet organ, for the diVusely distributed islets have usually only three distinct cell types with (B, D, and A/F) and the distribution of the D cells is not euteleost in character (i.e., no D1 and D2 cells regionally distributed). Observations of islet organs in basal teleosts, such as the eel and the bonytongues, provide a picture of a situation of transition between the basal actinopterygian situation in gars and bowfin and the derived euteleosts with either principal islets

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and/or Brockmann bodies. There is evidence that these basal teleosts have an islet organ with some large islets, some concentrated islets, and that there are distinct A‐, B‐, D‐, and F‐cell types in the islets with a further distinction of types of D cells. The discussion above with respect to the various peptides generated by the GEP cells also show some phylogenetic relevance. It can be concluded that the GEP system in the ‘‘primitive fishes’’ under discussion in this section can be broadly viewed as representing steps in the phylogeny, and in some cases the ontogeny, of the islet organ in other members of this ancient group and in more advanced bony fishes. 3. THYROID GLAND 3.1. Background The term thyroid gland for this chapter includes all organs in fishes that synthesize and secrete thyroid hormones (THs), namely 3,5,30 , 50 ‐tetraiodothyronine or thyroxine (T4) and 3,5,30 ‐triiodothyronine (T3). Generally, the majority of T3 is converted from T4 in extrathyroidal tissues by loss of an outer‐ring iodine or T4 outer‐ring deiodination (T4ORD). This definition of a thyroid gland and the principal products means that the endostyle, found in larval lampreys and believed to have some interesting ontogenetic and phylogenetic significance, is included (Figure 8.7). As indicated by Eales (1997), a survey of the animal, and even plant, kingdoms indicates that a follicular thyroid gland might not be a prerequisite for TH synthesis. This statement is particularly relevant even with teleosts, given the argument by Raine et al. (2005), based on their histological evidence from juvenile rainbow trout (Oncorhynchus mykiss), that in many species of teleosts, the thyroid cells may be arranged in a tubular configuration. Since Gorbman (1969) provided an account of thyroid tissue for this series in Fish Physiology, there have been some important reviews on this tissue over the past 40 years that have included more than passing attention to the fish thyroid. Notable among these reviews are those of Eales (1979, 1988), Leatherland (1982, 1987, 1988, 1994), Dent (1986), Grau (1987), and the most recent by Yamano (2005). As stated by Plohman et al. (2002a) and Yamano (2005), thyroidology in fishes has mainly concentrated on neopterygians, and in particular, salmonid teleosts. This section of the chapter will emphasize the need to expand our studies to include the fishes with an ancient heritage. For the most part, the thyroid glands of adult fishes resemble those of higher vertebrates in consisting of thyroid follicles and they are located in a basibranchial or a ventro‐pharyngeal region. According to Leatherland (1994), however, nonpharyngeal ectopic or heterotopic thyroid tissue is not

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Medial chamber

A

Hypobranchial duct

Posterior lateral chambers

Anterior chambers B

Type 4

Type 5

Type 3

Core substance

Type 1

Type 2c Dorsal glandular tract Type 2b Plug

Type 2a

Ventral glandular tract

Fig. 8.7. Diagrammatic representation of the gross (A) and histological structure of the anterior chamber (B) of the larval lamprey endostyle. [Originally from Barrington and Sage (1972), but modified by Manzon (2000).] See the text for further description.

uncommon, at least in teleosts. The apparent diVerences in histology of the thyroid follicles between species are not necessarily of any functional significance, for they can vary between individuals of the same species and even between follicles within the same individual. Raine et al. (2005) have emphasized that there may even be diVerences in thyroid histology that are consequences of development in the region of the pharynx that ultimately houses the definitive thyroid. The appearance of a thyroid follicle, however, is often reflective of a physiological state of a gland or an individual follicle at the time the animals were prepared for observation. For example, large follicles with much luminal colloid and a flattened epithelium suggest an unstimulated gland, while follicles with a columnar epithelium lining a narrow lumen indicate stimulation, perhaps by thyroid‐stimulating hormone (TSH) from the adenohypophysis. The point being made here is that in the past we have

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not been able to rely on thyroid histology when we wish to document phylogeny of the fish thyroid gland. On the other hand, the tubular profiles of thyroid tissue may provide another morphological parameter in phylogeny of the fish thyroid gland (Raine et al., 2005). This section of the chapter will provide a review of what we presently know about the thyroid gland and its function in the groups of fishes that have been considered in Section 2 of this chapter. Thyroid follicles of vertebrates are consistent in their form and function (Eales, 1997). The proteins are synthesized in the rough endoplasmic reticulum (RER) of follicular cells, and they are glycosylated in the RER and the Golgi apparatus before being transported in vesicles and released for storage into the follicular lumen as a glycoprotein. The luminal colloid stains with periodic acid‐SchiV (PAS). Iodination of the glycoprotein, thyroglobulin, occurs primarily at the apical surface of follicular cells through the activation of iodine by iodoperoxidase, a protein also packed into vesicles at the Golgi apparatus. Inorganic iodide is transported from the blood across the basal membrane of the follicular cell (usually with Naþ) and binds with tyrosines of the thyroglobulin molecule. Iodothyronines are stored in the iodinated thyroglobulin within the colloid of the follicular lumen. Iodinated thyroglobulin is incorporated into the cell by endocytosis and forms colloid droplets. The colloid droplets unite with primary lysosomes to form electron‐dense, endolysosomes and in these bodies the thyroglobulin is hydrolyzed by the lysosomal enzymes to form T4, and some T3, 3‐monoiodotyrosine (MIT), 3,5‐diiodotyrosine (DIT), and amino acids. There are thyroid deiodinases in the RER to hydrolyze MIT and DIT and to convert some T4 to T3, but the majority of TH is released into the vascular system at the base of the cell as T4. TH is mainly transported in the blood in a bound form, primarily to thyroid‐binding globulin. With the exception of the endostyle, this is the general description and pattern for TH production in thyroid follicles of all species to be discussed. Exceptions to this mechanism will be emphasized. 3.2. Agnatha The thyroid glands of both hagfishes and lampreys and the action of their hormones have been studied extensively, mainly because of the influence of two of the founding members of modern comparative endocrinology, E. J. W. Barrington and Aubrey Gorbman. The most recent reviews that specifically focus on agnathan thyroid glands are those of Hardisty (1979) and Hardisty and Baker (1982). The most recent investigations in agnathan thyroidology focus on the role of the thyroid gland in lamprey metamorphosis (Youson, 1997) and the assessment of homologies between endostyles of protochordates and larval lampreys using molecular tools (Ogasawara et al., 1999).

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Hagfishes have direct development and thyroid follicles are present in the embryo (i.e., before hatching). Lampreys have indirect development and their thyroid follicles do not appear until during a metamorphosis that is divided into seven (one, earliest, to seven, final) stages (Youson, 1988). Although diVerences in the ontogeny of the thyroid glands of lampreys and hagfishes has often been used as an example of the early divergence of these two agnathans in their evolutionary history, Gorbman (1997) found that the method of development of thyroid follicles from a broad area of pharyngeal epithelium in hagfish embryos is similar to that seen during follicular development during lamprey metamorphosis. In the latter case, follicles arise from clumps of cells from the transforming epithelium of the endostyle (Wright and Youson, 1976, 1980b) that extends throughout much of the length of the pharyngeal region (Hardisty, 1979). Does hagfish embryology reflect a step in the development of agnathan thyroid follicles that occurred later in lampreys, when metamorphosis was ‘‘selected’’ as part of a late stage of ontogeny (Youson, 2004)? 3.2.1. Hagfish Light microscopic and particularly fine structural observations of the hagfish thyroid gland show a follicular epithelia in Eptatretus stouti and Eptatretus burgeri similar to that of higher vertebrates (Henderson and Gorbman, 1971; Fujita and Shinkawa, 1975). The follicles are embedded in adipose tissue ventral to, and between, the two rows of branchial pouches. All studies are consistent in their view that the hagfish thyroid is poorly vascularized; it is estimated that E. stouti has only 20–30% of its thyroid follicles bound by vascular tissue (Henderson and Gorbman, 1971). Although RER is extensive in hagfish follicular cells, it does not have the wide, dilated cisternae seen in mammalian cells. Scanning electron microscopy also reveals the follicular arrangement of the epithelium, poor vascularization, and both a central cilium and some apical protrusions on the luminal surface of the cells (Suzuki and Kawabata, 1988). The apical protrusions may correspond to the pseudopodia (filopodia) profiles described as part of the process of incorporation of luminal colloid into the cells (Henderson and Gorbman, 1971). The number of electron‐dense inclusions (presumably condensed or hydrolyzed versions of colloid droplets) in follicular cells of hagfish far exceeds the numbers seen in mammalian cells. Autoradiography with 125I shows that, like in mammals, the main site of iodination of thyroglobulin is the luminal colloid (Fujita and Shinkawa, 1975). X‐ray microanalysis also indicates that the electron‐dense inclusions contain large amounts of iodine (Fujita, 1975) and confirms that they likely are a consequence of absorbed colloid and coalescence with primary lysosomes. The general opinion from morphological observations is that the hagfish thyroid gland does not reflect high activity and this view is consistent with

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functional studies of radioiodine uptake and T4 secretion rates (Tong et al., 1961; Waterman and Gorbman, 1963). On the other hand, Henderson and Lorscheider (1975) were surprised at the level of T4 in the plasma (2–3 mg/ 100 ml) of E. stouti relative to that present in the plasma of other fish species. Packard et al. (1976) measured 3.4 mg/100 ml in the plasma of the same species compared to only 0.5 mg/100 ml in adult lamprey, Lampetra (Entosphenus) tridentata. Hagfish plasma levels of T4 and T3 are not aVected by prolonged fasting (Henderson, 1976; Plisetskaya et al., 1983a), and the thyroid glands show higher radioiodine uptake and response to TSH than freshly caught animals (Kerkof et al., 1973). The existence of a hypothalamic‐pituitary‐thyroid axis in hagfish is still under investigation. When cocultured with pituitary tissue, the hagfish thyroid tissue shows a significant increase in T4 in the medium over levels in thyroids incubated alone (DickhoV and Gorbman, 1977). On the other hand, a response is not immediately evident at the fine structural level in the hagfish thyroid gland 6 h after injection of TSH (Fujita and Shinkawa, 1975). It has been suggested in an immunohistochemical study of the adenohypophysis of M. glutinosa and E. burgeri, where antisera to the glycoprotein hormones (TSH and gonadotropins) immunoreacted in the same cells, that a distinct TSH is a derived hormone that permitted expansion of vertebrates into environments diVerent from those occupied by the ancient species (Nozaki et al., 2005). A hypothalamic component of the thyroid axis in hagfish, that is a thyrotropin‐releasing hormone (TRH), is questionable (DickhoV et al., 1978) and its presence seems unlikely given the apparent absence of a distinct TSH in the adenohypophysis. However, we still need to resolve why the hagfish thyroid gland responds to some thyrotropic factor located within the pituitary (DickhoV and Gorbman, 1977; DickhoV et al., 1978). Some earlier (Suzuki and Gorbman, 1974; Suzuki et al., 1975) and more recent (Ohmiya et al., 1989) studies on hagfish thyroid iodoproteins indicate four subfractions with iodine, hormonal iodine, and carbohydrate content strikingly diVerent from those present in thyroglobulin. For example, the amino acid composition of hagfish iodoprotein indicates a cysteine content of less than 1%, much less than that in thyroglobulin and perhaps influencing molecular stability. The implication is that tyrosine residues within hagfish thyroid glycoprotein with a nonrigid structure are potentially problematic for iodination (Ohmiya et al., 1989). The process of deiodination is critical for peripheral fine‐tuning of TH bioactivity in fishes (Orozco and Valverde‐R, 2005), and hagfishes demonstrate extrathyroidal deiodination activity (McLeese et al., 2000). In fasting hagfish (M. glutinosa), liver, muscle, intestine, and brain exhibit low‐substrate enzymatic activity for T4ORD and for inner‐ ring deiodination of T4 (T4IRD) that usually produces inactive, 3,30 ,50 ‐triiodothyronine or reverse T3 (rT3). Inner‐ring deiodination of T3 (T3IRD), but no deiodination of rT3, occurs in these tissues to produce 3,30 ‐diiodothyronine

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(3,30 ‐T2), another inactive product. Hagfish pathways of deiodination require a thiol cofactor (dithiothreitol) and are inhibited by established deiodinase inhibitors and by TH analogues and, therefore, are not unlike the pathways in teleosts (McLeese et al., 2000). In contrast to teleosts, however, T4ORD activity in hagfish is tenfold higher in the intestine than in the liver. This high intestinal T4ORD activity over liver is also characteristic of larval and adult lampreys (Eales et al., 1997). T3IRD activity in the liver and intestine of hagfish has characteristics similar to those in teleosts and led McLeese et al. (2000) to suggest that this pathway is the most highly conserved of the deiodinating systems. Although the need for TH in hagfish has been questioned, there seems to be an important interrelationship between insulin and TH levels and their regulatory action on intermediary metabolism (Plisetskaya et al., 1983a,b,c). There is a decrease in plasma T3 in situations of insulin insuYciency after anti‐insulin administration (Plisetskaya et al., 1983b). T3 injection is followed by an elevation of plasma fatty acids, suggesting a lipolytic eVect of the hormone (Plisetskaya et al., 1984). T3 administration elevates serum levels of this hormone significantly above preinjection and controls levels in M. glutinosa even after just 3 h (Leary et al., 1997). After this same postinjection interval, low and high dosages of T3 have the eVect of elevation of glucose‐6‐phosphatase dehydrogenase in the liver that the authors suggest may be an indication of increased rates of biosynthesis of lipids, DNA, RNA. or protein. In addition, there is an increase in liver carnitine palmitoyl transferase, an enzyme of lipid catabolism, 3 h after a low T3 dose, but the significance of the increase is not clear due to some suspected stress‐related response in a control group (Leary et al., 1997). However, there seems to be suYcient evidence from the short‐term studies on M. glutinosa (Leary et al., 1997) and the long‐term studies on E. stouti (Plisetskaya et al., 1984) to infer that T3 has some lipolytic eVect in hagfishes. Unlike in higher animals, however, the lipolytic eVect of administered T3 is not mediated through the potentiation of the fat‐mobilizing action of catecholamines (Plisetskaya et al., 1984). To the present date, all data on the thyroid and TH in hagfish support an earlier hypothesis that pituitary control of hagfish thyroid through TSH is absent in this organism, and in some other lower vertebrates, and that a phylogenetically ancient mechanism is in place. That is, regulation of function in the thyroid gland is through its involvement in metabolism and target tissue sensitivity (DickhoV and Darling, 1983). 3.2.2. Lamprey a. Larval (Ammocoete) Endostyle and THs. As referenced above, the thyroid gland of larval lampreys is not a gland of follicles. Instead, the larval gland is composed of a pair of straight, adjacent anterior and posterior

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lateral chambers and a coiled posterior medial chamber located in the floor of the pharyngeal region of the alimentary canal between the first and fifth gill arches (Figure 8.7A). There is a communication between the lumina of the pharynx and the chambers through the hypobranchial duct. This subpharyngeal gland, better known as the endostyle, has been of great interest from both ontogenetic and phylogenetic points of view because of the presence of a gland of similar structure in protochordates. As a consequence, the structure and function of the endostyle in larval lampreys has been discussed in many previous reviews of the fish thyroid gland (Gorbman, 1969; Eales, 1979; Dent, 1986; Leatherland, 1994) and in specific reviews of lampreys or cyclostomes (Barrington and Sage, 1972; Hardisty, 1979; Hardisty and Baker, 1982). The reader should reference these earlier reviews for detailed descriptions of the larval lamprey endostyle and for the list of the many classical studies on this subject matter. The present chapter will briefly review the structure of the endostyle with the objective of providing a baseline to permit a report of more recent data on the structure and function of the gland (Figures 8.7B and 8.8A). Each chamber consists of glandular tracts of type 1 cells that are believed to secrete mucus (Barrington and Sage, 1972). Ventral and dorsal to an opening of the glandular tracts into the lumen of the chamber are columnar‐shaped type 2a and b cells, but it is type 2c and 3 cells that are the primary iodide‐binding cells of the endostyle (Suzuki and Kondo, 1973; Wright and Youson, 1976). Type 4 and 5 cells have a limited capacity to bind radioiodine (Wright and Youson, 1976). Localization of antisera against human thyroglobulin within and at the luminal surface of type 2c and 3 cells (Figure 8.8A) is a strong support for these cells as the primary site for TH production (Wright et al., 1978a,b). These cells also have peroxidase activity (Tsuneki et al., 1983). Studies with radioiodine indicate that iodine is incorporated into iodoproteins in the endostyle that correspond to the homodimer (19S) and monomer (12S) of mammalian thyroglobulin (Monaco et al., 1978). Moreover, the hydrolysis of endostylar iodoprotein (Suzuki and Kondo, 1973) shows that they contain T4, T3, MIT, and DIT. Homogenates of endostyles from the brook lamprey, Lampetra appendix, have a lamprey thyroglobulin with estimated molecular mass of 226 kDa (Manzon and Youson, 2002). Studies (also see Section 2.4) have used in situ hybridization to show the localization of thyroid transcription factors in specific areas of the endostyle as a means of establishing homology of the protochordate and larval lamprey endostyles (Ogasawara et al., 2001). Goitrogens, known inhibitors in other vertebrates of thyroid activity through inhibition of either iodide uptake or peroxidase‐catalyzed iodination, act directly on the endostyle (Manzon and Youson, 2002) to lower serum levels of THs (Youson et al., 1995; Manzon et al., 1998; Holmes et al., 1999). The above information serves to illustrate the point that the larval

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Fig. 8.8. Light micrographs of a portion of the endostyle of the larval lamprey (A), the transforming endostyle during metamorphosis (B), and the newly formed follicular thyroid near the end of metamorphosis (C) showing immunostaining for thyroglobulin (TG). P, pigment. (A) The apical surface and cytoplasm of type 2c and 3 cells and some granules of type 3 cells (arrow) stain for TG

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lamprey endostyle, although a nonfollicular thyroid gland, has the features of a gland capable of TH synthesis and iodination. Like the hagfish follicular thyroid, however, vascularization is poor, but it is likely that TH can also be released into the alimentary canal for absorption by the intestinal epithelium. As will be discussed below, the primary site for deiodination of T4 in larval lampreys is the intestine (Eales et al., 1997). Since there is no colloid for storage of synthesized products in the larval endostyle, larval lampreys either synthesize and release TH at times of need or they store TH elsewhere. Given the large concentrations of circulating TH in the plasma of larval lampreys (Wright and Youson, 1977, 1980a; Lintlop and Youson, 1983a; Leatherland et al., 1990a; Youson et al., 1994b, 1995; Manzon and Youson, 1997; Holmes et al., 1999), the plasma storage seems to be the most likely fate for newly synthesized hormone. Larval life in lampreys lasts from 2 to 7 years with the duration of this phase varying between species and within diVerent populations of the same species (Potter, 1980). This larval growth phase is terminated when the metamorphic phase commences (Youson, 1980, 1988, 1994, 2003, 2004). In P. marinus, serum concentrations of both T4 and T3 increase during larval life (Figure 8.9) to reach their peak prior to metamorphosis (Youson et al., 1994b). There is also some seasonal variation in serum levels of both T4 and T3 in P. marinus (Wright and Youson, 1980a; Lintlop and Youson, 1983a) and G. australis (Leatherland et al., 1990a). In both species, TH levels can be altered by subjecting the animals to temperatures (lowered or elevated) that are opposite to those to which they normally would experience in their natural environment at that time of the year. Manipulations of water temperature can also influence the incidence of metamorphosis in P. marinus (Youson, 2003) and are reflected in diVerences in serum levels of TH. Thus, the seasonal variation in TH can likely be explained by the known variation of serum TH in larvae in response to changes in environmental temperature (Lintlop and Youson, 1983a; Youson et al., 1994b; Manzon and Youson, 1999), yet it cannot be ignored that the peak of serum T4 occurs at a time of the year when the food source is most prevalent and is assimilated (Wright and Youson, 1980a). Although it is diYcult to compare data from the various studies on the eVects of temperature on larval TH levels because of their diVerent experimental conditions and interassay variation (Wright and Youson, 1980a; Lintlop and Youson, 1983a; Leatherland et al., 1990a; Youson et al., 1994b), in general, it but type 1, 2b, and 4 cells are nonimmunoreactive to antibovine TG. (B) Staining for TG is apparent within many cells (arrows) of the transforming endostylar epithelium and within the lumina (L) of some of the new follicles. (C) Staining for TG is apparent in the follicle lumen (L) and at the apex of follicular cells (arrows) but not in the cellular mass (C) of an adjacent follicle. (A) 350 is from Wright et al. (1978a) (B) and (C) both 300 are from Wright et al. (1980).

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Climax Leptin-like ir-protein Brain GnRH I and III

Relative increase TH

Thyroid inhibitor

Lipogenesis

Lipolysis lipid Temperature

0

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II

III

Larval year classes

IV+

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2

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6 7

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Fig. 8.9. Diagrammatic representation of the factors involved in metamorphosis of the sea lamprey, P. marinus. There is a gradual increase in the serum levels of TH over the IV þ larval year classes but metamorphosis (stages 1–7) is marked by a sharp decline in both T4 and T3 at the initial stage. The TH decline occurs at the time of the year when stream water temperature sharply elevates and body lipid in the older larvae is at peak level. A phase of lipogenesis in older, premetamorphic larvae is followed by a lipolytic phase when the lipid is utilized as an energy source during the nontrophic phase of metamorphosis. There is some suggestion from leptin immunoreactivity (IR) that this protein in fat‐storing tissues might be an initial trigger for metamorphosis and is influenced by, or influences, changing TH levels. Gonadotropin‐releasing hormones (GnRH‐I and ‐III) in the brain elevate at the time of lowered TH levels. A putative inhibitor of thyroid gland activity is indicated in this scheme. [Modified from Youson (1994).]

seems that T4 levels are higher at elevated temperature (or summer months), whereas T3 serum concentrations tend to be elevated during times of colder temperature (or winter months). In year class III larvae (3þ years old) of P. marinus, serum concentrations of T4 and T3 are
70 and 22–32 nmol/liter (Youson et al., 1994b, 1995), respectively, which represent values at a minimum tenfold higher than most other vertebrates (Norris, 1997). Therefore, the blood plasma seems to be a reservoir for TH in larval lampreys. The enormous capacity for TH storage in the blood is emphasized in experiments where exogenous T4 and T3 are added to the holding water of larval lampreys, with or without KClO4, and is ultimately incorporated into the bloodstream (Manzon and Youson, 1997; Youson et al., 1997; Manzon et al., 1998). In these cases, the serum concentration of T4 becomes elevated far beyond what one would consider physiological levels (
1600 nmol/liter). The levels of T3, however, seem to be more carefully regulated and are not elevated significantly beyond physiological levels. Given the high capacity for storage

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of TH in the blood of larval lampreys, it would seem feasible to search for TH‐ binding proteins. Since it seems that thyroxine‐binding globulin is absent in nonmammalian vertebrates (Bentley, 1998), albumin, with usual high TH‐ binding capacity but low aYnity, would be the most likely serum protein candidate. It is noteworthy that larval lamprey albumin has been identified in P. marinus and it is replaced by an adult albumin at metamorphosis (Filosa et al., 1992, 1998). Is it possible that larval and adult albumin have varying binding capacity and aYnity for TH and a switch in aYnity and capacity between the two albumin forms commences during metamorphosis? On the other hand, some recent preliminary data indicate that the transthyretin gene exists in P. marinus and this oVers another possibility of transthyretin binding of TH in a temporal manner (Manzon and Smith, 2006). The properties of T4ORD activity (pH optimum, dithiothreitol cofactor requirement, apparent Km, substrate preference, and potency of potential inhibitors) in intestine and liver of larval and upstream‐migrant adults of P. marinus are similar in most respects to those for teleosts, that is, there is a low‐Km T4ORD enzyme that most closely resembles mammalian type II deiodinase (Eales et al., 1997). In larvae, the highest T4ORD activity is present in intestine followed in order by liver, kidney, and muscle but inner‐ring deiodination pathways are negligible in these tissues (Figure 8.10). The apparent absence, or low level of activity, of T4IRD and T3IRD indicates that there is little, if any, degradation of T4 or T3, respectively. Since the extrathyroidal deiodinase pathways in larvae are geared toward T3 production, rather than degradation of either T4 or T3, this may be an explanation for the high concentrations of these hormones at this period of lamprey development. It is of interest that the larval intestine should have the highest level of T4ORD activity, for this organ is presumed to be the primary route of TH into the bloodstream after newly synthesized hormone is released from the poorly vascularized endostyle. Support for the view that the larval intestine may be the site of regulation of T3 production in larval lampreys is found in some protochordates that have an endostyle and deiodinate T4 to T3 in the intestine (Fredricksson et al., 1993). b. THs and Metamorphosis. A dramatic drop in serum levels of both T4 and T3 is coincident with the first signs of metamorphosis (Figure 8.9) in lampreys (Youson, 1994, 1997). This feature of metamorphosis suggests that the output of TH from the endostyle has greatly lessened and/or that peripheral regulation and utilization of TH has greatly increased or modified. Hoheisel and Sterba (1963) induced incomplete metamorphosis in larval Lampetra planeri with the goitrogen, potassium perchlorate (KClO4), and similar results were seen in larval Lampetra reissneri with this and several other goitrogens (Suzuki, 1986, 1987, 1989). KClO4 also induces metamorphosis in P. marinus

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III

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a

a

3

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a

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0 14 12 10 8 6 4 2 0

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Stage Fig. 8.10. Intestinal deiodination activities during various intervals of the life cycle of the lamprey, P. marinus. Data are from four diVerent studies (I–IV). (A) T4‐outer‐ring deiodination (T4ORD). (B) T4‐inner‐ring deiodination (T4IRD). (C) Ratio of T4IRD to T4ORD. (D) T3‐ inner‐ring deiodination (T3IRD). Abbreviations: A, adults in immediate prespawning phase; L, larvae of nonmetamorphic size; PR, premetamorphic larvae; 1–7, stages of metamorphosis; PM, postmetamorphic lampreys. Within a given study, means with similar letters do not diVer significantly. Note that T4ORD activity is prominent in premetamorphic and early stages of metamorphosis and T4IRD activity gains prominence at the end of metamorphosis. T3IRD activity is only detectable in adult intervals. [From Eales et al. (2000).]

and L. appendix larvae and lowers the concentrations of the TH in the serum of nonmetamorphosing and metamorphosing animals (Holmes and Youson, 1993; Youson et al., 1995; Manzon and Youson, 1997; Holmes et al., 1999). Both inhibitors of peroxidase‐catalyzed iodination (methimazole) and anionic

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competitors of iodide uptake (KClO4, sodium perchlorate, potassium thiocyanate) show a correlation of incidence of metamorphosis with lowered serum TH, but another inhibitor of iodination, propylthiouracil, does not induce metamorphosis despite lowering TH levels (Leatherland et al., 1990a; Holmes et al., 1999; Manzon et al., 2001). Since the eVective (metamorphosis‐inducing) goitrogens act directly on the endostyle to reduce iodoprotein output (Manzon and Youson, 2002), and they also result in lowered serum TH levels, they might mirror the eVect of an endogenous factor (Figure 8.9) that triggers metamorphosis by suppressing TH synthesis (Youson, 1994). This view is supported by the observation that spontaneous metamorphosis can be retarded or prevented by adding T3, but not T4, to the holding water of immediately premetamorphic animals (Youson et al., 1997). Furthermore, KClO4‐induced metamorphosis can be blocked by adding either T4 or T3 along with the inducer (Manzon and Youson, 1997; Manzon et al., 1998). In blocked‐spontaneous and induced metamorphosis, the addition of TH results in elevated serum TH. It is clear that suppression of TH synthesis and a decline in circulating TH is an important early event of lamprey metamorphosis, but it is still uncertain that this is the event that is the primary cue for initiation of this developmental phenomenon (Youson, 1997). The extensive investigations with P. marinus have indicated that water temperature and physiological preparation for the nontrophic event of metamorphosis through fat stores (Figure 8.9) are key parameters for a successful initiation and completion of development (Youson, 1994, 1997, 2003, 2004). It is now well established in P. marinus that the initiation of metamorphosis is strictly regulated by a spring warming of the water temperature (Holmes and Youson, 1994, 1997) and that lowering of the optimal temperature for metamorphosis (21  C) by as little as
10  C will completely inhibit this event (Holmes and Youson, 1998). That an elevation of the water temperature is an important cue to initiate metamorphosis is illustrated by the fact that animals kept at the optimum temperature throughout the fall and winter months and into spring do not undergo metamorphosis (Holmes et al., 1994); however, at the critical time of spring warming, even a short‐term exposure to colder water of 1 month can drastically reduce the incidence of metamorphosis (Youson et al., 1993). That water temperature is a critical cue is also seen in induced metamorphosis (Figure 8.11A–C). For example, KClO4 is ineVective as an inducer of metamorphosis at temperatures of 3  C despite the fact that T4 and T3 serum concentrations are 73% and 78% lower, respectively, than those in untreated control larvae at the same temperature (Manzon and Youson, 1999). In comparison, 18  C and KClO4 induces metamorphosis in all larvae and lowers T4 and T3 serum concentrations by 66% and 95%, respectively, relative to those in untreated larvae in holding water of the same temperature. Furthermore, lipogenesis and lipolysis are critical events for the nontrophic metamorphic phase in P. marinus (Figure 8.9), and they are

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Cold KCIO4

Untreated warm Warm KCIO4

Groups Fig. 8.11. The eVect of 23 weeks of exposure to warm (18  C) and cold (3  C) water on the incidence of KClO4‐induced metamorphosis and serum thyroxine (T4) and triiodothyronine (T3)

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controlled by a hormonal cascade including somatostatin, insulin, and TH (Kao et al., 2003; Youson, 2003). Lipid metabolism related to spontaneous and goitrogen‐induced metamorphosis can be reversed through administration of TH (Kao et al., 1999). There is also some suggestion that leptin released from adipocytes and gonadotropin‐releasing hormones from the brain are corelated with the changing TH profile during early metamorphosis (Figure 8.9) (Youson and Sower, 2001; Youson, 2003). The studies of the role of TH in lamprey metamorphosis have been complicated by the fact that the larval TH‐synthesizing organ, the endostyle, undergoes a major transformation into thyroid follicles during this event (Wright and Youson, 1976, 1980b). The transformation of the larval endostyle begins at the first signs of external metamorphosis (stages 1–2) and the adult form of the gland is reached by stage 5. The glandular tracts and their type 1 cells undergo autolysis and are removed through phagocytosis by macrophages. The follicular epithelium seems to arise primarily from type 2c and 3 cells of the endostyle but type 5 are also likely involved. The obvious question is whether it is the collapse of the endostyle that results in the decline of serum levels of TH during metamorphosis (Leatherland, 1994). The endostylar collapse seems an unlikely explanation for lowered serum TH, for the thyroidal tissues of lampreys continue to bind radioiodine and to show immunoreactivity to thyroglobulin antisera (Figure 8.8B) throughout the entire process of transformation (Wright and Youson, 1976; Wright et al., 1980b). If in larvae the route for newly synthesized TH into the circulation is through absorption in the intestinal epithelium, then changes in this route could be a more likely cause of declining serum TH. The entire alimentary canal undergoes a complete reorganization and included is a severance of the pharyngeal lumen from any connection to that of the intestine. The larval oesophagus is replaced by an adult oesophagus that is independent of the pharynx; there is also a transformation of the larval intestinal mucosa to an adult mucosa with diVerent epithelial cell types and elaborate folds (Youson, 1981; Youson and Horbert, 1982). Extrathyroidal regulation of TH such as deiodination and changes in binding capacity and aYnity of TH receptors (TRs) could also influence circulating levels of both T4 and T3. As will be discussed in detail below, however, the intestine remains

levels in larval sea lampreys (P. marinus). (A) T4 levels drop significantly from control values to similar levels in treated animals in both cold and warm water. (B) T3 levels are significantly lower in treated animals in the two temperatures compared to their controls but T3 in the treated group in warm water is significantly lower than the value in the cold‐treated group. (C) Induced metamorphosis only occurs in treated animals in warm water. DiVerent letters indicate significant diVerence (p < 0.05). [From Manzon and Youson (1999).]

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eYcient in iodination despite the major transformation of its mucosa during metamorphosis (Eales et al., 2000). Knowledge of receptors is a key piece of information in the total picture of the action of any hormone, and receptors for TH in the context of lamprey metamorphosis are no exception. Since there is a major reorganization during metamorphosis of the larval liver into an adult liver with no gall bladder and bile duct system (Youson, 1993), the binding capacity of T3 to hepatocyte nuclei was studied in vitro during this developmental phase and during the rest of the life cycle of P. marinus (Lintlop and Youson, 1983b). The binding aYnity of T3 to the hepatocyte nuclei is of high aYnity (Kd ¼ 2.9  10–10 M) and the maximum binding capacity is 1.89, 2.40, 0.78, and 0.12 pg T3/mg DNA1 in larvae, metamorphosing individuals, juveniles, and upstream migrants, respectively. The maximum binding capacity in larvae and metamorphosing individuals is about 10 times the average value in higher vertebrate species, and it is noteworthy that larval serum T3 levels are in the order of 10 times those of most other vertebrates (Lintlop and Youson, 1983a,b). The maximum binding capacity is highest during metamorphosis but the diVerence from that of the larva is not significant. However, the retention of the high binding capacity during metamorphosis does suggest that T3 may exert some influence during this interval of the life cycle. The decline in serum T3 during metamorphosis is not accounted for by increased binding of the hormone to hepatocyte nuclei. On the other hand, it must be understood that these data are generated from in vitro observations and we cannot preclude the possibility that in vivo there may be cellular mechanisms in larvae that prevent T3 binding to hepatocyte nuclei and, hence, are partially responsible for the high levels of serum T3 (Lintlop and Youson, 1983b). Two TRs, PmTR1 and PmTR2, and three retinoid X receptors cDNAs have been cloned in P. marinus (Manzon, 2006; L.‐A. Manzon and Youson, unpublished data). Expression data using the cDNA probes and both Northern blot and relative quantitative RT‐PCR indicate that PmTR1 might be a component of the peripheral regulation system that operates in a tissue/organ‐specific fashion to mediate TH action during metamorphosis. Besides the involvement of the tissue/organ TRs in contributing to the TH serum profile during metamorphosis, there are some interesting changes at this developmental interval in the deiodinase pathways that perhaps explain serum TH levels and diVerences in the deiodinase profiles in larvae and adults (Eales et al., 2000). In the intestine, T4ORD activity is highest in microsomes from stages 1 and 2 of metamorphosis but is very low between stages 3 to the last stage (7) of this event and into immediately postmetamorphic juveniles (Figure 8.10). T4IRD activity, which is not recognized in larvae (Eales et al., 1997, 2000), appears at stage 3 and increases thereafter to reach a peak at stage 7 and still is appreciable in postmetamorphic adults.

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At stage 6, T4IRD activity is 14 times that of T4ORD. T3IRD activity correlates with T4IRD activity at the times it is measured. Liver, for the most part, has lower deiodination activity than the intestine throughout all lamprey developmental stages (Eales et al., 2000). T4ORD activity is negligible after stage 2 and shows slight recovery in postmetamorphic adults. At stage 2, T4ORD activity in intestine is 20 times that in the liver. T4IRD and T3IRD activities are very low in the liver whenever they appear in development. T3ORD activity is not detectable in liver and intestine throughout development. The general conclusion is that in the intestine there is a reciprocal relationship between T4ORD and T4IRD activities with the former dominating larval and early metamorphic life and the latter predominating middle and later metamorphosis (Figure 8.10). It is noteworthy that the highest T4ORD activity is in immediately premetamorphic (expected to metamorphose) and stages 1 and 2 of metamorphosis, for this T4ORD profile suggests that at these times there is a major increase in T3 production. On the other hand, the T4IRD profile, and perhaps to lesser extent the T3IRD profile, implies that there is a shutdown of T4 conversion to T3 and an induction of IRD of both these hormones. That is, with T4IRD, T4 is converted to inactive rT3 and with T3IRD, T3 is converted to 3,30 ‐T2. The induction of IRD in conjunction with depressed T4ORD activity during metamorphosis in P. marinus may contribute to decreased serum TH levels that are characteristics of early stages of this event and seemingly are critical for developmental processes to proceed. It is curious, however, that KClO4‐ induced metamorphosis does not alter intestinal T4ORD activity (Manzon et al., 1998). In this case, the decline in serum levels of TH may be attributed to the aVect of the goitrogen in decreasing T4 production by the endostyle and the deficiency of T4 substrate to produce T3 (Eales et al., 2000). c. Adult Thyroid and Hormones. Comparisons of serum levels of both T4 and T3 between larval and adults of all species of lampreys studied to date indicate that, following the metamorphic decline, there is no recovery in adults to the hormone levels seen in larvae. In the immediately postmetamorphic juvenile, prior to feeding, there is evidence of T4ORD, T4IRD, and T3IRD activities (Eales et al., 2000) and at this time serum T4 and T3 concentrations are similar to those seen at the end of metamorphosis (Youson et al., 1994b). After a short period of feeding in the laboratory, following their nontrophic metamorphic phase of
10 months, both T4 and T3 show a significant decline in concentration from those in the immediately postmetamorphic, nonfeeding juveniles (Youson et al., 1994b). With the exception of G. australis, where there is some increase in TH during later stages of metamorphosis with a continued increase in early juveniles (Leatherland et al., 1990a), the trend in Holarctic species is for a progressive

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decline in TH concentrations to juveniles through to upstream‐migrant adults (Wright and Youson, 1977; Lintlop and Youson, 1983a; Holmes et al., 1999). In landlocked P. marinus, for example, T4 and T3 serum concentrations in year class IV larvae of
130 and
51 nmol/liter, respectively, are compared with values of
5 for T4 and 0.4 nmol/liter for T3 in feeding juveniles (Youson et al., 1994a,b) at roughly the same time of the year. An analysis of T4 and T3 levels during the adult‐feeding period of freshly captured, landlocked P. marinus, when there is a 200‐fold increase in mass and 60‐fold increase in length, shows that there is a constancy of systemic levels of these TH (Youson et al., 1994a). These data, along with those from serum concentrations of insulin (Youson et al., 1994a), indicate the metabolic peculiarities of feeding adult lampreys relative to teleosts (Plisetskaya, 1985). In salmonids, fluctuations in serum levels of TH and insulin reflect a consistency with the participation of these hormones in body growth and metabolism at times of active feeding (Murat et al., 1981; Plisetskaya et al., 1986; Eales, 1988; Leatherland, 1994). It seems that feeding adult lampreys do not require or utilize TH (and insulin) in the manner that is demonstrated for salmonids. It appears that at least T3 levels do not change substantially from those in juveniles during the upstream (prespawning) migration of P. marinus but are lower than those in metamorphosis (Lintlop and Youson, 1983a). Hornsey (1977) studied small numbers of upstream‐migrant P. marinus to come to some provisional conclusion that T4 levels increase in females during spawning from levels seen earlier in the migration. The values of 70–139 nmol/liter for T4 in three females seem extraordinarily high considering that the hormone was not detected earlier in the migration, that a study at a similar time frame showed low levels of T4 (Packard et al., 1976), and that the T3 concentrations are consistent with those obtained by others (Lintlop and Youson, 1983a). In contrast, T4 levels during the more protracted upstream migration in G. australis remain constant throughout the migration and are similar to those at the end of metamorphosis, while T3 levels show a progressive decline from levels at the same metamorphic interval (Leatherland et al., 1990b). Clearance kinetics of T4 and T3, to and from the circulation and tissues, during the upstream migration of P. marinus are roughly comparable to those reported in teleosts (Brown et al., 1982). Experimental investigations have shown that the thyroid gland of the river lamprey, L. fluviatilis, is not under pituitary stimulation during the upstream, spawning migration (Pickering, 1972) and state‐of‐the‐art immunohistochemistry of the adenohypophysis of P. marinus during this migration shows no cells immunoreactive to TSH antisera (Nozaki et al., 2001). Yet there is still some belief and expectation that thyroid activity at this time is related to reproduction (Hardisty, 1979; Hardisty and Baker, 1982). Data from Sower et al. (1985) in P. marinus seem to

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support this view in that in both males and females there is a peak of T3 concentration coinciding with a significant drop in T4 early in the final maturation period. It is still not clear whether these changes in the TH profile are directly related to changes in reproductive hormones or are indirectly related to the reproductive event (Sower, 2003). Estradiol‐17b induces a lowering of T4 serum concentrations and a rise in serum T3:T4 ratios in upstream‐migrant G. australis (Leatherland et al., 1990b), suggesting that this steroid either reduces T4 secretion from the thyroid and, thus, conforming to its eVect in salmonids, or that it increases clearance of this hormone. It has also been suggested that changes in circulating levels of TH, such as that indicated by Hornsey (1977) for T4 in female P. marinus, could be associated with changes in metabolic rhythms and behavioral patterns, such as daytime activity replacing nocturnal habits (Hardisty and Potter, 1982). The review by Plisetskaya et al. (1983b) still remains as the only compilation and evaluation of studies on intermediary metabolism and TH during the upstream migration of lampreys. At that time, and up to the present date, there are no clear views of the roles of hormones in intermediary metabolism that could explain the shifts in TH profile shown by Sower et al. (1985) during final sexual maturation. It seems that TH has some involvement, perhaps through interaction with other hormones, in regulation of glycemic levels during the nontrophic phase of upstream migration in L. tridentata (Plisetskaya et al., 1983a). The authors of a study on G. australis account for the decline in T3 levels as linked to metabolic changes rather than related to reproduction and provide data from spawning salmonids to support their viewpoint (Leatherland et al., 1990b). Eales and Brown (1993) have emphasized that even short‐term starvation in teleosts results in markedly reduced hepatic T4ORD activity and, hence, potentially reduced T4 to T3 conversion. A total absence of hepatic monodeiodinase activity in upstream migrating G. australis after 5 months of their 16‐month migration is provided as a possible explanation for the progressive declining levels of T3 and the constancy of T4 in the serum (Leatherland et al., 1990b). Similarly, T4ORD activity is undetectable in microsomes of liver of upstream‐migrant P. marinus, but instead is highest in microsomes from intestine and low in muscle and kidney (Eales et al., 1997). Therefore, it is possible that T4 to T3 conversion also takes place in the intestine of upstream‐migrant G. australis; a drop in T3 production would occur over the long migration as the intestine undergoes atrophy. Upstream‐migrant P. marinus also shows T4IRD and T3IRD activities in kidney, but particularly in the intestine; no T3ORD is detectable in any tissue (Eales et al., 1997). Recalling that T4ORD was the only deiodinase found in larval P. marinus, the shift in tissue distribution of deiodinase activity and the potential for inner‐ring deiodinase pathways

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provides a mechanism for regulating the various forms of TH in upstream migrants. Since there is an apparent absence of pituitary control of the thyroid in adult lampreys, the control of the tissue supply of the more biologically active TH, T3, may be achieved through peripheral regulation by the deiodination pathway (Eales and Brown, 1993). As was mentioned with hagfishes, adult lampreys seem to support the hypothesis that thyroid gland function is regulated by metabolism and target tissue response, rather than by TSH and, thus, they use an ancient mechanism (DickhoV and Darling, 1983). 3.3. Gnathostomes It has been stressed by others, for example Leatherland (1994), that, in general, a follicular thyroid gland is similar in form throughout the jawed fishes. Unlike the mammalian thyroid, however, the teleost thyroid does not usually form a discrete mass and supposed functional heterotopic follicles can be found within kidney tissue, heart, brain, intestine, and several other regions (Chavin, 1956). Thus, knowledge of the distribution of thyroid follicles in non‐teleost bony fishes would seem to be of great value to our understanding of the phylogenetic development of the thyroid gland, particularly in light of what we are beginning to understand about the homologies between thyroid tissue function in vertebrates and protochordates (Section 2.4). The thyroid follicular cells of fish operate in the production of TH in a manner that is found in mammals and all the biologically active and inactive forms of vertebrate TH are identical (Eales, 1997). Despite this, we have already observed some unusual characteristics of agnathan thyroglobulin to that seen in mammals, but less information is available on this subject in jawed fish with an ancient lineage. The following subsections on the groups of ancient jawed fishes that have been considered in earlier parts of this chapter (Section 2) will not be uniform in the breadth of their content, or will there be the depth of subject matter provided for agnathans, because, simply, there is no information on certain subjects. An attempt has been made to search the literature on the thyroid gland of ancient bony fishes for the past 70 years and the following sections provide the results of this search. 3.3.1. Sarcopterygii Although there were a few investigators who preceded him in their curiosity about the dipnoan thyroid gland, Chavin (1976) provided the most comprehensive, multispecies study of this endocrine gland in lungfishes. Australian (N. forsteri), South American (Lepidosiren paradoxa), and African (P. annectens) lungfishes show radioiodine accumulation in only a cephalic region underneath the pharynx that corresponds to a discrete and

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compact thyroid gland; on the basis of radioiodine location, there are no heterotopic follicles like in many teleosts. In N. forsteri, there is a single, ovoid medial mass, but in the other two species, the thyroid is flattened and bifurcated into two connected lobes. The glands are highly vascularized and the thyroid follicles are of various sizes but with a luminal colloid staining with PAS. Chavin (1972, 1976) also examined the thyroid gland of a single coelacanth, L. chalumnae, and the gland is encapsulated and has an ovoid form like that of N. forsteri. The gland is also highly vascularized with various sized follicles. It is not known whether there are heterotopic follicles. Chavin (1976) has proposed that the sarcopterygian thyroid is a forerunner to the tetrapod gland and that the single ovoid gland, as presented in N. forsteri and L. chalumnae, preceded the more advanced bifurcated glands, as represented in L. paradoxa and P. annectens. We have no information on thyroid physiology in the coelacanth and that from lungfishes has primarily been focused on interesting aspects of development and evolution in N. forsteri (Joss, 1998, 2006). Studies of the lungfish thyroid during development indicate that the gland appears only at hatching and this information, in conjunction with data from methimazole‐treated animals, suggests that the thyroid axis acts like that seen in premetamorphic amphibians and that lungfish may be neotenic (Joss et al., 1997; Joss, 1998). In a review, Joss (2006) has indicated that N. forsteri shows all the characters of the thyroid axis that are responsible for neoteny in urodele amphibians. That is, there are no detectable levels of TH in serum of juvenile N. forsteri and there is an inability of pituitary thyrotropes in several age classes to respond to these low levels of serum TH by releasing TSH. In contrast, 1.0 IU of bovine TSH is stimulatory to radioiodine metabolism in the thyroid of the African lungfish (Gorbman and Hyder, 1973). The picture of TRs and the role of hypothalamic, TRH are less clear in N. forsteri. Immunohistochemistry, with heterologous antibodies, on the liver of 40‐week‐old N. forsteri indicates that TRa, and not TRb, may be a character of a lungfish neoteny (Joss et al., 1997). Joss (2006) did not discuss TRH in N. forsteri, but Gorbman and Hyder (1973) showed no stimulatory action of synthetic TRH on iodine metabolism of the thyroid of the African lungfish. Autoradiographic studies indicate that TRH receptors are located mostly in acellular sites throughout the central nervous system of P. annectens with the higher concentrations in the olfactory bulb and telencephalon rather than in the diencephalon, the general region of the pituitary (Pack et al., 1989). The role of iodothyronine deiodinases in N. forsteri has received considerable attention (Joss, 2006; Sutija and Joss, 2006). There are three well‐ characterized deiodinase enzymes in vertebrates: type I (D1) that has both ORD and IRD activity, type II (D2) that has exclusive ORD activity, and type III (D3) that has IRD activity (Sutija and Joss, 2006). Full‐length cDNAs of D2 and D3 have been cloned from liver of juvenile N. forsteri and their

426

JOHN H. YOUSON

Killifish

100

Rainbow trout Lungfish Frog 100

99 African clawed toad Chicken

98 95

Mouse 100 100

Rat Human

Fig. 8.12. Phylogenetic analysis of deiodinase type II sequences emphasizes the relationship of the Australian lungfish (bold) with amphibians, relative to the teleosts and birds and mammals. The tree was generated by the Neighboring‐Joining method and bootstrap values (100%) are shown as percentage. [From Sutija et al. (2003).]

deduced amino acid sequences compared with those of other vertebrates (Sutija et al., 2003, 2004). A phylogenetic analysis of amino acid sequences of lungfish D2 with those of other vertebrates shows taxonomic and evolutionary relationships that are similar to those that arise from a variety of nonmolecular parameters (Sutija et al., 2003). Lungfish D2 is grouped most closely with frog and Xenopus (Figure 8.12). There is a truncated form of D2 in the brain of N. forsteri and diVerent length D2 mRNA transcripts in liver, kidney, heart, and gills. Northern blot and real‐time PCR indicate low expression of the lungfish D2 mRNA transcripts in a variety of tissues in juveniles. In contrast, lungfish D3 mRNA is expressed in liver, lung, kidney, brain, heart, and gills of juvenile N. forsteri and has all the characteristics of this exclusive IRD enzyme seen in other vertebrates (Sutija et al., 2004). T3IRD activity is highest in microsomes from liver with lesser activity in the brain and kidney and no activity in other tissues showing D3 mRNA expression (Sutija et al., 2003). These data that show a higher activity of D3 over D2 in juvenile N. forsteri are interpreted as indicating a possible low conversion of T4 to T3 and a higher conversion of any T3 that is being formed to T2 that is likely cleared from the organism (Joss, 2006). The impact of this iodinase activity, likely in conjunction with the other elements of the thyroid axis described above, would be a

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limited amount of T3 available to trigger many prominent developmental changes in juveniles. To Joss (2006), this thyroid profile places juvenile N. forsteri in a comparable position to urodele amphibians that are obligate neotenes. 3.3.2. Actinopterygii a. Polypteriformes and Acipenseriformes. Chavin (1976) quoted a study by Thomopoulos (1951) that the thyroid gland of the polypterid, Polypterus senegalus, is an encapsulated focal mass without heterotopic follicles. In this way, the thyroid of the polypterid is closer to that of dipnoans and the coelacanth, rather than that of teleosts. To the author’s knowledge, there are no detailed descriptions of thyroid tissue in sturgeons and paddlefish, the Acipenseriformes. All the available information on thyroidology of acipenserids seems to be focused on sturgeon. Plohman et al. (2002a) have indicated that the thyroid gland is localized (as seen by accumulation of radioiodide) in the Lake sturgeon, Acipenser fulvescens, ‘‘in the same basibranchial region as in salmonids.’’ This latter description of ‘‘localization,’’ however, suggests a compact thyroid for sturgeon that would be unlike the thyroid in most teleosts, including salmonids. In the context of iodide in sturgeon, Leloup (1970) recorded that A. fulvescens has some of the lowest levels of plasma iodide for any fish; these data are used to explain some of the peculiarities of deiodinase activity in this species (Plohman et al., 2002b). As might be expected from the long‐established interest in Russian sturgeon, Acipenser gu¨ldenstadti, as a food commodity and then for survival of its populations, that most of the early studies on sturgeon thyroid came from this species or Acipenser stellatus (Gerbil’ski and Zaks, 1947). In this latter study, the eVects of T4 on postembryonic development are shown. Yakovleva (1964) was cited by Boiko et al. (2004) as providing some detail of the ontogeny of the thyroid gland during the larval period of the sturgeon. At hatching, the thyroid gland is an undiVerentiated anlage (Yakovleva, 1964), despite the fact that tissue levels of TH increase at this time (Boiko et al., 2004). A measurement of tissue levels of T4 and T3 in eggs, embryos, prelarvae of A. gu¨ldenstadti indicates that eggs supply TH necessary for early development but eventually their TH content decreases (Boiko et al., 2004). A rise in T4 levels occurs prior to their active feeding so, therefore, the thyroid gland must be actively secreting this hormone. The authors of this developmental study summarize their data by inferring that the TH profile of developing sturgeon more closely resembles that in teleost fish and amphibians that have a metamorphosis (Boiko et al., 2004). In the context of high TH content in sturgeon eggs as indicated above, it is noteworthy that T3 has some influence on ripening of oocytes in stellate sturgeon, A. stellatus (Detlaf and Davydova, 1974).

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Reversing of degenerative changes in the ovary due to captivity takes place following TH administration (Norris, 1997). Prior to the recent major studies on thyroidology in both cultured, immature‐phase and wild, spawning‐phase lake sturgeon, A. fulvescens (Plohman et al., 2002a,b), there was only some limited information that serum T4 and T3 are of low levels and show seasonal variability in the white sturgeon, Acipenser transmontanus (McEnroe and Cech, 1994). In both immature‐ and most spawning‐phase A. fulvescens, T4 (
0.3 ng/ml) and T3 (
0.2 ng/ml) serum concentrations are low but the high values of T4 (2.9 ng/ml) and T3 (
9.7 ng/ml) in some spawning‐phase animals suggest a role of TH in sturgeon during spawning (Plohman et al., 2002a). Plasma‐free T4, T3, and rT3 in sturgeon are higher than in rainbow trout and suggest that there is less TH binding to plasma carrier proteins in acipenserids (Figure 8.13). Furthermore, most extrathyroidal tissue levels of T4 and T3 are much (10–100 times) lower than in salmonids. On the other hand, there is an extremely high T3:T4 ratio (T3 exceeds T4 content by 10.6‐fold) in the thyroid implying that, in contrast to present teleost data, the gland may be a direct source of T3 for transport in the serum; that is, extrathyroidal T4ORD activity may be a less important source of T3 (Plohman et al., 2002a). Lake sturgeon, A. fulvescens, demonstrates T4ORD, T4IRD, T3IRD, and rT3ORD activities in the liver and intestine and T4IRD and T3IRD activities appear in the brain (Plohman et al., 2002b). The thyroid gland shows only

4.0 Free plasma hormone (%)

*

Lake sturgeon Rainbow trout

3.5 3.0 2.5 2.0 1.5

*

1.0

*

0.5 0.0

T4

T3

rT3

Fig. 8.13. Mean (SEM) percentages of thyroxine (T4), triiodothyronine (T3), and reverse T3 (rT3) in the free fraction of plasma from immature, cultured sturgeon (A. fulvescens), solid bars, and rainbow trout (O. mykiss), open bars. Asterisk (*) indicates a diVerence ( p < 0.05) between trout and sturgeon. [From Plohman et al. (2002a).]

8.

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PERIPHERAL ENDOCRINE GLANDS. I.

B

A

3.0

0.8 0.7 0.6 0.5 0.4

*

0.3 0.2 0.1

Thyroid hormone content (ng/g)

Plasma thyroid hormone (ng/ml)

0.9

2.5 2.0 1.5

*

1.0 0.5 0.0

0.0 T4

T3

T4

T3

Fig. 8.14. The eVects of diets of trout pellets (solid bars) and ocean zooplankton (open bars) on thyroxine (T4) and triiodothyronine (T3) levels in the (A) plasma and (B) diet of immature, cultured lake sturgeon, A. fulvescens. DiVerences (p < 0.05) between the two diets are indicated by an asterisk (*); n ¼ 12. [From Plohman et al. (2000b).]

T4ORD activity and explains the source of high tissue T3 (Plohman et al., 2002a). Administration of T3 in the diet doubles plasma levels of this hormone and decreases hepatic T4ORD activity but aVects no deiodinase pathway in the liver, intestine, and brain. A change in diet from trout pellets to ocean zooplankton (Plohman et al., 2002b) reduces serum T3 levels (Figure 8.14) and increases hepatic and intestinal T3IRD and hepatic rT3ORD activities but does not change activity of T4ORD in the intestine or liver (Figure 8.15). Thus, reduced serum T3 in a diet change is due to an increase in T3IRD in the liver and intestine, but particularly in the latter organ, that results in production of T2 for clearance. Many of the peculiarities of deiodinase activity in the lake sturgeon, relative to those in teleosts, may be explained by the freshwater habitat throughout the life cycle of the former and its need to conserve iodine for reutilization (Plohman et al., 2002b). b. Amiiformes and Semionotiformes. To the author’s knowledge, there are no detailed morphological descriptions of the thyroid glands of adult bowfin, Amia calva, or any of the gar (Lepisosteus) species. In a description of the early development of the thyroid gland in the bowfin, the adult gland is referred to as a group of follicles scattered in a venous plexus in the pharyngeal region, around the ventral aorta (Hill, 1935). This site for the thyroid gland in adult bowfin conforms to that for teleosts, but it does not tell us whether there are heterotopic follicles like many members of this latter group. Hill (1935) has described how the thyroid arises as a solid median primordium from the pharynx, detaches, and migrates to its definitive position ventral to

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JOHN H. YOUSON

the aorta and between the third branchial pouches. Primary follicles appear during this migration of the detached primordium and secondary follicles seem to arise after some later fragmentation of the primordium. Now that accurate staging is available for bowfin embryogenesis to hatching (Ballard, 1986), it would be interesting to have a more detailed account of thyroid ontogeny in this species. We have no details on function of the bowfin thyroid gland, the levels of circulating TH, or on a hypothalamic‐pituitary‐thyroid axis. However, intraperitoneal injections of T3 reduce oxidation of some substrates by mitochondria isolated from liver (Ballantyne et al., 1992). Therefore, we might anticipate that, based on the eVects of T3 after 3 h postinjection in this experiment, this hormone has at least some short‐term eVect on metabolism in bowfin. It was mentioned above that we know little about the thyroidology of gar. However, this void is likely to change given the interest in Lepisosteus spp. in the past few years. There has been increased focus on the plight of gar species in the southern United States and in Mexico, Cuba, and Central America

A

2.0

*

pmoles hormone deiodinated/h/mg protein

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4

*

0.2 0.0 T4ORD

T3IRD

T4IRD

rT3ORD

8.

431

PERIPHERAL ENDOCRINE GLANDS. I.

B

0.20

pmoles hormone deiodinated/h/mg protein

0.18 0.16 0.14 0.12 0.10

*

0.08 0.06 0.04 0.02 0.00 T4ORD

T3IRD

T4IRD

rT3ORD

Fig. 8.15. The eVects of diets of trout pellets (solid bars) and ocean zooplankton (open bars) on mean (SEM, n ¼ 12) T4ORD, T3IRD, T4IRD, and rT3ORD activities in (A) liver and (B) lower intestine of immature, cultured lake sturgeon, A. fulvescens. An asterisk (*) indicates a diVerence (p < 0.05) between the diets for the diVerent deiodination pathways. [From Plohman et al. (2000b).]

due to overfishing and environmental pollution (Orlando and Guillette, 2001; Mendoza et al., 2002a; Theodorakis et al., 2006). The alligator gar (Atractosteus spatula), for example, is the largest freshwater fish inhabiting the rivers around the Gulf of Mexico, and it is prized for its size and the quality of its meat. The population is drastically declining because of commercial and sport overfishing (Mendoza et al., 2002a). As a means of counteracting this decline, there have been eVorts to understand the factors that aVect growth and development in laboratory‐reared animals and the physiological eVects of the pollutants. Some information on the thyroid system is coming from these latest investigations. Unfortunately, we still lack some base‐line information on thyroid physiology, such as that recently provided by another threatened ancient fish, the sturgeon (Section 3.2.2.a). Mendoza et al. (2002a,b) referred to A. spatula has having a metamorphosis in its life cycle, but the direct development seen between the newly hatched

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JOHN H. YOUSON

fry and the juvenile would not be considered a true fish metamorphosis (Youson, 1988). Despite this matter of semantics, exogenous TH can influence development and body concentrations of TH increase after hatching in this species. T3 concentrations in eggs and embryos just before hatching are negligible but are 2.22 ng per larva 10 days after hatching (DAH). A subsequent decline (0.3 ng per larva) in concentrations of T3 by 13 DAH is the rationale for the authors to suggest that 10 DAH is the ‘‘metamorphic climax.’’ It is quite possible that T3 levels at 10 DAH reflect the beginning of TH production by a thyroid gland that has just completed development and a subsequent decline reflects peripheral use and/or regulation of the hormone. T3 when administered to fry increases body concentrations of this hormone and there is some increase in the rate of development (Mendoza et al., 2002b). Thiourea reduces the length of the developing snout, whereas T3 accelerates snout development. Since thiourea does not suppress growth and survival but a shorter snout reduces the ability to feed on larger prey, this goitrogen is suggested as a means of preventing cannibalism in culturing of gar fry (Mendoza et al., 2002b). In contrast, another putative goitrogen in the environment, perchlorate, accumulates in the body musculature of the spotted gar, Lepisosteus oculatus, in a lake in east‐central Texas, United States. The concentrations of perchlorate found in this sturgeon are suYcient to prevent metamorphosis in amphibians, and they impact on the normal histology of the thyroid gland of other fish species (Theodorakis et al., 2006). c. Basal Teleosts. Descriptions of thyroid tissue and hormones of Osteoglossiformes are not presently available. Although one would anticipate that the thyroid gland would have significance to studies of metamorphosis in bonefish, there does not seem to be any information on thyroidology at any period of the life cycle of the Elopiformes. Data from eels, the Anguilliformes, are abundant and I refer the readers to the early literature cited in the reviews on this fish thyroid gland at the beginning of this section. The early literature will be referenced when it is required to provide baselines before introducing the data from more recent studies. Interpretations of the earlier literature on the fish thyroid by Raine et al. (2005) give the impression that the thyroid gland of Anguilla spp. is composed of ovoid follicles that are often large and branching. Although the gland is primarily confined to the subpharyngeal region in the conger eel, Conger myriaster (Yamano et al., 1991), heterotopic thyroid follicles are reported in the renal tissue of the Indian mud eel, Amphipnous cuchia (Srivastava and Sathyanesan, 1967). Larval eels undergo a true fish, first metamorphosis (Youson, 1988) and TH plays a principal role in this event (Yamano et al., 1991; Ozaki et al., 2000). In the conger eel (C. myriaster), the thyroid gland is activated in early metamorphosis as evidenced by increased

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cell height (Yamano et al., 1991), while in the Japanese eels, Anguilla obscura and Anguilla bicolor pacifica, the thyroid follicular cells of metamorphosing animals immunostain for T4 before there is evidence of TSH immunoreactivity in the pituitary (Ozaki et al., 2000). By the end of metamorphosis, the follicular epithelial cells are squamous in shape and reflect a low level of activity (Yamano et al., 1991). The influence of TH in development is shown in conger eel by the dramatic increase in whole‐body T4 concentration between premetamorphosis (5 ng/g) and early metamorphosis (30 ng/g) and whole‐body T3 levels between premetamorphosis (0.15 ng/g) and late metamorphosis (2.0 ng/g). A similar pattern for these TH concentrations occurs in the European eel, A. anguilla (Jegstrup and Rosenkilde, 2003). Cortisol levels in C. myriaster decline over this period as well, but the biggest decline occurs before metamorphosis. In A. anguilla, the final stages of metamorphosis are marked by a characteristic pigmentation pattern and change from pelagic to benthic behaviors that are strictly controlled by TH (Jegstrup and Rosenkilde, 2003). The immersion of metamorphosing larvae in T4 increases the rate of pigmentation while thiourea reduces the rate. One of the primary explanations for the presence of metamorphosis in the life cycle of fishes is larval dispersal. A recent laboratory investigation concludes that TH and its influence on locomotor activity have had a central role in colonization of glass eels in its continental habits (Edeline et al., 2005). T4 immersion increases whole‐body levels of this hormone (from
11.5 to
105 ng/g) and this hyperthyroid state increases locomotor activity, whereas thiourea immersion does not eVect body T4 levels but has the converse eVects on locomotion (Figures 8.16 and 8.17). In addition, T3 levels are significantly elevated in T4‐treated fish indicating that exogenous T4 must be converted to T3, whereas thiourea‐treated fish have lower whole‐body T3 compared to controls, indicating inhibitory action of this goitrogen on T4ORD activity (Figures 8.16 and 8.17). The data show a strong influence of TH on rheotactic behavior in experimental flume tanks (Figure 8.18) suggesting that TH may operate by controlling migration through a direct eVect on olfactory processes (Edeline et al., 2004, 2005). There is a second marked period during the life cycle of an eel (yellow to silver eel) during which time the animal undergoes some morphological and physiological changes that prepare the organism for migration back to a marine environment. This period has often been equated with the salmonid, parr‐smolt transformation that involves THs (Hoar, 1988) and has been referred to as a second metamorphosis or a second type of metamorphosis (Youson, 1988). However, a recent investigation on A. anguilla at this silvering interval of the life cycle has concluded, from examining profiles of various hormones of the reproductive and thyroid axes, that it is more of a pubertal event than one of a genuine metamorphosis (Aroua et al., 2005).

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A 150 b

T4 ng/g

100

50 a 0

B

C

a T4

TU

9 8 7

b

T3 ng/g

6 5 4 3

a c

2 1 0

C

T4

TU

Fig. 8.16. Whole‐body thyroxine (T4) and triiodothyronine (T3) levels (mean  SD) in glass eels of A. anguilla in control (C), T4‐treated (T4), and thiourea‐treated (TU) groups; n ¼ 15 for each group. Significant diVerences between the groups are indicated when the letters are diVerent. [From Edeline et al. (2005).]

In contrast to the many stimulations to the reproductive axis (e.g., increases in GTH mRNA, estradiol, several androgens, and vitellogenin), no pronounced changes are evident in the thyroid axis (relatively stable TSH mRNA and plasma TH levels). Furthermore, chronic treatment with T4 has no eVect, but testosterone stimulates developmental change in the eye and digestive tract. Pradet‐Balade et al. (1998) showed from implants of T4 and T3 in silver eel, A. anguilla, that these TH decrease TSHa‐ and TSHb‐mRNA levels. Support for the view that TH exerts a negative feedback on the expression of mRNAs

8.

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PERIPHERAL ENDOCRINE GLANDS. I.

Percentage of locomotor activity

60 50

b

40 30

a

c

20 10 0

C

T4

TU

Fig. 8.17. EVect of thyroxine (T4) and thiourea (TU) on locomotor activity of glass eels of A. anguilla in experimental flume tanks. Data are expressed as mean  SD of the average percentages of locomotor activity (upstream and downstream swimming/total number of glass eels; n ¼ 8 trials per treatment or control and 40 glass eels per trial. Significant diVerences between the groups are denoted by diVerent letters. [From Edeline et al. (2005).]

encoding TSH subunits is found in experiments where silver eels are treated with thiourea for 3 months. These thiourea‐treated eels show depressed circulating TH but increased a‐ and TSHb‐mRNA levels. In vitro studies on cultured eel pituitary indicate that the main regulation of TSH expression is exerted by control of the specific b‐subunit (Schmitz et al., 1998). There is no information on deiodinase activity in the eel but cDNA cloning has provided a picture of the sequences of two distinct TRs, cTRb1 and cTRb2, in the conger eel, C. myriaster (Kawakami et al., 2003). Each of these TRs possess a splice variant so there are at least four TR isoforms in conger eel. The cTRb1 mRNA is widely expressed but is most prominent in the brain, pituitary, and kidney, while cTRb2 mRNA expression is less widespread but abundant in brain and kidney. To avoid confusion with mammalian TRb2, Kawakami et al. (2007) proposed that the conger eel TRs, cTRb1 and cTRb2, be redefined as cTRbA and cTRbB, respectively. The Japanese eel, Anguilla japonica, possesses a TRbB that has high sequence homology with that of the conger eel and also shows most pronounced expression in the brain and the pituitary (Kawakami et al., 2007). It is noteworthy that TRbB in conger and Japanese eel has an amino acid insertion that is also seen only in the TRbs in the Japanese flounder, Paralicththys olivaceus (Yamano et al., 1994; Yamano and Inui, 1995), when sequences comparisons are made with other vertebrates. Since the eels (Anguilliformes) and the Japanese flounder (Pleuronectiformes) are representative of a basal

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JOHN H. YOUSON

A Percentage of upstream swimmers

20 b 15 a 10

a

5

0

C

T4

TU

40 Percentage of downstream swimmers

B

b 30

20

a

c

10

0

C

T4

TU

Fig. 8.18. EVect of thyroxine (T4) and thiourea (TU) on rheotactic behavior of glass eels of A. anguilla in experimental flume tanks. Data are expressed as mean  SD of the average percentages of upstream (left panel) and downstream (right panel) swimmers/total number of glass eels; n ¼ 8 trials per treatment or control and 40 glass eels per trial. DiVerent letters indicate a significant eVect of the treatments on rheotactic behavior. [From Edeline et al. (2005).]

and a derived order, respectively, of division Teleostei, this amino acid insertion might be common to all teleosts and/or have either phylogenetic or functional significance. Some of the studies listed above for the eel, A. anguilla, stress the importance of endocrine knowledge of this species because of decline in populations (Edeline et al., 2005). The eel has not escaped the eVects a variety of contaminants in their natural environment. Heavy metals such as chromium

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(Cr) and copper (Cu) are essential for a variety of physiological processes. However, a study on yellow eels of A. anguilla shows that T4 decreases are more prominent than any alterations that are evident in either cortisol, TSH, or T3 levels when the animals are exposed to concentrations of Cr and Cu; these heavy metals are known to inhibit some metabolic activity of hepatocyte microsomes (Teles et al., 2005). Although the target of the heavy metals is not known, the lack of eVect on T3 suggests that they inhibit TH production and/or disrupt deiodination or clearance rates (Teles et al., 2005). 3.4. Phylogenetic Considerations The follicular thyroid glands of hagfish, adult lamprey, and the bony fishes of ancient lineage discussed in this chapter are of similar structure. DiVerences lie within the extent of their vasculature, whether there are heterotopic follicles, and the degree of compactness of the gland. The poor vascularization of the agnathan glands is likely reflecting an ancient feature of reliance on intestinal absorption and deiodination of synthesized hormone. Heterotopic thyroid tissue seems to be a more derived feature in bony fishes as it still questionable in the basal teleosts (e.g., eel), it is present in many euteleosts and is likely absent in the non‐teleost, basal actinopterygians such as the Amiiformes, Semionotiformes, Polypteriformes, and the Acipenseriformes. The phylogenetic significance of the tubular profiles of thyroid tissue in bony fishes (Raine et al., 2005) needs further study. There has been insuYcient study on thyroid physiology of the basal actinopterygians, but it is noteworthy that the sturgeon has higher levels of free TH in the blood than salmonid teleosts (Plohman et al., 2002a). There is also some suggestion that there may be greater reliance on a thyroid gland T4 to T3 conversion than in the thyroid of salmonids (Plohman et al., 2000b). Whether there is any phylogenetic significance to these features needs to be explored. The feature of the thyroid tissue structure and function in the ancient fishes under study in this chapter that has the most relevance to the phylogeny of the vertebrate thyroid gland is the presence of the endostyle in larval lampreys. This gland undergoes transformation to a follicular thyroid gland during lamprey metamorphosis. Although another ancient fish, the eel, has a metamorphosis in the life cycle, the leptocephali do not have an endostyle but a follicular thyroid. Some extended discussion of the phylogenetic significance of the lamprey endostyle and the evolution of lamprey metamorphosis is provided below. As the author has emphasized elsewhere, metamorphosis is not a developmental strategy that is well represented among the fishes (Youson, 1988, 2004). There does not seem to be any clear explanation for the selection of

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metamorphosis during the evolutionary history of those fish species that have this developmental strategy in their present life history. Metamorphosis is present in both basal (Anguilliformes, Elopiformes, Notacanthiformes) and derived (Pleuronectiformes) teleosts and in lampreys but not hagfishes or more generalized teleosts. The most prominent common feature of fish metamorphosis is their dependency on TH, but in a somewhat diVerent manner in lampreys and teleosts, and that it is proceeded by a free‐living, larval phase where some growth and larval dispersal is obtained (Youson, 2004). The fossil record of lampreys has not provided any clue of when metamorphosis appeared in the life history of this agnathan group. Eales (1997) and Johnson (1997) have emphasized the long history of dependence of multicellular organisms for some ability to concentrate iodide in one or several tissues; ultimately, there was the production of iodoproteins. Others have illustrated how hormones, such as thyroxine, function and have guided development and evolution, particularly in coordinating life‐history transitions and their evolution (Heyland et al., 2005). It appears that thyroxine first appeared in animals near the base of the animal kingdom as a metabolic waste product in a process that evolved to produce iodide and tyrosine from iodine to form an eYcient oxidant defense system shielding tissues against damage by reactive oxygen species (Berking et al., 2005). This latter study supports the long‐held view that the potential for evolution of a thyroid gland may have had its origins 3 billion years ago in blue‐ green algae (Cyanobacteria) where seawater iodine species, iodate (IO3–) and iodide (I–), were used in a protective antioxidant action (Venturi and Venturi, 1999). The fact that the larval lampreys likely absorb TH through the intestinal epithelium is not surprising since sea urchin larvae obtain TH from ingested plant diatoms (Chino et al., 1994). The most direct connection of the lamprey thyroid to thyroid‐like functions in invertebrates is the larval endostyle, for it is also the primary iodide‐binding organ of extant protochordates (Eales, 1997). T4 has been located within extracts of the urochordate, Ciona intestinalis (Patricolo et al., 2001). However, the most convincing evidence of homology between the protochordate and larval lamprey endostyles has come through molecular biology (Ogasawara et al., 1999; Ogasawara, 2000), although there is still a question whether some molecular pathways might be recruited from diVerent regions of the pharyngeal endoderm (Mazet, 2002). More recent evidence using in situ hybridization and probes for thyroid‐related transcription factors, such as TTF‐1, TTF‐2/FoxE4, and Pax2/5/8, and forkhead transcription factors (FoxQ1 and Fox A) to name just a few, suggest that the ascidian and amphioxus endostyles reflect that the genetic basis of thyroid function was in place before the appearance of the first vertebrates (Ogasawara

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et al., 2001; Hiruta et al., 2005). In essence, the endostyles of protochordates and lampreys contain the same protothyroid (follicular thyroid) regions. On the basis of evidence from the endostylar homologies between protochordates and larval lampreys and on the plasticity of lamprey metamorphosis with respect to environmental cues (Youson, 1999), it has been proposed that the first lampreys that predated those in fossil records (Potter and Gill, 2003) were free‐living, marine, and larval‐like in form (Youson, 2004). They had an endostyle and were possibly paedomorphic (Youson and Sower, 2001). Thus, the first lampreys were very much like the primitive urochordates, the Larvacea, which are free‐swimming adults but possessing some larval features. According to Eales (1997), the follicular thyroid evolved in aquatic vertebrates at the time that they moved from iodide‐rich saltwater to freshwater that was relatively poor in iodide. The lumen of the follicles provided the site for storage of the essential iodine necessary for tyrosine iodination. Many of the fossil lampreys are present in marine deposits but because of their ‘‘soft anatomy’’ we know little of their internal morphology or have any absolute certainty of whether they represent pre‐ or postmetamorphic animals (Potter and Gill, 2003). We are aware of experimental evidence and serum TH data from extant larval lampreys, with an endostyle, that they have great capacity for production and ‘‘storage’’ of high concentrations of TH (Section 3.2.2.a.). We also have evidence from other vertebrates, particularly anuran amphibians, that high concentrations of serum TH elicit developmental change (Rose, 2004) when the conditions are appropriate. Lamprey metamorphosis is influenced by water temperature and appropriate physiological preparation (Section 3.2.2.b). Putative, ancient lampreys, that were marine and larval‐like, entered warmer freshwater with high concentrations of serum TH, and consequently (based on evidence from extant larval lampreys), a later stage of ontogeny was initiated and developmental change took place. Since the protothyroid region of the pharyngeal endoderm was present even in ancestors of lampreys (Hiruta et al., 2005) and challenges of the need for iodine were compelling in the freshwater environment (Eales, 1997), eventually metamorphosis was ‘‘selected’’ as a developmental strategy. This developmental strategy permitted the appearance of a thyroid with follicles in postmetamorphic individuals. Because developmental change, such as that described in the endostylar transformation to a follicular thyroid gland, does not occur in isolation but is part of an integrated system of change aVecting other body parts, many other organ systems were influenced by this late stage of ontogeny (Youson, 1980, 2004). Included among these aVected systems in lampreys was the reproductive system (Youson and Sower, 2001) that has a long evolutionary relationship with the thyroid gland, and it was stimulated into further development at

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metamorphosis just like it does in extant lampreys (Youson and Sower, 1991; Youson et al., 2006). Also, there would be changes in the gills, alimentary canal, and kidney that would permit, such as in anadromous species, osmoregulation and feeding of an adult in a marine environment. These developmental changes would explain why many fossils, assumed to be juvenile adults, are present in marine deposits (Potter and Gill, 2003). 4. SUMMARY AND CONCLUSIONS This chapter provides the first review of the extensive research that deals specifically with the thyroid gland of the Agnatha and the ancient bony fishes. In addition, some recent views of the ontogenetic and phylogenetic development of the GEP endocrine system in agnathan, cartilaginous, and bony fishes are discussed in the context of the extant agnathan and ancient bony fishes. As the taxonomic scale is ascended from the agnathans through the basal actinopterygians to the basal teleosts, the tendency is for a simplified, nearly single‐cell type (insulin) islet organ with scattered small islets in hagfishes and larval lampreys to become specialized into a more compacted islet organ with four‐peptide, principal islets in Brockmann bodies in basal teleosts. In between are the basal, non‐teleost actinopterygians, such as the bowfin and gar, that provide an intermediate islet organ with diVusely scattered, three‐peptide islets. The classical distribution of the islet cell types, particularly the D cells, does not seem to reach the derived euteleost profile until the generalized teleosts such as the salmonids. Since insulin is often the first GEP peptide to appear in ontogeny of the pancreas with the later development showing the progressive appearance of other peptides, there has been reference to phylogeny of the fish pancreas reflecting ontogeny of the endocrine pancreas in more advanced species (Berwert et al., 1995). There are some relevant features of the primary structures of the GEP peptides that reflect both on intra‐ and inter‐group conservation and on intergroup diVerences that are consistent with phylogeny. For example, phylogenetic analysis of both insulin and SST in osteoglossomorph species provide support for their monophyly and their common origins prior to continental drift (Al‐Mahrouki, 2001; Youson et al., 2006). The general conclusion is that the GEP system has some utility as a parameter for studying the phylogeny and interrelationships of ancient fishes. The thyroid glands and THs of agnathans provide several interesting features that reflect on their ancient origins. Both adult lampreys and hagfishes have poorly vascularized, follicular thyroid glands and the intestine is the primary site of deiodination; this is the primary site of deiodination of TH in protochordates, whereas liver is a primary site in euteleosts. The endostyle

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of the larval lamprey has no follicles and is homologous to the endostyle of protochordates, but it concentrates thyroglobulin, binds radioiodine, and is the principal target for goitrogens that lower serum levels of TH. The absence of both thyroid follicles and good vascularization in the endostyle is compensated for by high deiodination activity of the intestinal epithelium and high capacity for storage of TH in the blood. Metamorphosis, which is speculated to be a derived feature in the lamprey life cycle, is characterized by an initial, dramatic drop in circulating TH, changes in deiodination pathways, and transformation of the endostyle to a follicular thyroid gland. Changes in the thyroid systems in lamprey metamorphosis are in concert with changes in lipid metabolism, the influence of GEP peptides, changing water temperature, and the diVerentiation of part of the reproductive axis. Existing data indicate that the thyroid glands of hagfishes and lampreys may not be controlled by a hypothalamic‐pituitary directive but are regulated by a more ancient mechanism concerned with the response of TH target tissues and the state of the intermediary metabolism (DickhoV and Darling, 1983). In contrast to the studies on the agnathan thyroid gland which have been driven by interest in the evolution of the gland and the role of TH in development, metabolism, and reproduction, recent interest in thyroid glands and hormones of other ancient fishes has been stimulated by a need to prevent the demise of the species. In both the sarcopterygians and the basal actinopterygians, we are just beginning to gain some information on their thyroidology. What we are seeing in the case of the dipnoans is a thyroid profile more closely akin to urodele amphibians. As we might expect from examining other endocrine parameters in the sturgeon, both the free hormone profile and deiodination pathways are not the same as even generalized teleosts. Information on the thyroids of polypterids, the paddlefish, the bowfin, the bonefish, and the bonytongues is virtually nonexistent. Data from the gar thyroid is focused on the eVects of environment and how we can improve laboratory culture of the Semionotiformes. There is a need to provide basic information on the structure, distribution, and products of the thyroid glands of these species under what we might consider a natural environment without the eVects of pollutants. The European eel, A. anguilla, has been a focus of attention for comparative endocrinologists for over a century but some recent studies, driven by both curiosity and a concern for species survival, have just shown how the thyroid profile eVects, or has no influence, on their very important migrations. At the same time, TH profiles in this eel species are influenced by specific concentrations of heavy metals that have lesser eVects on other endocrine systems. Needless to say, we need to include thyroid analysis of any studies that are examining hormones as a measure of stress in fish as a consequence of environmental contaminants.

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ACKNOWLEDGEMENTS I am most grateful to have had such devoted graduate and postdoctoral students who contributed much to the research from my laboratory that is reported in this chapter. I am also most appreciative of the long collaborations and friendships with Michael Conlon, Robert Dores, GeoV Eales, Michael Filosa, Hiroshi Kawauchi, John Leatherland, Erika Plisetskaya, Mark Sheridan, and Stacia Sower. I thank the Natural Sciences and Engineering Research Council of Canada and the Great Lakes Fishery Commission for research support over the years.

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Berwert, L., Segner, H, and Reinecke, M. (1995). Ontogeny of IGF‐1 and the classical islet hormones in the turbot, Scophthalmus maximus. Peptides 16, 113–122. Boiko, N. E., Vorob’eva, O. A., Grigor’yan, R. A., and Kornienko, G. G. (2004). Dynamics of thyroid hormones at early stages of development of the sturgeon, Acipenser gu¨ldenstadti. J. Evol. Biochem. Physiol. 40, 176–181. Brinn, J. E., Jr. (1973). The pancreatic islets of bony fishes. Am. Zool. 13, 653–665. Brown, C. L., Dashow, L., Epple, A. W., and Stetson, M. H. (1982). The thyroid hormone clearance kinetics in adult sea lampreys, Petromyzon marinus. Gen. Comp. Endocrinol. 47, 333–339. Chavin, W. (1956). Thyroid distribution and function in the goldfish, Carassius auratus L. J. Exp. Zool. 133, 259–279. Chavin, W. (1972). Thyroid of the coelacanth, Latimeria chalumnae Smith. Nature 239, 340–341. Chavin, W. (1976). The thyroid of the sarcopterygian fishes (Dipnoi and Crossopterygii) and the origin of the tetrapod thyroid. Gen. Comp. Endocrinol. 30, 142–155. Chino, Y., Saito, M., Yamasu, K., Suyemitsu, T., and Ishihara, K. (1994). Formation of the adult rudiment of sea urchins is influenced by thyroxine. Dev. Biol. 161, 1–11. Conlon, J. M. (1995). Peptide tyrosine‐tyrosine (PYY)‐An evolutionary perspective. Am. Zool. 35, 466–473. Conlon, J. M. (2000). Molecular evolution of insulin in non‐mammalian vertebrates. Am. Zool. 40, 200–212. Conlon, J. M., and Larhammar, D. (2005). The evolution of neuroendocrine peptides. Gen. Comp. Endocrinol. 142, 53–59. Conlon, J. M., Askensten, U., Falkmer, S., and Thim, L. (1988a). Primary structures of somatostatins from the islet organ of the hagfish suggest an anomalous pathway of posttranslational processing of prosomatostatin‐1. Endocrinology 122, 1855–1859. Conlon, J. M., Deacon, C. F., Hazon, N., Henderson, I. W., and Thim, L. (1988b). Somatostatin‐related and glucagon‐related peptides with unusual structural features from the European eel (Anguilla anguilla). Gen. Comp. Endocrinol. 72, 181–189. Conlon, J. M., Goke, R., Andrews, P. C., and Thim, L. (1989). Multiple molecular forms of insulin and glucagon‐like peptide from the pacific ratfish (Hydrolagus colliei). J. Exp. Zool. 73, 136–146. Conlon, J. M., Andrews, P. C., Thim, L., and Moon, T. W. (1991a). The primary structure of glucagons‐like peptide but not insulin has been conserved between the American eel, Anguilla rostrata and the European eel, Anguilla anguilla. Gen. Comp. Endocrinol. 82, 23–32. Conlon, J. M., Bjenning, C., Moon, T. W., Youson, J. H., and Thim, L. (1991b). Neuropeptide Y‐related peptides from the pancreas of teleostean (eel), holostean (bowfin) and elasmobranch (skate) fish. Peptides 12, 221–226. Conlon, J. M., Youson, J. H., and Whittaker, J. (1991c). Structure and receptor‐binding activity of insulin from a holostean fish, the bowfin (Amia calva). Biochem. J. 276, 261–264. Conlon, J. M., Youson, J. H., and Mommsen, T. P. (1993). Structure and biological activity of glucagon and glucagon‐like peptide from a primitive bony fish, the bowfin (Amia calva). Biochem. J. 295, 857–861. Conlon, J. M., Platz, J. E., Nielsen, P. F., Vaudry, H., and Vallarino, M. (1997). Primary structure of insulin from the African lungfish, Protopterus annectens. Gen. Comp. Endocrinol. 107, 421–427. Conlon, J. M., Fan, H., and Fritzsch, B. (1998). Purification and structural characterization of insulin and glucagons from the bichir Polypterus senegalis (Actinopterygii: Polypteriformes). Gen. Comp. Endocrinol. 109, 86–93. Conlon, J. M., Basir, Y, and Joss, J. M. P. (1999). Purification and charcterization of insulin from the Australian lungfish, Neoceratodus forsteri (Dipnoi). Gen. Comp. Endocrinol. 116, 1–9.

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9 PERIPHERAL ENDOCRINE GLANDS. II. THE ADRENAL GLANDS AND THE CORPUSCLES OF STANNIUS JOHN H. YOUSON

1. Introduction 2. Adrenal Glands 2.1. Background 2.2. Adrenocortical Homologue 2.3. ChromaYn Tissue 2.4. Prospective on the Adrenal Glands of Ancient Fishes 3. Corpuscles of Stannius 3.1. Background 3.2. Amiiformes 3.3. Semionotiformes 3.4. Basal Teleosts 3.5. Phylogenetic Considerations of the CS and STC in Fishes 4. Summary and Conclusions

The adrenal glands and the corpuscles of Stannius (CS) are two endocrine tissues that have at least one primary component of mesoderm derivation, and they are both intimately associated with the kidneys in neopterygian, ray‐ finned fishes. The first section of this chapter describes the distribution and products of the steroid‐synthesizing, adrenocortical homologue (AH) and of the catecholamine‐secreting, chromaYn tissue in agnathans (hagfish and lamprey) and in bony fishes of ancient lineage. A scheme is provided of the phylogeny of the AH among these fish groups with the broad distribution of putative AH in lampreys being closest to the ancestral condition. Emphasis is placed on the lack of evidence of the functional significance of the AH–chromaYn tissue topographical relationship among these and other bony fishes and the need for more data on catecholamines and corticosteroids as a measure of stress in threatened species. The second section on CS indicates the presence of these glycoprotein‐secreting glands in only neopterygians and 457 Primitive Fishes: Volume 26 FISH PHYSIOLOGY

Copyright # 2007 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(07)26009-1

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shows a phylogenetic trend in reduced numbers and concentration of CS from the basal, non‐teleost actinopterygians (bowfin and gar) toward the basal teleosts. Stanniocalcin (STC), the homodimeric product of the CS, has certain consensus amino acid sequences in glycosylation sites and cysteine residues in most fish species; the latter are important for intra‐ and intermonomeric linkages. This section describes the presence of a consistent cysteine mutation of the STC molecule among geographically isolated species of the ancient teleosts, the Osteoglossiformes. The value of these types of morphological and molecular data to support other taxonomic parameters in phylogenetic analyses is discussed. 1. INTRODUCTION This is the second of two chapters for this volume on the peripheral endocrine glands. As indicated in the previous chapter, peripheral is referring to the endocrine glands outside of the nervous system and the four glands under discussion are paired in the two chapters based on their common origins from specific germ layers. The topics in this chapter, the adrenal gland and the corpuscles of Stannius (CS), have at least a part of their definitive structures arising in mesoderm. The steroid‐producing cells of the adrenal gland are derived from lateral or splanchnic mesoderm, whereas the CS develops from embryonic renal tissue, which itself is a product of intermediate mesoderm. In fishes, the steroid‐producing cells that comprise one component of their adrenal tissue, likely arise from mesoderm‐derived cells that are part of or are near, the peritoneal epithelium (Chester Jones and Mosley, 1980); the CS of salmonids originates in the epithelium of the pronephric duct (Kaneko et al., 1992), but that of the Amiiformes appears from distal renal epithelium even in adult life (Youson et al., 1976). While the two glandular systems in the previous chapter, the gastroenteropancreatic system and the thyroid gland, produce hormones that sometimes interact in intermediary metabolism in fishes, there is no evidence of direct interaction of the principal products of steroid‐producing cells of the adrenal and the cells of CS. The adrenal glands and CS of fishes have an intimate association with the kidneys and, in some species, they are directly apposed to one another within the kidney tissue proper near the renal or posterior cardinal veins. Even the person for whom the CS is named, Stannius (1839), believed that they were the fish adrenals. Given their embryonic origins, topography with respect to the kidneys, and research history, it seems fitting that the fish adrenal glands and CS of ancient fishes should be discussed within the same chapter. A second component of adrenal gland of fishes will also be discussed in this chapter. Despite the origins of chromaYn tissue from neural

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tube/neural crest, there is a phylogenetic, and perhaps even a physiological, relevance to the association of the adrenal, steroid‐producing cells, and the catecholamine‐secreting cells that in higher vertebrates makes up the adrenal medulla. To date, there has not been a review that has been specifically concerned with the phylogenetic signficance of the distribution and function of chromaYn tissue in fishes of ancient lineage. Therefore, the section on the adrenal glands will include subsections on the adrenosteroid tissue and the adrenochromaYn tissue. 2. ADRENAL GLANDS 2.1. Background The term adrenal gland in vertebrates refers to a steroid‐secreting component (adrenal cortex in mammals or the AH equivalent) and catecholamine‐secreting cells (adrenal medulla in mammals or an equivalent chromaYn tissue). All vertebrates share the feature of having these two tissues near or within renal tissue, hence, the name adrenal or suprarenals. Although in amphibians, reptiles, birds, and mammals, the adrenocortical and chromaYn tissues are usually in close association to one another, in fishes there can be a wide dispersal of these two components (Balment et al., 1980; Henderson and Kime, 1987). In fact, if one ascends the taxonomic scale from agnathans, through jawed fishes and amphibians to the amniotes, there is a trend of highly diVuse intrarenal adrenocortical and vascular‐associated chromaYn cells to eventually form more compacted suprarenal glands where the two tissues are confined within a gland separate from the kidney. This phylogenetic trend has been highlighted through an examination of some of the more ancient of the extant fishes. 2.2. Adrenocortical Homologue Many researchers in fish and amphibian biology refer to the AH in these groups as the interrenals. This term arose following the observation of the AH ‘‘between’’ the kidneys of cartilaginous fishes (Chester Jones, 1987). To date, this interrenal position seems to be unique to cartilaginous fishes, for in all other fishes (and in amphibians) the AH is mostly ‘‘within’’ the kidneys, that is intrarenal. Like Henderson and Kime (1987), this author prefers the term AH and recommends that it should be adopted as the more correct designation once the homology has been established. Besides the recognition of the fine‐ structural features of steroid‐secreting cells, positive identity of the AH cells comes through their possession of the enzyme, 3b‐hydroxy‐
5‐steroid

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JOHN H. YOUSON OH

HO

HO Cholesterol

22a-Hydroxycholesterol OH OH

OH

HO 20a-Hydroxycholesterol

HO 20a,22a-Dihydroxycholesterol CH3

P450scc

C=O CH3 3b-HSD

C=O

HO Pregnenolone O

Progesterone

CH3 C=O HO

HO

CH3 C=O

P450c21

CH2OH C=O

O O O 17a-Hydroxyprogesterone 11b-Hydroxyprogesterone 11-Deoxycorticosterone P450b21

CH2OH

CH3

C=O HO

C=O HO

O 11-Deoxycortisol

HO

O

HO

Cortisol

CH2OH

CH2OH

CH2OH C=O

HOH2C C=O

HO

O 21-Deoxycortisol

P450c11

O

P450c11

O Corticosterone 18,21-Dihydroxyprogesterone

CH2OH

P450c11

HOH2C C=O HO

C=O OH

O 18-Hydroxycorticosterone P450c11 HO

CH2OH O

C=O CH

O Aldosterone

Fig. 9.1. Schematic presentation of the principal pathway for corticosteroid synthesis in adrenocortical tissue from cholesterol and through progesterone. Key enzymes are 3b‐hydroxysteroid dehydrogenase (3b‐HSD) and the cytochrome P450 enzymes, particularly P450c11 (11b‐hydroxylase) and P450c21 (21‐hydroxylase). [Reproduced from Norris (2007).]

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dehydrogenase (3b‐HSD). The latter is the catalyst for the conversion of pregnenolone to progesterone in corticosteroid synthesis (Figure 9.1). With suitable controls, a positive 3b‐HSD reaction during incubation of frozen sections in histochemistry is considered to be strong proof of the potential for steroidogenesis in presumptive adrenocortical tissue and that it is the AH. This particular character was emphasized by Nandi (1967) in his comparative survey of steroids in vertebrates, but particularly in species where the existence of the AH was questionable because of its diVuse distribution. There have been many reviews in the past on the vertebrate adrenal cortex, which have included the AH and its products in fishes. For the most part, the earliest reviews originated in the laboratories of the late Ian Chester Jones and David Idler. The reviews of Nandi (1967), Butler (1973), Chester Jones and Mosley (1980), Balment et al. (1980), and Henderson and Garland (1980) provide references to this earlier literature. To date, there has not been a review of the literature on the AH that has concentrated specifically on fishes with an ancient lineage. The review of the hypothalamic‐pituitary‐adrenal (HPA) axis and corticosteroids in fish concentrates on HPA organization and the physiological functions of the steroids in teleosts (Norris and Hobbs, 2006). 2.2.1. Agnathans a. Lamprey. The AH of the lamprey received considerable attention in at least four laboratories between 1970 and 1985. Identity of the cells and their products was the primary focus. Prior to 1970, there had been a preliminary identification of both cortisol and corticosterone in both lamprey and hagfish plasma (Chester Jones and Phillips, 1960; Chester Jones et al., 1962a,b), and it was concluded that corticosteroids appeared early in vertebrate evolution (Nandi, 1967). However, Weisbart and Idler (1970) undertook a more detailed analysis of lamprey and hagfish plasma, and incubated presumptive AH tissue from these organisms. These latter authors concluded that there is no cortisol and corticosterone in the plasma and the presumptive AH has no ability to transform radioactive precursors to these corticosteroids. These authors did recognize, however, that a major diYculty in their interpretation was their inability to identify AH cells in both lampreys and hagfishes. The reviews of lamprey AH are provided by Hardisty and Baker (1982) and Youson (1985). Hardisty (1972a, 1979) has summarized in two earlier reviews his findings of lamprey ‘‘interrenals’’ in the brook lamprey, Lampetra planeri, where the tissue is primarily localized within the pronephric region. Seiler and Seiler (1973) have shown a close relationship between the presumptive AH and chromaYn tissue in the pronephric region of L. planeri (Figure 9.2). Hardisty and Baines (1971) and Seiler et al. (1973) have shown that the putative AH cells are located in cords or follicles and they have an ultrastructure typical of steroid‐producing cells, namely liposomes, extensive

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Ch Vs

Vd

A Gl

Kn

N

At

Ve

7.Bbr

7.Bbr

Ba

Vj Fig. 9.2. Diagrammatic representation of the distribution of the presumptive adrenocortical tissue (PAT, dark dots) and chromaYn tissue (þ) in the pronephric region of adult brook lamprey, L. planeri. ChromaYn tissue is also seen in the atrium (At) and ventricle (Ve) and in the walls of dorsal aorta (A) and posterior cardinal veins (Vs and Vd); the PAT is also associated with these vessels. Ch, notochord; Gl, glomus of pronephros; N, nephrostome of pronephros; 7.Bbr, gills. [From Seiler and Seiler (1973).]

smooth endoplasmic reticulum, and many mitochondria with tubular or transverse cristae (Figure 9.3). Seiler et al. (1970) have claimed that these ‘‘intrarenal’’ cells of the pronephros show 3b‐HSD activity. In a more extensive examination throughout the life cycle of the sea lamprey, Petromyzon marinus, it was shown that the putative AH cells are located throughout both the pronephric and opisthonephric regions of the coelomic cavity, both in the walls of the great dorsal vessels (dorsal aorta, posterior cardinal veins) and within the renal tissue (Youson, 1972). The intrarenal steroid tissue of the opisthonephros follows the arterial circulation (aVerent and eVerent arterioles) and is even present in the large compound glomerulus (glomus). Although this putative AH tissue has the cellular fine structure of steroid‐ producing tissues described above, repeated histochemistry with positive

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Fig. 9.3. The presumptive adrenocortical tissue (PAT) in the pronephros of a larval sea lamprey, P. marinus. (A) Light micrograph of a 0.5‐mm thick plastic section shows the islets of vacuolated PAT cells (arrows) among kidney tubules (t).
1200. (B) Mitochrondria (M), with cristae (arrowheads) and matrix inclusions (In), and lipid droplets (LD) in a PAT cell.
42,000. (C) PAT cells show numerous mitochondria (M) and lipid droplets (LD).
14,000. [From Ellis (1993).]

controls (anuran AH) did not reveal 3b‐HSD activity in the lamprey tissues. As a result of these findings, and in particular because of the absence of evidence for 3b‐HSD activity, the expression ‘‘presumptive interrenal tissue (Youson, 1972)’’ and, eventually (Weisbart, 1975), ‘‘presumptive or presumed adrenocortical tissue (PAT)’’ was coined for this tissue in lampreys. This precautionary designation, originally used by Weisbart and Idler (1970), is applied to the present day despite the fact that lamprey PAT undergoes hypertrophy following injections of mammalian adrenocorticotropin (ACTH), and the cells follow a pattern of fine‐structural change that parallels with that seen in cells of the mammalian adrenal cortex following ACTH administration (Sterba, 1955; Youson, 1973a,b).

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This broad distribution of PAT in lampreys throughout the length of the nephric region (including both pronephric and opisthonephric kidneys and around the great dorsal vessels) is considered to represent an early step in the evolution of the kidney–AH relationship (Figure 9.4) that culminates in the compact suprarenals of many amniote vertebrates (Youson, 1972, 1985; Hardisty, 1979). Some of the chromaYn cells of lampreys (discussed in more detail in Section 2.3.1) are also located in the vicinity of the great dorsal

Agnatha

Chondrostei

Dipnoi

Neopterygii

Acipenserid Polypterid ?

?

?

?

a⬘

a

b

c

d

d⬘

e

f

g

Fig. 9.4. The distribution of the presumptive adrenocortical tissue (PAT) in Agnatha and of the adrenocortical homologue (AH) in bony fishes (Chondrostei, Dipnoi, and Neopterygii). (a0 ) PAT in hagfish is suspected to be in the pronephros and is unknown (?) in the more caudal opisthonephros. (a) PAT in larval lamprey is a diVuse population of islets in the pronephros, the opithonephros, and in the walls of the great dorsal vessels but it is unknown (?) whether it exists with the undiVerentiated (oblique lines) nephric region. (b) PAT islets in adult lampreys are present in the regressed (hatched line) pronephric region, in the walls of the great dorsal vessels, and now in adult opisthonephros that developed from the undiVerentiated tissue in (a) It is uncertain (?) whether PAT is still present in the site of the former larval opisthonephros (crosses). (c) The sturgeon AH is in the form of intrarenal yellow corpuscles almost throughout the length of the kidney but near the posterior cardinal veins. (d) Polypterus has the AH in the same form as the sturgeon but the corpuscles extend about two‐third the length of the kidney. (d0 ) The lungfish AH is present as large intrarenal patches like those in urodele amphibians. (e) The bowfin AH is confined to yellow corpuscles within the anterior one‐half of the kidney and close to the posterior cardinal veins. (f) The gar AH is like that of the bowfin except that yellow corpuscles have a more cranial location with respect to the opisthonephros. (g) The AH of teleosts is confined to the head kidney (pronephric region) as patches of variable size and location but AH is absent from the more caudal opisthonephros. [Modified from Youson (1985).]

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vessels (Paiement and McMillan, 1975) and in the pronephros (Figure 9.2). This close topographical association may be physiological advantageous, particularly in a stress response (Hardisty and Baker, 1982). However, this relationship presumes that PAT cells produce corticosteroids and, as discussed below, this is still an open question. That the PAT cells in P. marinus are steroidogenic is supported by the observation through autoradiography that they incorporate 3H‐cholesterol (Youson, 1975) and through incubations that they manifest 171‐ and 21‐hydroxylase and 20‐desmolase activity in vitro (Weisbart and Youson, 1975) in larvae and parasitic‐phase adults. Upstream‐migrant (prespawning) adults of this species fail to manifest 21‐ or 11b‐hydroxylase (Weisbart and Idler, 1970; Weisbart, 1975). In vitro studies on P. marinus PAT show the formation of 11‐deoxycortisol, 171‐hydroxyprogesterone, and androstenedione but no cortisol, corticosterone, 11‐deoxycorticosterone (DOC), or testosterone (Weisbart and Youson, 1975). Hence, these results suggest the apparent absence of a key enzyme, 11b‐hydroxylase or P450c11 (Figure 9.1). In contrast, in vivo analysis of steroidogenesis following injections of [1,2,6,7‐3H]‐progesterone provides no products suggesting the absence of 17‐hydroxylase activity but the formation of DOC, indicating the presence of 21‐hydroxylase (Weisbart and Youson, 1977) or P450c21 (Figure 9.1). Weisbart et al. (1978) have provided spectrophotometric, but not histochemical, evidence for 3b‐HSD in PAT of mature (upstream‐migrant) P. marinus and no in vitro conversion of [1,2‐3H] cholesterol into known types of steroids. There is histochemical and spectrophotometric evidence for 3b‐HSD in PAT during various phases of the life cycle of L. planeri (Seiler et al., 1981, 1983). Although ACTH stimulation fails to produce convincing levels of known corticosteroids in upstream‐migrant female Lampetra fluviatilis (Buus and Larsen, 1975), Weisbart et al. (1980) indicated through double‐isotope derivative assays (DIDA) that it stimulates a product they identify as 171‐hydroxy‐20 b‐hydroxyprogesterone. The DIDA technique also demonstrates cortisol, 11‐deoxycortisol, corticosterone, 11‐dehydrocorticosterone, and testosterone, but not cortisone or DOC, in the serum of upstream‐migrant P. marinus (Weisbart et al., 1980). The search for circulating products of PAT steroidogenesis was aided by heterologous radioimmunoassay (RIA) but at this date we are not much clearer on what the products are in lampreys. Ackermann et al. (1984) identified both pregnenolone and androstenedione in opisthonephric PAT homogenates from sexually mature L. planeri. This finding supports earlier observations (see above) that 3b‐HSD is present for pregnenolone to progesterone and for dehydroepiandrosterone to androstenedione conversions. This observation was confirmed in a subsequent study over 3 months of the reproductive phase, but it is also noteworthy that PAT homogenates in both the pronephric and opisthonephric regions contain testosterone

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(Seiler et al., 1985). Given the previous observations from in vitro studies described above, it is surprising that Dashow et al. (1984) have reported progesterone, corticosterone, cortisol, androstenedione, testosterone, dihydrotestosterone, estrone, and estradiol in the plasma of larval sea lampreys, P. marinus; the appearance of some of these hormones is altered when ‘‘the bodies of the decapitated larvae are squeezed during blood collection.’’ Androstenedione, and perhaps estradiol, may be the stress‐related hormones in upstream‐migrant P. marinus (Katz et al., 1982). Kime and Callard (1982) propose, primarily from their observations on testis, that future studies on products from PAT incubations should focus on 15‐hydroxylated derivatives. Larsen (1987) has always contended that products of PAT are involved in the initiation of sexual maturation. Since definitive identity of the products of lamprey PAT has been allusive, it is diYcult to deal with the issue of the physiological role of this tissue (Hardisty and Baker, 1982). Most attempts at determining the function of lamprey PAT have used, as the primary research tool, changes in cellular morphology under various conditions of stress (Hardisty, 1972b; Hardisty et al., 1976). Hyperplasia of the tissue and some changes in nuclear diameter occur after a sham operation of the pituitary, constant light, saline injection, and osmotic stress in upstream‐migrant L. fluviatilis (Hardisty, 1972b). McKeown and Hazlett (1975) have indicated, through observations of decreased cellular and nuclear diameters of PAT cells in juvenile P. marinus, that lamprey PAT is involved in osmoregulation. PAT cells of the opisthonephric kidney of this species are more prominent in juveniles in full‐strength seawater (Youson, 1982), so there may be some credence to this view of the importance of PAT products in lamprey osmoregulation. Molecular studies on the lamprey adenohypophysis have identified two proopiomelanocortin (POMC) genes, proopiocortin (POC ) and proopiomelanotropin (POM ) in lampreys (Heinig et al., 1995; Takahashi et al., 1995a, 2001, 2005a, 2006a,b; Youson et al., 2006a). These preprohormones were the first to be identified in the adenohypophysis, primarily because they are highly expressed in this tissue. Although the POC and POM genes likely arose from a common ancestor by gene duplication, the absence of promoter activity in the POC gene suggests that the promoter region of the ancestral gene may have been deleted with functional diVerentiation of the two genes (Takahashi et al., 2005b). POC codes for ACTH and this gene shows variable expression during the lamprey life cycle (Ficele et al., 1998; Heinig et al., 1999; Youson et al., 2006a). Increased expression of POC occurs at a time when the animals are demonstrating both final gonadal maturation and prespawning behavior such as the cessation of feeding and upstream migration (Heinig et al., 1999; Youson et al., 2006a). Since ACTH is a primary posttranslational productive of POC, and synthetic lamprey ACTH

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promotes in vitro steroidogenesis in PAT cells from spawning phase lampreys (Takahashi et al., 1995b), it is suggested that the high expression of POC is to produce ACTH for stimulation of PAT into steroidogenesis (Youson et al., 2006a). Steroid synthesis in the PAT might be upregulated at this time in response to stresses surrounding the reproductive event or, as has been suggested by Larsen (1987), PAT‐derived steroids may be directly involved in stimulating the gonads. Although Sterba (1955) reported a gradual increase in numbers of PAT cells during larval life up to metamorphosis, there is no evidence that any products of PAT cells have any involvement in this phenomenon. However, it was noted that PAT cells in the pronephros and larval opisthonephros retain their viable appearance even while the renal epithelium surrounding them undergoes degeneration (Youson, 1980). Furthermore, a detailed ultrastructural analysis of PAT cells in the pronephros during metamorphosis in P. marinus indicates that changes occur in mitochondria and lipid droplets that, in other vertebrates, reflect steroidogenesis (Ellis, 1993). This observation is consistent with histochemical and spectrophotometric evidence in L. planeri that 3b‐HSD activity is increased during metamorphosis (Seiler et al., 1981, 1983). b. Hagfish. The identity of hagfish PAT has been allusive. Most investigators have searched the pronephric area of the coelomic cavity and only Idler and Burton (1976) are convinced of their finding of PAT cells in Myxine glutinosa. They used 14C‐isoxazole, a radiolabeled synthetic steroid inhibitor of 3b‐HSD activity, to locate cells they call presumptive interrenal cells in the ‘‘central mass region of the pronephroi.’’ With a Bouin fixation and Mallory‐Heidenhain stain they describe cells with ‘‘osmiophilic cytoplasmic inclusions,’’ a description usually employed after osmium tetraoxide fixation. Descriptions and the accompanying micrographs do not suggest cells in this region that have the light microscopic morphology of AH cells of other vertebrates. Furthermore, the present author examined through electron microscopy the pronephric regions of 12 individuals of each of M. glutinosa and Eptatretus stouti and was unable to locate cells that resemble in any way PAT or AH cells of other vertebrates, including those in the lamprey. Under conditions with positive controls, extensive histochemical testing for 3b‐HSD activity in the pronephric regions of E. stouti met with failure (Youson and Gorbman, unpublished data). This is not to say that a PAT is absent in the pronephric region of hagfish, but this author is convinced that an identity cannot be made on the basis of the normal light and electron microscopic morphology or histochemistry of PAT cells as has been employed in other vertebrates. Chester Jones and Mosley (1980) came to a similar conclusion after a histological examination of the pronephric region of M. glutinosa. Idler and Burton (1976) provided photographs and some morphometric

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data to illustrate that the PAT of hagfish undergoes hypertrophy and hyperplasia after animals receive ACTH injections. Antibodies to ACTH and both [125I]‐ and [131I]‐ACTH localize mostly to cells in the central mass of the pronephros. Although these authors could find no evidence of their tracers localizing in any large degree in more posterior positions, such as in the opisthonephros or along the great dorsal vessels, they did not eliminate the possibility of a wider distribution of PAT in hagfish. As it stands, the hagfish PAT is presumed to be confined to the head kidney (pronephros) region that is also the typical site for AH in some teleosts (Figure 9.4). Unlike lampreys, hagfish PAT does not illustrate the pattern of widespread distribution of this tissue that is considered to be close to the ancestral condition (Youson, 1972, 1985). Idler and Burton (1976) felt that the small numbers of cells in the PAT of the hagfish pronephric mass would make the tissue unsuitable for incubation studies to show cholesterol to corticosteroid conversions. However, earlier it had been reported that the plasma of hagfish contains corticosteroids. The evidence from Chester Jones and Phillips (1960) and Phillips et al. (1962), using paper chromatography, that plasma of M. glutinosa contains cortisol and corticosterone at concentrations similar to those of mammals, was later criticized by Weisbart and Idler (1970). The latter authors used the more sensitive DIDA technique and could not provide rigorous proof of these steroids in the plasma of the same species. Later, these two authors were part of a team that identified cortisol, 11‐deoxycortisol, corticosterone, 11‐dehydrocorticosterone, and testosterone in the sera of E. stouti using both DIDA and RIA (Weisbart et al., 1980). These authors also indicated that multiple injections of ACTH elevated serum concentrations of corticosterone. The potential for a pituitary‐PAT axis in hagfish is supported by evidence of ACTH bioactivity in brain, but mainly in the pituitary, of E. stouti (Buckingham et al., 1985) and by the recognition of small numbers of ACTH‐immunoreactive cells in the adenohypophysis of both E. stouti and M. glutinosa (Nozaki et al., 2005). 2.2.2. Gnathostomata a. Sarcopterygii. The AH of the lobe‐finned fishes, the order Coelacanthiformes, and the two orders within family Dipnoi, has received only cursory attention. For the coelacanth, Latimeria chalumnae, the PAT is located within the kidney and shows some morphological features of steroidogenetic tissue (Lagios and Stasko‐Concannon, 1979). Chester Jones and Mosley (1980; Lagios and Stasko‐Concannon, personal communications) have described the ‘‘presumptive interrenals’’ as consisting of numerous bright yellow corpuscles (0.1‐ to 2‐mm diameter) distributed along the course of the posterior cardinal veins (PCV) and the major tributaries within the kidney. They also

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quote Lagios and Stasko‐Concannon (personal communications) that the coelacanth AH is ‘‘primitive’’ in being like that of chondrostean and holosteans, which had been described prior to their study (Youson and Butler, 1976a; Youson et al., 1976). In another study, frozen tissue removed from an area of the kidney and ‘‘. . . presumed to be the site of adrenocortical cells. . .’’ was analyzed for corticosteroids by DIDA (Truscott, 1980). There is evidence for DOC, cortisol, and corticosterone but not for 11‐deoxycortisol or cortisone (for the pathway, see Figure 9.1). Incubation of this tissue with radioactive steroid precursors and identification of products implied the presence of key enzymes such as 3b‐HSD, 201‐HSD, and 11b‐HSD. Amino acid sequence analysis of hormones that are derived from POMC in an extract of L. chalumnae pituitary shows the existence of the ACTH domain and concludes that most hormonal segments of this precursor are closer to their tetrapod rather than to their lungfish counterparts (Takahashi et al., 2003). Some of the earliest reports of the adrenal glands of dipnoans indicate that the AH and chromaYn tissue have separate distribution with the latter tissue adjacent to the dorsal aorta (Holmes, 1950; Ge´rard, 1951). The distribution of the AH in lungfishes seems to resemble the pattern of distribution in some amphibians, particularly urodeles (Janssens et al., 1965; Call and Janssens, 1975). In both the African (Protopterus sp.) and Australian (Neoceratodus forsteri) lungfishes, the AH appears in gross dissection as small, yellow‐pigmented regions within the kidney proper. Histological preparations show these pigmented regions to consist of irregularly, organized clusters of lipid‐containing cells bordering on the renal veins (Figure 9.5A). In N. forsteri, the cells stain positively for 3b‐HSD (Figure 9.5B) and their sudanophilic lipid droplets contain cholesterol and its esters (Call and Janssens, 1975). Although Chester Jones and Mosley (1980) quoted earlier preliminary findings, Idler et al. (1972) appeared to be the first to provide a thorough measure of corticosteroid levels in the plasma of lungfish, in this case in the South American species, Lepidosiren paradoxa. Cortisol, aldosterone, corticosterone, and 11‐deoxycortisol, but not cortisone and 11‐dehydrocorticosterone, are detected in plasma by DIDA. A more recent analysis of plasma of N. forsteri by RIA shows similar findings, except that 11‐deoxycortisol is not measured due to cross reactivity with the antibody that is detecting corticosterone (Joss et al., 1994). These latter authors demonstrate corticosteroids at higher levels than those previously reported for the species (Blair‐West et al., 1977). Whereas Idler et al. (1972) emphasized aldosterone and cortisol concentrations in L. paradoxa as being tetrapod‐ and bony fish‐like, respectively, corticosterone is the dominant corticosteroid in N. forsteri plasma (Joss et al., 1994). However, both studies conclude that the corticosteroid profile in lungfish plasma is more reflective of a tetrapod than a fish.

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Fig. 9.5. (A) Diagrammatic representation of a transverse section of the kidney (K) of the Australian lungfish, Neoceratodus forsteri, showing the distribution of lipid‐containing cells (L) near the renal veins (RV). (B) Histochemistry for 3b‐HSD by diformazan deposition (F) is indicated in the lipid‐containing cells of figure 4A. K, kidney parenchyma; RV, renal vein.
60. [From Call and Janssens (1975).]

Dores et al. (1990) suggested from pituitary extracts that ACTH is present in the pituitary of N. forsteri, thus indicating the existence of a hypophysial‐adrenal axis in this lungfish species. The cDNA for the preprohormone, POMC, that encodes for ACTH, has now been cloned for both N. forsteri and P. annectens (Amemiya et al., 1999; Dores et al., 1999; Lee et al., 1999). It is noteworthy in the context of the discussion above on corticosteroids that sequence parsimony analysis of POMC from individuals representing two lungfish genera indicates a closer relationship of lungfishes to tetrapods than to ray‐finned fishes (Dores et al., 1999). Studies also indicate that control of corticosteroid secretion by a functioning renin‐ angiotensin system was likely important in the aquatic to terrestrial transition in vertebrate evolution. Both angiotensin I and II can stimulate the release of aldosterone in N. forsteri (Joss et al., 1994, 1999).

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b. Actinopterygii. When considering the AH of the ray‐finned fishes, it is important to emphasize at the outset that there is potential for a second endocrine tissue to be present within, or directly associated with, the kidneys. This endocrine tissue is called the CS and sometimes it is directly apposed to the AH. The CS was referenced in Section 1. In early literature, it was referred to as the AH, but now we know that the CS are not steroidogenic. To date, this tissue has only been described in members of subclass Neopterygii. A separate section (Section 3) of this chapter is devoted to the CS and their products. i. Polypteriformes. It is beyond the scope of this chapter to argue for the correct taxonomic placement of the bichirs and reedfish. However, this author tends to favor the term ‘‘lower actinopterygians’’ that includes the two distinct, nonmonophyletic orders, Polypteriformes and Acipensiformes (Grande and Bemis, 1996). de Smet (1960) was probably the first to recognize ‘‘interrenal tissue’’ in his study of the nephron of Polypterus retropinnis. However, it was Youson and Butler (1985) who provided the first positive identity of the AH in Polypterus palmas through histochemistry for 3b‐HSD and ultrastructural of the cells. The AH appears in dissection as about 40–60 yellow corpuscles that are equally divided between the two kidneys but directed toward the mid‐anterior (cranial) one‐half of each kidney (Figure 9.4). The spherical to cigar‐shaped corpuscles are usually confined to the dorsal side of the kidneys, close to the posterior cardinal and renal veins, and often adjacent to the vertebral column. The cells show an ultrastructure similar to steroid‐synthesizing cells, namely numerous mitochondria with tubulovesicular cristae, aggregations of lipid droplets, and an extensive network of smooth endoplasmic reticulum. An unusual feature of the AH cells is their arrangement into pseudofollicles or pseudotubules with lumina; this is a feature often equated with hyperactivity of AH tissue. This same profile of steroidogenic‐looking cells around lumina (Figure 9.6B) is also seen in the reedfish, Calamoichthys calabaricus (Youson et al., 1988). As in the bichir, the reedfish AH is positive for 3b‐HSD. However, in the reedfish 175 yellow corpuscles that contain these groupings of AH cells are found throughout the length of the kidneys on their dorsal surface directly apposed to the PCV (Figure 9.6A). In both the reedfish and the bichirs, the sinusoids surrounding the cell groupings empty directly into the lumen of these large veins and perhaps provide a mechanism for the rapid dispersal of newly synthesized corticosteroids. To the knowledge of the author, there has been no study that has measured corticosteroid production of the AH tissue or has performed an analysis of corticosteroid levels in the plasma of Polypteriformes. However, there has been a description of a cDNA clone for POMC from Polypterus senegalus (Bagrosky et al., 2003). The melanocortin sequence, ACTH/

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Fig. 9.6. (A) A yellow corpuscle (YC) of the polypterid, the reedfish (C. calabaricus), is in an intrarenal position next to the posterior cardinal vein (cv). a, artery; h, hemopoietic tissue.
100. (B) Epithelial cells in the reedfish YC have apical nuclei (n) are separated by wide intercellular spaces (small arrow), and seem to border a narrow lumen (large arrow). The cells are surrounded by sinuoids (s), hemopoietic tissue (h), and kidney tubules (t).
435. [From Youson et al. (1988).]

a‐MSH, implies that there is at least the potential for a hypophysial‐adrenal axis in this group. A parsimony analysis of POMC sequences places P. senegalus closer to sturgeon (Acipensiformes) than to the neopterygian ray‐finned fishes (Bagrosky et al., 2003).

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ii. Acipenseriformes. The AH of the sturgeons, Acipenser oxyrhynchus and Acipenser fulvescens, appears as yellow corpuscles that extend throughout the length of the kidney region (Figure 9.4), often just below the kidney capsule, and near the great dorsal vessels (Idler and Sangalang, 1970; Youson and Butler, 1976a). Although it seems that these corpuscles are deeply embedded and scattered within the kidney proper in A. oxyrhynchus (Idler and O’Halloran, 1970), only a few of the 80 or more corpuscles are present in this location in A. fulvescens (Youson and Butler, 1976a). That the corpuscles represent the sturgeon AH tissue is confirmed through 3b‐HSD activity, ultrastructure of the cells, and by in vitro transformation of labeled steroid precursors into identifiable corticosteroids (Idler and O’Halloran, 1970; Idler and Sangalang, 1970; Youson and Butler, 1976a). The cells are richly endowed with lipid droplets, mitochondria with tubulovesicular cristae, and smooth endoplasmic reticulum (Youson and Butler, 1976a). There seems to be no detailed morphological analysis of the AH of the paddlefish in the published literature. Fortunately, however, the distribution, ultrastructure, and steroidogenic activity of ‘‘interrenal tissue’’ of Polyodon spathula has been examined and reported in a graduate thesis (Rahn, 1997). Like the sturgeon, the AH cells of the paddlefish are richly endowed with lipid droplets (Figure 9.7). Gundersen et al. (2000) agreed with Rahn (1997) that the AH is dispersed throughout the kidney of the P. spathula. This description is consistent with the distribution of the AH in the other acipenseriforme, the sturgeon (Figure 9.4). Gundersen et al. (2000) reported hyperplasia of ‘‘interrenal tissue’’ and hypertrophy of the cells as a consequence of contamination of paddlefish with polychlorinated biphenyl (PCB) and chlordane. The plasma of A. oxyrhynchus contains small amounts of cortisol, cortisone, and corticosterone and there is a tentative identification of deoxycortisol and DOC (Sangalang et al., 1971). A thorough analysis of corticosteroids in paddlefish plasma has not been undertaken, but cortisol is believed to be the main corticosteroid in the Acipenseriformes (Idler and Truscott, 1972). There has been a considerable eVort to look at circulating levels of corticosteroids, usually cortisol, in fish under a diversity of stresses, and the sturgeon and paddlefish have been included in these studies (Barton, 2002). The incentive for studies of sturgeon is based on the fact that their numbers have declined dramatically in North America, and they do not exhibit the same levels of physiological response to exhaustive exercise as other fishes (Barton et al., 2000; KieVer et al., 2001). In particular, the plasma cortisol levels do not elevate from basal levels to the degree seen in teleosts under similar stress‐related conditions of handling and transport in both Scaphirhynchus spp. and Acipenser spp. sturgeons. Both Barton et al. (2000) and

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Fig. 9.7. An electron micrograph of lipid‐rich cells from the adrenocortical homologue in the paddlefish, P. spathula.
7000. [From Rahn (1997).]

KieVer et al. (2001) suggested that the broad distribution of the AH throughout the length of the kidney in sturgeons compared to the concentration of AH in the teleost head kidney may be an explanation for the reduced physiological response of the sturgeons as reflected by plasma cortisol levels. In contrast, Bayunova et al. (2002) demonstrated elevated plasma cortisol in stellate (Acipenser stellatus) and Russian (Acipenser gueldenstaedtii) sturgeon during capture, forced swimming, and air exposure. Also, Belanger et al. (2001) reported significant plasma cortisol increases over prestress and poststress levels in white sturgeon (Acipenser transmontanus) after stresses of reducing the volume of the holding water, transportation, and handling. Furthermore, these latter authors provide the first evidence that AH in sturgeon responds to exogenous ACTH in a dose‐dependent manner through elevation of levels of plasma cortisol. Since two distinct POMC cDNAs, sturgeon POMC A and sturgeon POMC B, both of which encode ACTH, have been cloned from A. transmontanus (Amemiya et al., 1997; Alrubaian et al., 1999), it is most probable that a hypophysial‐adrenal axis exists in the sturgeons. Two isoforms of ACTH are recognized in the cDNAs of

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POMC (A and B) from the green sturgeon, Acipenser medirostris (Lankford et al., 2006). Antisera, generated against the C‐terminal amino acids of green sturgeon ACTH when injected into a stressed fish of the same species, can moderate the in vivo response to the stressor (Lankford et al., 2006). The antiserum neutralizes the stimulatory eVect of endogenous ACTH on cortisol synthesis in stressed fish but does not aVect the cortisol levels in resting fish. There has been no detailed analysis of the types of plasma corticosteroids in paddlefish. However, Idler and Truscott (1980) report in their review article that the plasma from the one paddlefish they examined contained cortisone, corticosterone, and large quantities of cortisol. As in the case with sturgeon, plasma cortisol has been measured in paddlefish as a parameter of stress following various physical disturbances. Barton et al. (1998) have used appropriate standards for detection of aldosterone, cortisone, cortisol, corticosterone, and 11‐deoxycortisol but cortisol is the only measurable steroid in their study. Exposure to salinized water, to handling, to continuous chasing, and to high‐density confinement causes elevations in plasma cortisol; the latter stessor is the most prominent elevator of the corticosteroid (Barton et al., 1998). As described above for sturgeon, all the stressors on paddlefish elicit a lower magnitude physiological response than has been shown for teleosts. The ‘‘less stressed’’ labeling for paddlefish may be a consequence of the low response of their AH cells to ACTH, as has been indicated in some preliminary in vitro studies (Rahn, 1997). Barton et al. (1998) have suggested that the diVerence in capacity to respond to stressors between teleosts and the paddlefish may be found in the hypothalamic‐pituitary‐AH axis. As with the sturgeon, there has been a duplication of the POMC gene in paddlefish with the cloned cDNAs implying that each has all the functional domains, including ACTH (Danielson et al., 1999). iii. Amiiformes. As recently as 1986, it was indicated that so little was known about the AH of holostean fishes, that is the bowfin and the gars, that it was not worth considering in a review of the distribution of the tissue in vertebrates (Chester Jones and Phillips, 1986). However, de Smet (1962) provided a clear description of the presence of the ‘‘interrenals’’ and the CS in the bowfin, Amia calva. Later, Youson et al. (1976) mapped the distribution and structure of the AH, CS, and the chromaYn tissue in the bowfin using histochemical methods; the AH was definitively identified by 3b‐HSD activity in this study and eventually also by its fine structure (Youson and Butler, 1976b). A 50‐cm long, 450‐g animal has about 140 yellow corpuscles within the anterior (cranial) two‐third of the kidney and associated with the posterior cardinal and renal veins (Figure 9.4). White corpuscles, numbering well over 300, are randomly distributed throughout the length of the kidneys and show no 3b‐HSD activity but have granules that are positive with

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periodic acid‐SchiV (Youson et al., 1976). The white corpuscles represent the CS and they will be described in a later section. The AH cells possess abundant lipid‐containing vacuoles, tubular smooth endoplasmic reticulum, and mitochondria with tubular cristae; a usual feature is cytoplasmic processes of AH cells into the lumina of adjacent sinusoids (Youson and Butler, 1976b). Idler et al. (1971) were the first to report on cortisol and corticosterone identification in bowfin plasma. RIA was used by Hanson and Fleming (1979) to measure 0.49 mg 1001 ml1 cortisol in the serum of juvenile (15–30 g) bowfin. Hypophysectomized juveniles show elevated serum cortisol but no change in serum cortisol after ACTH injection, changes that are opposite to those in most other fishes (Butler, 1973). Steroidogenesis occurs in vitro in yellow corpuscles but not in white corpuscles of bowfin (Butler and Youson, 1986). Incubated yellow corpuscles convert [7‐3H]pregnenolone to radioactive 11‐deoxcortisol, cortisol, and corticosterone; cortisol is the dominant steroid after 3 h. Aldosterone is not detected. It seems, therefore, that bowfin plasma likely contains the same corticosteroid profile as that of the more advanced ray‐finned fishes, the teleosts, and that the corticosteroids are a product of the AH tissue. A partial sequence of the bowfin POMC gene has been reported (Venkatesh et al., 2001), and the ACTH domain has been detected in extracts of the pituitary by RIA (Dores et al., 1994). iv. Semionotiformes. As is the case with the bowfin, the gar kidney contains both yellow and white corpuscles but they are greatly reduced in numbers and have a diVerent distribution than the bowfin. In two species of gar, Lepisosteus osseus and Lepisosteus platyrhynchus, the yellow corpuscles outnumber the white corpuscles by a minimum of 2 to 1 and a maximum of 7 to 1, respectively, with 55 being the maximum number of yellow corpuscles present (Bhattacharyya et al., 1981). Furthermore, the yellow corpuscles are distributed relatively evenly in the anterior (cranial half) of both kidneys (Figure 9.4) and close to the posterior cardinal and renal veins. The yellow corpuscles are of oblong shape, and in a Florida gar, L. plathyrhynchus, one was 5‐mm long. Only the yellow corpuscles show 3b‐HSD activity and the fine structure of their cells suggests steroidogenesis (smooth tubules of ER, mitochondria with tubular cristae, and many lipid inclusions). Their recognition as cells of the AH is further substantiated by electron microscopic observations after the animals are either perfused with digitonin (deposition of cholesterol‐digitonide crystalline spicules) or administered ACTH. In the latter case, the cells are stimulated into hyperactivity with lipid droplet depletion, increases in dense bodies, and cells surrounding lumina producing pseudofollicles (Bhattacharyya et al., 1981). A POMC cDNA has been cloned from the pituitary of the gar and an ACTH domain has been identified (Dores et al., 1997).

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There does not appear to be any studies where gar corticosteroids have been measured in plasma or following incubation of the AH with radioactive steroid precursors. v. Basal Teleosts. There have been numerous studies of the AH of teleosts, including many euteleost species and some basal teleosts. Most of the latter studies have been on the anguilliforme, the eel. The eel AH has often been used as an example of the teleost glandular tissue, for it is present in the form of islets embedded in a region called the ‘‘head kidney’’ just caudal to the last branchial arch. Therefore, the position of the AH in the basal teleost, the eel, represents according to this description (Chester Jones and Mosley, 1980) an apparent endpoint to a progressive trend for a more cranial location of the AH in ray‐finned fishes (Figure 9.4). On the other hand, Bhattacharyya and Butler (1979) have described the AH in Anguilla rostrata as islands or lobules of epithelial cells forming two or more layers as a collar around the anterior and posterior cardinal veins but confined inside the vein tunics. The AH tissue of A. rostrata appears around the posterior cardinals in a position that is cranial to the head kidney and extends to this kidney before disappearing in this tissue. In contrast, the AH around the anterior cardinal veins is present as large masses within the head kidney region. The reader is referred to Chester Jones and Mosley (1980) for an extensive summary of the teleost AH and, in particular, for references to the studies of this glandular tissue in the eel. Also, Bhattacharyya and Butler (1979) have described ultrastructural changes in the AH cells of A. rostrata that indicate stimulated steroid secretion as a manifestation of saltwater acclimation. Nandi (1962) provided a scheme for describing the morphological location of the AH with respect to its relationship with the PCV, renal veins, and venous sinuses. Chester Jones and Mosley (1980) used this scheme of types I–IV and intermediates to describe the AH of some basal teleosts. With the exception of the Elopiformes, the tenpounders, where there is a concentrated mass of AH tissue (type IV), the usual distribution for AH in basal teleosts (the Anguilliformes, Osteoglossiformes, and Clupeiformes) is smaller groups of AH tissue surrounding the PCV or largest branches (type I) or around small or medium‐sized branches of the vein (type II) or intermediates of these two types. 2.2.3. Phylogenetic Development of the Fish AH As has been stated previously, there is a general trend toward both a focal and a cranial concentration of the AH in the kidneys from agnathans to gnathostome fishes (Youson, 1985). We still know very little about the distribution of the AH in hagfishes, but the distribution in the lamprey, as small islets of cells throughout the entire nephric region (including the pronephric and opisthonephric kidneys) both within the kidneys and associated with the great dorsal vessels, represents the least specialized in terms of structural

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organization of the gland among vertebrates. Given the taxonomic position and antiquity of the Acipenseriformes, it may not be surprising that in the sturgeon, and apparently in the paddlefish, the AH is also widespread throughout the kidney region but now as more compacted bodies, yellow corpuscles. The bowfin (Amiiformes) has many yellow corpuscles but they are found in the cranial two‐third of the kidney, while small numbers of yellow corpuscles in the gar (Semionotiformes) are located in the cranial one‐half of the kidneys. The Polypteriformes, represented by the bichirs and the reedfish, show both a semionotiforme‐like and an acipenseriforme‐like distribution of AH, respectively. The generalized and more derived teleosts with their AH in the most anterior region of the kidney region of the coelomic cavity, the so‐ called head kidney region, represent a close to final stage of this progressive trend toward focal and regional (cranial) concentration in ray‐finned fishes. However, there seems to be a wide diversity in the distribution of AH in the teleost kidney (types I–IV) with a sold mass of cells in a localized area, such as the type IV of Chester Jones and Phillips (1986), being the one most resembling the yellow corpuscles of more basal, non‐teleost actinopterygians. It would be interesting in the future to examine the distribution of AH in some basal teleosts, such as the Osteoglossiformes, for they may show some intermediate steps between the situation in the gar and the more general and derived teleosts. On the other hand, the AH distribution in this group of bonytongues may prove to be divergent from any tendency for head kidney concentration in teleosts. The Anguilliformes, another order of basal teleosts, seem to depict a more forwardly directed distribution of AH that is markedly divergent from even the more generalized euteleosts (Henderson and Kime, 1987). The dipnoans provide an AH distribution that may have resulted from an evolution from the most ancient condition (lamprey‐like?) that was parallel to that of the ray‐finned fishes and led eventually to the urodele‐like distribution among tetrapods. It has been emphasized that the distribution of AH in fishes, and likely other vertebrate groups, is a consequence of ontogeny (Chester Jones and Mosley, 1980), although physiological relationship also likely played a role. As in studies of the patterns in phylogeny of other hormone systems, for example the gastroenteropancreatic system (Youson and Al‐Mahrouki, 1999), the ontogeny of the AH tissue and the evolution of physiological responses to its products are intimately related. 2.3. ChromaYn Tissue For most of the species and orders of fishes described in Section 2.2 , there is a relatively close relationship of AH with chromaYn tissue. Tables have been created that specify whether the orders of teleosts have single or mingled

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chromaYn and AH elements to their adrenal tissue (Chester Jones and Mosley, 1980). ChromaYn tissue can be broadly classed in fish, and other vertebrates, as one that produces catecholamines, namely epinephrine (adrenaline) and norepinephrine (noradrenaline), under various internal and environmental stressors (Reid et al., 1998). There is a considerable body of literature on chromaYn tissue and catecholamines in elasmobranchs and euteleosts (Mazeaud et al., 1977; Nilsson, 1984; Randall and Perry, 1992; Grassi Milano et al., 1997; Reid et al., 1998). A chapter in a previous volume of this series provides the biosynthetic pathway of catecholamines and all the metabolic and degradative processes related to these in fishes (Randall and Perry, 1992). A follow‐up review by Reid et al. (1998) gives an account of the complexity of the controls of catecholamine storage and release in fish chromaYn cells and emphasizes the role of other hormones, neurotransmitters, and second messenger systems. The reader is referred to these earlier reviews for specific information on catecholamine synthesis and its control. Although there is some reference to fishes of ancient lineage in these earlier reviews, there has not been a study or review that specifically focuses on chromaYn tissue of the adrenal glands of ancient groups of fishes. Furthermore, many of the existing studies have concentrated on this tissue and the function of its products located within the vicinity of the heart and far removed from the AH. In the big picture of adrenal gland function, perhaps all chromaYn tissues irrespective of its location should be considered as part of the gland. It is not clear whether the question about chromaYn tissue distribution, regional specialization, and relationship to the adrenal gland AH has ever been addressed to any large extent. An excellent example to illustrate this point is the study by Perry et al. (1993) that compares catecholamines in chromaYn tissue of the PCV and the heart in the hagfish, M. glutinosa. Although equal quantities of epinephrine and norepinephrine are stored in the heart, the much smaller quantity of catecholamine in the PCV is predominantly norepinephrine. This introduces the question of whether the tissues of these two regions synthesize and release catecholamines under diVerent stimuli, perhaps because of diVerences in innervation or cholinergic receptor capacity. Regional diVerences in storage levels and types of catecholamines are emphasized as potentially significant features of the chromaYn system in fishes (Reid et al., 1998). Since one of the primary roles of plasma catecholamines is to control cardiac and respiratory physiology, the eVerent adrenergic response and actions of these hormones will undoubtedly be dealt with in other chapters of this volume. The description below attempts to focus, as much as possible, on information from those groups and species where the tissue has been identified as part of an adrenal gland, that is, in close physical association to the AH.

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2.3.1. Agnatha Although we do not have a clear view of the location of hagfish AH, should it ultimately be found near the pronephric region and heart cavity, ¨ stlund et al., 1960; Bloom et al., then the chromaYn tissue in the heart (O 1961) may be part of its adrenal gland. On the other hand, Perry et al. (1993) have shown from an analysis of catecholamines in heart and PCV of M. glutinosa that the chromaYn tissue is well represented in the latter region. Since Idler and Burton (1976) did not eliminate the possibility that some of the hagfish AH might be present more posteriorly around the PCV, this more caudal collection of chromaYn tissue of the PCV is possibly also a part of a diVusely distributed adrenal gland. The morphology of hagfish chromaYn tissues has only been described in the ventricles and atrium of the heart but ¨ stlund et al., 1960; Bloom et al., 1961), catecholamines are also found in (O and regulate (Johnsson and Axelsson, 1996), the portal heart. The cells have the typical granulation of catecholamine‐containing cells but they are not innervated (Bloom et al., 1961). It is noteworthy (and see discussion in the previous paragraph) that the PCV stores mainly norepinephrine and in the heart equal amounts of norepinephrine and epinephrine are present; the heart stores about three times the catecholamine of the PCV (Perry et al., 1993). Previously, Bloom et al. (1961) indicated that the ventricle contains mostly epinephrine and the atrium mostly norepinephrine. Plasma catecholamine levels are similar in M. glutinosa and the Pacific hagfish, E. stouti (Bernier et al., 1996). An in situ saline‐perfused systemic heart/PCV preparation (Perry et al., 1993) shows that boluses of either ACTH, serotonin, or carbachol, and perfusion of high‐[Kþ] saline stimulate the release of both epinephrine and norepinephrine (Bernier and Perry, 1996). However, injections of angiotensin II, at dosages known to elicit catecholamine secretion in other vertebrates, have no eVect. There is strong evidence in hagfishes that adenosine modulates catecholamine release, in particular it stimulates ACTH‐induced catecholamine release (Bernier and Perry, 1996; Bernier et al., 1996). Given the apparent absence of extrinsic innervation of hagfish chromaYn cells and, therefore, no neuronal release of acetylcholine, it is likely that catecholamine release is achieved through endocrine (ACTH or even corticosteroids from nearby AH cells) and/or by paracrine control (Bernier and Perry, 1996). There does not appear to be any potentiation by catecholamines in thyroid hormone‐induced lipolysis in E. stouti (Plisetskaya et al., 1984). As in hagfish, there have been many studies on the nature and function of cardiac chromaYn cells in lampreys (Otsuka et al., 1977; Dashow and Epple, 1985). The fine structure of the heart chromaYn cells resembles that of typical catecholamine‐secreting cells but with no specific morphology

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directed toward a particular type of catecholamine (Bloom et al., 1961). Histochemical methods on the hearts of the paired lamprey species, L. fluviatilis and L. planeri, indicate epinephrine and norepinephrine in specific cells of the atrium and sinus venosus, whereas only epinephrine is present in specific cells of the ventricles (Dahl et al., 1971). The distribution of the extracardiac chromaYn tissue in lampreys has been described for larvae of L. planeri (Figure 9.2) (Seiler and Seiler, 1973) and for P. marinus and the nonparasitic species, Lampetra appendix (Paiement and McMillan, 1975) in the Northern Hemisphere and for the Southern Hemisphere species, Geotria australis (Epple et al., 1985). In the Northern Hemisphere species, the chromaYn cells are located within the tunica adventitia (outer layer) of the major arteries (Figure 9.8) such as the coeliac artery as it branches from the dorsal aorta near the pronephros. The cells actually appear beneath the endothelium of the thin‐walled veins that accompany the arteries at this site

Fig. 9.8. Schematic drawing of a cross section through the adjacent walls of an artery and vein from larval lampreys to show the location of chromaYn cells (CC). The CC are embedded in the tunica adventitia of the artery, near pigment cells (PC), but are directly beneath the endothelium of the vein (VE). AE, arterial endothelial cells; EM, elastic membrane; MC, smooth muscle cells. [From Paiement and McMillan (1975).]

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and between the dorsal aorta and the PCV along the rest of the trunk (Figure 9.8). PAT cells can also be found in these locations (Youson, 1973a). Various tests for catecholamines in lampreys by Paiement and McMillan (1975) confirm the monoamine nature and lead the authors to speculate that the single type of granulated cell likely elaborates norepinephrine or dopamine (Figure 9.8). Using a silver technique, Epple et al. (1985) described a large and discrete body of extracardiac chromaYn cells in the lateral wall of right PCV of adult G. australis. These authors go on to suggest that this large body of chromaYn cells resembles the precardiac axillary bodies of elasmobranchs and, as such, it represents an intermediate condition of chromaYn cell distribution between the cartilaginous fishes and Northern Hemisphere lampreys. Plasma from the ventral aorta of prespawning P. marinus contains high concentrations of free norepinephrine and epinephrine and low levels of dopamine but no conjugated catecholamines, whereas perfused sinus venosus contains the free catecholamines but also conjugated dopamine and norepinephrine (Epple et al., 1995). The reader is referred to earlier reviews (Hardisty, 1979; Hardisty and Baker, 1982) where hypertrophy of the generalized chromaYn cell population, but mostly the cardiac population, and elevated plasma catecholamine levels are seen in lampreys following various stress conditions. Cardiovascular and respiratory activity seems to be the primary target of dopamine and norepinephrine that is released from the general chromaYn cell population (Dashow and Epple, 1985). Hardisty (1979) has provided a viewpoint that close approximation of the PAT and chromaYn tissue in lampreys is physiologically advantageous for the potential of corticosteroids to control the synthesis and activity of a key enzyme, phenylethanolamine‐N‐methyl transferase (PNMT), involved in the methylation of norepinephrine into epinephrine in the catecholamine pathway. 2.3.2. Gnathostomata a. Sarcopterygii. Call and Janssens (1975) confirmed in N. forsteri that lungfishes contain no chromaYn tissue within the kidney (Holmes, 1950). Most of the morphological (Larsen et al., 1994; Chopin and Bennett, 1995) and physiological (Abrahamsson et al., 1979) studies have been performed on tissues within the myocardium (atrium) from which the circulating catecholamines likely arise (Perry et al., 2005). However, some extracardiac chromaYn tissue occurs around the dorsal aorta (Holmes, 1950) and both dopamine‐b‐hydroxylase (DbH, conversion of dopamine into norepinephrine) and PNMT activity is located in intercostal arteries (Abrahamsson et al., 1979) in Protopterus spp. Perry et al. (2005) summarized that the major sites of catecholamine storage in the three genera of lungfishes also include the anterior region of the PCV. Adrenergic nerves seem to be absent in lungfishes (Nilsson, 1983) and catecholamines released from the atrium

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are likely critical for adrenergic control of the heart (Fritsche et al., 1993). The plasma profile for catecholamines of P. dolloi in various hypoxic states indicates that aerial, but not aquatic, hypoxia stimulates the release of these monoamines (Perry et al., 2005). To the knowledge of this present author, extracardiac chromaYn tissue and its products have not been described in the coelacanth. b. Acipenseriformes and Polypteriformes. There appear to be no studies of either the morphology or function of extracardiac chromaYn tissue in Polypterus spp. or the reedfish, C. calabaricus. In fact, there is no report of plasma catecholamine levels in either species. There is one report that the Russian sturgeon, Huso huso, has both epinephrine and norepinephrine in the cardinal veins (Balashov et al., 1981). As has been indicated above in the discussion of AH in sturgeons and paddlefishes, however, most of the investigations on the adrenal gland tissues and their products in these species have been concerned with the topic of a stress response, mainly because they are threatened species. Unfortunately, the AH and plasma cortisol have been the primary focus of the measurement of an endocrine response in these species (Barton et al., 1998; Bayunova et al., 2002). The one exception was the study by Gundersen et al. (2000) in the Ohio River paddlefish, where hyperplasia of chromaYn tissue is spread throughout the length of the kidney. The foci (nodules) of chromaYn tissue are larger and more common in fish that have a known high content of PCB. This is the first report that chromaYn tissue is found throughout the length of the kidney in an acipenseriforme. Future studies should investigate the distribution of chromaYn tissue and measure plasma catecholamine levels in normal and stressed Acipenseriformes. c. Amiiformes and Semionotiformes. The activity of the enzymes, DbH and PNMT, in chromaYn tissue is known in the Florida gar, L. platyrhynchus (Abrahamsson et al., 1981) but, although we know that epinephrine is the primary catecholamine in the PCV chromaYn cells (Nilsson, 1981), there has not been a thorough study of the distribution of this tissue in gars. In contrast, the distribution and structure of this tissue in the bowfin has been well documented (Youson, 1976; Youson et al., 1976). At the cranial end of each kidney chromaYn tissue is present in equal abundance in the walls of the right and left PCV but the left vessel gradually loses the cells until they are absent about the kidney midpoint. A similar gradual reduction in chromaYn cells occurs in the right vessels from the midpoint to the caudal end of the kidney but they persist here and also in the walls of the renal veins. The bowfin chromaYn tissue reacts with the ferric ferricyanide method but does not show the classical reaction with dichromate. Despite this, the fine structure of the cells is reflective of those involved in the synthesis and release of

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catecholamines; in particular, the cells resemble those involved in the elaboration of norepinephrine (Figure 9.9). As in many other fishes, bowfin chromaYn tissue seems to have one morphologically distinct cell type. The cells are strategically located beneath the endothelium of the renal and posterior cardinal veins to release their product directly into the blood stream (Figure 9.9). In contrast to the gar, however, there is no information on enzyme activity in the catecholamine pathway or data on plasma catecholamine levels in the bowfin. Since consecutive injections of norepinephrine increase dorsal aortic blood pressure in a dose‐dependent manner, this

Fig. 9.9. ChromaYn cells in the wall of the posterior cardinal vein of the bowfin, A. calva. Inset (top left corner): light micrograph of 0.5‐mm thick plastic section demonstrating the location of granulated chromaYn cells (G) beneath the endothelium (E).
1200. The main figure is an electron micrograph of chromaYn cells beneath the endothelium with only collagen microfibrils (C) in the subendothelial space (S). The chromaYn cells are separated by wide lateral intercellular spaces (IS) and contain many electron‐dense granules (G), rough endoplasmic reticulum (RE), a Golgi apparatus (GA), and a few mitochondria (M). N, nucleus; NT, nerve terminals.
8400. [From Youson (1976).]

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indicates that the chromaYn system is likely involved in regulation of the cardiovascular system of the bowfin (Butler et al., 1995). d. Basal Teleosts. Among the basal teleosts, the chromaYn system of the eel (Anguilliformes) has received the most attention. There does not seem to be any data on the chromaYn system of the Osteoglossiformes, Elopiformes, Albuliformes, and Saccopharyngiformes or even the Clupeiformes. This void in data on chromaYn tissue from these orders is unfortunate, for the present view is that the pattern of distribution of tissue in generalized and derived euteleost represents an intermediate state between that observed in non‐teleost actinopterygians and tetrapods (Grassi Milano et al., 1997). That is to say that the phylogenetic trend is from the more ‘‘primitive’’ broad distribution of chromaYn cells near cardinal veins of pronephric and opisthonephric regions to tissue concentrated within an adrenal medulla. One cannot but wonder whether there is a further phylogenetic stage of chromaYn tissue distribution in these basal teleost orders. The eel extracardiac chromaYn tissue is very much teleost‐like in being distributed within the walls of the PCV and within the hemopoietic tissue of the head kidney (Reid et al., 1998). Often there is intimate association of the chromaYn and AH cells (Bhattacharyya and Butler, 1979). The anterior two‐third of the PCV in A. rostrata seem to be the primary site for catecholamine storage with epinephrine dominating over norepinephrine (Reid and Perry, 1994) and with cholinerigic release mediated by a nicotinic receptor (Reid and Perry, 1996). However, a systematic analysis of the vascular system of this species shows high concentrations of dopamine, norepinephrine, and epinephrine throughout the length of the PCV from their caudal origin in the opisthonephric kidneys to their termination at the duct of Cuvier (Hathaway and Epple, 1989). Since this latter site contains the highest concentrations of the three catecholamines, it is considered by the authors to be the presumed adrenomedullary equivalent. Immunohistochemistry for DbH, PNMT, and tyrosine hydroxylase (TH, hydroxylation of tyrosine to L‐DOPA) shows all but PNMT in every cell of the chromaYn system of A. rostrata; this immunohistochemistry also shows a gradual reduction in numbers of cells of the system caudally to only a few in the opisthonephric kidney (Hathaway and Epple, 1990). The reader is referred to an earlier review for information on the various endogeneous and external factors that influence catecholamine release in eels (Reid et al., 1998). 2.4. Prospective on the Adrenal Glands of Ancient Fishes The paragraphs above outline that there are still some vacancies in our basic knowledge of distribution, structure, and function of both the AH and the chromaYn tissue. With respect to AH the positive identity of the hagfish

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tissue is needed, and there is little definitive information on the circulating corticosteroids in both hagfishes and lampreys. We know nothing about the AH in Osteoglossiformes. We also need to know more about the plasma corticosteroid profiles of Polypteriformes, the paddlefish, and the Semionotiformes. Information is totally absent on corticosteroid receptors in all the fish groups described above. Molecular characterization of corticosteroid receptors, both glucocorticoid (GR) and mineralocorticoid (MR), is a new and important research direction in general and derived teleosts (Baker, 2003; Greenwood et al., 2003; Gilmour, 2005; Sturm et al., 2005; Norris and Hobbs, 2006; Prunet et al., 2006). Although cortisol proves to be the physiological ligand for GR, studies show that, given the apparent absence of aldosterone in fishes, DOC might be the physiological ligand for MR (Sturm et al., 2005). As emphasized by Gilmour (2005) and Prunet et al. (2006), these data give us a slightly diVerent view of corticosteroids and osmoregulation in fishes. Future studies on corticosteroid receptors in fish should include sampling of fishes with an ancient lineage to elucidate the phylogenetic development of the various types of receptors; these fishes serve as examples of organisms with an appropriate internal mechanism to permit them to survive many changes in their external environment over their protracted evolutionary history. Many of the species dealt with in this section are showing a decline in numbers in wild populations. One of the parameters being utilized to find a cause for this decline is the stress response, mainly through examination of changes in blood values of glucose, lactate, and cortisol (Barton, 2002). As has been illustrated above, the chromaYn tissue is strategically located within the heart and/or in the PCV to provide catecholamines that aVect, among other functions, heart rhythm and respiration. There is a need for better knowledge of the chromaYn system in some of these species and to include blood catecholamine levels in evaluations of stress. A case in point is the hypertrophy of chromaYn tissue in PCB‐contaminated paddlefish (Gundersen et al., 2000), yet we have no known knowledge of plasma catecholamine levels. Despite the many years of study of the adrenal glands of fishes, there is still no clear explanation as to the functional significance of the close relationship between chromaYn cells and the AH. Also what is the functional relevance for the unequal regional distribution of epinephrine and norepinephrine, and, often, the activity levels of key enzymes in the catecholamine pathway? Although these questions can also be addressed in studies of more advanced bony fishes, any future studies should also include the more ancient fishes. In certain cases, this latter group provides more suitable preparation for investigation because of the more concentrated regions of AH (yellow corpuscles) and chromaYn cell nodules in the PCV. Finally, adrenomedullin was initially isolated from the adrenal medulla of humans

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and there is an independent adrenomedullin family consisting of five paralogous peptides in fish (Ogoshi et al., 2003). Since there is interest in the molecular evolution and function of these peptides in fishes, because of their diverse biological actions on circulating hormones and as paracrine factors in other vertebrates (Takei et al., 2004), it seems reasonable to suggest that some of the more ‘‘primitive’’ bony fishes referenced above should be considered in future studies. 3. CORPUSCLES OF STANNIUS 3.1. Background Nearly 170 years ago, Stannius (1839) identified cream‐colored glands in the kidneys of bony fishes. Stannius thought that these glands, now referred to as the Stannius bodies, Stannius corpuscles, or the CS, might be the AH, but it was left to Fontaine (1964) to show a clear relationship between the CS and calcium homeostasis. Hypercalcemia in the European eel (Anguilla anguilla), as a result of stanniectomy, is eliminated following the injection of a CS extract. A few years later, Chester Jones et al. (1966) showed some pressor eVect of a CS extract in the same eel species. Previously, Garrett (1942) emphasized the origin of the CS from kidney tubules but Ogawa (1967) showed that the CS cells have many secretory granules and a fine structure typical of cells involved in protein synthesis. That the CS are confined to just holostean (bowfin and gar) and teleost fishes was confirmed in a study that used period acid‐SchiV (PAS) stain to show that all CS secretory granules contain glycoprotein and there may be two cell types (Krishnamurthy and Bern, 1969). During the 1970s there was a flurry of research activity in several laboratories resulting in data showing that the environment is the source of calcium following stanniectomy because of an increase in Ca2þ influx mainly across the gill epithelium (for review see, Mayer‐Gostan et al., 1987 and Fenwick, 1989). Since the hypercalcemic eVects of stanniectomy are relieved by administration of CS extracts, the active principal was originally named hypocalcin (Pang et al., 1974). The histophysiology of the teleost CS has been reviewed (Krishnamurthy, 1976). There have been two previous reviews of the fish literature on CS in this book series. The first, in Volume 2, was part of a chapter that also discussed the early literature on the distribution, structure, and function of the AH (Chester Jones et al., 1969). The chapter (Wagner, 1994), in Volume 13, had as its primary focus the history of development and recognition of the principal product of the CS, which came to be known as STC. Wagner

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Fig. 9.10. Amino acid sequences of smallmouth bass stanniocalcin (STC) are compared with STC‐1 sequences from flounder, white sucker, arawana, butterfly fish, elephantnose, bowfin, gar, and mouse. Hyphens indicate amino acids identical to those in smallmouth bass STC. Gaps

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(1994) described the history of discovery of STC in fishes from the points of view of molecular structure and gene expression. At that time there was only a limited amount of information about STC in Amiiformes (Marra et al., 1992) and nothing was known about the molecule in Semionotiformes or basal teleosts, other than the eel. Since that time, STC has been shown in annelids (Tanega et al., 2004) and mammals (Chang et al., 1995, 1996; Wagner et al., 1995; Gerritsen and Wagner, 2005) and more data is now available on the diversity in structure of the CS and STC in actinopterygians of ancient lineage. This section of the chapter reviews only the early literature on the CS of these ancient extant fishes and provides data on STC from those species that are new since the review by Wagner (1994). As was stated above, the CS are only found in Amiiformes, Semionotiformes, and the teleosts among the ray‐finned fishes. There have been no reports of CS or STC in Polypteriformes, Acipenseriformes, or the Sarcopterygii. The reader is also referred to other reviews on the fish CS and STC (Wendelaar Bonga and Pang, 1986, 1991; Hirano, 1989; Wagner, 1993) and on the STC family of proteins (Wagner and DiMattia, 2006). Wagner and DiMattia (2006) reviewed the history of discovery of STC, a homodimeric glycoprotein, in fishes that has now become known as fish STC‐1. Protein sequencing and cloning of teleost STC cDNAs and deduced amino acid profiles indicate a mature monomer with 11 cysteine residues (Figure 9.10) with the one at position 169 for intermonomeric disulfide linkage to produce the dimer (Hulova and Kawauchi, 1999). STC‐1 is present in salmonid plasma in several forms (Wagner, 1993) and the gene is expressed in many tissues other than the CS (McCudden et al., 2001). This wide expression of STC‐1 is also seen in the Pleuronectiformes (Hang and Balment, 2005; Shin et al., 2006). A second STC, referred to as STC‐2, was first recognized in Atlantic salmon but it is a less eVective inhibitor of gill Ca2þ transport in fish (Wagner et al., 1998). There are STC‐1 and STC‐2 in mammals but they show only 50% and 35% sequence identity, respectively, with fish STC‐1 (Ishibashi and Imai, 2002). On the basis of sequence homology to human STC‐2, an STC‐2 ortholog in fishes was isolated by searching the Fugu rubripes genome and amplification from a zebra fish (Danio rerio) cDNA library (Luo et al., 2005). Fish STC‐2, now described in Fugu and the

(asterisks) were introduced to maximize sequence identity. The numbers at the right indicate the position of the amino acids and the shaded box shows the common glycosylation site. The position of cysteine (C) residues is denoted by open boxes and the five common intramonomeric disulfide linkages are indicated by lines. The site of the intermonomeric linkage at position 169 (dimer) is indicated in open boxes in most species except the osteoglossomorph species (arawana, butterfly fish, and elephantnose) where cysteine has been substituted. [From Amemiya et al. (2006).]

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zebra fish, has less than 30% identity to fish STC‐1 but it has paracrine bioactivity in rat ovary (Luo et al., 2005). Although fish STC‐1 seems to have systemic actions, mammalian STC‐1 is more often referenced as a paracrine hormone (Luo et al., 2005). Since only one form of STC has been recognized in the groups of fishes to be described below, the term STC will be used throughout and these STCs have highest identity with teleost STC‐1. 3.2. Amiiformes de Smet (1962) quoted the work of Giacomini (1933) as the first to distinguish between the AH and the CS in the bowfin, A. calva, on the basis of both their distribution and embryological origins. Garrett (1942) and de Smet (1962) noted that unlike teleosts, the AH and CS in bowfin can often be positioned in close apposition. In teleosts, the small number of CS in adults (Figure 9.11) arise from the pronephric duct during embryogenesis (Kaneko et al., 1992) but in the bowfin the CS continue to form, likely from the distal renal tubules, during adult life (Youson et al., 1976). Perhaps as a consequence of this continued development of CS, mature adults have over 300 CS appearing as white corpuscles throughout the opisthonephric kidney, but more heavily concentrated in the bulbous, caudal portion of the kidney. In contrast, 140 yellow corpuscles (AH) are present in the cranial two‐third of the kidney (Youson et al., 1976). As stated in Section 2.2.2.b.iii, only the yellow corpuscles show in vitro conversion of radiolabeled steroid precursors into corticosteroids (Butler and Youson, 1986). The CS cells of bowfin are all type 1 cells, as defined in teleosts (Wendelaar Bonga and Greven, 1975;

Fig. 9.11. Light micrograph showing immunoreactivity for anti‐chum salmon stanniocalcin in the paired corpuscles of Stannius (CS) but not the renal tissue (R) in arawana (O. bicirrhosum). Scale bar ¼ 100 mm.

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Wendelaar Bonga et al., 1980). They show all of the fine‐structural features of protein‐synthesizing and protein‐secreting cells, and staining of their granules with PAS (Figure 9.12A) implies that they store a glycoprotein (Youson and Butler, 1976b; Youson et al., 1976; Marra et al., 1992). The granule content is immunoreactive with salmon STC antiserum in both light (Figure 9.12B) and electron microscopic preparations and CS extracts show, with Western blotting using the salmon antiserum, the presence of immunoreactive material at 47 and 41 kDa under nonreducing conditions; the putative STC protein is reduced with b‐mercaptoethanol (Figure 9.13). Although bowfin STC appears to be a glycoprotein, it does not bind to concanavalin A (ConA) and it cannot be eluted from ConA sepharose, as is the case with STC‐1 for many other fishes (Wagner, 1994). More recently, two forms of STC were identified in Atlantic salmon, and one of these forms does not have

Fig. 9.12. Adjacent sections of the bowfin (A. calva) kidney to show a distal segment of the renal tubule (T) near the corpuscle of Stannius (arrows). (A) Section stained with periodic acid‐SchiV shows the glycoprotein nature of granules (arrows) in the corpuscle of Stannius (CS). (B) Immunohistochemistry with anti‐salmon stanniocalcin shows immunoreactivity (arrow) in the CS but not the tubule or the surrounding hemopoietic tissue. The dark material in each section is melanin pigment.
800 (courtesy of L. Marra).

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Arawana NR R

Bowfin NR R

51 kDa 47 kDa

White sucker R NR

48 kDa

34 kDa 28 kDa

29 kDa

24 kDa 21 kDa

R = reduced condition NR = nonreduced condition Fig. 9.13. Western blots of crude extracts (100‐mg protein per lane) of corpuscles of Stannius from arawana (O. bicirrhosum), bowfin (A. calva), and white sucker (C. commersoni) under reducing (b‐mercaptoethanol) and nonreducing conditions. Incubation was with anti‐chum salmon stanniocalcin (STC). Under nonreducing conditions the putative STCs of bowfin and white sucker exist as dimers with the molecular weights (kDa) indicated, whereas arawana has a single 21‐kDa band. Under reducing conditions both bowfin and white sucker putative STCs migrate as lower molecular weight bands, whereas arawana putative STC migrates to a molecular weight similar to that in nonreducing conditions. [Modified from Amemiya et al. (2002).]

an aYnity for ConA (Wagner et al., 1998). Perhaps there is a second, ConA‐ binding, form of STC in bowfin or, given the antiquity of this species, this ConA‐void form is a more ancestral form of STC in fishes. The simplest explanation is that bowfin STC and the second form in Atlantic salmon have unique carbohydrate moieties compared to other known fish STCs. Eventually, a bowfin STC cDNA was cloned from the CS and a deduced amino acid sequence was determined (Amemiya and Youson, 2004). The mature STC monomer is 220 amino acids and shows a 60–70% identity with the STCs of other fishes, with the exception of gar where identity is 87%. Figure 9.10 shows a sequence alignment of bowfin STC and other fish species with emphasis on the fact that there are 11 cysteine residues in the bowfin molecule and that it shares a common N‐glycosylation consensus amino acid sequence. We still do not know the compositional makeup of the carbohydrate moiety in this bowfin glycoprotein. RT‐PCR amplification of bowfin STC mRNA shows bowfin gene expression in brain, gill, pituitary, heart, liver, intestine, muscle, ovary, testes, kidney, and the CS with the final two having the most

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pronounced signal. As is the case with the STC gene in rainbow trout (McCudden et al., 2001a,b), the bowfin STC gene is widely expressed. However, the functional significance of this broad pattern of STC expression in fishes and in mammals needs to be further considered (Wagner and DiMattia, 2006). It was mentioned earlier that two functions have long been attributed to the active principal(s) secreted by the CS in fishes. These are the elaboration of a hypocalcemic hormone (Wagner, 1994) and/or one that acts with pressor eVects on the cardiovascular system (Chester Jones et al., 1966). There are no studies of the CS and calcium regulation in bowfin, principally because ablation of the activity of CS by stanniectomy would take too long or involve the removal of the entire kidneys due to the large number of CS. Studies by Butler et al. (Butler and Oudit, 1994, 1995; Butler and Zhang, 2001) on eels have measured cardiovascular events following stanniectomy. Data from these studies are interpreted as reflecting that the hypercalcemia subsequent to stanniectomy is not due to the loss of a hypocalcemic hormone but instead to changes in blood flow to the organs and tissues that regulate ion fluxes. Bowfin were one of three fishes used in a study to illustrate how degranulation of CS cells accompanies experimental hypotension or hypovolemia caused by blood withdrawal (Butler et al., 2003). In the case of the bowfin, the number of cytoplasmic granules in CS cells decreases by 39%. Using the connection with data showing that in eels both dorsal aortic and caudal venous blood flows increase following injections of angiotensin II or CS extracts (Butler and Zhang, 2001), the degranulation of CS cells in bowfin, eel (A. rostrata), and white sucker (Catastomus commersoni) is explained as a release of renin or isorenin from these glands (Butler et al., 2003). Since in at least the eel there is no correlation between granule depletion and plasma Ca concentrations following the hypotensive/hypovolemic state, it seems that the CS in bowfin, eel, and white sucker elaborate an active principal that acts like renin in a renin‐angiotensin system. 3.3. Semionotiformes As was indicated in the section of this chapter on AH, the gar (Lepisosteus spp.) has greatly reduced numbers of white corpuscles, representing the CS, compared to the bowfin. Five to seven round to oval corpuscles are present at the midpoint of each kidney and close to the posterior cardinal and renal veins, and sometime adjacent to the yellow corpuscles of the AH (Bhattacharyya et al., 1982). Unlike the yellow corpuscles, the white corpuscles are sometimes deeply embedded in the hemopoietic tissue. The convoluted cords and islets of cells are richly vascularized and the cells stain positively with PAS. Unlike the bowfin, the gar CS has both type I

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and type II cells as defined in teleosts (Wendelaar Bonga and Pang, 1986). The type I cells have the fine‐structural profile of cells synthesizing and elaborating protein, while the type II cells appear less active in these functions. Both types of cells, and specifically their granules, are immunoreactive with antisera against either trout or salmon STC (Marra et al., 1994). Western blot analysis of CS extract from gar reveals the STC‐immunoreactive material as a single 68‐kDa band under nonreducing (b‐mercaptoethanol) conditions and three bands (29, 31, and 34 kDa) under reducing conditions. An STC cDNA was cloned from gar CS tissue and the deduced amino acid sequence has high (87%) sequence identity with bowfin STC (Figure 9.10). In fact, this level of identity supports a view (Gardiner et al., 1996) that gar and bowfin form a monophyletic holostean clade (Figure 9.14). The mature STC protein in gar is 220 amino acids, has the same‐sized signal peptide (32 amino acids), an identical glycosylation site, and 11 cysteine residues like that seen in the bowfin (Figure 9.10). RT‐PCR amplification shows that the gar STC gene is expressed in all the same tissues as in the bowfin, except for muscle and testis; kidneys and CS show the most pronounced expression (Amemiya and Youson, 2004). There have been no studies of CS function in Lepisosteus spp. However, the N‐terminal of the gar STC is similar to the bioactive portions (residues 1–20) of the teleost STC molecule (Milliken et al., 1990; Verbost et al., 1993). Therefore, it is possible to speculate that gar, and likely the bowfin, STCs carry out the same roles in mineral metabolism and/or cardiovascular control as is being proposed for teleosts. Confirmation of these, or novel, functions to gar and bowfin STC awaits future study. 3.4. Basal Teleosts The CS and STC of the eel, an Anguilliformes, have received considerable attention for nearly 50 years as representing this endocrine tissue and hormone, respectively, in teleosts. Much of the past literature in earlier reviews has concentrated on this fish model. Both the first physiological studies (for review see Chester Jones et al., 1969; Wagner, 1993, 1994) and the first description of a fish STC cDNA (Butkus et al., 1987) were performed in the eel. Above in the description of the Amiiformes CS (Section 3.2), there are references to recent studies on eel where the CS is believed to release renin‐like products that are involved in cardiovascular control. It is suggested that the reader should examine earlier reviews (references above and also others provided in Section 2.1) and papers by Butler et al. (Butler, 1993, 1999; Butler and Oudit, 1994, 1995; Butler and Zhang, 2001; Butler et al., 2003) for a synopsis and discussion of the pressor, calcaemic, and osmoregulatory roles of the CS in Anguilliformes. For the most part, studies of CS

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Smallmouth bass

51

Puffer fish

100

Turbot 98

72

European flounder

Coho salmon

98 100 91

Rainbow trout White sucker

100 90

Zebra fish 1a Austrailian shortfin eel Freshwater butterfly fish Elephantnose

100 94

Silver arawana

Zebra fish 1b Bowfin

85 83

Longnose gar African clawed frog Cow

100 100

Mouse

0.05 Fig. 9.14. The phylogeny of prestanniocalcin sequences from diverse fish species is estimated using the neighbor‐joining algorithm: frog and mammalian sequences are used as an outgroup. Numbers on each lineage indicate the percentage of 500 bootstrap replications that support each lineage. [Modified from Amemiya et al. (2006), with the exception of the turbot, all GenBank Accession Numbers used in this analysis are provided in this publication.] Accession No. DQ156537 for turbot added in this present modification. Note the groupings of the gar and bowfin, osteoglossomorph species, and other teleost orders (see text for further discussion).

structure and function in eels have not taken into account that it is a basal teleost and most data have been interpreted in the context of teleosts in general. Until physiological studies have been performed for a more derived teleost at a level of magnitude that has been provided for the eel, we will not be able to interpret the present eel data in a phylogenetic context. Support for this viewpoint is found in phylogenetic analysis of fish STC. It is interesting that when eel STC is placed within a parsimony analysis with STC from other fishes, it is grouped closer to the more generalized and basal teleosts than with the more derived fishes (Figure 9.14). Another basal teleost group, the Osteoglossiformes (the bonytongues), represent a collection of extant ancient fishes with diverse distribution

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whose common ancestry can be traced back to a time before the splitting of the tectonic plates (Li and Wilson, 1996). Although there is still some controversy on the subject, there have been claims that the osteoglossomorphs are among the most primitive living teleosts (Lauder and Liem, 1983). Given this background on the osteoglossomorphs, and the fact that the CS is found only in the Neopterygii of the ray‐finned fishes, it seems important that there should be some consideration of the CS in this important teleost group. In the silver arawana, Osteoglossum bicirrhosum, a white corpuscle, 1–4 mm in diameter, is located immediately anterior to the caudal, bulbous portion of each kidney (Figure 9.11). These two white corpuscles constitute the entire mass of the CS tissue. This distribution pattern is representative of the more advanced actinopterygian condition, relative to the broad distribution entailing hundreds of CS in the bowfin and five to seven CS in the mid‐ region of each kidney in the gar. Although only two white corpuscles (one per kidney) of similar size to those in arawana are usually present in the kidneys of other osteoglossomorphs, such as the butterfly fish (Pantodon buchholzi), knifefish (Chitala chitala), and the elephantnose (Gnathonemus petersii), they tend to be found more toward the mid‐portion of the kidneys on the dorsal surface (Marra, 2000). The CS cells of all species show PAS‐positive staining and are arranged into follicles or cords. The granules of types I and II cells immunostain with both salmon‐ and trout‐STC antisera in light and electron microscope preparations but the staining is abolished if the antisera are preabsorbed with a crude CS extract (Marra et al., 1995; Marra, 2000; Amemiya et al., 2002). It is particularly noteworthy that some cells in th distal renal tubules of arawana, butterfly fish, and knifefish show immunoreactivity to STC antisera, but this is not the case in gar, eel, or also elephantnose. Electron microscopic immunocytochemistry reveals that the immunoreactive cells are mitochondrial‐rich or renal chloride cells with the label primarily localized on cytoplasmic membrane (Amemiya et al., 2002). STC immunoreactivity is found in certain cells of the mammalian kidney (Wagner et al., 1995), but there are no CS in mammals. On the other hand, we see a connection of CS with distal tubules in bowfin but no STC immunoreactivity in the renal cells (Marra et al., 1992). The question arises as to whether the immunoreactive profile described above in the kidneys of certain Osteoglossomorpha may represent an intermediate phylogenetic arrangement between the more ancient (holostean) vertebrates and the more derived vertebrates such as mammals. A second question is whether this renal distribution of STC is related to the ontogeny of CS from pronephric ducts in teleosts (Kaneko et al., 1992). Since the morphology of the immunoreactive renal cells does not suggest synthesis and secretion of STC, the cells likely have the ability to concentrate or bind the ligand, perhaps by way of receptors.

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That a function for STC may be present here in the renal epithelial membrane and/or the cytoplasmic matrix is supported by the established role in both fish and mammals that STC is involved in renal phosphate transport (Lu et al., 1994; Wagner et al., 1997; Gerritsen and Wagner, 2005). The view that STC receptors and ligand accumulation in target renal cells account for their immunoreactivity is supported by the inability in arawana kidney to obtain any hybridization signal on Northern blots with an STC cDNA probe or to detect STC mRNA in RT‐PCR (Amemiya et al., 2002). The deduced amino acid sequence from a cloned STC cDNA from arawana shows a pre‐STC of 249 amino acids and a mature STC of 218 amino acids (Amemiya et al., 2002). Although the consensus glycosylation site is present, arawana STC has only 10 of the 11 cysteines present in monomers of STCs of other vertebrates (Figure 9.10). Whereas all of the five intramonomeric disulfide linkages are present in the arawana molecule, the location of arginine rather than a cysteine at position 169 precludes the possibility of an intermonomeric disulfide linkage to create the dimer. Western blot of arawana CS extracts under reducing (24‐kDa band) and nonreducing (21‐kDa band) conditions confirms that arawana STC exists as a monomer, that is, since a band lower than 21 kDa does not appear in reducing conditions (Figure 9.13). Subsequent cloning of STC cDNAs from the butterfly fish and elephantnose (Figure 9.10) revealed that these two other members of Osteoglossiformes also have a cysteine substitution at position 169 and their STCs also likely exist as monomers (Amemiya et al., 2006). In one case the cysteine substitution is arginine (elephantnose) and in the other it is histidine (butterfly fish). There is only about a 3% diVerence in sequence identity of STC between the three Osteoglossiformes and a phylogenetic analysis of STC sequences has Osteoglossomorpha in a well‐ supported monophyletic group (100% bootstrap support). The phylogenetic analysis of STC sequences (Figure 9.14) places arawana and elephantnose in close relation, however, present taxonomy has butterfly fish and arawana in suborder Osteoglossoidei and elephantnose in suborder Notopteroidei (Li and Wilson, 1996). Given that there is still controversy over osteoglossomorph taxonomy, and as a phylogenetic analysis using somatostatin sequences of similar species produced a like placement of osteoglossoforme species to STC (Youson et al., 2006b), perhaps there is some reason to consider the STC sequence as a reasonable tool in taxonomic analysis. 3.5. Phylogenetic Considerations of the CS and STC in Fishes The above discussion on the CS in ancient fishes hopefully raises a few curiosities about this gland. Some of these curiosities are not new but have not been broadly addressed herein or in previous reviews. First, and

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foremost, is the absence of the gland in Sarcopterygians, Polypteriformes, and the Acipenseriformes and its presence in all neopterygian ray‐finned fishes. Since the glands are derived from embryonic renal tissue in most cases and they reside within or closely apposed to the kidneys, one would suspect that the absence of the CS is a consequence of diVerences in either kidney/CS ontogeny or in kidney morphology. However, from what we know of the development and morphology of the kidneys of the CS‐devoid groups of bony fishes listed above, they share much in common in these two parameters with the groups possessing CS. The same can be said for the well‐ documented role of the CS active principal in mineral metabolism, for STC would be just as important for the CS‐devoid groups that operate under much the same environmental conditions as members of the CS group. Similarity of osmoreglatory challenges between teleosts and leeches was the driving force behind the discovery of sites of STC‐1 immunoreactivity in cells of the epidermis and dermis of the freshwater leech (Tanega et al., 2004); high‐aYnity STC receptors also seem to be present in the leech integument (Wagner and DiMattia, 2006). Perhaps these CS‐devoid fish species express and utilize STC in a manner similar to that seen in higher vertebrates such as mammals (Ishibashi and Imai, 2002; Chang et al., 2003; Gerritsen and Wagner, 2005; Wagner and DiMattia, 2006); this view needs to be explored in the future. These CS‐devoid fishes either lost their CS during their long evolutionary history or followed an ‘‘STC evolution’’ that paralleled that which led to the present mammalian, and invertebrate (?), STC functional profile. The extracorpuscular distribution of STC immunoreactivity in the renal epithelium of some Osteoglossiformes may reflect a step in an evolutionary trend for a diVerent or modified role for STC. This group is the only one to show a monomeric STC as a principal product of the CS gland; perhaps the future will show that this monomer has no function or maybe a novel function in this ancient group of teleosts. If we accept the increasing evidence that the CS of fishes produces a renin‐like factor, then these CS‐ devoid species should be examined for juxtaglomerular cells as their source of renin. Our current knowledge implies that STC has an ancient lineage (Tanega et al., 2004). Furthermore, the family of STC proteins is proving to have a significant role in metabolism, reproduction, and development in both mammals and teleosts (Gerritsen and Wagner, 2005; Wagner and DiMattia, 2006). It seems reasonable to assume that future investigations will show that the STC family of peptides and/or the STC gene is present in the CS‐devoid group of bony fishes. In those fish species that have CS, there is a tendency for smaller numbers of corpuscles from the hundreds in Amiiformes to the usual two in basal teleosts and beyond to the derived euteleosts. What is the explanation for the large numbers of CS in bowfin with a particular concentration in the

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posterior kidney and reduced, but still substantiative numbers, in the mid‐ kidney of the gar? Is there functional significance to the CS tissue becoming more concentrated in the teleosts or is it just a consequence of diVerences in ontogeny of the glands between Amiiformes and Semionotiformes and the teleosts? The molecular evolution of STC is worthy of some comment given the rather recent description of the primary structure of the molecule in an Amiiformes, a Semionotiformes, several Osteoglossiformes, a Cypriniformes, and several Perciformes (Amemiya et al., 2002; Amemiya and Youson, 2004, 2006; Hang and Balment, 2005; Shin et al., 2006). As was indicated above with respect to STC sequences in Osteoglossiformes species representing diVerent suborders, STC amino acid sequences have some utility as a taxonomic tool when used with other parameters. When the STC sequences of the ancient species are considered in a phylogenetic analysis with those of euteleosts, the diVerent clades that are created closely align with their present taxonomy that is based on characteristics, principally of the skeleton and soft anatomy (Figure 9.14). For instance, turbot, European flounder, puVer fish, and smallmouth bass are all Percomorpha and they are present in the same clade. Despite being members of two separate superfamilies in order Cypriniformes, zebra fish and the white sucker are closely aligned, as are all the osteoglossomorph species. The close alignment of bowfin and gar supports a view of a holostean monophyly (Gardiner et al., 1996). The Osteoglossiformes are referenced as one of the oldest groups of extant fishes (Lauder and Liem , 1983) and old enough that their distribution was likely influenced by movement of tectonic plates (Li and Wilson, 1996). In the context of the CS and STC, it is interesting that the individuals studied to date, butterfly fish (West Africa), silver arawana (South America), elephantnose (Africa, Nile) represent two continents but they are the only group to have a monomeric form of STC (STC‐1) among the neopterygian fishes. Since it is unlikely that a mutation occurred in both cases at position 169 to replace cysteine in geographically isolated species, this information suggests that extant osteoglossifome species originated from a common ancestor that possessed the mutation before continental drift. A search for the time when the STC mutation might have taken place in fish evolution included the Amiiformes and the Semionotiformes, for there has been much controversy over an amid‐teleost monophyletic group known as the Halecostomi (Patterson, 1973) and even a lepisosteid–teleost (Olsen and McCune, 1991) connection to explain the ancestors to the modern teleosts. As indicated above, however, both the bowfin and gar STCs have the cysteine at position 169 and they have the dimeric form of the molecule (Figure 9.15). These bowfin and gar STC sequence data allow us to postulate

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Cypriniformes D Salmoniformes D Pleuronectiformes D Mammalia Euteleostei Anguilliformes D

D

Perciformes D Osteoglossomorpha M

Teleostei Amiiformes D Semionotiformes D Neopterygii

Actinopterygii

Tetrapoda

D= Dimer M= Monomer

Sarcopterygii Fig. 9.15. A schematic tree of bony fish and the tetrapod lineages to illustrate species where stanniocalcin (STC) has been characterized. In particular, STC‐1 is present as a homodimer (D) in all species except the Osteoglossiformes where only a monomer (M) has been identified. The presence of a dimeric STC‐1 in Amiiformes and Semionotiformes suggests that a dimer is the ancient form of the molecule and that the monomeric form appeared in osteoglossomorphs independent of the rest of the teleosts (see text for further discussion of the phylogenetic relevance of these data).

that dimeric STC was the original form of STC and the 11th cysteine (position 169) was mutated early in Osteoglossiformes evolution (Figure 9.15). These data also suggest that, although they are ancient, the Osteoglossiformes are likely not ancestral to at least the orders of teleost in which STC has been examined, that is salmonids, cyprinids, pleuronectids, percids, and tetraodontids (Figure 9.15). 4. SUMMARY AND CONCLUSIONS The preceding pages have reviewed in fishes with ancient lineage the present state of information on two endocrine glands, the AH and the CS, which develop from mesoderm and have some intimacy with renal tissue. The AH of fishes, like the adrenal cortex of birds and mammals, also has a close association with catecholamine‐secreting, chromaYn tissue. Although the review of literature has emphasized some deficiencies in our knowledge in some fish groups, the distribution and functional morphology of both the AH and the chromaYn tissue in available data indicate that there are some

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phylogenetic trends. In two extremes, an agnathan, the lamprey, shows a diVuse distribution of small AH islets throughout the renal tissue and the basal teleost, the eel, has the AH concentrated in or near the pronephric (head kidney) region, like the situation in euteleosts. In between these groups are basal actinopterygians first with a tendency for a broad, intrarenal distribution of larger AH corpuscles and then a more cranial, intrarenal distribution of the corpuscles. The distribution of the AH in dipnoans, and also its corticosteroid profile, reflects the evolutionary relationship of this group with tetrapods. As with the AH, the chromaYn tissue is in close apposition to the PCV, likely for rapid dispersal of catecholamines. There are some data indicating regional specialization of cardiac and extracardiac chromaYn tissue in ancient fishes, but there is need for future study. Also, we need to know whether there is any functional, developmental, or evolutionary significance to the close positioning of AH and chromaYn tissue. Since many of the fish species considered in this chapter are considered endangered or threatened, it is recommended that catecholamines, as well as cortisol or another prominent corticosteroid from the AH, be monitored as indicators of stress in these species. The CS are present in only Neopterygii, that is, Amiiformes, Semionotiformes, and teleosts. Although there are considerable data from teleosts indicating that the CS are responsible, through their product STC, for adjusting internal calcium and phosphate levels in fish in hypercalcemic environments, there is increasing evidence from Amiiformes, basal teleosts, and euteleosts that CS products have a pressor, renin‐ or isorenin‐like, eVect on cardiovascular physiology. These functional data from fishes are becoming increasingly important given the fact that STC is ubiquitous in mammals, even though there are no CS. The data emanating from studies of mammalian STC are an incentive for studying STC or STC gene expression in the Polypteriformes, Acipenseriformes, and the Sarcopterygii that have no CS. There is a tendency for small numbers (2–5) of caudally positioned CS in the basal teleosts from the situation in Amiiformes, where there are hundreds throughout most of the kidney. Phylogenetic analysis of the primary STC in fishes, STC‐1, indicates that it has some utility as a taxonomic parameter. In particular, although STC‐1 in most fishes and mammals has 11 cysteine residues in each monomer to produce the homodimeric mature peptide, all bonytongues, the osteglossomorphs, have a cysteine substitution at the intermonomeric bonding site. The presence of a dimeric STC‐1 in both Amiiformes and Semionotiformes and the monomeric STC‐1 in species of Osteoglossiformes that have been geographically isolated since continental drift provide a useful data on both the ancestral form of STC and on basal actinopterygian and teleost relationships.

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ACKNOWLEDGEMENTS I am most grateful for the many undergraduate students who have worked with me on studies of the adrenocortical homologue in fishes. Luciano Marra and Yutaka Amemiya were the primary contributors to the study of the corpuscles of Stannius. Almost throughout my academic career I have had a highly rewarding and stimulating collaboration with my Department of Zoology colleague, David Butler, on the subject matter of this chapter, and I thank him for his part in this continued relationship. Aubrey Gorbman and Mel Weisbart gave me much needed support in my early studies of agnathan adrenal tissue. I thank Sjord Wendelaar Bonga, Gert Flik, Hiroshi Kawauchi, and particularly, Graham Wagner for encouragement, advice, and antisera in studies of the CS. Data generated in my laboratory were from studies supported by the Natural Sciences and Engineering Research Council of Canada.

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Truscott, B. (1980). Corticosteroids of the coelacanth Latimeria chalumnae smith: A provisional study on their identity. Gen. Comp. Endocrinol. 41, 287–295. Venkatesh, B., Erdmann, M. V., and Brenner, S. (2001). Molecular synapomorphies resolve evolutionary relationships of extant jawed vertebrates. Proc. Natl. Acad. Sci. USA 98, 11382–11387. Verbost, P. M., Butkus, A., Atsma, W., Willems, P., Flik, G., and Wendelaar Bonga, S. E. (1993). Studies on stanniocalcin: Characterization of bioactive and antigenic domains of the hormone. Mol. Cell. Endocrinol. 93, 11–16. Wagner, G. F. (1993). Stanniocalcin: Structure, function and regulation. In ‘‘Biochemistry and Molecular Biology of Fishes’’ (Hochachka, P. W., and Mommsen, T. P., Eds.), Vol. 2, pp. 419–434. Elsevier, Amsterdam. Wagner, G. F. (1994). The molecular biology of the corpuscles of Stannius and regulation of stanniocalcin gene expression. In ‘‘Fish Physiology’’ (Farrell, A. P., and Randall, D. J., Eds.), Vol. XIII (Sherwood, N. M., and Hew, C. L., Eds.), pp. 273–306. Academic Press, San Diego. Wagner, G. F., and DiMattia, G. E. (2006). The stanniocalcin family of proteins. J. Exp. Zool. 305, 769–780. Wagner, G. F., Guiraudon, C. C., Milliken, C., and Copp, D. H. (1995). Immunological and biological evidence for a stanniocalcin‐like hormone in human kidney. Proc. Natl. Acad. Sci. USA 92, 1871–1875. Wagner, G. F., Vozzolo, B. L., Jaworski, E., Haddad, M., Kline, R. L., Olsen, H. S., Rosen, C. A., Davidson, M. B., and Fenfro, J. L. (1997). Human stanniocalcin inhibits renal phosphate excretion in the rat. J. Bone Miner. Res. 12, 165–171. Wagner, G. F., Jaworski, E. M., and Haddad, M. (1998). Stanniocalcin in the seawater salmon: Structure, function, and regulation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 274, R1177–R1185. Weisbart, M. (1975). In vitro incubations of presumptive adrenocortical cells from the opisthonephros of the adult sea lamprey, Petromyzon marinus. Gen. Comp. Endocrinol. 26, 368–373. Weisbart, M., and Idler, D. R. (1970). Re‐examination of the presence of corticosteroids in two cyclostomes, the Atlantic hagfish (Myxixne glutinosa L.) and the sea lamprey (Petromyzon marinus L.). J. Endocrinol. 46, 29–43. Weisbart, M., and Youson, J. H. (1975). Steroid formation in the larval and parasitic adult sea lamprey, Petromyzon marinus L. Gen. Comp. Endocrinol. 27, 517–526. Weisbart, M., and Youson, J. H. (1977). In vivo formation of steroid from (l, 2, 6, 7, ‐ 3H)‐ progesterone by the sea lamprey, Petromyzon marinus L. J. Steroid Biochem. 8, 1249–1252. Weisbart, M., Youson, J. H., and Wieb, J. P. (1978). Biochemical, histochemical and ultrastructural analyses of presumed steroid producing tissues in sexually mature sea lamprey, Petromyzon marinus L. Gen. Comp. Endocrinol. 34, 26–37. Weisbart, M., DickhoV, W. W., Gorbman, A., and Idler, D. R. (1980). The presence of steroids in the sera of the Pacific hagfish, Eptatretus stoutii and the sea lamprey, Petromyzon marinus. Gen. Comp. Endocrinol. 41, 506–519. Wendelaar Bonga, S. E., and Greven, J. A. (1975). A second cell type in stannius bodies of two euryhaline teleost species. Cell. Tissue. Res. 159, 287–290. Wendelaar Bonga, S. E., Vander Maij, J. C. A., and Pang, P. K. T. (1980). Evidence for two secretory cell types in the stannius bodies of the teleosts Fundulus heteroclitus and Carassius auratus. Cell Tissue. Res. 212, 295–306. Wendelaar Bonga, S., and Pang, P. K. T. (1986). Stannius corpuscles. In ‘‘Vertebrate Endocrinology: Fundamentals and Biomedical Implications, Vol. 1, Morphological

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Considerations’’ (Pang, P. K. T., and Schreibman, M. P., Eds.), pp. 439–464. Academic Press, Orlando. Wendelaar Bonga, S., and Pang, P. K. T. (1991). Control of calcium regulating hormones in vertebrates: Parathyroid hormone, calcitonin, prolactin, and stanniocalcin. Int. Rev. Cytol. 128, 139–213. Youson, J. H. (1972). Structure and distribution of interstitial cells (presumptive interrenal cells) in the opisthonephric kidney of the sea lamprey, Petromyzon marinus L. Gen. Comp. Endocrinol. 19, 56–68. Youson, J. H. (1973a). A comparison of presumptive interrenal tissue in the opisthonephric kidney and dorsal vessel region of the larval sea lamprey, Petromyzon marinus L. Can. J. Zool. 5l, 796–799. Youson, J. H. (1973b). EVects of mammalian corticotrophin on the ultrastructure of presumptive interrenal cells in the opisthonephros of the lamprey, Petromyzon marinus L. Am. J. Anat. 138, 235–252. Youson, J. H. (1975). Radioautography of presumptive interrenal cells in the sea lamprey after 3 H‐cholesterol injection. Acta Zool. (Stockh.) 56, 219–223. Youson, J. H. (1976). Fine structure of granulated cells in the posterior cardinal and renal veins of Amia calva L. Can. J. Zool. 54, 843–85l. Youson, J. H. (1980). The morphology and physiology of lamprey metamorphosis. Can. J. Fish. Aquat. Sci. 37, 687–1710. Youson, J. H. (1982). The morphology of the kidney in young adult anadromous sea lampreys, Petromyzon marinus L adapted to sea water. 2. Distal and collecting segments, the archinephric duct, and the intertubular tissue and blood vessels. Can. J. Zool. 60, 2367–2381. Youson, J. H. (1985). Organ development and specialization in lamprey species. In ‘‘The Evolutionary Biology of Primitive Fishes’’ (Foreman, R. E., Gorbman, A., Dodd, J. M., and Olsson, R., Eds.), pp. 141–164. Plenum Press, New York. Youson, J. H., and Al‐Mahrouki, A. A. (1999). Ontogenetic and phylogenetic development of the endocrine pancreas (islet organ) in fishes. Gen. Comp. Endocrinol. 116, 303–335. Youson, J. H., and Butler, D. G. (1976a). The adrenocortical homolog in the lake sturgeon, Acipenser fulvescens Rafinesque. Am. J. Anat. 145, 207–224. Youson, J. H., and Butler, D. G. (1976b). Fine structure of the adrenocortical homolog and corpuscles of Stannius in Amia calva L. Acta Zool. (Stockh.) 57, 217–230. Youson, J. H., and Butler, D. G. (1985). Distribution and structure of the adrenocortical homolog in Polypterus palmas Ayres. Acta Zool. (Stockh.) 66, 131–143. Youson, J. H., Butler, D. G., and Chan, A. T. C. (1976). Identification and distribution of the adrenocortical homolog, chromaYn tissue, and corpuscles of Stannius in Amia calva L. Gen. Comp. Endocrinol. 29, 198–211. Youson, J. H., Butler, D. G., and Bawks, B. A. (1988). Distribution and structure of the adrenocortical homolog in the reed‐fish (Calamoichthys calabaricus Smith). Acta Zool. (Stockh.) 69, 77–86. Youson, J. H., Heinig, J. A., Khanam, S. F., Sower, S. A., Kawauchi, H., and Keeley, F. W. (2006a). Patterns of proopiomelanotropin (POM) and proopiocortin (POC) gene expression and of immunohistochemistry for gonadotropin‐releasing hormones (lGnRH‐I and ‐III) during the life cycle of a nonparasitic lamprey: Relationship to this adult life history type. Gen. Comp. Endocrinol. 148, 54–71. Youson, J. H., Al‐Mahrouki, A. A., Amemiya, Y., Graham, L. C., Montpetit, C. J., and Irwin, D. M. (2006b). The endocrine pancreas: Review, new data, and future research directions in ontogeny and phylogeny. Gen. Comp. Endocrinol. 148, 104–115.

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10 WHY HAVE PRIMITIVE FISHES SURVIVED? K. L. ILVES D. J. RANDALL

1. Introduction 2. Life During the Early Phanerozoic 3. The Teleosts 3.1. Feeding and Locomotion 3.2. Genome Duplication 4. Primitive Fishes: Relationships Between Groups 4.1. Agnathans 4.2. Elasmobranchs 4.3. Ratfishes/Chimaeras 4.4. Lungfishes 4.5. Coelacanths 4.6. Bichirs/Reedfishes 4.7. Sturgeons 4.8. Paddlefishes 4.9. Gars 4.10. Bowfin 5. Why Have These Primitive Fishes Survived? 5.1. Role of Physiology 5.2. Role of Genomics and Prospects for Future Research 6. Conclusions

In this chapter, we provide a description of life during the early Phanerozoic, a summary of what may have contributed to teleost diversity, followed by a brief overview of the major characteristics of the extant groups of primitive fishes. We conclude with a discussion of possible reasons why these fishes have not gone extinct, with some thoughts on the role of physiology and genomics in addressing this as yet unanswered question in the evolution of fishes. Our main argument is that extant primitive fishes have survived because they inhabit niches where teleostean advantages in feeding and locomotion are not important. 515 Primitive Fishes: Volume 26 FISH PHYSIOLOGY

Copyright # 2007 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(07)26010-8

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1. INTRODUCTION It is clear that most species that have existed at one time on the earth are now extinct and that the biosphere has evolved and animals have evolved along with those changes, often having a marked influence on the biosphere in the process. The total biomass, as well as the number of species, has varied over time. Changes such as continental drift and the composition of the atmosphere have resulted in the extinction of species and the evolution of other species through natural selection. Whole groups of organisms have prospered and then been replaced by others that, in turn, have disappeared. Several species, including some primitive (see Chapter 1, this volume, for discussion of ‘‘primitive’’) fishes, appear to have survived many of these changes. The question is: why have these primitive fishes survived where others have not?

2. LIFE DURING THE EARLY PHANEROZOIC The diversity of life in the sea and on land has increased exponentially (Benton, 1995) and the rate of species extinction has gradually declined throughout the Phanerozoic, interrupted by periodic episodes of mass extinction, referred to as crises. There are five main crises recognized, namely the end‐Ordovician, late Devonian, end‐Permian, end‐Triassic, and end‐ Cretaceous crises (Raup and Sepkoski, 1982). These crises were associated with climatic events such as a sharp temperature drop and extensive glaciation and, in some cases, collision with asteroids. Changes in UV radiation (Visscher et al., 2004), temperature change, and hypoxia were probably proximate causes of extinction of some species, with subsequent degradation of habitats and food chains being the ultimate cause of extinction of others. McGhee (1996) concluded that survivors generally represented more primitive or ancestral morphologies. Interestingly, generalist species have survived the large anthropogenic pulses of aquatic hypoxia seen in present‐day coastal waters (Diaz and Rosenberg, 2001). It appears that generalists are more likely to survive an ecological crisis than specialists. Each crisis was often not a single event but represents several mass extinctions of varying magnitude coupled together. The end‐Permian was the largest and most extensive mass extinction in which nearly 90% of marine species and around 70% of land vertebrates disappeared. The late Devonian, end‐ Ordovician, and end‐Triassic all had similar losses of around 25% in the marine environment (Table 10.1) when measured in terms of percent decrease in familial diversity in a broad range of organisms (McGhee et al., 2004).

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Table 10.1 Percent Loss in Familial Diversity Based on Data From Benton (1995), Including Both Flora and Fauna, Following the Five Mass Extinctions (Crises) That Have Occurred During the Phanerozoic (After McGhee et al., 2004) Crisis

Marine

Continental

End‐Permian Late Devonian End‐Ordovician End‐Triassic End‐Cretaceous

47.5 27.8 24.3 23.4 14.7

61.5 43.6 NA 21.7 6.3

The mass extinction at the end of the Cretaceous was the smallest in terms of percent loss of familial diversity, but was second only to the Permian crisis in terms of ecological damage because many key species were lost (McGhee et al., 2004). Extensive glaciation probably precipitated the end‐Ordovician crisis, resulting in the loss of many species but with minimal ecological impact because most of the key species were not aVected. The much smaller losses at the end of the Cretaceous, involving dinosaurs, marine reptiles, and ammonites, had a much larger ecological impact because of the removal of key species, making way for the mammals (McGhee et al., 2004). Temperatures were high and increasing throughout the Devonian (Budyko et al., 1987) and, by the end of this period, plants had covered the land and there were trees 30‐m tall. These are the oldest known trees and constituted the first forests. This rapid appearance of many plant groups and growth forms has been called the ‘‘Devonian explosion.’’ The greening of the continents with plants may have reduced carbon dioxide levels in the atmosphere and produced a colder climate. Evidence such as glacial deposits in northern Brazil (located near the Devonian South Pole) suggest widespread glaciation at the end of the Devonian, as a large continental mass covered the polar region. Massive glaciation tends to lower eustatic sea levels, which may have exacerbated the late Devonian crisis. Because glaciation appears only toward the very end of the Devonian, it is more likely to be a result rather than a cause of the drop in global temperatures. The Devonian was an important period in the evolution of aquatic vertebrates. Sea levels were generally high, and the flourishing ostracoderms were joined in the mid‐Devonian by the first jawed fishes, the armored placoderms, as well as the first sharks, and ray‐finned fishes. They became abundant and diverse. The lobe‐finned fishes also appeared in the Devonian, subsequently giving rise to the first tetrapods.

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It is not clear if the late Devonian crisis was two or many events over a period of about 3 million years. The Devonian extinction primarily involved marine biota inhabiting shallow warm‐water rather than cool‐water organisms. The great Devonian reef‐systems, which extended for thousands of kilometers, were decimated and major reef‐building did not recover until the Mesozoic era. The first pulse of the late Devonian extinction occurred about 364 mya, when fossil agnathans disappeared. A second strong pulse closed the Devonian period. The late Permian crisis, about 251 mya, was the largest and most extensive, aVecting both marine and terrestrial biota. This rapid and catastrophic event was precipitated by very extensive volcanic activity, the most widespread the world has ever experienced. This crisis probably also involved a meteorite collision, indicated by the presence of fullerines, large soccer ball‐ shaped compounds of 60–200 carbon atoms containing helium and argon in ratios indicating that they had come from space (Basu et al., 2003; Poreda and Becker, 2003). The Permian crisis resulted in the loss of nearly 90% of marine biota, clearing the way for the evolution of new forms, especially the teleosts. 3. THE TELEOSTS Since their first appearance in the fossil record in the early Mesozoic (200 mya), the teleosts (perfect bone) have evolved to become, arguably, the most successful group of fishes ever in terms of species numbers, range of habitats and climates in which they live, variation in body form, feeding, and reproductive habits (Helfman et al., 1997). With almost 26,000 named species (Eschmeyer, 2006), teleosts compose upwards of 95% of extant fish species (Helfman et al., 1997). The teleost lineage is generally divided into four major radiations, Osteoglossomorpha, Elopomorpha, Clupeomorpha, and Euteleostei. Of these, the euteleosts, which are generally broken down into four lineages (Ostariophysi, Protacanthopterygii, Paracanthopterygii, and Acanthopterygii), are by far the most diverse with over 22,000 species (Helfman et al., 1997; Moyle and Cech, 2004). Despite their incredible diversity, teleosts are generally accepted to be a monophyletic group of lineages, defined primarily by a number of characteristics of the caudal skeleton and skull (Patterson and Rosen, 1977; de Pinna, 1996). In particular, teleosts have uroneural bones in the upper caudal fin, derived from the fusion of the most posterior ural neural arches, and an urostyle (derived from fused vertebrae) at the posterior of the vertebral column, which function as supports (Helfman et al., 1997). In the skull, the supramaxillary bone, unpaired vomer, interopercular bone, and a mobile enlarged premaxillary bone are among characteristic teleost traits (Patterson and Rosen, 1977; de Pinna, 1996).

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3.1. Feeding and Locomotion Explanations for why teleosts have become the dominant group of fishes fall into two broad, interrelated, categories: improvements in (1) feeding and (2) locomotion. Many of the general feeding and locomotory trends in teleost evolution have appeared in earlier groups of both agnathans and gnathostomes and have figured prominently in explanations for the success and/or demise of many of these earlier groups. Helfman et al. (1997) have identified five general trends in morphology throughout teleostean evolution that in combination likely explain their current dominance among fishes in most aquatic systems. These include a trend toward reducing the number of bony elements, changes in the position and function of the dorsal fin, trends in the placement and function of the paired fins, correlated changes in the shape and function of the caudal region, and a shift in gas bladder function from a breathing apparatus to a buoyancy regulation device, and perhaps most significantly, major changes in the skull and buccal regions that have led to improvements and elaborations of jaw function. All of these changes can be related to either locomotion or feeding. These improvements are not mutually exclusive, rather in many cases they are likely synergistic, for example, improvements in locomotion could aid in food capture, as well as reduce the chance of being eaten. The evolution of protrusible jaws and the associated modifications that followed have been argued to be the initial ‘‘key innovation’’ that allowed teleosts to exploit a wide variety of novel niches, thereby facilitating their amazing diversification (Northcutt and Gans, 1983; Romer and Parsons, 1986; Mallatt, 1997). A phylogenetic analysis of trends in fin placement, associated spines, and upper jaw specializations showed that specializations in the jaw arose first, followed by modifications in the position of paired fins and finally by the appearance of spines (Rosen, 1982). The euteleosts, considered the most advanced teleosts, generally show the most advanced states in all of these categories, suggesting that refinements in locomotion and feeding ability together promoted the incredible diversity seen in teleosts today. 3.2. Genome Duplication Genomic explanations for why teleosts display such remarkable phenotypic diversity have received much recent attention. Because one of the three postulated whole‐genome duplications appears to coincide with the emergence of the teleosts, it has been argued that the availability of extra genetic material, particularly the developmentally important Hox gene clusters, allowed rapid phenotypic evolution and adaptive radiation (Meyer and Schartl, 1999; Chiu et al., 2004; Hoegg and Meyer, 2005). A review of these hypotheses, however,

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casts doubt about the causal connection between genome duplication and diversification in teleosts, as patterns of phenotypic change do not correlate with the timing of the duplication when fossil taxa are included (Donoghue and Purnell, 2005). An alternative model to that of rapid diversification following duplication suggests that duplications result in decreasing extinction rates by reducing the eVect of otherwise deleterious mutations as one gene copy can maintain current function(s) (Crow et al., 2005). It has also been suggested that evolutionary rates in paralogs increase following duplication (Crow et al., 2005; Brunet et al., 2006), on which natural selection can then act. This process may then explain the phenotypic diversity and adaptive radiations witnessed in teleost lineages. Understanding the eVects of genome duplication remains an active area of research and may yet lead to increased understanding of why the teleosts have been so successful. 4. PRIMITIVE FISHES: RELATIONSHIPS BETWEEN GROUPS Chapter 1 (this volume) details the current state of understanding of extant agnathan and gnathostome relationships; therefore, disagreements about particular nodes will not be discussed further in this chapter. Here we leave aside the debate about relationships between hagfishes, lampreys, and jawed vertebrates, and consider sturgeons and paddlefishes as a monophyletic group, with the bowfin and gars sister to the teleosts and the lungfishes as the tetrapods’ closest living relatives (see Chapter 1, this volume). A reasonably well‐accepted phylogeny is shown in Figure 10.1. 4.1. Agnathans 4.1.1. The Demise of the Agnathans Of the 26,000 extant fish species, only 108, represented by 2 lineages, the hagfishes (Myxinidae; 67 spp.) and the lampreys (Petromyzontidae; 41 spp.), lack jaws (Froese and Pauly, 2006). Most agnathans disappeared in the late Devonian extinction, some 364 mya, after the evolution of jawed fishes, and it has been suggested by many that these events were linked. Most hypotheses to explain the drastic turnover from jawless‐ to jawed fishes from the Devonian to today centered on the idea that competition between the bulky, awkward jawless fishes with unsophisticated feeding mechanisms and the early gnathostomes drove all, but the most specialized agnathans extinct (Gans, 1989; Long, 1995; Pough et al., 1996; see also Purnell, 2001). Not surprisingly, many of the arguments for why so few agnathans remain today follow along the same lines as explanations for why teleosts have been so successful at the apparent expense of most early gnathostomes. Namely, basal, less‐specialized lineages were

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Hagfishes Lampreys Sharks Skates/rays Ratfishes Coelacanths Lungfishes Tetrapods Bichirs/reedfishes Sturgeons Paddlefishes Gars Amia Teleosts Fig. 10.1. Phylogeny showing relationships between major groups of extant fishes. (Adapted from Chapter 1, this volume.)

outcompeted by lineages with improved feeding and/or locomotory abilities, thereby leading to their extinction. Advocates of this hypothesis refer to patterns of diversity through geologic time, which purportedly show that agnathan diversity declines precipitously with the increasing diversity of gnathostomes. Because many ostracoderm lineages coexisted with early gnathostomes for a significant period of time (100 million years), others have found fault with the assumption that competition from jawed fishes led to the extinction of the agnathans. By mapping agnathan and gnathostome diversity from the Cambrian through the Triassic, Purnell (2001) has showed that the relationship between the decline of agnathans and rise of gnathostomes is not as straightforward when agnathans are considered as separate lineages. Further, Purnell (2001) has argued that the minimum requirements for showing competition between the two groups have not been met. For the competition hypothesis to be supported, it must at least be shown that these groups came into contact in both space and time and that they utilized the same limiting resource such as food or habitat. Only stratigraphic data are available to indicate that agnathans and early gnathostomes overlapped

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in time, whereas there is relatively little information about geographic overlap, habitat use, trophic levels, and food sources for these groups (Purnell, 2001). Future work compiling distributional data and further analyses of functional morphology related to feeding may provide the necessary evidence to support or refute the competition hypothesis. Alternative explanations for the massive extinction of agnathans by the late Devonian include a significant role for climatic changes. Maisey (1996) has argued that changes in climate and associated sea level drops in the late Devonian probably reduced shallow‐water habitats, thereby leading to a general increase in competition between organisms restricted to these continental shelf areas, which includes most of the ostracoderms, but also many gnathostomes. There is also evidence that coincident with the change in nearshore habitats, bottom waters of the world’s oceans became increasingly anoxic, further reducing the potential habitat of many Devonian taxa (Maisey, 1996). Maisey (1996) has contended, therefore, that the decline of the agnathans was not because of their inferiority relative to gnathostomes, but was brought about by increased competition between all taxa following habitat loss. The two hypotheses are not mutually exclusive: hypoxia and temperature change in the late Devonian could have put stress on all animals and only the better feeders survived. There was probably some competition between jawed and jawless fishes and hypoxia and the probable climate change associated with the late Devonian crisis may have exacerbated the situation, promoting the demise of the agnathans. Regardless of the exact reason(s) for the demise of the majority of agnathan species, the fact remains that two lineages have survived both the evolution of the gnathostomes and the radiation of the teleosts: hagfishes and lampreys. The relatively few fossil representatives of these groups date to the Carboniferous, although it is postulated that the lineages actually date much further back into the Ordovician, making them basal to the now extinct Ostracoderms (Chapter 1, this volume). Explanations for their persistence hinge on a competition hypothesis, which in their case states that because of their specialized lifestyles, hagfishes and lampreys did not directly compete with any early gnathostomes and were able to survive the onslaught of jaws and locomotory advances throughout gnathostome evolution. 4.1.2. Survival of Hagfishes and Lampreys All extant hagfish species are marine and distributed in temperate marine regions of the Northern Hemisphere. Hagfishes have an eellike body, lack scales and image‐forming eyes, although they have eyespots that can detect light and dark. They have a benthic existence and a predatory and scavenging lifestyle where they feed on a variety of invertebrates and burrow into decaying matter on the seafloor using their lateral tooth plates (Martini, 1998). Hagfishes and their eggs are preyed on by some fish and marine mammals

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(Martini et al., 1997; Martini, 1998), although aspects of their behavior may reduce predation pressure. Most famous is their ability to produce copious amounts of slime when agitated, which has been shown to clog fish gills (Lim et al., 2006). The fact that hagfishes can survive when surrounded by slime, when oxygen delivery must be impaired, indicates that they are hypoxia/ anoxia tolerant (see also Bernier et al., 1996; Forster, 1998). Their bodies are incredibly flexible and they have a tendency to tie themselves into knots, which could make them diYcult for a potential predator to handle, but may also be an adaptation to remove excess slime (Martini, 1998). Overall, the hagfishes appear to fill a niche unexploited by teleosts and have probably survived as a result of a combination of their feeding and defensive specializations, as well as hypoxia tolerance. Lampreys, found mainly in temperate regions of the world, have much more varied lifestyles than hagfishes, with anadromous and freshwater‐ resident life histories. Further, there exist both parasitic (anadromous, freshwater) and nonparasitic (freshwater only) forms. Like the hagfishes, lampreys appear to fill an exclusive niche. Lampreys can swim well (Beamish, 1978) and some species make upstream migrations following similar routes to those of salmon (see Chapter 7, this volume). Lampreys also have an eellike body form, and the parasitic forms have well‐developed eyes and an oral disc with teeth and a rasping tongue, which they use to attach to the sides of a wide variety of host fishes and feed on their bodily fluids (Hardisty and Potter, 1971a). The larval stage (ammocoete) of lampreys, which remains buried in sediments of lakes and rivers filter feeding, is extensive, comprising more than 3/5 of the life span in parasitic forms and 7/8 of the life span in nonparasitic forms (Hardisty and Potter, 1971a). There is an evidence that ammocoetes are able to withstand relatively low oxygen levels (Hardisty and Potter, 1971b). Predation risk appears greatest at early larval stages but is comparatively low throughout most of the ammocoete life cycle (Hardisty, 1961). There are known fish predators of adult lampreys (Froese and Pauly, 2006), and Pacific lampreys may be under threat by introduced species such as small mouth bass, although the impact of natural predation on adult lampreys is unclear. Given their unique ammocoete larval stage, swimming abilities and the predatory habits of the anadromous and many freshwater species, it seems unlikely that the feeding and locomotory abilities of teleosts would have compromised the survival of the lampreys. 4.2. Elasmobranchs With 800 species, elasmobranchs have managed to survive the teleost radiation. Elasmobranchs have jaw protrusion, and it is not clear that teleosts have an advantage in terms of feeding mechanisms. Sharks are thought to be some of the fastest swimming extant fishes but they do not swim as well as

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some teleosts, at least in swim tunnels (Lai and Graham, 1990). Elasmobranchs hatch at a much bigger size than teleosts and some recently hatched sharks (e.g., Mako) may be 60–70 cm in length. This increased size at birth immediately confers speed on the young compared with newly hatched teleosts, simply because the sharks are much larger. Sharks eat other fish because they can chase and catch them due to a combination of size and innate swimming ability. Sharks, however, are unable to brake, reverse, or turn as well as teleosts and, as a result, are not found in environments that require great maneuverability. They have survived in habitats, such as the open ocean, where speed rather than maneuverability is important. Only a few elasmobranchs have invaded freshwater, notable examples being the Amazonian stingray and the Lake Managua shark. Once again these are wide‐open waters (the Amazon is 5‐km wide at Manaus) where elasmobranchs can compete with teleosts in terms of swimming speed and maneuverability is of less importance. It would seem that the osmotic problem is probably not the barrier to the radiation of elasmobranchs in freshwater, we suggest that the lack of maneuverability is the key element. Wherever locomotion and maneuverability are required in the aquatic environment, teleosts dominate. Fin placement and design have been central in teleost radiation, small changes in these elements are possible without the collapse of the whole system and have resulted in species that can swim in reverse as well as forward, turn rapidly, and move up and down in the water column. The basic bony structure of teleosts has allowed these developments. A large muscle mass is typical of both elasmobranchs and teleosts, especially white fibers, for high burst speeds, to catch prey or escape being eaten. The whale shark (Rhiniodon typus) and the basking shark (Cetorhinus maximus) are the largest extant fishes and both are filter feeders. These elasmobranchs achieve much larger size than both teleosts and carnivorous elasmobranches. Presumably size confers speed on carnivores, but increased size reduces the ability to turn quickly. The upper size limit for carnivores is presumably determined by the size and agility of prey, the upper limit being influenced by the requirements to change direction quickly in order to catch prey. No such limitation is placed on filter feeders, size is probably an advantage in filter feeding, the bigger the better. There are small filter‐feeding teleosts but no large filter‐feeding teleosts that approach the size of the basking and whale sharks. Presumably, the agility of teleosts is not significant in competing with these large filter feeders but is of significance in avoiding predation in small filter feeders. Size, rather than agility, is used to avoid predation in large filter feeders. Thus, teleosts dominate among small filter feeders but elasmobranches dominate among large filter feeders. Whale sharks are viviparous and elasmobranchs in general hatch at much

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larger sizes than teleosts, increasing the chance of small basking and whale sharks reaching large size. The elasmobranchs have a somewhat diVerent design of oxygen and carbon dioxide transport from that of teleosts (Randall, 1998; see also Chapter 5, this volume). In teleosts, there is tight coupling between oxygen and carbon dioxide transfer in the blood (Brauner et al., 2000a,b), but this is less so in elasmobranchs. Elasmobranch hemoglobin does not have a Root shift and there is little or no pH regulation of erythrocytes (Tufts and Randall, 1989). The absence of these features does not seem debilitating for elasmobranchs, and they appear to be able to hold their own against teleosts in terms of swimming speed. The presence of a Root shift hemoglobin in teleosts is important for buoyancy control using a swim bladder (Pelster and Randall, 1998). Sharks must swim to maintain position, and it may be that buoyancy control using a bladder reduces cost in teleosts, compared with elasmobranchs, but this remains speculation. 4.3. Ratfishes/Chimaeras These close relatives of the elasmobranchs are cartilaginous fish sometimes called ghost sharks. The 30 species, representing 3 families, are found in temperate oceans. Chimaeras have claspers for internal fertilization of females and they lay eggs with leathery cases. They lack the sharp, replaceable teeth of sharks, but have large grinding tooth plates. They appear not to swim as well as teleosts and lack their maneuverability. They occupy benthic regions, and perhaps their feeding habits and movements are such that the advantages that teleosts have in feeding and locomotion are not a factor in their environment. Ratfishes have a poisonous dorsal spine, and humans do not eat them. Perhaps other animals do not eat ratfishes, contributing to their success. Little is known of the physiology of these animals. 4.4. Lungfishes Extant lungfishes fall into three families: the monotypic Ceratodontidae of Australia (Neoceratodus fosteri), Lepidosirenidae of South America (Lepidosiren paradoxa), and the four species of Protopteridae of central and southern Africa (Protopterus spp.). Of the extant species, the Australian lungfish is considered the most primitive as it has retained more ancestral characters, such as the flipper‐like fins, large scales, and an unpaired lung (Long, 1995). The first lungfishes (400 mya) were marine, although since the end of the Carboniferous (340 mya) all lungfishes, extant species included, have apparently been restricted to freshwater (Long, 1995). While the extant species are restricted to parts of Australia, South America, and

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Africa, there is an extensive fossil record that shows they were once widely distributed in all continents (Helfman et al., 1997). As suggested by their name, all lungfishes are able to breathe air but diVer in the extent to which they use this ability. The South American and African lungfishes are obligate air‐breathers as adults and live in aquatic environments prone to drying up (Moyle and Cech, 2004). Estivation has been extensively documented in the African species, but less so in the South American species. This ability appeared early in lungfish evolution as fossil burrows have been identified from the late Paleozoic (Long, 1995). By contrast, the Australian lungfish is a facultative air‐breather during periods when little oxygen is available and, unlike the other extant species, does not appear to have any specific adaptations to avoid desiccation (Helfman et al., 1997). In terms of morphology, the African and South American species have eellike bodies with small scales, potentially making them more maneuverable than the comparatively bulky Australian lungfish. All extant species generally inhabit slow‐moving or swampy regions prone to either drying up completely or becoming oxygen depleted. Their adaptation to air breathing has thus allowed them to exploit an environment relatively depauperate of teleost fishes. Another characteristic of lungfishes is their massive tooth plates, which they use to feed on hard‐shelled invertebrates such as mollusks (Bemis, 1987). The unfavorable habitats in which they live and their ability to eat hard‐bodied prey may have allowed them to avoid competition with more motile and possibly more trophically restricted teleosts. 4.5. Coelacanths Coelacanths first appeared in the fossil record in the mid‐Devonian, but no fossils have been located that postdate the late Cretaceous (Helfman et al., 1997). The only remaining species of a formerly species‐rich group of large predators are Latimeria chalumnae and L.menadoensis, found oV the coast of eastern Africa and in the Indian Ocean, respectively. Their most unique morphological characteristic is the presence of an intracranial joint, which allows the skull to move upward when the jaws are opened (Thomson, 1973; Moyle and Cech, 2004). This results in a wide gape and allows somewhat independent movement of the head and body (Thomson, 1973). Another interesting trait is that their swim bladder is filled with fat as opposed to air, which likely permits the maintenance of neutral buoyancy (Moyle and Cech, 2004) at depths between 100 and 700 m. This fat‐filled bladder will also allow them to move in the water column much more easily than fish with air‐filled bladders. Like lungfishes, coelacanths appear to inhabit an environment (depth) less well exploited by teleost fishes, and as with sharks, their large body size

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and predatory habits may limit predation pressure. Further, Smith et al. (1975) and Wourms et al. (1991) have shown that coelacanths are ovoviviparous, meaning that the young hatch and develop from eggs inside the female’s body, which may further reduce predation on the juvenile stage. 4.6. Bichirs/Reedfishes The 10 species of extant bichirs (Polypterus spp.) and the single reedfish (Erpetoichthys calabaricus) are generally confined to shallow vegetated regions of lakes and rivers in west and central tropical Africa (Helfman et al., 1997), although some Polypterus species have been found in fast‐ flowing streams and oVshore in lakes (Greenwood, 1984). Within this region they are confined to river systems emptying into the Atlantic or associated with the Nile and they do not occur in any drainages that flow into the Indian Ocean (Greenwood, 1984). All fossil and extant species are of freshwater origin (Greenwood, 1984). Their phylogenetic aYnities have been much debated due to their possession of characters shared by a number of other primitive fish groups, such as sarcopterygians (lobed pectoral fins) and chondrosteans (spiracle, ganoid scales, maxillary fused to skull). Their most distinctive characteristics are the 5–18 independent dorsal finlets that comprise the dorsal fin (Moyle and Cech, 2004). While the extant bichirs are morphologically and ecologically similar, the reedfish has lost the pelvic fins and has a more eellike body (Greenwood, 1984). Both the bichirs and the reedfish are facultative air‐breathers, using their heavily vascularized swim bladder as a lung. As with lungfishes, this allows them to live in low‐oxygen environments where there are few actinopterygian fishes (Greenwood, 1984). 4.7. Sturgeons The 24 species in 4 genera of the Acipenseridae are found in rivers, lakes, and coastal waters, but only in the Northern Hemisphere. Sturgeon are usually benthic but they do migrate and have a bladder for buoyancy. They have a widespread distribution due to dispersion along coastal routes, lakes, and rivers (Bemis et al., 1997). Some sturgeons are reported to live for over a century and achieve a very large size. They do not breed until around 10 years old but then produce large numbers of eggs (caviar). Sturgeons are heavily armored and their protective scales are said to be able to deflect bullets. The defensive nature of these scutes is supported by the fact that they are proportionally larger and sharper on the more heavily predated juvenile sturgeon than they are on adults (Findeis, 1997). The fossil record indicates

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an estuarine habitat and a conserved life history with little change in morphology. This conserved lifestyle associated with polyploidy is inconsistent with the view that gene duplication promotes species diversity. The evolutionary history of the sturgeons is more consistent with the view that duplications result in decreasing extinction rates by reducing the eVect of otherwise deleterious mutations as one gene copy can maintain current function (Crow et al., 2005). The distant geographical separation of closely related species has been explained in terms of climatic and geologic events in the Tertiary (Choudhury and Dick, 1998) rather than there being a widespread distribution that has since diminished, leaving widely separated pockets of closely related sturgeon. The absence of sturgeon from the Southern Hemisphere is probably due to climatic and geographical barriers throughout the tropics. A few species are found in freshwater only, but many species live in the sea and then migrate up rivers to spawn. Sturgeon stir up the soft bottom mud and feed on exposed crabs, shellfish, and other small fish. They can tidally ventilate their gills via the opercular opening (Burggren, 1978) when their head is buried (see also Chapter 5, this volume). The specialized mouth of teleosts appears not to give them an advantage over sturgeon in this benthic feeding mode as sturgeons also have protrusible jaws. The sturgeons swim less well than salmonids (Chapter 7, this volume) but their heavy armor, triangular cross section with a flattened ventral surface, and benthic lifestyle appear to oVset any detriment in swimming. Once again the sturgeons occupy a niche where the teleost advantages in feeding and locomotion appear not to be a factor. In the Fraser River in British Columbia, Canada, there were large stocks of both sturgeon and Pacific salmon in the river 150 years ago. The Fraser sturgeon was decimated by fishing in the late nineteenth century and the early part of the twentieth century (Semakula and Larkin, 1968). The demise of the sturgeon in the first years of the twentieth century had no obvious eVect on the sockeye salmon populations at that time. Thus, as generally accepted, there appears to be little eVect of sturgeon on the salmon population in the river. It may be, however, that the subsequent demise of the salmon population in the last half of the nineteenth century has contributed to the limited recovery of the sturgeon population. 4.8. Paddlefishes There are only two freshwater species of paddlefish, one in North America (Polyodon spathula) the other in China (Psephurus gladius). They can be easily recognized by their long snout (rostrum, bill, spoon, or paddle), which creates lift as they move forward, lifting the head, and allowing the fish to filter plankton from the water flowing over the gills. Like the sturgeon,

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they can achieve large size and longevity and do not start to breed until around 10 years old, producing large numbers of eggs. They have appeared in the fossil record for 300 million years and have changed a little. They were very abundant a few hundred years ago, but like the sturgeon, they have been decimated by overfishing. Paddlefishes have clear diVerences in feeding habits from, and probably do not compete with, sturgeon. They are filter feeders and agility is required to avoid predation rather than to obtain food. In filter feeding, the animal moves through the water and filters out plankton, the feeding structures of teleosts do not give them any special advantage. Like the basking shark, size reduces predation pressure in paddlefishes. Once size is achieved, swimming speed and agility are no longer significant in avoiding predation. Thus, the teleosts do not have a competitive edge and the paddlefishes have survived. How paddlefishes avoid predation when small is not clear to us, but some make it to large size and have essentially negated the teleost advantage in locomotion and agility. 4.9. Gars Five of the seven extant species of Lepisosteus are generally found in rivers, large streams, and lakes associated with the Mississippi River drainage. One species is distributed in estuarine and coastal marine regions from Costa Rica, and another is found in Cuba (Helfman et al., 1997; Moyle and Cech, 2004). Despite their restricted distribution in North and Central America, fossil gars had a wide Pangean distribution (Wiley, 1976). Gars are ambush (sit and wait) predators, which is reflected in their body shape (cylindrical, depth and fins oriented posteriorly) and extended snout with large teeth (Moyle and Cech, 2004). Their bodies are heavily armored with ganoid scales (Helfman et al., 1997), and their large swim bladders can be used as a lung for facultative air breathing when water temperatures increase and oxygen availability decreases (Burleson et al., 1998), permitting them to survive in environments in which other fishes cannot. While gars appear morphologically and ecologically similar to esociforms (e.g., pikes), their current distributions do not appear to overlap (Froese and Pauly, 2006). The gars’ ability to tolerate low oxygen levels in combination with their lurking predator lifestyle and eastern North and Central American distribution may help explain their persistence. 4.10. Bowfin Amia calva, or bowfin, is a freshwater fish found in the waters of eastern North America, extending from Florida to the lakes of central Canada. They live in slow‐moving water and if the temperature is high or the water hypoxic,

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then they will breathe air using their bladder. They breed in winter when temperatures are low and air breathing is not required. When full with eggs, there is not enough room to fill the air bladder and air breathing is not possible. They lay eggs in a small mud depression and the male defends the eggs and fry in a very aggressive manner. They are carnivores with a mouth full of sharp teeth. They bury in the mud in summer when water levels are low and are called mudfish in the Southern States but they do not estivate (MacKenzie and Randall, 1990). The Amiiformes have appeared in the fossil record since the Jurassic largely unchanged, Amia is the only survivor of a much larger group, including some very large species. It is not clear to us why Amia has survived, the characteristics of this fish are found in teleosts, but not in this specific combination in a single species. Perhaps it is the particular combination of aggressive behavior, and temperature and hypoxia tolerance that has kept Amia extant despite the teleost radiation. 5. WHY HAVE THESE PRIMITIVE FISHES SURVIVED? 5.1. Role of Physiology The role of physiology in the survival of primitive fishes can be asked in two ways: first, what has been the contribution of physiology to our understanding of fish evolution and second, what was the role of their physiology in the survival of extant primitive fishes? Regarding the first question, the contribution of molecular biology and physiology to our understanding of the evolution of fishes has been minimal. This is, in part, a reflection of the eVort made compared with that based on analysis of the morphology of extant and extinct species. Form and function are tightly coupled but function cannot always be designated from a hard structure alone and is, therefore, of limited value in analysis of the fossil record. Attempting to derive evolutionary patterns from studies of extant groups is diYcult (see Chapter 5, this volume) because of convergent evolution. Is a particular set of characteristics due to common ancestry or to convergence because of living in a similar environment? The marbled swamp eel, Synbranchus marmoratus, and the lungfish, Lepidosiren paradoxa, live in similar environments and have many similar characteristics, but these are clearly not due to common ancestry but rather because these animals have adapted to similar environments. In addition, there are often several solutions for the same problem. For example, gas transfer to maintain fast swimming is somewhat diVerent in sharks and teleosts (Randall, 1998) but both groups can swim well. Once a certain solution has been adopted, this then limits subsequent possible changes.

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An understanding of physiology, however, can help in formulating questions in evolution; for example, from a functional point of view, filling the swim bladder with air will enhance the oxygen stores within the body and increase buoyancy. These functions are not mutually exclusive and enclosing a volume of air within the body will achieve both eVects. Thus, the question in the paleontological literature concerning whether the bladder initially evolved as an air‐breathing or buoyancy organ is superfluous, its initial appearance must have had an eVect on both buoyancy and oxygen stores, as well as altering the transmission of sound through the fish. The selective forces operating to retain the bladder could have been one or all of these factors. What is clear is that extant fishes have evolved a variety of modifications of the swim bladder to either enhance buoyancy, air breathing, hearing, or sound production from this more primitive state. The evolutionary question of which of these came first can be answered functionally, they all appeared together and then diVerent animals modified the swim bladder to enhance one or more of these features. The second question of the role of physiology in the survival of primitive fishes is diYcult to address. It is tempting to conclude, however, that many extant primitive fishes share potentially significant physiological traits. For example, although air breathing has evolved independently in a number of teleost lineages (e.g., gobies, arapaima, galaxiids), this ability may be particularly significant in the survival of a number of the primitive fish lineages (e.g. lungfishes, bichirs, gars, bowfin). Several, but not all, primitive fishes are known to tolerate hypoxia (lungfishes, bowfin, gars, hagfishes) and dehydration (lungfishes), and other primitive fishes may also possess these properties. Many teleosts, however, can tolerate hypoxia and some can move onto land (e.g., catfishes and eels). Interestingly, most primitive fishes also have electrosensory abilities (coelacanth, sturgeon, paddlefishes, elasmobranchs, ratfishes), but these are also found in some teleosts. Of our list of primitive fishes, only hagfishes, ratfishes, and coelacanths are strictly marine. Lungfishes were a predominantly marine group, but now only freshwater species are extant. Presumably, the capacity to survive dehydration and hypoxia/anoxia confers less of an advantage in the more constant and contiguous marine environment. It is clear that the teleosts have radiated into much of the aquatic environment at the expense of other vertebrates, whereas primitive fishes appear to be decreasing in numbers of species. Primitive fishes have survived in environments where the main features of the teleost advantage, namely feeding, locomotion, and agility, have little significance. It may be that a capacity to breathe air and tolerate hypoxia has allowed many of these primitive fishes to survive environmental crises where some other species have disappeared. Of course, chance can also play an important part in survival. Each species

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undoubtedly has an ‘‘Achilles heel’’ such that a certain set of environmental and biotic factors will lead to the demise of that species. To determine the nature of the ‘‘Achilles heel’’ is usually impossible, particularly if only the fossil record is available. Finally, much of what we say is speculative and not easily prone to experimentation. We hope, however, that better minds than ours will lead to a more formal understanding of these problems. 5.2. Role of Genomics and Prospects for Future Research The field of evolutionary genomics is rapidly expanding and will likely provide great insights into the roles of gene and genome duplications in the evolution of vertebrates. Understanding the eVects of such duplications will be particularly informative in the context of explaining why teleosts are so incredibly successful, but is less likely to help address the question of why primitive fishes have survived. Given the impossibility of comparative genomics of extinct and extant primitive fishes, we contend that further work in fields such as ecology, comparative physiology, and functional morphology will be more productive avenues of research. For instance, as suggested by Purnell (2001), compiling distributional data of extinct taxa will allow a more direct test of the competition hypothesis as it pertains to the demise of the agnathans. Such an approach may also be fruitful for addressing consequences of the emergence of teleosts. There is relatively little ecological data on virtually all primitive fishes, and as such there is much to be learned about their roles as competitors and predators in their respective ecosystems. Comparative biogeographic analysis of potential ecological analogues (e.g., gars and esociforms in North America; bichirs and lungfishes in Africa) may also prove useful in explaining the evolution of these groups. 6. CONCLUSIONS From our overview of the extant groups of primitive fishes, we conclude that, while still speculation, the most likely explanation for their survival of the teleost onslaught in the Mesozoic is due to their inhabiting environments and/or trophic niches in which the teleosts’ feeding and locomotory abilities are not major advantages, or where their distributions do not overlap with apparent ecological analogues. In other words, we contend there is little or no direct competition between teleosts and extant primitive fishes. As has been previously stated (Rosen, 1982; Janvier, 1996; Purnell, 2001), however, explanations for why major vertebrate groups have gone extinct may be nothing more than untestable speculation. Even with greater understanding of the extent, or lack, of competition between extant primitive fishes

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and teleosts, it is fallacious to extend this argument to explain why primitive fishes have survived. We agree with Purnell (2001) that it is only with further understanding of the distribution, ecology, feeding, and functional morphology of extinct fishes that we may begin to properly address the question of the survival of extant primitive fishes. With this in mind, further research in these areas on extant species will provide the opportunity to understand the significance of particular features, which can then be cautiously applied to those that have gone extinct. ACKNOWLEDGMENTS We would like to thank Colin Brauner, Anthony Farrell, David MacKenzie, Graham Scott, and Eric Taylor for their comments on an early version of this chapter and Stephen Latham for providing historical Fraser River salmon data.

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INDEX A A. naccarii, ionic and osmotic regulation, 289 ABO mechanoreceptors, in respiratory control systems, 205 Acanthodians, 33–6, 41 Acanthopterygii, 518 Acanthostega, 18, 29 Achilles heel, 532 Achoania, 38 Acipenser baeri, 137, 150 ammonia and urea excretion, 301 blood O2 transport characteristics, 241–2 O2 and CO2 exchange across respiratory surfaces, 226 respiratory control systems in, 199 whole‐blood and hemoglobin characteristics of, 219 Acipenser brevirostrum ammonia and urea excretion, 301 ammonia tolerance, 292 burst swimming and recovery from exhaustion, 364 O2 and CO2 exchange across respiratory surfaces, 226 Acipenser fulvescens adrenocortical homologue of, 473 auditory system in, 155–6 critical speed swimming, 349, 352 exercise‐related respiratory metabolism in, 353 swimming performance measurements, 341 thyroid gland, 427–9, 431 Acipenser gueldenstaedti, 301 Acipenser gueldenstaedtii, 110 adrenocortical homologue of, 474

swimming performance measurements, 341 thyroid gland, 427 Acipenserids, 10, 12, 24, 427 Acipenseriformes, 12, 14–15, 127, 138 adrenocortical homologue of, 474–8 blood O2 transport characteristics, 241–2 chromaYn cells, 483 corpuscles of stannius (CS) in, 498, 501 insulin, 392, 394 internal gills ventilation in, 192 Mauthner cell neurons in, 335 O2 and CO2 exchange across respiratory surfaces in, 225–6 ontogenetic and phylogenetic development of GEP in, 392–4 thyroid glands in, 427–9, 437 Acipenser medirostris, 475 critical speed swimming, 349–50, 352 exercise‐related respiratory metabolism in, 353 metabolic responses to exhaustive exercise in, 363 Acipenser naccarii, 67, 127 blood O2 transport characteristics, 241–2, 257 circulatory anatomy, 109–10 circulatory control, 111 critical speed swimming, 350, 352, 356, 359, 361 exercise‐related respiratory metabolism in, 353 metabolic responses to exhaustive exercise in, 363 O2 and CO2 exchange across respiratory surfaces, 226 whole‐blood and hemoglobin characteristics of, 220 537

538 Acipenser nudiventris, swimming performance measurements, 341 Acipenser oxyrhynchus/oxyrinchus adrenocortical homologue of, 473 ammonia and urea excretion, 301 metabolic responses to exhaustive exercise in, 363–4 O2 and CO2 exchange across respiratory surfaces, 226 Acipenser oxyrinchus desotoi, ammonia and urea excretion, 301 Acipenser ruthenus, 147 ammonia excretion, 299 blood O2 transport characteristics, 241, 254 whole‐blood and hemoglobin characteristics of, 220 Acipenser sp., 132 adrenocortical homologue of, 473 branchial and coronary circulation, 59, 127 swimming performance measurements, 341 Acipenser stellatus adrenocortical homologue of, 474 blood O2 transport characteristics, 242 circulatory anatomy, 109–10 thyroid gland, 427 Acipenser sturio, 110 Mauthner cell neurons in, 335 Acipenser transmontanus, 110, 134 adrenocortical homologue of, 474 blood O2 transport characteristics, 241 critical speed swimming, 349 exercise‐related respiratory metabolism in, 353, 357 gill surface area, 217 internal gills ventilation in, 192 O2 and CO2 exchange across respiratory surfaces in, 225–6 respiratory control systems in, 199 role of heterocercal tail in axial swimming of, 324 swimming performance measurements, 341 thyroid gland, 428 whole‐blood and hemoglobin characteristics of, 219 Acipensiformes, 122, 471 Actinistian (coelacanth), 6–9, 35 phylogeny, 26 Actinistian‐lungfish‐tetrapod relationships, 14 Actinopterans, 15, 35

INDEX

Actinopterygian fishes, 14, 22–5, 36–7, 41, 141, 266 brains of, 127–9 fast‐start behavior modes, 329 fast‐start performance, 366 fin function in, 327 ionic and osmotic regulation in, 284 M‐cell and startle response, 334, 337 monophyly, 11, 38 phylogeny, 24 STC in, 489 swimming modes and morphological adaptations, 321 thyroid gland, 440–1 ventilation of ABOs in, 194 Actinopterygii, 122 adrenocortical homologue of, 471–7 blood O2 transport characteristics, 239–43 CO2 transport and elimination, 254–7 Mauthner cell neurons in, 335 nitrogen excretion during early development, 305 O2 and CO2 exchange across respiratory surfaces in, 214–26 thyroid glands in, 427–37 whole‐blood and hemoglobin characteristics of, 218 Adaptive convergences (homoplasies), 3 Adenohypophysis, 382 Adrenal gland, 459 adrenocortical homologue, 459–78 of agnathans, 461–8 of gnathostomata, 468–77 phylogenetic development of, 477–8 of ancient fishes, 485–7 chromaYn tissue, 478–85 of agnatha, 480–2 of gnathostomata, 482–5 Adrenocortical homologue (AH), 457 of agnathans, 461–8 of gnathostomata, 468–77 of lampreys, 461–7 phylogenetic development of, 477–8 Adrenocortical tissue, corticosteroid synthesis in, 460–1 African lungfishes, see Lungfishes Agnathans, 6, 9, 64, 122, 309 adrenocortical homologue of, 461–8, 477 ammonia tolerance, 292 auditory system in, 156

.

INDEX

axial undulation in, 322 blood O2 transport characteristics, 250–3 brains of, 124–5 burst swimming and recovery from exhaustion, 362, 364 chromaYn tissue of, 457, 480–2 feeding and locomotion, 519 gills, 184 internal gills ventilation, 190–2 ionic and osmotic regulation in, 283–4 locomotor muscles in, 331 Mauthner cell neurons in, 335 O2 and CO2 exchange across respiratory surfaces in, 229–30 ontogenetic and phylogenetic development of GEP, 385–90 origins in seawater, 284 phylogeny of islet organ in, 404 respiratory control systems in, 196–7 skin, 186 survival of, 520–3 thyroid glands in, 407–24, 440 urea excretion, 303 Air‐breathing organs (ABOs), ventilation of, 193–6 Albuliformes, chromaYn system in, 485 Allenypterus, 26–7 Amia (Bowfins), 9–10, 12–14, 25, 121–2, 125, 127, 132, 137, 149, 234, 266, 521 adrenocortical homologue of, 475–6, 478 ammonia and urea excretion, 301 ammonia synthesis, 294 auditory system in, 154–5 blood O2 transport characteristics, 239–40 branchial circulation, 59 bulbus arteriosus in, 58 burst swimming and recovery from exhaustion, 365 cardiac activity control in, 62 chromaYn cells, 483–4 circulatory systems in, 105, 107–9 critical speed swimming, 358–60 CS in, 487–90, 493, 496 estivation challenges, 309 fast‐start behavior modes, 329 fast‐start performance, 366 fin function in, 327 GEP system of, 396–7 insulin, 392, 399 ionic and osmotic regulation in, 284, 289

539 islet organ in, 395, 398, 402 lungs and respiratory gas bladders, 188 M‐cell and startle response, 337 neuromotor coordination, 332 nitrogen excretion in, 309 phylogeny of islet organ in, 404 respiratory control systems in, 199–200, 200–2, 204–5 respiratory strategies, 183, 185 skin, 187 STC in, 491–4, 499 survival of, 529–32 swimming modes and morphological adaptations, 321 thyroid gland, 429–30 urea synthesis, 297–8 Amia calva, 25, 67, 132–3, 137, 147, 149, 152, 263, 269, 529 adrenocortical homologue of, 475 ammonia and urea excretion, 301 ammonia synthesis, 294 auditory system in, 154–5 blood O2 transport characteristics, 239–40, 242–3, 254, 257–8, 261 chromaYn cells, 484 critical speed swimming, 350, 360 CS in, 490–2 estivation challenges, 309 fast‐start behavior modes, 328–9 fast‐start performance, 367–9 GEP system of, 396 gill surface area, 217 Mauthner cell neurons in, 335 metabolic responses to exhaustive exercise in, 363 O2 and CO2 exchange across respiratory surfaces, 214–18, 224 respiratory control systems in, 199–201, 205 swimming modes and morphological adaptations, 321 thyroid gland, 429 whole‐blood and hemoglobin characteristics of, 218 Amiids, 24 Amiiformes, 127, 437 adrenocortical homologue of, 475–6, 478 blood O2 transport characteristics, 239–40 chromaYn cells, 483–5 corpuscles of stannius (CS) in, 458, 490–4, 498–9, 501

540 Amiiformes (continued ) insulin, 392–3, 399–400 Mauthner cell neurons in, 335 ontogenetic and phylogenetic development of GEP in, 394–400 STC in, 489 thyroid glands in, 429–32 Ammonia synthesis, in fishes, 294–5 Amphibians, Mauthner cell neurons in, 338 Amphipnous cuchia, thyroid gland, 432 Anaspids, 30, 40 Ancient taxa, 4–6 Andreolepis, 36 Anglerfish, insulin, 398 Anguilla anguilla blood O2 transport characteristics, 254 critical speed swimming, 351, 361 exercise‐related respiratory metabolism in, 354 hypercalcemia in, 487 Mauthner cell neurons in, 335 thyroid gland, 433–7, 441 Anguilla eel, axial undulation in, 322 Anguilla rostrata adrenocortical homologue of, 477 axial undulation in, 322, 401 chromaYn system of, 485 CS cells in, 493 Anguilla sp., thyroid gland, 432 Anguilla bicolor pacifica, 433 Anguilla japonica, 435 Anguilla obscura, 433 Anguilliformes, 265 adrenocortical homologue of, 477–8 islet organ in, 400 Mauthner cell neurons in, 335 metamorphosis in, 438 STC in, 494 Anguilloids, 15 Antiarchs, 34 Apodans, electroreception, 160 Arandaspids, 30 Arapaima, ventilation of ABOs in, 196 Arapaima gigas skin, 186 Arapaimids, 15 Arterio venous O2 diVerence, factors aVecting arterial and venous PO2, 237 blood Hb concentration, 231 and blood O2 transport characteristics, 231–8

INDEX

CO2, 235 Hb multiplicity, 232 O2 equilibrium curve and its modulators, 231–7 organic phosphates, 232–3 pH, 233–5 RBC pH regulation, 236–7 specific Hb buVer capacity, 237 temperature, 233 Arthrodires, 34, 43 Astraspids, 30 Atractosteus, 25 Atractosteus spatula blood O2 transport characteristics, 240 thyroid gland, 431 Atractosteus tristoechus, whole‐blood and hemoglobin characteristics of, 218–19 Australian lungfishes, see Lungfishes Australosomus, 24, 36

B Basal neopterygians, 33–6 Basal teleosts, see also Teleosts adrenocortical homologue of, 477 chromaYn cells, 485 corpuscles of stannius (CS) in, 494–7, 501 Batomorphs, 9–10, 12, 14–15, 20–1 Batrachoididae, ammonia tolerance, 293 Bdellostoma cirrhatum, urea synthesis, 297 Bichirs, 9, 41, 121–2, 125, 138, 148, 471 auditory system in, 154–6 blood O2 transport characteristics, 242–3 calcitonin, 382 circulatory systems in, 105–6 ionic and osmotic regulation in, 284, 290 lateral line, 160 locomotion in, 320 neuromotor coordination, 332 O2 and CO2 exchange across respiratory surfaces in, 226 survival of, 521, 527, 531–2 urea synthesis, 298 Birchirs internal gills ventilation in, 192 nitrogen excretion in, 309 respiratory control systems in, 199

.

541

INDEX

Birgeria, 24–5 Blood O2 transport characteristics, 230–53 arterio venous O2 diVerence, 231–8 extant fishes survey, 239–43 Hb function, 230–1 jawless fishes (agnatha), 250–3 hagfishes (myxiniformes), 251–3 lampreys (petromyzontiformes), 250–1 lobe‐finned fishes (sarcopterygii), 243–9 coelacanths, 243–7 lungfishes (dipnoi), 247–9 ray‐finned fishes (actinopterygii), 239–43 bowfin (amiiformes), 239–40 gars (lepisosteiformes), 240–1 reedfish and bichirs (polypteriformes), 242–3 sturgeons and paddlefishes (acipenseriformes), 241–2 Body and caudal fin (BCF) swimming, 322–5, 369 axial undulation, 322–3 heterocercal tail role in axial swimming, 323–4 skin and axial undulation, 324–5 Body bending, in elongate fishes, 322–3 Bohr eVect, 233–4, 237, 239, 248–9, 252, 254, 265–6 Bohr–Haldane eVect, 214 Bony fishes, internal gills ventilation, 192–3 Bony‐tongues, 9 phylogeny of islet organ in, 404 Boxfishes, 33 Brains of actinopterygians, 127–9 of hagfishes and lampreys, 124–5 of sarcopterygians, 125–7 Brockmann body, 385, 400–2, 404–5, 440 Bulbus arteriosus, 58, 61 Burst swimming and recovery from exhaustion, 361–6 Butterfly fish, CS in, 496

C CA activity and location, in CO2 transport and elimination, 260–2 CA in ABO, 261–2 Calamoichthys, 127

Calamoichthys calabaricus, 139, 147, 472 chromaYn cells, 483 islet organ of, 392 lungs and respiratory gas bladders, 188 Mauthner cell neurons in, 335 Callorhinchidae, 22 Callorhinchids, 23 Carassius auratus, 146 Mauthner cell neurons in, 335 Carcharhinus amblyrhincos, 145 Carcharhinus menisorrah, auditory system in, 154 Cardiac anatomy in dipnoi (lungfishes), 91–3 of hagfishes, 64–6 in lampreys, 80–1 in Lepidosiren, 91–3 in Protopterus, 91–3 in sturgeon, 109–10 Cardiac chromaYn tissue, in catecholamines, 62 Cardiorespiratory control, in tropical fishes, 54 Cardiovascular system in Amia, 107–9 anatomical patterns, 58–60 in coelacanth, 86–8 in dipnoi, 88–105 Doppler flow probes, 55 in garfishes, 106–7 in hagfishes, 64–80 in lampreys, 80–6 in lobe‐finned fishes, 86–105 measurement systems, 55–7 physiological patterns, 60–4 in polypterids, 105–6 in primitive fishes cyclostome circulatory systems, 64–86 evolutionary progression, 57–64 sarcopterygii circulatory systems, 86–105 sturgeon circulatory systems, 109–11 Cartilaginous fishes, M‐cells in, 334 Catastomus commersoni, CS in, 492–3 Catecholamines, in cardiac chromaYn tissue, 62 Catecholamine‐secreting cells, 459 Catfishes, 33, 41, 531 Central CO2 chemosensitivity, in respiratory control systems, 204

542 Central nervous system (CNS), 122 development of, 123–4 functional classification of, 129–31 olfactory input to, 147–8 of primitive fishes, 122 visual input to, 140–1 Cephalochordates, 5, 28 Cetorhinus maximus (Basking shark), 524 Cheirolepis, 17, 24, 36 Chemoreceptive systems common chemical sense, 152 gustation, 148–51 olfaction, 144–8 in primitive fishes, 144–52 solitary chemoreceptor systems, 151 Chemosensory systems, 144 Chiloscyllium punctatum, role of heterocercal tail in axial swimming of, 324 Chimaera monstrosa, 145 Chimaeras survival of, 525 urea retention in, 304–5 Chimaeriforms, 9–10, 12, 20, 22, 35, 44 Chinook salmon, arterial blood pressures for, 57 Chitala chitala, 403 CS in, 496 Chlamydoselachus, 9, 21 Chondrenchelyids, 22, 23 Chondrichthyans, 9, 11–12, 14, 20–2, 39, 141 swimming modes and morphological adaptations, 321 Chondrostean acipenseriformes, ionic and osmotic regulation in, 284 Chondrosteans auditory system in, 156 coronary circulation in, 59 electroreception, 160, 162 respiratory strategies, 183, 185 Chondrostei Mauthner cell neurons in, 335, 338 respiratory control systems in, 199 Chondrosteus, 24 ChromaYn system, 479, 485 ChromaYn tissue, 457, 459 of agnatha, 480–2 of gnathostomata, 482–5 Ciona intestinalis, 438 Circulatory control in hagfishes, 78–80

INDEX

in lampreys, 86 in Protopterus, 100–3, 105–6 in sturgeon, 111 Circulatory dynamics in hagfishes, 70–8 in lampreys, 84–5 in Lepidosiren, 95–7, 99 in Protopterus, 95–100 in sturgeon, 110–11 Circulatory patterns in hagfishes, 66–70 in lampreys, 81–4 in Lepidosiren, 93–5 in Protopterus, 93–7 in sturgeon, 110 Circulatory systems in coelacanth, 86–9 of hagfishes, 64–80 in lampreys, 80–6 in polypterids (bichirs and reedfish), 105–9 in sarcopterygii (lobe‐finned fishes), 86–105 in sturgeon, 109–11 Cladistians bichirs, 9, 15, 38 electroreception in, 162 Polypteriformes, 10, 12, 14–15 Cladistic revolution, 2 Cladoselache, 21 Clupeiformes adrenocortical homologue of, 477 chromaYn system in, 485 Clupeomorpha, 518 islet organ in, 400 Coccosteus, 34 Coelacanthiformes, 122 adrenocortical homologue of, 468 O2 and CO2 exchange across respiratory surfaces in, 227 Coelacanth‐lungfish‐tetrapod node, 14 Coelacanths, 14, 41, 54, 121–2, 138, 266, 391 adrenocortical homologue of, 468–9 auditory system in, 153 blood O2 transport characteristics, 243–7 circulatory systems, 86–9 electroreception, 162 fin use and coordination in maneuverable swimming of, 326–7 heart and its inflow and outflow vessels, 87, 89

.

543

INDEX

ionic and osmotic regulation and balance in, 283–4, 286 Mauthner cell neurons in, 335 nitrogen excretion in, 309 O2 and CO2 exchange across respiratory surfaces in, 227 phylogeny of islet organ in, 404 survival of, 521, 526–7, 531 swimming modes and morphological adaptations, 321 swimming performance measurements, 340 thyroid gland, 425, 427 urea retention in, 304–5 urea synthesis, 297–8 Conger myriaster, thyroid gland, 432–3, 435 Conus arteriosus, 58, 61 Corpuscles of stannius (CS), 457–8 in amiiformes, 490–3 in basal teleosts, 494–7 phylogenetic development of, 497–500 in semionotiformes, 493–4 Corticosteroid synthesis, in adrenocortical tissue, 460–1 Coryphaena hippurus, blood O2 transport characteristics, 254 CO2 transport and elimination CA activity and location, 260–2 hemoglobin and RBC function, 254–60 jawless fishes (agnatha), 259–60 lobe‐finned fishes (sarcopterygii), 258–9 model, 253–62 in primitive fishes, 253–62 ray‐finned fishes (actinopterygii), 254–7 bowfin (amii formes), 254–6 gars (lepisosteiformes), 256–7 reedfish and bichirs (polypteriformes), 257 sturgeons and paddlefishes (acipenseriformes), 257 Critical speed swimming, 347–61, 369 Crossopterygians, 6–7 electroreception, 160 Crossopterygii (crossopterygians), 6 Crown‐group teleosts, 24 Crown‐group tetrapods, 29 Ctenacanthiforms, 20, 21 Ctenurella, 34

Cyclostome circulatory systems hagfishes, 64–80 lampreys, 80–6 Cyclostomes, 5, 13, 32–3, 54 branchial circulation, 59 cardiac activity control in, 62 heart, 58–9 monophyly, 13 oxygen supply to heart, 63 Cypriniformes, Mauthner cell neurons in, 335 Cyprinus carpio, blood O2 transport characteristics, 245

D Danio rerio, 489–90 Deuterostomes, 5 Devonian explosion, 517 Diabolepis, 27, 37 Dialipina, 24, 35–7 Dicentrarchus labrax critical speed swimming, 351, 361 exercise‐related respiratory metabolism in, 354 Dipnoans, 9, 54 adrenocortical homologue of, 468–9 branchial circulation, 59 cardiac activity control in, 62 electroreception, 160, 162 gills, 185 respiratory control systems in, 197–8 thyroid gland, 427 Dipnoi, 122, 138 blood O2 transport characteristics, 247–9 circulatory systems, 88, 90–105 gills, 186 heart, 90 Mauthner cell neurons in, 335 O2 and CO2 exchange across respiratory surfaces in, 227–9 Dipnomorphs, 26, 35, 37 phylogeny, 27 Dipterus, 27, 41 Dollo’s Law, 2 Doppler flow probes, 55–6 Duyunolepis, 42

544

INDEX

E Early ray‐finned fishes, brains of, 127–9 Echinochimaera, 22–3 Eels, 9, 16, 27, 437, 531 calcitonin, 382 chromaYn system in, 485 coronary circulation in, 59 CS cells in, 493 fast‐start behavior modes, 328 hypercalcemia in, 487 islet organ in, 400–1 M‐cell and startle response, 337 phylogeny of islet organ in, 404 skin and axial undulation in, 324 STC in, 494 thyroid gland, 432–6, 441 Elasmobranchs, 35, 54, 138–9, 143, 151 ammonia and urea excretion, 300 auditory system in, 153–4, 156–7 bulbus arteriosus in, 58 burst swimming and recovery from exhaustion, 365–6 cardiac filling, 61 catecholamines in, 479 coronary circulation in, 59 critical speed swimming, 360–1 electroreception, 160–2, 164 fast‐start performance, 366 gills, 184–5 ionic and osmotic regulation in, 283 locomotor movements, 320 locomotor muscles in, 328, 331 osmoregulation and nitrogen excretion sites in, 285 phylogeny, 20–1 physiological patterns in, 60 survival of, 523–5, 531 swimming performance measurements, 345 urea retention in, 304–5 urea synthesis, 297–8 Electromagnetic probes, 55 Elephantnose, CS in, 496 Elongate fishes, body bending in, 322–3 Elopiformes chromaYn system in, 485 metamorphosis in, 438 Elopomorphs, 9–10, 15–16, 25, 265, 518 islet organ in, 400

Elops, 17 Elpistostegalians, 6, 37 End‐Cretaceous crisis, 516–17 Endocrine pancreas, gastrointestinal endocrine system and, 383–405 End‐Ordovician crisis, 516–17 End‐Permian crisis, 516–18 End‐Triassic crisis, 516–17 Entosphenous tridentatus, 84–5, 134 ammonia and urea excretion, 300, 302 Entosphenus, whole‐blood and hemoglobin characteristics of, 223 Entosphenus japonicus, 147 auditory system in, 156 Eptatretus burgeri, 135, 143 adenohypophysis of, 409 thyroid glands in, 408–10 Eptatretus cirrhatus (Pacific hagfish), 66, 70, 73, 76–7, 79–80 blood O2 transport characteristics, 251 burst swimming and recovery from exhaustion, 362 cardiac performance, 75, 79 metabolic responses to exhaustive exercise in, 363 O2 and CO2 exchange across respiratory surfaces in, 229 urea synthesis, 297 whole‐blood and hemoglobin characteristics of, 223 Eptatretus stouti, see Eptatretus stoutii Eptatretus stoutii, 70–1, 73, 75–8, 135, 151 ammonia and urea excretion, 300 blood O2 transport characteristics, 252–3 chromaYn tissue, 480 O2 and CO2 exchange across respiratory surfaces in, 229 PAT cells in, 467–8 thyroid glands in, 408–9 urea excretion, 303 whole‐blood and hemoglobin characteristics of, 223 Erpetoichthys, 22, 127 lungs and respiratory gas bladders, 188 respiratory control systems in, 198 ventilation of ABOs in, 195 Erpetoichthys calabaricus, 127, 132, 527 blood O2 transport characteristics, 243, 254, 257

.

545

INDEX

fast‐start behavior modes, 328–9 M‐cell and startle response, 337–8 O2 and CO2 exchange across respiratory surfaces in, 226 respiratory control systems in, 198–9 whole‐blood and hemoglobin characteristics of, 220 Esociformes, Mauthner cell neurons in, 335 Esox lucius, Mauthner cell neurons in, 335 Esox masquinongy, 327 Esox sp., critical speed swimming, 351 Euchondrocephalans, 22–3, 35 Eugeneodontids, 20, 23 Euphanerops, 42 Eusthenopteron, 17 Euteleostei, 518 Euteleosts, 437, 518 catecholamines in, 479 feeding and locomotion, 519 islet organ in, 399 locomotor movements, 320 phylogeny of islet organ in, 404 Exercise‐related respiratory metabolism, 353–4 Extinct fishes, phylogeny relationships between, 521 Extinct sarcopterygian taxa, 37–8

F Fick Principle, 55–6 Fish circulatory system, 54, see also Circulatory systems Fishes, see Primitive fishes Fossils and physiology, 39–41 Frank‐Starling eVect, 61, 74 Fugu rubripes genome, 489 urea synthesis, 297

G Gadus morhua (Atlantic cod), 66 Ganoids bulbus arteriosus in, 58 circulatory systems in, 104–9

Gar‐bowfin‐teleosts node, 13–14 Gars, 9, 13–14, 41, 54, 121–2, 125, 137–8, 141, 148–9, 266 adrenocortical homologue of, 475 blood O2 transport characteristics, 240–1 bulbus arteriosus in, 58 burst swimming and recovery from exhaustion, 365 calcitonin, 382 chromaYn cells, 483 circulatory systems in, 106–7 corpuscles of stannius (CS) in, 487, 493–4 critical speed swimming, 358–60 fin function in, 327 insulin, 392, 398–9 ionic and osmotic regulation in, 284, 290 locomotion in, 320 lungs and respiratory gas bladders, 188 nitrogen excretion in, 309 O2 and CO2 exchange across respiratory surfaces in, 224–5 phylogeny of islet organ in, 404 respiratory control systems in, 198, 199–200 respiratory strategies, 183, 185 skin and axial undulation in, 325 STCs, 499 survival of, 520–1, 529, 531–2 thyroid gland, 429, 431, 441 urea synthesis, 297 Gastroenteropancreatic (GEP) system, 458 in euteleosts, 384 phylogenetic and ontogenetic development, 381–5 thyroid gland and, 383 Gastrointestinal endocrine system, and endocrine pancreas, 383–405 Gemuendina, 34 Geotria australis, 84–6, 134–6, 142, 151, 388–9 chromaYn tissue, 481–2 O2 and CO2 exchange across respiratory surfaces in, 229 thyroid hormones and, 413, 421–3 Gill CA, in CO2 transport and elimination, 261 Gill mechanoreceptors, in respiratory control systems, 205 Ginglymods (lepisosteids), 10, 12, 24 Gnathonemus petersii, 403 CS in, 496 Gnathostomata, see Gnathostomes

546

INDEX

Gnathostomatous, auditory system in, 152–3 Gnathostomes, 5, 9, 13, 16–18, 33, 41, 521–2 adrenocortical homologue of, 468–77 chromaYn tissue of, 482–5 feeding and locomotion, 519 jaws, 3 locomotor muscles in, 329–30 Mauthner cell neurons in, 335 ontogenetic and phylogenetic development of GEP in, 391–403 thyroid glands in, 424–37 Goldfish M‐cell and startle response, 337 neuromotor coordination, 332 Gray reef shark, auditory system in, 154 Great Devonian reef‐systems, 518 Gryphognathus, 27 Gymnarchids, electroreception, 160, 162 Gymnotiform, electroreception, 162 Gyroptychius, 29

H Hadronector, 26 Hagfishes, 5–6, 9–13, 19–20, 30–1, 33, 41, 44, 57, 112, 121–2, 141–3, 151, 267 adrenocortical homologue of, 461, 467–8 AH in, 485–6 ammonia excretion, 299 ammonia tolerance, 292 auditory system in, 152–3, 157 axial undulation in, 322–3 blood O2 transport characteristics, 251–3 brains of, 124–5 branchial circulation, 59 burst swimming and recovery from exhaustion, 362 calcitonin, 382 cardiac activity control in, 61–2 cardiac filling, 61 cardiovascular performance for, 75–7 chromaYn tissue in, 457, 479–80 circulatory system arrangement of, 65 blood volume of, 69 cardiac anatomy, 64–6 caudal heart, 69–71, 78

circulatory control and dynamics, 70–8, 78–80 circulatory pattern, 66–70 critical speed swimming, 348 insulin, 392, 399 internal gills ventilation, 190–1 ionic and osmotic regulation and balance in, 283, 285–8 islet organ in, 395 lateral line, 159 locomotor muscles in, 328 marine origins, 284–5 M‐cells in, 334 metamorphosis in, 438 nitrogen excretion during early development, 305–6 O2 and CO2 exchange across respiratory surfaces in, 229–30 olfactory organ of, 145, 147 ontogenetic and phylogenetic development of GEP, 385–7, 389 phylogeny of islet organ in, 404 physiological patterns in, 60–1 presumed adrenocortical tissue (PAT) in, 467 respiratory control systems in, 196–7 skin, 186 survival of, 520–3, 531 swimming modes and morphological adaptations, 321 thyroid glands in, 407–10, 437 urea excretion, 300, 303 urea synthesis, 297–8 veins and sinuses arrangement in, 68 ventral aortic blood pressure of, 63 whole‐blood and hemoglobin characteristics of, 223 Hagfish‐lamprey‐gnathostome node, 13 Haikouichthys, 30 Haldane eVects, 254, 268–9 Halecostomi (halecostomes), 13 Hardistiella, 19–20 Hb function and blood O2 transport characteristics of fishes, 230–1 and RBC function, in CO2 transport and elimination, 254–60 Heterostracans, 17, 30, 41 Hexanchiform sharks, 9 Holocephalans, urea retention in, 20, 304–5

.

547

INDEX

Holopterygius, 26–7 Holostei (holosteans), islet organ in, 13, 394 Homoplasies, see Adaptive convergences (homoplasies) Hormones, and thyroid glands, 421–4 Hox gene, 123 Huso huso, 110 chromaYn cells, 483 ionic and osmotic regulation, 289 Hybodontiforms, 21

I Ichthyomyzon, 389 Ichthyomyzon gagei, 389 Ichthyomyzon hubbsi, respiratory control systems in, 197 Ichthyomyzon unicuspis, 139, 147, 149, 157 Ictalurus catus, 146 Iniopterygians, 20, 23 Ionic and osmotic regulation alternative strategy, 288–9 in freshwater, 289–90 in seawater, 285–9 in water moving between river and sea, 291 Islet organ, definitions of, 384 Isurus oxyrinchus, swimming performance measurements, 342

J Jawed fishes, evolution of, 517, 521 Jawed vertebrates, 5, 30, 35 Jawless fishes, 6, 41, 264 blood O2 transport characteristics, 250–3 CO2 transport and elimination in, 259–60 O2 and CO2 exchange across respiratory surfaces in, 229–30 hagfishes (myxiniformes), 229–30 lampreys (petromyzontiformes), 229 Jawless vertebrates, 9

K Katsuwonus pelamis, gill surface area, 217 Kenichthys, 29, 37–8

Knifefish, CS in, 496 Krox20 gene, 123 L L. tridentatus, 264 Lampetra appendix chromaYn tissue, 481 thyroid hormones and, 411, 416 Lampetra (Entosphenus) tridentata, thyroid glands in, 409 Lampetra fluviatilis, 67, 84, 123–4, 141–3, 268, 389, 422 blood O2 transport characteristics, 250–1, 259 brains of, 124 chromaYn tissue, 481 gill surface area, 217 ionic and osmotic regulation, 290 O2 and CO2 exchange across respiratory surfaces in, 229 PAT cells in, 465–6 respiratory control systems in, 197 whole‐blood and hemoglobin characteristics of, 222 Lampetra japonica, 81 Lampetra planeri, 81, 84, 147, 151 adrenocortical homologue of, 461–2 chromaYn tissue, 462, 481 ionic and osmotic balance in, 290 Mauthner cell neurons in, 335 presumed adrenocortical tissue (PAT) in, 465–6 thyroid hormones and, 415 Lampetra reissneri, thyroid hormones and, 415 Lampetra sp., 135 whole‐blood and hemoglobin characteristics of, 223 Lampetra tridentata blood O2 transport characteristics, 250–1 critical speed swimming, 349 electroreception, 164 metabolic responses to exhaustive exercise in, 363 thyroid hormones and, 423 Lampreys, 5–6, 9–13, 16–20, 30–1, 41, 112, 121–3, 132–4, 141–3, 145–6, 151, 236–7, 267, 269, see also Cyclostomes

548 Lampreys (continued ) adrenocortical homologue of, 461–7 AH in, 486 ammonia and urea excretion, 300 ammonia excretion, 299, 302–3 ammonia synthesis, 294 ammonia tolerance, 292 auditory system in, 152–3, 156–8 axial undulation in, 322–3 blood O2 transport characteristics, 250–1 blood volume of, 81 brains of, 124–5 burst swimming and recovery from exhaustion, 362, 370 calcitonin, 382 cardiac activity control in, 62 cardiac anatomy, 80–1 cardiac filling, 61 chromaYn tissue in, 457, 464, 481 circulatory system, 80–6 cardiac anatomy, 80–1 control and dynamics, 84–6 circulatory pattern, 81–4 critical speed swimming, 348 electroreception, 160, 162, 164–5 fast‐start behavior modes, 328 gills, 83–4, 185 insulin, 398–9 internal gills ventilation, 190, 192 ionic and osmotic regulation in, 283–6, 288–91 lateral line, 159 locomotion in, 320 locomotor muscles in, 328 M‐cells in, 334 metamorphosis in, 438–41 neuromotor coordination, 331–2 nitrogen excretion during early development, 306 O2 and CO2 exchange across respiratory surfaces in, 229 olfactory epthelium of, 146–7 ontogenetic and phylogenetic development of GEP, 385–90 photoreceptors sensitivity, 136 phylogeny of islet organ in, 404 presumed adrenocortical tissue (PAT) in, 463–5 respiratory control systems in, 196–7 rhythm generation center in, 333–4

INDEX

skin, 186 survival of, 520–3 swimming modes and morphological adaptations, 321 swimming performance, 340 thyroid glands in, 406–7, 410–24, 437 urea excretion, 303 urea synthesis, 297–8 whole‐blood and hemoglobin characteristics of, 222 Lancelet, 143 Larval (ammocoete) endostyle, thyroid hormones and, 410–5 Late Devonian crises, 516–18 Latimeria, 6–10, 12, 26, 37, 41, 44–5, 57, 112, 149 auditory system in, 153 blood O2 transport characteristics, 258 coronary circulation, 59 locomotor muscles in, 328 Latimeria chalumnae, 14, 86, 88–9, 126, 142, 147, 264, 391–2, 526 adrenocortical homologue of, 468–9 auditory system in, 153 blood O2 transport characteristics, 243–7, 258 electroreception, 162 fast‐start performance, 367–8 fin use and coordination in maneuverable swimming of, 326–7 gill surface area, 217 ionic and osmotic regulation, 288 Mauthner cell neurons in, 335 O2 and CO2 exchange across respiratory surfaces in, 227 osmoregulation and nitrogen excretion sites in, 285 swimming performance measurements, 341 thyroid gland, 425 urea synthesis, 298 whole‐blood and hemoglobin characteristics of, 220–1 Latimeria menadoensis, 14, 526 ionic and osmotic regulation, 288 Lchthyostega, 29 Lepidosiren, 10, 12, 14, 27 air‐breathing, 64, 88 brains of, 125 branchial circulation, 59 cardiac anatomy, 91–3

.

INDEX

circulatory control, 100–1 circulatory dynamics, 95–7, 99 circulatory patterns, 93–5 estivation challenges, 307 lateral line, 160 lungs and respiratory gas bladders, 188–9 oxygen supply to heart, 63 respiratory control systems in, 197 skin, 187 Lepidosiren paradoxa, 126, 186, 264, 268, 525, 530 adrenocortical homologue of, 469 blood O2 transport characteristics, 247, 249, 258 gill surface area, 217 O2 and CO2 exchange across respiratory surfaces in, 227–9 thyroid gland, 424–5 whole‐blood and hemoglobin characteristics of, 221 Lepisosteiformes, 9 blood O2 transport characteristics, 240–1 O2 and CO2 exchange across respiratory surfaces in, 224–5 Lepisosteus oculatus, 148, 150 blood O2 transport characteristics, 240 circulatory control, 106–7 critical speed swimming, 358 gill surface area, 217 O2 and CO2 exchange across respiratory surfaces in, 224–5 respiratory control systems in, 200, 205 thyroid gland, 432 whole‐blood and hemoglobin characteristics of, 218–19 Lepisosteus osseus, 145, 263, 398 adrenocortical homologue of, 476 blood O2 transport characteristics, 256 critical speed swimming, 350 fast‐start behavior modes, 328 fast‐start performance, 367–8 Mauthner cell neurons in, 335 metabolic responses to exhaustive exercise in, 363 respiratory control systems in, 200, 204 skin and axial undulation in, 324–5 whole‐blood and hemoglobin characteristics of, 219 Lepisosteus osseus oxyurus, 133

549 Lepisosteus platyrhinchus, 107, 132, 137, 139–40, 149 electroreception, 161 neuromasts in, 160 Lepisosteus platyrhincus, 67 blood O2 transport characteristics, 240, 254, 257 whole‐blood and hemoglobin characteristics of, 219 Lepisosteus platyrhynchus adrenocortical homologue of, 476 chromaYn cells, 483 Lepisosteus sp., 25, 149, 529 branchial circulation, 59 cardiac activity control in, 62 CS cells in, 493–4 insulin, 393 lungs and respiratory gas bladders, 188 O2 and CO2 exchange across respiratory surfaces in, 224 respiratory control systems in, 198–9 respiratory strategies, 183, 185 skin, 187 thyroid gland, 429–30 Leptocephalous larva, 16 Living fossil, see also Living primitive fishes concept of, 6–9 Living primitive fishes, 2 fossil relatives actinopterygians, 22–5 chondrichthyans, 20–3 hagfishes and lampreys, 19–20 sarcopterygians, 26–8 naming and dating taxa, 16–28 in vertebrate phylogeny, 9–16 coelacanth‐lungfish‐tetrapod node, 14 gar‐bowfin‐teleosts node, 13–14 hagfish‐lamprey‐gnathostome node, 13 interrelationships of, 10 mitochondrial DNA‐based vertebrate tree, 12 other problematic node, 14–16 Lobe‐finned fishes, 122 adrenocortical homologue of, 468 blood O2 transport characteristics in, 243–9 brains of, 125–7 evolution of, 517 GEP endocrine system of, 391 M‐cells in, 6, 122, 334

550 Lobe‐finned fishes (continued ) O2 and CO2 exchange across respiratory surfaces in, 227–9 coelacanths (coelacanthiformes), 227 lungfishes (dipnoi), 227–9 Locomotor muscles, 328–31 Locomotor performance and physiology burst swimming and recovery from exhaustion, 361–6, 370 continuous swimming, 339–45 critical speed swimming, 347–61, 369 endurance curves, 345–7, 369 exercise‐related respiratory metabolism, 353–4 fast‐start performance, 366–7 field measurements of, 339–45 laboratory studies, 345–66 Lophosteus, 36 Lungfishes, 6–9, 12, 14, 27–8, 37, 40–1, 57, 112, 121–2, 132–3, 139, 141, 146, 148–50, 266, see also Dipnoi adrenocortical homologue of, 469 air‐breathing, 64 ammonia and urea excretion, 300–1 ammonia tolerance, 292–3 auditory system in, 153, 156–7 blood O2 transport characteristics, 247–9 calcitonin, 382 cutaneous gas exchange, 189 electroreception, 164 estivation challenges, 307 fast‐start behavior modes, 328 hypoxic bradycardia, 62–63 ionic and osmotic regulation in, 283–4 islet organ of, 391–2 lateral line, 159–60 locomotor performance, 370 lungs and respiratory gas bladders, 188 nitrogen excretion in, 305, 308 O2 and CO2 exchange across respiratory surfaces in, 227–9 osmoregulation and nitrogen excretion sites in, 285 respiratory control systems in, 197–8 respiratory strategies, 183, 186 skin, 187 survival of, 520–1, 525–6, 531–2 swimming modes and morphological adaptations, 321 swimming performance, 343, 370

INDEX

systemic circulation in, 60 thyroid gland, 424–6 urea synthesis, 297–8

M M. mordacia, 388–9 Macropoma, 26 Macrosemiiforms, 13 Marlin, coronary circulation in, 59 Mass extinction of species, causes of, 516–18 Mawsonia, 26 Mayomyzon, 19–20 Mechanoreceptors, in respiratory control systems, 204–5 Median and paired fins (MPF) swimming, 325–7 fin function, 327 fin use and coordination in, 326–7 pectoral fin specialists, 325–6 Meemannia, 35, 38 Metamorphosis, thyroid hormones and, 415–21 Micropterus salmoides, critical speed swimming, 351 Miguashaia, 26 Misggurnus anguillicaudatus, 146 Monopterus, cutaneous gas exchange, 189 Mordacia mordax, 138, 142 ontogenetic and phylogenetic development of GEP, 385, 388 Mormyrids, electroreception, 15, 160, 162 Moythomasia, 23–4, 36 Mustelus mustelus, Mauthner cell neurons in, 335 Myllokunmingia, 30 Myllokunmingiids, 28–32 Myxine glutinosa (Atlantic hagfish), 66–7, 70, 80, 135, 146, 264, 268, 284 adenohypophysis of, 409 ammonia and urea excretion, 299–300 blood O2 transport characteristics, 252–3, 259 brains of, 124 chromaYn tissue, 479–80 insulin, 392 O2 and CO2 exchange across respiratory surfaces in, 230

.

551

INDEX

olfactory organ of, 145 PAT cells in, 467–8 skin, 186 whole‐blood and hemoglobin characteristics of, 223 Myxineides, 19, 44 Myxine sp., 66, 69–70, 73, 78–9, 85, 132, 135, 151 auditory system in, 153, 157 cardiac performance in, 74–7 lateral line, 159 Myxiniformes, 64, 122 blood O2 transport characteristics, 251–3 O2 and CO2 exchange across respiratory surfaces in, 229–30 Myxinikela, 8, 19 Myxinoformes, Mauthner cell neurons in, 335

N Negaprion brevirostris auditory system in, 154 critical speed swimming, 350 Neoceradatus, air‐breathing, 88 Neoceratodus, 7, 10, 12, 14, 27–8, 149 air‐breathing, 64 brains of, 125 branchial circulation, 59 cardiac anatomy, 91–3 circulatory control, 100–2 circulatory dynamics, 95–7, 99 circulatory patterns, 93–5 cutaneous gas exchange, 189 estivation challenges, 307 lateral line, 160 lungs and respiratory gas bladders, 188–9 respiratory control systems in, 197 skin, 187 Neoceratodus forsteri, 125, 132, 140, 146, 149, 266, 525 adrenocortical homologue of, 469–70 blood O2 transport characteristics, 247, 249 chromaYn tissue, 482 electroreception, 164 estivation challenges, 307 gills, 186 gill surface area, 217 islet organ of, 391–2

O2 and CO2 exchange across respiratory surfaces in, 227–8 thyroid gland, 424–7 whole‐blood and hemoglobin characteristics of, 221 Neopterygians, ionic and osmotic regulation in, 15, 284 Neopterygii, 496 corpuscles of stannius (CS) in, 501 Mauthner cell neurons in, 335 respiratory control systems in, 199–202 respiratory strategies, 185 Nesides, 7 Neurohypophysis, 382 Nitrogen excretion in primitive fishes, 291–309 toxic ammonia, 291–4 Normocythemic primitive fishes, 56 Notacanthiformes, metamorphosis in, 438 O Obaichthys, 25 Octavolateralis system audition, 152–6 inner ear and hair cells, 153–5 sound source localization, sensitivity, and frequency tuning, 155–6 lateral line, 152, 158–60 frequency sensitivity and object localization, 160 neuromasts, 159–60 in primitive fishes, 152–60 vestibular control, 156–8 semicircular canals and balance, 157 vestibulo‐ocular control, 157–8 Old Red Sandstone deposits, 42, 44 Oncorhynchus gorbuschka, swimming performance measurements, 342 Oncorhynchus kisutch critical speed swimming, 361 exercise‐related respiratory metabolism in, 354 Oncorhynchus mykiss, 428 ammonia excretion, 302 ammonia tolerance, 292 blood O2 transport characteristics, 245, 254, 259 critical speed swimming, 351

552

INDEX

Oncorhynchus mykiss (continued ) exercise‐related respiratory metabolism in, 354 M‐cell and startle response, 337 metabolic responses to exhaustive exercise in, 363 thyroid glands in, 405 urea synthesis, 297 Oncorhynchus nerka critical speed swimming, 350–1 exercise‐related respiratory metabolism in, 354 swimming performance measurements, 342 Ontogenetic and phylogenetic development of GEP gnathostomes, 391–403 acipenseriformes and polypteriformes, 392–4 amiiformes and semionotiformes, 394–400 basal teleosts, 400–3 sarcopterygii, 391–2 Ontogeny, definitions of, 384 Onychodontiforms, 35 Opsanus beta, nitrogen excretion during early development, 306 Opsanus tau, gill surface area, 217 O2‐sensitive chemoreceptors, in respiratory control systems, 202–3 Osteichthyans, 11, 27, 33 Osteichthyes, urea retention in, 305 Osteoglossiformes, 265, 495 adrenocortical homologue of, 477 AH in, 486 chromaYn system in, 485 corpuscles of stannius (CS) in, 499–501 Osteoglossomorphs, 9–10, 15–16, 25, 518 islet organ in, 400–2 Osteoglossum bicirrhosum, 403 CS in, 490, 492 Osteolepiformes, 6, 37 Osteostracans, 17, 30, 32 Ostracoderms, 31–3, 40

P Paddlefishes, 9, 121–2, 125, 137 AH in, 486 auditory system in, 155

blood O2 transport characteristics, 241–2 chromaYn tissue, 483, 486 critical speed swimming, 348, 352, 355–8 electroreception, 162–5 insulin, 392–3, 399 ionic and osmotic regulation in, 284, 290 islet organ of, 393 nitrogen excretion in, 309 O2 and CO2 exchange across respiratory surfaces in, 225–6 plasma corticosteroids in, 475, 477 respiratory strategies, 183, 185 skin, 187 survival of, 520–1, 528–9, 531 swimming performance measurements, 344 thyroid gland, 427, 441 urea synthesis, 298 Paleoniscoids, 6, 33–6 Paleospinax, 21 Panderichthys, 29, 37 Pantodon buchholzi, 403, 496 Paracanthopterygii, 518 Paralicththys olivaceus, thyroid gland in, 435 Paramyxine, 132 Paramyxine atarii, 69 Parasemionotids, 13–14, 24 Pax6 gene, 123 Perciformes, Mauthner cell neurons in, 335 Percina caprodes, respiratory control systems in, 198 Periophthalmodon schlosseri, ammonia tolerance, 293 Peripheral CO2‐sensitive chemoreceptors, in respiratory control systems, 203–4 Petalodontids, 20, 22–3 Petromyzon, 389 Petromyzon fluviatilis, Mauthner cell neurons in, 284, 335 Petromyzon marinus, 67, 85–6, 122, 132–5, 142, 146, 149, 157, 165, 388–90 adrenocortical homologue of, 462–3 ammonia and urea excretion, 300, 303 ammonia synthesis & tolerance, 292, 294 blood O2 transport characteristics, 250–1, 260–1 burst swimming and recovery from exhaustion, 361–2 chromaYn tissue, 481–2 critical speed swimming, 349, 352

.

INDEX

exercise‐related respiratory metabolism in, 353 ionic and osmotic regulation, 291 Mauthner cell neurons in, 335 metabolic responses to exhaustive exercise in, 363 nitrogen excretion during early development, 306 O2 and CO2 exchange across respiratory surfaces in, 229 presumed adrenocortical tissue (PAT) in, 465–7 respiratory control systems in, 197 swimming performance measurements, 341 thyroid hormones and, 413–16, 418–23 urea excretion, 303 whole‐blood and hemoglobin characteristics of, 223 Petromyzontiformes, 64, 122 blood O2 transport characteristics, 250–1 Mauthner cell neurons in, 335, 338 O2 and CO2 exchange across respiratory surfaces in, 229 Phaneropleuron, 27 Phanerozoic, life and crises during, 516–18 Pholidophorus, 24–5 Phoxinus, 147 Phylogenetic development of adrenocortical homologue, 477–8 of corpuscles of stannius (CS), 497–500 Phylogeny definitions of, 384 physiological signatures in, 3 Physiology, see also Vertebrate phylogeny fossils and, 39–41 Pikaia, 29 Pimephales promelas, ammonia tolerance, 292 Pipiscius, 20 Piscine circulatory system, 54 Placoderms, 17, 33–5, 41 Pleuronectiformes Mauthner cell neurons in, 335 metamorphosis in, 438 Polybranchic vertebrates, 42 Polyodon sp., 127, 149, 162, 394 Polyodon spathula, 110, 127, 140, 528 adrenocortical homologue of, 473–4 auditory system in, 155–6 critical speed swimming, 350 electroreception, 162–4

553 exercise‐related respiratory metabolism in, 353, 358 insulin, 393 O2 and CO2 exchange across respiratory surfaces in, 225 swimming performance measurements, 341–2 Polyodontids, 10, 12, 24 Polypterids, 54 bulbus arteriosus in, 58 circulatory systems in, 105–6 electroreception, 160 gills, 185 lungs and respiratory gas bladders, 188 respiratory control systems in, 198–9 ventilation of ABOs in, 195–6 Polypteriformes, 9, 122, 127, 138, 265–6 adrenocortical homologue of, 471–2 blood O2 transport characteristics, 242–3 chromaYn cells, 483 corpuscles of stannius (CS) in, 498, 501 internal gills ventilation in, 192 ionic and osmotic regulation in, 284 Mauthner cell neurons in, 335 O2 and CO2 exchange across respiratory surfaces in, 226 ontogenetic and phylogenetic development of GEP in, 392–4 thyroid glands in, 427–9 Polypterus bichir auditory system in, 147, 156–7 Polypterus bichir, 335 Polypterus delhezi, 139 Polypterus ornatipinnis, blood O2 transport characteristics, 243 Polypterus palmas, 147, 471 fast‐start behavior modes, 329 fast‐start performance, 367–9 fin function in, 327 Polypterus retropinnis, 471 Polypterus senegalus, 130, 148, 471–2 blood O2 transport characteristics, 242–3, 257 critical speed swimming, 359 fast‐start performance, 367–9 O2 and CO2 exchange across respiratory surfaces in, 226 respiratory control systems in, 199 thyroid gland, 427 whole‐blood and hemoglobin characteristics of, 220

554 Polypterus sp., 22, 24, 44, 127, 527 air‐breathing, 104 branchial circulation, 59 fast‐start behavior modes, 328–9 insulin, 392 internal gills ventilation in, 192 lateral line, 160 lungs and respiratory gas bladders, 188 M‐cell and startle response, 337 neuromasts in, 160 respiratory control systems in, 202 ventilation of ABOs in, 195 Porolepiforms, 6, 27, 37 Powichthys, 37 Prestanniocalcin sequences, phylogeny of, 495 Presumed adrenocortical tissue (PAT) L. fluviatilis, 465–6 in lampreys, 463–5 in P. marinus and L. planeri, 465–7 Primitive characters (symplesiomorphies), 4–6 Primitive fishes, see also Specific fishes agnathans, 520–3 ammonia excretion, 299–303 ammonia synthesis, 294–5 anatomical patterns atrium and ventricle, 58 branchial circulation, 59 caudal and cephalad coronaries, 59 heart, 58–9 systemic circulation, 60 bichirs, 521, 527 blood O2 transport characteristics, 230–53 arterio venous O2 diVerence, 231–8 extant fishes survey, 239–43 Hb function, 230–1 blood pressure among, 60–1 bowfin, 529–30 brains of, 124–9 central nervous system of, 122–4, 129–31 development of, 123–4 functional classification of, 129–31 chemoreceptive systems, 144–52 chimaeras, 525 coelacanths, 521, 526–7 CO2 transport and elimination, 253–62 elasmobranchs, 523–5 electroreception, 160–5 ampullary receptors, 161–3 passive electrolocation role, 163–5 estivation challenges, 307–9

INDEX

evolutionary progressions, 57–64 garfishes, 520–1, 529 genomics role, 532 hagfishes, 520–3 lampreys, 520–3 locomotion in, 319–21 locomotor performance and physiology, 338–67 lungfishes, 520–1, 525–6 Mauthner cell neurons in, 334 metabolic responses to exhaustive exercise in, 363 MPF–BCF coordination swimming modes pattern, 322–7 neural cardiac excitatory mechanism in, 60 neuromotor coordination, 331–8 axial rhythm generation circuits, 332–4 Mauthner neurons, 332, 334–8 startle neural circuit evolution, 334–8 nitrogen end‐products synthesis, 294–9 nitrogen excretion, 291–309 O2 and CO2 exchange across respiratory surfaces, 214–30 in jawless fishes (agnatha), 229–30 in lobe‐finned fishes (sarcopterygii), 227–9 in primitive ray‐finned fishes (actinopterygii), 215–26 octavolateralis system, 152–60 audition, 152–6 lateral line, 158–60 vestibular control, 156–8 origins in seawater, 284 osmoregulation and nitrogen excretion sites in, 285 other fishes and, 262–4 paddlefishes, 520–1, 528–9 phylogeny of islet organ in, 404–5 physiological patterns in, 60–4 physiology role in survival, 530–2 pumping mechanisms, 189–96 ratfishes, 521, 525 reedfishes, 521, 527 relation between groups, 520–30 respiratory control systems, 196–205 respiratory organs, 184–9 respiratory strategies, 183–4 skates, 521 sturgeons, 520–1, 527–8

.

INDEX

swimming modes and morphological adaptations, 321–8 urea excretion, 301–4 urea synthesis, 295–9 vertebrate blood O2 and CO2 transport characteristics and, 264–9 visual system, 132–43 whole‐blood and hemoglobin characteristics of, 218–23 Primitive lobe‐finned fishes (sarcopterygii), 263–4 Primitive ray‐finned fishes (actinopterygii), 6, 263 Principal organ, definitions of, 385 Priscomyzon, 20 Pristiophoriforms, 14 Proopiocortin (POC ) gene, 466 Proopiomelanocortin (POMC ) genes, 466 Proopiomelanotropin (POM ) gene, 466 Protacanthopterygii, 518 Protopterus aethiopicus ammonia and urea excretion, 301 blood O2 transport characteristics, 248–9, 258 O2 and CO2 exchange across respiratory surfaces in, 227–9 respiratory control systems in, 198, 203 urea synthesis, 298 whole‐blood and hemoglobin characteristics of, 222 Protopterus amphibius blood O2 transport characteristics, 248–9 O2 and CO2 exchange across respiratory surfaces in, 229 respiratory control systems in, 198 whole‐blood and hemoglobin characteristics of, 222 Protopterus annectens, 146, 148 adrenocortical homologue of, 470 ammonia and urea excretion, 301 blood O2 transport characteristics, 249 fast‐start behavior modes and performance, 328, 367 islet organ of, 391–2 Mauthner cell neurons in, 335 O2 and CO2 exchange across respiratory surfaces in, 229 respiratory control systems in, 203 thyroid gland, 424–5

555 urea synthesis, 297–8 whole‐blood and hemoglobin characteristics of, 222 Protopterus dolloi, 139, 142–3, 147 ammonia and urea excretion, 300–3 ammonia tolerance, 293 blood O2 transport characteristics, 262 chromaYn tissue, 483 estivation challenges, 307, 309 ionic and osmotic regulation, 290 islet organ of, 392 Mauthner cell neurons in, 335 nitrogen excretion in, 308 O2 and CO2 exchange across respiratory surfaces in, 228 whole‐blood and hemoglobin characteristics of, 222 Protopterus sp., 10, 12, 14, 22, 27, 67, 525 adrenocortical homologue of, 469 air‐breathing, 64, 88 auditory system in, 156 brains of, 125 branchial circulation, 59 cardiac anatomy, 91–3 chromaYn cells, 482–3 circulatory control, 100–3, 105–6 circulatory dynamics, 95–100 circulatory patterns, 93–7 estivation challenges, 307 external gills ventilation, 189 GEP system in, 392 gills, 186 heart, 90 islet organ of, 391–2 lateral line, 160 lungs and respiratory gas bladders, 188–9 O2 and CO2 exchange across respiratory surfaces in, 227, 229 respiratory control systems in, 197, 202 respiratory strategies, 183 skin, 187 systemic circulation in, 60 ventilation of ABOs in, 196 whole‐blood and hemoglobin characteristics of, 222 Psarolepis, 35, 38–9 Psephurus, 127 Psephurus gladius, 528 Pucapampella, 22, 35, 39

556

INDEX

R Rainbow trout, 56 air‐breathing, 63 physiological patterns in, 61 thyroid glands in, 405 Raja asterias, Mauthner cell neurons in, 335 Raja clavata, auditory system in, 154 Raja erinacea ammonia and urea excretion, 300 urea retention and synthesis in, 298, 304 Ratfishes, 138 insulin, 399 survival of, 521, 525, 531 Ray‐finned fishes, 121, 214 auditory system in, 154 blood O2 transport characteristics, 239–43 CO2 transport and elimination, 254–7 CS in, 496 evolution of, 517 O2 and CO2 exchange across respiratory surfaces in, 214–26 amiiformes, 215–24 bowfin, 215–24 garsfishes (Lepisosteiformes), 224–5 reedfish and bichirs (polypteriformes), 226 sturgeons and paddlefishes (acipenseriformes), 225–6 Rays, 9, 15, 21, 33, 138, 325–6 RBC CA, in CO2 transport and elimination, 260–1 Reedfishes, 121–2, 125, 132, 139, 471–2, 478 blood O2 transport characteristics, 242–3 chromaYn cells, 483 circulatory systems in, 105–6 ionic and osmotic regulation in, 284 islet organ of, 392 lungs and respiratory gas bladders, 188 O2 and CO2 exchange across respiratory surfaces in, 226 respiratory control systems in, 198 survival of, 521, 527 Respiratory control systems hypoxic and hypercarbic ventilatory responses and reflex pathways, 196–202 in agnathans, 196–7 in chondrostei, 199

in dipnoans, 197–8 in neopterygii, 199–202 in polypterids, 198–9 in primitive fishes, 196–205 receptors involved in reflex ventilatory control, 202–5 ABO mechanoreceptors, 205 central CO2 chemosensitivity, 204 gill mechanoreceptors, 205 mechanoreceptors, 204–5 O2‐sensitive chemoreceptors, 202–3 peripheral CO2‐sensitive chemoreceptors, 203–4 Respiratory organs air breathing, 187–9 water breathing, 184–7 Respiratory strategies, in primitive fishes, 183–4 Rhabdoderma, 26, 41 Rhiniodon typus (Whale shark), 524 Rhizodontids, 6, 29, 37 Rivulus marmoratus, ammonia excretion, 302 Root eVects, 234, 241, 266, 269 Ropefish, fast‐start behavior modes, 328

S S. pallidus, critical speed swimming, 350 S. trutta, metabolic responses to exhaustive exercise in, 363 Saccopharyngiformes, chromaYn system in, 485 Salamandra, 17 Salmon, 127 insulin, 398 islet organ in, 395 Salmonids, corpuscles of stannius of, 458 Salvelinus alpinus, critical speed swimming, 351 Sarcopterygian fishes, 7, 26–8, 37–8, 41, 266 brains of, 125–7 CS in, 498 fast‐start performance, 366 fin use and coordination in maneuverable swimming of, 326–7 ionic and osmotic regulation in, 284 monophyly, 38 ventilation of ABOs in, 193–4

.

INDEX

Sarcopterygii (lobe‐finned fishes), 122 adrenocortical homologue of, 468–71 blood O2 transport characteristics, 243–9 chromaYn tissue, 482–3 circulatory systems, 86–105 coelacanth, 86–9 dipnoi (lungfishes), 88, 90–105 corpuscles of stannius (CS) in, 501 Mauthner cell neurons in, 335, 338 O2 and CO2 exchange across respiratory surfaces in, 227–9 ontogenetic and phylogenetic development of GEP in, 391–2 thyroid glands in, 424–7 whole‐blood and hemoglobin characteristics of, 220 Sawfishes, 9, 15, 21 Scaphirhynchus albus, 394 insulin in, 393 Scaphirhynchus platorynchus, 110, 149 auditory system in, 154–7 critical speed swimming, 350 exercise‐related respiratory metabolism in, 355 Scaphirhynchus sp., 127, 394 adrenocortical homologue of, 473 Scaumenacia, 27 Scyliorhinus canicula, 123, 304 Scyliorhinus stellaris gill surface area, 217 Mauthner cell neurons in, 335 Semionotids, 13–14, 24 Semionotiformes, 122, 127 adrenocortical homologue of, 476–7, 478 chromaYn cells, 483–5 corpuscles of stannius (CS) in, 493–4, 499, 501 insulin, 392, 399–400 Mauthner cell neurons in, 335 ontogenetic and phylogenetic development of GEP in, 394–400 STC in, 489 thyroid glands in, 429–32, 437 Serenoichthys, 22, 24 Sharks, 10, 12, 15–16, 20, 138, 141, 523–5 auditory system in, 154 evolution of, 517, 521 role of heterocercal tail in axial swimming of, 323–4

557 skin and axial undulation in, 324 urea excretion, 303 Siluriforms, electroreception, 160, 162 Silver arawana, CS in, 496 Silver lamprey, 139 Skates, 9, 15, 21, 138 locomotion in, 325–6 survival of, 521 urea retention in, 304 urea synthesis, 298 Solea vulgaris, Mauthner cell neurons in, 335 Species extinction, causes of, 516–18 Sphyrna lewini critical speed swimming, 350 exercise‐related respiratory metabolism in, 353 swimming performance measurements, 342 Squalus, 17 Squalus acanthias (Dogfish), 66, 123 ammonia and urea excretion, 300 blood O2 transport characteristics, 261 burst swimming and recovery from exhaustion, 366 cardiac filling, 61 fast‐start performance, 366–9 Mauthner cell neurons in, 335 metabolic responses to exhaustive exercise in, 363 role of heterocercal tail in axial swimming of, 323 urea excretion & retention, 303, 305 Stanniocalcin (STC), 458, 488–9, 491–4 phylogenetic development of, 497–500 Stem dipnoans, 6 Stethacanthids, 21–2 Sturgeons, 9, 54, 121–2, 125, 137, 141, 149, 266 adrenocortical homologue of, 472–5 ammonia and urea excretion, 299, 301 ammonia tolerance, 292 auditory system in, 154–7 blood O2 transport characteristics, 241–2 burst swimming and recovery from exhaustion, 364–5 calcitonin, 382 chromaYn cells, 483 circulatory systems anatomy, 109–10 control, 111 dynamics, 110–11

558

INDEX

Sturgeons (continued ) patterns, 110 critical speed swimming, 348, 352, 355–8 insulin, 392–4 internal gills ventilation in, 192 ionic and osmotic regulation in, 284, 288–9, 291 locomotion in, 320, 328 O2 and CO2 exchange across respiratory surfaces in, 225–6 respiratory strategies and control systems in, 183, 185, 199 role of heterocercal tail in axial swimming of, 323–4 skin, 187 survival of, 520–1, 527–8, 531 swimming performance measurements, 343–4 thyroid gland, 427–8 urea synthesis, 297–8 Styloichthys, 35, 38 Suprarenal glands, 459 Swimming modes and morphological adaptations body and caudal fin (BCF) swimming, 322–5 continuous swimming and physiology, 339–45 critical speed, 347–61, 369 fast‐start behavior modes, 327–8 median and paired fins (MPF) swimming, 325–7 fin function, 327 fin use and coordination in, 326–7 pectoral fin specialists, 325–6 Swordfishes, 33 Symmoriids, 21–2 Symplesiomorphies, see Primitive characters (symplesiomorphies) Synbranchus marmoratus, 264, 530

T Takifugu rubripes, urea synthesis, 297 Tarpon (Megalops), air‐breathing, 63 Teleostei ionic and osmotic regulation, 289 Mauthner cell neurons in, 335

Teleosts, 9–10, 12–16, 54, 133, 137, 143, 149, 214, 234–5, 237, 266, 269 adrenocortical homologue of, 477 ammonia synthesis and tolerance, 293–4 auditory system in, 154–5 axial undulation in, 322 branchial circulation, 59 bulbus arteriosus in, 58 burst swimming and recovery from exhaustion, 365–6 chromaYn cells, 485 circulatory system of, 69–70 coronary circulation in, 59 corpuscles of stannius (CS) in, 487, 490, 494–7, 501 critical speed swimming, 360–1 electroreception, 160, 162, 164 evolution of, 518–21 fast‐start behavior modes, 328 fast‐start performance, 366–9 feeding and locomotion, 519 genome duplication, 519–20 gills, 185, 191, 217 ionic and osmotic regulation in, 283, 288–9, 291 islet organ of, 391 lineage, 518, 532 locomotor muscles in, 328, 330–1 Mauthner cell neurons in, 338 M‐cells in, 334 metamorphosis in, 438 nitrogen excretion during early development, 306 O2 and CO2 exchange, 214–15 ontogenetic and phylogenetic development of GEP in, 400–3 phylogeny of islet organ in, 404 physiological patterns in, 60 skin and axial undulation in, 186, 324–5 STC in, 489 swimming performance measurements, 345 thyroid glands in, 405–6, 427, 432–7, 440–1 thyroid hormones, 437 urea synthesis, 297 Telesteomorph, Mauthner cell neurons in, 338 Tetrapodomorphs, 26, 28–9, 35, 41 Tetrapods, 6–7, 10, 12, 14, 16, 37, 54, 520–1 respiratory control systems in, 197

.

559

INDEX

Thelodonts, 30, 40 Thyroid glands, 458 in agnathans, 407–24 adult thyroid and hormones, 421–4 hagfishes, 408–10 lampreys, 410–24 larval (ammocoete) endostyle, 410–5 metamorphosis, 415–21 in fishes, 405–7 and GEP system, 383 in gnathostomes actinopterygii, 427–37 amiiformes and semionotiformes, 429–32 basal teleosts, 432–7 polypteriformes and acipenseriformes, 427–9 sarcopterygii, 424–7 phylogenetic considerations, 437–40 Thyroid hormones G. australis and, 413 L. appendix and, 411, 416 L. planeri and, 415 L. reissneri and, 415 L. tridentata and, 423 metamorphosis and, 415–21 P. marinus and, 413–16, 418–20 Tiktaalik, 29, 37 Toadfish, nitrogen excretion, 306 Torpedoes, 9, 15, 21 Transit time flow probes, 56 Triakis semifasciata/semifasciatus critical speed swimming, 350, 352 role of heterocercal tail in axial swimming of, 324 Tristichopterids, 29, 37, 41 Tropical fishes, cardiorespiratory control in, 54 Truncus cordis, 58 Tunicates, 5, 28

U Undichna, 40 Uranoscopus faber, Mauthner cell neurons in, 335 Urea synthesis, in fishes, 295–9 Uric acid excretion, in sauropsid amniotes, 3

Urodeles circulatory systems, 88 electroreception, 160 V Ventilation of ABOs, 193–6 in actinopterygian fishes, 194 in polypterids, 195–6 in sarcopterygian fishes, 193–4 Ventilatory mechanism (pumps) cutaneous gas exchange, 189 external gills ventilation, 189 internal gills ventilation, 189–93 in acipenseriformes and polypteriformes, 192–3 in agnatha, 190–2 in bony fishes, 192 phylogenetic perspectives, 189–90 in primitive fishes, 189–96 ventilation of ABOs, 193–6 Ventilatory systems, 182, see also Ventilatory mechanism (pumps) Ventricular myocytes, physiological patterns in, 60–4 Ventricular outflow tract, 58 Vertebrate blood O2 and CO2 transport characteristics and in primitive fishes, 264–9 trends in evolution of, 267–9 Vertebrate phylogeny extinct fish taxa position acanthodians, 33–6 basal neopterygians, 33–6 extinct sarcopterygian taxa, 37–8 myllokunmingiids, 28–32 ostracoderms, 31–3 paleoniscoids, 33–6 placoderms, 33–5 yunnanozoans, 28–32 stability, 38–9 Vis‐a‐tergo cardiac filling, 61 Visual system nonvisual photoreception, 141–3 deep‐brain photoreceptors, 143 deermal photoreceptors, 143 pineal and parapineal organs, 142–3 optical apparatus, 132–3 retina and visual function, 133–7

560

INDEX

Visual system (continued ) spectral filters, 138 visual input to CNS, 140–1 visual resolution, 139–40 visual sensitivity, 138–9

Y Youngolepis, 37 Yunnanozoans, 28–32

X Xenacanthiforms, 21 Xenacanthus, 17 Xenopus, 426 rhythm generation center in, 333–4 Xenopus laevis, 234, 268–9 Xenotransplantation, 383, 385

Z Zebra fish, 489–90 neuromotor coordination, 332 rhythm generation center in, 334 Zhaotongaspis, 42

OTHER VOLUMES IN THE FISH PHYSIOLOGY SERIES VOLUME 1

Excretion, Ionic Regulation, and Metabolism Edited by W. S. Hoar and D. J. Randall

VOLUME 2

The Endocrine System Edited by W. S. Hoar and D. J. Randall

VOLUME 3

Reproduction and Growth: Bioluminescence, Pigments, and Poisons Edited by W. S. Hoar and D. J. Randall

VOLUME 4

The Nervous System, Circulation, and Respiration Edited by W. S. Hoar and D. J. Randall

VOLUME 5

Sensory Systems and Electric Organs

VOLUME 6

Environmental Relations and Behavior

Edited by W. S. Hoar and D. J. Randall Edited by W. S. Hoar and D. J. Randall

VOLUME 7

Locomotion Edited by W. S. Hoar and D. J. Randall

VOLUME 8

Bioenergetics and Growth Edited by W. S. Hoar, D. J. Randall, and J. R. Brett

VOLUME 9A

Reproduction: Endocrine Tissues and Hormones Edited by W. S. Hoar, D. J. Randall, and E. M. Donaldson

VOLUME 9B

Reproduction: Behavior and Fertility Control Edited by W. S. Hoar, D. J. Randall, and E. M. Donaldson

VOLUME 10A

Gills: Anatomy, Gas Transfer, and Acid-Base Regulation Edited by W. S. Hoar and D. J. Randall

VOLUME 10B

Gills: Ion and Water Transfer Edited by W. S. Hoar and D. J. Randall

VOLUME 11A

The Physiology of Developing Fish: Eggs and Larvae Edited by W. S. Hoar and D. J. Randall 561

562 VOLUME 11B

OTHER VOLUMES IN THIS SERIES

The Physiology of Developing Fish: Viviparity and Posthatching Juveniles Edited by W. S. Hoar and D. J. Randall

VOLUME 12A

The Cardiovascular System

VOLUME 12B

The Cardiovascular System

Edited by W. S. Hoar, D. J. Randall, and A. P. Farrell Edited by W. S. Hoar, D. J. Randall, and A. P. Farrell

VOLUME 13

Molecular Endocrinology of Fish Edited by N. M. Sherwood and C. L. Hew

VOLUME 14

Cellular and Molecular Approaches to Fish Ionic Regulation Edited by Chris M. Wood and Trevor J. Shuttleworth

VOLUME 15

The Fish Immune System: Organism, Pathogen, and Environment

VOLUME 16

Deep Sea Fishes

Edited by George Iwama and Teruyuki Nakanishi Edited by D. J. Randall and A. P. Farrell

VOLUME 17

Fish Respiration Edited by Steve F. Perry and Bruce Tufts

VOLUME 18

Muscle Growth and Development

VOLUME 19

Tuna: Physiology, Ecology, and Evolution

Edited by Ian A. Johnson Edited by Barbara A. Block and E. Donald Stevens

VOLUME 20

Nitrogen Excretion Edited by Patricia A. Wright and Paul M. Anderson

VOLUME 21

The Physiology of Tropical Fishes Edited by Adalberto L. Val, Vera Maria F. De Almeida-Val, and David J. Randall

VOLUME 22

The Physiology of Polar Fishes Edited by Anthony P. Farrell and John F. SteVensen

VOLUME 23

Fish Biomechanics Edited by Robert E. Shadwick and George V. Lauder

VOLUME 24

Behaviour and Physiology of Fish

VOLUME 25

Sensory Systems Neuroscience

Edited by Katherine A. Sloman, Rod W. Wilson, and Sigal Balshine Edited by Toshiaki J. Hara and Barbara S. Zielinski

VOLUME 26

Primitive Fishes Edited by David J. McKenzie, Anthony P. Farrell, and Colin J. Brauner