P. J. Butler, J. D. Metcalfe (Auth.), Dr. Trevor J. Shuttleworth (Eds.) - Physiology of Elasmobranch Fishes (1988, Springer-Verlag Berlin Heidelberg)

Trevor 1.
Shuttleworth (Editor)
Physiology of
Elasmobranch Fishes
With 116 Figures
Springer-Verlag Berlin Heidelberg NewYork

London Paris Tokyo
Dr. Trevor J. Shuttleworth
The University of Rochester Medical Center
Department of Physiology
601 Elmwood Avenue
Rochester, N.Y. 14642, USA
ISBN -13: 978-3-642-73338-3 e- ISBN-13 :978-3-642-73336-9

DOl: 10.1007/978-3-642-73336-9
Library of Congress Cataloging in Publication Data.

Shuttleworth, Trevor J. Physiology of Elasmobranch Fishes. Includes index.
1. Chrondrichthyes Physiology. 2. Fishes Physiology. I. Title. QL638.6.S.58 1988597' .304188-1989
ISBN-13:978-3-642-73338-3
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concerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation, broadcasting,
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© Springer-Verlag Berlin Heidelberg 1988
Softcover reprint of the hardcover 1st edition 1988
The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific
statement, that such names are exempt from the relevant protective laws and regulations and therefore free for
general·use.
213113020/543210
To
Professor R. F. H. "Roy" Freeman

for sharing his enthusiasm
with patience and good humour
Preface
There can be little doubt that, to use the parlance of the advertising world,
the elasmobranch fishes have a "high profile image" in today's world. To most mem-
bers of the general public they are seen as terrors of the deep, perfect aquatic
predators, and the stars (or more acurately, the villains) of major Hollywood movie
films and innumerable television nature programmes. Such an image belies the fact
that the vast majority of elasmobranch species feed on invertebrates and that, for
man, the threat from shark attack is infinitesimal compared with even being struck
by lightning! Similarly, there can be few biologists who have not carried out the
classic vertebrate dissection of the dogfish at some stage early in the formative
years of their scientific education. Yet elasmobranch species make up only a small
proportion, perhaps little more than I %, of all vertebrates, and there are probably
nearly 50 times as many teleost species as there are elasmobranchs.
It is also curious that, as subjects for modern research, elasmobranchs seem
to be chosen sometimes for their unique physiological characteristics and at other
times because they represent excellent model systems for the study of some general
process. Equally, it is for both these, seemingly contradictory, reasons that this
book was proposed. It is perhaps because the elasmobranchs are so different in
many ways from other fish species, that they often receive rather scant or
piecemeal attention in otherwise excellent texts on fish physiology, and this is
another justification for the current text. The intention was to present a broad,
but comprehensive, review of our current understanding of the functioning of the
major physiological systems of elasmobranch fish, with the aim of providing a con-
cise background for those interested in elasmobranchs, an insight for those interested
in the comparative aspects of a particular physiological system, and a reference
text for those interested in working on elasmobranchs perhaps for· the first
time. The desire to avoid a multi-volume treatise has inevitably led to some selection
of the areas to be covered, and I know some readers will be disappointed that
their specific interests are not catered for. In defence, I can only say that
making such a selection is always hard, often partly arbitary, and that I am no
less aware of the omissions than they.
Rochester, January 1988 Trevor J. Shuttleworth

Contents
Chapter 1 Cardiovascular and Respiratory Systems

P. J. Butler and J. D. Metcalfe ..................................... .
1.1 Functional Morphology of the Cardiovascular and Respiratory Systems ......... .

1.1.1 The Heart and Coronary Circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.2 Branchial Circulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.1.2.1 The "Respiratory" Blood Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.1.2.2 The "Non-Respiratory" Blood Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.1.3 The Systemic Circulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10
1.1.4 The Respiratory System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.2 Gas Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.2.1 Properties of Oxygen and Carbon Dioxide in Water and Blood. . . . . . . . . . . . . . . . . 15
1.2.2 Analysis of Gas Exchange in Relation to the Morphology of the Gills ........... 20
1.2.2.1 Direction of Blood Flow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21
1.2.2.2 Diffusion Resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21
1.2.2.3 Shunts ....... , ................................... " ..... , .... , .. " .. '" " 24
1.2.2.4 Unequal Distribution of Water and Blood Flow. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 26
1.2.3 Effectiveness of Gas Exchange. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 27
1.2.4 Use of the Fick Equation for Calculating Cardiac Output. . . . . . . . . . . . . . . . . . . . .. 27
1.3 Control of the Cardiovascular and Respiratory Systems ...................... " 28
1.3.1 The Heart. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 28
1.3.2 The Branchial Circulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 32
1.3.3 The Systemic Circulation ................................................. " 34
1.3.4 Ventilation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 35
1.4 Supply of and Demand for Oxygen: Integrated Responses of the Respiratory and
Cardiovascular Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 36
1.4.1 Temperature ............................................................. 36
1.4.2 Exercise ................................................................. 37
1.4.3 Hypoxia ................................................................. 38
References "............................................................. " 42
Chapter 2 The Central Nervous System

B. L. Roberts ............................... " ....................... 49
2.1 The Plan of the Elasmobranch Central Nervous System .. , .... , ... , ........... , 49
2.1.1 The Environment of the Brain ........................ " ..... " .......... , ., 51
2.2 Control of Motor Behaviour ............................................... 52
2.2.1 Locomotion in the Spinal Dogfish. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 52
VIII Contents
2.2.2 Is There a "Central Pattern Generator" for Locomotion? . . . . . . . . . . . . . . . . . . . . .. 52

2.2.3 The Relationship Between Sensory Input and Control Circuits . . . . . . . . . . . . . . . . .. 56
2.2.4 The Organization of the Spinal Cord ................. . . . . . . . . . . . . . . . . . . . . . .. 56
2.2.5 Spinal Cord and Brainstem ............................................. '.' .. 58
2.2.6 The Reticular Formation ................................................... 58
2.2.7 Control of Extrinsic Eye Muscles ............................................ 59
2.2.8 Control of Intrinsic Eye Muscles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 59
2.2.9 Control of Heart Muscle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 59
2.2.10 Control of Jaw and Gill Musculature During Respiration. . . . . . . . . . . . . . . . . . . . .. 60
2.2.11 Control of Sensory Hair Cells .............................................. 60
2.2.12 The Cerebellum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 61
2.2.13 Function of the Cerebellum ................................................ 63
2.2.14 Properties of Cerebellar Neurons. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 65
2.2.15 The Structure of Motor Programmes ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 65
2.3 Central Analysis of Sensory Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 66
2.3.1 Central Processing for Vestibular Function. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. 66
2.3.2 Central Processing for Audition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 67
2.3.3 Central Processing for Lateral Line Mechanoreception. . . . . . . . . . . . . . . . . . . . . . . .. 68
2.3.4 Central Processing for Electroreception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 68
2.3.5 Central Processing for Tactile Information ................................... 70
2.3.6 Central Processing of Vision. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 72
2.3.7 Central Processing of Olfactory Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 72
2.3.8 Sensory-Motor Integration ..................... , . . . . . . . . . . . . . . . . . . . . . . . . . . .. 73
2.4 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 73
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. 74
Chapter 3 Sensory Physiology

J. C. Montgomery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 79
3.1 Olfactory System............. ........ ...... .... ............................ 80
3.1.1 Behavioural Studies.............. .......... ............................... 80
3.1.2 Anatomy...... ... .. .. ....... ... ........ ............................... .. 81
3.1.3 Electrophysiology.............. .... ... ....... ............................. 82
3.2 Visual System ............................................................. 83
3.2.1 Behavioural Studies.. .. .. . . . . . . . . . . . . . . . . . . . . . . .. . . . .. . .. . . .. .. .. ... . . .. . . 83
3.2.2 Anatomy of the Visual System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.2.2.1 Extraocular Muscles/Eye Movements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 83
3.2.2.2 Eyelids.................................................................. 84
3.2.2.3 Optics................................................................... 84
3.2.3 Retinal Anatomy and Electrophysiology ..................................... 85
3.2.4 Visual Pigment ........................................................... 87
3.2.5 Retinal Electrophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 87
3.3 Octavolateralis System .......'. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 88
3.3.1 MechiLIioreceptors ........................................................ ~ 88
3.3.1.1 Behavioural Studies .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
3.3.1.2 Hair Cell Mechanoreceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 89
3.3.1.3 Anatomy of the Vestibular System ............................. ,. . . . .. . .. . .. 90
3.3.1.4 Anatomy of the Lateral Line Canals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. 91
3.3.1.5 Electrophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 92
3.3.2 Electroreception ........................................................... 93
3.3.2.1 Behavioural Studies ................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 93
3.3.2.2 Anatomy of Ampullae of Lorenzini ........... . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 94
3.3.2.3 Electrophysiology. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 95
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 96
Contents IX
Chapter 4 Muscles and Locomotion

Q. Bone ............................................................. 99
4.1 General Organization of the Locomotor System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 100

4.1.1 Sharks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 100
4.1.2 Batoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 104
4.2 The Locomotor Muscle Fibres .............................................. 105
4.2.1 Structure ................................................................. lOS
4.2.2 Motor Innervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. III
4.2.3 Sensory Innervation ....................................................... 113
4.3 Physiology ................................................................ 115
4.3.1 Electrophysiology ......................................................... 115
4.3.2 Mechanical Properties ..................................................... 118
4.3.3 Biochemical .............................................................. 120
4.4 Buoyancy and Lift. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 123
4.5 Swimming ............................................................... 128
4.5.1 Swimming Speeds ......................................................... 128
4.5.2 Body Form and Fin Distribution in Sharks ................................... 129
4.5.3 Kinematics of Shark Swimming ............................................. 131
4.5.4 Drag-Reducing Adaptations ................................................ 134
4.5.5 Warm-Bodied Sharks ...................................................... 136
4.6 Concluding Remarks ...................................................... 137
References ............................................................... 138
Chapter 5 The Autonomic Nervous System

S. Nilsson and S. Holmgren. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 143
5.1 Anatomy of the Autonomic Nervous System ................................. 144
5.1.1 Terminology ............................................................. 144
5.1.2 Cranial Autonomic Nerves ................................................. 144
5.1.3 Spinal Autonomic Nerves .................................................. 146
5.1.4 Enteric Autonomic Nerves ................................................. 146
5.1.4.1 Anatomy of the Elasmobranch Gut ......................................... 147
5.1.4.2 Arrangement of the Enteric Nervous System .................................. 147
5.1.4.3 Extrinsic Nerves .......................................................... 148
5.1.4.4 The Myenteric Plexus ...................................................... 148
5.2 Chromaffin Tissue ........................................................ 149
5.3 Circulatory System ........................................................ 150
5.3.1 The Heart. ............................................................... 150
5.3.2 The Branchial Vasculature ................................................. lSI
5.3.3 The Systemic Vasculature .................................................. lSI
5.4 The Spleen ..... '.' ........................................................ 153
5.4.1 Nerve Supply to the Spleen ................................................. 153
5.4.2 Physiological Functions of Splenic Autonomic Nerves ......................... 153
5.5 Gut ..................................................................... 154
5.5.1 Neuron Types of the Enteric Nervous System ................................. .155
5.5.2 Functions of Extrinsic Nerves .............................................. 155
5.5.2.1 Vagal Innervation ......................................................... 156
5.5.2.2 Splanchnic Innervation .................................................... 157
5.5.3 Transmitter Functions in the Gut ........................................... 158
5.5.3.1 Cholinergic Innervation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 158
X Contents
5.5.3.2 Adrenergic Innervation ................................ . . . . . . . . . . . . . . . . . . .. 158

5.5.3.3 Serotonergic Innervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 159
5.5.3.4 Peptidergic Innervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 159
5.6 Uro-GenitalOrgans . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . .. .. . . . .. . .. 163
5.7 The Iris. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. 163
5.8 . Concluding Remarks.................................... .................. 163
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 165
Chapter 6 Salt and Water Balance - Extrarenal Mechanisms

T. J. Shuttleworth ................................................... 171
6.1 Overall Hydromineral Status ............................................... 172
6.2 The Retention of Urea and TMAO .......................................... 172
6.3 Water Fluxes and Permeabilities ............................................ 175
6.4 Ion Fluxes and Permeabilities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 176
6.5 Elimination of Excess Ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 177
6.6 Rectal Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 179
6.6.1 Structure and Histology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 179
6.6.2 Secretion Rate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . .. 180
6.6.3 Mechanism of Secretion ................................................... 183
6.6.4 Mechanism of Cyclic AMP Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 185
6.6.5 Control of Secretion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 186
6.6.5.1 Hormonal Control - Peptides and Adenosine ................................ 186
6.6.5.2 Hormonal Control - Steroid Hormones. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 188
6.6.5.3 Gland Blood Flow and Secretion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 188
6.6.5.4 Initiation of the Secretory Response In Vivo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 189
6.7 Euryhaline and Freshwater Elasmobranchs ................................... 189
6.8 Osmoregulation During Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 192
References .... . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . .. 194
Chapter 7 Kidney Function

I. W. Henderson; L. B. O'Toole and N. Hazon ..................... 201
7.1 Gross Morphology ........................................................ 201
7.2 Blood Supply ............................................................. 201
7.3 Microanatomy of the Nephron ............................................. 203
7.4 Kidney Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 205
7.4.1 Urea Reabsorption ........................................................ 207
7.4.2 Reabsorption and Secretion of Ions ......................................... 208
7.5 Control of Kidney Function ................................................ 209
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 212
Chapter 8 Acid-Base Regulation

N. Heisler ........................................................... 215
8.1 Steady-State Acid-Base Regulation .......................................... 216
8.1.1 Release of Acid-Base-Re1evant Substances .................................... 216
Contents XI
8.1.2 Steady-State Acid-Base Status .............................................. 218

8.1.3 Imidazole Alphastat Regulation ............................................. 221
8.2 Acid-Base Stress Conditions ............................................... , 222
8.2.1 Temperature Changes ..................................................... 222
8.2.1.1 Transients of Extracellular Acid-Base Regulation .............................. 223
8.2.1.2 Bicarbonate - Equivalent Ion Transfer Processes ............................. 223
8.2.1.3 Contribution of Buffering and Ion Transfer to the Acid-Base Regulation ......... 228
8.2.1.4 Adjustment Kinetics of Intracellular pH ..................................... 229
8.2.2 Hypercapnia ............................................................. 230
8.2.2.1 Environmental Hypercapnia ................................................ 230
8.2.2.2 Hyperoxia - Induced Hypercapnia .......................................... 235
8.2.3 Lactacidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 239
8.3 Site and Utilization of Transepithelial Ion Transfer Mechanisms ................ 245
8.4 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 248
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 248
Chapter 9 Nitrogen Metabolism

Deborah F. Perlman and L. Goldstein ......................... " ... 253
9. F N'\ture and Routes of Excretion ............................................ 253

9.1.1 Urea ......... , ............ , .................. " ., ............... , .. " .,. 255
9.1.2 Trimethylamine Oxide (TMAO) ............................................. 255
9.\.3 Ammonia .... '" . " ..... " ................................. , ............. 256
9.1.4 Amino Acids .. , .. " ..... " .................... " .................... '" .. 256
9.2 Biochemical Pathways of Formation ......................................... 256
9.2.1 Urea .................................................................... 256
9.2.2 Trimethylamine Oxide (TMAO) ............................................. 258
9.2.3 Ammonia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 259
9.3 Urea Toxicity and Counteraction by Trimethylamine oxide ............. , ....... 261
9.4 Physiological and Evolutionary Adaptations of Nitrogen Metabolisni. for
Osmoregulation .......................................................... 262
9.4.1 Physiological Adaptations ................................................. 262
9.4.2 Evolutionary Adaptations .................................................. 265
9.5 Amino Acids and Intracellular Osmoregulation ............................... 267
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 273
Chapter 10 Reproductive Physiology

1. P. Callard and L. Klosterman (Part A); Gloria V. Callard (Part B)
ParI A The Female. , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 277

10.1 Gonadal Cycles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 277
10.1.1 The Little Skate, Raja erinacea. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 277
10.1.2 The Spiny Dogfish, Squalus acal1lhias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 278
10.2 Hormones and the Ovary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 279
10.2.1 In Vitro Ovarian Steroidogenesis .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 279
10.2.1.1 Follicular Wall ........................................................... 279
10.2.1.2 Corpus Luteum ... " ................................. " ................... 281
10.2.2 Regulation of Steroid Secretion and Synthesis in Vivo and in Vitro. . . . . . . . . . . . .. 282
10.2.3 Ovarian Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 285
10.2.3.1 Relaxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 285
XII Contents
10.3 Gametogenesis and Vitellogenesis 286

10.4 The Reprodnctive Tract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 287
10.4.1 Gross Morphological and Histological Changes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 287
10.4.2 Hormonal Regulation of the Reproductive Tract. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 288
10.4.3 Viviparity ................................................................ 291
Part B The Male .. .............................................................. 292
10.5 Gross Anatomy of the Adult Male Reproductive Tract ........................ 292
10.6 Organization of the Testis ................................................. .
10.7 Germ Cells and Spermatogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 295
10.8 Spermatozoa, Spermatophores and Fertilization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 299
10.9 Leydig Cells, Sertoli Cells and Steroidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 30 I
10.9.1 Leydig Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 30 I
10.9.2 Sertoli Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 302
10.9.3 Steroidogenesis .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 305
10.10 The Brain-Pituitary-Testicular Axis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 306
10.11 Targets of Testicular Hormone Action ....................................... 307
10.12 Seasonal Cycles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 308
10.13 Sex and Sex-Related Behaviours ............................................ 309
10.14 Sex Differentiation, Growth and Maturation ................................. 310
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 312
Subject Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 319

Chapter 1 P. J. BUTLER! and J. D. METCALFE2
Cardiovascular and Respiratory Systems
Most of what follows can be regarded as a general "ground plan" for the elasmo-
branchs as we understand them today. For practical reasons, the bulk of the detailed
physiological studies has been performed on the smaller and more sedentary species
such as the larger- and lesser-spotted dogfishes (Scyliorhinus stellaris and Scyliorhinus
canieula respectively) which are found widely in the Atlantic and Mediterranean,
and the spur-dog (Squalus acanthias, now believed to be the same species as Squalus
suekleyi, Wells and Weber 1983) which is found in the Atlantic and Pacific coastal
waters of the U.S.A. In Australia and New Zealand the Port Jackson shark
(Heterodontus portusjaeksoni) is the commonly used elasmobranch in physiological
studies. Although these species may be typical of elasmobranchs, there are notable
exceptions, such as the warm-bodied lamnid sharks. Yet, apart from a few valuable
studies on their anatomy and blood pigments, we know little of their physiological
control processes. Hopefully, more detailed studies of these species in the future
will greatly enrich our understanding of the elasmobranchs as a whole.
1.1 Functional Morphology of the Cardiovascular

and Respiratory Systems
Like that of most water-breathing fishes, the so-called single circulation of e1asmo-
branchs consists of a four-chambered heart which pumps deoxygenated blood via
the ventral aorta directly to the gills where gas exchange occurs. Oxygenated blood
then flows on to the systemic circulation before returning, now deoxygenated, to the
heart. The following description of the functional morphology of this cardio-respira-
tory system has accordingly been divided into four parts, namely: the heart and
coronary circulation, the branchial circulation, the systemic circulation, and the
respiratory system.
1 Department of Zoology and Comparative Physiology, University of Birmingham, Birmingham

B 15 2TT, U.K.
2 Directorate of Fisheries Research, Fisheries Laboratory, MAFF, Lowestoft, Suffolk, NR33 OHT,
U.K.
2 Chapter 1. Cardiovascular and Respiratory Systems
1.1.1 The Heart and Coronary Circulation
The elasmobranch heart (Fig. 1.1) is composed of four principal chambers: the
thin-walled sinus venosus, which possesses only a delicate lining of cardiac muscle
and which receives venous' blood from the great venous sinuses via the Cuverian duct
and the hepatic veins. From the sinus venosus venous blood flows via the valved
Cuv . duct
Vent r a I a o::::r:..:t.::a_.::$.~~0'Vi,o.\1. C ompacta
Conus art.
Ventricle
Fig. 1.1. Diagram of a generalized elasmobranch heart showing the position of the four principal
chambers and the position of the heart valves. S-A sino-atrial; A-V atrio-ventricular
sino-atrial ostium into the atrium. The atrium is also a fairly thin-walled structure,
but with a volume much larger than that of the sinus venosus. Blood is forced from
the atrium by its contraction into the single ventricle via the atrio-ventricular ostium.
The ventricle is a substantial pyramidal structure, the myocardium of which consists
of an outer compact layer (the compacta) which surrounds the larger, inner trabecular
layer (the spongiosa). The compacta is found in the ventricles of all elasmobranchs
but in only a few teleosts, and makes up between 15 and 43 % of its mass (Santer and
G reer-Walker 1980). These authors have suggested that the compacta is an adaptation
to either a periodically or generally active life style. In a recent study of the hearts
of warm-bodied and poikilothermic sharks, Emery et al. (1985) have shown that
the ratio of compacta to spongiosa in the ventricle is almost twice as large in
endothermic species such as great white ( Carcharodon carcharias ) and mako
( Isurus oxyrinchus) sharks at it is in poikilothermic species such as blue ( Prionace
glauca ) and tiger (Galeocerdo cuvieri) sharks. However, the size of the ventricles is
about the same in both groups of fish. It appears that in elasmobranchs, high-energy
endothermic habits are made possible by an alteration of ventricular tissue composition
rather than overall size ; this is in contrast to the pattern in fish generally (Poupa
and Ostadal 1969). Contraction of the ventricle ejects blood into the conus arteriosus.
This is surrounded by a layer of cardiac muscle and possesses two or three internal
ridges on each of which there is one or more tiers of valves (Satchell 1970).
A conspicuous feature of e1asmobranch hearts is the presence of coronary arteries
which run over the surface of the conus and the ventricle. Generally the coronary
arteries are derived from the hypobranchial arteries (see Santer 1985), but in some
species, particularly the rays, additional caudal coronary arteries are derived from
parietal branches of the dorsal aorta which supply the pericardium (Grant and
Regnier 1926). From the coronary blood vessels arise arteries, arterioles and capillaries
1.1 Functional Morphology of the Cardiovascular and Respiratory Systems 3
which supply both the outer compacta and the inner spongiosa with oxygenated
blood (Tota et al. 1983). Thus in elasmobranchs generally, the ventricular myocardium
does not rely solely on the venous blood in its lumen for its oxygen supply. The
capillaries of the spongiosa drain into the intertrabecular spaces of the ventricle
lumen while the venous drainage of the compacta is via the subepicardial veins into
the sinus venosus.
The heart lies within a more or less rigid cartilaginous pericardium which plays
an important part in cardiac function. The caudal wall of the pericardium is formed
by the fibrous pericardio-peritoneal septum while its ventral and lateral surfaces are
integral with the cartilage of the pectoral girdle. As long ago as 1895, Schoenlein,
working on the torpedo (Torpedo ocella), had recorded intrapericardial pressures
which were below ambient, and suggested that blood was aspirated into the heart.
More recent studies on Mustelus canis (Sudak 1965a, b) and S. acanthias (Johansen
1965) have confirmed and extended these early observations. It is now apparent
that the elasmobranch heart operates both as a pressure pump and a suction pump
in the following way. Ventricular contraction, which propels blood under pressure into
the ventral aorta, also results in a decrease in cardiac volume and since the
pericardium is a rigid structure, sub-ambient intrapericardial pressures develop and
blood is aspirated into the sinus venosus and atrium from the cardinal sinuses. The
mechanism of cardiac filling is enhanced by the presence of the large and compliant
cardinal sinuses which provide a reservoir of blood. This empties directly into the
sinus venosus and allows a large inflow of blood to both the sinus venosus and
atrium during the brief period of ventricular systole. In this respect the cardinal
sinuses of the elasmobranch venous system appear to be an essential component of
a system which incorporates a suctorial heart (Satchell 1970). As the atrium
contracts, blood moves into the ventricle but, since no blood leaves the pericardium
during this phase, there is no substantial change in intrapericardial pressure. Ven-
tricular blood pressure remains above that in the atrium throughout diastole (Sudak
1965 b) and blood passes into the ventricle only during atrial systole. This situation
is different from that found in the mammalian heart, where the atrio-ventricular
valves open following ventricular systole allowing the passive flow of blood into the
ventricle.
The pericardium is not entirely sealed offfrom the rest of the body. A pericardio-
peritoneal canal interconnects these two cavities, running caudally from the pericar-
dium along the ventral surface of the oesophagus and opens into the abdominal
cavity. Although the integrity of the pericardium is essential for its proper function
(Sudak 1965a), the presence of this canal does not compromise this, since it appears
to act as a one-way valve which allows the passage of fluid from the pericardium into
the peritoneum, but not the other way (Satchell 1971).
Initiation of the elasmobranch heart beat originates from the sino-atrial region
(see later), although the contraction of the sinus venosus itself is only very weak.
This is followed by sequential contraction of the atrium, ventricle, and finally the
conus arteriosus. Contraction of the cardiac muscle in the conus may contribute to
overall cardiac output, but in addition it serves to close the conal valves and
prevent any back flow of blood from the ventral aorta (Satchell and Jones 1967).
From the heart, deoxygenated blood is pumped to the gills via the ventral aorta and an
appropriate afferent branchial artery.
4 Chapter I. Cardiovascular and Respiratory Systems
1.1.2 Branchial Circulation
In recent years there have been a number of studies of the microvascular anatomy
of the gills from a variety of elasmobranch species using elaborate microscopical and
vascular casting techniques (Wright 1973; Cooke 1980; Olson and Kent 1980; De
Vries and De Jager 1984; Metcalfe and Butler 1986) and it is now possible to
present a general account of the branchial vascular anatomy.
Fig. 1.2. A semi-diagrammatic representation of the gill vascular anatomy of the dogfish, S. canicula
presented as a solid cast of the blood vessels. The figure illustrates portions of two consecutive 'gill
filaments from one hemibranch. afa afferent filament artery; ec corpus cavernosum ; ala afferent
lamellar arteriole; I lamella; ela efferent lamellar arteriole; efa efferent filament artery; eff. ava
efferent arterio-venous anastomosis; cvs central venous sinus; aev afferent companion vessel;
eev efferent companion vessel; me marginal channel; ss septal sinus. (Metcalfe and Butler 1986)
Each gill arch or holobranch consists of a sheet of muscular and connective tissue
(the inter branchial septum) which is supported by lateral rods of cartilage (the gill
rays) which radiate out from the cerato-branchials (Marshall and Hurst 1905). On
both the anterior and posterior surfaces of the inter branchial septum are attached,
for most of their length, numerous gill filaments which run radially out along the
gilL The tip of each gill filament is usually free of the inter branchial septum. On
both the dorsal and ventral surface of each of these gill filaments are arranged a row
of (secondary) lamellae and these are the site of gaseous exchange (Figs. 1.2 and 1.4).
Between the proximal edge of the lamellae and the interbranchial septum lies a water
channel which runs the entire length of the filament. In a number of elasmo-
branch species (e.g. S. acanthias and Centrophorus scalpratus) , the lamellae of the more
distal part of each gill filament possess one or more finger-like appendages which
project from their distal edge (Cooke 1980; Olson and Kent 1980; De Vries and De
Jager 1984) and these projections appear to link the lamellae of adjacent filaments,
so reducing the ventilatory dead space. De Vries and De Jager (1984) have also
observed button-like outgrowths on both sides of the lamellar epithelium at their
distal edge. These outgrowths on successive lamellae touch each other and serve as
devices to keep the interlamellar space open.
Each holobranch receives deoxygenated blood direct from the heart via its afferent
branchial artery. Within the gill there are two distinct yet extensively interconnected
blood pathways. One appears to supply blood to the lamellae, where respiratory gas
exchange occurs, and has become known as the "respiratory" blood pathway, the
other is called the "non-respiratory" blood pathway and probably has a mixed
nutritive and associated venous drainage function. For the sake of clarity, the
anatomies of these two blood pathways will be described separately.
1.1.2.1 The "Respiratory" Blood Pathway

Each gill filament receives blood from the afferent branchial artery (Fig. 1.3) via
an afferent filament artery which may itself divide and supply several adjacent
filaments. The afferent filament artery passes through the tissues of the inter-
branchial septum and joins the filament about one third of the way along its
length, where it divides into two branches. One of these branches supplies the
distal two thirds of the filament, while a recurrent branch supplies the basal third.
These two branches can functionally be considered as one continuous afferent filament
artery. Adjacent to the afferent filament artery on the filament side lies the corpus
cavernosum (Figs. 1.2, 1.3 and 1.4), which is an irregular structure almost uniquely
confined amongst fishes to the elasmobranchs. This runs the entire length of the
filament and receives blood from the afferent filament artery via numerous con-
nections. Wright (1973) reports that in S. canicula these junctions possess smooth
muscle sphincters, but this has not been confirmed in more recent studies on this
species (Dunel and Laurent 1980) or in S. acanthias (De Vries and De Jager 1984).
Towards the tip of each filament, as it separates from the interbranchial septum,
the distinction between a separate afferent filament artery and corpus cavernosum
becomes less clear and they appear to merge into a single structure in both
S. acanthias (De Vries and De Jager 1984) and S. canicula (Metcalfe and Butler
1986).
The separation of the corpus cavernosum into lateral and medial parts (Olson
and Kent 1980) does not appear to be a functionally meaningful description (De
Vries and De Jager 1984). The physiological role of the corpus cavernosum is not
fully understood and it seems likely that it may perform three important functions.
Acrivo (1935) suggested that it is a site of erythrocyte destruction and although
Kempton (1969) could find no evidence for this, Wright (1973) describes endothelial
cells which appear to be phagocytic. Wright (1973) has also suggested that the
corpus cavernosum may act as a pulse-smoothing capacitance vessel and De Vries
and De Jager (1984) support this idea of the corpus cavernosum exerting a Wil1d-
Fig. 1.3. A light micrograph of a

transverse section through a dogfish
gill cast with latex showing the
general form of the vascular anato-
my. aba afferent branchial artery;
eba efferent branchial artery; bv
branchial vein; other captions as in
Fig. 1.2. (Metcalfe and Butler 1986)
kessel effect on gill blood flow. Cooke (1980) suggested that the corpus cavernosum
may act as a hydraulic skeleton providing support for the gill filament, since the
skeletal cartilage usually observed in the gill filaments of teleost fishes is absent
from those of elasmobranchs. This idea is supported by the recent studies of De Vries
and De Jager (1984) on S. acanthias, in which they observed the effects on the position
of the gill filaments of artificially applied ventral aortic pressures. Those pressures
towards the top of the physiological range caused the filament tips to move into the
position normally observed in the live animal. It may therefore be functionally
significant that the corpus cavernosum occupies the same position in the gill
filaments of elasmobranchs as the skeletal cartilage in the gill filaments of teleosts.
The lamellae, of which there are approximately between 10 and 20 per cm along
the length of the filament, receive blood from the corpus cavernosum via afferent
lamellar arterioles (Fig. 1.4). Each lamella consists of a thin (some 15- 25 J..lm)
blood-filled lacuna bounded on its outer surface by thin epithelia which are kept
separate by pillar cells (Fig. 1.5). The blood/water diffusion distance across the
lamellae is very variable, but on average is about 11 J..lm in dogfishes , whereas in the
bottom-dwelling rays it is somewhat shorter, being about 6 J..lm (Hughes and Morgan
1973). Blood leaves the lamellae via efferent lamellar arterioles and enters the
Fig. 104. A light micrograph of a

longitlJdinal section through two
consecutive gill filaments of -a dog-
fish gill cast with latex. Abbrevia-
tions as in Fig. 1.2. (Metcalfe and
Butler 1986)
Fig. 1.5. A scanning electron micro-

graph of a transverse section
through a dogfish gill (secondary)
lamella. Tissue fixed in gluteralde-
hyde. bs blood space; p pillar cell.
(Metcalfe and Butler 1986)
8 1.1 Functional Morphology of the Cardiovascular and Respiratory Systems
efferent filament artery (Fig. 1.2). Both the afferent and efferent lamellar arterioles
are reported to possess smooth muscle sphincters (Wright 1973 ; Dunel and Laurent
1980). The blood, now oxygenated, flows along the efferent filament artery and enters
the appropriate efferent branchial arch artery of which there are two in each
holobranch, one receiving blood from the filaments on its anterior surface, and one
receiving blood from those on its posterior surface (Fig. 1.3). Blood flows from the
gill arch via its efferent branchial artery into the dorsal aorta from which it is
distributed to the systemic circulation. In S. canicula smooth muscle sphincters have
been observed in the efferent filament artery just prior to its junction with the efferent
branchial arch artery, but these sphincters do not exist in Raja clavata (Dunel and
Laurent 1980).
1.1.2.2 The "Non-Respiratory" Blood Pathway

In addition to the vascular structures described above, which are directly concerned
with blood flow to and from the gas exchange surface, there is also an extensive
vascular network which is composed of a number of components and which appears
not to be involved with gas exchange.
As well as supplying blood to the lamellae via the corpus cavernosum and afferent
lamellar arterioles, the afferent filament artery also gives rise to a network of blood
vessels within the interbranchial septum. These extend for a short distance on both
the ventral and dorsal sides of the afferent filament artery at its more distal end
before it separates from the inter branchial septum . As the gill filaments separate
from the inter branchial septum, this vascular network ' becomes more extensive and
joins with similar networks from adjacent filaments in such a way that the vascular
network is continuous throughout the distal portion of the inter branchial septum
(Fig. 1.6). The function of this network is not fully known but presumably it must,
Fig. 1.6. A light micrograph of the

distal edge of a portion of a dogfish
gill cast with latex showing the net-
work of blood vessels (11) which
originate from the distal end of each
afferent filament artery. The major
part of the gill filaments has been
dissected away.fgill filament; other
abbreviations as in Fig. 1.2. (Met-
calfe and Butler 1986)
Fig. 1.7. A scanning electron micro-

graph of a methyl methacrylate cast
of a dogfish gill filament showing
the connections between the cen-
tral venous sinus and the afferent
companion vessels. Abbreviations
as in Fig. 1.2. (Metcalfe and Butler
1986)
in part at least, serve a nutritive function for the tissues of the interbranchial
septum.
Within the central part of the gill filament, between the corpus cavernosum and
the efferent filament artery, lies an extensive central venous sinus (Figs. 1.2, 1.4 and
1. 7). In a number of species, e.g. S. acanthias, C. scalpratus and R. clavata, the
central venous sinus receives both pre- and post-lamellar blood via anastomoses with
the corpus cavernosum and the efferent filament artery respectively (Olson and Kent
1980 ; De Vries and De Jager 1984 ; Cooke 1980; Dunel and Laurent 1980), but in
other species, e.g. S. amicula and Raja erinacea (Metcalfe and Butler 1986 ; Dunel
and Laurent 1980 ; Olson and Kent 1980), the central venous sinus only receives post-
lamellar blood from the efferent filament artery. Blood which has entered the
central venous sinus drains both into the large blood sinuses within the inter-
branchial septum via vessels which have variously been termed " afferent companion
vessels" (Figs. 1.2, 1.4 and 1.7) (Cook 1980; Metcalfe and Butler 1986) or a "venous
web" (De Vries and De Jager 1984), and into efferent companion vessels which run
parallel to the efferent filament artery (Fig. 1.2) and empty into subepithelial sinuses
in the gill arch (Fig. 1.3). The efferent companion vessels are also reported to
receive a small blood supply directly from the efferent filament artery. The venous
sinuses of the interbranchial septum and the gill arch drain dorsally into the anterior
cardinal sinus and ventrally into the inferior jugular sinuses.
Although the functional role of the central venous sinus has yet to be resolved, it
is now generally accepted (Cook 1980; De Vries and De Jager 1984; Metcalfe and
Butler 1986) that, even in those species where it connects with both the afferent and
efferent sides of the filament circulation, it does not constitute a route by which
blood may bypass the exchange surface of the lamellae in the way proposed by Steen
and Kruysse (1964). Since the central venous sinus is openly connected to the large
venous sinuses of the gill arch and inter branchial septum, it must presumably operate
at blood pressures below those of the efferent filament artery, thus precluding a role
as a functional bypass. Although efferent arterio-venous anastomoses will allow
oxygenated, post-lamellar blood to be diverted via the branchial venous system back
to the heart, it seems unlikely that in elasmobranchs this is an important route for
supplying cardiac muscle with oxygen, since there is frequently a well-developed
coronary blood supply (Tota et al. 1983).
1.1.3 The Systemic Circulation
The "non-respiratory" blood pathways in the gills are perfused in parallel with the
systemic circulation and should be considtired as part of it. However, the following
description will be restricted to that part of the systemic circulation which receives
post-branchial blood. -It is not the intention of this review to present a detailed anato-
mical description of the systemic circulation in elasmobranchs; for this the reader is
referred to a number of the older, but still excellent, studies from a number of elasmo-
branch species: Burne (1923) (Lamna cornubica, now Lamna nasus), Carazzi (1905)
Table 1.1 Oxygen consumption, respiratory and cardiovascular variables of elasmobranchs at different
environmental temperatures
Species and source Mass Temperature Yentilation Cardiac Vw

(kg) (oq volume (Vw) output (Vb) -Y·
(ml kg- i min-i) (ml kg- i min-i) b
S. stellaris
Baumgarten-Schumann
and Piiper 1968 2.2 15-17 195 22 8.9
Piiper et al. 1970 2.3 15-17 305 40 7.6
Piiper et a!. 1977 2.6 16-18 320-590 52 11.3
Negaprion
brevirostris
Bushnell et a!. 1982 2.0-3.4 25
Dasyatis sabina
Cameron et al. 1971 0.49 23 572 64.5 9.2
Squalus suckleyt'
Hanson and Johansen
1970 1.6-2.5 6-7 327 32 12
Lenfant and Joliansen
1966 2.5-6.0 11
S. canicula
Short et a!. 1979 0.75 15 760 32 24
Butler and Taylor 1975 0.86 7 19

a Now thought to be the same as S. acanthias (Wells and Weber 1983)

b Adjusted for temperature, where necessary
C Calculated by any of the methods described on p. 23
(Selache maxima), Marshall and Hurst (1905) (Scyllium catalus, now S. stellaris),
Parker (1887) (Mustelus antarcticus).
As blood flows through the gills, blood pressure is reduced by some 25-30% from
pre branchial values which range from 3 to 5 kPa to post-branchial values of between
2 to 4 kPa (Table 1.1). It is presumably because of these low post-branchial blood
pressures that the arteries which branch off the dorsal aorta and supply the skeletal
muscles and viscera are valved close to their origin. These valves prevent any
backflow of blood that would otherwise occur when blood pressure in the peripheral
arteries rises as a consequence of the compression of the blood vessels in the post-
pelvic trunk during locomotion.
After the blood has flowed through the capillaries of the systemic vascular beds,
where it delivers its oxygen and nutrients and picks up carbon dioxide, it enters the
venous system en route back to the heart. Apart from those of the hepatic portal
system which convey venous blood from the gut, pancreas and spleen to the liver,
the veins are either rigid tubes which run through connective tissue (e.g. the lateral
cutaneous vein), or mQre generally are large voluminous sinuses in which there is
only a thin endothelial lining and no proper vessel wall. Since post-branchial arterial
G vcnt Oxygen Diffusing Respiratory Heart Mean blood

consumption b capacityC frequency rate pressures in
G perf
(J.lmol kg-I min-I) (Gdiff ) (min-I) (min-I)
(J.lmol kg-I) Ventral Dorsal
kPa- 1 min-I) aorta aorta
(kPa) (kPa)
0.42 27 2.75 d 41 31
33 40 39 3.1 2.1
49-58 5.25 39-50 42 3.4 2.5
44 60
34 3.63
1.7 36 4.12 59 32 5.1 3.9

(34)'
15 24 19 3.1 2.2
20 39 29 4.2 3.3
31 61 41 4.6 3.8
d Calculated by Piiper and Baumgarten-Schumann (1968 a)

e From unrestrained unoperated fish (Metcalfe and Butler 1984b)
Table 1.1 (continued)
Species and source Pi 0 2 Pe 0 2 % Extr Pa 0 2 Pv0 2 p.C0 2 PvC0 2 pHa pHv

(kPa) (kPa) (kPa) (kPa) (kPa) (kPa)
S. stellaris
Baumgarten-Schumann
and Piiper 1968 19.9 7,5 62 6.5 1.3 0.27 0.35 7.78 7.71
Piiper et al. 1970 18.9 7.80
Piiper et al. 1977 7.5 64 8.5 0.28
Negaprion brevirostris
Bushnell et al. 1982 4.3 0.95 7.72 7.54
Dasyatis sabina
Cameron et al. 1971 12
Squalus suckleyt'
Hanson and Johansen
1970 19.5 12.4 36 11.1 1.3
Lenfant and Johansen
1966 17.7 25 9.2 1.1 0.31 0.45 7.47 7.36
S. canicula
Short et al. 1979 19.7 14.8 25 12.7 3.1 7.76 7.71
Butler and Taylor 1975 18.7 12.1 2.8 7.88 7.83
blood pressures are only a few kPa, it is not surprising to find that venous blood pressu-
res in elasmobranchs are very low and often fall below ambient as a consequence of
the aspiratory action of the heart. To maintain a unidirectional flow of venous
blood from the periphery back to the heart, many of the venous vessels, especially
in the sharks, are equipped with valves. The arrangement of these valves may be
quite elaborate and may, in conjunction with body movements, operate as auxillary
pumps which serve to drive venous blood into the great venous sinuses (Birch et al.
1969).
The description of the functional anatomy of the elasmobranch cardiovascular
system given above describes fairly well the situation found in most of the dogfishes,
sharks and rays. However, the lamnid sharks (Lamnidae) such as the porbeagle
(L. nasus) and mako (I. oxyrinchus) exhibit a substantially different vascular anatomy
which is worthy of mention. In these fishes the oxygenated blood supply to the
alimentary canal and gonads comes, not from the dorsal aorta, but from the ventral
ends of the efferent branchial vessels via large lateral hypobranchial connectives and
the pericardial arteries; these vessels run along the floor of the pharynx alongside
the ventral aorta (Burne 1923). In most other elasmobranchs the hypobranchial
arteries are comparatively much reduced.
The muscles of the trunk in the lamnid sharks receive their blood supply not from
the dorsal aorta but from the large cutaneous arteries. These vessels arise from the
chain of connecting vessels of the efferent branchial system (see Burne 1923) arid
run down the body level with the lateral line canal. These arteries supply the large
red (slow oxidative) swimming muscles of the trunk. Many small vessels arise from
the lateral cutaneous arteries and form a complex mesh, the rete mirabile, which
intermingle with the veins returning to the lateral cutaneous vein from the muscles.
l.l Functional Morphology of the Cardiovascular and Respiratory Systems 13
This rete arrangement acts as a counter-current heat exchanger which functions

as a thermal barrier and allows these sharks to maintain muscle temperatures of
between 7 and 10 °C above that of their environment (Carey and Teal 1969).
This ability to maintain high muscle temperatures is necessary to obtain the
power required for high-speed swimming which is a feature of lamnid sharks. These
fishes also possess rete in the venous sinus of the eye orbit which also function as
heat exchangers. Since these animals have reduced internal carotid arteries, the major
part of the blood supply to the brain, eye and ocular muscles flows through this rete.
This vascular arrangement can maintain retina and brain temperatures up to 6 °C
above ambient (Block and Carey 1985).
1.1.4 The Respiratory System
Oxygen is taken up by the blood from the respiratory water flowing across the
epithelium of the lamellae. This water flow is maintained by the double pumping
action of the orobranchial and parabranchial cavities (Fig. 1.8). Water is drawn into
Orobranchia l
cavity
cavities
,....Holobranch
Fig. 1.8. A diagrammatic representation

of the gills of a dogfish showing the water
movements (arrows) from the oro bran-
chial cavity into the first two parabran-
chial cavities on the left side
the orobranchial cavity via the mouth and, especially in the skates and rays, via the
spiracles. In actively ventilating fish, gill ventilation is mainly brought about by the
superficial sheet of constrictor muscles which contract against the elastic visceral
skeleton. These muscles contract in a peristaltic sequence with the closure of the jaw
preceding the main phase of contraction which travels caudally down the branchial
basket. As these muscles cor.tract, the volumes of both the oro- and parabranchial
cavities are reduced and water is forced across the gill sieve; the increase in pressure
in both cavities is almost simultaneous (Fig. 1.9). When the constrictor muscles relax,
I.d.
a Cor.b.
1 s
Oro branchial 0.1
pressure (kPa)
0
Closed
Lower jaw
Parabranchial 0 .1
Open
pressure (kPa)
0
Closed
3rd gill slit
Open
A. m.
L.p.
L. h.
C. h.
C. b.
I. d.
Cor. h.
b Cor. b.
Fig. 1.9a. A diagram of the dogfish head (Scyliorhinus canicula ) showing the skeleton and the
main muscles involved in ventilation . (The superficial constrictor muscles have been omitted for
clarity) ; b a diagram of the oro branchial and 3rd parabranchial pressures in relation to movements
of the lower jaw and the 3rd gill slit together with the main phase of activity of the muscles involved
in ventilation in the dogfish S. canicula. A.m. Adductor mandibulae; L.p. Levator palatoquadrati;
L.h. Levator hyomandibulae; C.h. Constrictor hyoideus; C.b. Constrictor branchialis; I.d. Inter-
arcualis dorsalis; Cor.h . Coraco-hyoides; Cor.b. Coraco-branchialis. (Redrawn from Hughes and
Ballintijn 1965)
the parabranchial cavIties expand passively due to the elasticity of the visceral
skeleton. However, since the external gill flaps are closed, water is drawn across the
gill sieve from the orobranchial cavity. During this phase of the ventilatory cycle, the
main muscle to be active is the adductor mandibulae (Fig. 1.9), which functions
to maintain the position of the mandible and hyoid arch. This muscle regulates the
amount of water that enters the or 0 branchial cavity by adjusting the degree of opening
of the mouth (Hughes and Ballintijn 1965). Thus an almost continuous flow of water
is maintained across the gills by the combined action of an orobranchial pressure pump
and a parabranchial suction pump. However, there is no anatomical basis for consider-
ing these as being two separate pumps.
1.2 Gas Exchange 15
In the dogfish, S. canicula, water which enters the mouth leaves via the three
posterior gill slits, while that which enters via the spiracles leaves by the more
anterior gill slits (Hughes 1960), indicating that there is little mixing of water in the
orobranchial cavity. This separation of water flows is less marked in the skate
R. clavata. The differential pressure gradient between the oro branchial and para-
branchial cavities in the dogfish indicates that the parabranchial suction pump is at
least as important as the oro branchial pressure pump in maintaining water flow,
whereas in the skate, the parabranchial pump may be of more importance
(Hughes 1960). The lack of even a slight reversal of water flow across the gills of the
skate suggests that the gill flaps may be controlled actively, rather that passively.
Hughes (1960) has suggested that this may be functionally significant in preventing
the entry of sand into the branchial apparatus of this bottom-dwelling species.
1.2 Gas Exchange
There are many factors which affect gas exchange in fishes and their interactions are
complex. So, before discussing some of the models which have been developed to
describe gas exchange in elasmobranch fish and relating them to the morphology
of the gas exchange organs, some of the physical properties of oxygen and carbon
dioxide in water and blood will be considered.
1.2.1 Properties of Oxygen and Carbon Dioxide in Water and Blood
The two respiratory gases have different properties from one another in water and
blood and these differences have a profound influence on the exchange of the gases
across the respiratory surface. The capacitance coefficient (P) of a gas in a liquid is the
change in concentration (C) of that gas per unit change in its partial pressure (P),
LlC
i.e. ~ = LlP' For oxygen in water, ~ is equivalent to the physical solubility of the
gas, and this decreases with increasing temperature and salinity, but is independent
of partial pressure. The capacitance coefficient for CO2 in sea water at 10°C is given
as being some 33 times greater than that for oxygen (Dejours 1981). However, at
low P C02 in carbonated water, such as seawater, the addition of CO2 leads to the
formation of bicarl;lonate:
CO 2 + CO;- + HzO ¢ 2 HCO;
Consequently, the increase in P C02 is less than it would be if CO 2 went only into
solution, i.e. Pis greater than the physical solubility. When PC02 is above approximately
0.2 kPa there is not much carbonate available to convert to bicarbonate, and any
additional CO 2 goes into solution. This gives rise to a non-linear relationship between
P C02 and CC02 over the physiological range of P C02 (Fig. 1.1Oa) such that the
difference between PC02 in expired water (P e CO 2 ) and inspired water (Pi CO2 ) is greater
at higher values of P iC0 2 than at lower values (Truchot et al. 1980).
12
...
~
8
"0
E
.§
0'"
c.Y --------
------------
0
0 2 3
a
2.5
2.0
:;:.
~
"0 1.5
E Fig. 1.10. Equilibrium curves of blood
.§ from Scyliorhinus stellaris ( - - ) and
1.0
cJ'" of a sea water (---) at 17 °C for
carbon dioxide (a) and oxygen (b).
0.5 (Based on data from Piiper and Baum-
-------- garten-Schumann 1968 band Dejours
- - - - - - - ------ 1978)
4 12 16
b
The fact that CO2 is so much more soluble in water than is 02 means that even
if equal amounts of these two gases are exchanged across the gills (i.e. If the
respiratory exchange ratio, RE, equals 1), the decrease in P02 in the water will be many
times greater than the increase in PCO2. Rahn (1966) pointed out that this difference
is approximately 30-fold (i.e. the ratio of ~co2 to ~o2 in water). Thus, even if all of the
oxygen were extracted from the water, PC02 in the water leaving the gills would not
exceed 0.67 kPa (5 mm Hg). This applies only to distilled or non-carbonated water.
For seawater the
.
change in Pco2 is even less (Fig. 1.11) as a result of the higher value
of ~C02 at low PC02 (Dejours 1978).
The capacitance coefficients of 02 and CO2 in blood are also dependent on the
partial pressures of the gases, while the relationships between content and partial
pressure (the equilibrium curve) of these two gases in blood are influenced by a
number of factors such as pH, organic phosphates and temperature. In elasmo-
branchs, the chemical combination of oxygen with haemoglobin gives a slightly
sigmoid PO)C02 curve. In other words, Hill's coefficient, n, is relatively small,
ranging from 1.3 to 1.8 (Table 1.2). The oxygen-carrying capacity of elasmobranch
blood is between 1.9-2.1 mmoll- 1 at a haematocrit of 16-22% (Table 1.2), which
1.2 Gas Exchange 17
means that at a Po2 of 13 kPa there is approx. ten times more oxygen III
elasmobranch blood than in seawater (Fig. l.10b). The blood is halfcsaturated at a

Po 2 (Pso) of 1.1 kPa-3.2 kPa at physiological temperatures and pH (Table l.2).
Exceptions to the above values are the thornback ray, R. clavata, and the electric ray,
Torpedo marmorata (Hughes and Wood 1974; Hughes 1978). In the former, n = 2.5
and P 50 = 4 kPa, while in both species oxygen carrying capacity = 1.6 mmoll- 1 .
Despite these differences, the Bohr coefficient (Lllog Pso/LlpH) for these rays and the
sting ray, Dasyatis sabina, is small and within the range for other elasmobranchs
(-0.25 to -0.43). In the Atlantic electric ray, Torpedo nobiliana, there is no Bohr
effect (Bonaventura et al. 1974a). The blood pH in the dogfishes S. canicula and
S. stellaris is dependent on temperature (LlpH/LlT = -0.014 (Butler and Taylor
1975) and -0.012 (Heisler et al. 1980) respectively, so an increase in temperature
would tend to reduce the affinity of the haemoglobin for oxygen via the Bohr effect.
Temperature has a direct effect on P so in the carpet shark Cephaloscyllium isabella
and T. marmorata (Table l.2). However, there is little or no direct effect of
temperature on Pso of the blood in the porbeagle and mako sharks (Andersen et al.
1973). This may be related to the fact that temperature of the blood changes by several
degrees in these fish as it passes from the gills, to the warmer gut and muscles.
1.0
0.8
-;;; 0.6
a.
6
a
a..() 0.4
2
0.2
0
0 5 10 15 20
P02 (kPa)
Fig. 1.11. P cO2 vs. P 02 diagram for expired water of an aquatic animal with a respiratory exchange
ratio of 1 in distilled water or non-carbonated freshwater (1) and in seawater (2). The values of
P 02 and P co 2 in inspired water are given at point i. In seawater the increase in PcO2 accompanying
the fall in Po during gas exchange at the gills is much smaller than in non-carbonated water
2 .
because at such low values of PC02 the CO2 capacitance of seawater is high (see Fig. 1.10). (After
Dejours 1978)
The predominant organic phosphate in elasmobranch erythrocytes and the relative

sensitivity of the haemoglobin to each phosphate varies from species to species.
In the Japanese shark, Triakis scyllia, and the smooth dogfish, M. canis, GTP
is the major erythrocytic trinucleotide (Kono and Hashimoto 1977; Borgese et al.
1978). In two Australian sharks and a ray (school shark, seven-gilled shark and fiddler
ray) the predominant organic phosphate is inositol monophosphate (IMP) (Coates
Table 1.2. Respiratory characteristics of the blood of elasmobranchs 00
Species Authors Pso Bohr Hill Oxygen Haldane Haematocrit

coefficient coefficient carrying effect ('l
capacity ::r
I»
(kPa) (0) (n) (mmoll- 1 ) 'S
(I)
...,
Torpedo Bonaventura 0 :-
<1.5 ('l
nobiliana et al. 1974a ...,I»
3.2 (25°C) 14 0..
Dasyatis sabina { Cameron et al. 1971 -0.28 to -0.38 1.5 o·
Mumm et al. 1978 -0.38 <
~
()
Cephaloscyllium Tetens and Wells 1984 0.64 (5 "C, pH 7.67) -0.49
isabella l.l (15 DC, pH 7.7) -0.32 1.5 15 ...,0;
'"
Torpedo Hughes 1978 2.7 (15 DC, pH 7.8) -0.32 1.3-\.8 I»
::l
marmorata 3.7 (20°C) 0..
Raja clavata Hughes and Wood 1974 4.0 (15°C, pH 7.7) -0.25 2.5 1.6 Present :>::I
(1)
Squalus acanthias Wells and Weber 1983 1.8 (15°C, PcO2 -0.28 1.6 Weak 11.5 ~
0.3 kPa, pH 7.85) I»
".
Squalus suckleYl~ Lenfant and Johansen 1966 20
8'
...,
2.3 (11 DC, P co2 0 I.3 b 1.9 None '<
()')
0.1 kPa, pH 7.6)
Scyliorhinus Piiper and Baumgarten- 2.1 (17 DC, P C02 1.8 1.9 16 ~
(1)
stellar is Schumann 1968 b 0.2 kPa) ~

{ Pleschka et al. 1970 2.9 (17°C, PcO 2 -0.43 1.7 2.1 None 20-22
S. canicula
Butler and Taylor 1975 0.3 kPa, pH 7.58) found
Negaprion Bushnell et al. 1982 1.6 (24°C, pH 7.7) -0.36 2.1 16
brevirostris
a Now thought to be the same as S. acanthias (Wells and Weber 1983).

b Calculated by Piiper and Baumgarten-Schumann (1968 b).
1.2 Gas Exchange 19
et al. 1978), whereas in the dogfishes, S. canicuia, S. acanthias, the shark,

C. isabella, the ray, Narcacion nobiliana, and the skate, R. ciavata, ATP is
at the highest concentration (Borgese and Nagel 1978; Leray 1979; Wells and Weber
1983; Tetens and Wells 1984). Strangely, IMP has no effect on P50 in the Australian
sharks, although ATP, which is present at a slightly lower concentration, does increase
the P50 in the school shark (Coates et al. 1978). In contrast, ATP has no effect on P50
in the Atlantic electric ray and sting ray (Bonaventura et al. 1974a; Mumm et al.
1978). To make matters more complex, although both ATP and GTP depress the
oxygen affinity of stripped haemoglobin from S. acanthias (with GTP being the more
potent) urea, which is present in the blood at approximately 0.45 M, increases
affinity and so antagonizes the influence of the trinucleotides in vivo (Weber et al.
1983). This relationship appears to be temperature-dependent, for in C. isabella the
concentration of erythrocytic-organic phosphates is unaffected by acclimation temper-
ature, whereas blood urea is higher in warm-acclimated fish (Tetens and Wells 1984).
In contrast, the affinity of haemoglobin for oxygen is relatively insensitive to urea con-
centration in the clearnose skate, Raja egianteria, the electric ray, the sting ray and
the smooth dogfish (Bonaventura et al. 1974b; Mumm et al. 1978). The inclusion of
the dogfish in this list suggests that, contrary to the claim of Weber et al. (1983),
there is not a distinction between sharks on the one hand and the skates and rays on the
other with respect to the effect of urea on P50 . This merely illustrates how difficult it
is to make general statements about the effects of the various factors that modify
the oxygen equilibrium curve in elasmobranch blood.
Carbon dioxide is transported in blood predominantly in the form of bicarbonate
and the curve describing the relationship between PC02 and CC02 is particularly
steep at low PC02 (Fig. 1.10a). Thus, ~C02 for blood is high and some 50--70 times
greater than ~02 for blood (Piiper and Baumgarten-Schumann 1968 b). Thus, in
fishes in general, the CO2 equilibrium curve is very steep and the difference between
arterial and venous PC02 is small. At a PC02 of 0.4 kPa the difference between CC02 in
blood and seawater is approximately two fold (Fig. 1.10a). Surprisingly, in view of the
presence of a Bohr effect in most elasmobranchs, there is little or no Haldane effect
(the change in CC02 with changing CO2 at constant PCO) in any of the species studied
(Table 1.2) except for R. clavata (Hughes and Wood 1974). Possible reasons for the
inability to demonstrate the Haldane effect in S. canicuia are discussed by Pleschka
et al. (1970).
Temperature has a significant effect on CO2 transport in elasmobranchs, total
CO 2 in whole blood being reduced by approximately 2.5 %per °C increase (Albers and
Pleschka 1967).
The formation of CO2 from bicarbonate
CO2 + HzO ~ H+ + HCO;
takes several seconds or even minutes if not catalyzed (Dejours 1981), but blood
resides in the gills for 1-3 s (Randall 1982). The enzyme carbonic anhydrase is
present in the erythrocytes of fishes (Maren and Rittmaster 1977) and it appears that
plasma HCO; enters the erythrocyte, in exchange for Cl-, where its dehydration is
catalyzed by carbonic anhydrase. The CO2 which is produced then diffuses into the
water across the gill epithelium (Obaid et al. 1979; Randall and Daxboeck 1984).
1.2.2 Analysis of Gas Exchange in Relation to the Morphology of the Gills
A theoretical analysis of gas exchange in fishes was presented by Hughes and Shelton
in 1962 and their assessments of the effectiveness of the system are based on the
studies made by engineers of heat-exchange systems. These concepts have been applied
to experimental data and developed further basically by two groups of workers (see
Randall 1970; Piiper and Scheid 1984 for reviews) studying teleosts and elasmo-
branchs respectively.
The exchange of gases across the respiratory surface and at the metabolizing
tissues is fundamentally by the passive process of diffusion, although the conditions
that are necessary to ensure that this process proceeds adequately are maintained by
the activity of the respiratory and cardiovascular systems, i.e. by convection. The
factors affecting the rate of diffusion of a gas across a permeable membrane are
described in Fick's law of diffusion:
. KA i1P
M -
--x
-' (1)
where M = amount of gas diffusing per unit time (mmol s -I)

K =. Krogh's diffusion constant (mmol s - I mm - I kPa -1)
A = area of exchange surface (mm 2 )
i1P = difference in partial pressure of gas on either side of the exchange surface
(kPa)
x = thickness of the exchange surface (mm).
Krogh's diffusion constant is proportional to the solubility of the gas in the tissue
(or fluid) and inversely proportional to the square root of the molecular weight of the
gas, i.e. for the two respiratory gases in distilled water at 20°C:
Since there is little difference between the solubility of either gas in water or
in tissue (Dejours 1981), CO 2 diffuses much faster than 02 through both.
The respiratory and circulatory systems maintain i1P at as high a level as possible
and at the same time transport, by convection, oxygen to the gills and then to the
tissues (vice versa for carbon dioxide). Actual gas exchange across the gills can be
described in terms of convective transport by the respiratory system
M = V (C. W 1
- Ce ) = Vw . PAw (P.
1
- Pe ) , (2)
and by the circulatory system
M = Vb(Ca - C~) = Vb . ~b(Pa - P~) , (3)
where Vwand Vh = volume of water and blood passing over and through the gills
in unit time (\ min -I)
C.1 and C e = concentration of gas in inspired and expired water (mmoll- I )
C a and C~ = concentration of gas in arterial and mixed venous blood
(mmoll- I )
1.2 Gas Exchange 21
Pi and P e = partial pressure of gas in inspired and expired water (kPa)

Pa and P-;; = partial pressure of gas in arterial and mixed venous blood (kPa)
~w and ~b = capacitance coefficient of gas in water and blood
(mmoll- 1 kPa- 1 ).
The effectiveness of the gills in exchanging gas depends on a number of factors. These
include: the relative direction in which the blood and water flow; the resistance to
diffusion which exists at the gills, in the water and in the blood; the proportion of
each fluid which is effectively not involved in gas exchange, thus forming shunts;
unequal distribution of water and blood to the gills.
Piiper and Scheid (1975) and Scheid and Piiper (1976) have analyzed gas exchange
across the gills of fishes in terms of three different conductances, which can be
derived from Eqs. (1)-(3). Ventilation conductance, G vent = Vw~w. = MjP i - P e
[see Eq. (2)], perfusion conductance, G perf = Vb . ~b = M/Pa - Pv [see Eq. (3)]
and diffusion conductance, G diff = MjAP. The latter is derived from Eq. (1) and
is equal to KAjx. It is, therefore, related to the effective surface area of the
gills and to the thickness of the diffusion barrier as well as to Krogh's diffusion
constant.
1.2.2.1 Direction of Blood Flow

If there is no diffusion barrier between blood and water and if G vent = Gperf' then
with blood and water flowing in opposite directions (counter-current) it is theoretically
possible that Pa0 2 would equal PP2 and that P e0 2 would equal P-;;02 (Fig. 1.12a).
This situation has never been found to occur in any aquatic animal; however, the
fact that Pa02 can be > Pe02 in a number of elasmobranchs (Baumgarten-Schumann
and Piiper 1968; Hanson and' Johansen 1970; Grigg and Read 1971; Short et al.
(1979) indicates that there is not a parallel flow arrangement (Fig. 1.12b). It
was suggested that the presence of the inter-branchial septum in the gills of elasmo-
branchs would tend to produce a multicapillary or cross-current type of exchange
system, similar to that in the lungs of birds (Piiper and Schumann 1967). It has
since been demonstrated that at least in S. acanthias and the Jackson shark,
H. portusjacksoni, there is a septal canal between the ends of the secondary lamellae
and the inter-branchial septum (Kempton 1969; Grigg 1970a; De Vries and D Jager
1984). The latter two studies have presented evidence indicating that water follows
this route, thus allowing water and blood to flow in opposite (counter-current)
directions. It is now generally agreed that a counter-current arrangement exists in
elasmobranchs (Piiper and Scheid 1984), but despite the fact that G vent does almost
equal G perf in some cases, i.e. Vw • ~wjVb . ~b ~ 1 (Table 1.1), gas transfer is far from
being 100% effective. It is worth pointing out at this stage that because of the
approximately ten times differences between ~02 in blood and in water, Vw/Vb is
usually around 10 (Table 1.1).
1.2.2.2. Diffusion Resistance

This includes not only the resistance offered by the tissue separating the blood from
the water (Fig. 1.12c), but also the diffusion resistance of the water and blood
themselves. For the latter this also includes the reaction time of the oxygen with
(e (e
--
j
-- J
r --
Pa l Pi
11 v P pJ
v
P.I
I) Pa
20
,....
I.1l
Pe
Cl.
.>L-
10 Pa
Pv
a 0 b
P.
I
P.I
20
,.... Pa
I.1l
Cl. 10 Pe
~
Pv
c 0 e
Fig. 1.12. Models for exchange of oxygen across the gills. a Ideal conditions (i.e., no diffusion
limitation, G vont = G e,r) with counter-current flow of blood and water. Length of arrows in
exchange unit indicate G vent or Gp"r' If G vent i= G ped the partial pressure profiles are exponential);
b ideal conditions with co-current flow; c counter-current with diffusion resistance; d counter-current
\\Cith blood and water shunts. In this case some blood and water are thought of as equilibrating
completely and, therefore, as being effective (Vb and V respectively), while the rest of the blood
and water, which undergo no gas exchange, can ebe tho:ght of as being ineffective and constituting
shunts (Vb, and Vw ) ; e counter-current with combination of diffusion resistance and shunts.
Arrows represent gas exchange by diffusion with the thickness indicating the degree of eqnilibration
over the distance denoted by the length. Beyond a certain distance no gas exchange occurs within
the time that the liquids are in the exchange unit. The graphs indicate changes in PO 2 in water and
blood under each of the above conditions. Pi and P e are partial pressures in inspired and expired water
respectively while Pa and P v are partial pressures in arterial and mixed venous blood respectively.
(Redrawn from Butler 1976 and based on Piiper and Baumgarten-Schumann 1968a)
1.2 Gas Exchange 23
the haemoglobin, which will be slow at low temperature. As the width of the inter-
lamellar space is large (20-100 ~m) compared with the thickness of the tissue
barrier (up to 12 ~m), a sizeable part of the resistance to diffusion is expected to
reside in inter-lamellar water. An indication of overall resistance to diffusion can
be obtained by calculating its reciprocal, diffusion conductance, G diff or D, i.e.
~. This was first done by Randall et al. (1967) when they calculated G diff for
oxygen (or oxygen transfer factor, T 02 , as they called it) in the trout. They
calculated dP02 as
(4)
This assumes that the oxygen equilibrium curve is linear rather than sigmoid and that
the oxygen partial pressure profiles in both water and blood are linear along the
lamellae (see Fig. 1.12). The latter is only the case if G vent = G perf (Piiper and
Scheid 1982). Any non-linearity (when G vent #- G perf )' but still assuming linearity
of the 02 equilibrium curve, can be taken into account by calculating dP02 as
(P i0 2 - Pa 0 2) - (P e0 2 - P~02)
(Scheid and Piiper 1976) (5)
In (PiOZ - Pa 0 2)/(Pe0 2 - P~02)
A more accurate way of calculating dP02' which also takes account of the shape of
the 02 equilibrium curve, is the modified Bohr integration technique (Piiper and
Baumgarten-Schumann 1968a; see Piiper and Scheid 1982 for details). In practice,
G vent is close enough to G perf for Eqs. (4) and (5) to give identical values of
Do (Piiper et al. 1977; Short et al. 1979) and, depending on the values of the
2
blood gases, Do2 calculated by the Bohr integration technique may be identical to
that calculated by Eq. (4) (Piiper and Baumgarten-Schumann 1968a) or it may be
slightly less (Piiper et al. 1977).
Thus it is possible to obtain an indication of overall resistance to gas exchange
across the gills by measuring 1\1:, Pi' P~, Pe and Pa and calculating liD 02 , at
least under resting normoxic conditions. Using physiological data on gas transfer
across the gills and morphometric data on the gills themselves, Scheid and Piiper
(1976) have estimated that in the resting normoxic dogfish, S. stellaris, 42 % of the
total resistance to 02 transfer resides in the inter-lamellar water and 49 % in the
water-JJlood barrier. The small remainder (9 %) may be accounted for by factors such
as diffusion and chemical reaction within the blood, cyclic variations of water and
blood flow and shunting of water and/or blood.
The ratio GdifriGperf (i.e. Do/Vb . ~02) has been used to give an indication of the
relative importance of diffusion limitation and perfusion limitation in various gas
exchange organs (Scheid 1982). For large values of this ratio (>3) gas exchange
is perfusion-limited, while for small values «0.1) it is diffusion-limited. When the
ratio is < 0.5 there is little difference in gas exchange between counter- and co-current
systems (Piiper and Scheid 1982). From the data of Baumgarten-Schumann and Piiper
(1968) on S. stellaris, the value of GdifriGperf is 0.5, indicating that in these animals
oxygen exchange is predominantly diffusion-limited. Although diffusion limitation is
generally thought to be relatively unimportant for CO2 because of its greater solubility
in water and tissues, consideration of Gdiff/Gperf for CO2 indicates that this may
not be the case (Piiper and Scheid 1982).
A problem with the physiological assessment of Gdiff(D) is that the measurement
of Pe in dogfish involves the attachment of collecting devices around the gill slits, a
procedure which appears to prevent the fish from increasing ventilation sufficiently
to cope with environmental hypoxia (see Butler and Taylor 1975; Short et al. 1979).
In an attempt to circumvent this problem Metcalfe (1983) has shown that G diff for
oxygen can be reliably determined in the dogfish, S. canicuia, from measurements of
1\10:\' Pa 0 2 and P i0 2 , providing that G vent = G perf ' This does not, unfortunately,
obVIate the need to me~sure Pe 0 2 in order to calculate percentage extraction (% Ext)
which indicates the ability of the system to remove oxygen from the water
P.O -PO
%Ext = 1 2pP2 e 2 X 100 .
1.2.2.3 Shunts
Some water may not pass through the sieve formed by the gill lamellae and therefore
may never come into contact with the gas exchange surface of the gills. This represents
an anatomical or true shunt. A similar anatomical shunt exists when water passes
between lamellae that are not being perfused. In resting trout in well-aerated water,
some 40 % of the lamellae are not perfused with blood (Booth 1978). Such a pheno-
menon may exist in elasmobranchs. For water that does flow between the lamellae
that are perfused, its degree of participation in gas exchange will depend upon
its distance from the lamellar surface, and another way to look at the effect of
diffusion resistance in water is to think in terms of a shunt.
For water more than a given distance away, the diffusion resistance of the water
may be too high for any significant gas exchange to occur during the given transit
time through the gills (Fig. 1.12d). Such water can be regarded as constituting a shunt
(Piiper and Baumgarten-Schumann 1968a). Water participating in gas exchange, but
equilibrating incompletely, can be considered as consisting of a component that
completely equilibrates and of another component that takes no part in gas exchange
at all (i.e. functionally similar to the water that completely by-passes the gills or is
too far away to partake in gas exchange). Thus total water flow (Vw ,.) can be divided
into ineffective or shunted water (Vw 'S ), and effective water (V). W' e
In otherwise
ideal conditions, the deviation of Pe from its ideal value (i.e. P~) can be attributed to
the fraction of total water flow that is effectively shunted:
VW'S
(Piiper and Scheid 1984).
Abnormally high ventilation rates, possibly as a result of the animals being

disturbed, would reduce the contact time of the water with the lamellae (thus
increasing the effective shunt component) and maybe increase the proportion of water
by-passing the lamellae (true shunt). This would be manifest as a high ratio of
G vent : G perf and could at least partly account for the fact that Pa 02 is not always
1.2 Gas Exchange 25
greater than P e 02 in a number of species of elasmobranchs (Lenfant and Johansen

1966; Baumgarten-Schumann and Piiper 1968; Hanson and Johansen 1970; Short
et al. 1979). Even in the absence of any diffusion barrier, P e will not equilibrate to
P-v if G vent > G per f (Scheid and Piiper 1976). It would appear that operative procedures
do have a profound effect on ventilation in elasmobranchs, raising it substantially
above the true resting level and close to the maximum. Only if the animals are
left undisturbed for several days does ventilation reach a low resting value (Metcalfe
and Butler 1984 b). The difference in time given for the fish to recover from
anaesthesia could be one explanation for the low Gvent/Gpcrf obtained from the data
of Baumgarten-Schumann and Piiper (1968) for S. stellaris compared with the higher
value obtained by Short et al. (1979) from S. canicula (see Table 1.1).
Experimentally induced variations in ventilation below and above the normal
levels in S. suckleyi cause accompanying, although not matched, changes in
cardiac output so that at low values of Vw (27 % normal), Vw;V b is 5, whereas at high
values of Vw (2.6 times normal), the ratio is 21 (Hanson and Johansen 1970). At
low Vw, P a02' ]\1 02 and T02 are less than normal, despite a near doubling of
percentage extraction of oxygen from the water (% Ext) to 62 %. Conversely, at
high Vw' % Ext is approximately half the normal value at 17 % but P a0 2' MOl and
To are close to normal, indicating a large increase in the water shunt component
2
(true and/or physiological).
There is no anatomical evidence for the existence of direct connections between
afferent and efferent filament arteries in elasmobranchs which would allow blood from
the ventral aorta to by-pass the lamellae and enter the dorsal aorta (Metcalfe and
Butler 1986). It is possible, however, that anatomical (true) blood shunts do occur in
elasmobranchs if, as in teleosts, the basal channels in the lamellae are so far from the
lamellar surface as to make it unlikely that the blood within them takes part in gas
exchange (Tuurala et al. 1984). Also, in the Endeavour dogfish, C. scalpratus, the
basal lamellae of each gill filament are covered by a sheet of tissue formed from
the gill arch (Cooke 1980). This branchial canopy prevents ventilatory water flowing
over the basal lamellae, thus placing about 20 %of the gill surface area in a ventilatory
dead space. Whether blood actually perfuses these lamellae, thus constituting an
anatomical shunt, remains to be seen. In S. acanthias the branchial canopy covers
4 % of the proximal lamellae which are not thought to be in a dead space (De Vries
and De Jager 1984).
As well as these possible true shunts, it is also conceivable that effective shunting
of blood could occur within the lamellae in a fashion similar to that described
above for water passing between the lamellae (Piiper and Baumgarten-Schumann
1968 a). As with water, these shunts would incorporate diffusion resistances within
the blood (Fig. 1.12d). Again, under otherwise ideal conditions, deviation of Pa from
Pi can be attributed to the fraction of total blood flow that is effectively shunted:
Vb., Pi - P a
V b•t Pi - P"
Excessively high rates of perfusion (G vent < G perf ) could increase the effective shunt
and any true shunts that exist on the blood side of the exchanger, and even in the
absence of any diffusion limitation, Pa will not equilibrate with Pi (Piiper and Scheid
1984).
In reality, and in addition to anatomical shunts, the limitations of the gas exchange
system consist of a combination of diffusion resistance and physiological shunts
(Fig. 1.12 e) and there is a continuous transition from one to the other.
1.2.2.4 Unequal Distribution of Water and Blood Flow
If some lamellae are perfused but not ventilated, or vice versa, there is a blood or
water shunt. Between these two extremes and the optimum ratio between ventilation
and perfusion, i.e. when Vw. ~w/V b . ~b = I, there are intermediate conditions where
some lamellae may be underperfused and others may be underventilated (Fig. 1.13).
Fig. 1.13. Model showing unequal distribution of water and blood

among parallel gas exchange units, i.e., G vent is greater than or less than
Gpc<f in different units. Lengths o.larrows in the exchange units indicate
relative magnitude of G vent or Gpc<f' Abbreviations as in Fig. 1.12.
(After Piiper and Scheid 1982)
Such inequalities of water and blood flow between different lamellae can reduce
the overall effectiveness of the gas exchanger, but the extent to which this occurs
depends on the overall ratio of G vent : G perf (see Piiper and Scheid 1984). Data
obtained from the dogfish, S. suckleyi, and the sting ray, D. sabina, suggest that
elasmobranchs have little control over the distribution of water to various gill
arches if some of the arches are deprived of blood, although they do seem able to
readjust blood flow in response to removal of water flow to some gill arches
(Cameron et al. 1971). The latter may be the direct result of hypoxia causing
local constriction of the blood vessels in the unventilated gills (cf Satchell 1962).
The pulsatile nature of the flow of water and blood through the lamella may
produce effects that are similar to those of unequal distribution of water and blood.
This would be particularly so if slow flow oscillations of the two fluids are out of phase
(Piiper and Scheid 1984). The data on synchrony between respiration and heart beat
from restrained dogfish are inconsistent. In Squalus lebruni and M. antarcticus, the
heart tends to beat during a particular phase of the respiratory cycle (Satchell 1960),
whereas in S. canicula there is no lasting synchrony between the ventilation and cardiac
cycles (Taylor and Butler 1971; Hughes 1972). More recent studies on unrestrained
and undisturbed electric ray, T. marmorata, and dogfish in well-aerated water have
1.2 Gas Exchange 27
demonstrated a clear 1: 1 synchrony between the two rhythmus (E. W. Taylor and
D. J. Barrett in prep.) which may maximize the effectiveness of gas exchange in the
resting fish.
1.2.3 Effectiveness of Gas Exchange
The overall effectiveness of gas exchange on each side of the gills can be quantified
by calculating the ratio of actual to maximum possible transfer of gas to or from the
blood. The formula for effectiveness of transfer to or from the water is
E = Pw(Pj - Pe)
W Pw(Pj - P~) ,
and that for effectiveness of transfer from or to the blood (Eb) is
Eb = Pw(Pa - P~) .
Pb(P j - P~)
As mentioned earlier, Pw for O 2 is independent of Po2 ; but Pw for CO 2 and
Pb for 02 and CO 2 are all dependent on the partial pressure of the gas. Thus, if
Pw and Pb are cancelled out in each case, leaving just the relative partial pressure dif-
ferences, Lip, (piiper and Scheid 1972, 1975) errors may be introduced. These authors
have, nonetheless, introduced the term P e - P)P j - P~, which theyeonsider to be a
useful indicator of the overall effectiveness of gas transfer between blood and water
and have given it the abbreviation Lip, tr.
1.2.4 Use of the Fick Equation for Calculating Cardiac Output
On the face of it, it would seem possible to use Eq. (3) to calculate cardiac
output CV b) from measurements of total oxygen uptake (Mo) and the concentration
of oxygen in arterial and mixed venous blood (C a 02 and C~02)' This assumes,
however, that all of the blood leaving the heart reaches the dorsal aorta, that all of
the oxygen transfer occurs across the gills and that the gills consume an insignificant
proportion of total oxygen uptake. None of these conditions may actually exist. Gills
may have a relatively high oxygen consumption (see Johansen and Pettersson 1981
for teleosts), the skin of S. canicula may be responsible for approximately 5 %
of total oxygen uptake, even if it is for its own use (Toulmond et al. 1982) and
some of the blood leaving the heart may immediately enter the venous system
before traversing the lamellae and/or return to the venous system from the
lamellae before entering the dorsal aorta (Cooke 1980; Olson and Kent 1980; De
Vries and De Jager 1984; Metcalfe and Butler 1986). Differences between directly
measured and calculated value for cardiac output have been demonstrated for
S. canicula, particularly in animals exposed to hypoxia (Metcalfe and Butler 1982).
28 Chapter!. Cardiovas.cular and Respiratory Systems
1.3 Control of the Cardiovascular and Respiratory Systems
The functional division of the elasmobranch cardiovascular system into heart,

branchial circulation and systemic circulation used in describing its morphology also
provides a useful framework from which also to describe its control. However, it
must be borne in mind that this anatomical arrangement of the cardiovascular
system means that changes in one area will affect the function of others. Present
knowledge of cardiovascular control in fish generally is still only rudimentary in
comparison with that of mammals; it is therefore not surprising that there are still
large gaps in our understanding of cardiovascular control in elasmobranchs.
1.3.1 The Heart

Elasmobranchs possess a myogenic heart which is formed of typical vertebrate-
type cardiac muscle fibres. Contraction of the heart is initiated by an action potential
propagated across the myocardium from a rhythmically depolarizing pacemaker.
Pacemaker activity is reported to be diffuse in fishes and the typical nodal pace-
maker tissue of mammals is not apparent. In S. canicula, Rybak and Cortot
(1956) report that the wall of the sino-atrial valve continues to contract when isolated,
and presumably this is the site of pacemaker activity, at least in this species. The
ECG generated by the pacemaker is generally similar to that of other vertebrates
(Satchell 1971) although there is an initial "V" wave caused by the depolarization of
the sinus venosus which precedes the PQRS complex. Although this myogenic pace-
maker causes the heart to beat continuously in the absence of any external stimuli,
the rate and force of contraction in the intact animal is continuously influenced
by neural, humoral and intrinsic effects. Since in general most elasmobranchs do not
control their body temperature, changes in environmental temperature will affect
heart rate directly because pacemaker depolarization increases with temperature.
The elasmobranch heart receives only inhibitory, parasympathetic innervation via
the vagus (Xth cranial) nerve (Young 1933; Short et al. 1977). Consequently, direct
nervously mediated cardioacceleration can only be achieved in these fish by a release
of inhibitory vagal tone. Although there is no accepted physiological evidence of any
sympathetic innervation, an adrenergic influence may be exerted by specialized
catecholamine - containing cells in the endothelium of the sinus and atrium
which are innervated by cholinergic fibres (Nilsson 1983). Two distinct branches of the
vagus innervate the heart, one arises from the visceral branch (visceral cardiac
vagus) and the other arises from the 4th branchial division (branchial cardiac vagus).
These terminate in a plexus of fibres which appears to be restricted to the sinus
venosus. Nerve stimulation experiments have demonstrated that the branchial cardiac
vagus (bcv) is more effective in inhibiting the heart than is the visceral cardiac
vagus (vcv) which may mainly have a sensory function (Short et al. 1977). The
inhibitory effects of the vagus are mediated by the release of acetylcholine, which acts
via muscarinic receptors and is blocked by the action of the antagonist atropine.
The level of tonic cardiac inhibition by the vagus increases with temperature. In
the dogfish, S. canicula, at 7 °C (and presumably below) there is little or no inhibitory
vagal influence acting on the heart; injection of atropine causes heart rate to
1.3 Control of the Cardiovascular and Respiratory Systems 29
increase from 20 to 22 beats min - 1. At progressively higher temperature there is an

accompanying increase in vagal tone and at 17 DC, injection of atropine causes a rise
in heart rate from 37 to 48 beats min- 1 (Taylor et al. 1977).
Taylor and Butler (1982) recorded efferent activity in the bcv and found two
distinct types of motor unitactivity. Some small units fire sporadically (non-bursting)
while other, larger units fire in rhythmical bursts which are synchronous with
ventilatory movements. Furthermore, hypoxia increases the firing frequency of the
non-bursting units while decreasing the firing frequency of the bursting units
(Fig. 1.14). These observations led these authors to suggest that it is the non-
Normoxia
b' ~., f +Iil.lb

" 14 t '"1~ "I·
··t '41~"t
I
Hypoxia
1 ~lIlItl>
2s
I*,
I
a Respirator y movements b 2s
Fig. 1.14. Recordings of efferent activity from the left branchial cardiac branch of the vagus nerve
in the dogfish (S. canicula) . a Activity related to respiratory movements. Contractions of the walls of
the pharynx (upward deflection) are associated with a burst of action potentials in the cardiac·vagus.
Smaller action potentials are present between and within the bursts ; b activity in response to
ventilation of the fish with normoxic and hypoxic water. The bursts of activity are slower
and contain fewer action potentials during hypOXIa, whereas the activity in the continuously firing
units is markedly increased. (Taylor and Butler 1982)
bursting units in the bcv motoneurons which play the major role in cardio-inhibition
in response to hypoxia, while the bursting units may loosely couple the respiratory
and cardiac pumps. The bursting activity in the bcv continues after paralysis of the
skeletal muscles of the branchial basket and this bursting is synchronous with
efferent activity in the branchial vagus which innervates the gill muscles (Barrett
and Taylor 1985 a) . This indicates that the bursting activity in the bcv is central in
origin and does not originate from branchial mechanoreceptors.
Detailed studies using retrograde transport of horseradish peroxidase (HRP)
to locate vagal cardiac motoneurons and electro physiological recordings from the
vagal motor column have greatly enhanced our understanding of the neural control
of the elasmobranch heart. Branchial cardiac vagal motoneurons in the medulla are
distributed in the vagal motor column over about 1 mm rostral to obex (Fig. 1.15).
Like other vagal motoneurons, the majority of the bcv motoneurons are located
medially, close to the wall of the fourth ventricle, while some bcv motoneurons are
located in a ventrolateral group (Barrett and Taylor 1985 b). Extracellular recordings
from these two groups of cardiac vagal motoneurons (cvm) have revealed that the
bursting activity originates from the medial cvm's, while the non-bursting activity
originates from the ventrolateral cvms (Barrett and Taylor 1985c). However, both
groups of neurons will fire in response to mechanical stimulation of the gill septa
which, in intact animals, will cause bradycardia (Taylor and Butler 1982). Mechanical
stimulation of the gill septa will also cause normally silent neurons in the ventrolateral
group to fire.
-f(~--A
,l
I
WVI~
BCVM
,
I
I
,,
I
4th Ventricle
\ I
,
'v'
a Caudal
4th Ventricle
Ventral >--f
b 1mm
Fig. 1.15. A diagrammatic illustration of the location of cardiac vagal motoneurons in the hind
brain of the dogfish S. canicula. a A "longitudinal" section through the hind brain showing the
location of the visceral cardiac vagal motoneurones (VCVM), together with the medial (m) and
ventrolateral (vi) branchial cardiac vagal motoneurones (BCVM) relative to the obex. (Based on
data from Barrett and Taylor 1985b). b A "transverse" section through the hind brain at the level
A showing the location of the medial and ventrolateral branchial cardiac vagal motoneurons. (After
Barrett and Taylor1985 b)
Although we do not yet fully understand the functional significance of the separate
origins of the motor activity in the bcv, the observation that the bursting activity
in the medial cvm's is centrally entrained with respiration is interesting in the
context of the central modulation of cardiac activity in phase with ventilation, since
this is similar to the sinus arrhythmia observed in mammals. As long ago as 1895,
Schoenlein had described 1 to 1 synchrony between the heart beat and breathing
movements in T. marmorata. As mentioned earlier, the functional significance of such
1.3 Control of the Cardiovascular and Respiratory Systems 31
a synchrony may be to maintain a match between the relative flows of blood and
water across the gas exchange surface of the gills thereby enhancing the effectiveness
of the gas exchange process. Although subsequent studies have demonstrated a
coupling between ventilation and heart rate (Satchell 1960; Hughes 1972) which
involves the vagus (Lutz 1930), this coupling was often only loose and was frequently
out of phase (Butler and Taylor 1971), particularly in restrained animals. Consequent-
ly, the evidence for cardio-respiratory coupling was not overwhelming. However,
the clear and maintained 1 to 1 synchrony between heart rate and ventilation
recently described in unrestrained, resting dogfish is abolished by atropine, which
demonstrates the role played by the vagus in this phenomenon (E. W. Taylor and
D. J. Barrett in prep.). The coupling is also abolished by activity and disturbance,
which causes an increase in ventilation and a reduction in heart rate.
In addition to the direct neural control of the heart via the vagus, the rate of the
heart beat and the force of its contraction can also be influenced by substances
borne in the blood (humoral control) and by mechanisms which arise from the pro-
perties of the myocardium itself (intrinsic control). Since the nervous control of the
elasmobranch heart is less well developed than that of higher vertebrate species, it
seems probable that humoral and intrinsic control mechanisms may play a more
important role in the control of cardiac function in these animals.
Although a number of blood-borne substances are known to affect cardiac
function, it is the catecholamines, adrenaline and noradrenaline, that have received
most attention. The most important extra-neuronal site of catecholamine storage and
release in elasmobranchs is the axillary bodies, which are located, in association with
sympathetic ganglia, on the anterior dorsal surface of the posterior cardinal sinus
(Abrahamsson 1979). It has previously been pointed out (Satchell 1971 ; Gannon et al.
1972) that this location is ideally suited for adrenergic control of the heart via
circulating catecholamines, since the blood into which these hormones are relased
will be aspirated directly into the heart. The effect of adrenaline on the isolated
perfused dogfish heart is to increase cardiac frequency (positive chronotropic effect)
while noradrenaline decreases it. However, both these hormones increase the force
of contraction (positive inotropic effect) (Capra and Satchell 1977 a). The positive
inotropic and chronotropic effects are mediated by ~- (probably ~z-) adrenergic
receptors, while the negative chronotropic effect of noradrenaline is mediated by
a.-adrenergic receptors and does not appear to be associated with a cholinergic link
of the type proposed by Hnge and Ostlund (1954) (Capra and Satchell 1977 a). The
little evidence that is available from in vivo studies of the cardioregulatory effect of
circulating catecholamines suggests that there is a ~-receptor-mediated augmentation
of cardiac stroke volume during both normoxia and hypoxia in the dogfish,
S. canicula (Short et al. 1977). The comparatively high levels of catecholamines in the
blood of elasmobranchs (Butler et al. 1978) may in some way compensate for the lack
of cardiac sympathetic innervation.
Recently it has been reported that the atrium of S. canicula possesses PI-type
inhibitory purinoreceptors. Adenosine, ATP and ~, y-methylene ATP all produce
negative inotropic and chronotropic effects. These are not blocked by atropine,
indicating that their action is not caused by the release of acetylcholine (Meghji and
Burnstock 1984).
Of the intrinsic control processes which regulate cardiac function, one of the
physiologically more meaningful is the Frank-Starling relationship. Like vertebrate

hearts generally, those of elasmobranchs possess the property that the more they are
stretched, the more vigorously they subsequently contract. Thus an increase in
ventricular filling, as a result either of an increase in venous return to the heart, or
of an increased diastolic filling time during a reduction in heart rate, results in an
increase in cardiac stroke volume. This mechanism allows the automatic matching of
cardiac output to venous filling without any need to involve the central nervous
system.
Since the combined function of the respiratory and circulatory systems is the ex-
change of oxygen and carbon dioxide between blood and water, it seems almost
axiomatic that changes in the levels. of these gases in either the environment or the
blood should affect these two systems. Even so, our knowledge of the location and
function of chemoreceptors sensitive to oxygen and CO2 in elasmobranchs is only
very' poor. In the dogfish S. canicula, externally located 02 receptors which, when
stimulated by low levels of oxygen, mediate a reflex bradycardia, appear to be
distributed diffusely in the orobranchial and parabranchial cavities and are innervated
by cranial nerves V, VII, IX and X (Butler et al. 1977). There may also be
receptors in the venous system which, when stimulated by high levels of oxygen, also
cause a reflex bradycardia (Barrett and Taylor 1984).
1.3.2 The Branchial Circulation
Elasmobranch gills receive parasympathetic innervation via the glossopharyngeal

(IX cranial) and vagus (X cranial) nerves. The glossopharyngeal innervates hemi-
branchs 1 and 2 of the 1st gill slit, while branchial branches of the vagus innervate
the more posterior gills. Each branchial nerve is divided into pre- and post-trematic
branches; the pre-trematic branch is almost entirely sensory, while the post-trematic
has mixed sensory and motor functions (Nilsson 1983). Unlike those of teleost
fishes, the gills of elasmobranchs receive no sympathetic motor innervation (Metcalfe
and Butler 1984a). Although it does not circulate in the blood, acetylcholine causes
vasoconstriction in isolated perfused gills (Davies and Rankin 1973; Metcalfe and
Butler 1984a) and histological studies (Dunel and Laurent 1980) report a motor inner-
vation in S. canicula, which is probably cholinergic, of sphincter muscles in the efferent
filament artery just prior to its junction with the efferent arch artery. However, direct
observations from nerve stimulation studies in both isolated perfused gills and in the
whole animal have not shown any physiological evidence for vasomotor innervation
of the major gill blood vessels in this species (Metcalfe and Butler 1984a)
(Fig. 1.16). Presumably the branchial motor nerves innervate only the skeletal muscles
of the gill arch (Gaskell 1886). The situation with regard to vasomotor innervation
of the gill blood vessels in other .eIasmobranch groups is so far not known.
Since branchial vasomotor innervation appears to be absent in elasmobranchs,
any control of the branchial vascular· bed must be either by humoral and/or
intrinsic mechanisms. As with the heart, the only hormones to have received any
detailed study with respect to their ability to affect gill blood flow are the catechol-
amines. Although the early work of Ostlund and Fiinge (1962) on S. acanthias
failed to demonstrate any vasomotor effects with adrenaline, numerous subsequent
c ard.vag card. vag
bran c. vag y branc.vag branc.vag y branc.vag branc.vag
y y y y y y y
At~ Pan~
~ ~
VAbp 4 wmi\\\I,\\~~~\\~\\\~W,~\~~,Q~~\I,\\~~~\~~\\~\\
J w
(kPa ) :
()
o
~
~
o
...,
;.
('1)
()
0..
'..."
DAbp 2 4j o·
~
~
'"
(kPa) 0 n g
_ _ _ -.JnL-_ __ _
....--JI I'l......-- .-I1 n'--_ _ _
~
Time (min) i:l
'"
0..
;x:l
('1)
Fig. 1.16. Traces of ventral aortic blood pressure ( V Abp) and dorsal aortic blood pressure ( D Abp) obtained from an anaesthetized ~
~.
dogfish (S. canicula) during electrical stimulation of either both branchial vagal roots (branc. vag.) (30-50 Hz) or one branchial
cardiac vagus (card. vag.) (50 Hz) before atropine, after atropine (At. 0.15 mg kg-I), and after the skeletal muscle paralyzing drug S
...
'<
pancuronium (Pan. 2 mg kg ' ). Before atropine, stimulation of the branchial vagi results in an increase in ventral aortic blood C/l
pressure with little change in dorsal aortic blood pressure, which indicates an increase in resistance to blood flow through the gills. 'a
Stimulation of the cardiac vagus causes a marked bradycardia. After atropine, the bradycardia, in response to stimulation of the
cardiac vagus, is aboli shed, indicating the effectiveness of the dose in blocking muscarinic transmission. However, the increase in ~
blood flow resistance in the gills in response to stimulation of the branchial vagi is unaffected. This is only abolished when the
skeletal muscles of the gill arch are paralyzed with pancuronium, indicating that the changes in blood flow resistance are due to
w
contraction of the skeletal muscles of the gill arch rather than being due to any vasoconstriction. (Metcalfe and Butler \984a) w
studies have shown that both adrenaline and noradrenaline cause overall vasodilata-
tion in perfused branchial preparations of S. canicula (Davies and Rankin 1973;
Metcalfe and Butler 1984a) and S. acanthias (Carpa and Satchell 1977b; Evans and
Claiborne 1983). This effect is mediated via ~-adrenergic receptors and masks a smaller
ex-adrenergic receptor-mediated vasoconstriction. Kent and Pierce (1978), however,
found no evidence of branchial vasodilatation in response to 8 x 10- 3 mg kg- 1
adrenaline in intact S. acanthias.
Although circulating hormones appear to be the only route by which extrinsic
control may be exerted on the branchial blood vessels, there is limited evidence
to suggest that there may be some degree of autoregulation. Satchell (1962) demon-
strated that brief (2 min) periods of anoxia caused branchial vasoconstriction in
S. acanthias and subsequent studies on S. stellaris (Piiper et al. 1970) and S. canicula
(Short et al. 1977) have reported branchial vasoconstriction in response to hypoxia.
Satchell (1962) suggests that localized intrinsic vasoconstriction in response to hypoxia
would divert blood from poorly ventilated areas to areas with a better oxygen supply,
thus maintaining post-branchial blood oxygen levels as high as possible. This may
well be what happens in vivo, since sealing off up to half the gill slits does not
result in a significant reduction in Pa 0 2 (Cameron et al. 1971).
1.3.3 The Systemic Circulation
Our knowledge of the mechanisms which regulate the distribution of blood flow to
the various organs of the systemic circulation in elasmobranchs is extremely poor and,
in general, limited to the smaller and less active species. Catecholamines injected in
vivo produce a dominant systemic vasoconstriction (Kent and Pierce 1978; Opdyke
et al. 1982), which is mediated via ex-adrenergic receptors, although ~-adrenergic
receptors which mediate vasodilatation are also present. Despite the fact that cate-
cholamines have been shown to affect the systemic circulation, we know little about
either their functional role, or the method of their release. Since ganglionic blockade
reduces catecholamine secretion in response to physical disturbance (Opdyke et al.
1983a), while ganglionic stimulation increases it (Opdyke et al. 1983b), it would
appear that there exists a neurogenic mechanism which may account for at least
part of the increase in circulating adrenaline and noradrenaline in response to hypoxia
(Butler et al. 1978) and exercise (Butler et al. 1986). However, both potassium
(Opdyke et al. 1981 a) and angiotensin (I and II) (Opdyke et al. 1981 b) are also able
to stimulate catecholamine release. In fact, the pressor response in S. acanthias to
angiotensin ·appears to be entirely due to the induced increase in circulating
catecholamines (Opdyke and Holcombe 1978) rather than to the direct effect of the
angiotensin itself. The effects of both potassium and angiotensin in the release of
catecholamines are not prevented by ganglionic blockade, indicating a direct action
on the chromaffin tissue rather than a neurally mediated one.
In addition to circulating catecholamines there is histological evidence that the
major systemic arteries of S. acanthias receive an adrenergic innervation (Nilsson
et al. 1975). However, although there is some evidence for baroreceptor activity in
the gill blood vessels of elasmobranchs (Lutz and Wyman 1932; Irving et al. 1935),
the evidence that exists from spinal cord stimulation studies on intact animals
1.3 Control of the. Cardiovascular and Respiratory Systems 35
suggests that there is functionally little neural control of the systemic circulation
(Opdyke et al. 1972) and that it may act as a simple pressure-volume system
(Bagshaw 1985). Indeed in tilting experiments, Ogilvy and Dubois (1982) found that,
unlike in teleost fishes, the circulatory system of unanaesthetised M. canis was
unable to compensate for a tilt of 30°, and that over a 30-min period ventral aortic
blood pressure fell from about }.2 kPa to about 1.5 kPa, while the pulse pressure
fell to almost zero as blood accumulated in the posterior portion of the fish and
compromised cardiac function.
1.3.4 Ventilation
The ventilatory muscles of the oro- and parabranchial cavities of elasmobranchs which
power ventilation (see Hughes and Ballintijn 1965) (Fig. 1.9) are innervated by cranial
nerves V, VII, IX, and X and these nerves have their motor nuclei in the medulla
oblongata. In each respiratory cycle, the onset of motor activity appears first in the
mandibular branch of the Vth cranial nerve which supplies the muscles of the jaw.
This is followed by activity in the IXth cranial nerve, which innervates the 1st gill arch,
and this is then followed by simultaneous activity in the four branchial branches of the
Xth cranial nerve innervating the posterior gill arches (Barrett and Taylor 1985 a).
These authors have pointed out that the independent innervation of the 1st gill arch
may be functionally significant, since in some species is has been shown that the 1st
gill slit may be used for inspiring, rather that expiring, water (Grigg 1970b), which
may be an important route for inspired water, in addition to the spiracles, when the
mouth is obstructed during feeding. If the medulla is isolated from the rest of the
brain and spinal cord, rhythmic ventilation continues (Satchell 1959), indicating the
presence of central respiratory rhythm-generating neurons which appear to be located
in the reticular formation. However, the rate, rhythmicity, and amplitude of ventila-
tory movements are continuously influenced by information afferent to the central
nervous system which presumably acts to optimize the effectiveness of gas exchange.
The gill arches and filaments possess numerous mechanoreceptors (Satchell and
Way 1962) which not only detect extraneous material which interferes with gill
function and elicits the "cough" reflex, but also provide afferent information to the
central nervous system about both the load upon and the distortion of the
respiratory apparatus and are important in controlling the rate and amplitude of
ventilation. Both inflation of the oro branchial cavity and direct stimulation of the
central cut ends of the branchial nerves inhibit ventilation in S. lebruni (Satchell 1959),
while branchial nerve section increases respiratory frequency as a result of a reduction
in the duration of the pauses between each ventilation. Paralysis of the ventilatory
muscles increases the rate of efferent activity in the branchial nerves of S. canicula
(Barrett and Taylor 1985a), indicating that branchial mechanoreceptor stimulation
generally inhibits the central respiratory rhythm generator. This may be similar to the
Breuer-Hering reflex of mammals. The oxygen-sensitive receptors which appear to
be diffusely located in the oro branchial and parabranchial cavities and are innervated
by cranial nerves V, VII, IX and X (Butler et al. 1977) are not involved in the
control of ventilation. However, there may be internally located 02 receptors, possibly
in the brain (Bamford 1974), which are able to monitor oxygen levels in the blood,
and which may be responsible for controlling ventilation.
Although the basic ventilatory rhythm is maintained by a central rhythm generator,
it would be incorrect to assume that ventilation is always continuous. The Port Jackson
shark will, for instance, exhibit respiratory pauses when left undisturbed in well-
aerated water (Capra 1976).
1.4 Supply of and Demand for Oxygen:

Integrated Responses of the Respiratory and Cardiovascular Systems
It is the combined function of the circulatory and respiratory systems to maintain

an adequate flow of oxygen to the metabolizing tissues in response to varying
demand and/or reduced supply. At the same time, of course, these two systems
are also responsible for the removal of CO 2, Demand for oxygen can be influenced
by a change in environmental temperature as well as by variations in the level of
activity. Although not generally a problem in the marine environment, hypoxia (a
reduction in the availability or supply of oxygen) may occur in certain environmental
niches or if there is damage to the gas exchange surface.
The ways in which the respiratory and cardiovascular systems deal with these
situations are basically similar. Increased usage of oxygen by the tissues causes
internal hypoxia if no compensatory adjustments are made, so with increased demand
for oxygen and when supply is reduced, ventilation must increase to maintain Pa0 2,
and hence Ca0 2. At the same time, delivery to the tissues can be increased (e.g. with
increased temperature or exercise) or maintained (if Ca02 falls during environmental
hypoxia) by extracting more oxygen from the venous blood and/or by increasing
cardiac output [see Eq. (3)].
1.4.1 Temperature
Most elasmobranchs are poikilotherms, and any change in environmental temperature

is rapidly transferred to the body of the animal, predominantly across the gills. An
increase in environmental temperature not only causes an increase in metabolism of
fishes but also reduces the amount of oxygen in the water at a given Po , i.e. ~w falls,
and may reduce the affinity of blood for 02' i.e. P50 increases. A c~mprehensive
study of the response of the dogfish, S. canicuia, to a variation in acclimation
temperature of lO °C (7-17 0c) was made by Butler and Taylor (1975). Oxygen
uptake, respiratory frequency, cardiac output and heart rate all increase with a rise
in acclimation temperature. Over the fulllO °C range, the QlO values for these variables
are 2.1, 2.5, 2.1 and 2.1 respectively. Thus there are no changes in cardiac stroke
volume or in (Ca-C~)o2' Although ventilation stroke volume was not measured, it is
probable that both respiratory and cardiac pumps cope with the increased demand
for oxygen by frequency changes only. Despite the reduced amount of oxygen in
1.4 Supply of and Demand for Oxygen 37
the warmer water, both Pa02 andCa 0 2 increase and the latter is partly the result
of a rise in haematocrit. Over the full 10°C range mean dorsal aortic pressure
increases by 72 % and mean ventral aortic pressure by 50 %. However, resistance
to blood flow in the systemic circulation does not change, whereas there is a progressive
decrease in resistance in the branchial vascular bed.
Not all elasmobranchs are poikilothermic; the highly active lamnid or mackerel
sharks are able to retain heat by way of counter-current heat exchangers (rete). Mako
sharks may pass through the thermocline at regular 2-3-hourly intervals, but there
are no corresponding fluctuations in stomach temperature, which may remain 5 °C
or more above ambient (Carey et al. 1981 ; Carey 1982). This, no doubt, enables a high
rate of digestion to proceed (Stevens and McLeese 1984). Mako, porbeagle and white
sharks are able to maintain muscle temperature several degrees higher than that of
the environment (Carey and Teal 1969; Carey et al. 1982) and this is, no doubt,
related to their high level of muscular activity. It has been calculated that the heat
exchangers need to be extremely efficient to maintain the observed temperature
differences. This may be facilitated by the extraordinarily high haematocrits and oxy-
gen carrying capacities in the blood of these fish (approx. 35% and 6.0 mmoll- 1
respectively), which would allow blood flow to be reduced to a minimum (Carey et al.
1981). These high values (see Table 1.2) are yet another indication of the extent to
which these animals are adapted to an extremely active life-style.
1.4.2 Exercise
Dogfish will not readily swim in a water channel (Brett and Blackburn 1978; Butler
et al. 1986), although they do swim spontaneously, particularly at night (Metcalfe
and ButJer 1984 b), and this behaviour has been utilized by one group of physiologists.
The responses of the cardiovascular and respiratory systems to exercise are different
in detail from those seen in response to an increase in temperature. During spontaneous
swimming activity in S. stellaris, at a speed of 23 em S-1 (0.27 body lengths S-1),
oxygen uptake increases by 50-75 % (piiper et al. 1977). Ventilation volume is
approximately three times the resting value, mainly resulting from a rise in stroke volu-
me, but there are slight reductions in Pa 0 2 and Ca0 2. There is no substantial change in
(Ca-C;)02' Cardiac output is 70% above the resting value, again largely as the
result of a rise in stroke volume; heart rate increases by a mere 7-15%. Reductions
in systemic and branchial vascular resistance match the increased cardiac output so
that ventral and dorsal aortic pressures are unchanged during exercise. Any tendency
for branchial blood pressure to increase during exercise could stimulate J-type recep-
tors in the gills which elicit a number of reflex responses, including cessation of
swimming activity, thus preventing possible branchial oedema (Satchell 1978).
Calculations indicate that D02 increases by 20 % during the bouts of spontaneous
swimming. This is not much, but if there is any shunting of respiratory water outside
the interlamellar space,.then a greater increase in Do actually occurs. The relatively
G 2
low G diff ratio during swimming indicates the presence of strong diffusion limitation
perf
(piiper et al. 1977). It may be that the respiratory apparatus of the dogfish is not
able to cope with the increased oxygen demand of exercise, even of a moderate
nature. Certainly Piiper et al. (1977) recorded a large oxygen debt in these animals
following such activity. On the other hand, this limitation may be the result of the
method used to collect expired water. The attachment of rubber bags is thought to
impede respiratory activity. This may be part of the explanation for the different
effect of exercise on blood oxygen in the relatively sedentary dogfish and the more
active lemon shark Negaprion brevirostris. In the lemon shark, without collecting
devices around its gill slits, Pa 02 is comparatively low at rest, but it, Ca 02 and
haematocrit increase during exercise (Bushnell et al. 1982). Venous oxygen reserve
is low in this fish and so cannot be used during exercise as it is in the dogfish.
Bushnell et al. (1982) suggest that a reduction of blood shunts may be partly
responsible for the increased blood oxygen levels during exercise. Such a reduction
in vascular shunts, as well as other changes in the branchial circulation, could
result from an increase in circulating catecholamines, as these are known to rise in
the dogfish S. canicula during spontaneous exercise and to reach levels that could
affect the branchial vasculature (Butler et al. 1986). The more active sharks transfer
the work of ventilating the gills from the respiratory muscles to the locomotor
muscles by swimming with their mouth permanently open ("ram" ventilation) (Hughes
1960; von Wahlert 1964). The receptors on the gills which inhibit ventilation (Satchell
and Way 1962) could be important in the switch to ram ventilation in some fishes.
To what extent the dogfish utilizes this method is not known (Piiper et al. 1977).
Satchell (1968) has discussed the neurological basis for the co-ordination of
swimming movements with ventilation in S. acanthias, although no evidence for such
co-ordination was seen in spontaneously swimming S. stellaris (Piiper et al. 1977).
1.4.3 Hypoxia
Bottom waters along the inner continental shelf in certain parts of the world may
become hypoxic (Po 2 < 6.7 kPa) or even anoxic (Leming and Stuntz 1984). It is
possible that the bottom few cm of larger areas of seas and oceans may be
hypoxic as a result of oxygen consumption of benthic fauna. Skates and rays in
particular, but also dogfish, spend much of their time inactive on the bottom.
Even if hypoxia is not a common problem in the marine environment, the response
of fish when experimentally exposed to it tells us much about how they control their
respiratory and circulatory systems.
Environmental hypoxia has little effect on the dogfish S. canicula at 7 °C whereas
at 12°C there are reductions in oxygen uptake and heart rate, which are more severe
at even higher temperatures (Butler and Taylor 1975). Because temperature has such
a profound effect on the response of these animals to hypoxia, it is not really
justifiable to refer to their being either regulators or conformers to hypoxia (see
Dejours 1981). At relatively low temperatures they regulate, while at higher tempera-
tures they conform. The critical temperature at which the transition between being
a regulator or conformer occurs will vary from species to species.
There is no clear sign of large increases in ventilation in dogfish during hypoxia.
In S. canicula and S. stellaris at 15° and 17 DC respectively, ventilation volume increases
by approximately 50 %with little or no change in respiratory frequency (Piiper et al.
1970; Short et al. 1979). These animals were, of course, heavily instrumented with
collecting devices around the gills. In undisturbed, un instrumented fish, resting
respiratory frequency is substantially lower than that in instrumented fish and increases
by 50 %during hypoxia (Metcalfe and Butler 1984 b). It is essential, therefore, to ensure
that the animal is as undisturbed as possible before commencing the experimental
procedure. Collecting devices around the gill slits may affect respiratory water flow
during hypoxia particularly in fish such as the Port Jackson shark in which water
enters the first gill slit when environmental Po is at 4 kPa (Grigg 1970b).
2
It would appear, however, that the elasmobranchs that have been studied are
not able to increase ventilation by a sufficient amount to maintain P a 0 2 as P i 0 2
declines (Butler and Taylor 1975). Once the shoulder of the 02-equilibrium curve is
reached, Ca 02 begins to fall and oxygen uptake is maintained by an equivalent
reduction in C;02' Although the reduction in heart rate (bradycardia) during
hypoxia is accompanied by an increase in cardiac stroke volume, this is only
sufficient to maintain overall cardiac output (Fig. 1.17). Thus, once reduction of
Pi O2 18.7 kPa Pa0 2 12.7 kPa

a Heart rate 25 beats min- 1 Stroke volume 0.31 ml
,
c:
'E
E
Pi O 2 4.8 kPa
b Heart rate 13 beats min- 1 Stroke volume 0.6 ml
Fig. 1.17. Traces from a female dogfish, S. canicula, at 12°C and weighing 0.85 kg, showing blood flow
to the first two pairs of branchial blood vessels during normoxia (a) and hypoxia (b). In each series, the
traces from top to bottom are: pulsatile blood flow, time marker(s) and ventral aortic blood
pressure. Values of Pi02' Pa 0 20 cardiac stroke volume and heart rate are given beneath each series.
(After Butler and Taylor 1975)
°
C~02 can no longer match the fall in Ca 2 , there is a fall in oxygen uptake
(Fig. 1.18).
Short et al. (1979) analyzed gas exchange in S. canicula in response to environmental
hypoxia in some detail. Percentage extraction of oxygen from the water (% Ext)
and Ew are maintained, whereas Eb declines, presumably as PaOZ falls below the
shoulder of the O?- equilibrium curve. Also, Do 2 remains constant. The increase in
G
ventilation and constant cardiac output means that G::: t increases slightly (from
1.7 to 2.5). The authors were surprised to find that vagotomy, which abolishes the
bradycardia, has no effect on the efficacy of gas transfer during hypoxia. This
compares with an earlier finding, that at 17°C P a02 is higher during hypoxia in
untreated fish than it is in fish which exhibit no bradycardia as a result of injection
of atropine (Taylor et al. 1977). Again, it may be that the less traumatic procedure
in the latter study enabled the animals to respond more naturally. Indeed, in a
1: 40
'E
'7
Ol
""'0
E 30
3-
ell
""C.as 20
:::J
C
I
ell
Ol
»
x 10
0 4 8 12 16 20
a
2.5
:;:"
2.0
1.5
!
'0
E
.§
C,)
ON 1.0 f
0.5 '2
0 •
4 8 12 16 20
b Pi 02 (kPa)
Fig. 1.18. Mean changes in a oxygen uptake and b oxygen content in arterial (0) and mixed venous
(.) blood in 7 dogfish, S. canicula, during progressive hypoxia at 17°C, The first and last points
of each curve are given ± S.E. mean. (After Butler and Taylor 1975)
recent study Taylor and Barrett (1985) used atropine instead of vagotomy to prevent
the hypoxic bradycardia and the method of Metcalfe (1983) to calculate Do . They
z
reaffirmed the lower PaOZ in those animals without bradycardia during hypoxia and
calculated that D oz and Eb are also lower in these than in untreated fish (i.e. those
exhibiting a hypoxic bradycardia).
It has been suggested that the increased cardiac stroke volume and pulse pressures
in the ventral aorta associated with the hypoxic bradycardia in S. canicula (see
Fig. 1.17) may be important in producing inter- and intra-lamellar recruitment
and in reducing diffusion distance, thus maintaining gas transfer (Butler and Metcalfe
1983). It is, nonetheless, strange that fish do not respond to environmental hypoxia
by increasing cardiac output, unless it is important for them to prevent the power
output of the heart from increasing (Taylor et al. 1977). It is worth noting here that
the initial level of hypoxic bradycardia is dependent on the rate of fall in Ppz (Butler
and Taylor 1971). Rapidly induced hypoxia causes an initial intense bradycardia,
but as PiOZ reaches a steady level heart rate increases to a value characteristic of the
Ppz. The functional significance of the initial intense bradycardia is not clear.
The bradycardia that occurs upon gradual exposure to hypoxia is not present after
3 days (Butler et al. 1979) and this may be indicative of the animal adapting to low
oxygen. It would be interesting to know whether in elasmobranchs the level of
organic phosphates decreases during hypoxia, thus reducing P so ' and whether
haemoglobin concentration increases as is the case in some teleosts (Wood and
Johansen 1972, 1973).
The hypoxic bradycardia is mediated predominantly by the two branchial cardiac
vagi (Taylor et al. 1977), whereas the increased cardiac stroke volume appears to be
the result of the intrinsic Starling relationship (Short et al. 1977). The receptors
responsible for the reflex bradycardia are widely distributed in the oro branchial and
parabranchial cavities and are innervated by cranial nerves V, VII, IX and X
(Butler et al. 1977). These receptors are also most likely responsible for the resting
vagal tone under normoxic conditions, as exposure to hyperoxia causes an increase in
heart rate (tachycardia) in S. canicula of similar magnitude to that seen after
injection of atropine (Barrett and Taylor 1984). These authors also present evidence
for the existence of oxygen receptors in the venous system. At elevated values of
P~Oz there is a vagally mediated bradycardia, presumably so that Vb matches the
decrease in Vw that occurs during hyperoxia (Heisler 1984) (i.e. to maintain
Vw~w cIose to umty
-.-- . ) an d to ml11lmlZe
. . , card'laC energy expend'lture.
Vb~b
Circulating catecholamines increase in S. canicula in response to hypoxia (Butler
et al. 1978) and even after 3 days' exposure, when the catecholamines have returned
towards resting levels (Butler et al. 1979), they are still high enough to have
profound effects on the branchial circulation (Davies and Rankin 1973; Butler et al.
1986). In fact the catecholamines and the increased cardiac stroke volume may
both counteract the tendency for the bran,chial blood vessels to constrict in response
to hypoxia (Satchell 1962). There is, however, no evidence to suggest that the
elevated levels of catecholamines play any part in maintaining gas transfer during
hypoxia (Metcalfe and Butler 1988a and b).
It has been stated that hypoxia causes an increase in activity in resting fish
(Randall 1970). This is not the case for S. canicula (Metcalfe and Butler 1984b);
in line with the observations on teleost fish (Kutty 1968), hypoxia inhibits spontaneous
activity in the dogfish, which indicates that these animals attempt to reduce the
demand for oxygen when it is in short supply.
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Chapter 2 B. L. ROBERTS
The Central Nervous System
The elasmobranch brain, although well known to most biologists as a subject for
dissection, has seldom been subjected to investigation and little is known of its phy-
siology. Early anatomical work on the elasmobranch nervous system concentrated
on the question of possible head segmentation in the 'vertebrates. Subsequent studies
continued with an evolutionary perspective and were primarily concerned with the
establishing of homologies. Physiological workers have also imbi bed the evolutionary
spirit and have examined the nervous systems of these "primitive" fishes in the
hope of uncovering organizations that are more simple than those of "higher"
vertebrates.
The present survey shows that of necessity much of the physiological work on the
central nervous system has also been concerned with the establishing of connections
and pathways within the brain. This work is effectively "physiological neuroanatomy"
and many of the findings have been subsequently confirmed or extended by the
use of modern neuroanatomical techniques. In only a few cases has there been an
'attempt to interpret neural mechanisms in action. Much of the physiological work
has been at a gross level and, except for studies on certain eye-catching neurons,
little has been done with individual cells. The data are often fragmentary, perhaps the
result of a summer visit to a marine station, and so much more research needs to be
done.
Operational analysis of the nervous system has been hampered in the past by the
difficulties of obtaining and keeping living elasmobranchs, by the lack of objective
information about the behaviour of these fishes, and by the limited amount of neuro-
anatomical information.
Elasmobranch behaviour is expressed as a limited repertoire of body movements
that are triggered by sensory inflow. I shall review the physiology ofthe central nervous
system, therefore, in relation to these two main themes: the control of motor
behaviour and the central analysis of sensory information.
2.1 The Plan of the Elasmobranch Central Nervous System

Although the external form of the e1asmobranch brain is well known, it will be helpful
here to review a few general features, to place the physiological work in a
Author's address: Department of Experimental Zoology, Biological Centre, University of Amsterdam,
Kruislaan 320. Amsterdam Netherlands
50 Chapter 2. The Central Nervous System
i i
b
13
17 16
c d
28
e f
Fig. 2.13. Dorsal view of the brain of Scyliorhinus; arrows mark the location of the transverse
sections shown in b-f. The position of some of the nuclei and brain regions referred to in the
text are marked on the sections. J olfactory bulb; 2 telencephalic hemispheres; 3 epiphysis; 4 dience-
phalon; 5 tectum; 6 cerebellum; 7 auricle; 8 pallium; 9 striatum; IO hypophysis; 11 infundibular lobe
of the hypothalamus; 12 thalamus; 13 tectum; 14 mesencephalic V nucleus; 15 Edinger-Westphal
nucleus; 16 oculomotor nucleus; 17 tegmentum; 18 molecular layer; 19 granule cells; 20 cerebellar
nucleus; 21 molecular layer; 22 dorsal nucleus; 23 intermediate nucleus; 24 octavus column; 25
descending V nucleus; 26 branchiomotor column; 27 medial longitudinal fasciculus; 28 reticular
formation
2.1 The Plan of the Elasmobranch Central Nervous System 51
morphological context. A detailed survey of the anatomy of the elasmobranch

central nervous system has recently appeared (Smeets et al. 1983) and the reader
should turn to that work for more information, and for full coverage of the
anatomical literature.
A diagram of the brain of the dogfish Scyliorhinus in dorsal view is provided in
Fig. 2.1 along with tracings of selected transverse sections. The telencephalon is
large and its organization is complex. It is not easily separated into cell masses
which can be homologized with known systems in other vertebrates; in fact, the
entire region constitutes a "cytoarchitectonic puzzle" (Smeets et al. 1983). It can
be divided into the olfactory bulb and pallial and subpallial portions (Fig. 2.1 b),
but none is as markedly laminated as the cerebral cortex of amniotes.
The diencephalon is divided into epithalamus, thalamus and hypothalamus. It
expands ventrocaudally to form the conspicuous inferior lobes of the hypothalamus
(Fig. 2.1 c). The dorsal surface of the mesencephalon is provided by the large laminated
tectum (Fig. 2.1 d), which is thought to be homologous to the superior collicu1us.
The tegmental floor of the mesencephalon merges with that of the rhombencephalon
and houses the motor systems of the brainstem. A conspicuous component of the
tegmentum is the reticular formation. The brainstem contains the centres of origin
and termination of all ten pairs of cranial nerves (except for 0 and I). The lateral
walls of the rhombencephalon are enlarged to house the primary nuclei (Fig. 2.1 f)
associated with the octavolateralis sensory systems. The cerebellum is a striking
feature of the elasinobranch brain. It consists of the cerebellar body, which is
highly convoluted in some species, and the auricles, that protrude laterally and are
associated with the octavolateralis region.
2.1.1 The Environment of the Brain
The highly vascular choroid plexuses are the sites of formation of the cerebrospinal
fluid that circulates through the ventricular spaces of the brain and the cavity of the
cord. Comparisons between the electrolyte composition of this fluid and of blood
plasma show that in elasmobranchs these media are independently regulated (Bund-
gaard and Cserr 1981), as in other vertebrates. Moreover, there exists in these species
a permeability barrier between the plasma and the extracellular cerebral space, as
shown for example by the limited penetration of inulin into the brain (Cserr et al.
1978).
In most vertebrates this blood-brain barrier consists of the endothelial wall of the
capillaries and the associated glial sheath, the actual barrier depending on tight junc-
tions between the endothelial cells. However, in elasmobranchs the endothelium
is permeable (Brightman et al. 1971) and the barrier results from tight junctions
between glial cells (Bundgaard and Cserr 1981). Elasmobranchs provide, therefore,
an opportunity to compare the properties of glial and endothelial barriers. Micro-
electrode recordings from cerebral blood vessels in Scyliorhinus implicates the role
of this glial barrier in ion homeostasis (Abbott and Butt 1986).
2.2. Control of Motor Behaviour
The behaviour of elasmobranchs is expressed as sequences of movements. The sharks,

sawfishes, guitarfishes, and torpedoes swim by undulation of the tail. The skates
and sting-rays send undulations down the pectoral fins, and the eagle-, cow-nosed-
and devil rays swim by a bird-like flapping of the pectoral fins. These movements use
fewer separate muscles and are more stereotyped and predictable than are those of
mammals. They occur, moreover, even after lesions have been made within parts
of the central nervous system. Thus decerebrated rays (Dasyatis) swim with the
same patterns of muscular activity as do intact animals (Droge and Leonard 1983 a)
and locomotory-like movements continue even after complete destruction of the
brain in dogfishes. The latter observation, first reported by Steiner in 1888, has
attracted several workers to look at the control of locomotion in spinal preparations
of these fishes. These studies have contributed to a general understanding of loco-
motory coordination and, in particular, have emphasized that the vertebrate spinal
cord is more than a substrate for reflex actions.
2.2.1 Locomotion in the Spinal Dogfish
The undulations of the spinal dogfish are produced by a rhythmic sequence of

contractions of the lateral body muscles that passes from head to tail (Fig. 2.2). There
has been much debate about the source of the rhythm and whether the pattern is
produced entirely within the central nervous system or depends on sensory feedback
from the moving body. There have been reports which claimed to demonstrate
conclusively the importance of either central circuits or peripheral inputs. No single
experiment, however, reveals what is happening in the intact animal, or is without
some criticism.
2.2.2 Is There a "Central Pattern Generator" for Locomotion?
Repetitive motor activity has been observed to occur in the absence of sensory activity
in the nervous systems of several phyla (for examples see Roberts and Roberts 1983).
This general finding is the basis for the view that discrete "pattern generators" exist
within the nervous system which are responsible for locomotion. In the 1960's and
1970's, prorriinence was given to the idea that these generators provide the basic
pattern required for locomotion. Sense organs were considered to play no part in
pattern formation but only corrected the generators if the locomotory environment
changed. The review by Delcolmyn (1980), which proposes central pattern generation
as a principle of neural organization, exemplifies this interpretation. It is probable,
however, that this is too simple a view of nervous system operation and that within the
intact animal there is no separable entity that can be designated as a "central
pattern generator".
The regular undulations of the spinal dogfish seem to be very suitable for produc-
tion by central generators and, indeed, the early experiments by Gray and Sand (1936)
2.2 Control of Motor Behaviour 53
suction electrode electromyogram transducer attachment
I I
a 100 mm
decerebrate fish
,~"""~"~i+'_~"~t,,· J..
spinal cord cut
1s
Fig. 2.2. The swimming spinal dogfish. a shows a deccerebrated dogfish held in a holder in a tank
of seawater and attached to a mechanotransducer. A spinal motor nerve is drawn into a suction
electrode and an enamelled wire is inserted in the lateral musculature; b recording from the
lateral muscle in a non-swimming fish ; c shows the same recording after the spinal cord has been
transected (at level of arrow in a)
demonstrate the importance of central circuits in spinal swimming. Spinal cord

circuits must also be able to generate rhythmical movements in the dogfish embryo,
for regular movements under neural control are seen even before sensory nerves
form (J. E. Harris 1962).
The tendency for spinal cord neurons to discharge rhythmically, in the absence
of regular sensory activity, was first shown by recording from the motor nerves
of spinal dogfish ( Scyliorhinus) paralyzed with curare (Roberts 1969a); the rhythms
observed, however, were much slower than are required for locomotion. The ability
of the spinal cord of paralyzed spinal dogfish to generate rhythmical activity of a
more normal pattern was shown for the dogfish Squalus by Grillner et al. (1976) who
concluded that "peripheral signals are apparently not required for a proper coordi-
nation of the swimming movements".
The presence of a sustained rhythm, with appropriate patterning, has now been
confirmed for Scyliorhinus (Williamson and Roberts 1980; 1986) and established
for decerebrate preparations of Dasyatis (Droge and Leonard 1983 b). The basic
finding is illustrated in Fig. 2.3, which shows recordings from motor nerves in a
swimming spinal preparation (a) and then after an injection of curare has abolished
all movement (b); a neural rhythm continues in the absence of body movement
and this must be produced within spinal cord circuits, without any rhythmical
sensory input. Does this spontaneous activity arise from a central pattern genera-
tor?
}omv
c 25
Fig. 2.3. Rhythmical activity of spinal cord motoneurons. a Suction electrode recording from a spinal
inotor nerve of a swimming spinal dogfish. Bottom trace mechanotransducer record; b recordings
from same fish after an injection of curare had paralyzed the musculature; c intracellular
recording from a motoneuron discharging spontaneously in a paralyzed spinal dogfish
A critical point for the central pattern generator concept is that the neural
output of the isolated nervous system must resemble that seen in moving animals .
In Squalus, Scyliorhinus and Dasyatis, the basic locomotory requirement of an
alternating motor pattern, produced in a rostrocaudal sequence, is still observed in
the isolated spinal cord (Grillner et al. 1976; Droge and Leonard 1983 b ; Williamson
and Roberts -1986). Moreover, the relationship between the segmental activity and
the construction of the rhythmical bursts remains well defined in the absence of
sensory input. However, there do seem to be changes in rhythm frequency depelJdent
on the species of elasmobranch studied. Grillner et al. (1976) reported the rhythm in
Squalus as falling within the normal range of frequencies, but a wider range was
found in Dasyatis and the rhythm of the isolated cord in Scyliorhinus has always
been found to be slower than that recorded from swimming fish (Figs. 2.3 and 2.4).
In fact, if Scyliorhinus swam according to the motor pattern seen after paralysis it
would always descend to the sea bed! The difference in rhythm frequency between
swimming and paralyzed preparations does suggest at least a tonic function for
sensory input, because the slowing of the neural rhythm is seen as paralysis
begins and as movement ceases (Williamson and Roberts 1986). In recent years
evidence has been accumulating that the role of sensory input may be more than this,
for sensory stimulation has very striking and immediate effects on the pattern of
locomotion.
R E
IE
L~
a 1s
b 2s
0·6
o o.g
C input frequency (Hz)
Fig. 2.4. Impact of sensory stimulation on locomotory performance. a Recordings from motor
nerve (top trace ) in a swimming spinal dogfish (bottom trace transducer); b same recording site after
curare paralysis. At arrow an oscillation was applied to the body; c frequency of the output motor
rhythm plotted against the frequency of the imposed oscillation. Dotted line indicates the frequency
in the absence of oscillation
2.2.3 The Relationship Between Sensory Input and Control Circuits
Deep receptors in the skin, probably the Wunderer's corpuscles, are stimulated when
the body bends and their excitation has a general and very striking effect on the
locomotory rhythm. This can be seen even in electromyograms taken from swimming
fish when an oscillatory movement is imposed on the body (Roberts 1969b), but is
particularly clear in neural recordings from paralyzed spinal preparations (Grillner
and Wallen 1982; Wallen 1982; Williamson and Roberts 1986). The impact of
oscillatory body movements on the output rhythm is shown in Fig. 2.4 b. Over a
certain range of frequency of oscillation the timing of the motor output recorded
from a motor nerve follows exactly tl?at of the imposed oscillation (Fig. 2.4c). Outside
this range the output is still synchronized with the input frequency, but misses some
inputs or provides additional outputs. Hence the exogenous sensory signals dominate
the endogenous pacemaking and effectively determine the motor output. However,
this entrainment is limited to a range which is in some way related to the frequency
of the rhythm of the isolated spinal cord, for the sensory input can certainly follow
wider ranges of oscillation frequency (Williamson and Roberts 1986) than that of
entrainment.
Sensory stimulation can also exert more specific and limited effects on the spinal
cord. Thus cutaneous stimulation will modify the swimming rhythm of the spinal
dogfish. The effect is largest in the region of the stimulus and has all the characteristics
of a reflex action that moves the body away from the stimulus (Lissmann 1946).
Reflex responses of this type, however, are not independent of the ongoing activity
of the spinal cord. Thus a stimulus to the tail in a swimming fish will evoke a
reflex response from whichever side of the body is active (Grillner et al. 1977), and
the result is determined by the activity of the central circuits (Wallen 1980).
Further insight· into the mechanism of interaction of sensory input with the
central circuits depends on knowledge about spinal cord organization.
2.2.4 The Organization of the Spinal Cord
Spinal cord motoneurons lie within the ventral horn (about 700 per segment in
Scyliorhinus) and have very extensive dendrites that are flattened in the transverse
plane and spread throughout much of the ipsilateral cord. Labelling the different
muscle fibre types with horseradish peroxidase has shown that motoneurons supplying
the lateral red musculature are generally smaller and more laterally situated than those
that innervate the white musculature (Mos and Williamson 1986). It is tempting
to think that these differences reflect a size "principle" that might be regulating the
order of neuronal firing. Small motoneurons would be used for steady locomotion,
while larger neurons, activated later because of their higher threshold, would discharge
during phasic movements.
The motoneurons are driven by brain stem neurons via the descending pathway,
by other spinal cord neurons, and by sensory inputs. Sensory fibres enter through
the dorsal root, bifurcate and ascend and descend the ipsilateral spinal cord. In the
dogfish no dorsal root fibres enter the ventral horn, nor is there evidence of
monosynaptic relay onto motoneurons. However, recordings from motoneurons of
Dasyatis (Leonard et al. 1978) during stimulation of sensory nerves has demon-
strated monosynaptic as well as polysynaptic activation. This difference from the
dogfish may relate to the different types of receptors found peripherally in these
two species, for specialized muscle receptors are absent from the dogfish, but are
found within the fin muscles of rays (see Roberts 1978). In mammals monosynaptic
inputs to mononeurons from muscle spindles are, of course, well known. The study in
Dasyatis also showed that small afferent fibres evoke activity in the most dorsal
part of the dorsal horn, whereas larger afferents are effective in deeper regions,
closer to the motoneurons.
Intracellular recordings have been made from the motoneurons of the paralyzed
spinal dogfish when the spinal cord is spontaneously active (Roberts and Williamson
1983). The motoneurons can be grouped into two broad classes on the basis of their
activity. Some cells are spontaneously active and show oscillating membrane potentials
that have the same rhythm as motor nerve activity; they discharge action potentials
if the depolarizations are sufficiently large (Fig. 2.3 c). The number of potentials
and the firing frequency depend on the rhythm of the spontaneous activity. Similar
.oscillating potentials have been found in the motoneurons in other vertebrates and
have been shown to consist of alternating excitatory and inhibitory potentials (e.g.
Jordan 1983). We assume that the spontaneously active cells are innervating the
lateral musculature which is used in spinal "swimming". Silent cells form the second
class of motoneurons and these sometimes discharge to cutaneous stimulation;
presumably they supply inactive lateral and white muscle fibres.
The rhythmical activity of the motoneurons depends on inputs from premotor
interneurons, but at present little is known about these cells. Some are spontaneously
active, bursting in time with the output rhythm, while others are silent or continuously
active (Roberts and Williamson 1981). The data available at present provide no
insight into the mechanisms underlying either rhythm generation or sensory entrain-
ment. How the rhythmical activity of the spinal cord neurons arises remains to be
determined. It may involve discrete pacemaker neurons, or it may arise from the
interaction of specific networks. It is also unknown at present whether the source
of the rhythm constitutes a separable component, a "generator", which can be removed
from the organism somewhat like a beating heart. But just as the isolated heart can
tell us much about the mechanism of cardiac function and little about cardiovascular
control, so the isolation of a generator, should it exist, will still provide us with an
incomplete view of locomotory coordination. In the case of elasmobranch fishes,
the impact of sensory inflow seems so strong that it is likely that this input is
contributing in some way to the motor output pattern in the intact animal. More-
over, as we shall see, inputs from descending systems also affect spinal cord
circuits, so the pattern of the motor output cannot be considered to be produced at
anyone location in the nervous system but must involve the coordinated action of
many systems.
The debate over the significance of central generators and peripheral reflexes
has probably told us more about the way we think and polarize issues than it has
informed us about the control of fish locomotion. It is to be hoped that in the
future emphasis will be given to experiments that expose the mechanisms underlying
spinal cord operation and the way sensory, descending, and spinal cord neurons
interact to produce the motor performance of the real animal.
2.2.5 Spinal Cord and Brainstem
The projections to the spinal cord have been examined experimentally by Smeets and
Timerick 1981, in Scyliorhinus and Raja. The axons arise from different portions
of the reticular formation and descend to the spinal cord in the medial longitudinal
funiculus and in the funiculus of Stieda. Stimulation of these axon tracts in
decerebrate dogfish provokes large unilateral body contractions, of the type used
in rapid turning movements, because of the synaptic excitation of many spinal
motoneurons by the descending axons (Roberts and Williamson 1983). The descending
pathways also strongly influence interneurons in the cord, and can affect the motor
rhythm.
Spinal "shock" follows the transection of the spinal cord of mammals, but tlns
is not seen in elasmobranchs, as a wide range of reflex movements can still be evoked
after spinal cord transection and in sharks spontaneous undulatory swimming is
released. This result not only indicates that the basic programme for locomotion
is produced within the spinal cord, but that descending influences must normally
control its expression. The location of these control regions is unknown. Swimming
is released after the medial longitudinal fasciculus is sectioned (Paul and Roberts
1984) or after small bilateral lesions are placed in the lateral rhombencephalon
(Roberts and Williamson 1983). In the cat, activation of a lateral continuous strip
of cells in the brainstem (mesencephalic locomotor region, MLR) elicits controlled
locomotion and in the carp, an equivalent region has been identified in the
caudal part of the mesencephalic tegmentum, where local stimulation evoked
swimming (Kashin et al. 1974). Droge and Leonard (l983b) and Grillner and Wallen
(1984) report that in elasmobranchs midbrain stimulation will evoke swimming
movements, but more histological information is required before a homologue to the
mammalian MLR can be designated in the fish brain.
2.2.6 The Reticular Formation
The central core of the brain stem is occupied by the reticular formation. This
consists of loosely arranged cells of different types and sizes and their associated
fibre systems. In elasmobranchs it constitutes a histologically striking component of
the brain, as some of the neurons are large and have very extensive dendritic
arborizations. The neurons form loose aggregations which on cytoarchitectonic
grounds can be divided into three columns: a median zone (raphe nuclei), an
extensive medial zone (superior, medial and inferior divisions) and a diffuse lateral
zone. Equivalent regions in mammals receive and process information from other
parts of the nervous system. They participate in the control of the limbs and body
during locomotion, movements of the eyes, sleep-wakefulness states, sensorimotor
activity and nociception. We can only surmise about these functions in elasmo-
branchs, as our knowledge of the brain is so incomplete.
Some features of reticular neurons in Squalus have been examined with micro-
electrode recordings (Restieaux and Satchell 1958). The conduction velocities of the
reticulospinal neurons fall within the range of 40 to 60 m S-l, while a few reach
80 m S-l which places them, in terms of speed, alongside Mauthner cell axons of
teleost fishes. Reticulospinal neurons were found by Satchell (1968) to discharge

rhythmically with a pattern that was clearly related to respiratory movements, and
which ceased when these were abolished by curare paralysis. The rhythmical driving
of these cells came from sensory endings of the head, as stimulation of cranial
nerves V and VII, or mechanical stimulation of the pharynx, always excited these
cells. Many reticulospinal cells are also synaptically activated by spinal cord
stimulation.
The axons descending to the spinal cord from the reticular formation comprise
one of the motor systems of the brain. Other motor systems lie within the brain and
include the nuclei of the somatomotor, visceromotor andbranchiomotor nuclei.
These cranial outputs control eye movements, jaw and gill movements and some
autonomic functions.
2.2.7 Control of Extrinsic Eye Muscles
The eye muscles are innervated via cranial nerves III, IV and VI from neurons
located in the brain stem, close to the ventricles (Rosiles and Leonard 1980;
Montgomery and Housley 1983; Graf and Brunken 1984). The nuclei for nerves III
and IV contain neurons that innervate eye muscles of both eyes, but the abducens (IV)
nucleus innervates only the ipsilateral lateral rectus muscle. The presence in elasmo-
branchs of a contralateral supply to the medial rectus muscle contrasts with the
situation known for all other vertebrates.
The compensatory movements of the eyes that accompany locomotor movements
in the dogfish, Squalus, were investigated by A. J. Harris (1965), who distinguished
eye movements driven from the labyrinth from those that required spinal circuits.
Nothing is known about the integration of these systems for the connections between
the extraocular motor nuclei, and the octavus column neurons are still unknown.
A circuit that might underlie eye movements in these fishes, involving vestibular
activation, has been proposed recently (Graf and Brunken 1984).
2.2.8 Control of Intrinsic Eye Muscles
The light reflex, a constriction of the pupil in response to eye illumination, is a para-
sympathetic reflex which is observed in elasmobranchs and involves both an inner-
vation via nerve III from the Edinger-Westphal nucleus (Kuchnow 1971) and a direct
sensitivity of the iris'sphincter muscles to light (1. Z. Young 1933).
2.2.9 Control of Heart Muscle
The location and some properties of the parasympathetic innervation to the heart
have been described recently for Scyliorhinus (Barrett and Taylor 1985b). The supply
to the heart is carried by the branchial and visceral cardiac branches of the vagus
nerve from motoneurons that lie in lateral and medial divisions of the vagal motor
column. The lateral cells are either inactive, or spontaneously irregularly active,
whereas the medial cardiac neurons are rhythmically active and discharge in time
with respiratory movements (Barrett and Taylor 1985a, c). This relationship to the
respiratory rhythm suggests that there must be input from the respiratory rhythm-
generating neurons to the medial cardiac motoneurons. A relationship between these
two systems could account for the coupling between heart rate and respiration that
has been frequently reported for these fishes, and the functional significance of which
is still debated (see Chap. this Vol. 1).
2.2.10 Control of Jaw and Gill Musculature During Respiration
Vagal motor neurons, along with those in the Vth, VIIth, and IXth motor nuclei, are
involved in the control of the muscles of the jaws and gills during respiration. A
study of the organization of the vagal motor column has recently been published
(Withington-Wray et al. 1986).
Electromyographic recordings from the respiratory muscles show their patterns
of activity (Hughes and Ballintijn 1965), while recordings from the peripheral branches
of these nerves confirm their involvement in respiratory control (Barrett and Taylor
1985 a). A motor rhythm, although of changed frequency, persists when all move-
ments are stopped by curare paralysis, indicating that central circuits are able to
produce patterned outputs. This rhythm can be entrained by stimulating the
sensory fibres in the vagus nerve. Details of the circuits involved in sensory-motor
interactions are presently unknown; it is probable, however, that the rhythm involves
activity within reticular neurons.
In the electric rays (Torpedo, Narcine) some of the branchiomotor neurons
activate the electric organs that are derived from gill musculature. These electromotor
neurons, which are very large and multipolar, comprise the enormous electric lobes
(Roberts and Ryan 1975). There are no distinctive physiological features of these
cells (Saito 1966). In Torpedo the electromotor neurons are relay cells. They are
controlled by bilateral nuclei in the rostral rhombencephalon (Albe-Fessard and
Szabo 1954), which when sufficiently excited, trigger the neurons of the electric lobe
to discharge.
2.2.11 Control of Sensory Hair Cells
The motoneurons of the rostral pole of the facial (VIIth) nucleus overlap and inter-
mingle with the neurons of the octavolaterlis efferent nucleus, which provide an
efferent innervation to the sensory hair cells of the ear and lateral line (Meredith and
Roberts 1986). This efferent innervation is a consistent feature throughout the
vertebrates, yet its functional significance remains uncertain. Stimulation of auditory
and lateral line efferent neurons has an inhibitory effect on the hair cells (Roberts
1978), but stimulation of vestibular efferents can have an excitatory action (Highstein
and Baker 1985). Little is known about how the efferent nuclei are organized or
when they are nonnally active. A recent study by Highstein and Baker (1986) on a
teleost, the toadfish, shows that posterior semi-circular canal efferent neurons are
electrically coupled.
A possible function of efferent neurons is that they provide some kind of feedback
regulation of the hair cells. However, in the dogfish, attempts to activate these cells
with natural lateral line stimulation were unsuccessful (Roberts and Russell 1972).
Electrical stimulation of lateral line afferent fibres will, however, reflexly drive the
efferent neurons (paul and Roberts 1977c).
Lateral line efferent neurons discharge in response to several types of peripheral
stimulation which have in common the fact that they evoke movement. Efferent
cell activity seems consistently to be correlated with movement, either of the gills or
of the body, and one of its functions may be to modify lateral line sensitivity during
locomotion. Vestibular efferent neurons of the toadfish increase their rate of firing
when the fish is aroused (Highstein and Baker 1985). Further information on the
role of the efferent system is provided elsewhere (Roberts 1978).
2.2.12 The Cerebellum
The cerebellum of elasmobranchs resembles that of other vertebrates, both in external

form and in internal construction (Fig. 2.5 a). Thus its wall contains four layers:
granule cell, fibre, Purkinje cell, and molecular (Nicholson et al. 1969). The output
of the cerebellum is provided by the Purkinje cells, whose axons terminate on and
inhibit neurons in. the cerebellar nuclei that are located, one on each side, in the
cerebellar peduncles.
Because of its resemblance to the mammalian cerebellar cortex, and its ordered
cellular organization, the elasmobranch cerebellum has been the subject of several
electrophysiological studies (Eccles et al. 1970 a, b; Nicholson et al. 1969; Paul 1969 ;
W. Young 1980a). When the surface of the cerebellum is stimulated, a series of
potentials can be recorded from a microelectrode placed in the molecular layer.
These potentials probably represent as voll(lY of parallel-fibre activity, conducting
slowly across the cerebellum, as well as the subsequent excitation of the Purkinje
and stellate cells (Paul 1969; W. Young 1980a).
In the mammal, input to the cerebellum comes via two main afferent pathways:
the mossy and the climbing fibres. In elasmobranchs, mossy fibres arise from
several sources (Smeets et al. 1983) and presumably give Hie simple spike discharge
of Purkinje cells (Fig. 2.5c). The existence of climbing fibres, however, is much less
secure, although as they are an essential component of the mammalian cerebellum
we would expect, on comparative grounds, to find them in the cerebellum of fishes.
They are not seen, however, in good Golgi-impregnated material which displays all
other cerebellar elements. Climbing fibres in mammals originate from the inferior
olive, a nucleus which has been recognized in elasmobranchs. Horseradish peroxidase
injections made into the cerebellum retrogradely label cell bodies of the olive, thus
supporting the view that a climbing fibre system is present. The complex electrical
responses obtained sometimes in the molecular layer (Fig. 2.5b, c) resemble climbing
fibre responses of the mammal and have been considered to be so (Eccles et al.
1970a). However, as Nicholson and Llinas (1969) have shown, these responses can
be blocked by intracellular hyperpolarization of the Purkinje cell, and evoked from
the cell by current injection, indicating that the responses are a property of the
Purkinje cells themselves and not of their inputs. The most satisfactory interpretation
62 Chapter _.
? The CentraI Nervous System
t-------I
b c 20 ms
Is
+
mV
d
of these data would be to assume that a climbing fibre system is found in these fishes,
but that the axons of olivary cells do not "climb" molecular layer dendrites and
terminate only on Purkinje cell somata. More precise anatomical data are needed
to test this idea. A thorough discussion of the climbing fibre problem in fishes has
been provided recently by Paul (1982).
"Spreading depression", a phenomenon observed in various brain structures and
particularly the mammalian cerebral cortex, has been reported in the cerebellum
of Raja (W. Young 1980 b). This consisted of a predominantly negative extracellular
shift of some 40 m V, which propagated throughout the cerebellum in response to
DC stimulation of the cerebellar surface; ongoing neural activity ceased during
this event (Fig. 2.5). The mechanisms underlying this phenomenon are unknown,
although Young believes that the potentials have a glial rather than a neuronal
source.
2.2.13 Function of the Cerebellum
The physiological and anatomical studies on the elasmobranch cerebellum all support
the view that the neural circuit is similar in construction and function to that of other
vertebrates. The mossy fibre input drives granule cells which then excite Purkinje
cells and stellate cells via the parallel fibre pathway. The climbing fibre input
excites the Purkinje cells. Stellate and Golgi cells are interneurons that presumably
regulate Purkinje cell activity. The output of the cerebellum is provided only by
Purkinje cells, which inhibit cerebellar nuclear neurons, which excite neuronal
groups in the brain stem.
The functional significance of the cerebellar circuit remains uncertain. Cerebellar
ablation in fishes produces few motor defects (see Healey 1957), whereas in mammals
profound motor disturbances follow cerebellectomy. Closer examination of move-
ments in decerebellectomized fishes, however, does indicate a role for the cerebellum
in motor control. One study of this problem was made recently in Scyliorhinus,
using a reflex elevation of the pectoral fin as the test movement (Paul and Roberts
1979). This reflex is generated by skin stimulation over the fin and involves a
contraction of the elevator muscles. The response is a graded increase to stimulus
strength which has two components - an initial rapid elevation followed by a longer-
lasting tonic phase (Fig. 2.6a). Removal of the cerebellum does not abolish this
movement, but changes its form. There is a marked increase in threshold (Fig. 2.6d),
and a reduction of the tonic phase. As the complete form of the reflex is seen in spinal
preparations, we can conclude that the cerebellum does not contribute to the
~
Fig. 2.5. The cerebellum. a Drawings of Golgi-impreghated neurons from the cerebellum of
Platyrhinoidis. 1 Purkinje cell; 2 stellate cell; 3 Golgi cells; 4 granule cells; b "simple" and
"complex" spikes recorded from cerebellum of Scyliorhinus; c "simple" spikes and "conplex"
burst evoked by parallel fibre stimulation in Platyrhinoidis; d Slow potential recorded from three
sites in the cerebellum of Raja in response to surface DC stimulation. (a, c from Nicholson et aJ. 1969;
d from W. Young 1980b)
detailed structure of the movement, but only to the way it is expressed. This
could be brought about by a cerebellar regulation of the amount of descending
inhibition and excitation sent by the brain stem to spinal cord circuits.
-~~~ ......................
a
t 500 ms
b
500 ms
,
~
J~II~I~IIIIIIIIIII~ 11~11I1~111~1111111~1111~lll
f----i r----;
c 500 ms e 500 ms
1000
>
Qj
N
Ui
Qj 500
(/)
c
o
a.
(/)
Qj
a:
50 100
d Stimulus Size ( V )
Fig. 2.6. Cerebellar function. a Electromyographic (EMG) recordings from pectoral fin elevator
muscle during reflex movement. Top trace decerebrate fish ; bottom same, after cerebellectomy;
b Discharge of Purkinje cell during fin reflex; c Purkinje cell ( bottom ) discharge in swimming fish.
Top trace EMG from body muscle; d Size of the pectoral fin reflex measured from the EMG
plotted against the size of the stimulus to the fin. Circles decerebrate ; squares decerebellate ;
triangles spinal fish; e Cerebellar nuclear neurons discharging during fin reflex. All bars 500 ms ;
arrows time stimulus given to fin
2.2.14 Properties of Cerebellar Neurons
An understanding of the operation of the cerebellum requires an analysis of the type

and form of the inputs, and information about the interactions between Purkinje
cells, nuclear cells, and nuclear outputs.
Visual, electrosensory, mechanosensory and tactile-proprioceptive inputs were
shown to project to distinct zones of the cerebellum of immobilized Platyrhinoidis
(Tong and Bullock 1982). These zones showed only slight overlap. Tactile responses
of Purkinje cells were observed by Paul and Roberts (1984) in the caudal portion of
the cerebellum of Scyliorhinus, but only when there was some accompanying body
or fin movement.
In spontaneously swimming dogfish, rhythmical activity in time with body move-
ment was recorded from about one third of the Purkinje cells (Fig. 2.6c) distributed
throughout the cerebellum (paul and Roberts 1984). This activity probably arose
from spinal cord generator circuits, for rhythmical activity persisted after muscle
paralysis had stopped all body movement. It was suggested that this input provides
a monitor of the state of spinal cord circuits during movement.
During the pectoral fin reflex, Purkinje cell activity was limited to the caudal
half of the cerebellum (Paul and Roberts 1981). Only 26% of the Purkinje cells in
that region responded to stimuli that evoked pectoral fin elevation (Fig. 2.6b). The
latency of the response was long (40 ms) and most cells increased their rate of
firing (only 16% were inhibited). The Purkinje cell discharge could not be initiating
the reflex, but the timing is appropriate for it to influence the later components.
The cerebellar nuclear neurons in Scyliorhinus discharge spontaneously, mostly
with a very regular pattern (paul and Roberts 1983 a). During pectoral fin stimulation,
most of these neurons are strongly excited (Fig. 2.6e), but with latencies that
are longer than those of the fin movement. A few nuclear cells, however, were
inhibited during fin stimulation (Fig. 2.6e) and these were considered to be connected
to those Purkinje cells that were excited during the reflex. This response pattern of
nuclear cells, when considered together with that shown by Purkinje cells, provides
us with a mechanism for cerebellar action during the pectoral fin reflex. The set of
Purkinje cells activated during the reflex is monitoring spinal cord motor circuits
and inhibits a set of nuclear neurons which are driving brain stem descending
systems to the same spinal cord circuits. Their inhibitory action is removed,
allowing the movement to be expressed fully, while simultaneously, via the action
of other sets of cerebellar nuclear neurons, descending inhibition is increased to other
motor systems so as t~ prevent their unwanted expression.
2.2.15 The Structure of Motor Programmes
We may consider motor behaviour in an elasmobranch fish such as the dogfish to

be organized as shown in Fig. 2.7. The basic programme is developed within the
spinal cord and is controlled by descending systems that are regulated by "higher"
centres under the general supervision of the cerebellum.
Although it is conceptually helpful to regard the nervous system as operating in
this way, it is doubtful whether it really does so, or that the nervous system is
CEREBELLUM
'HIGHER'
SPINAL
..... BRAINSTEM .....
CENTRES CORD
Fig. 2.7. A schematic interpretation of motor control in the dogfish
arranged in such a hierarchical fashion with regions of distinct function. This

interpretation of neural action is very much the product of our experimental appro-
aches in which we isolate pieces from the nervous system. It is unlikely, however,
that the nervous system of an intact animal operates merely as the sum of the
properties seen in these experimental preparations. We have seen that there is good
evidence of central neuron rhythmicity, underlying both locomotion and respiration,
but this is regulated in the intact animal by descending systems and by sensory input.
In addition, the presence of corollary discharges within the central nervous system,
and efferent modification of sensory function, makes the distinction between
"central" and "peripheral" events hard to make, and probably meaningless.
The independence of the spinal cord in the dogfish, expressed in the rhythm of
the spinal preparation, may reflect a phylogenetic history or an ontogenetic require-
ment, but it may also be a way of simplifying descending control. If the basic response
pattern is incorporated into spinal cord design, then relatively simple commands
from the brain may be sufficient.
Sensory activity can have several consequences for the pattern of movement.
The effect can be direct, as in a reflex response operating locally. For example a
movement of the tail away from the stimulus, an abrupt gill closure or a "startle"
response to vibration. Other regions of the nervous system may be involved,
however, as we have seen with the cerebellum in the pectoral fin reflex.
Some sensory inputs operate indirectly via generating circuits. These then have
a stabilising, modulating and entraining role (Roberts 1983); while others are not
immediately translated into a motor action but require further analysis and integra-
tion with other sensations and motor programs.
2.3 Central Analysis of Sensory Information
2.3.1 Central Processing for Vestibular Function
The labyrinth is innervated by cranial nerve VIII which projects to the octavus
column of the rhombencephalon. The primary fibres terminate in ascending, magno-
cellular and descending octavus nuclei, and project also to the granule cells of the
2.3 Central Analysis of Sensory Information 67
pars medialis of the auricle. Stimulation of the primary fibres of the VIIIth nerve
evokes large, predominantly negative, field potentials throughout the octavus column
(Montgomery and Roberts 1979; Boord and Roberts 1980; Plassmann 1982).
Electrical stimulation of both octaval nerves shows that the nuclei are interconnected,
giving rise to both excitatory and inhibitory responses of octaval neurons (Mont-
gomery 1980).
The outputs of the octavus column have not been completely studied, but those
that project to the spinal cord are known from anatomical studies in the ray and
dogfish (Smeets and Timerick 1981). The vestibulospinal projection originates mainly
from the large-celled nucleus octavus magnocellularis which sends axons into
the fasciculus of Stieda and the medial longitudinal fasciculus. Stimulation of these
fasciculi, and of the VIIIth cranial nerves, evokes clear responses in the pectoral fins
(Timerick 1983). Timerick (1982) examined the fin reflexes in response to stimulation
of individual semi-circular canals and showed that stimulation of the vertical canals
evoked activity predominately from the ipsilateral depressor and contralateral leva-
tor pectoral fin muscles. This is a pattern of response which would compensate
for a roll of the fish about the longitudinal axis towards the stimulated side, and
just these responses were seen when fish were exposed to sinusoidal rolls (Timerick
1983). The fin movements during roll were abolished after bilateral labyrinthectomy .
At low frequencies of roll (less than 0.1 Hz) the muscle response is in phase with
angular position but as the frequency increases there is a phase advance so that at
1 Hz the response is in phase with angular velocity. The simplest explanation for this
would be to assume that at low frequencies the otoliths provide the dominant input,
but that input from the semi-circular canals becomes progressively more important
at higher frequencies.
Recordings have been made in Scyliorhinus from primary afferent fibres, and
secondary vestibular and cerebellar neurons during horizontal canal stimulation
(Montgomery 1980). Sinusoidal head rotation produces a smooth sinusoidal modula-
tion of the discharge frequency of the primary afferent fibres. This pattern is
followed by the neurons of the octaval nuclei and the auricles, but with some
differences. For example, some secondary neurons discharge completely out of
phase with the afferent fibres (type II neurons) and many cease to fire during the
inhibitory portion of the rotation.
Electrophysiological recordings from the pars medialis of the auricles (Montgo-
mery 1982) have shown that stimulation of the VlIIth nerves excite the granule
cells and this is followed by more complex excitatory and inhibitory responses
of the Purkinje cells. These cells responded to horizontal rotation with various
patterns of response (types I, II and III), ind with phase relationships to the
stimulus which are different from those of the primary fibres. Purkinje cells may
project back to the octavus column and so they could playa significant role in
regulating octaval (vestibular) actions.
2.3.2 Central Processing for Audition
Elasmobranchs are known to respond to sound stimulation, although the auditory

endorgan is not well defined. The macula neglecta is important, but there is
probably some involvement of the sacculus as well. Consequently, it is unknown if

a portion of the octavus zone forms a discrete acoustic nucleus, or whether vestibular
and auditory inputs intermingle.
Corwin and Northcutt (1982) examined this problem using 14C-2-deoxyglucose
autoradiography and evoked recordings in Platyrhinoidis during sound stimulation.
They found areas of high activity in the ascending octavus nucleus and around a
group of cells called cell plate X (nuclei C1 and C2 of Smeets et al. 1983), in the
nucleus of the lateral lemniscus and in the lateral mesencephalic nucleus. In bony
fishes, a mesencephalic region, the torus semicircularis, is an important centre
where auditory, lateral line and visual information is collated. Evoked activity
in response to air-borne and water-borne sound stimulation can be recorded from
the region of the torus of the elasmobranch brain (Bullock and Corwin 1979). Unit
activity has been recorded in response to vibrational stimuli in the octavus magno-
cellular nucleus, the torus semicircular is and the oculomotor nuclei. Variations in the
responsiveness of the units may indicate that the inputs come from more than one end
organ of the labyrinth (Plassmann 1983).
2.3.3 Central Processing for Lateral Line Mechanoreception
Mechanoreceptive lateral line afferent fibres enter the brain through the ventral
division of the anterior lateral line nerve (Bodznick and Northcutt 1980) and terminate
in the intermediate nucleus, dorsal to the octavus region. The principal neurons of
this nucleus, which resemble Purkinje cells, project to the mesencephalon in the lateral
lemniscus (Boord and Northcutt 1982). Presumably this system also projects further,
to the telencephalon via a thalamo-telencephalic pathway.
The similarities in sense organ morphology, the neural pathway and the
evoked potential records (Paul and Roberts 1977 a) suggest that mechanoreceptive
lateral line processing resembles that performed within the electroreceptive system,
about which we have more information.
2.3.4 Central Processing for Electroreception
The electro receptive behaviour of elasmobranch fishes is striking (see Chap. 3,

this Vol.). Electrophysiological studies have yet to give us detailed information on
electrorecept.ive processing. Evoked potential recordings clarify which regions are
active, although the distinction between excitatory and inhibitory events is often not
clear. In unit studies the element recorded from has not always been identified and so
it is possible to confuse primary afferent fibres with the soma and axons of the
secondary neurons.
The first stage in the central analysis of the electro receptive system takes
place in the dorsal nucleus of the lateral wall of the rhombencephalon (Fig. 2.8 a).
The principal neuron of this nucleus is a large multipolar cell which has dorsal
dendrites that extend into the overlying molecular layer (Fig. 2.8 b). Stimulation
of the afferent nerve exerts a powerful excitatory action on these cells (Fig. 2.8 c),
most likely on their extensive, ventrally oriented dendrites (Paul and Roberts 1977 b).
Incoming afferent fibres ascend also to the granule cells of the auricles which in
turn project their axons, as fine unmyelinated parallel fibres , through the molecular
layer. This circuit is very reminiscent of that of the cerebellum (Roberts 1981),
but its significance for electroreception remains unexplored.
1AL. .. ~
~, r-"·~""·"'I"'I=&.
c 5 ms
I11I1 , 111111I IUI!~ InI

III1I1II1
........ IIII~ IIIIII11111
d 100 ms
•
•••
e
Fig. 2.8. Central analysis of electroreception. a diagram of pathway. e incoming electro receptive
information ; d dorsal nucleus; In mesencephalic nucleus ; t thalamus ; p pallium. (After Boord and
McCormick 1984); b diagram of principal cell of dorsal nucleus ; c evoked potentials recorded in
dorsal nucleus of Scyliorhinus in response to stimulation (at arrow) of superficial ophthalmic
lateral line nerve. Five superimposed sweeps; d electroreceptive unit in dorsal nucleus responding
to an electric field in water around fish. When head of animal is positive, the firing rate
decreases ( top trace) ; firing rate increases when the polarity is reversed. Arrows mark stimulus.
(Nicholson et a\.. 1969); e evoked responses recorded from the telencephalon of Raja following
stimulation of dorsal nucleus. Dots indicate on the telencephalic section the location of the
potentials. (After Bodznick and Northcutt 1984)
Each branch of the anterior lateral line nerve collects fibres from different groups
of ampullae and telminates in discrete regions of the nucleus (Bodznick and
Schmidt 1984). However, individual neurons in these regions can be excited by more
than one branch of the lateral line nerve (Paul and Roberts 1977b). This convergence
of input probably results because the extensive ventral dendrites of the principal
cells intermingle with incoming afferent fibres. Functionally the convergence would
serve to reduce "noise" of the kind generated by the animal's own breathing
movements (Montgomery 1984a), but inevitably some resolution must be lost,
although the directional sensitivity of secondary neurons is apparently retained
(Andrianov et al. 1974). The axons of the large principal neurons carry their output
to the contralateral dorsal nucleus and forwards in the lateral lemniscus to the
mesencephalon. Information is then relayed on to the thalamus and the telence-
phalon.
In the study by Platt et al. (1974), recordings were made from regions of the brain
of Torpedo in response to nerve stimulation and to the presentation of electric fields
around the fish. This study established the importance of the mesencephalon in
central analysis, for the largest fields were observed in that region. The complex
series of waves recorded from the mesencephalic tegmentum changes in form
depending on electric field orientation, current direction and the temporal aspects
of the stimulus (Bullock 1979). Evoked responses were also recorded in the telen-
cephalon.
Bodznick and Northcutt (1984) recorded from the telencephalon of Raja and
identified a pallial electro sensory area within the telencephalon. The latency to
electric field stimulation was 130 ms or longer and the response habituated
totally to stimuli given at 0.8 stimuli per second. An interesting finding was
that the electro receptive area overlapped very closely with the region in which promi-
nent visually evoked potentials were recorded.
Electroreceptive responses were recorded from the mesencephalon and dien-
cephalon of the ray, Platyrhinoidis, by Schweitzer (1983), in response to fields
established around the fish. Strong responses were recorded in the lateral mesence-
phalic nuclear complex, and in the anterior nucleus and posterior lateral thalamic
nucleus of the diencephalon. Multiunit evoked activity was also recorded in these
reglOns.
2.3.5 Central Processing for Tactile Information
Tactile meclianoreceptors on the head are supplied by the trigeminal nerve.

The central sensory trigeminal system consists of sensory, descending and mesen-
cephalic nuclei. There have been no experimental studies on the sensory and
descending nuclei, but the mesencephalic nucleus has been examined physiologically
(Roberts and Witkovsky 1975). The neurons of the mesencephalic nucleus form a
striking midline group in the tectum at the edge of the ventricle. They are large
and spherical (Fig. 2.9b) and associate closely with the tectal blood supply
(Witkovsky and Roberts 1975). Many of them make direct contact with the
ventricular cerebrospinal fluid (Kemali and Miralto 1979; MacDonnell 1980). The
axons of these cells supply high-threshold, non-spontaneously active, slowly adapting
mechanoreceptors of the teeth and perioral skin (Fig. 2.9a). There is no jaw muscle
receptor supply, however, as is found in mammals. In mammals the mesencephalic
trigeminal system is involved in a "jaw-jerk" reflex contraction of the jaw closing
muscles. Roberts and Witkovsky (1975) showed that stimulation of the pathway of
mesencephalic V axons in the brain also evoked a jaw-jerk, and that the basis for
this was a monosynaptic activation of the motoneurons of the trigeminal nucleus,
which innervate the adductor mandibulae. Stimulation of the peripheral branches of
V, not only antidromically activated mesencephalic V cells, but drove them synaptic-
ally through a pathway that was presumed to arise from the sensory trigeminal
nuclei (Fig. 2.9c, d). This observation provides an explanation for the unusual
a 500 ms b 50 lLm
~
120 mV 12 mV
~
10 ms 10 ms
.",.
c d
Fig. 2.9. Mesencephalic V (Mes V) neurons. a Mes V discharge to teeth stimulation ; b Golgi-
impregnated mes V neuron ; c intracellular response of mes V neurons to stimulation of mandibular
Vth nerve. Note third spike; d EPSP's recorded as in c. (Roberts and Witkovsky 1975 ;
Witkovsky and Roberts 1975)
location of the cell bodies which, being sensory cells, would be expected to lie
in extracranial gangiia. The presence of synaptic terminals on mesencephalic V cells
was established in an electron microscope study of these neurons (Witkovsky and
Roberts 1976), which also showed that many of the neurons are coupled through
gap junctions that are often associated with conventional chemical synapses.
Intracellular injection of Lucifer yellow shows that dye will pass between cells in
this nucleus (Roberts and Witkovsky, unpub1.). The sources of the synaptic input
to these neurons have not been identified, but probably include the tectum,
telencephalon and diencephalon, for axons from these regions penetrate the deeper
layers of the tectum. A very recent report for the r(tt shows that mesencephalic V
neurons receive synaptic inputs from the magnocellular nucleus of the hypothalamus
(Nagy et al. 1986), and this is a very interesting finding in view of the role of the
hypothalamus in feeding behavior (see below) and the impact of mesencephalic V
neurons on jaw closure.
2.3.6 Central Processing of Vision
The mesencephalic tectum has traditionally been considered as the site of visual
processing. Visual information is carried to the brain in the optic nerve by the axons
of the ganglion cells of the retina. Axons arising from the contralateral tectum project
back to the retina, also in the optic nerve (Luiten 1981), and provide an efferent inner-
vation (Witkovsky 1971). An efferent retinal supply is found throughout the verte-
brates but its function is unknown.
Afferent optic nerve fibres cross completely in the optic chiasma and pass to the
contralateral thalamus, pretectal area and tectum. The tectum is a laminated cortex,
comprising six cell zones, which receives afferent input from several sources and
provides efferent pathways to all other regions of the brain, except the telencephalon.
Studies in teleosts have shown the visual projection to be ordered, terminating
topographically within the tectum. Although no experiments have been done on
mapping within the elasmobranch tectum, it seems reasonable to assume that
its organization is similar to that of other vertebrates. Field potentials recorded in
Raja in response to optic nerve stimulation show that the primary visual input
is restricted to the dorsal layers of the tectum (Witkovsky et al. 1980). Some other
inputs to the deeper layers were shown in the study by Platt et al. (1974) on
Torpedo to be driven by stimulation of the spinal cord and by the electrosensory
nerves.
Visual information reaches the telencephalon, for visually evoked potentials have
been recorded there (Cohen et al. 1973; Bodznick and Northcutt 1984). Some
behavioural evidence also implicates the telencephalon in visual function, as nurse
sharks with complete tectal ablations can learn to perform simple visual discrimina-
tions, but not after telencephalic ablations (Graeber 1980).
No studies have yet been carried out on the physiology of individual tectal neurons,
or of other neurons in the visual pathway.
2.3.7 Central Processing of Olfactory Information
Elasmobranchs show pronounced behavioural responses to olfactory stimuli which

are processed by the telencephalon. Hodgson and Mathewson (1978) recorded
from the brain of paralyzed nursesharks and obtained large responses in the rostral
regions of the telencephalon when olfactory stimuli were perfused through the
olfactory sac. They also recorded from the brains of free-swimming nurse sharks and
attempted to correlate brain responses to behavioural events. They observed modifi-
cations of respiratory as well as locomotory movements and showed that protein
breakdown products were particularly effective stimuli, in evoking both neural
responses and behavioural changes.
2.4 Concluding Remarks 73
Experimental anatomical studies in the nurseshark (Ebbesson and Heimer 1970)

demonstrated that only a limited part of the telencephalon receives an olfactory
input, and subsequent anatomical studies on other species have confirmed this view.
Electrophysiological studies have also shown that olfactory activity is restricted
within the telencephalon (Bruckmoser and Dieringer 1973). These findings raise the
important question of the function of the telencephalon, particularly in view of its
large size in elasmobranch fish. As we have already seen, evidence is increasing
that it receives inputs from sensory modalities other than olfaction and it is clearly
more than just an olfactory receiving station.
2.3.8 Sensory-Motor Integration
The significance of the telencephalon has received relatively little attention. This
is partly because of the belief that it was merely an olfactory processing centre, and
in part because it was taken to be much less complex than the mammalian
cerebral cortex. Changing ideas within evolutionary neurobiology, plus more under-
standing of telencephalic function in teleost fishes, have shown that the telencephalon
plays an integrative role, associating inputs with output programs. Indeed, similarities
to the mammalian limbic system are beginning to emerge.
The telencephalon outputs to, and receives its inputs from the diencephalon.
In mammals the hypothalamus, in association with the limbic centres of the telen-
cephalon, maintains homeostatic control of bodily functions and regulates behaviour
such as feeding, escape, attack, aggression and sex. Although we know little of
diencephalic function in elasmobranchs, what evidence we do have suggests that it
plays a similar role.
An example of the role of the hypothalamus in homeostatic control is provided
by Wilson et al. (1974), who showed the importance of this region in controlling
colour change in Scyliorhinus. Small lesions made in the mesencephalon and dien-
cephalon located areas that are involved in the control of release of melanophore-
stimulating hormone from the neurointermediate lobe of the pituitary gland. The
controlling circuit involves both a monoaminonergic innervation of the pituitary
and visual input. Wilson and Dodd (1973) have also demonstrated that colour
changes in this fish may involve the epiphysis (pineal organ). The epiphysis has
been shown electrophysiologically to be a very sensitive photoreceptor (Hamasaki
and Streck 1971).
2.4 Concluding Remarks
In the past, biologists have been attracted to work with elasmobranchs for two main
reasons. The first is the practical one that fish with large brains and cartilaginous
skulls offer certain experimental advantages. This feature of elasmobranch biology
remains attractive, whether the approach is physiological or phylogenetic. The
second reason is more abstract and is based on the belief that elasmobranchs are
"primitive" vertebrates that can reveal basic features of the design of the vertebrate
nervous system. For example, the cerebellum is a brain structure that is strikingly
similar both in elasmobranchs and mammals, and this similarity must say something
about the operation of the cerebellar circuit. Nevertheless, elasmobranchs are not just
simplified mammals and the way the cerebellum operates in elasmobranchs and mam-
mals may not be similar, particularly as their movements are so different. This appro-
ach is limited by the difficulty of knowing how far similarities and differences in struc-
ture are reflected in function. Hopefully, as information about the elasmobranch
nervous system accumulates, the recognition of features of similarity and difference
will become easier. There is no doubt that the ability to make these distinctions will be
very important in determining how far we can go in using the elasmobranch fishes
as "model" systems.
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Williamson RM, Roberts BL (1980) The timing of motoneuronal activity in the swimming spinal
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Chapter 3 J. C. MONTGOMERY
Sensory Physiology
The cranium of an elasmobranch fish encloses the brain, and encapsulates the
olfactory, optic and otic sense organs (Fig. 3.1 a). In addition to these three major
paired sensory systems of the head, this chapter will deal with the lateral line and
electrosensory systems which are distributed over the surface of the body (Fig. 3.1 b).
Vestibular sense organs of the otic capsule, lateral line and electro sensory systems all
share anatomical and developmental features, and are often linked together under
the heading "octavolateralis sensory systems". This chapter will summarize our
knowledge of elasmobranch sensory organs, but refer particularly to papers published
since the extensive review of the sensory biology of sharks, skates and rays edited
by Hodgson and Mathewson (1978). .
Eye - - - -f+- - J
Fig. 3.1. Elasmobranch sense organs. a Diagrammatic dorsal view of a dissection of the head of
Sey liorhinus amicula showing the olfactory, optic and otic (vestibular) senspry systems and the brain.
F forebrain ; M midbrain; C cerebellum; H hindbrain; In extraocular musCles; he horizontal semicir-
cular canal; pvc posterior vertical semicircular canal; ave anterior vertical semicircular canal;
b diagrammatic dorsal view of the head of Scyliorhinus eanicula showing the electrosensory pores
(isolated black dots) and the pores and canals of the lateral line system (open circles). (After
Dijkgraaf and Kalmijn 1963). Illustrated are the lateral canal which extends down the lateral
flank of the animal ( 1), the supratemporal canal (2) and the supraorbital canal (3)
Author's address: Department of Zoology, University of Auckland, Auckland, New Zealand

80 Chapter 3. Sensory Physiology
Our appreciation of the structure and function of sense organs depends on a

knowledge of the naturally occurring signals in the environment, the biologically
relevant information they contain, and behavioural evidence as to the uses to
which this information is put. It i~ only on this basis that the physiologist is
able to ask the most appropriate questions of the system under study. Each
sensory modality will be dealt with in turn, giving the relevant details of the
sensory milieu of elasmobranch fish and the behavioural evidence of their sensory
capabilities, detailing the anatomical structure of the sense organs involved, and the
physiology of sensory transduction and encoding. It is important to remember,
however, that under normal conditions of operation the sensory input from one
sensory modality will not be treated in isolation, but that the animal's response will
be determined by the composite input over the whole spectrum of its sensory
systems.
Physiological studies have concentrated on the relatively few species that can be
captured and kept under laboratory conditions conveniently. Elasmobranchs live
in a wide variety of habitats and each species exhibits different degrees of development
and specialization of its sense organs related to its habitat and life style. At
present we have only begun to sample the diversity found within the sensory
systems of this fascinating group of animals.
3.1 Olfactory System
3.1.1 Behavioural Studies
The olfactory sense is well developed in elasmobranchs and has been shown to play
an important role in the detection and localization of food, and it is also implicated
in reproductive behaviour. The olfactory system can be distinguished both anatomical-
ly and functionally from the gustatory, or taste senses of the oral cavity. For
instance, fish bait impregnated with quinine is quickly located by sharks and taken
into the mouth but then spat out, indicating that the quinine stimulated the
chemical sense of the mouth, but not that of the olfactory surface. Gustation in
elasmobranch fish has been studied very little, and will not be considered further
here.
Field studies of the attraction of sharks to stationary olfactory stimuli have
shown that the overwhelming majority of approaches are made from a downstream
direction. This is to be expected in that the olfactory stimulus is carried downstream,
forming a so-called "olfactory corridor". Experiments with nurse sharks in enclosures
under controlled current conditions (Hodgson and Mathewson 1978) reveal that some
sharks show a true gradient searching, or klinotaxis. The shark approaches the origin
of the olfactory stimulus along an "s" -shaped track presumably initiating turns
in the direction of the nostril that receives the strongest olfactory stimulation. Evidence
for this view comes from the early observation that experimental animals, which
had one nostril obstructed with a cotton plug, turned predominantly toward
the side of the functional nostril when activated by an olfactory stimulus (see
Kleerekoper 1978). Other species of shark, notably lemon sharks, do not follow the
3.1 Olfactory System 81
olfactory gradient directly, but on encountering an olfactory stimulus in the water

turn and swim upstream. Normally this would bring them close to the source of the
olfactory stimulus, but in experimental situations where the water current and the
olfactory trail can be separated, the behaviour is revealed to be a chemically released
rheotaxis. In contrast to other species, lemon sharks were never observed to localize
the source of the olfactory stimulus in open sea tests, but continued swimming against
the current past the stimulus source. Having been attracted into the general area by
the chemically released rheotaxis, lemon sharks apparently rely on other cues to
identify potential food items (Hodgson and Mathewson 1978).
Reproductive behaviour has not been extensively studied in e1asmobranchs (see
Dodd 1983), but recent studies of the potential role of a pheromone receptor system
in vertebrates (Demski and Northcutt 1983), and the presence of this system in elasmo-
branchs (Bullock and Northcutt 1984) indicate a possible role for chemosensory com-
munication in reproductive behaviour.
One of the motivating forces for the study of olfaction in sharks has been the
interest in developing shark repellants capable of deterring shark attacks. Tests of
chemical repellants have met with only limited success, and this approach is presently
considered impractical. There is, however, evidence that other marine animals have
chemical defenses against shark attack, and investigations of these should prove
interesting both from the point of view of potential shark repellants, and as an
approach to understanding the elasmobranch olfactory system (Hodgson and
Mathewson 1978).
3.1.2 Anatomy
The anatomy of the elasmobranch olfactory system has been described by Kleere-
koper (1978). The olfactory sacs are located in the nasal capsules of the skull, and
consist of a series of septa, or leaves, over which cilia maintain a constant current
of water between incurrent and excurrent apertures (Doving et al. 1977). The
olfactory epithelium is located mainly at the base of the troughs formed by the
septa, and consists of receptor cells and their axons, supporting cells and basal
cells (Fig. 3.2). Receptor and supporting cells typically have both cilia and micro-
villi.
Elasmobranch receptor cells have relatively large somata (15-20 J..lm) and typically
a peripheral dendritic process with cilia projecting into the lumen of the olfactory
sac. In some species microvilli are present in addition to cilia (Bakhtin 1977),
whereas in Rhinobauisonly microvilli are found. It is thought that the cilia and
microvilli carry the receptor sites for chemical stimulation. The axons of the bipolar
receptor cells aggregate into bundles of various sizes which penetrate the base of the
septae and pass directly into the olfactory bulb which is opposed to the outer surface
of the olfactory sac. On entering the olfactory bulb, the olfactory nerve fibres synapse
with the mitral cells in the olfactory glomeruli (Fig. 3.2c). In elasmobranchs
each mitral cell is connected to several glomeruli, and their axons exit from the
bulb as the olfactory tract which in turn connects to the brain. Within the bulb
itself, granule cells are connected to the mitral cells through a variety of synaptic
types, including reciprocal synapses (axodendritic and dendroaxonic) between the
Fig. 3.2. Anatomy of the olfactory system. a Ventral aspect of the head of Scyliorhinus. On the
right side of the drawing the nasal flap has been retracted to show the entrance (anterio-lateral naris)
and exit (posterio-medial naris) of the olfactory chamber. (After Kleerekoper 1978); b schematic
diagram illustrating the principle of water flow in the olfactory organ of dogfish. The olfactory
chamber is divided into a series of corridors by leaves of tissue on which is located the receptor
epithelium. Water currents (arrows) are directed through the corridors by the beating of cilia on the
supporting cells. E entrance; S exit. c diagram of neural circuitry of the olfactory epithelium and
bulb. The bipolar sensory cells (b) have a dendrite which extends into the lumen of the olfactory
chamber and an axon which links with other axons to synapse with the dendrites of the mitral
cells (m ) of the olfactory bulb. Also shown are local inhibitory cells, or granule cells (g) , and
efferent fibers' from the brain. The supporting cells of the receptor epithelium (S) are intersperced
between the receptor cells. After Kleerekoper 1978)
axons of the granule cells and the mitral cell dendrites, and a major input onto the
granule cells from mitral cell axon colaterals.
3.1.3 Electrophysiology
Electrophysiological studies of the olfactory system in elasmobranchs have focussed
on the questions of sensitivity and selectivity. Because of the small size of the neural
3.2 Visual System 83
elements, most workers have used a compound electrical response, or electro-

olfactogram (Hodgson and Mathewson 1978; Silver 1979), but recently single unit
responses have been obtained from olfactory tract fibres (Broun and Fesenko 1982).
Single unit responses show the sensitivity and degree of selectivity of the olfactory
system particularly well. Some fibres have a very low threshold response to
specific amino acids (e.g., 10- 14 to 10- 13 moll- 1 for serine). Responses are elicited
by other amino acids, but only at considerably higher concentrations (10- 10 mol 1- 1
for asparagine and 10- 8 moll- 1 for lysine and valine). This degree of selectivity
tends to indicate that the receptors for different amino are probably located
on different receptor cells, and that the specificity of information provided by the
receptors is not lost in the processing of information by the olfactory lobe. Although
some olfactory tract fibres could be found which responded selectively to specific
chemical stimuli, responses were most frequently elicited by natural stimuli such as
the cutaneous mucus and blood of marine animals. It is to be expected that these
stimuli, encountered in the animal's natural habitat, play an important role in
behavioural responses. Which chemical components of these stimuli produce the
responses, details of the molecular nature of the receptors and their transduction
processes, and the details of processing of information by the olfactory lobe in elasmo-
branchs are, as yet, unanswered questions.
3.2 Visual System

3.2.1 Behavioural Studies
Elasmobranchs inhabit a wide variety of environments, from the clear, well-lit,

epipelagic zone, to turbid inshore waters. Their visual systems show a corresponding
variety of form and importance in behaviour. Field studies of visually mediated
behaviour are difficult to interpret, and our best indications of the visual capability
of elasmobranchs come from psychophysical experiments conducted in the laboratory.
Experiments utilizing operant conditioning techniques have shown that the visual
system of lemon sharks is at least as sensitive as humans, and can detect light
from all parts of our visible spectrum. Discrimination between targets that differ in
brightness is roughly comparable to ours, as is the range of light intensities over
which vision is possible (Gruber and Cohen 1978). Form discrimination has not been
extensively studied, but sharks learn simple discrimination tasks, such as distinguishing
upright versus inverted triangles, without difficulty (Graeber 1978).
3.2.2 Anatomy of the Visual System
3.2.2.1 Extraocular Muscles/Eye Movements

Elasmobranch fish possess three pairs of extraocular muscles which control the
movement of the eye within its orbit. Each muscle c(,mtains an inner core of large
white muscle fibres, which presumably subserve quick saccadic movements, and an
outer layer of smaller red fibres used for slower movements (Housley and Mont-
gomery 1984 ; Graf and Brunken 1984). Rapid eye movements occur in anticipation
of turns in freely swimming fish (Harris 1965), in the nystagmus produced by
prolonged vestibular stimulation (Paulin and Montgomery, unpubl. observation)
and as a protective reflex in some species. Slow compensatory movements which serve
to stabilize images on the retina occur during swimming or passive head move-
ments.
3.2.2.2 Eyelids
Elasmobranchs have well-formed eyelids that are mobile in some species, for example
Ginglymostoma and Cephaloscyllium. In some other species, particularly the Car-
charhinidae, the lower lid is secondarily folded into a third eyelid, the original lower
lid forming a structure similar to the nictitating membranes of some higher verte-
brates. In sharks, the nictitating membrane is dense and opaque and serves a protective
function, closing in response to stimulation of the skin around the eye, and during
feeding activity.
3.2.2.3 Optics
The organization of the elasmobranch eye is fairly typical of vertebrate eyes, but
with some distinctive features (Fig. 3.3). The sclera is supported by a thick cartilaginous
layer, and the nutritive choroid contains a tapetum lucidum, which in pelagic species
can be occluded in bright light by the migration of dark pigment granules over
the reflecting plates. The retina is not vascularized and contains no obvious landmarks
other than the optic disc, or blind spot, marking the point of exit of the optic nerve
from the retina. The crystalline lens is supported by a dorsal suspensory ligament and
the ventral pseudocampanule, which may function as a protractor 1entis muscle to
Dorsal
Suspensory Sclera
Ligament
Iris Scleral
Aqueous Cartilage
Humor
Lateral Medial
Cornea Choroid
Retina
Optic
Nerve
Ventral
Fig. 3.3. Anatomy of the visual system. Diagrammatic transverse section through the eye of a lemon
shark. (After Hueter and Gruber 1982)
produce accommodatory movements of the lens in some species (Sivak 1978).

The iris has sphincter and dilator muscles which respond directly to light but are also
under neural control, allowing a tenfold change in the area of the pupil. The
upper margin of the iris in many rays is modified into a so-called operculum
pupillare which descends Over the pupil during light adaptation to reduce the pupil to
a series of "pinhole" apertures.
The ellipsoidal crystalline lens is the most important optical structure due to the
relative ineffectiveness of the cornea in contributing refractive power underwater.
Working with juvenile lemon sharks Negaprion brevirostis Hueter and Gruber (1982)
have shown that the lens has a high overall equivalent refractive index (1.66),
resulting in a principle power of about 140 diopters (compared to only about 20 diop-
ters in the human lens). With no evidence of accommodation in this species, the
power of the lens is insufficient to bring objects into focus on the photoreceptor
layer, but these workers argue that this small degree of hypermetropia (+2.76
diopters) would probably produce little functional deficit in the murky visual environ-
ment of lemon sharks.
3.2.3 Retinal Anatomy and Electrophysiology
The retina is an embryological extension of the brain. Its receptors transduce visual
stimuli into electrochemical signals and the other cellular elements process this visual
information before transmitting it to the brain. Understanding of this process first
Receptor
Rods and -35mV ~ <D
Cones
BIPOI"~
I
Horizontal
Cells
Bipolar
Cells
~- --- -- -- --
IPL
-45mV 5 mV ®
----='9i;;;;;;:z:~~=I==== GCL Ganglion Cell
Ganglion
Cells ~~~==~= ~-- --- -- --- H--i-H!::iiii!I~-++- ®
a
t· LIGHT
t t t b
o
-LIGHT
2 3 s
Fig. 3.4. Retinal anatomy and electrophysiology. a Organization of the elasmobranch retina
(amacrine cells not shown). (After Gruber and Cohen 1978). EPL external plexiform layer; INL inner
nuclear layer; IPL internal plexiform layer ; GeL ganglion cell layer ; b electro physiological responses
of retinal cell types to a I s pulse of light shone on the centre of their receptive fields. Intracellular
recording would reveal a hyperpolarizing reponse to light in receptor cells, .and a larger magnitude
depolarizing response in bipolar cells. These responses increase in amplitude with increasing light
intensity. Ganglion cells are spiking neurons so their responses can be recorded extracellularly .
In this example light shone on the centre of the receptive field increases the spike discharge rate,
light intensity is encoded not by an increase in spike size, but by an increase in spike fre-
quency.
requires a description of the cellular organization of the retina which is facilitated by

its highly ordered structure of three cellular and two synaptic layers (Fig. 3.4a).
The receptor cells occupy the outer cellular layer, which means that light must
travel through the entire retina before impinging on them. The majority of elasmo-
branchs so far studied have been shown to have both rods and cones distinguishable
on histological and ultrastructural criteria. However, the relative number of rods and
cones varies widely from a ratio of about 4: 1 in some pelagic species to the
pure rod retina of some skates. Regional specialization of receptor density within
the retina has been noted in a number of species.
The external plexiform layer is the site of contact between the receptor cells and
the horizontal and bipolar cells of the internal nuclear layer. In Mustelus three
types of horizontal cells are recognized: the large cells of the distal layer probably
contact all of the rods within their field, the flattened horizontal cells of the inter-
mediate layer form clusters of terminals which contact rods within the field of that
cluster, and the flattened stellate-shaped cells of the proximal layer which send relative-
ly thick processes into the external plexiform layer to make exclusive contact with
cones. The horizontal cells of each layer are electrotonically coupled by way of
specialized contacts called gap junctions, but the coupling does not extend to the
horizontal cells of other layers, or the bipolar cells. A similar pattern of horizontal
cell organization has also been found in the sting ray Dasyatis, but in this species
the distal horizontal cells make synaptic contacts with both rods and cones
(Toyoda et al. 1978), a finding which may be related to the much higher cone
density in Dasyatis when compared to that of Mustelus.
Bipolar cells are neurons of the internal nuclear layer which send processes out
to the receptor cells and inwards to the ganglion cell layer. There are two basic types of
bipolar cells, one type associated with each of the photoreceptor types, but the two
basic types can be further subdivided on the basis of dendritic morphology and the
presence of clublike processes extending into the receptor layer, to form five
recognizable bipolar cell types. The inner plexiform layer is divided into three sub-
layers, which appear to segregate specific bipolar types with specific ganglion cells
types. Amacrine cells are cells of the inner nuclear layer with long bifurcate
processes spanning up to 5 rom. These cells apparently receive input from bipolar
cells and spread information laterally across the retina.
Ganglion cells form the most proximal retinal layer, their axons make up the
optic tract and communicate the retinal output to the brain. As with other retinal
neurons, ganglion cells may be divided into several subgroups characterized by size,
location, and dendritic arborization. In addition, there are many small ellipsoidal
cells in the ·ganglion cell layer which are thought to be glial cells associated
with the myelination of the optic tract fibres. Some of the ganglion cells are
displaced into the inner plexiform layer, and in Heterodontus these cells have been
shown to be concentrated in the far temporal retina presumably subserving frontal
vision, whereas the outer ganglion cells are concentrated in a horizontally oriented
visual streak which would provide for a panoramic view of the lateral visual field
(Peterson and Rowe 1980).
3.2.4 Visual Pigment
Outer segments of the receptor cells contain a visual pigment which absorbs
light as the first step of the sensory transduction process. The absorbance maximum
of ordinary elasmobranch visual pigment is around 500 nm, which is typical of
rhodopsin. However, deep water elasmobranchs have been shown to have a pigment
chrysopsin with an absorbance maximum of about 480 nm which matches the blue
quality of the ambient light at depth. Elasmobranch retinas have provided good
experimental preparations for studies of the biophysics and molecular biochemistry
of rhodopsin (e.g., Pepper berg et al. 1978; Clack and Pepper berg 1982; Catt et al.
1983), but the results of this work pertain more to fundamental visual science than to
the sensory biology of elasmobranchs.
3.2.5 Retinal Electrophysiology
The gross electrical activity of the retina, or electroretinogram (ERG), can be recorded
relatively easily, and is useful in biophysical studies and some aspects of sensory
biology, such as studies of spectral sensitivity and time course of dark adaptation.
The physiology of sensory transduction and encoding is better understood from intra-
cellular microelectrode studies of each cell type (Fig. 3.4b). The receptor cells are
very difficult to penetrate with microelectrodes, but based on the results obtained in
other animals it is reasonable to assume that the absorption of light by rhodopsin
produces an isomerization of the visual pigment which causes a hyperpolarization
(increases the internal negativity) of the receptor cell. The sensitivity of the rod
receptors is such that bleaching of a single rhodopsin molecule by a photon of light is
sufficient to produce a 2 m V hyperpolarization of the receptor. The internal potential
of the receptor cells modulates the release of a neurotransmitter substance at the
receptor terminals such that the high rate of release which occurs in the dark is
reduced by hyperpolarization; however, the precise chemical nature of the transmitter
is, as yet, unknown (Shiells et al. 1981).
Bipolar, horizontal and ganglion cells are more amenable to microelectrode
work, and recently their electrophysiological response characteristics have been
extensively studied (Ashmore and Falk 1980a, 1980b, 1981, 1982). The main result
of these, and earlier studies (see Gruber and Cohen 1978), is that horizontal cells
respond to light with a graded hyperpolarizing potential and a sensitivity about four
times that of rod receptors, whereas bipolar cells predominantly show a graded
depolarizing response to light with a remarkable sensitivity about 135 times that
of the rod receptors. Horizontal cells have a receptive field of up to 10 mm due
to the lateral extent of their dendrites and electrotonic junctions with neighbouring
horizontal cells. Bipolar cells have a relatively small receptive field of about 0.15 mm.
Ganglion cells represent the output of the retina, and respond to increases or
decreases in light with all-or-none action potentials. The receptive field of ganglion cells
is organized into a central region and a concentric surround. Some ganglion cells
show an increase in their firing rate when light is shone on the central region of
their receptive field, whereas light shone on the surrounding area inhibits the back-
ground firing rate. Other ganglion cells show the reverse pattern of organization.
Receptive fields are quite large, with a centre diameter of about 1.5 rom and the
surround extending a further 3 mm from the border of the centre. The precise
way in which the antagonistic "centre-surround" response of ganglion cells is produced
by the other cells of the retina is as yet unknown, but the basic principle of
organization of the retina is apparent, with the bipolar cells providing the direct
pathway of activation of the ganglion cells from the receptors, and the horizontal
and amacrine cells mediating lateral interactions.
The major features of visual information processing by the retina in elasmobranchs
are thus similar to those in other vertebrates. The array of retinal receptors encode the
retinal image as point-by-point variations in light intensity, in a manner analogous
to a photographic plate. However, the retina then processes this information to provide
an output at the ganglion cell level which is relatively insensitive to gradual changes
in overall illumination, but designed to measure differences within the receptive
field of each ganglion cell by comparing the illumination of the centre and the
surround. This makes the output of ganglion cells particularly sensitive to contrasts
such as the edge of an image, or the movement of images across the retina.
3.3 Octavolateralis System
3.3.1 Mechanoreceptors
3.3.1.1 Behavioural Studies

The sense organs of the vestibular labyrinth provide information on the orientation of
the head in space and its angular velocity. In addition to providing input for stabilizing
reflexes of the eyes (Sect. 3.2.2.1), this information is used in controlling locomotion,
particularly by initiating righting reflexes in response to passive disturbance. The
way in which signals from the vestibular apparatus are converted into righting reflexes
will vary with circumstance, particularly with the velocity of forward movement.
However, vestibular fin reflexes have been studied only in stationary animals
(Roberts 1978), but even in this case, body pitch and roll produce pectoral fin
movements which would tend to restore an upright position if the animal were
moving.
In the section on olfaction it has already been noted that some species of shark
show a chemically released rheotaxis, and that orientation to a current is important
in searching for food. In addition to responding to water currents, elasmobranchs show
a variety of responses to vibratory water motion or acoustic stimulation. The most
dramatic of these is the attraction of sharks to low frequency (40-300 Hz) pulsed
sound, simulating the sounds produced by struggling and wounded fish. Well-oriented
responses were observed in sharks at distances of up to 250 m from the sound source.
Sudden, or loud, acoustic stimulation, on the other hand, produce withdrawal, or
escape responses (see reviews by Myrberg 1978 and Corwin 1981).
Laboratory studies of hearing also confirm that elasmobranchs are sensitive
to low frequency, and that they are able to discriminate between sounds on the
basis of frequency alone. Most studies have assumed that elasmobranchs are
3.3 Octavolateralis System 89
sensitive only to particle motion in a sound field because they lack a gas bladder that
could act as a pressure-to-displacement transformer. Recent psychophysical studies,
however, show that sharks are also sensitive to acoustic pressure (van den Berg and
Schuijf 1983).
3.3.1.2 Hair Cell Mechanoreceptors
The sensory cells of the vestibular system and lateral line are termed hair cells. At the
top of each hair cell is a bundle of cilia, one of which has the familiar 9 + 2 '
configuration of internal tubules and is called a kinocilium. The rest of the cilia
-------... ~
k Ampulla
Nerve
Fig. 3.5. Octavoloateralis mechanoreceptors. a Hair cells. Diagrammatic cross-section through a

mechanoreceptive sensory epithelium. Two hair cells are shown with associated supporting cells.
The apex of the hair cell has a ciliary bundle with I kinocilium ( k ) and a number of steriocilia (Sf ) .
Afferent and efferent synapses are found on the base of the hair cells. Mechanical stimulation
in the direction shown by the arrow will depolarize the left hand cell and increase the firing rate
in its afferent fibre ; b ampulla of semicircular canal. Relative movement between the canal
and the endolymph displaces the cupula and activates the hair cells of the sensory epithelium (S)
located on the crista ; c otolith organ. Linear acceleration and vibration set up shearing forces
between the otoconia laden cupula and the hair cells of the sensory epithelium (S); d neuromast
of the lateral line. Differential movement between the sensory epithelium ( S) and the canal fluid will
stimulate the hair cells
lack any internal structure, and are termed stereocilia. They are graded in height,
with the tallest of them being adjacent to the kino cilium (Fig. 3.5 a). The base of the
hair cell forms a synaptic contact with an afferent sensory fibre, and also
receives a synaptic contact from an efferent nerve fibre. The hair cell, the associated
supporting cells, and the peripheral secretory cells form a sense organ called a neuro-
mast. Neuromasts are capped by a gelatinous structure, or cupula, which couples the
ciliary bundles to fluid movements, or to the motions of dense particles embedded in
the cupula. Movements of the cilia alter the electrical potential within the hair cell
(by an unknown mechanism), which in turn modulates the release of neurotransmit-
ter at the afferent synapse. In this way displacement of the cilia in the direction of the
kinocilium causes an increase in afferent firing rate, while displacement in the opposite
direction decreases the firing rate of the sensory afferent below its spontaneous level
(Roberts 1978). The hair cell is similar in structure and function throughout the
mechanosensory systems. It is the ancillary structure which determines whether the
effective stimulus is head rotation, gravity, or water movement.
3.3.1.3 Anatomy of the Vestibular System

The location of the vestibular system is shown in Fig. 3.1 a. A lateral view of the
left labyrinth (Fig. 3.6) shows the basic structure of three semi-circular canals
mutually at right angles and the centrally located otolith organs. Each semi-circular
Fig. 3.6. Vestibular labyrinth. Lateral view of left labyrinth. AV anterior vertical semicircular
canal; PV posterior vertical canal ; He horizontal canal; U utriculus; S sacculus; L lagena; A
ampulla of a semicircular canal. The dense white areas are otoliths
3.3 Octavolat~ralis System 91
canal is a fluid-filled duct opening off the central vestibular sac, with an ampullary
swelling at one end which houses a saddle-shaped ridge (crista) on which are located
the sensory hair cells (Fig. 3.5b). Cilia of the hair cells project up into a gelatinous
cupula which spans across the centre of the ampUlla. Within each crista, the ciliary
bundles are all oriented with their kinocilium on the same side. The canals in
opposite labyrinths form antagonistic pairs, for example input from the anterior
vertical canal of one side is equal and opposite to the input from the posterior
vertical canal of the controlateral side.
There are three otolith organs, the utriculus, sacculus and lagena. Each has a
sensory epithelium called a maculus which contains the sensory hair cells, with up to
3 X 105 hair cells reported in the saccular macula of the juvenile lemon shark. The
hair cells in the sacculus are oriented in a regular fashion with the kinocilia ends
of the hair cell bundles facing outwards from the midline of the macula. The utricular
hair cells show a wide range of directional orientation, while in the lagena, where
the maculus is vertical, the hair cells have their kinocilium either on the ventral, or
dorsal side of the ciliary bundle (Barber and Emerson 1980). The maculae are
covered by cupulae in which are embedded many small (10 Ilm) calcium carbonate
granules or otoconia (Fig. 3.5c). In some elasmobranchs the otolith material is of
exogenous origin (Hinge 1982) and the presence of magnetite in the saccular
otolith of some elasmobranchs raises the possibility of the sacculus providing informa-
tion suited to geomagnetic orientation (Vilches-Troya et a1. 1984).
In addition to the three otolith organs, there is another sense organ, the macula
neglecta, which is located near the junction of the posterior vertical canal and the
sacculus. It has a cupula but no otoconia. The macula neglecta is situated close
to the fenestra ovalis, or dorsal opening in the otic capsule cartilage. In sharks
this organ is particularly well developed with up to 2.6 X 105 hair cells all aligned
perpendicular to the plane of the fenestra ovalis. A high degree of hair cell to afferent
fibre convergence is indicated by the observation that there are fewer than 5000 axons
innervating the macula neglecta (Corwin 1981).
3.3.1.4 Anatomy of the Lateral Line Canals
Scattered over the surface of the head and the body of elasmobranchs are a
series of free-standing neuromasts or pit organs (Roberts 1978). The lateral line proper
is a series of subepidermal canals which distribute over the surface of the head, and
along the sides of the body (Fig. 3.1 b). Typically, there is a lateral canal running from
the head to the tail, Ii supraorbital canal passing over the eye and an infraorbital canal
from which branches the hyomandibular canal, a supratemporal canal which crosses
the head to unite the two lateral canals and a discrete mandibular canal on the side of
the lower jaw. There are variations in this pattern, particularly within the skat.es and
rays. In most cases the canals open to the exterior via pores, and neuromast organs
are found within the canals opposite these openings (Fig. 3.5 d).
The spiracular organ, which is a small pouch opening off the spiracle, has recently
been shown to be a mechanoreceptor closely related to the lateral line system of
sense organs (Barry and Boord 1984).
3.3.1.5 Electrophysiology
Afferent fibres from the semicircular canals generally have a fairly rapid and regular
rate of spontaneous discharge. The firing rate of all the afferents from one canal is
elevated by head rotations in one direction and decreased by rotations in the opposite
direction. This is consistent with the morphological finding that within one crista, all
the kinocilia are on the same side of the ciliary bundle. Electrophysiological
studies of canal inputs have concentrated on determining the dynamic properties of
the firing rate response to head rotation. The canals operate as inertial angular
accelerometers, with the fluid inertia of the endolymph tending to deflect the cupula
during head movement, but being opposed by the elasticity of the cupula, and the
frictional contact between the endolymph and the canal wall. Actual cupula movement
is small during normal operation, and has been estimated as less than 3 J.lID in
the skate (Oman et al. 1979). For head rotations· within the normal frequency range,
the interplay of these factors results in a maximum firing rate of afferent fibres which
is in phase with head angular velocity (Lowenstein and Compton 1978; Montgomery
1980). There is considerable variation in the response characteristics of fibres
which innervate different regions of the crista, which may indicate that different
trajectories of head rotation will be best encoded by particular populations of afferent
fibres.
The density of the otoconia enables the otolith organs to respond as differential
density linear accelerometers. Gravity is a form of linear acceleration, and single unit
recordings 'show that the posterior portion of the sacculus, the main body of the
utriculus, and the lagena are gravity receptors. The hair cell orientation in the
utricular maculus is pluridirectional and utricular afferent units can be found which
respond strongly to deviations in all directions from the normal axis of the resting
animal. The lagena afferents all respond maximally when the animal is in a normal
position (Roberts 1978). .
Afferent fibres from the utriculus, anterior sacculus, and macula neglecta are
sensitive to vibration and acoustic stimulation (Corwin 1981). The macula neglecta,
in particular, is thought to play an important role in sound localization. Two
suggestions have been made to account for the directional specificity: firstly that it is
determined by the directional properties of the parietal sound transmission path
(Corwin 1981), secondly that the macula neglechi operates as an acoustic pressure
transducer and provides a coherent phase reference against which local particle
acceleration can be compared to provide unambiguous localization (van den Berg
and Schuijf 1983).
Electrophysiological experiments show that the lateral line is very sensitive to
water displacements. Free-standing neuromasts have a lower sensitivity than canal
organs and may provide information on water currents. From its structural layout
the lateral line should be ideally suited to localize disturbances created by nearby
moving objects, but there seems to have been little work on the frequency response
characteristics of elasmobranch lateral line units, or their receptive field properties.
Most of the electrophysiology has been concerned with the modulation of the lateral
line inputs during the animal's own movements, and possible efferent control of
lateral line sensitivity. During violent movements the efferent system is activated, and
the sensitivity of lateral line afferents depressed (Roberts 1978).
3.3.2 Electroreception
3.3.2.1 Behavioural Studies

Behavioural studies of electroreception have been reviewed by Kalmijn (1978, 1982).
Initial indication of the exquisite sensitivity of elasmobranch electroreceptors came
from the study of unconditioned cardiac responses to presentation of low frequency
uniform square wave fields. Responses were evident down to voltage gradients of
0.01 J.lV cm -1 and were dependent on the receptors known as the ampullae of
Lorenzini. The significance of this electric sense in predation has been shown in
laboratory and field experiments. Aquatic animals produce dc and low frequency
bioelectric fields in the surrounding water, which enable sharks to locate and attack
prey even in the absence of other sensory cues. Simulation of the prey by a small
dipole field, at frequencies between 0 and 8 Hz, will also provoke well-aimed feeding
responses. Field experiments have shown that sharks attracted into an area by an
olfactory stimulus almost invariably attack a set of active electrodes located close
by, rather than the odour source, or a set of control electrodes (Fig. 3.7 a). In these
experiments, the threshold voltage gradient which provoked a feeding attack was
estimated to be 5 nV cm -1. Having access to special sensory information is of
considerable advantage, as illustrated by the ambush strategy of swell sharks (Tricas
1982). These lethargic sharks lie motionless on the bottom during the night, and are
able to snap up small fish which, unaware of their presence, drift close to them. The
sharks use their electro sensory system to determine the location of the prey.
The electrosensory system may also provide information for orientation and navi-
gation. Animals may be able to estimate their passive drift with a flow of water from
the electrical fields that ocean currents produce by interaction with the earth's
magnetic field. Secondly, when actively swimming, an animal may be able to derive
its magnetic compass heading from the electric field it generates by interaction with
the earth's magnetic field (Kalmijn 1984). Conditioning experiments have shown the
co
a b
Fig. 3.7. Behavioural responses mediated by electrosensory system. a Feeding attacks of the dogfish
on an electrically simulated prey. os odour source; dl electrodes pass a current of81lA d2: control
electrodes. b Orientation of the sting-ray to a uniform electric field of 5 nV em -1. The sting ray is
trained to enter the corral (co) on the left relative to the direction of the field. (After Kalmi:jn 1982)
sting-rays are able to distinguish imposed uniform electric fields from the usually
much stronger fields that they induce by moving through the earth's magnetic field
(Fig. 3.7b). Yet field experiments are still required to show that e1asmobranchs
actually use this electro sensory information for orientation and navigation.
3.3.2.2 Anatomy of Ampullae of Lorenzini
The distribution of electro sensory pores on the dorsal surface of the head in
dogfish are shown in Fig. 3.1 b, and for the ray in Fig. 3.8. The electro sensory pores
are the external openings of canals which are filled with a highly conductive jelly,
and which have thin, electrically resistive walls. The canals end in blind sacs, called the
ampullae, and in the ray the ampullae are grouped into four clusters from which the
canals radiate as a series of rosettes. The ampulla contains the sensory and supporting
cells of the sensory epithelium (Fig. 3.9). At the luminal surface, the cells are
connected by occluding junctions that cause electrical current to flow through the cells
rather than between them. The receptor cells are thought to be related to the mechano-
receptive hair cells and do have an apical cilium. The basal surface of the receptor
=--';-- 4
Fig. 3.8. Electrosensory system of the ray. Distribution of canals and ampullary clusters on the
dorsal surface of the thornback ray. Right side canals which open onto the dorsal surface and
their origins in the supraorbital and hyoidean ampullary clusters (buccal and mandibular ampullary
clusters are located ventrally). Left side: dots show openings of the ampullary canals ; lines show
lateral line canals. 1 supraorbital ampullae and canals ; 2 anterior canals; 3 hyoidean ampullae ; 4 outer
canals; 5 inner canals ; 6 posterior canals; 7-10 lateral line canals, supraorbital, infraorbital,
hyomandibular, and scapular respectively
ca.al SA
~_ J
~~~ ])
Nerv e
a 4Hz
lumen
2 Hz
~------WlUII.IIUWWIIIww.lmUA~-
-------- - -
1 Hz
WlIllllllm
mWmmuuu____- -
b c
Fig. 3.9. Anatomy and electrophysiology of ampullae of Lorenzini. a Ampulla of Lorenzini. The
ampulla consists of a cluster of alveoli , one of which is shown in cross-section. The receptor
cells are innervated by about five afferent nerve fibers that ramify profusely over the surface of the
alveoli. (After Bennett and Clusin 1978) ; b diagram of the cell types of the sensory epithelium. Two
receptor cells are adjoined by supporting cells. Occluding junctions (0) partition the cell membranes
into luminal and basal faces. In the basal region there are characteristic ribbon synapses with
the afferent fibers. (After Bennett and Clusin 1978) ; c electrophysiological recording from an
electro sensory primary afferent fibre. SA spontaneous activity, lower traces show response to uniform
field stimulation (50 IlV cm - 1 peak to peak) at modulation frequencies between I and 4 Hz. Top
trace of each pair is the spike record , bottom trace the stimulation wave form
cell makes synaptic contact with an afferent fibre, but there are no efferent fibres in the
elasmobranch electrosensory system. Bodznick and Boord (1986) provides a more
detailled review of the anatomy and physiology of electroreception.
3.3.2.3 Electrophysiology
In the ampullae of Lorenzini, a lumen-negative stimulus depolarizes and excites the

lumenal face of the receptor cells. This depolarizing response is regenerative, and
depolarizes the basal face of the receptor elevating the release of transmitter
[possibly glutamate (Akoev et al. 1980)], and increasing the rate of afferent fibre
discharge. The evidence for these mechanisms is given in Bennett and Clusin (1978).
The natural stimulus for the receptors is the difference between the voltage at
the opening of the canal, and that in the basal face of the receptor cell.
There is evidence that inputs from canals on opposite sides of a rosette are subtracted
by the central nervous system allowing the system to operate differentially (Mont-
gomery 1984a). In this case it would be the voltage difference between pores on
opposite sides of a rosette which would provide the natural input to the system .
Electrophysiological recordings from single electro sensory afferents (Mont-
gomery 1984b) show them to have a relatively regular spontaneous activity of
around 15 impulses S-1 with a sensitivity of about 4 impulses S-1 IlV-1 cm- 1 .
Good responses to sinusoidally modulated fields were obtained within the frequency
range 0.05 to 8 Hz, which agrees well with the behaviourally determined frequency
range.
Given the many factors involved in the electrical sensitivity of the receptors, and
their high sensitivity, it is not surprising that the ampullae respond to a variety of
other modes of stimulation. In this instance, behavioural experiments have provided
the evidence that the remarkable sensitivity to electrical fields is the biologically
important response of these receptors.
Acknowledgements. I am grateful to Dr AJ Kalmijn for his helpful comments on the manuscript.
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Chapter 4 Q. BONE
Muscles and Locomotion
Like all fish, elasmobranchs swim in a dense viscous fluid which has at once profound
effects upon the design of their locomotor system, and by buoying them up, enables
them partially or completely to escape the effects of gravity. So far as the locomotor
system is concerned, the main design constraint is that in such a fluid the drag
opposing forward motion increases rapidly as swimming speed increases, so that to
operate over a wide speed range, the locomotor muscle has to provide a rather wide
range of power output. Just how wide this range may be is not certainly known for
any elasmobranch species, since little information is available about the speed range
over which any given species operates. In the small dogfish Scyliorhinus, minimum
cruising speed (below which insufficient dynamic lift can be generated to maintain
horizontal position) is around 25 cm s- 1, whilst burst speed is probably around I m s -1.
Like most sharks and rays, Scyliorhinus is relatively well-streamlined (though not so
superbly as fast-swimming lamnids) and skin friction drag is much the most
important drag component opposing forward motion: pressure drag and lift-associated
vortex drag are of minor consequence.
Thus, since skin friction drag is proportional to the square of the forward speed
(V2), the power required from the muscles will be proportional to y3, and for the
dogfish, this means that around 64 times cruising power must be provided at burst
speed. For several reasons, (e.g. the flow regime around the fish is likely to alter at
different swimming speeds, or again, at different contraction velocities, the muscle
fibres may operate at different points along their force/velocity curves), the power
required is unlikely to be exactly proportional to y3, but it is manifest from the
considerations above that the propUlsive machinery must be adapted to provide a very
wide range of power output.
Elasmobranchs solve this problem (as do other fish and cephalopods) by dividing
the locomotor systetp. into a small portion specialized for low speed cruise economy,
and a much larger portion specialized for shorts bursts of high speed operation, where
economy is not mandatory. In the locomotor muscles of all elasmobranchs therefore,
there are two main muscle fibre types which are normally organized into discrete
zones within the myotomes or, (in batoids) in the pectoral fin muscles. Because they
are zoned, and because the two main fibre types are different in colour, it is a simple
matter to place EMG, electrodes in one or other fibre type, or to isolate each
Author's address: The Marine Biological Association of the UK, The Laboratory, Citadel Hill,
Plymouth PLl 2PB U.K
100 Chapter 4. Muscles and Locomotion
for physiological and biochemical studies. Indeed, it was in the electric ray Torpedo
that Lorenzini (1678) first described red and white muscle fibres in vertebrates.
Experimental proof of this functional division of the elasmobranch locomotor
system only came nearly three centuries later.
In this chapter, after considering the general organization of the locomotor system
in sharks and rays, the structure and properties of these two types of locomotor
muscle fibre will be described; and finally, what is known of the swimming patterns
they drive will be examined; and how elasmobranchs generate lift and thrust.
4.1 General Organization of the Locomotor System
4.1.1 Sharks
The locomotor fibres in sharks are myotomal, for in none do those of the paired fins
contribute to forward propulsion, as they do in the other shark-like elasmobranchi-
morph group, the Holocephali, where slow swimming is brought about by the pectoral
fins. Several authors have examined aspects of the myotomal organization of sharks
(e.g. Alexander 1969; Willemse 1972), but it is not yet clear just how they
operate to oscillate the body and tail in swimming.
The red muscle fibres in the superficial zone of the myotome are aligned along
the long axis of the fish, and it seems at first sight obvious that tension in these fibres
in one myotome is transmitted to the next adjacent via the myosepta, and so on
along the body, so bending the incompressible vertebral column. But as Wain-
wright (1983) emphasizes, it is unlikely that tension is transmitted from con-
tracting muscle in one myotome via inactive compliant muscle in those adjacent,
and it is much more probable that the connective tissue elements of the myo-
tome are actually transmitting tension as well as simply limiting the range of
deformation permitted to the myotome. Unfortunately, although it is relatively
easy to determine the orientation of connective tissue fibres within the peri-
mysium, myosepta, :and skin by simple dissection (Willemse 1972; Motta 1977;
Wainwright et al. 1978), it is a very much more difficult matter to monitor tensions
and changes in myoseptal and myotomal form during movements of the living fish.
Wainwright (1983) should be consulted for an illuminating discussion of the problems
and possible approaches to their solution. At present, it is only possible to make some
inferences from the orientation of the fibres within the connective tissue systems of
the myotome about the directions in which forces are exerted during swimming.
Willemse· (1972) has examined the orientation of connective tissue fibres in Squalus,
and has shown that the myotome is divided into packets or rectangular bundles of
muscle fibres, separated by thin perimysial sheets (Fig. 4.1). This arrangement is also
found in Scyliorhinus and in some deep-sea squaloids such as Centroscymnus, but
other species (in particular the fast-swimming lamnids) have not been examined. It is
a fortunate feature of the system, pre-adapting the shark myotome for physiological
studies, since it is an easy matter to take thick slices of the myotome at an appropriate
angle (as shown by the shaded areas in Fig. 4.1), when undamaged muscle fibres can
be obtained simply by picking off the perimysium with fine forceps, giving access to
4.1 General Organization of the Locomotor System 101
an undamaged layer of fibres in the sandwich. Within the perimysial sheets, which
cross the myotome from its surface to the vertebral column, the connective tissue
fibres are aligned at angles between 60° and 70° to the longitudinal axis . A similar
arrangement is found in Agnatha, but teleosts and other bony fish do not have their
myotomes divided up in this manner. Willemse determined the cross-sectional area
of the myotome occupied by connective tissue elements, and found that, although
this was about the same in Squalus and in various teleosts, in Squalus this was made
up of the myosepta and the perimysial component; in teleosts of myosepta alone.
From this he concluded that in sharks, both components must, at least in part, have
the same function as that of the teleost myoseptum, viz. to connect the musculature
a
Fig. 4.1. a Transverse section of post-anal region in Squalus acanthias, showing myosepta (m) and
outer (dotted) border of red muscle fibres . The darker hatched rectangles indicate position of
muscle slices for isolation of fibres ; b enlargement of area indicated in a showing perimysial
partitions within myotome. Red muscle zone dotted. (After Willemse 1972)
Fig. 4.2. Arrangement of muscle fibres within shark myotome. a Orientation of fibres in white zone
with-reference to myosepta I tendons; b schematic diagram to show corresponding overlapping cones
of myotomes. (After Alexander 1969 and Wainwright 1983)
to the vertebral column. The obliquity of the connective tissue fibres in the perimysial
sheets is not far from the angle of 54°44' (arctan 112
shown by Jarman (1961) to be
that at which fibres are not in tension or compression when bending occurs) and
according to Willemse, represents a compromise between any restriction on the
contraction of the myotomal muscle fibres and sufficient connection with the axial
skeleton. The muscle fibres in the myotome (apart from those in the superficial red
muscle) are not aligned along the longitudinal axis. Alexander (1969) has shown
that those in the white portion of the myotome form a series of cones, within which
some fibres make angles of almost 40° to the axis of the body (Fig. 4.2). The
functional significance of this arrangement is that it enables fibres at different positions
within the myotome to contract to the same extent as the body flexes. The white muscle
portion of the myotome is specialized for maximum power production, hence it is
a necessary requirement that all its fibres contract to the same extent and can operate
at the maximum power point on their force/velocity curve. Each muscle cone in the
white portion of the myotome consists of muscle fibres attaching at its tip to a
horizontal septum (an extension of the myoseptum) and at its broad base to the
oblique myoseptum, and to the vertebral column. Considering this arrangement,
Wainwright (1983) suggests that tension in the myotomal muscles is transmitted to
the collagen fibre tracts in the myoseptal cones and thence backwards and inwards
to the vertebral column via the horizontal and medium septum. This view has two
consequences; it avoids the transmission of force from active muscle fibres to inactive
compliant fibres, and it implicates the connective tissue fibre system of the skin in
transmission of force along the body. Motta (1977) showed that in several shark
species the connective tissue fibres of the stratum com pactum were arranged in
helices around the body, making angles between 50° and 77° to the long axis (Fig. 4.3).
In the main part of the body, in some other species, Wainwright et al. (1978) observed
angles between 50° and 70° (Fig. 4.3). At the caudal peduncle, angles between 45°
Fig. 4.3. Orientations of dermal connective tissue fibres in Sp/zyrna lewini (a) and Negaprion brevi-
rostris (b). (After Motta 1977 and Wainwright et al. 1978)
4.1 General Organization of the Locomotor System 103
and 50° were found. Motta suggested that the helical collagen fibre system of the
skin functioned partly as a mechanical protection, and partly to distribute muscular
forces to the skin; Wainwright et al. extended this latter hypothesis with the
ingenious and attractive idea that the connective tissue system of the skin not only
transmitted forces, but also might act as an elastic energy store.
By direct measurement (using a simple pressure probe under the skin of a small
lemon shark), they showed that intramyotomal pressures rose in active myotomes as
the shark bent its body, to as much as 20 times the values recorded at rest.
When the shark was sprinting, this pressure rise (which results from the increase in
myotomal muscle fibre diameters as they contract) imposes a stress on the skin which
is mainly circumferential, rising to 2.8 MPa during maximum swimming speed. Since
the fibre angle of the connective tissue fibres in the dermis is greater than 55° (Fig. 4.3),
the stress is shed diagonally and contributes to the shortening of the skin also
brought about directly by the shortening of the myotome as its muscles contract;
whilst at the same time the contractile force is transmitted via the skin to the vertebral
column at head and tail. Biaxial stress tests on pieces' of shark skin using length
changes similar to those observed directly during swimming show that during cruise,
little energy is stored in the helical fibre system of the skin, but during rapid swimming,
the skin stiffness presumably greatly increases, and a significant amount of elastic
energy may be stored (Fig. 4.4). As Wainwright (1983) points out, it is not known
~4
(II
I
E
z3
::E
C/)
C/)
....E2
C/)
iti
c
:c 1
B
.s,
c
..2
-20 o +20 +40
(%) longitudinal elongation
Fig. 4.4. Curves showing longitudinal stress-extension behaviour of Negaprion skin. Lower curves
show loading and unloading stresses when the specimen was pre-stressed to 03 MN m- 2 to
simulate slow-swimming conditions; upper curves when the specimen was pre-stressed to 2.8 MN m - 2
to simulate fast-swimmi~g conditions, (After Wainwright et aL 1978)
whether the elastic energy stored in the skin at the extreme position of bending is
utilized by the shark to contribute to the acceleration of unbending as the myotomal
muscles on the opposite side of the vertebral column contract. To decide if this sensible
trick is indeed used requires measurement of skin stiffness during swimming (still
lacking), but it seems unlikely that the shark design has not taken advantage of the
possibility. Since Wainwright's calculations (Wainwright 1983) indicate that elastic
energy storage is only likely to be of consequence during burst swimming, if this
were a major function of the skin system it might be supposed that fast-swimming
sharks would have a thicker stratum compactum than slower-swimming forms.
Insufficient data exist to test this, but Motta's measurements on the pelagic Sphyrna
and benthic Ginglymostoma of similar size show that in the former it is less than half
as thick as in the latter. It would certainly be interesting to carry out stress tests on the
skin of a range of sharks of different habit.
4.1.2 Batoids
The general organization of the locomotor pectoral fin musculature in batoids has
been little investigated. Not all swim with their pectoral fins, for example electric
rays, such as Torpedo , cruise by oscillating the tail (Roberts 1969a) as do some
guitarfishes, but in most rays the pectoral fin (and to a lesser extent the pelvic fin)
musculature is the only source of forward thrust. Preliminary work on a variety of
batoids (Holst and Bone, in prep) has shown that the pectoral fin musculature is
surprisingly complex, and rather different in different batoids, presumably because
there is a range of swimming patterns in batoids. In all, however, the basic pattern
for dorsal and ventral pectoral fin musculature is similar, and consists (Fig. 4.5) of
deep and superficial fibre bundles arranged in a pinnate fashion. Constraints of space
within the fin have presumably influenced this arrangement, since Alexander (1968)
has shown that pinnate-fibred muscles exert more force than an equivalent volume
of parallel-fibred muscles.
/#,~/~7"""~"'cA ".-
..- ~
Fig. 4.5. Schematic diagram to show arrangement of muscle fibres in batoid pectoral fin. Red
fibre bundles dotted. Note that both deep ( d), and superficial (s ) fibre bundles are larger dorsally
than ventrally
The fibres of the deep bundle take origin from the pectoral girdle or from the
jointed fin rays, and attach to long tendons which form an arch around the bundle;
these tendons finally attach to the outer fin rays, thus forming long arcs along the fin
ray axes. The superficial muscle bundle of each fin ray is arranged in a similar manner,
except that its fibres may take origin not only from the pectoral girdle, but also from
4.2 The Locomotor Muscle Fibres 105
the edges of the arch of connective tissue fibres formed by the tendons of the deep bund-
le. The superficial bundle does not extend as far out along the fin rays as does the
deep bundle.
Both superficial and deep bundles are mainly composed of white large-diameter
fibres which resemble those of the deep portion of the shark myotome. They also
contain a small amount of small-diameter red fibres which are SDH-positive
(Fig. 4.8), and resemble those of the superficial zone of the shark myotome. It seems
probable that the two locomotor fibre types in batoids have the same functions as
the two main fibre types in the shark myotome, (viz. that the small-diameter fibres
are used in cruising, the large during burst swimming) but their role has not been
examined by experiment. What is striking in the batoid pectoral fin is not only that
the tendons of the bundles form overlapping arcs along the fin-rays, but that there is
a considerable extent of longitudinally arranged collagen fibres forming tendon
bundles at the outer parts of the fin.
It seems very probable that these may represent a significant elastic energy store
quite independent of any storage in connective fibres of the dermis, and that this
could be utilized during slow swimming as well as in fast swimming. In almost all
batoids which swim by using the pectoral fins, the dorsal deep and superficial fibre
bundles are significantly larger in cross-sectional area than are the ventral, despite
the fact that almost all batoids are relatively dense fish (Bone and Roberts 1969).
In R. clavata, for example, the ventral pectoral musCles weigh only one third of the
dorsal whilst the fish is much denser than seawater, weighing in water 5.5 % of its air
weight.
4.2 The Locomotor Muscle Fibres
4.2.1 Structure
As in other fish, elasmobranch locomotor fibres are broadly divided into small-
diameter mitochondria-rich fibres, with an abundant capillary supply and myoglobin
content, and into large-diameter mitochondria-poor fibres with a poor capillary supply
and lacking myoglobin. A detailed study of the structure of these contrasting fibre
types has so far only been made in Scyliorhinus, although scattered observations
on other species indicate that this small shark is likely to be typical of elasmobranchs
in general. The special case of "warm" sharks where red fibres are internalized in
the myotome is considered in a later section (4.5.5). However, in Scyliorhinus, a third
fibre type is also found, which is not seen in other elasmobranchs.
The dogfish myotome in transverse section when stained with techniques for
oxidative mitochondrial enzymes (Fig. 4.6) shows from the superficial to the deep zones
a sequence of fibre types which can also be distinguished by staining for Ca2+ -activated
myofibrillar ATP-ase (Bone and Chubb 1978) as seen in Fig. 4.7. First, just under
the connective tissue fibres of the stratum compactum there is a single layer of large-
diameter mitochondria-poor superficial fibres. This fibre type is peculiar to dogfish
and will be considered later.
Next, there is the zone of small-diameter mitocondria-rich red fibres, which over-
lies the large-diameter mitochrondria-poor white fibres making up the major portion
of the myotome. Histochemical and ultrastructural studies show that these red and
white zones each contain two sub-types offibre, grading into each other, with respect
to their mitochondrial content and Ca2+ myofibrillar ATPase activity (Bone and
Chubb 1978). Thus, the most superficial or type I red fibres contain a higher
proportion of mitochondria and show a lower ATPase activity than those deeper ,
next to the white fibre zone, the type 2 red fibres. Similarly, the most superficial
(or type I) white fibres next to the type 2 red fibres have a higher mitochondrial
content and lower ATPase activity than the type 2 white fibres lying deeper in the white
zone. The type I white fibres of several sharks are termed intermediate fibres by
Totland et al. (1981), but this term is less suitable, since both types of white fibre
are entirely distinct from both red fibre types . Figures 4.9-4.11 illustrate the ultra-
structure of the main fibre types as seen in transverse section. The electro-
physiological and mechanical studies described in Section 4.3 have not investigated
Fig. 4.6. Transverse section of outer edge of Scyliorhinus myotome showing SDH-positive red
fibres; superficially there is an interrupted layer of superficial fibres and internally (below ) white
fibres , both of which are SDH-negative
Fig. 4.7. Similar section stained for myofibrillar ATP-ase showing lack of reaction in superficial
fibres and intermediate staining in type 2 red fibres
Fig. 4.8. Variation in fibre diameter and SDH activity in red fibre bundle from pectoral fin of
Raia clavata
Figs. 4.9.-4.11. Representative areas
of Scyliorhinus myotomal fibres in
transverse section 9 Superficial fibre
10 Type I red fibre 11 White fibre
Table 4.1. The relation of capillaries to surface area and volume of distinct fibre types in Galeus, Etmopterus and Scyliorhinus. (Totland et al. 1981)
Species Fibre No. No. Fibre No. of Fibre Capillary Vascularized Mean fibre
type of of circumference capillaries cross-sectional contact length fibre surface volume
fibres capillaries (Ilm) surrounding area (Ilm) . per fibre (Ilm) (% of total served by one
(mean ± S.D) each fibre (mean ± S.D.) (mean ±. S.D.) surface) capillary
(mean ± S.D.) (Il m3 )
G=FxIOO E
A K B C E F H=-
B C
Galeus Red 258 222 164.3 36.8 2.5 1.2 1778 815 30.0 17.4 18.3 10.4 711.2
Int. 92 32 267.4 66.2 0.8 0.9 4869 2965 10.2 15.7 3.8 5.7 6086.3
White 124 17 433.4 97.7 0.4 0.6 12217 5016 5.6 10.2 1.4 2.5 30542.5
Etmopterus Red 446 165 204.0 38.5 1.4 0.7 2617 976 46.3 30.7 22.7 14.9 1869.3
Int. 139 25 247.4 45.6 0.4 0.6 3507 1128 11.2 20.9 4.5 8.4 8767.5
White 418 8 357.8 111.7 0.04 0.19 6993 2255 0.6 3.6 0.2 0.9 174825.0
Scyliorhinus I Superf. 20 11 377 58 1.2 1.3 6923 1549 8.8 10.3 2.6 3.7 5769.0
Red 425 434 191.1 34.3 2.8 1.4 2416 748 30.9 13.2 16.3 9.6 878.5 ~
Int. 44 59 398.9 37.7 3.0 1.2 10241 1877 30.3 13.2 7.6 3.3 3436.6 tv
White 100 168 479.2 49.0 3.9 1.1 15114 2757 33.3 11.9 7.0 2.6 3875.4 >-:l
!f
Scyliorhinus II Superf. 22 37 277 68 1.6 1.3 4914 2506 22.1 19.7 8.0 7.1 3071.0 t"'
0
Red 658 1467 181.4 46 4.7 2.1 1879 719 62.4 36.1 33.5 16.9 399.8 n
0
Int. 94 186 291.2 52.8 3.7 1.6 5089 1206 49.9 21.3 17.3 6.8 1375.4 S
~
White 96 209 343.3 52.8 3.0 1.1 7476 2069 36.8 13.9 11.1 4.9 2534.2 ...,0
~
=
~
::!1
Sf
'"'"
0\0
differences between the two types of fibre found in the red and white zones, but it seems
likely that the division (or rather, gradation) of the two types in each zone indicates
a functional gradation across each zone. For example, it is reasonable to suppose that
during low-speed cruise swimming, the type I red fibres are alone active, and that
as cruising speed increases, progressively more and more of the type 2 red fibres are
recruited until maximum sustainable cruising speed is reached.
In line with the differences in mitochondrial content and oxidative enzyme activity
of the different fibre types, their capillary bed is different. As yet, the capillary distri-
bution of the shark myotome has been studied in detail only in some small sharks
(Totland et al. 1981), with the results shown in Table 4.1.
The curious superficial fibres forming a single layer external to the red fibre zone
in Scyliorhinus show an unusual combination of characters of the fibres of the red and
white zones (Bone et al. 1986). They are large-diameter and mitochondria-poor
like the white fibres, but are low in Ca2+ -ATPase (next section) and are multiply-
innervated and apparently do not propagate action potentials. On each side, in a
single myotome, at the immediate post-anal level, there are around 8000 red fibres
and 11,000 white fibres but only 80-90 superficial fibres, forming a band along the
flank of the fish.
Thus the superficial fibres make up less than 0.5 % of the total myotomal fibre
number, but since they are larger than the red fibres, they form some 0.6% of
the total myotomal cross-sectional area, compared with 24.4 %for the red fibres and
75 % for the white fibres. During ontogeny, red and white fibres can be distinguished
in embryos' of 48 mm total length, superficial fibres make their appearance at
Table 4.2. Percentage mitochondrial volume of the different fibre types at various stages of
development. (Bone et al. 1986)
Fibre type Body length (mm)
48 94 100 450
Superficial 8.3 ± 3.98 3.27 (I only) 2.39 ± 0.49

Red 10.14 ± 1.77 13.79 ± 3.12 18.07 ± 3.55 21.55 ± 3.39
White 5.67 ± 2.42 0.81 ± 0.26 1.11 ± 0.29 0.99 ± 0.16
Table 4.3. Ultrastructural characteristics of fibre types in the dogfish myotome. Values represented
mean ± S.E. (Bone et al. 1986)
Parameter Muscle fibre types
Superficial Red White
Relative volume density 75.7 ± 2.5 62.2 ± 2.2 77.8 ± 1.2

Myofibrils [Vv(m .ol
Mitochondria [Vv(mt,Ol
Sarcotubular system
94 mm, when their mitochondrial content is intermediate between the white and red
fibres. As development proceeds, the mitochondrial content of the red fibres increases
and that of the white fibres declines, so that by 100 mm the mitochondrial
content of the three fibre types resembles that of the adult (Table 4.2). Apart from
the differences mentioned, M-lines are lacking, and the myofilament array and sarco-
tubular systems occupy different proportions of the fibre volume as shown in
Table 4.3.
The ultrastructure of batoid locomotor fibres has not been examined. Preliminary
histochemical studies (Bone and Chubb 1975; Horst and Bone, unpubl.) of pectoral
fin muscles in various batoids have shown that although large-diameter SOH-
negative fibres fonn the larger part of the locomotor musculature, there is some
variability in the smaller-diameter SOH-positive fibre bundles (Fig. 4.8), and that
there are greater differences in size and SOH-reactivity within this system than in
the range between type 1 and type 2 red fibres in shark myotomes.
In adult batoids, it may be assumed that this reflects functional differences along the
same lines as those suggested for the dogfish type 1 and type 2 fibres but there is
no experimental evidence for this assumption.
4.2.2 Motor Innervation
Both superficial and red fibres in dogfish and red fibres in other sharks are
multiply-innervated by large en grappe terminals along the length of the fibre.
At least some of the terminals on the superficial fibres are derived from axons
supplying terminals also on type I red fibres (Fig. 4.12). In dogfish, the sarcolemma is
infolded below the tenninals, in this respect differing from teleosts examined where
there are no sub-junctional folds below nerve terminals on red fibres. Acetylcholin-
esterase is present at the neuromuscular junctions, and the vesicles contained within
the nerve terminals are almos! all around 50 nm diameter, like those observed in
cholinergic terminals in other animals.
The innervation of SOH-positive small-diameter fibres in the pectoral fins of
batoids is similar (Bone and Chubb 1975).
The innervation of the white myotomal muscle fibres is entirely different, for they
are·focally innervated by large basket-like terminals which enfold one end of the fibre
where it attaches to the myoseptum. There are extensive sUbjunctional folds, and
the terminals lie amongst the myotendinous infoldings of the fibre attachment.
Curiously, it appears that these terminal neuromuscular junctions are derived from
two separate axons. At the uItrastructurallevel (Fig. 4.17), two types of terminal can be
distinguished by the"ir vesicle content (Bone 1972 a). One contains the electron-lucent
50-nm vesicles typical of cholinergic terminals; the other much larger vesicles, many
of which possess dense cores, up to 100 nm in diameter. At the light microscope
level, it is often possible to see in whole mount preparations, in both shark and
ray myotomal white fibres, two axons supplying a single basket terminal that are
entirely separate and indeed, reach the terminal from different directions from the
myoseptal plexus (Fig. 4.14). A similar dual innervation of fast fibres has been
described histologically in lampreys (Kashapova and Sakharov 1976), in sturgeons
(Kashapova and Sakharov 1978), and in Oipnoi, holocephali and possibly in clupeid
Fig. 4. 12
Fig. 4.13
50~m
Fig. 4.14 Fig. 4.15

Fig. 4.16
50 11m
teleosts (Ono 1983). In none is the significance of the arrangement understood, and
the situation in elasmobranchs certainly deserves pharmacological investigation.
The white fibres in the pectoral fins of batoids are focally innervated but in the
middle of the fibre rather than at one or other of its insertions, and unlike
the white myotomal fibres in the batoid tail, only a single axon forms the terminal.
The end-formations are large (Fig. 4.13) and enwrap the fibre. The white muscle fibres
of shark paired fins are similarly innervated, and as in rays, only a single axon
contributes to the end-formation. It seems probable, as Ono (1983) suggests, that
dual innervation of myotomal white fibres is the primitive condition and that, as
might be supposed on other grounds, body oscillation rather than fin propulsion is
the primitive mode of locomotion in the" group.
In Torpedo, alone of the elasmobranchs that have been examined, a few of the
myotomal white fibres (used in this animal for forward propulsion) are innervated in
the same way as those in the pectoral fins, viz. in the middle of the fibre, rather
than at the end (Bone 1964). These fibres correspond in position to the type 1
white fibres of the dogfish myotome. Barets (1961) has shown that a similar medial
innervation of the outermost white fibres is found in the myotome of the teleost
Ameirus, where as in sharks, the major part of the white fibres are terminally inner-
vated. Once again, the significance of this innervation pattern is not understood in
functional terms.
4.2.3 Sensory Innervation
Amongst the motor axons passing to the locomotor muscles, there are a few
large-diameter (20-25 11m) sensory fibres, which terminate in batoids as elongate
beaded terminals lying between slow muscle fibres (Bone and Chubb 1975) (Fig. 4.16),
and in sharks as coiled corpuscular endings just under the skin, between the stratum
compactum and the outer muscle fibres of the myotomes (Bone and Chubb 1976)
(Fig. 4.15). Both types of ending almost certainly provide information about swimming
movements, and in rays, Ridge (1977) has shown that although they are not encapsu-
lated and are only loosely attached to the neighbouring muscle fibres, their responses
to ramp stretches resemble those of the muscle spindles of higher forms (Fig. 4.18);
they are slowly adapting length and velocity receptors. In dogfish, the coiled
endings are slowly adapting mechanoreceptors, and Roberts (1969b) has shown that
~
Fig. 4.12. Motor termimlls on superficial (above) and type 1 red fibre (below) in Scyliorhinus
myotome. Note branching of axon to both terminals. Whole mount, reduced silver
Fig. 4.13. Large motor terminals from central region of pectoral fin white fibre from Raia
microocellata. Methylene blue
Fig. 4.14. Two axons contributing to motor terminal on Scyliorhinus white mytomal fibre. The
muscle fibre itself is not visible in this methylene blue preparation
Fig. 4.15. Coiled corpuscular ending from Scyliorhinus myotomal surface, reduced silver
Fig. 4.16. Elongate beaded stretch receptor ending from superficial red fibre layer of Raia clavata
pectoral fin. The small fibre (m) supplies a motor terminal on one of the red fibres. Reduced silver
Fig. 4.17. Electron micrograph of motor terminal on Scyliorhinus white myotomal fibre , showing
subjunctional folds and myo-tendinous infoldings ; note that larger terminal contains many dense-
cored vesicles which are not present in the smaller terminal profiles. (Bone 1972a)
4.3 Physiology 115
they are activated as they are compressed during the swimming cycle by the lateral
movements of the fish. As the endings in the batoid pectoral and pelvic fins are not
found in those fins in sharks (nor in holocephalans which swim with the pectoral
fins), and the coiled corpuscles are not found in batoids, perhaps the undulations
of batoid fins require to be controlled more precisely than the lateral oscillatory
movement of sharks. However, endings similar to those found amongst the slow fibres
of batoid pectoral and pelvic fins are seen along the batoid tail in the same
superficial position below the stratum compactum as the coiled endings of the
shark myotome: their function is not clear.
"
400
300
!:!L.
QJ 200
:::c
100
1---1
0.1 5
Fig. 4.18. Discharge frequency (frequency meter output) of stretch receptor in red fibre bundle of
pectoral fin of Raia clavata in response to four ramp stretches (lower lines) at velocities of 2.5; 5;
10; and 20 mm s -1. (Ridge 1977)
4.3 Physiology
4.3.1 Electrophysiology
The membrane properties of red and white muscle fibres in dogfish have been
studied by Stanfield (1972), and Hagiwara and Takahashi (1967, 1974) have examined
the white fibres of several species of tropical batoids. In both studies, a high Cl-
conductance was found in the resting membrane, in Taeniura some eight to ten times
that of the K + conductance at pH 7.7 (Hagiwara and Takahashi 1974). By altering
external Cl- concentration, it was found that the membrane behaved similarly to
a perfect Cl- electrode, with a small deviation at low external Cl- explained
by cation permeability of the membrane.
In dogfish white fibres, Stanfield found with a two-electrode clamp technique that
the approximate values for K+ and Cl- conductances were 0.12 mho cm- 2 and
0.51 mho cm -2 respectively. Ringer solutions in which urea is replaced by NaCI,
or NaCl is replaced by Na methylsulphate, therefore have significant effects upon
muscle resting potential. The normal Ringer solutions used by Stanfield and
Hagiwara and Takahashi contained 314 mmoll- 1 Cl-, whilst the simple Ringer
solution given by Young (1933) contains 380 mmoll- 1 Cl. It is important to
notice also that elasmobranchs confined in aquaria prior to experiment. may show
Table 4.4. Changes in composition of cerebrospinal fluid in Scyliorhinus in captivity (in mmoll- 1 ).
(Bone unpubl.)
Freshly caught fish Fish probably up to 2 months in aquarium

circulation
Na+ K+ Cl- Na+ K+ Cl-
254 4.4 273 295 3.3 261.5

206 4.2 239 293 3.8 286
229 4.1 297 297 3.3 269
273 3.9 284 307 3.9 279.5
229.3 2.7 226.5 303 2.4 264
323.3 4.6 269
275.7 6.6 263
266.9 6.4 190
257.5 3.7 279
257.2 ± 10.2 4.6 257.8 ± 31.6 299 ± 5.2 3.3 273 ± 9.3
Table 4.5. Membrane constants of elasmobranch muscle fibres. (Stanfield 1972; Hagiwara and
Takahashi 1967)
Species and R A- r1 rm Rl Rm tm Cm
fibre type (m) mm x 106 cm- 1 ) (x 105 cm) (cm) (cm2 ) (ms) (JlF cm- 2 )
Scyliorhinus
canicula
White 0.14 2.36 0.62 0.34 108 1588 15.9 10.3
fibres
Red 0.75 2.27' 6.92 3.44 136 5410 47 10.2
fibres
Himantura
uarnak
White 0.36-
fibres 0.46 3.0-3.5 6-7
Taeniura
lymma
White 0.06-
fibres 0.08 2.5-3.0 1.5-2.5
Table 4.6. Characteristics of the action potential of white muscle fibres in Scyliorhinus canicula.
(Stanfield 1972).
RP RP after AMP Total Max. rate Max. rate Rise time Fall time
insertion AP of rise of fall (20-100%) (100-50%)
of current
electrode
(mV) (mY) (mY) (mV) (VS-l) (V S-I) (ms) (ms)
Mean (14 fibres) -85.7 -80.6 +54.1 134.7 409 166 0.90 0.79
S.E.ofmean ± 1.1 ± 1.6 ± 3.4 ±2.3 ±20 ±8 ±0.07 ±0.04
Mean temperatures 20.3 DC

4.3 Physiology 117
significant changes in the ion content of the serum. Thus Hartman et al. (1941)
have shown that in R. eglanteria kept for some days in captivity, urea decreases and
Na + and Cl- concomitantly increase. Freshly caught dogfish at Plymouth were
found to show similar changes (Table 4.4). For critical work, therefore, it may be
necessary to determine the Cl- composition of the serum or cerebrospinal fluid.
Since Hagiwara and Takahashi (1967) have shown that the spike in white fibres is
Na + -dependent and that overshoot depends upon external Na +, it may also be neces-
sary to determine Na +.
The cable constants of the white fibres are shown in Table 4.5 with those from
dogfish red fibres for comparison. Since they are focally innervated, the white
fibres propagate action potentials, those of dogfish white fibres are characterized in
Table 4.6. Action potentials similar to those evoked by current injection are seen
with nerve stimulation, but critical experiments which would attempt to stimulate
one or other axon of the dual nerve supply to the white fibres have not been
performed.
The multiply-innervated red fibres in dogfish present an interesting situation for
although they would not be expected to propagate action potentials, Stanfield found
an abortive spike on one occasion, and 8 of 27 slow fibres showed sufficiently large
100
/ '
/ II
10-8 A
SO
300
60
II 10- 8 A
/.
//
200
40
./
20 v· 100
/1
-120 -100
~
4--
. . .J
L-so
)
-60 -40
I I
-20 o 20
._.
-120 -100 __p:!~7"~
-SO -6,0 -40 -20
I I
a -20L mV mV
-100
60
_........ /
10-8 A b -200
40
Fig. 4.19. Voltage-current relations from Scy-
liorhinus myotomal muscle fibres. The dotted
20 ~ line in a is the voltage-membrane current rela-
-120 -100 ....A T tion obtained by Cole's theorem. a Red fibre
~",""I
.__- Y
III
showing no signs of inward Na+ current;
.. -SO -60,, -40 20 b white fibre, filled "circles currents flowing at
,, end of 100 ms pulse; open circles currents flow-
-20 ,
,, ing 2-3 ms after start of depolarising pulse;
c red fibre showing large inward Na + currents
-40 on depolarization. Open and filled circles as
for b. Abscissae membrane potential; ordinates
c -60 total current. (Stanfield 1972)
inward Na + currents as soon as depolarized, to suggest that they were capable of

propagating action potentials. Six showed no inward Na + currents, on depolarization,
and the remainder showed only a small Na + current. Figure 4.19 shows active
voltage-current relations obtained from a slow fibre without inward Na + current;
from a slow' fibre in which there was a large inward Na + current; and from a white
fibre.
As Stanfield emphasizes, all red fibres had a high input resistance and strikingly
long time constant (compared to the white fibres), and could be easily obtained
uncontaminated by white fibres; so there is no reason to suppose that his results
do not show a genuine heterogeneity in the electrical responses of the slow fibres.
A single superficial fibre was examined, and showed no inward Na + current. It is
difficult to evaluate the significance of the presence of a Na + current in some red
fibres sufficient to fulfil the conditions for a propagated depolarization. Stanfield
found no evidence that fibres with and without Na + currents were superficial or deep
in the red fibre zone, hence it does not seem that they may correspond to the type 1
and type 2 red fibres distinguished morphologically. Extracellular EMG. records
from swimming spinal fish when the electrode tip is placed in the red fibre zone show
small (200 ~V) potentials, which contrast with the much larger (1-2 mY) spike po-
tentials ob~ained from the white muscle when it is active, and bundles of red fibres
stimulated with depolarizing agents such as acetylcholine or KCl show sustained
contractures rather than twitches as do bundles of white muscle fibres.
Moreover, recent studies of the mechanical properties of single red and white
fibres from the dogfish myotome (next section) suggest that the former are adapted for
slow contractions, the latter for twitches.
The evidence at present favours the view that no red fibres propagate action
potentials in vivo; evidently it would be interesting to study the system in dogfish
in more detail, in particular to examine the histology and location within the red
zone of those fibres showing a large inward Na + current. It would also be
interesting to examine the presumed slow fibres of batoid pectoral fins, which show
a wider range of diameter and mitochondrial enzyme activity than those of the
dogfish myotome.
4.3.2 Mechanical Properties
Although the mechanical properties of the "jaw-muscle" in dogfish were examined

long ago (Levin and Wyman 1927), it is only recently that the properties of the more
"difficult" preparation of the myotomal muscle have been examined by Johnston
and his colleagues, using isolated fibres or small fibre bundles chemically "skinned"
with detergent (Altringham and Johnston 1982; Bone et al. 1986). This technique
avoids the difficulty of the compliance of the myosepta, and of attaching them to
transducers. The experimental set up and procedure are described in Johnston and
Sidell (1984). Since tension and length can be separately measured and step releases
and load clamping can be applied, force velocity curves and unloaded contraction
velocities can be readily determined. Table 4.7 summarizes the contractile properties
of the three types of dogfish myotomal muscle fibres, and Fig. 4.20 compares their
force-velocity characteristics.
4.3 Physiology 119
0.8
_ 1.5
";"
'en en
;, •
.:J 0.6 ....I
;> ;>
>. ~1.0
:!::
u 'u
~0.4
.9 •
> t
OJ
>
c:
•
c: 0
0
:;::
+ •
- -
u gO.5 •
eO.2 + ....
+
••
c:
u
c:
0 u
0 • •
+ •
+ •
0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.1 0.2 0.3 0.4 0.5
a Relative load (P/Pol b Relative load (PI Pol
t
4
'en
0 •
....I
?::
,
3
>.
Tj
.9
OJ
> 2
c: +
0
:;::
+
-
u
....
0
++
•
c:
0 Fig. 4.20. Force-velocity curves for Scyliorhinus myoto-
u
mal muscle fibres. a superficial fibre; b red fibre; c white
fibre. Experiments carried out at 12°C. (Bone et al.
1986). Note different scales for ordinates
0 0.1 0.2 0.3 0.4
c Relative load (PI Pol
As would be expected from the roles of the red fibres in slow cruising, and the
white in burst swimming established by electromyography and other approaches
(Bone 1966), the maximum force generated and the unloaded contraction velocity
are much higher in the white fibres than in the red. Both are lowest in the
enigmatic superficial. fibres. Since the different types of fibre contain different
amounts of mitochondria, and thus have different myofibrillar densities, the ratios
of the maximum forces produced by the three given in Table 4.7 may be corrected for
this, using the values given in Table 4.2, and are then 1: 1.74: 3.55 for superficial:
red: white.
The relative power outputs (W kg-I) for the three fibre types are 1.4,8.5, and 55.
The low values for the maximal force and power output for the superficial fibres,
taken together with the paucity of these fibres in the myotome where they represent
only around 0.6 % of the cross-sectional area, shows that they can hardly play any
significant role in locomotion. Their force/velocity curve resembles that of frog tonic
Table 4.7. Contractile properties of dogfish muscle fibres (12 QC). Values represent mean ± S.E.
Numbers of fibres used are expressed in brackets. (Bone et al. 1986)
Parameter Fibre type
Superficial Red White
Maximum
Ca2+ -activated force 49 ±4 70 ±8 180 ±5
(Po, kN m- 2 )
Unloaded 0.47 ± 0.03 1.44 ± om 4.4 ± 0.3
contraction velocity (11) (9) (8)
V,lack(LOs-')
V max(LO s-') 0.58 . 1.53 4.5
(6) (2) (4)
0.08 0.12 0.17
0.05 0.22 0.69
fibres (Uinnergren 1978) in being more curved than that of the other fibre types, and
values for Hill's constant (a/Po) are similar for frog tonic fibres and dogfish
superficial fibres. It seems therefore that the dogfish superficial fibres may be con-
sidered (paradoxically) as non-locomotor myotomal fibres, equivalent to tonic fibres
in amphibia and reptiles. Thus they presumably playa postural role, although the fact
that they share motor innervation with the type I red fibres means that they are
activated during slow swimming.
One feature of these fibres which deserves further investigation is that they
produce, on maximum activation, much lower isometric tensions than white twitch
fibres, even when correction is made for different myofibrillar volume density. It
seems, therefore, that in the different myosin isotypes of the two fibres the tension
generated per myosin head, or the proportion of bound cross-bridges must differ.
The size,ease of dissection, and relative stability of dogfish isolated single fibre
preparations make them suitable material in which to examine this difference in more
detail.
The mechanical properties of batoid fibres have not been examined in any detail.
Johnston (1980) found tetanic fusion frequency in white fibres of the pectoral fin in
R. naevus to lie between 5-10 Hz (as compared with 10 Hz for dogfish white
myotomal fibres), but apart from these preliminary results, batoids have not been
examined.
4.3.3 Biochemical
The striking differences between the two main types of locomotor fibres in mito-
chondrial content, histochemistry and vascular supply (Sect. 4.1) naturally imply
that their metabolism must be very different. As Johnston (1981) emphasized in a
useful review, white fibre metabolism during burst swimming is much better under-
stood than that of the red fibres operating during sustained swimming. Most of the
work on fish muscle metabolism has been done on teleosts, but sufficient is known
of the biochemistry of elasmobranch muscle to show that in most respects muscle
4.3 Physiology 121
metabolism in the two groups is similar, although there are some interesting differen-
ces. These arise in part from the fact that in all elasmobranchs the white fibres are
focally innervated, and as far as is known, play no part in sustained swimming, unlike
the situation in the majority ofteleosts, where the white fibres are multiply-innervated
and may be involved in sustained as well as in burst swimming. In part also,
as Zammit and Newsholme (1979) suggest in their comparative study of lipid meta-
bolism in teleosts and elasmobranchs, teleosts have developed the ability to store and
mobilize non-esterified fatty acids, which elasmobranchs lack, and thus the lipid
metabolism of the two groups is very different.
As would be expected from its role in rapid swimming, and from its poor vascularity
and low mitochondrial content, the white muscle is fuelled by anaerobic glycolysis,
and the glycolytic pathway shows adaptations for high ATP turnover. Thus, for
example, the activities of the rate-limiting enzymes phosphorylase and phosphofructo-
kinase are around five times higher in dogfish white muscle than in red (Crabtree and
Newsholme 1972). It is notable that dogfish phosphorylase is not regulated by
circulating catecholamine levels, but its activity is directly Ca2 + -dependent (Fischer
et a1. 1975; Fischer et a1. 1978). Perhaps this reflects the poor vascular supply to the
white muscle, as 10hnston (1983) suggested, but in any event, the result is that
glycolytic activity in the white muscle is entirely dependent on Ca2 + release from the
sarcoplasmic reticulum triggered by motor nerve activity. Elasmobranch white muscles
(like those of other fish) contain large amounts of the Ca2 + -binding parv,t1hllmins
(e.g. Gerday and Teuwis 1972) which are assumed to function as Ca 2 ' huffers
between the Ca2 + binding sites of the contractile proteins and the sarcoplasmic
reticulum.
In red muscle, where parvalbumins are at much lower levels than in the white
muscle, a similar role is played by the abundant mitochondria, the paucity of mito-
chondria in the white muscle explaining the presence of the parvalbumin system.
This scheme of parvalbumin function (see Gillis and Gerday 1977) means that they
not only operate diIring relaxation by removing Ca2 + from the contractile proteins,
but also concomitantly decrease phosphorylase activity.
Burst swimming is necessarily brief, for glycogen stores in the white fibres are
rapidly diminished during such activity. For example, in dogfish, after 1-2 min
vigorous activity, white muscle glycogen declines to 1/20 of the level in rested fish
(Bone 1966), and the fish are then incapable of further rapid swimming (Giaja and
Markovic-Giaja 1957). The metabolic fate of the lactate resulting from anaerobic
glycolysis in the white fibres is not yet clear. The poor vascularity of the white muscle
suggests that it will not enter the circulation rapidly, and Zammit and Newsholme
found that blood lactate levels determined in dogfish 2 h after capture (when the
fish presumably used the white muscle system) were ten times those seen in rested
fish maintained for long periods in aquaria. The low activities of pyruvate carboxylase
and glucose-6-phosphatase in white muscle (Crabtree et a1. 1972) exclude the possiblity
of significant gluconeogenesis from pyruvate and lactate in situ, hence it seems most
probable that lactate passing into the circulation is oxidized to pyruvate at the
gills, liver and other peripheral sites. Further work is certainly needed here.
Red muscle metabolism is evidently very different, for it is highly aerobic, and
since red muscle contains both glycogen and lipid in greater quantity than the white
fibres, it seems likely that both will be utilized during sustained swimming.
Hexokinase activity in dogfish red muscle is almost 20 times that in the white
(Crabtree and Newsholme 1972), and as these authors have shown, there is a good
correlation between this activity and the calculated maximum capacities for glucose
oxidation. However, no significant difference in glycogen content was observed in
dogfish fibres after 50 h continuous slow swimming (Bone 1966). Lipid metabolism
in the red fibres is particularly interesting, for as Zammit and Newsholme (1979)
showed, non-esterified fatty acids are not important as fuel for the red fibres (as
they are for teleost red fibres). Rather, ketone bodies seem to be the fuel for the red
fibres, and are found in the serum, whilst D-3-hydroxybutyrate dehydrogenase is
present in the red fibres. This striking difference in lipid metabolism between teleosts
and elasmobranchs deserves further investigation, as does the relative importance of
lipid and glycolytic metabolism in the red muscles. Small sharks, such as dogfish,
offer excellent material for studies of metabolite utilization during slow swimming,
for they will swim continuously for long periods when made spinal and appropriately
set up, but this approach has only been used in a preliminary way so far.
An interesting feature of all elasmobranchs apart from the freshwater rays such
as Potamotrygon (Thorson et al. 1967) is that the tissue fluids contain amounts of
urea sufficient to denature mammalian proteins and inhibit mammalian muscle
enzymes. Whilst some elasmobranch enzymes are apparently adapted to this urea
concentration (e.g., Mt + -myofibrillar ATPase activity in vitro is maximal with
physiological concentrations of urea in the assay medium, Nishimoto 1981), many
others, such as creatine kinase and pyruvate kinase, are inhibited. Yancey and
Somero (1979, 1980) have shown that the perturbing effects of urea on proteins
and on enzyme function in vitro can be counteracted by methylamine compounds
such as trimethylamine oxide (TMAO), betaine and sarcosine provided that these
are in the ratio urea: methylamine compounds of 2: 1, the ratio in which these
compounds are actually found in elasmobranchs. Altringham et al. (1982) examined
the effects of urea and TMAO on the mechanical responses of dogfish skinned
fibres, finding that urea alone depressed maximum isometric tension, which could
be almost completely restored by the addition of TMAO (Table 4.8).
At present, the manner in which TMAO and other methylamine compounds act
as protein stabilizers compensating for the effects of urea is not understood (see
Table 4.8. The effect of physiological concentrations of osmoregulatory solutes on maximum

isometric tension. (Altringhan et aJ. 1982)
Fibre type Temperature Po (solute)jPo) x 100 %

(no. of fibres) . COC)
Urea TMAO Urea + TMAO
(no. of observations)
Fast (n = 4) 0.5 68.5 ± 2.29 106.5 ± 2.51 90.4 ± 0.95

(n = 6) (n = 4) (n = 6)
Slow (n = 3) 0.5 64.0 ± 2.5J' 108.7 ± 2.85 89.5 ± 1.50
(n = 3) (n = 3) (n = 3)
Slow (n = 6) 8 77.6 ± J.73 a 104.0 ± 1.29 95.1 ± 1.43
(n = 9) (n = 4) (n = 9)
a P < O.oJ.
4.4 Buoyancy and Lift 123
Yancey and Somero 1979). Evidently, where mechanical experiments are carried out
in Ringer solutions containing urea, the appropriate amount of TMAO should also
be added to the Ringer (for dogfish at Plymouth this involves addition of 5.6 g 1-1
TMAO.HCl).
4.4 Buoyancy and Lift
Elasmobranchs are constructed from materials that are denser than the water in
which they swim (Table 4.9) and hence either have to generate dynamic lift, or in some
cases, may gain sufficient static lift by storing low-density lipid that they achieve
neutral buoyancy and need not generate dynamic lift during horizontal swimming.
The lift-generating foils resemble aeroplane wings in several respects, since the
principles of lift generation in a fluid apply equally to both. Usually the foils involved
are the pectoral fins which, in fast-swimming sharks, are of high aspect ratio
span)
( AR: - - to minimize lift-associated vortex drag, and elliptical in planform for
area
the same reason. They are invariably set low on the body, thus avoiding pendulum
stability, and operate with pronounced anhedral, presumably again to avoid inherent
stability which would limit manoeuverability. Sphyrnid sharks have, in addition,
foils produced by lateral elongations of the head, most pronounced in Eusphyra
bloch ii, where the width of the hammer head is almost half the total body length.
In batoids, the flattened body and pectoral fins provide a large lifting surface,
since both are of aerofoil section.
But these lifting surfaces, (and that of the flat ventral surface of the head in many
sharks) which are anterior to the centre of gravity cannot by themselves provide the
sole lift required, for the couple about the centre of gravity involved must be
weight in water )
Table 4.9. Percentage weight in W<lter of tissues in different species ( ~. . . x 100 .
(Bone and Roberts 1969) weight In air
Cetorhinus Prionace Squalus Scyliorhinus Squatina
Whole fish 0.6 1.8 2,8 4.6 5.4

Fish less liver 2.9 2.5 3.6 5.8 5.5
Red muscle 3.5,2.7 2.9 2.8 4.8 a 5.4b
White muscle 2.6,2.5 1.9, 1.2 3.4 3.8 4.6
Pectoral cartilage 3.4 4.4 7.9 8.5 15.3
Vertebrae 12.9c 5.3 10.5 7.4 14.9
Skin 5.2,7.6 l5.8 d 11.2, 11.1 15.0 12.2
Subcutis 2.1 2.6
Gelatinous layer 0.6 0.6
a Including subcutaneous connective tissue network.

b Including horizontal septum.
c For calcified portion of centrum; the rest of the column is less dense.
d Determined after a long period deep-frozen.
balanced by an equal couple behind the centre of gravity when the fish swims
horizontally. In those batoids which swim by oscillating the tail (such as Torpedo or
Rhinobatos) the centre of lift of the disc is apparently anterior to the centre of
gravity, and hence has to be balanced by the motion of the tail, but in others,
where the tail is either not oscillated during forward swimming (Raja) or is
miniscule or even absent (Himantura, M obula) the centre of lift of the disc or triangle
must be assumed to be coincident with the centre of gravity, although this has not
been determined experimentally.
In sharks and those batoids where the tail provides forward thrust, its movement
must also provide dynamic lift if the fish are denser than water. Two possible
mechanisms have been suggested whereby the tail may generate lift. It has long been
supposed that the heterocercal tail generates lift as it sweeps across because the
hypochordal lobe (unstiffened by the vertebral column) will adopt an angle of attack,
so the tail generates lift because of its form. Alexander (1965) and Simons (1970)
have examined the action of isolated tails (using modifications of an apparatus
originally devised to determine the muzzle velocity of cannon !), and have shown
that lift is indeed generated as the tail moves through the water. Yet the neutrally
buoyant deep sea squaloids have heterocercal tails similar to those of dense fish,
and it is manifest that in these experiments, the intrinsic muscles of the tail are not
operating, so that the angles of attack need not be what it is in life, and the results
obtained do not necessarily represent what occurs in the living fish. Another possibi-
lity was raised by Simon's work, for he showed that in two very different sharks
requiring to generate dynamic lift (the fast-swimming Squalus and the slow Hetero-
dontus) the line of thrust from the tail passed above the horizontal (26 in the0
former and 12° in the latter), i.e., above the position of the centre of gravity.
Removal of the hypochordal lobe increased the deviation from horizontal of the
thrust line (up to 47° in Squalus). It seems improbable that in life, the thrust line
can be greatly above the horizontal, but even if it passed closely above the centre
of gravity, this would produce a couple tending to raise the tail and depress the head,
(quite apart from any lift generated by the tail being at an angle of attack as it
sweeps across), to counter the lift generated by the pectoral fins anterior to the
centre of gravity.
Thomson (1976) and Thomson and Simanek (1977) have considered the form
of the tail in a comparative study of over 50 sharks, and have extended Simon's
views about the angle of the line of thrust, concluding that the heterocercal tail
in its different forms can generate forward thrust over a range of angles in the
vertical plane; in forward swimming this passes through the centre of gravity.
As Blake (l983) points out, if the line of thrust passes through the centre of
gravity, then there is no lifting couple produced by the tail to compensate for
the lift provided by the pectoral fins, so that level swimming would not be possible
without lift generation by the former method. Further work is necessary to disentangle
the contribution of the two tail mechanisms in providing the required lift, and
kinematic studies of normally neutrally buoyant sharks (or those experimentally
made so) would be of interest here. It does seem clear, however, that the consequence
of this rather complex interaction between the direction of forward thrust and the
moment produced about the centre of gravity, and the lift generated by angling
the tail obliquely, is that it makes the fish highly manoeuevrable in the pitching
plane by slight changes in the rotation of the tail, coupled with changes in the angle
of attack of the pectoral fins.
Two kinds of special case should be mentioned. First, the neutrally buoyant deep
sea squaloids (Corner et al. 1969) might be expected to have symmetrical tails,
whose line of thrust would pass through the centre of gravity. In fact, their tails are of
a form similar to those of denser sharks (see Fig. 4.23) although they have much
reduced pectoral fins (needed only for manoeuvering, not for lift generation).
Presumably the line of thrust here passes directly through the centre of gravity. Other
sharks that are close to neutral buoyancy (e.g. Cetorhinus, Rhincodon and probably
Carcarhodon) have near-symmetrical tails, as has Torpedo. Secondly, the bottom-
living dense angel sharks (Squatinidae) have remarkable tails, for they are either
symmetrical or the hypochordal lobe is larger than the dorsal! Here, there are two
lifting surfaces, the pectoral and the pelvic fins, and it is likely that the latter lie
behind the centre of gravity, this acting as the tail planes of an aircraft and per-
mitting the tail to provide horizontal thrust only, without rotation as it moves trans-
versely.
The amount of dynamic lift which elasmobranchs require to generate depends
on their density. As will be seen below, although elasmobranchs vary widely in
density, the density of different species is relatively constant, and is largely deter-
mined by the amount of static lift provided by oil stored in the liver. The density of
different species can be related to their habits if we consider the consequences of
dynamic lift genenition (Bone and Roberts 1969).
Reduction in density and the amount of dynamic lift required permits the fish
either to reduce the speed at which the lift can be generated or to reduce the size
of the lifting surfaces. Such lifting foils as the pectoral fins of sharks incur skin friction
drag (proportional to y2) and lift-associated vortex drag (proportional to Ijy2).
Reduction in pectoral fin area with reduced density will significantly reduce the total
drag opposing forward motion [and as Corner et al. (1969) showed, neutrally buoyant
sharks have relatively small pectoral fins], but there will be a minimum size set by
the need for their use in manoeuvering. These considerations suggest that reduction
in density will be useful for fast-swimming pelagic sharks, since this will lead to
reduction in the skin friction drag incurred by the reduced lifting surfaces, but that
neutral buoyancy will not lead to important benefits since vortex drag is relatively
unimportant at higher swimming speeds, and the pectoral fins (which cannot be furled
like those of teleosts) have to be of a certain minimum size. On the other hand,
neutral or near-neutral buoyancy is appropriate both for those sharks which require
to swim slowly as part of their feeding pattern (like Cetorhinus), where vortex drag
is relatively more important and skin friction drag is also reduced, and for sharks which
swim very slowly or hover, (like the deep sea squaloids).
These inferences accord well with the density of the different sharks (Baldridge
1972; Bone and Roberts 1969) that have been measured, with some exceptions
discussed by the latter authors.
Elasmobranchs gain static lift from oil stored in their livers, sometimes sufficient
to confer neutral buoyancy. The use of lipid rather than gas (as in bony fish) has
advantages and disadvantages. A relatively large volume of lipid must be stored to
provide a significant reduction in density of the fish, and the regulation of lipid
metabolism is likely to be complex, but at the same time, the lipids stored are of a
compressibility comparable to seawater, so that the lift provided varies little with
changes in depth and ambient pressure.
Deep sea squaloid sharks are very close to neutral buoyancy because they have
greatly enlarged livers (up to 30 % of the total weight of the fish) which contain
enormous quantities of oil (Corner et a!. 1969). These vast oil tanks in some cases
consist of 90 % of oil by weight, and not only is there so much lipid, but it is of
especially low density, being almost entirely composed of the low density hydrocarbon
squalene (s.g. 0.86). In dense sharks, such as Scyliorhinus, where static lift from liver
oil is unimportant, the oil in the liver is not only much less abundant, but it is of much
higher density (Specific gravity 0.93).
After the discovery of neutral buoyancy in the deep sea squalids by Corner
et a!. (1969), comparative studies of the density and livers of a range of elasmo-
branchs were carried out by Bone and Roberts (1969) and Baldridge (1972). These
showed that different species varied widely in density, although for a single species
only a small range of density was found (Table 4.10).
Table 4.10. Mean percentage weight in water of different species of elasmobranch

weight in water )
( --=------ x 100. (Bone and Roberts 1969)
weight in air
Species Mean % wt Range No. of

in water detelminations
Cetorhinus maximus 0.33 0.09-0.56 2

Lanum nasus 3.2 2.5 -4.3 3
Prionace glauca 2.5 1.6 -3.4 6
Squalus acanthias 2.7 2.4 -3.3 7
Dalatius licha -0.1 1
Galeorhinus galeus 3.S 2.9 -4.1 5
Mustelus asterias 3.9 3.8 -4.6 5
Cephaloscyllium uter 4.1 1
Scyliorhinus canieula 4.7 3.7 -S.9 30
S. stellaris S.O 4.S -S.S 3
Squatina squatina S.S 4.7 -6.9 8
Dasyatis pastinaca 3.1 1
Raja clavata S.S 3.9 -7.6 4
R. 1110ntagui 4.8 4.3 -S.2 4
R. microocellata 4.8 1
Torpedo nobiliana 0.4 0.1 -0.8 14
T. marmorata 2.2
With a few exceptions, such as Torpedo (where the low density of the electric organ
is significant), this variation in density depends entirely upon the size of the liver,
directly related to its lipid content (Fig. 4.21). It is therefore feasible to estimate the
density of the fish from the relative size of the liver, if direct density measurements
are not available. For example, two deep sea rays, R. richardsonii and Breviraja pallida
(Forster 1965, 1967), have very large livers, and are probably neutrally buoyant.
----
o
~ 80
-
•• • •
•
--
'0 Ui" •
>
• ••
••I- • •
'+-
o :=
••
1: '0 60
Ol .....
'w -§,
•
• •• -
?: 'w
?: •• • •
~40
'0
<IJ
Ol
o
C 20
<IJ
U
L..
<IJ
0..
o 5 10 15 20 25
Percentage liver (L /W ·1001
Fig. 4.21. The relation between relative liver size and amount of oil within the liver in a range of
elasmobranchs. Solid line is based on the assumption that increase in liver size is due solely to
increase in oil content, liver tissue after oil extraction being a constant % of the weight of the
fish. W wt. of fish; L. wt. of liver. (Bone and Roberts 1969)
The role of the liver in many elasmobranchs in providing static lift obviously may
conflict with its other roles of providing reserve lipid for the adult or for the
embryos, and it seems probable that starvation and pregnancy will in most cases alter
the density of the fish by diminishing the lipid stored in the liver. For example, Ranzi
and Zezza (1936) observed that in Dasyatis violacea the liver at full term might be
only one third of the size prior to mating.
In other cases, however, as in Squalus (Bone and Roberts 1969), the fish regulates
liver and muscle lipid during pregnancy to maintain constant density and here it is
obvious that the regulation of lipid metabolism and the control of density must not
only be complex, but also relatively rapid.
Malins and Baron (1970) compared liver lipids in Squalus that had been weighted
to increase their density with those of unweighted controls, and showed that the
experimental animals, whose density had been increased, altered liver lipid by in-
creasing the amount of diacyl glyceryl ethers (DAGE) with respect to triglycerides.
Since the specific gravity of DAGE was (at 25°C) 0.908, and that of the triglyceride
0.922, this change has the effect of increasing static lift from the liver to com-
pensate for experimentally increased density. It is noteworthy that significant
changes in the DYGEjtriglyceride ratios were observed after only 50 h. In vitro and
in vivo studies (Malins 1968; Malins and Baron 1970; Malins and Sargent 1971)
and Sargent et al. (1971) have shown that triacyl glycerols are rapidly synthesized in
the intestinal mucosa and liver, and that turnover is much less rapid for alkyl
diacyl glycerols, which are therefore relatively conserved. It is evident that lipid
metabolism in elasmobranchs and its role in controlling density presents very
interesting physiological and biochemical problems, and deserves further investigation.
4.5 Swimming
Analysis of swimming in elasmobranchs has lagged behind that in teleosts [as is sadly
evident from the scarcity ofreferences to elasmobranchs in the index of Blake's (1983)
useful book on fish locomotion], partly because the important results obtained for
teleosts from fish swimming in tunnel respirometers (e.g. Webb 1971) are not available
for elasmobranchs. The large amplitude lateral movements of the tail in small sharks,
together with the flexibility of the body, make them unsuitable for such experiments
(Brett and Blackburn 1978), for if the tunnel is wide enough for normal tail beat,
it is also wide enough for the fish to turn around. However, it would seem feasible to
use small spinal sharks in tunnel respirometers and this approach should yield
useful data.
Thus analysis of swimming in elasmobranchs has been either from the theoretical
viewpoint (Weihs 1981); by the comparative study of sharks of different habits
(Thomson and Simanek 1977), sometimes using model fish in wind or water tunnels
(Harris 1936), or has depended on kinematic studies of free-swimming sharks in large
aquaria. As Webb and Keyes (1982) point out, it is very difficult to induce free-
swimming sharks to swim over a wide speed range, and so this last approach has
largely been confined to sustained cruising speed swimming; little is known of burst
swimming in sharks. Exit velocity of jumping makos is around 10m s -1 (Carey and
Teal 1969), presumably close to burst maximum.
4.5.1 Swimming Speeds
For some species, cruising speeds measured from sharks in large aquaria or by
satellite or sonar tracking of free-swimming fish accord well with the theoretical
expectations based on minimal energy expenditure for distance travelled, given by
Vopt = 0.503 LO. 43 (Weihs 1977). For example, Weihs et al. (1981) found 2-m sharks
of two carcharinid species to swim at speeds close to the expected 68 cm s -1 ;
Scyliorhinus 50 cm long swim close to the expected 28 cm S-I; and a 9-m Cetorhinus
swam at 94 cm S-1 (Harden Jones 1973), close to the expected 1 m S-I. The shark
tracked by Harden Jones was swimming 8 m below the surface, whereas that tracked
by Priede (1984) using the ARGOS satellite swam at a maximum speed of 74 cm S-1
at the surface, where wave drag will increase the total drag incurred (Blake 1983); it
was feeding with wide-open mouth. The shark species examined by Webb and Keyes
(1982) apparently swam somewhat above the predicted optimal cruising speeds,
whilst the wlrite shark tracked by Carey et al. (1982) swam a little more slowly, but
nevertheless, Weihs's formula provides a useful guide to the expected cruising
speed of sharks where body length is· the only measurement known. Klimley and
Brown (1983) have described a stereophotographic technique for determining the
length of free-swimming sharks (which they used with Sphyrna lewini); in principle,
by taking stereo pairs at known time intervals, their technique could also be used to
determine swimming speed. An alternative estimate of cruising speed, (where the den-
sity of the shark is known), can be obtained by estimating the minimum speed at
which sufficient dynamic lift can be provided by the pectoral fins, for Scyliorhinus
swimming speed so estimated is in good agreement with those observed.
4.5 Swimming 129
4.5.2 Body Form and Fin Distribution in Sharks
Thomson and Simanek (1977) have made an interesting comparative study of body
form in a wide range of sharks, and have grouped them on the basis of body form
and tail shape into four groups (Table 4.11); typical representatives of each group
are illustrated in Fig. 4.22. The first group consists of sharks characterized by a high
aspect ratio tail with large heterocercal angle; with deep bodies and lateral flukes on
the caudal peduncle. The second is rather similar, but the heterocercal angle is
smaller, lateral flukes are lacking, and the body is less deep. The head is flatter
ventrally than in the first group. The third group contains sharks with large blunt
heads, relatively elongate tails of low heterocercal angle, and with posteriorly
attached first dorsal fins and anteriorly attached pelvic fins.
Table 4.11. List of shark by genus in the four groups defined in the text on the basis of general body
and tail shape. (Thomson and Sinaret 1977)
Group 1 Group 2 Group 3 Group 4
Carcharodon A lop ias Apristurus Centrascyllium

Cetorhinus Aprionodon Galeus Centroscymnus
Isurus Carcharias Ginglymostoma Dalatias
Lamna Carcharhinus Mustelus Echinorhinus
Rhincodon Galeucerdo Pseudotriakis Etmopterus
Hypoprion Scyliorhinus Isistius
Negaprion Triakis Somniosus
Paragaleus Squalus
Prionace
Scoliodon
Sphyrna
The final group is the squaloid sharks, distinguished by the absence of the anal
fin, and a large epicaudal lobe on the tail.
As Thomson and Simanek observe, the taxonomic position of the sharks in
each group (apart from the last) is diverse, and the groups fit better with differences
in mode of life. The first group, however, contains both very fast and rather
slow-swimming sharks. It seems probable that both types require to minimize drag
(to increase speed, or for the filter feeders, to reduce the cruising power needed)
and that this has led to similar body form. The second group are generalists, whilst
the third are regarded as slow-swimming demersal and benthic species. There are a
number of exceptions to this broad division into four categories, e.g. the thresher
sharks (Alopias) whose tail is apparently specialized for a particular feeding mode,
or the six- and seven-gilled sharks (Hexanchias and Heptranchias) which resemble
those of the third group, but lack a second dorsal fin and have a large ventral
hypochordal lobe on the tail. Unlike the other sharks in the third group, it seems
likely that both Hexanchias and Heptranchias are close to neutral buoyancy, (Bone
and Roberts 1969) and their caudal fins resemble those of the neutrally buoyant
deep sea squaloids.
Fig. 4.22. Representative sharks from the four groups recognized by Thomson and Simanek (1977).
A Carcharodon carcharias; B Galescendo curvier; C Centroscylli!im fabricii; D Ginglymostoma
brevicaudatum. (After Compagno 1984)
Thomson and Simanek (1977) found that the posItIOns of the paired and
unpaired fins in the sharks that they examined were strikingly uniform in the
position of insertion on the body, whatever the shape of the caudal fin, as might be
expected from their function in controlling pitch and yaw (Harris 1936). However,
in the deep-sea squaloid sharks, and the small pelagic squaloids, the position and
size of the unpaired fins varies greatly (Fig. 4.23). Both groups are either known or
assumed to be very close to neutral buoyancy and presumably swim slowly, hence do
not require large control surfaces.
Weihs (1981) has observed that many sharks, particularly those of the second and
third group above, show a marked change in cross-sectional shape along the body
(Fig. 4.24), which can be related to the amplitude oflateral motion at different distances
along the body; those regions with the largest lateral movements being "streamlined"
so that they show an elliptical section flattened in the horizontal plane, whereas
lateral movement which would lead to flow separation is minimized in the mid-region
of the body by lateral compression. He also noted that the "double tail" formed by
the second dorsal and ventral fins in front of the caudal fin would be advantageous
for rapid manoeuevring; this feature has been further examined by Webb and
Keyes (1982) in a kinematic study.
4.5 Swimming 131
c~
c
Fig. 4.23. Body form and fin position in some squa10id sharks. A Echinorhinus cookei; B Oxynotus
bruniensis; C Isistius philodus; D Euprotomicrops bispinatus; E Centroscymius coelolepis. (After
Compagno 1984)
Fig. 4.24. Body section variation in Triakis acutipinna. (After Weihs (981)
4.5.3 Kinematics of Shark Swimming
The only detailed information about the kinematics of swimming is provided by the
study of Webb and Keyes (1982) of six shark species belonging to groups 2 and 3
of Thomson and Simanek (1977). All swam in a sub-carangiform mode, but showed
significant differences to the teleosts swimming in this way.
In these sharks, the maximum rate of change of amplitude along the body occurs
around the region of the posterior margin of the first dorsal fin, and then declines
towards the tail, whereas in the more anguilliform teleosts the maximum is found
more anteriorly, and in the more fusiform the rate of change of amplitude
increases towards a maximum at the tail (Fig. 4.25). Evidently, it would be interesting
to examine the faster-swimming more fusiform sharks of group 1.
Webb and Keyes also found that in the blacktip sharks (which they examined in
most detail), change of swimming speed involved not only changes in tail beat
LLJ
0
~
I- 0.8
:J
Q. " fusiform
~ 0.6 " / ~ ~ sharks ....teleosts
U:I: / '" • <-'
i!;t;
frlz 0.4 .\~
,
Q.LLJ
(/)....1 " .... .... • "
11...>-
00 0.2 • ........ -~-
sharks
LLJo
(!lID
0
" •
~:I: Anguilla
:I: I-
u-
II...~
0
LLJ
-0.4 " I I I I I I
~ 0 0.2 0.8 1.0
0:: 0.4 0.6
(nose) LOCATION ALONG BODY (toil)
Fig. 4.25. Variation in amplitude of body movement along the body in sharks compared with Anguilla
and fusiform teleosts. (Webb and Keyes 1982)
1.4
:I:
t;
1.3 •• •
ifi....I •••
.... .
1.2
LLJ •
~
3=
1.1 ••
U 1.0 VL-1.29-0.17:1:0.06 U/L
iL ,/
(3
LLJ
Q.
0.9 •
(/)
I 0.8
....I
.....
;<. 0.7 •
•
0.6
0 2 3 4
U/L-SPECIFIC SWIMMING SPEED (L·s-I )
Fig. 4.26. Changes in wavelength with swimming speed in Carcharinus melanopterus (filled circles).
Open triangle Triakis .. filled square Sphyrna tiburo. Regression line fitted to C. melanopterus data.
(Webb and Keyes 1982)
4.5 Swimming 133
frequency (as in teleosts) but also changes in the wavelength of the propulsive wave
(Fig. 4.26) and changes in tail beat amplitude. They pointed out that two quite
different reasons might account for the fact that sharks modulate amplitude and
wavelength with speed whilst teleosts do not. First, because the caudal fin is flexible
in teleosts, they can vary the depth of the trailing edge of the caudal fin to change
the mass of water accelerated backward at each tail beat; sharks with fins that
are not of variable geometry cannot do so. Secondly, the more complex changes with
swimming speed in sharks may be related to advantages to be gained from flow
interactions between the first dorsal and caudal fins. Lighthill (1970, 1975) has
pointed out that if there is a large gap between the dorsal fins on an oscillating fish,
then the vortex sheet shed by the trailing edge of the anterior fin may interact with
the leading edge of the second fin, if the phase difference between the two fins is
appropriate, to increase thrust and efficiency. The requirement for an advantageous
interaction is that the phase difference 0 between the movements of the vortex sheet
and the leading edge of the second fin should be close to 0.5 7t. 0 is given by:
<p = 27t
(C
(a z - ad - - 1 ) (LighthillI975), where ai' az = trailing edge of anterior
Ie U
fin and anterior edge of second fin, c = backward speed of propulsive wave, and
U = forward speed of the fish.
Webb and Keyes found values for 0 between the first dorsal and the caudal fin
close to 0.5 7t, hence inferred that at constant speed, sharks indeed utilize flow
interactions between the first dorsal and caudal fin in the way suggested by Lighthill
(1970). As they observe, since the positions of the first dorsal and caudal fins are
fixed, the shark might vary the frequency of tail beat or the wavelength of the
propulsive wave to maintain 0 close to 0.5 7t at different speeds, but such changes
will affect the thrust generated. Thus if Ie is varied, compensatory changes in tail beat
frequency and/or amplitude must take place so that the necessary thrust can be
generated, and it is perhaps for this reason that sharks modulate these parameters as
they change their swimming speed. The kinematics of shark swimming have been
Fig. 4.27. Mobilia diabolis. Paths traced by inner and outer regions of the pectoral fin (shown on view
of fish) during a wing beat cycle. The angles of the two fin regions are shown at intervals during
the stroke. (After Klausewitz 1964)
studied only over a small speed range, effectively during cruising locomotion, and
Webb and Keyes suggest that most sharks are cruising designs, rather than designs
for rapid acceleration, and thus adaptations improving cruise efficiency (such as the
flow interaction outlined above) which improve thrust and efficiency in constant-
speed swimming are likely to be important. Other adaptations to increase cruise
performance are considered in the next section. Some sharks, however, notably some
of the deep sea squaloids like Echinorhinus cookei or the Oxynotidae, have large
posterior 2nd dorsal and pelvic fins which effectively make them "double-tailed"
and presumably are adaptations for rapid acceleration (Weihs and Webb 1983).
It is unfortunate that although Marey (1894) long ago provided excellent kine-
matic studies of swimming rays, almost nothing is known of batoid locomotion.
Klausewitz (1964) analyzed the swimming of Mobula from a film taken underwater
during the Xarifa expedition (1957-58), and described the elliptical path taken by the
tip of the pectoral fin and the different angles of attack during the cycle of
"wingbeats" (Fig. 4.27), but no quantitative observations have been made. Roberts
(1969a) made similar observations of a non-quantitative kind on the electric ray
Torpedo, which swims by oscillating the tail rather than with pectoral fin oscillations.
Further analysis of cruise swimming in rays should be of much interest, for the small
proportion of red fibres in the pectoral fins suggests that it is a very efficient
process.
4.5.4 Drag-Reducing Adaptations
Various mechanisms have been proposed which may reduce drag and thus increase
the efficiency of shark locomotion. Weihs (1973) suggested that fish which are denser
than water could advantageously adopt a two-phase swimming technique to obtain
a large saving in energy expended over a given distance. He suggested that alternate
gliding downwards and active swimming upwards would be advantageous, since the
drag incurred during active swimming is likely considerably to exceed that during
gliding (when lateral motions of the body are absent), and so such a swimming pattern
would be more economical. As yet, although sharks have been fitted with transmitting
devices to record depth, and have been followed for considerable distances, the time
resolution is not sufficient to know whether they adopt this technique of swimming
(e.g. Carey et al. 1982).
Other workers have suggested that sharks may control boundary layer conditions
around their bodies with suitably patterned dermal denticles. The denticles along the
flank of Scyliorhinus provide a surface roughness of approximately the appropriate
dimensions (those furthest downstream project around 0.12 mm) that they may be
employed to induce transition to a turbulent boundary layer. and hence delay
separation (Bone 1975). A more intriguing suggestion (which, unlike most suggested
drag reduction mechanisms for fish, has received some experimental support), has
been made by Reif and his co-workers (Reif 1978, 1985; Reif and Dinkelacker 1982;
Bechert et al. 1985). The suggestion is that the longitudinal sharp ridges and rounded
valleys found on the denticles of such fast-swimming sharks as carcharhinids and
isurids (Fig. 4.28) are designed to reduce drag within a turbulent boundary layer.
The experimental discovery of low speed "streaks" in the laminar sub-layer of a
4.5 Swimming 135
turbulent boundary layer, and the turbulent wall bursts to which they give rise,
suggested that longitudinal riblets could confine and reduce these bursts, so reducing
drag by reducing turbulent shear stress. Wind tunnel experiments on flat plates with
longitudinal riblets showed drag reductions up to 7 % compared with the drag on
smooth plates (Walsh 1980). The scale of the riblets on the denticles of isurids is
approximately correct for drag reduction by this means (there is uncertainty about
the burst swimming speed of the fish), and the spacing of the riblets is also
appropriate. On a short fin mako (!. oxyrhynchus) 2.2 m long the denticle riblets are
35 to 50 J.lm apart, which would give optimal drag reduction at swimming speeds
between 10 and 20 m S-I.
~
.. ..'" Fig. 4.28. Dermal dentic1es of isurid
sharks. a Lamna nasus; b Isurus
"
b
oxyrhinchus. (After Bigelow and
500 jJm Schroeder 1948)
Bechert et al. (1985) discuss the experimental data available, and what is known
of the theoretical basis for drag reduction by longitudinal riblets. These authors
also suggest that shark denticles may act as vortex generators, entraining fluid from
the free stream into the boundary layer and so preventing separation. Their model
experiments showed that the angle of attack of the denticles (which in some species
are anchored elastically) was important in such a role, and they suggest that in those
species where the denticles have "compliant" suspension, the angle of attack might
be controlled by the shear stress, so that close to separation, the angle of attack would
be increased and fluid entrainment enhanced. To this ingenious idea, they added the
further speculation that fluid ejection tangentially to the boundary layer flow could
result from water flowing along the skin between the denticles being ejected during
lateral movements of the body.
As they point out, both of these suggested mechanisms require experimental testing,
which does not seem an easy matter. Both mechanisms have previously been suggested
for the teleost Ruvettus (Bone 1972 b), but in that case, although experimental evidence
is also lacking, the structure of the integument ismuch more complex than in sharks,
and it is difficult to think of alternative reasons for the specializations found.
Obviously further experiments are required, but it is hard to think that all the devices
for boundary layer drag reduction or to prevent or delay flow separation that are
known to hydrodynamicists have not been employed by sharks designed for
maximum burst speed or maximum cruise economy.
4.5.5 Warm-Bodied Sharks
Carey and Teal (1969) made the remarkable discovery that two species of lamnid
sharks (Isurus oxyrhynchus and Lamna nasus) were able to conserve the metabolic
heat generated by the red muscle during cruising to maintain the myotomal muscle
7-10 °C above ambient water temperature, and subsequently Carey et al. (1982)
showed that Careharodon carcharias was also warm-bodied, although this shark did
not"thermoregulate. To conserve heat, a counter-current heat exchanger is required.
This is formed by the rete mirabile supplying the "internalized" red muscle
(Fig. 4.29), supplied by a large cutaneous artery (the dorsal aorta is much reduced),
and draining into the parallel cutaneous vein.
a b
Fig. 4.29. The retial system of [surus oxyrhinchus. a Position of internalized red muscle mass
(black ) at dorsal fin level ; b muscle rete ( r) formed by capillaries arising from the cutaneous
artery ( a) and passing to the cutaneous vein ( v) . Red muscle dotted. (After Carey et al. 1985,
and Carey and Teal 1969)
In the mako, the rete is confined to a narrow band along the flank of the fish,
whilst in the porbeagle and Carcharodon the retia are more diffuse. In the big eye and
common thresher sharks (Alopias superciliosus and A. vulpinus) there are simpler
retia, consisting of vascular bands similar to those supplying the white region of the
myotomes in the other species. The currently available data on lamnid shark tem-
peratures are reviewed by Carey et al. (1985).
The function of elevated muscle temperatures in these sharks (and indeed in
scombrids, which show remarkable parallel adaptations) is not yet clear. In such fish
it would seem a relatively simple matter to arrange for thermoregulation, and Carey
et al. (1971) have provided clear evidence that blue fin tuna can thermoregulate.
However, the experiment in which water temperature, muscle temperature and depth
were telemetered from a free-swimming Carcharodon (Carey et al. 1982) provided no
evidence for thermoregulation ; muscle temperature changed slowly as water tem-
perature changed (Fig. 4.30).
Night Day
25
20 ~
15
()
0
10
0
2400 1200 2400 1200 2400 1200 2400
2 3 4 July 1979
Dat e and Time
Fig. 4.30. Simultaneous records of ambient water temperature ( lower line) and white muscle tem-
perature from a free-swimming Carcharodon carcharias, followed for 4 days. The dotted horizontal
lines indicate average water temperature before and after a 2 °C increase at 2100 on 3 July.
The initial rate of increase in muscle temperature which followed is shown by the upper oblique line.
(Carey et al. 1982)
Evidently, if thermoregulation can be achieved, an obvious advantage is that

the muscles can be operated at a more or less constant temperature, and muscle
enzymes adapted to act at a single most efficient temperature. As yet, the thermal
sensitivity of muscle enzymes from these sharks has not been examined. Even in the
case of Carcharodon , where muscle temperature varies as ambient water temperature
alters, it does so relatively slowly, and is always above ambient (Fig. 4.30), so that
the muscle enzyme systems operate over a fairly narrow temperature range. The
mitochondrial content and diffusion distances are not known for "warm" red
muscle fibres and an ultrastructural study to compare such parameters with those of
"cold" sharks would be of interest.
4.6 Concluding R-emarks
This brief survey of elasmobranch locomotor muscle, buoyancy, and swimming has
shown that whilst in many respects elasmobranchs resemble teleosts, with which,
for all aspects considered, very much more work has been done, there are certain
notable differences, which in some ways make elasmobranchs more suitable for the
study of general fish physiology. For example, that the white fibres of all elasmo-
branchs are focally innervated, and play (so far as is known) no part in sustained
swimming, makes the study of the metabolism and physiology of the two motor
systems simpler than in higher teleosts. Again, the smaller elasmobranch species are
hardier than teleosts and it is easier to obtain suitable muscle preparations for
electrophysiological work. There are, however, some corresponding disadvantages.
Neither small sharks nor any batoids are well adapted for studies in tunnel respiro-
meters (though spinal sharks might be used); such studies have proven extremely
useful with teleosts for analysis of swimming patterns and for swimming metabolism.
Batoids have received much less attention than sharks, as far as neuromuscular
physiology and swimming is concerned, and there are many points which would
repay study here, for example, the role of ground effect (see Blake 1983) in
swimming, or the efficiency of pectoral fin swimming versus tail oscillation.
With sharks, the need is for studies of the larger fast-swimming forms such as
isurids and lamnids, not only kinematically, but also to see whether they have
achieved parallel adaptations to teleosts of similar habit in such features of the loco-
motor system as muscle fibre trajectories in the myotomes, and muscle fibre ultra-
structure.
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Chapter 5 S. NILSSON and S. HOLMGREN
The Autonomic Nervous System
The term autonomic nervous system was coined by Langley (1898) to describe such
nervous pathways outside the central nervous system which are involved in the control
of "involuntary" or "vegetative" functions. The terminology of Langley (1898, 1921)
provided a subdivision, on anatomical grounds, of the mammalian autonomic nervous
system into three parts: the sympathetic nervous system, comprising nervous pathways
with thoracic and lumbar connections with the spinal cord; the parasympathetic
nervous system, with pathways in the cranial and sacral nerves; and the enteric
nervous system comprising the intrinsic neurons of the gut. A simple definition of the
autonomic nervous system as all efferent nervous pathways which have a ganglionic
synapse outside the central nervous system (CNS) was offered by Campbell (l970a).
Autonomic pathways of the sympathetic and parasympathetic divisions generally
comprise two neurons, one preganglionic neuron which leaves the CNS to reach an
autonomic ganglion in the periphery, and one postganglionic neuron with its cell
body (soma) within the ganglion. The postganglionic neurons are innervated by the
nerve terminals of the preganglionic neurons.
In mammalian terminology, the anatomical term sympathetic has often been held
synonymous with the functional term adrenergic. This is because most postganglionic
sympathetic neurons are adrenergic, i.e. they release adrenaline or noradrenaline as
the transmitter substance. Similarly, the anatomical term parasympathetic is some-
times confused with the functional term cholinergic, since it was thought that most
of the postganglionic parasympathetic neurons act by release of acetylcholine. How-
ever, non-adrenergic postganglionic sympathetic pathways are well known, and
there are postganglionic parasympathetic pathways that are neither adrenergic, nor
cholinergic (see, e.g. Nilsson 1983). Therefore, it is crucial to maintain a clear
distinction between anatomical and functional terms in describing the autonomic
nervous system.
Autonomic nerves reach practically all parts of the vertebrate body, and serve in
the regulation of a number of functions essential for maintenance of homeostasis.
Autonomic fibres thus control smooth muscles (e.g. blood vessels, spleen, gut, bladder
and gonadal ducts, iris), the heart, a number of glands (including the catecholamine-
secreting adrenal tissue and enterochromaffin cells of the gut mucosa) and, in teleosts
and reptiles, the melanophores.
Authors' address: Comparative Neuroscience Unit, Department of Zoophysio1ogy, University of

Giiteborg, PO Box 250 59, 400 31 Giiteborg Sweden
144 Chapter 5. The Autonomic Nervous System
The pioneer work on the functional anatomy of the elasmobranch autonomic

nervous system was done by J. Z. Young in the early 1930's (Young 1933a, b), and
there are also early anatomical descriptions of autonomic nerves (Stannius 1849;
Chevrell887) and chromaffin tissue (Leydig 1853). Reviews dealing with the vertebrate
autonomic nervous system, including the elasmo branchs, are those by Nicol (1952),
Burnstock (1969), Campbell (1970b), Pick (1970) and, especially, by Nilsson (1983).
In later years, introduction of modern pharmacological and histochemical
techniques has furthered the knowledge of the structure and function of the
elasmobranch autonomic nervous system. The aim of this chapter is to summarize
the present knowledge of the functional anatomy of the autonomic nervous system,
and the related system of endocrine cells of the gut and the chromaffin tissue in
elasmobranch fish. Since the structure and function of the elasmobranch enteric
nervous system has not previously been reviewed in any detail, particular emphasis
is put on this system.
5.1 Anatomy of the Autonomic Nervous System
5.1.1 Terminology
The terms sympathetic, parasympathetic and enteric were originally used to describe
the autonomic nervous system of mammals, where the anatomical demarkation
between cranial, thoracic, lumbar and sacral is relatively clear. When applying the
same terms to describe the autonomic nervous system in the non-mammalian ver-
tebrates, it becomes difficult to distinguish between a sympathetic and a sacral
parasympathetic part of the autonomic n«rvous system. A simplified terminology is
therefore proposed (see Nilsson 1983, for discussion), in which the term cranial
autonomic describes the autonomic pathways leaving the CNS in the cranial
nerves, and spinal autonomic describes the autonomic pathways leaving the spinal
cord. The spinal autonomic system will thus comprise both the sympathatic pathways
and the sacral parasympathetic pathways in Langley's terminology. The term enteric
is retained in its original meaning to describe the intrinsic neurons of the alimentary
canal.
5.1.2 Cranial Autonomic Nerves
Cranial autonomic (parasympathetic) pathways in elasmobranchs run in the oculo-

motor (III), the facial (VII), the glossopharyngeal (IX) and the vagus (X) nerve
(Fig. 5.1).
A distinct ciliary ganglion is present close to the oculomotor (III) nerve, and
postganglionic fibres from this ganglion run to the eye in the short ciliary nerve.
The fibres innervate the iris (see Sect. 5.7), the retractor lentis muscle and blood
vessels of the eye (Young 1933a, b).
Autonomic nerve fibres have been described in the hyomandibular branch of the
facial nerve (VII), where they probably run to the pharyngeal smooth muscle (Young
v.
5'
o'"
a
'<
o....,
;-
(D
>-
<=
o::>
o
a
(i.
~
z
spiral intestine ""o<=en
<:I)
';;i
Fig. 5.1. The autonomic nervous system in an elasmobranch (based mainly on Young 1933b). Note chromaffin tissue (coarse stipple) associated with g
paravertebral ganglia. Abbreviations: an anastomosis between spinal autonomic and cranial nerves; ani spl n anterior splanchnic nerve; cil g ciliary ganglion;
mid spl n mid splanchnic nerve; post spl nn posterjor splanchnic nerves. Roman numbers refer to the cranial nerves. (Nilsson 1983)
v.
"""
1933 b). Glossopharyngeal (IX) and vagal (X) fibres innervate the anterior part of the
gut: vagal fibres reach as far as the pylorus and anterior part of the intestine.
Vagal fibres running in the most posterior branchial branch and in the cardiac
visceral branch reach the heart, where a vagal ganglion is present at the sino-atrial
border (Young 1933b). There is, however, no evidence for an autonomic innervation
of the branchial vasculature (Metcalfe and Butler 1984).
5.1.3 Spinal Autonomic Nerves
The spinal autonomic (sympathetic) system of elasmobranchs is less well developed

than that of the teleosts and tetrapods. The paravertebral ganglia are arranged
segmentally, except in the most anterior part, but longitudinal connections are irregular
and there are no distinct sympathetic chains of the teleost/tetrapod type. As in the
higher vertebrates, preganglionic myelinated fibres from the spinal cord enter the
paravertebral ganglia via white rami communicantes, but the existence of recurrent
grey rami has not been established, and a distribution of postganglionic spinal
autonomic nerve fibres together with the spinal nerves is thus unlikely (Stannius
1849; Chevre11887; Young 1933b; Nilsson 1983).
A special feature of the elasmobranch paravertebral ganglia is the occurrence of
clusters of catecholamine-containing chromaffin cells in association with the ganglia
(Fig. 5.1). Within the anterior venous sinuses in the anterior part of the abdomen,
a pair of particularly large chromaffin cell masses are associated with paravertebral
ganglia (the gastric ganglia) to form the axillary bodies (which were called Axillar-
Herz by Leydig 1853). In chimaeroids, the chromaffin cell masses of the axillary
bodies completely surround the subclavian arteries (Pettersson and Nilsson 1979).
The axillary bodies give off the anterior splanchnic nerves, which run along the
coeliac artery to the gut and, possibly, to the spleen. Contrary to the case in the
higher vertebrates, the splanchnic nerves of elasmobranchs and teleosts are composed
of postganglionic fibres only (Nilsson et al. 1975; Nilsson 1983; Nilsson and Holm-
gren 1983).
A mid splanchnic nerve leaves the middle trunk ganglia on the left side and runs
along the anterior mesenteric artery to the spleen, pancreas and anterior part of the
spiral intestine (Fig. 5.1). In the posterior part of the abdomen, several posterior
splanchnic nerves are given off from the segmental paravertebral ganglia and run to
the posterior part of the gut and the uro-genital organs (Young 1933 b; Nilsson
1983).
Anastomoses between the spinal autonomic system and the cranial autonomic
system occur as a thin connection between the vagus and the small paravertebral
ganglia anterior to the axillary bodies (see Fig. 5.1), and as connections betwet:n
the vagus and the anterior splanchnic nerve(s) (Young 1933b, 1980).
5.1.4 Enteric Autonomic Nerves
The enteric nervous system comprises all autonomic neurons intrinsic to the gut.
A large number of ganglion cells, mostly concentrated to the enteric nerve plexuses,
5.1 Anatomy of the Autonomic Nervous System 147
send axons to all parts and all layers of the gut. Cranial autonomic and spinal
autonomic extrinsic pathways interact with the enteric neurons in the plexuses and
at the neuromuscular effector site, modulating the activity of the enteric neurons.
Collaterals of sensory neurons may have the same function.
The general anatomy of the enteric nervous system obviously depends to a great
extent on the anatomy of its effector unit, the gut.
5.1.4.1 Anatomy of the Elasmobranch Gut
The elasmobranch gut (Fig. 5.2) can be divided into the oesophagus, the stomach,
the duodendum followed by the spiral intestine (ileum) with its spiral valves, and
the rectum. Histologically, the stomach can be subdivided into a cardiac stomach
and a pyloric stomach. The cardiac stomach may be further divided into a proximal
part (2/3) possessing a striated muscle wall, and a distal part with only a smooth muscle
wall (Pernkopf and Lehner 1937; Jacobshagen 1937).
Coel art Pyloric stomach
--~~~=
~ < \post mes art
Oesophagus Cardiac stomach
~~~=?~~d
st
) ) ) /7'7"7 ) \ I Rectum
Fig. 5.2. Diagrammatic representation of the gastrointestinal canal of an elasmobranch (Squa/us

acanthias). Note that the cardiac stomach comprises two portions: the striated muscle cardiac
stomach (st) and the smooth muscle cardiac stomach (sm). The major arteries entering the
gut are shown as follows: Coel art coeliac artery; Ant mes art, Post mes art anterior and posterior
mesenteric arteries, respectively. (Holmgren and Nilsson 1983)
The different layers in the gut wall of the elasmobranchs are arranged as described
for fish generally (see Fiinge and Grove 1979), which is essentially the' same as in
the higher vertebrates.
The alimentary canal of the elasmobranchs, as that in other vertebrates, possesses
along its full extension an outer smooth muscle coat of varying thickness. Characteri-
stically, this consists of an inner, comparatively thick layer of circularly oriented
smooth muscle fibres, and an outer longitudinal muscle layer. A submucosal layer
of connective tissue and larger vessels underlies the mucosa, which lines the luminal
surface of the gut wall.
5.1.4.2 Arrangement of the Enteric Nervous System

Earlier histological studies using, for example, methylene blue staining, silver nitrate
staining or gold chloride staining, have given a general picture of the anatomy of the
148 Chapter 5. The Autonomic Neryous System
elasmobranch enteric nervous system (Muller and Liljestrand 1918, Muller 1920;
Kirtisinghe 1940). Additional information can now be obtained from studies using
specific histochemical staining methods, such as Falck-Hillarp fluorescence histo-
chemistry for catecholamines or immunohistochemistry for neuropeptides (e.g.
Holmgren and Nilsson 1983).
The anatomy of the elasmobranch enteric nervous system (see Fig. 5.6a) follows
the pattern of the vertebrate enteric nervous system in general, with interconnected
ganglionated plexuses between the different functional layers of the gut. There is
thus a myenteric plexus present in the thick tunica intermuscularis between the circular
and longitudinal muscle layers along the gut (Jakobshagen 1937; Kirtisinghe 1940).
Concentrations of nerve cells and fibre bundles to the border between the submucosa
and the circular muscle layer can be compared to the submucous plexus described in
mammals (Gabella 1979). Thick bundles of nerve fibres, sometimes including ganglion
cells, run in the pockets of connective tissue between the bundles of smooth muscle
in the circular muscle layer and in the submucosa. Small bundles or single varicose
fibres run along the smooth muscle fibres of the circular muscle layer, while the
longitudinal muscle layer is mainly innervated by varicose fibres forming a sheet
of nerves on the inner surface of the longitudinal muscle layer. In Squalus acanthias
it was observed that the longitudinal muscle layer of the stomach and intestine was
split into two layers with an "outer" tunica intermuscularis, containing a well-
developed myenteric plexus, formed between the two layers (Holmgren and Nilsson
1983). Nerve fibres are also present in the mucosa.
5.1.4.3 Extrinsic Nerves
The enteric nervous system, although most likely sufficiently developed to maintain
an adequate gastrointestinal function on its own, receives a modulating input from
the central nervous system via the vagi and the splanchnic nerves (Fig. 5.1).
Preganglionic vagal fibres innervate the stomach and the anterior intestine via the
intestinal branches of the nn. Vagi. The anterior splanchnic nerve, running along the
coeliac artery, carries fibres from the most anterior pair of paravertebral ganglia
(gastric ganglia, axillary bodies) to the stomach and anterior part of the spiral
intestine. These fibres may be either postganglionic, originating from ganglion cells
in the paravertebral ganglia, or preganglionic, originating from spinal ganglion cells
and merely passing through the ganglia. A mid splanchnic nerve, running along the
anterior mesenteric artery, arises from the middle paravertebral ganglia and supplies
most of the spiral intestine. The posterior part of the spiral intestine and the rectum
receives fibres from the posterior paravertebral ganglia via posterior splanchnic nerves
(Muller 1920; Young 1933b, 1980; Nicol 1952; Nilsson 1983).
5.1.4.4 The Myenteric Plexus
The myenteric plexus, the part of the enteric plexus located in the tunica inter-
muscularis between the circular and longitudinal muscle layers, is the most extensively
studied part of the enteric nervous system. It consists of a network of bundles of
non-medullated, usually varicose nerve fibres crossing each other chiasmatically.
Nerve cells (ganglion cells), single or in small groups of two or three, are present at
5.2 Chromaffin Tissue 149
the points of crossing or along fibre bundles (Kirtisinghe 1940; Holmgren and
Nilsson 1983). This means that the elasmobranch myenteric plexus is considerably
less extensive than its mammalian counterpart, which contains a much larger number
of ganglion cells within the plexus.
The ganglion cells of the myenteric plexus in Scyliorhinus are all of Dogiel's type II
and are unipolar, bipolar or multipolar (see Fig. 5.6e). The multipolar cells may have
between three and eight processes (Kirtisinghe 1940).
Methylene blue staining of the myenteric plexus in fish, including the elasmo-
branch Scyliorhinus, shows the presence of a second type of cell, the so-called
interstitial cells of Cajal (see Fig. 5.6d). The cells are usually smaller than the
ganglion cells discussed above and of variable shape. They are arranged syncytically
in a network, i.e. they appear to have protoplasmic continuity with one or several
neighbouring cells, either through fused processes or a process from one cell fused
with the cell body of the next. The processes are long and thin and may carry swellings.
Processes not connecting with nearby cells have been seen penetrating into the muscle
layers. The network of interstitial cells does not make synaptic or syncytical connec-
tions with the "proper" ganglion cells or their neurons (Kirtisinghe 1940).
Two types of synaptic connections are described in the myenteric plexus of
Scyliorhinus. The most common involves a pericellular arborization. A nerve fibre
running close to a nerve cell may give off a branch, which in turn splits into a number
of smaller branches surrounding the nerve cell like a basket. The main nerve fibre
continues, and may give off arborizing branches to several ganglion cells along its
course.
In the second type of synaptic connection the nerve fibre (or branch of nerve fibre)
when reaching the ganglion cells whirls around the ganglion cell with a distinctly
varicose fibre, first at some distance from the cell but getting closer and closer
(Kirtisinghe 1940).
5.2. Chromaffin Tissue
A system intimately associated with the autonomic nervous system is the catecholami-
ne-releasing chromaffin tissue. Most of this tissue in elasmobranchs is associated with
the paravertebral ganglia (including, as mentioned in Sect. 5.1.3, the axillary bodies)
(Leydig 1853). In elasmobranchs the clusters of chromaffin cells (suprarenal bodies)
are anatomically separated from the steroid-secreting interrenal tissue (Coupland
1965).
Noradrenaline is the predominant catecholamine in the axillary bodies of all
elasmobranchs studied (see Nilsson 1983), and it appears that the biosynthetic pathway
for noradrenaline/adrenaline, as well as the catabolic pathways, is similar to that
of the other vertebrates (Mazeaud and Mazeaud 1973; Abrahamsson 1979; Jonsson
1982; Ungell and Nilsson 1983).
Electrical stimulation of the spinal cord produces release of catecholamines from
the chromaffin tissue into the posterior cardinal sinus, which suggests that the
chromaffin tissue is innervated by nerve fibres in a pattern resembling that of the
other vertebrates (Abrahamsson 1979).
5.3 Circulatory System
Elasmobranchs possess a typically piscine circulatory system, where the blood is

ejected from the heart into the vasculature of the gills before it enters the systemic
circulation. A detailed account of cardiovascular functions in elasmobranchs is
given elsewhere in this volume (see Chapt. 1), and the present short account will
focus on the arrangement and functions of the autonomic pathways controlling the
heart and vasculature.
5.3.1 The Heart
The elasmobranch heart consists of four chambers arranged in sequence: sinus veno-
sus, atrium, ventricle and bulbus cordis (conus arteriosus). There are two distinct
pairs of cardiac nerve branches from the vagi, one pair from the visceral branches
and one pair from the most posterior branchial branches (Marshal and Hurst 1905;
Norris and Hughes 1920). Although fibres from the spinal autonomic system could
enter the vagi via the anastomoses mentioned earlier (Sect. 5.1.3), there is so far
no evidence for a nervous control of the elasmobranch heart other than via the vagal
pathways (Young 1933b; Taylor et al. 1977; Nilsson 1983).
The vagal innervation of the heart is inhibitory, a situation similar to that in
other vertebrates with the exception of the cyclostomes. The inhibitory effect of
vagal stimulation is antagonized by atropine, which suggests that the innervation
is cholinergic as in the higher vertebrates. A variation in the tonic cholinergic vagal
influence on the heart is possibly the sole nervous control mechanism (Johansen et al.
1966; Butler and Taylor 1971; Taylor et al. 1977).
An adrenergic control of the elasmobranch heart could take place either via
catecholamines released locally from specialized endothelial cells in the sinus venosus
and atrium (Saetersdal et al. 1975; Pettersson and Nilsson 1979), or via catecholamines
released into the anterior venous sinuses from the chromaffin tissue of the axillary
bodies (Satchell 1970, 1971; Johansen 1971 ; Gannon et al. 1972; Abrahamsson 1979;
Laurent et al. 1983). It must be immediately stated, however, that the role of such
an adrenergic cardiac control in vivo has not been unequivocally demonstrated.
Adrenaline and noradrenaline produce positive chronotropic and inotropic
effects on the elasmobranch heart via a ~-adrenoceptor mechanism. There are also
some reports'of an initial bradycardia following the administration of catecholamines.
The bradycardia appears to involve a cholinergic element, since the response could
be blocked by atropine (Lutz 1930a, b; Hnge and Ostlund 1954; Ostlund 1954).
Furthermore, inhibition of the heart caused by noradrenaline, but not adrenaline or
isoprenaline, was demonstrated by Capra and Satchell (1977). These authors suggested
the involvement of Cl-adrenoceptors, since the response was abolished by large
quantities of the Cl-adrenoceptor antagonist phentolamine. The mechanisms behind
the discrepancies in the catecholamine responses obviously need further studies.
5.3 Circulatory System 151
5.3.2 The Branchial Vasculature
Stimulation of the branchial branches of the vagus produces a small increase in the
apparent vascular resistance of the gills of Scyliorhinus (J. C. Rankin, pers. commun.),
but later studies have demonstrated that the effect is due to.a passive obstruction
of the vascular pathways due to contraction of skeletal muscle of the branchial
arches (Metcalfe and Butler 1984). Thus there is no evidence for a direct vasomotor
innervation of elasmobranch gills.
A control of the branchial vasculature via circulating vasoactive agents such as
catecholamines is, however, possible. In Squalus acanthias and Scyliorhinus canicula
the responses of the gill vasculature to catecholamines is mainly a ~-adrenoceptor
mediated vasodilation, and in Squalus there is also evidence for an a-adrenoceptor-
mediated vasoconstriction (D. T. Davies and Rankin 1973; Capra and Satchell
1977). D. T. Davies and Rankin (1973) also demonstrated that the blood plasma
from "stressed" dogfish (Scyliorhinus canicula) contained catecholamines in sufficient
concentrations to affect the vascular resistance of perfused dogfish gills. The cate-
cholamines can be released from the strategically positioned chromaffin tissue within
the anterior venous sinuses (see Gannon et al. 1972 and Sect. 52), and will thus
rapidly affect the branchial vasculature.
5.3.3 The Systemic Vasculature
Adrenaline produces contraction of isolated systemic arteries from Squalus due to

an a-adrenoceptormediated effect, and there is also some evidence for the presence
of inhibitory ~-adrenoceptors in these vessels. Fluorescence histochemistry reveals
the presence of adrenergic nerves in the walls of the major arteries of Squalus, but
the importance of the adrenergic nervous control of the arteries is not clear (Fig. 5.3 a;
Nilsson et al. 1975; Holmgren and Nilsson 1983). In fact, most studies suggest that
the innervation of the systemic vasculature in elasmobranchs is scarce or even absent.
Instead, there may be an adrenergic control of systemic vascular resistance via cir-
culating catecholamines released from chromaffin stores (Opdyke et al. 1972, 1979,
1982; Short et al. 1977; Capra and Satchell 1977; Butler et al. 1977; Opdyke and
Holcombe 1978; Nilsson and Holmgren 1983). A hypertensive effect of injected
catecholamines has long been recognized in elasmobranchs (e.g. Wyman and Lutz
1932; Babkin et al. 1933; Capra and Satchell 1977), and more recent experiments
demonstrate a release of circulating catecholamines during hypoxia (Butler et al.
1978), during voluntary exercise and burst swimming in Scyliorhinus canicula (Butler
et al. 1986) and during stress induced by handling the animal (a stimulus which should
not be confused with exercise; see, e.g. Butler 1986; Abrahamsson 1979; Opdyke
et al. 1982).
Bombesin-, somatostatin-, substance P- and VIP-like immunoreactivity, but not
5-hydroxytryptamine-, neurotensin- or gastrinjCCK-like immunoreactivity was de-
monstrated in addition to catecholamine-containing nerve fibres within a nerve
plexus in the wall of visceral arteries of Squalus acanthias (Fig. 5.3a-g; Holmgren
and Nilsson 1983). The function of these nerves in vascular control remains to be
clarified.
Fig. 5.3. The innervation of blood vessels in an elasmobranch, Squalus acanthias , as demonstrated
with Falck-Hillarp fluorescence histochemistry for catecholamines (a) and immunohistochemistry
(b- g). a Catecholamine ( ca) fluorescence in paravascular nerves ( small arrows) and perivascular
varicose nerve fibres (large arrows) of the coeliac artery ; b Bombesin (bm) -like immunoreactivity
(IR) in the adventitio-medial border of the coeliac artery wall; c Somatostatin (som )-like IR in the
adventitio-medial border of the coeliac artery wall ; d Substance P ( sp ) -like IR in nerve fibres
along a submucosal vessel of the cardiac stomach ; e Somatostain (som)-like IR in the adventitio-
medial border of the anterior mesenteric artery ; f Vasoactive intestinal polypeptide ( vip i-like IR
in nerve fibres innervating a submucosal vessel in the rectum; g Bombesin (bm) -like IR in nerve
fibres innervating a submucosal vessel of the cardiac stomach. a and f are from sections, the others
from whole mount preparations. (g from Holmgren and Nilsson 1983)
5.4 The Spleen 153
5.4 The Spleen
Structure, function and innervation of the fish spleen have recently been reviewed
by Fiinge and Nilsson (1985). The functions of the elasmobranch spleen involve,
e.g. erythropoiesis as well as formation of other blood cells in the red pulp (Fey
1965; Zapata 1980) and participation of the white pulp in the immune defense
(Fiinge and Mattisson 1981; Pulsford et al. 1982). The major effector tissue for an
autonomic innervation of the fish spleen appears to be the vascular smooth
muscle, although the possibility of an innervation of capsular/trabecular smooth
muscle fibres cannot be ruled out (Fiinge and Nilsson 1985).
5.4.1 Nerve Supply to the Spleen
When innervated, the elasmobranch spleen receives autonomic postganglionic spinal

fibres from the paravertebrate ganglia via the mid splanchnic nerves. It is also
possible that fibres from the anterior splanchnic nerve reach the spleen along the
blood vessels connecting the spleen to the posterior part of the stomach. As in
mammals (B. N. Davies and Withrington 1973), there are no indications of a vagal
innervation of the elasmobranch spleen.
Species differences in the innervation of the spleen appear to exist. Thus no
nervous control of the spleen could be found in Scyliorhinus, and it is suggested that
the splenic smooth muscle in this animal is controlled by circulating agents, e.g.
catecholamines (Nilsson et al. 1975). In Squalus, on the other hand, electrical stimu-
lation of the splanchnic nerve caused a reduction of flow through the perfused
spleen. The flow reduction was in some cases followed by a phase of increased flow.
The response to nerve stimulation could be abolished by blockade of <x-adrenoceptors,
and it was suggested that the splanchnic nerve carries adrenergic autonomic fibres
controlling the flow through the Squalus spleen. Falck-Hillarp fluorescence histo-
chemistry for catecholamines confirmed the presence of adrenergic fibres in blood
vessel walls within the spleen, while other tissues of the spleen appear devoid of
adrenergic nerves (Nilsson et al. 1975).
The effect of acetylcholine on the spleen is weak, inconsistent and tachyphylactic
in Squalus and absent in Scyliorhinus. Furthermore, atropine does not reduce the
response to nerve stimulation in Squalus. It thus appears that the elasmobranch
spleen is devoid of a cholinergic innervation.
5.4.2 Physiological Functions of Splenic Autonomic Nerves
The major function of the autonomic innervation of the spleen is probably control
of blood flow through the spleen, thereby affecting its function. It is suggested that
erythropoieses is favoured by a relatively stagnant blood circulation with low
oxygen and high carbon dioxide levels (Jordan 1933).
The spleen of many animal groups has a function as a blood-storing organ, from
which red blood cells are expelled in situations of physiological stress. It is not
154 Chapter 5. The Autonomic Nervous Systenl
clear if this is a function of significant importance in the elasmobranchs. Opdyke

and Opdyke (1971) could not show an increase in haematocrit after adrenaline injection
in Squalus, while Nilsson et al. (1975), by optical measurement of the outflow from
the perfused isolated spleen in the same species, could demonstrate an expulsion of
erythrocytes, albeit small, after adrenaline administration (Fig. 5.4 ; Nilsson et al.
1975).
60] 120]
V ~
'NA 10- 10
inflow \ / inflow
20 40
"
"mfl::] ~
JI
120]
outflow -V- ~
20
40
outflow
drop
signal
5 min
a b
Fig. 5.4. The effects of A noradrenaline ( N A ) and B nerve stimulation on the flow of perfusion
fluid through the elasmobranch spleen. a Scyliorhinus canicula . The inflow decreases in response to
NA, while the outflow initially increases due to expulsion of fluid from the spleen and then decreases
during splenic constriction. During the constriction erythrocytes are expelled into the venous
effluent. The resulting increase in optical density of the venous effluent is detected by the drop
counter and recorded as an increase in amplitude of the outflow drop signal; b Squalus acanthias.
Effects of nerve stimulation with 15 Hz, 0.5 ms duration and 8 V for 30 s gives either a simple flow
decrease on both the inflow and outflow sides (left), or a complex response with a small initial
increase due to expulsion of fluid , a period of splenoconstriction and a period of splenodilatation
( right ) . (Nilsson et a1. 1975)
5.5 Gut
The gastrointestinal canal represents as large target area for the autonomic nervous
system. Many separate functions, such as secretion of digestive fluids , mucosal
transport, gastric receptive relaxation, mixing of stomach contents by segmentation
(annular contractions) and propulsion of contents by peristalsis, are controlled and
coordinated directly by the enteric nervous system, with a superimposed modulation
from the central nervous system via cranial and spinal autonomic pathways. The
innervation of the vascular bed allows control of the blood flow through the different
tissues, thus affecting tissue functions by a regulation of their blood supply.
If all nervous influence on the gut smooth muscle is inhibited, for example by
blockade with tetrodotoxin, a myogenic contractile activity - spasm - initiated
5.5 Gut 155
by myogenic pacemakers appears in the circular muscle layer. This is considered the
basic physiological drive for gut motility. Normal basic activity in the enteric
nervous system provides an inhibition of this myogenic activity, leading to the
normal resting state - ileus - of the gut. Controlled contractile activity is then
achieved by release of this inhibition by stimulatory nerves in a carefully controlled
pattern (Wood 1981).
5.5.1 Neuron Types of the Enteric Nervous System
The enteric nervous system as a part of the autonomic nervous system was defined
in mammals by Langley (1898, 1921). The presence of an enteric nervous system was
also described early in elasmobranchs and, although not as extensive as in the
higher vertebrate groups, it is well developed in this group (Muller and Liljestrand
1918; Muller 1920; Kirtisinghe 1940). Little was known until recently of the function
of the vertebrate enteric neurons and the nature of their transmitter substances.
Only during the last decade have the actions of the enteric nervous system been
better understood, and this has been due mainly to the development of immunological
techniques, which have led to the discovery of a number of neuropeptides in enteric
nerves in mammals (see Furness and Costa 1980) and subsequently also in other
vertebrates (see, e.g. Holmgren 1986). Only a few studies so far have dealt with the
presence of neuropeptides in elasmobranchs, and even less is known about the function
of these substances located in the enteric nerves.
Histochemical studies of enteric nerves in elasmobranchs have so far been made
in Squalus only. Immunohistochemistry reveals an extensive innervation by nerves
showing bombesin-, SP- and VIP-like immunoreactivity in the whole gut, and of
somatostatin- and gastrinfCCK-like immunoreactivity in the rectum. A sparse
innervation of nerve fibres showing 5-hydroxytryptamine-like immunoreactivity is
also reported. Fluorescence histochemistry according to the Falck-Hillarp method
has shown a rich innervation by adrenergic nerves. Of the possible transmittersf
neuropeptides mentioned, bombesin-, substance-P-, gastrinfCCK- and VIP-like
peptides were observed also in enteric ganglion cells, indicating their true enteric
nature. On the other hand, no ganglion cells showing catecholamine fluorescence
were observed, which indicates an extrinsic nature of the adrenergic innervation
(Nilsson et al. 1975; Holmgren and Nilsson 1983; Holmgren 1985). The distribution
and possible functi<?n of the identified substances will be discussed in more detail
below (Sect. 5.5.3).
5.5.2 Functions of Extrinsic Nerves
The studies of the function of extrinsic nerves have classically been performed by
electrical stimulation of the nerves. Recent histochemical work adds small details of
the identity of the extrinsic nerves, but little conclusive evidence as to the true
nature of the autonomic nerve fibres innervating the elasmobranch gut is available.
Several general problems occur on" the evaluation of the results of nerve
stimulation:
1. On electrical stimulation of a whole nerve it is most probable that several
types of nerve fibres (motor and sensory, excitatory and inhibitory, postsynaptic
and presynaptic) are stimulated at the same time. Although one type of fibre may
give a dominating response, this response may be modulated in several ways
by the simultaneous stimulation of other fibres, which are normally not activated
at the same time during physiological conditions.
2. An inhibitory response to nerve stimulation, i.e. abolishment of rhythmic activity
or decrease in tonus, has been shown, e.g. on vagal stimulation in Scyliorhinus and
Raja (Campbell 1975; Young 1983). Several authors have, however, been unable
to show an inhibitory effect during the stimulation period. This may be due to
low tonus and an inactive state adopted by the preparations during the experi-
mental conditions.
3. On cessation of an inhibitory stimulation, a so-called rebound contraction may be
obtained. This was first investigated in the guinea pig, and was suggested as
being due to an overshoot in the resting membrane potential when the inhibitory
hyperpolarizing effect of the stimulation ceased (Bennett 1966; Campbell 1966).
This effect may also be present in elasmobranchs, and could explain confusing
results reported from earlier studies (see review Campbell and Burnstock 1968
and below).
Later studies in mammals and birds have suggested that the rebound contraction
is instead due to the release (during stimulation) of a transmitter with a long-lasting
action, the effect of which is shaded by a more potent but rapidly degraded
inhibitory transmitter released at the same time. The transmitter causing the rebound
contraction has been suggested to be, for example, substance P (Brodin et al. 1981,
Leander et al. 1981).
5.5.2.1 Vagal Innervation
Botazzi (1902) working on Scyllium and Torpedo and Muller and Liljestrand (1918)
working on Squalus and Raja already observed that vagal stimulation could cause
inhibition of the spontaneously active stomach. Both studies, as well as that of Lutz
(1931) also report excitatory responses to vagal stimulation. The description of the
rebound phenomenon (Bennett 1966; Campbell 1966) led Campbell and Burnstock
(1968) to reevaluate the early elasmobranch studies, and to suggest that the vagal
innervation in most species is primarily inhibitory and may cause rebound contrac-
tions.
Campbell (1975) showed that electrical stimulation of the intracranial vagus
roots in Scyliorhinus canicula caused an inhibition of the spontaneous rhythmic
activity in the stomach of this species. An intragastric balloon mainly recording the
tension of the circular muscle layer was used in Campbell's experiments. Studies on
Scyliorhinus and Raja clavata with an experimental set-up mainly recording from
longitudinal muscle confirms the presence of an inhibitory innervation of the
elasmobranch stomach, although with the experimental design used the results are
5.5 Gut 157
less clear-cut (Young 1980, 1983). It is possible that the vagal influence on the two
muscle layers differs.
5.5.2.2 Splanchnic Innervation

All early studies report an excitatory response of the elasmobranch gut after
splanchnic nerve stimulation (Lutz 1931; Nicholls 1934; Babkin et al. 1935) and
Nicol (1952) in his review states that "the sympathetic innervation of the elasmo-
branch stomach is motor in effect and clearcut in action". However, Young (1980,
1983) in his studies on Raja and Scyliorhinus finds that, although stimulation of the
anterior splanchnic nerve with frequencies above 4 Hz causes only excitatory respon-
ses, stimulation below 4 Hz produces an inhibition. This inhibition is more prominent
than the inhibition caused by stimulation of the vagus, and it is followed by a
rebound contraction on cessation of the stimulation. On the other hand, Nilsson and
Holmgren (1983), in a study on the stomach of Squalus acanthias, report a purely
excitatory response to splanchnic nerve stimulation at all frequencies studied (Fig. 5.5).
It is possible that species differences occur in the anterior splanchnic innervation
of the elasmobranch stomach. It is also possible that vagal inhibitory fibres join the
splanchnic nerve before entering the stomach in some species, and that stimulation
of the splanchnic nerve therefore may involve stimulation of originally vagal
fibres.
The recent study by Young (1983) suggests that the splanchnic innervation of the
spiral intestine via the mid and posterior splanchnic nerves is excitatory and adrenergic,
but not via CJ.- or ~-adrenoceptors.
30
flOW)
20
- -
+Atropine
30 ]
flow ~---j\v-~_ _ _~~_ _ _ _ _ _ _ _ __
20
5 mil
~-----++~-~--~~-==~~~-~==~
+Phentolamine 10 min
Fig. 5.5. The effects of splanchnic nerve stimulation on the perfused stomach from the spiny dogfish,
Squalus acanthias. Stimulation with 10 Hz, 1 ms and 10 V for 60 s causes a direct as well as a rebound
contraction of the stomach, both unaffected by atropine but abolished by phentolamine, indicating
the adrenergic nature of the splanchnic innervation of the stomach in this species. (Nilsson and
Holmgren 1983)
5.5.3 Transmitter Functions in the Gut
5.5.3.1 Cholinergic Innervation

No physiological or histochemical evidence of a cholinergic innervation of the
elasmobranch gastrointestinal canal has so far been presented. The excitatory
effect obtained on splanchnic nerve stimulation could not be blocked by hyoscine
or at~?pine even in high, concentrations (Nicholls 1934; Babkin et al. 1935; von Euler
and Ostlund 1957; Yoong 1980, 1983; Holmgren and Nilsson 1983). Neither was
the excitatory effect obtained on vagal stimulation - whether primary or rebound
in nature - diminished by cholinergic blockade (Babkin et al. 1935; Nicholls
1934). If the elasmobranch gut lacks a cholinergic innervation this means that it
differs from the other vertebrate groups (see Burnstock 1969; Nilsson 1983).
Exogenous application of acetylcholine, however, produces a contraction of the
elasmobranch stomach, which can be blocked by atropine or hyoscine (Nicholls
1934 von Euler and Ostlund 1957) showing the presence of muscarinic receptors.
These receptors may be non-innervated and of little or no physiological importance.
It is also possible that cholinergic neurons are enteric, without direct central
connections, and therefore difficult to activate in experimental conditions by stimu-
lation of extrinsic pathways.
5.5.3.2 Adrenergic Innervation

Falck-Hillarp fluorescence histochemistry has demonstrated the presence of cate-
cholamines (adrenaline, noradrenaline) in dense nerve plexuses in all parts of the gut
of Squalus. Particularly prominent nerve nets were encountered in the myenteric
plexuses of the stomach and rectum, and numerous fibres were also present in
bundles between the smooth muscle bundles of the circular muscle in the stomach
(Holmgren and Nilsson 1983). This indicates a prominent adrenergic control of the
smooth muscle activity of the gut in Squalus. Frequently, the adrenergic fibres run
close to arteries and arterioles, possibly innervating the vessels (Holmgren and Nilsson
1983). Adrenergic fibres also innervate the blood vessels supplying the gut, and
bundles of fibres follow the course of these vessels to the gut. No adrenergic
ganglion cells have been observed within the stomach, while weakly fluorescent
ganglion cells, probably adrenergic, are present in the paravertebral ganglia, indi-
cating that the adrenergic innervation of the gut is mainly extrinsic (Nilsson et al.
1975; Holmgren and Nilsson 1983).
Nilsson and Holmgren (1983) showed that the splanchnic innervation to the
stomach of Squalus is adrenergic, i.e. electrical stimulation of the anterior splanchnic
nerve produced a primary contraction of the perfused stomach that could be
blocked by phentolamine but not by serotonergic or cholinergic antagonists. The
antagonists were used in concentrations which were proved to have a selective action.
The stimulation of the anterior splanchnic nerve in Squalus also produced a rebound
contraction (following the primary contraction). This was also selectively antagonized
by adrenergic blockade, indicating that an adrenergic agent is involved in the release
of the transmitter causing the rebound contraction in this case (Fig. 5.5).
The presence and pathways of an adrenergic innervation in other elasmobranch
species is less clarified. In most species the stomach contracts by exogenous
5.5 Gut 159
application of adrenaline (Lutz 1931; Young 1933b, 1980, 1983; Nicholls 1934;
Babkin et al. 1935; Dreyer 1949; Moore and Hiatt 1967; Holmgren and Nilsson
1983). However, several studies also report inhibitory responses in certain cases.
In Scyliorhinus and Raja the effect of adrenaline is described as excitatory with
signs of an inhibition, including a pronounced rebound on wash-out (Young
1983).
5.5.3.3 Serotonergic Innervation

The presence of nerves using 5-hydroxytryptamine (5-HT, serotonin) as transmitter
was proposed first for lower vertebrates (Baumgarten 1967; Baumgarten et al. 1973;
Watson 1979; Sakharov and Salim ova 1980), while the presence of such nerves in
the mammalian gut was accepted only much later (Gershon 1981; Costa et al. 1982;
Furness and Costa 1982). Amongst the elasmobranchs, histochemical evidence is
available from Squalus only. In this species a very sparse innervation by nerve
fibres trailing along the smooth muscle fibres could be demonstrated (Fig. 5.6g;
Holmgren and Nilsson 1983). If species similarities exist, the comparatively high
content of 5-HT in tissue extracts from the gut of Scyliorhinus canicula and
S. stellaris (Erspamer 1954; Piomelli and Tota 1983) could originate mainly from
endocrine cells, which occur in high numbers in the intestine and especially the rectum
of Squalus (Holmgren and Nilsson 1983).
It has been suggested that the inhibitory splanchnic innervation of the stomach in
Scyliorhinus is serotonergic in nature (Young 1983), a suggestion which requires
further confirmation.
5.5.3.4 Peptidergic Innervation

Bombesin. An extensive innervation of the gut of Squalus by nerves showing
bombesin-like immunoreactivity (lR) has been reported (Fig. 5.6a). Immunoreactive
ganglion cells (Fig. 5.6e) and numerous nerve fibres were present in the myenteric
plexus(es) and in the circular muscle layer. A ganglionated submucous plexus was
especially prominent in the cardiac stomach. Bundles of fibres were present in the
submucosa, and single fibres were seen in the mucosa (Fig. 5.6b; Holmgren and
Nilsson 1983). The true enteric (intrinsic) nature of this innervation is indicated by
the presence of the ganglion cells, but central connections are demonstrated by
immunoreactive fibres, albeit rather few, in the axillary body, in the splanchnic
nerve bundles running along the mid splanchnic nerve and in the vagus (Fig. 5.6h;
Holmgren and Nilsson, unpublished).
The immunohistochemical results thus indicate an important role of a bombesin-
like peptide in the control functions of the gut. Little is so far published on the nature
of this control, but a study on strip preparations of the Squalus rectum shows an
excitatory action of exogenous bombesin on the motility of the preparation (Fig. 5.7;
Lundin et al. 1984), which is in agreement with observations in the teleost stomach
(Holmgren 1983; Thorndyke et al. 1984).
Gastrin/Cholecystokinin (CCK). Gastrins and CCK's share a C-terminal penta-
peptide, and most immunohistochemical studies made so far do not differ between
these two peptides. In Squalus, gastrin/CCK-like IR occurs in a dense plexus of nerve
Fig. 5.6. Immunohistochemical demonstration of different types of nerves in the enteric nervous
system of an elasmobranch, Squalus acanthias. a Cross-section of the rectum showing the general
differentiation in layers of the elasmobranch gut and their innervation, represented by the occurrence
of bombesin (bm) -like immunoreactivity OR). eM circular muscle; LM longitudinal muscle.; M
mucosa; SM submucosa (from Holmgren and Nilsson 1983); b nerve fibres in the mucosa of the
cardiac stomach showing bombesin (bm)-like IR; c bundles of nerve fibres showing
5.5 Gut 161
fibres in the smooth muscle layers and the myenteric plexus of the rectum and
more sparsely in the stomach and intestine. IR ganglion cells were frequent in the
rectal myenteric plexus (Holmgren and Nilsson 1983).
The presence of gastrin/CCK-like peptides in the gut of Squalus has been further
confirmed by radioimmunoassay, and experiments with isolated strip preparations
show that different gastrin/CCK peptides cause an excitatory effect on the motility
to a varying degree (Falkmer et al. 1981; AIdman et al. 1986).
Somatostatin. Somatostatin has dual effects on the motility of the rectum of
Squalus, with a mainly inhibitory effect on circular muscle activity and a mainly
excitatory effect on longitudinal muscle (Fig. 5.7; Lundin et al. 1984). IR nerve
fibres are sparse in the stomach and intestine of SqlJalus, but occur more frequently
in all tissue layers of the rectum (Fig. 5.6f; Holmgren and Nilsson 1983; Reinecke
et al. 1984). It is thus possible that enteric neurons may affect rectal motility by
release of a somatostatin-like peptide (among others). Extracts from the intestine of
Torpedo marmorata contain somatostatin 14 (Conlon et al. 1985).
Substance P. A substance P-like peptide has been demonstrated to be present
in the gut of Squalus acanthias (von Euler and Ostlund 1956). This may originate
from one or several tachykinins (the group of substance P-related peptides) present
either in endocrine cells of the mucosa, or in the dense network of IR nerves
present throughout all layers (except the mucosa) of the stomach, intestine and rectum
(Fig. 5.6c; EI-Salhy 1984; Holmgren 1985). The distribution and density of nerves
and IR ganglion cells closely resembles the situation described for bombesin above.
The three tachykinins, substance P, physalaemin and eledoisin, all have an exci-
tataory effect on gut muscle from Squalus. The effect of substance P is in all
regions of the gut unaffected by tetrodotoxin and adrenergic, cholinergic or sero-
tonergic blockade, indicating an effect directly on the smooth muscle cells (Holm-
gren 1985). This direct mechanism of action is reported to be present in most other
vertebrates studied, but in representatives of other vertebrate groups usually in
combination with some other indirect mechanisms, e.g. via serotonergic neurons as in
Salmo gairdneri (Holmgren et al. 1985) or cholinergic neurons as reported for several
species of mammals (Hedqvist and von Euler 1975; Holzer et al. 1980; Daniel
et al. 1982).
VIP (Vasoactive Intestinal Polypeptide). The YIP-like peptide(s) present in the
gut of Scyliorhinus canicula shows properties in radioimmunoassay and radio-
receptorassay indicating a close relationship in the bioactive part of the molecule to
mammalian YIP (Fouchereau-Peron et al. 1980). The presence of YIP-like IR in
nerves has been demonstrated in the gut of Squalus (Holmgren and Nilsson 1983;
El Salhy 1984). The'YIP-nerves, along with bombesin- and SP-nerves, form the most
... Fig. 5.6. (Continued)

substance P (sp)-like IR in the submucosa of the pyloric stomach; d VIP-like IR in the myenteric
plexus of the intestinal wall. The immunoreactive nerve cell resembles those desc.ribed as Cajal cells
by Kirtisinghe (1940); e nerve cell showing bombesin (bm)-like IR in the myenteric plexus of the
cardiac stomach. Reproduced with permission from Holmgren and Nilsson 1983; f Somatostatin
(som)-like IR in the myenteric plexus of the intestine; g 5-hydroxytryptamine (5-ht)-like IR in
nerves of the myenteric plexus in the pyloric stomach; h nerve fibre in the intestinal branch of the
vagus showing bombesin (bm)-like IR. i VIP-like IR in the outer myenteric plexus of the cardiac
stomach. a and i are from sections, b--b from whole mount preparations
extensive nerve plexuses encountered in the gut, with a distribution of fibres and
ganglion cells to all layers of tissue similar to that described above for bombesin
(Fig. 5.6i; Section 5.5.3.4; Holmgren and Nilsson 1983). Also similar to bombesin,
a VIP-like peptide is present in a low number of nerve fibres in the vagus and
splanchnic nerves and in the axillary body, where also wea Uy fluorescent ganglion
cells are present (Holmgren and Nilsson, unpubl.).
Exogenous application of porcine VIP inhibits the rhythmic activity of spon-
taneously active strip preparations of Squalus rectum (Fig. 5.7; Lundin et al.
1984).
N
0.005
o
~ ~
10- 8 M SOMATOSTATIN 10 min
000: 1 .\\\\\\\\\\\\\\\\\\\\
~ 1-----1
10- 9 M VIP 10 min
N
0 .002
o
+ +
M
10- 8 10- 7 M 10- 6 M
~
10 min Fig. 5.7. The effects of somatostatin,
VIP and bombesin on the smooth mnscle
BOMBESIN
of the rectum from Squalus acanthias.
Somatostatin has mixed effects, VIP is
N
OOJ
inhibitory and bombesin excitatory, either
increasing the tonus of the preparation
(upper panel) or the rhythmic activity
( lower panel)
+
t------;
10- 7 M
+ +6 10 m in
10- M
BOMBESIN
5.6 Uro-GenitalOrgans
The walls of the urinary sinuses of Scyllium are richly supplied with spinal autonomic
nerves (Young 1933 b), but nothing is known about their nature.
Sympathetic nerves innervate the oviducts and vasa deferentia in Scyllium with a
complex network of fibres containing some ganglion cells, while there are no
indications of the presence of a vagal innervation of the gonads in this species
(Y oung 1933 b). Stimulation of the spinal cord produced excitatory motor effects on
the oviducts in Torpedo and Scyllium, an effect that could be mimicked by exogenous
application of adrenaline (Botazzi 1902, Young 1933 b).
5.7 The Iris
The pupillary diameter of the eye of most vertebrates is controlled by two sets
of smooth muscles, which form the circular iris sphincter (sphincter pupillae) and the
radially arranged iris dilator (dilator pupillae). Contraction of the sphincter causes
narrowing of the pupil (miosis), while contraction of the radially arranged muscle
causes a widening of the pupil (mydriasis).
The smooth muscles of the iris are innervated by autonomic nerve fibres, which
run in the oculomotor nerve (III) and/or in nerves from the spinal autonomic
(sympathetic) system. In some of the lower vertebrates, including the elasmobranchs
studied, there is a direct excitatory effect of light on the iris sphincter (see Nilsson
1983).
The control of the iris in several elasmobranch species (Scyllium catulus,
Scyllorhinus canicula, Mustelus laevis, Trygon violaceus) was investigated by Young
(1933 a). The iris sphincter in these species contracted in response to light due to a direct
effect on the light-sensitive smooth muscle. No autonomic innervation of the sphincter
could be demonstrated, nor was the sphincter sensitive to adrenaline or acetylcholine.
Stimulation of the oculomotor nerve (III) produced a widening of the pupil due to
contraction of the radially arranged muscles. The fibres involved are likely to be
cholinergic, since the cholinergic antagonist atropine produced narrowing of the
pupil.
The elasmobranch iris is thus controlled by the direct effect of light on the
sphincter muscle, which causes narrowing of the pupil, and the antagonistically
acting autonomic innervation of the dilator muscle causing widening of the pupil
(Young 1933a).
5.8 Concluding Remarks
The pioneering work of J. Z. Young in the early part of the 1930's opened up
research on the functional anatomy of the autonomic nervous system in elasmobranch
and teleost fishes. Since then, a trickle of new information regarding the patterns
;l~ ::::::J
~ ~ /1,. ~
~
'--/
~
'--/
/'.
"-./
**
,,*/
"--J "-./ "-1./ "--./ t
1 I'
I
/
/
/ I'
/
/
G'1
/
/
l~
/
/
/
/1
a+ /
a+
(~-)
a b
+ Excitation
Inhibition
* Chromaffin cells
~)C;lg
Cholinergic
Adrenergic
'" m+ NANC
""""';0"
m Muscarinic
a
c Adrenoceptors
;l~
•
"- i../ "--./ "-i../
•
1 1
1 1
1 i
i /
1 ;
1-----/ '"---_____
~? -;.'1
-----_/
d e
Fig. 5.8. .Diagrammatic figure summarizing the autonomic innervation (extrinsic nerves) of some
elasmobranch organs. (Nilsson 1983). a Spleen; b Heart; c Iris; d Intestine; e Stomach
References 165
and mechanisms of autonomic nerve control of various elasmobranch organs has

appeared, and new techniques such as immunohistochemistry, quantitative pharmaco-
logy and modern recording techniques have been applied also in research on the
non-mammalian vertebrates. An attempt to provide a general summary of the patterns
of autonomic innervation of various elasmobranch organs is made in Fig. 5.8.
There are two areas in which research on the autonomic nerve functions in
elasmobranchs offers a particular challenge. The first area deals with the mechanisms
for cardiovascular control in elasmobranchs, especially the function of cardiac control
during rest and exercise, and the question of whether the elasmobranch vasculature
is at all controlled by nerve fibres, or solely by circulating vasoactive agents or local
events (e.g. hypoxia). The nature of the cardiovascular innervation is also of great
interest, especially in view of the new ideas of peptidergic cardiovascular control in
other vertebrates.
The second challenge comes from the elasmobranch gut, where new techniques
make research into non-adrenergic, non-cholinergic innervation mechanisms possible.
The distribution and function of serotonergic and peptidergic nerves and the
related systems of endocrine and paracrine cells is a rapidly growing area of
research in mammals, and the elasmobranch gut, with its relatively simple arrange-
ment of the enteric nervous system, provides excellent models for research regarding
integrated enteric nerve functions. Such research is of great interest, not only from
the obvious comparative physiological point of view, but also in terms of a basic
knowledge of nerve functions where the relative simplicity of a primitive system can
be used to elucidate complex phenomena.
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Chapter 6 T. J. SHUTTLEWORTH
Salt and Water Balance - Extrarenal Mechanisms
Current theories of vertebrate evolution suggest that primitive fish-like vertebrates

originally evolved in a freshwater habitat and that the appearance of marine forms
resulted from a subsequent reinvasion of the sea. It appears that such a reinvasion
followed two basic patterns - whilst the bony fish retained a relatively low osmotic
concentration of their body fluids and developed in the sea as hypo-osmotic
regulators, the early cartilagenous fish elevated the osmotic concentration of their
body fluids so as to become isosmotic, or slightly hyperosmotic, to their marine
environment. The elevation of body fluid osmotic concentration in the latter group
was mainly achieved, however, not by large increases in inorganic ion concentrations,
but by the accumulation and retention of certain organic nitrogenous compounds,
such as urea and trimethylamine oxide (TMAO). The net result of this early divergence
in osmoregulatory strategy means that the two major groups of marine fish, the
teleosts and the elasmobranchs, are fundamentally different in their osmotic status,
although they do both face broadly similar problems of ionic balance. For elasmo-
branchs, the basic problems of hydromineral regulation therefore centre on the
retention of urea and related compounds, and the elimination of excess ions. This
latter process involves an unusual organ, the salt-secreting rectal gland, which has
attracted considerable interest, originally as an extremely rich source of the enzyme
Na-K-ATPase (Hokin et al. 1973), and more recently as a model system for a parti-
cularly widespread, and medically highly important, transport process - the secon-
dary active transport of chloride.
The purpose of this chapter is to provide a broad overview of the hydro-
mineral status of elasmobranchs and its consequences in terms of the resulting
fluxes of water, ions, urea etc., together with a discussion of the processes involved
in the maintenance of osmotic and ionic balance, specifically those operating at
extrarenal sites. (Por a description of the role of the kidney, see Chap. 7).
Considerable emphasis has been placed on a detailed description of the functioning
of the rectal gland, reflecting the considerable interest shown in recent years in the
transport processes occurring in this gland and their regulation. Finally, the more
specialized osmoregulatory problems faced by elasmobranchs inhabiting freshwater
environments, and those experienced during development of the young, are discussed.
Author's address: Department of Physiology, Box 642, University of Rochester Medical Center,
601 Elmwood Avenue, Rochester, NY 14642, USA
172 Chapter 6. Salt and Water Balance - Extrarenal Mechanisms
6.1 Overall Hydromineral Status
The hydromineral status of a range of elasmobranch species has been extensively

surveyed in the review by Holmes and Donaldson (1969). In addition, Robertson
(1975) provides a detailed analysis of the osmotic constituents of the plasma and
muscle of Squalus acanthias. Briefly, elasmobranch body fluids are characterized by
sodium and chloride concentrations that are somewhat higher than those typical of
other vertebrate groups, including marine teleosts, and by the presence of very high
concentrations of urea and trimethylamine oxide (TMAO). Th;e retention of these high
concentrations of organic nitrogenous substances results in an overall osmotic
concentration of the body fluids that slightly, but significantly, exceeds that of the
external medium. Thus, plasma concentrations of sodium and chloride are typically
230-270 mmoll- 1 ,i.e. some 60 % higher than those of marine teleosts. Despite this,
these ions together contribute only approx. 50 % of the total osmotic concentration
of the plasma, the majority of the remainder being made up of urea, which may reach
concentrations in excess of 400 mmoll-t, and TMAO (70-100 mmoll- 1). The total
osmotic concentration of the body fluids is thus usually in the range
1,000-1,100 mOsmoll- 1 , some 2-10 % higher than the surrounding seawater.
6.2 The Retention of Urea and TMAO
A detailed historical review of the discovery of the high concentrations of these

organic nitrogenous compounds in the blood and tissues of elasmobranch fishes,
together with speculations on the physiological implications of their presence, has
been given by Smith (1936). Although essentially solving the osmotic problems usually
experienced by vertebrates in a marine environment, the retention of urea and TMAO
in the body fluids does raise certain questions - in particular, how are these high
concentrations maintained, and what are the biochemical and physiological conse-
~ quences of their presence?
For example, it has been clainled that the urea gradient existing across the gills
of marine elasmobranchs represents one of the largest chemical gradients known
in the animal kingdom. Clearly the maintenance of such a gradient requires either
a high synthetic rate to replenish that inevitably lost by diffusion, or a low per-
meablity of the branchial epithelium to such compounds. Measured values for the rate
of urea efflu~ in elasmobranchs average 20-70 IlmollOO g-l h- 1 (Table 6.1). Rates
of TMAO excretion reported by Goldstein and Palatt (1974) appear to be similar
to those of urea (i.e. 10-30/lIDol 100 g-l h- 1 ) but, interestingly, in the four species
of elasmobranch studied by these authors, no correlation could be demonstrated
between the measured TMAO excretion rates and the apparent ability, or otherwise,
of the species to synthesise this compound. In those species apparently unable to
synthesise TMAO, e.g. Squalus acanthias, the ultimate source of this substance must
be in the diet. During prolonged starvation, plasma levels of TMAO are maintained
by a slow release from the large pool present in the muscle, supplemented by active
reabsorption in the kidney tubule (Goldstein et al. 1967).
6.2 The Retention of Urea and TMAO 173
Table 6.1. Rates of urea efflux in various elasffiobranch species
Species Urea efflux Reference

(llffiollOO g-l h-1)
Negaprion brevirostris 19 Goldstein et al. 1968

Scyliorhinus canicuia 25 Payan and Maetz 1970
Raja erinacea 24 Goldstein and Forster 1971 a
57 Payan et al. 1973
Ginglyrnostorna cirraturn 21-38 Carrier and Evans 1972
Poroderma afi'icanum 28 Haywood 1974
Hemiscylliumplagiosum 67 Wong and Chan 1977
Of the total urea efflux, renal losses represent only approximately 4--5 % (Payan
et al. 1973; Wong and Chan 1977), reflecting the extremely effective reabsorption of
this substance by the kidney tubule (see Chap. 7), and thus the gills are clearly
the major site of urea efflux from the animal. Measurements of the actual per-
meability of the gill epithelium to urea, taking into account estimates of the
relevant surface area, give values of 2.5-10 x 10- 8 cm S-l (Boylan 1967; Payan and
Maetz 1970; Payan et al. 1973) which, for example, is some 20-100 times less than
that of the toad bladder. It should be pointed out that the elasmobranch gill also
shows relatively low permeabilities to ions such as sodium (see Sect. 6.4 below) with
values approximately one tenth those of the toad bladder or frog skin. It is clear
that the disproportionately low permeability of the branchial epithelium to urea is
not simply a reflection of a general impermeability of elasmobranch cell membranes
to this molecule, as Fenstermacher et al. (1972) have shown in Squalus acanthias
that urea rapidly equilibrates with a space identical to the total body water
volume. Despite these interesting findings, relatively few studies have been made on
the actual nature of the unusually low branchial urea permeability in elasmobranchs
and almost everything we know on this topic is derived from the work of Boylan
and his colleagues in the 1960's. Boylan and Lockwood (1962) found that, in contrast
to the urea reabsorption mechanism in the elasmobranch kidney tubule, the gill
showed no detectable selectivity for urea over thiourea and that the branchial
permeability of this latter substance was similarly very low. Investigation of the
effect of temperature on branchial urea permeability (Boylan et al. 1963) revealed that
between 1 and 15°C the urea efflux remained constant, but between 15 and 30 °C
efflux increased dramatically in a linear fashion (Fig. 6.1). Although Boylan (1967)
has proposed various possible explanations for the increase in efflux at temperatures
above 15 DC including phase changes in the branchial membranes and/or failure of
a putative inward transport system for urea, it is, as pointed out by Goldstein
(1982), the lack of any change in efflux over the physiological temperature range
(i.e. up to 15°C) that is probably most relevant and revealing. The clear suggestion
is that the branchial impermeability does not involve any kind of urea transport
system, as this would be expected to demonstrate a marked temperature sensitivity.
Rather, the low branchial urea permeability appears to be the result of some form of
physical barrier to the diffusional movement of urea across the epithelium. Such
a conclusion was supported by studies of the urea efflux following elevation of the
plasma urea concentration by intravenous loading (Boylan et al. 1963). Rather than
17;l Chapter 6. Salt and Water Balance - Extrarenal Mechanisms
observing any saturation of a putative urea transport system, efflux increased in

an exponential fashion when plasma urea was elevated over the range 333 mmoll- 1
to 1,000 mmoll- 1 . Such an exponential increase could be explained by direct effects
of such extremely high urea concentrations on membrane permeability. Of course,
the evidence indicating that the unusually low permeability of the elasmobranch
branchial epithelium to urea is not the result of a urea transport system but is more
likely a "simple" physical property of the epithelium, does not detract in any way
from its interest. It is a pity that more detailed studies using modern techniques have
not been performed on this aspect of elasmobranch physiology.
200
I
/
/
./
/
/
I
~
/
,.
-i:: 150
.
/
/
/
OJ /
o /
o /
."
o
/
/
E I
2-
,2 100
.
/
CD /
.
cu /
OJ I
's I
/
~
.c ,'
u
~ 50
cD
Fig. 6.1. Rate of branchial urea efflux at diffe-

rent temperatures in Squalus acanthias. (After
Boylan et al. 1963)
10 20 30
Tempera lure ('[)
As urea is known to be a potent destabilizer of proteins, it might be expected

that the presence of such high concentrations of urea in the body fluids would have
severe consequences for metabolic processes in the animal, especially as the evidence
suggests that most elasmobranch enzymes are no less sensitive to urea than those of
other vertebrates. The answer to this apparent paradox lies in the simultaneous
accumulation of TMAO and other methylamines which act as general protein
stabilizers counteracting the adverse effects of urea. Hence, at least in vitro, the effects
of urea on the Vmax and Km of several different enzymes (Yancey and Somero 1980),
as well as the contractile properties of skinned muscle fibres (Altringham et a1.
1982), can be offset by TMAO when present in appropriate proportions (generally 2
urea: 1 TMAO) mimicking intracellular conditions. Hence, metabolic processes in
elasmobranchs may not depend on the development of unique urea-adapted or urea-
6.3 Water Fluxes and Permeabilities 175
resistant proteins, but rather on the accumulation of appropriate concentrations of

mutually counteracting organic osmotic solutes. However, this does not appear
to be always the case, as some elasmobranch proteins have been shown to be
urea-insensitive, and others actually require the presence of urea in order to
demonstrate normal properties or activities (see for example, Yancey and Somero
1978).
6.3 Water Fluxes and Permeabilities
The rate of total unidirectional water flux measured in various elasmobranch species
ranges from 65-170% of the body water per hour (Table 6.2), far larger than the
values of 10-20 % body water per hour typical of marine teleosts (Motais et al.
1969). Conversion of these measurements into diffusional permeability coefficients
(Pd)' taking account of estimates of the branchial surface area, gives values of
3.5-6 x 10- 5 cm S-l for the elasmobranch species (Payan and Maetz 1971), compared
with only 1-2 x 10- 5 em S-l for marine teleosts.
Table 6.2. Water fluxes in various e1asmobranch species
Species Water flux Reference

(% body water h- 1 )
Scyliorhinus canicula 156 Payan and Maetz 1971
Raja montagu 167
Torpedo marmorata 97
Ginglymostoma cirratum 81 Carrier and Evans 1972
Raja erinacea/ R. radiata 64.2 Payan et al. 1973
Poroderma africanum 97 Haywood 1974
This relatively high diffusional permeability to water, together with the fact that
the body fluids are slightly hyperosmotic to the environment, results in an uptake
of water which, with that produced metabolically, is sufficient to balance that lost
in the urine and in the fluid secreted by the rectal gland etc. As such, and in
marked contrast to marine teleosts, an osmotic requirement for ingestion of the
medium is absent and indeed Smith (1931 b) reported that drinking was negligible
in Raja. This was subsequently confirmed in Scyliorhinus canicuia, where drinking
rates of only 12.5 III 100 g-l h- 1 (payan and Maetz 1971) or 1.6 III 100 g-l h- 1
(Pang et al. 1980) were found. This compares with typical values of 500-1,000 III
100 g-l h- 1 seen in marine teleosts. It would seem that, although on occasions
seawater may be ingested, drinking in elasmobranchs is not a continuous or regular
process as it is in-marine teleosts.
6.4 Ion Fluxes and Permeabilities
Unidirectional sodium fluxes in elasmobranchs average 40-100 /lmol 100 g-1 h- 1

(Burger and Tosteson 1966; Maetz and Lahlou 1966; Chan et al. 1967; Horowicz
and Burger 1968; Payan and Maetz 1973), equivalent to approximately 0.5 % of the
total body sodium per hour. This is markedly different from the corresponding
values for marine teleosts which are typically in the range 1200--2600 /lmoll 00 g-1 h -1.
An usual feature of total ion fluxes in elasmobranchs is the finding in several species
that unidirectional chloride fluxes are some three to ten times larger than the
corresponding sodium fluxes (Maetz and Lahlou 1966; Carrier and Evans 1972;
Haywood 1974). This is also seen in measurements of those fluxes specifically
taking place across the gill (Bentley et al. 1976). As transepithelial electrical
potential gradients are small (Bentley et al. 1976), this presumably reflects a differential
permeability of the branchial epithelium to these two ions.
The presence of a large concentration gradient for sodium and chloride between
the environment and the body fluids, and the absence of any substantial electrical
potential gradient (Bentley et al. 1976) means that the animal is subject to a continuous
diffusional uptake of these ions across the permeable parts of the body surface.
The evidence suggests that the majority of such uptake occurs across the gills,
although the details of actual mechanisms involved are somewhat controversial.
From an analysis of unidirectional fluxes and simultaneous transepithelial electrical
potentials, Bentley et al. (1976) conclude that the branchial influx of chloride was
consistent with entirely passive processes, but that the influx of sodium was not.
Thus, branchial fluxes of sodium did not accord with the predictions of the Ussing
flux ratio equation and, when measured in external media of different concentrations,
the sodium influx showed saturation kinetics. The possible involvement of a Na + jH +
exchange mechanism was suggested by the reduction in branchial sodium influx
400
• •
. '
•
:::' 300
,.'.c •
OJ
• •••
o
o
~ 200
a- •
..
x
.2 ,,
.~ • ,,, , ,
•• •• ,
ro
Z 100
Fig. 6.2. Effect of plasma pH on sodium
••
influx rate in Scyliorhinus canicula. Plasma
pH was varied by the induction of a metabolic
acidosis by injection of HCI or ammonium
salts. (After Payan and Maetz 1973)
6.6 6.8 7.0 7.2 7.4 7.6 7.8
Blood pH
6.5 Elimination of Excess Ions 177
produced by a reduction in the external pH. In a similar way, Evans (1982) reported
that the branchial H+ efflux from fish made acidotic depended upon the presence of
external Na +, and Payan and Maetz (1973) found that total sodium influx was
inversely related to blood pH during metabolic acidosis (Fig. 6.2). The gills of elasmo-
branchs have been shown to contain carbonic anhydrase (Hodler et al. 1955) and the
involvement of this enzyme in such ion exchanges is suggested by the pronounced
inhibition of sodium uptake following injection of the inhibitor acetazolamide
(Payan and Maetz 1973). Evans (1984) has suggested that such a branchial Na+ /H+
ion pump exists in all fish, whether marine or freshwater, teleost or elasmobranch,
and probably plays a vital role in the maintenance of acid-base balance of the body
fluids (see Chap. 9, this Vol.). Whatever the precise nature of the branchial fluxes,
there can be no doubt that the gills are a significant and consistent site of a net
uptake of sodium and chloride ions for the fish. According to Bentleyet al. (1976),
in Scyliorhinus canicuia, this net uptake amounts to approximately 100-200 Ilmol
100 g -1 h -1. This, in fact, is higher than the values for total sodium influx noted above
but, as these authors point out, the fish in their experiments were likely to be in a
"stressed" condition and this is known to increase the rate of ion influx in dogfish
(Payan andMaetz 1973). A net branchial influx of around 10-30 llmol 100 g-1 h- 1
is more likely, as can be calculated from the data of Maetz and Lahlou (1966) and
Wong and Chan (1977).
The net uptake of ions at the gill referred to above is likely to present a continual,
relatively constant, salt load to the animal - the only factor likely to have a significant
influence on its rate being, as discussed above, disturbances in the acid-base balance
of the fish. The other major avenue for salt uptake for the fish is via the gut. As
stated above, available evidence suggests that elasmobranchs drink only very little, but
the ingestion and digestion of food will result in an additional salt load for the
animal - particularly as, for most species, invertebrates form the major part of the
diet. It is not possible to estimate accurately the contribution of this route to the
overall salt load for the animal, as very little is known about the feeding activity
of elasmobranch fishes other than the fact that specimens are clearly capable of
surviving for prolonged periods without feeding at all. Obviously, such a salt load can
be expected to be highly irregular and intermittent in nature.
6.5 Elimination of Excess Ions
The maintenance of ionic homeostasis of the body fluids in the face of the continual
net uptake of ions at the gill and, intermittently, via the gut, obviously requires
some mechanism for the elimination of the excess salt. Evidence suggests that,
in elasmobranchs, ion excretion can take place at three principal sites - the
kidney, the gills and the rectal gland.
In considering the relative contributions of these different sites to the efflux of
ions, it is important to distinguish between unidirectional effluxes and net fluxes
- especially as it is only the latter that are of physiological relevance to overall ionic
balance in the fish! Thus, several workers have shown that the kidney and rectal
gland combined contribute from 55 % to as little as 10% of the total sodium

efflux, the remaining 45-90% presumably being branchial (Maetz and Lahlou 1966;
Chan et al. 1967; Payan and Maetz 1970; Haywood 1975). However, it would be
quite wrong to conclude from this that the gills are a major site for the elimination
of excess ions as, whilst a large proportion of the unidirectional efflux of ions is
undoubtedly branchial, the gills will also be the site of a simultaneous unidirectional
ion influx. The net flux occurring at the gills will therefore be equal to the difference
between this influx and the measured efflux. As discussed above, the evidence suggests
that, in fact, the gills are the site of a significant net ion uptake (Maetz and Lahlou
1966; Horowicz and Burger 1968; Bentley et al. 1976) and cannot therefore contribute
to the overall elimination of the excess salt gained from the environment and in the
diet. Hence, the only routes available to the fish for the net excretion of excess ions are
the kidney and rectal gland.
The considerable confusion that appears to exist in the literature concerning the
contribution of the gills to the elimination of excess ions in elasmobranchs is in sharp
contrast to their unequivocal role in active ion regulation in marine teleosts.
Nevertheless, some evidence does· suggest that, at least for chloride, part of the
unidirectional branchial efflux in elasmobranchs does not result from entirely passive
processes (Bentley et al. 1976). Although convincing biophysical evidence for an active
branchial chloride efflux is lacking, support for such an active component comes from
reports describing the presence of so-called "chloride cells" (or Keys-Willmer cells) in
the gill epithelium of elasmobranchs (Doyle and Gorecki 1961; Garcia-Romeu and
Masoni 1970). In marine teleosts it is well established that these cells are the sites of
active ion extrusion, both in the gill and, more particularly, the opercular epithelia
(see, for example, Foskett et al. 1983). Whilst earlier reports suggested that the
appearance of the chloride cells of elasmobranchs showed no obvious \lifferences
from those of marine teleosts, more recently it has been noted that, in the
elasmobranch chloride cell, the basolateral membrane extends into the basal part of
the cell as a series of infoldings rather than the complex system of branched
tubules characteristic of these cells in teleosts (Laurent 1982), and certain differences
in their surface features have been claimed (Crespo 1982). However, it seems unlikely
that such relatively subtle ultrastructural differences would have any great physiologi-
cal significance and the overwhelming similarities between these cells in the two
groups of fishes would suggest that they playa similar role, namely the active extrusion
of ions, in both marine teleosts and elasmo branchs. Unfortunately, direct biophysical
and biochemical evidence for such a role in elasmobranchs is totally lacking.
What is clear, at least in those elasmobranch species studied, is that the numbers of
branchial chioride cells are far lower than is typical of marine teleosts and, at least in
Squalus acanthias, the activity of branchial Na-K-ATPase (an enzyme shown, in
teleosts, to be particularly concentrated in the chloride cell) is only one-tenth to one-
fifth of that seen in marine teleosts (Jampol and Epstein 1970). The conclusion
would seem to be that, if the ion efflux across the gills of elasmobranchs does
indeed contain an active component, then this could be achieved by the activity of
branchial chloride cells just as in marine teleosts. However, such an active branchial
efflux would be relatively small compared with that typical of marine teleosts and, it
must be emphasized, is clearly not sufficient to result in the gills being a site for the net
excretion of ions.
6.6 Rectal Gland 179
In summary then, it is clear that the kidney and the rectal gland are the only possible
sites for the net efflux of ions required by the fish to counterbalance the diffusional
and dietary uptake of ions. However, precise evaluation of their respective con-
tributions to this process is difficult partly because, even in the very few species
studied, in vivo cannulation, particularly of the rectal gland, is often technically
impossible. Even where cannulation is feasible, it is likely to involve considerable
stress to the animal, which itself has largely undefined, but probably profound,
effects on both renal and rectal gland function. What is clear is the fact that the
kidney of elasmobranchs is unable to produce a urine with ion concentrations greater
than those of the plasma, whilst the fluid secreted by the rectal gland contains
sodium and chloride at concentrations almost twice those of the plasma (see Sect. 6.7)
and is therefore specifically able to reduce plasma ion concentrations. As the physiolo-
gy of the kidney of elasmobranchs is described separately (see Chap. 7), only the role
of the rectal gland will be considered here.
6.6 Rectal Gland
Burger and Hess (1960) were the first to show that the rectal gland of elasmobranchs
functioned in the elimination of excess salt by excreting a fluid that was essentially
isosmotic with the body fluids but which consisted almost exclusively of sodium and
chloride at a concentration approximately twice that found in the body fluids, whilst
the urea concentration of the secreted fluid was only 10-20 mmoll- 1 . They were also
able to show that the gland produced this fluid in sufficient volume to effectively
remove significant amounts of sodium and chloride from the plasma.
6.6.1 Structure and Histology
Structurally, the rectal gland is a compound tubular gland consisting of many simple
and branched secretory tubules that drain into a central canal (Fig. 6.3). This central
canal continues into a duct opening into the intestine at a point posterior to
the spiral valve. The gland receives a blood supply via usually a single rectal gland
artery, derived from the posterior mesenteric artery, and is drained by the large dorsal
intestinal vein (Kent and Olson 1982). The histology and ultrastructure of the cells
that make up the seGretory tubules are extensively described in the studies of Doyle
(1962) and Bulger (1963). These cells possess the numerous mitochondria and greatly
expanded basolateral plasma membranes that typify cells engaged in ion transport
and, as such, are very similar in appearance to the salt-secreting cells of avian and
reptilian salt glands. Freeze-fracture studies have revealed that the so-called tight
junctions between the secretory cells of the rectal gland are relatively shallow, and
the complex interdigitation of the lateral surfaces of adjacent cells results in a high
value for the length of junction per unit of luminal surface, equivalent to some
70-100 m/cm2 (Ernst et al. 1981; Forrest et al. 1982). This may be of significance in
the proposed mechanism of transport as the secretion of sodium is believed to follow
a paracellular route (see Sect. 6.6.3).
Fig. 6.3. Transverse section of the rectal gland of Scyliorhiflus canicula showing the densely
packed arrangement of secretory tubules, each consisting of a single layer of secretory cells
6.6.2 Secretion Rate
As discussed above, accurate determinations of secretion rates in vivo are difficult

to obtain with reliability partly because of technical problems and partly because of
potential stress effects. Furthermore, the evidence indicates that secretion rates
in individuals, particularly under control conditions, may fluctuate quite markedly
(Burger and Hess 1960; Burger 1962; Holt and Idler 1975) and determinations must
therefore be made over periods of several hours. In any event, values have only been
reported for a very few species (Table 6.3). As discussed above, the finding that the
rectal gland is responsible for a small fraction of the total undirectional ion efflux
has frequently led to the erroneous conclusion that the gland plays only a minor
role in ion balance. It should be emphasized that, with the kidney, it is the only avenue
fable 6.3. Secretion rates from the rectal gland in various elasmobranch species. Values are given for
the concentration of sodium and chloride in the secreted fluid (in mmoll- ' ), the volume of
secretion produced (in J.lIIOO g-I h- I) and hence the rates of ion secretion (in J.lffio1100 g-l h- I)
Species Secreted fluid Secretion rate Reference
[Na] [Cl] Vol Na Cl
Squalus acanthias 540 533 0-190 0-103 0-101 Burger and Hess (1960)
Squalus acanthias 480-519 47 22- 24 Burger (1962)
Scyliorhinus canicula 554 8 4.5 Payan and Maetz (1970)
Hemiscyllium plagiosum 535 30.5 16.3 Chan et al. (1967)
Hemiscyllium plagiosum 461 445 51 23.5 22.7 Wong and Chan (1977)
Dasyaus sabina 583 13 7.6 Beitz (1977)
Raja ocellata 490 499-505 56-72 27-35 28- 36 Holt and Idler (1975)
for the net excretion of ions and is the only site available for the elimination of
those ions at concentration higher than that found in the body fluids.
It is also clear that attempts to define the role of the rectal gland in overall ion
balance have been further confused by some misinterpretation of studies in which the
rectal gland was removed or its function prevented. Thus, in his early studies on
Squalus acanthias, Burger (1965) found that plasma ion concentrations remained
unchanged for up to 21 days after surgical removal of the rectal gland, and no increase
in urinary chloride concentrations could be detected. However, a marked diuresis was
seen in the glandless fish, and this resulted in overall urinary chloride losses
increasing some three to five times. In addition, there appeared to be some indication
of a reduced ion influx into the glandless fish, which was tentatively ascribed to
some undefined change in branchial permeability. Following injection of a sodium
chloride load, the restoration of normal blood ion concentrations was very much
delayed in the glandless fish, taking longer than 5 days compared with the normal
48 h in unoperated fish. On the basis of these findings, Burger observed that,
without a rectal gland, sodium chloride is lost through increased urinary output
and not by concentration of the urine above plasma levels. Such a process would
necessitate a compensatory increase in water uptake presumably across the gills.
Burger (1965) concluded therefore that "the kidneys cannot serve as a substitute rectal
gland".
Similar findings have been reported by other workers. For example, in their study
on H emiscyllium plagiosum, Chan et al. (1967) found that removal of the rectal gland
markedly increases the rise in muscle sodium content seen following salt loading. In
addition, whilst salt loading in unoperated animals does not affect plasma sodium
concentrations, these become elevated under the same conditions in animals without
rectal glands. In Poroderma africanum ligation of the rectal gland produces increases
of 5-10% in plasma sodium and chloride concentrations which, although declining
with time, are still elevated by some 3-5 % even after 14 days (Haywood 1975).
Salt loading of such animals results in further increases in plasma sodium and
chloride concentrations which decrease over the following 4-6 h.
In view of the difficulties in evaluating rectal gland function in vivo, it is
perhaps not surprising that most workers have turned instead to the use of a variety
in vitro techniques. In particular, the relatively simple anatomy of the gland, its single
artery, vein and secretory duct, lends itself to the technique of perfusion in isolation as
first demonstrated by Palmer (1966). Early studies on the perfused preparation
(Hayslett et al. 1974; Siegel et al. 1975, 1976) were bedevilled by low secretion
rates which deteriorated further with time. A major advance came when Stoff et al.
(1977 a) showed that constant high rates of secretion could be obtained in the isolated
gland by perfusion with analogues of the cyclic nucleotide cyclic AMP (e.g. dibutyrl
cyclic AMP) together with the phosphodiesterase inhibitor theophylline. More recent-
ly, the precise relationships between perfusion conditions and secretory activity in the
isolated gland have been studied in some detail in both Squalus and Scyliorhinus
(Shuttleworth and Thompson 1983, 1986). Secretory parameters for the isolated
glands from both species under similar conditions are given in Table 6.4. Ion
Table 6.4. Parameters of secretion in the isolated perfused rectal glands of two species of elasmobranch.
(Data from Shuttleworth and Thompson 1983, 1986; Shuttleworth and Thorndyke 1984)
Scyliorhinus canicula Squalus acanthias
Control Stimulated" Control Stimulated"

eN) (5) (9) (4) (19)
Na concentration 516.8 504.0 520.6 530.6

±14.2 ±6.4 ±5.0 ±2.3
Secretion flow rate 3.6 78.9 2.5 46.7
± 1.9 ±2.6 ±0.9 ± 1.7
N a secretion rate 1.8 39.7 1.3 24.6
± 0.9 ±1.4 ±0.5 ±0.9·
Sodium concentration is in mmoll- 1, secretion flow rate in 1-11 g-1 min-I, and secretion rate in
I-Imol g-1 min-I. Values are means ± S.E.
a Glands perfused with cAMP (0.05 mmoll- 1) and theophytline (0.25 mmoll- 1).
=o
if
I
1.
600
-. • •
... :..., .
• ,.. • ,....... •• _ .....
~ 500-r- -~. - ~ -.-:,-::.-.-~----••--, -.-.- ~ .. -:-:.~--.- - --.~-
'--' I • • •
c • • ••••
.Q
~o 400
(j)
Ul
.<;
o
is 300
o
ro
Z
200~-----'-----'------r-----,---~~
o 10 20 30 40 50
Na secretion rate (~moj g-1 min-1)
Fig. 6.4. Concentration of sodium in the fluid secreted by the isolated perfused rectal gland of
Scyliorhinus measured at different rates of secretion
concentrations in the secreted fluid in these perfused preparations agree well with
those reported from in vivo studies, and remain essentially constant over the entire
range of observed secretion rates, which therefore simply reflect changes in the
volume of secreted fluid produced (see Fig. 6.4). Under appropriate conditions,
maximum sodium secretion rates in the perfused gland are approximately
25 Ilmol g-1 min- 1 in Squalus, and 40 Ilmol g-1 min- 1 in Scyliorhinus (Table 6.4).
Conversion to secretion rate per unit body weight gives 50-75 Ilffiol 100 g-1 h -1 for
Squalus and 20-25 Ilmol100 g-1 h- 1 for Scyliorhinus, values broadly similar to those
obtained in vivo.
Since 1977 the isolated perfused gland preparation has been widely used and,
together with tissue slices, membrane vesicles and, more recently, isolated perfused
segments of secretory tubule, has produced considerable insight into the actual
mechanisms of transport involved and their control. Nevertheless, it should be
emphasized that, despite 10 years of study, only two species (Squalus acanthias and
Scyliorhinus canicula) have been investigated in any detail.
6.6.3 Mechanism of Secretion
Early studies on the rectal gland had established that it contained very high levels of
the enzyme Na-K-ATPase and that this was probably involved in some way in the
mechanism of ion secretion (Bonting 1966; Jampol and Epstein 1970). However, the
finding that this Na-K-ATPase was specifically localized on the basolateral membrane
of the secretory cells (Goertemiller and Ellis 1976; Eveloff et al. 1979), where it
presumably functions to pump sodium out of the cell (i.e. in the direction opposite to
that required for sodium secretion), was difficult to reconcile with any direct role in
ion secretion. It was not until the studies of Silva et al. (1977) on the rectal gland,
together with those of Ernst and Mills (1977) on the very similar salt-secreting nasal
gland of birds, that this problem was resolved.
The early perfused gland studies of Hayslett et al. (1974) and Siegel et al. (1976)
had shown that, during secretion, the duct lumen was electrically negative with
respect to the perfusion medium, indicating that it was the transport of chloride,
rather than that of sodium, that occurred against the prevailing electrochemical
gradient. This was confirmed by Silva et al. (1977), using the perfused gland preparation
stimulated with cyclic AMP and theophylline. It was also shown that chloride
secretion was dependent on the presence of sodium in the perfusing fluid, and was
inhibited by ouabain and by the loop diuretic furosemide. Furthermore, intracellular
chloride concentrations were found to be some four to six times higher than
predicted on the basis of electrochemical equilibrium across the basolateral mem-
brane, a fact subsequently confirmed by micro electrode studies (Duffey et al. 1978;
Welsh et al. 1983). These data were incorporated into a model in which the uphill
entry of chloride into the cell across the basolateral membrane is coupled to the
simultaneous downhill entry of sodium via a common carrier or cotransporter that
is sensitive to inhibition by loop diuretics such as furosemide. The downhill
gradient for the entry of sodium into the cell is maintained by the ouabain-
sensitive sodium-potassium pump located on the basolateral membrane keeping
intracellular sodium activity low. The resultant accumulation of chloride intracel-
lularly to an activity greater than that predicted for electrochemical equilibrium

provides the driving force for the exit of this ion across the apical cell membrane
into the secretory lumen, with sodium following passively via a paracellular pathway
(Silva et al. 1977). Accordingly, this transepithelial transport of chloride is described
as a secondary active process.
In a wider context, it is important to note that the development of this model
owed much to related work on transport in the mammalian ileum (Nellans et al. 1973)
and gall bladder (Frizzell et al. 1975) and, as such, is essentially identical to that used
to describe the secondary active transport of chloride across a wide variety of
vertebrate epithelia (see Frizzell et al. 1979).
Subsequent studies on various preparations have generally confirmed this model
for secretion by the rectal gland. For example, more direct evidence for the close
coupling of sodium and chloride fluxes across the basolateral membrane, together
with their inhibition by loop diuretics, came from studies using isolated membrane
vesicles (Eveloff et al. 1978) and from micro electrode studies showing that the intra-
cellular accumulation of chloride was dependent on sodium in the perfusate (Welsh
et al. 1983). The only major modification to the original model has been that the
basolateral cotransport system now appears to involve, not only sodium and chloride
ions, but also potassium in a Na-2CI-K configuration (Hannafin et al. 1983),
blood lumen
Fig. 6.5. Diagram representing the current model

describing the mechanism of salt secretion in the elasmo-
branch rectal gland. See text for details
similar to that described, for example, in the thick ascending limb of the mammalian
loop of Henle (Greger and Schlatter 1983; Hannafin and Kinne 1985). Po-
tassium entering via this cotransporter, together with that entering on the Na-K-
pump, recycles via potassium channels in the basolateral membrane. Finally, from a
detailed eiectrophysiological study using isolated perfused fragments of secretory
tubule, Greger and Schlatter (1984) have produced a comprehensive biophysical
analysis of this model as applied to the rectal gland cell. Figure 6.5 illustrates the
essential features of this model for the mechanism of transport in the rectal gland
secretory cell.
It has long been known that the gland contains a high activity of the enzyme
carbonic anhydrase (Maren 1962), and this was considered to be implicated, in
some undefined way, in the secretory mechanism. However, attempts to demonstrate
effects on secretory activity resulting from inhibition of carbonic anhydrase were at

best equivocal (see Swenson and Maren 1984), and it is clear that the above model
does not include any role for this enzyme in the secretory mechanism. Recent
evidence suggests that its major function in the rectal gland lies in the rapid
elimination of metabolically produced carbon dioxide from the secretory cells, rather
than any direct involvement in the secretory process per se (Swenson and Maren
1984).
6.6.4 Mechanism of Cyclic AMP Action
As stated above, it was discovered quite early in the work on the perfused gland,
that secretion was sensitive to stimulation by membrane-permeable analogues of the
cyclic nucleotide cyclic AMP (cAMP), or by agents that might be expected to induce
a rise in the concentration of cAMP within the secretory cells. Such agents included
theophylline, which inhibits the phosphodiesterases responsible for the breakdown of
intracellular cAMP, and also forskolin, a direct stimulator of the enzyme adenylate
cyclase that is responsible for the synthesis of cAMP within the cell. Having established
the essential characteristics of the transport mechanism, the question arose as to
exactly how increases in intracellular cAMP could lead to an increase in secretion rate.
From the model illustrated, several possible sites of cAMP action exist - (a) an
increase in the activity of the basolateral sodium pump, (b) an increase in the
activity of the basolateral Na-2Cl-K co transporter, (c) an increase in the apical
chloride conductance, and (d) an increase in the basolateral potassium conductance.
Of course, in order to increase the secretory activity of the cell, it is likely
that all of the above must ultimately take place, but the question is which of
these processes is the primary site for cAMP action?
Cyclic AMP plus theophylline certainly results in an increase in the activity
of the sodium pump as, on stimulation, large increases are seen in ouabain-sensitive
oxygen consumption and in ouabain binding (Shuttleworth and Thompson 1978, 1979,
1980a; Silva et al. 1979). Although it has been claimed that these responses result, at
least in part, from direct effects of cAMP on the sodium pump (Epstein et al. 1983;
Silva et al. 1983), the bulk of the evidence suggests that the increase in sodium
pump activity is simply an indirect result of a cAMP-induced increase in sodium
entry into the cell (Shuttleworth and Thompson 1980 b; Shuttleworth 1982; Greger
et al. 1984). This cAMP-induced increased entry of sodium is clearly due to the
enhanced activity of the basolateral cotransporter (Shuttleworth and Thompson
1980b; Greger et aL" 1984), but evidence suggests that even this may not be the
primary site of cAMP action. Instead, detailed microelectrode and patch-clamp
studies on the isolated perfused secretory tubule have indicated that the specific action
of cAMP is to increase the chloride conductance of the apical membrane by
activating previously silent chloride channels (Greger et al. 1984, 1985, 1986).
It is hard to see, however, how such an action could lead to the marked increased
in activity of the basolateral Na-2Cl-K cotransporter, particularly as the net driving
force on this cotransporter actually decreases on secretion, largely because of an
increase in intracellular sodium activity (Greger et al. 1985, 1986). It is possible
that changes in intracellular calcium may be involved in this synchronization of apical
and basolateral events as it has been shown that, although not mediated directly by
calcium, the stimulation of secretory activity by cAMP does apparently show a calcium
dependency (Shuttleworth 1983a).
6.6.5 Control of Secretion
In view of the accepted role of cAMP as an intracellular second messenger for

many primary extracellular messengers, the demonstration that cAMP could stimulate
secretion in the isolated rectal gland was a clear indication that secretory activity was
potentially under the control of such an extracellular signal. As to the nature and
identity of this primary messenger, the early in vivo studies of Burger (1962)
had indicated that it was unlikely that the gland was under direct nervous control,
and this was supported by histological (Chan and Phillips 1967) and physiological
studies (Solomon et al. 1984a).
6.6.5.1 Hormonal Control - Peptides and Adenosine

Stoff et al. (1979) reported that, of a range of neurotransmitters and peptide
hormones tested, only vasoactive intestinal peptide (VIP) was effective in stimulating
secretion rate in the perfused gland of Squalus acanthias. VIP also increased intracel-
lular cAMP levels in this tissue, an effect considerably enhanced by the additional
presence of theophylline. However, despite this apparently clear effect, there is
now considerable evidence to indicate that the action of VIP in the isolated gland of
Squalus does not reflect a response of general physiological relevance in vivo. Firstly,
it appears that the response to VIP may be unique to Squalus as, even at high
concentrations (e.g. 10- 6 moll-i), VIP is completely without effect in the isolated
rectal glands of two other elasmobranchs species, Scyliorhinus canicula and Raja
clavata, (Shuttleworth and Thorndyke 1984). Furthermore, it appears that VIP
is even ineffective in Squalus when tested in vivo (Solomon et al. 1985a), and
attempts to relate secretory activity or ionic status to circulating levels of VIP
were, at best, inconclusive (Stoff et al. 1977b; Solomon et al. 1985a). In addition,
while VIP-like immunoreactivity can be demonstrated in elasmobranch tissues (Falk-
mer et al. 1980; Fouchereau-Peron et al. 1980), recent studies have clearly shown
that the VIP found in elasmobranchs shows significant structural differences from the
mammalian peptide used by Stoff et al. (1979), most notably in the amino acid
sequence of the C-terminal region (Dimaline and Thorndyke 1986; Dimaline et al.
1986). As these differences are likely to influence biological activity, this serves to
illustrate the potential dangers inherent in the use of non-homologous or non-
native molecules in comparative studies of hormone action. Investigations using the
native elasmobranch VIP again failed to show any stimulation of secretory activity
in Scyliorhinus or Raja (Thorndyke and Shuttleworth 1986), although this has not
been tested in Squalus. In summary, the reported effect of VIP is a response to a non-
native molecule that appears to be restricted to the Squalus gland, and even then is
only seen in vitro. In view of this, it does seem unlikely that VIP can be
considered the natural regulator of secretory activity in the rectal gland of elasmo-
branchs.
As an alternative approach, the study of peptide material directly extracted

from elasmobranch tissues has been rather more successful in the search for the regula-
tor of rectal gland function. A peptide molecule showing marked stimulatory activity
in the rectal glands from Squalus, Raja and Scyliorhinus has recently been isolated
and purified from the intestine of Scyliorhinus (Shuttleworth and Thorndyke 1984;
Thorndyke and Shuttleworth 1986). This peptide, provisionally named rectin, has
a molecular weight of approximately 2000-2500 and its amino acid composition,
as well as its biological activity and extraction characteristics, show it to be entirely
unrelated to VIP. Whilst the precise characterization of rectin must await further
work, including full amino acid sequencing, the fact that it has an endogenous
origin, together with its potent physiological action in all species tested to date,
strongly suggests that it is indeed the native regulator controlling secretion in the
elasmobranch rectal gland in vivo.
Another agent shown to have marked effects on secretory activity in the rectal
gland is the nucleoside adenosine, which has been shown to be released from the
gland cells during stimulation, presumably as a result of the increased metabolism of
ATP (Osswald et al. 1983). Erlij et al. (1978) and Forrest et al. (1980) showed
that at concentrations of 10- 5 moll- j and above, adenosine stimulated secretory
activity in Squalus, whilst at concentrations below 10- 6 moll- j , adenosine was found
to inhibit secretion (Poeschla et al. 1982). Both these responses apparently reflect
effects on the activity of adenylate cyclase and result from binding of adenosine to
specific receptors of the A2 (stimulatory) and Aj (inhibitory) subtypes respectively.
The interesting suggestion is that the major physiological role for adenosine is to act
during stimulation, as a feedback inhibitor, operating via the Aj receptors, thereby
preventing excessive tissue hypoxia and cell injury (Kelley et al. 1985). In support of
this is the finding that the secretory cells of Scyliorhinus appear to possess only the
inhibitory Aj-type adenosine receptor (Shuttleworth, unpubl. data). Furthermore,
in both Scyliorhinus and Squalus, the gland vasculature possesses vasodilatory adenosi-
ne receptors (Shuttleworth 1983b) and these may also playa role in this protective,
or "safety valve", function for adenosine by increasing blood flow to the gland during
stimulation.
It appears that secretory activity may also be under inhibitory regulation by the
peptide hormone, somatostatin (Stoff et al. 1979). In contrast with VIP, evidence
based on radio immunological and immunohistochemical techniques for demonstra-
ting the presence of this hormone in the tissues of elasmobranchs is much more
secure as the structure of this peptide has apparently changed little during vertebrate
evolution. However, there appears to be considerable confusion as to its mechanism
of action in the rectal gland. Thus, Stoff et al. (1979) and Epstein et al. (1982, 1983)
report that somatostatin, whilst inhibiting the VIP-induced stimulation of secretion
in Squalus, has no effect on the stimulation of secretion by cAMP plus theophylline,
or by adenosine or by forskolin. The suggestion was made that somatostatin
acted at a specific site on the VIP-receptor-adenylate cyclase complex to block the
VIP-induced production of intracellular cAMP (Epstein et al. 1982, 1983). Indeed,
Solomon et al. (1984 a) actually used the fact that the stimulation of secretion
following volume-loading was blocked by somatostatin as a specific indication for
the involvement of VIP in this response. However, in a more recent paper, the same
workers have shown that somatostatin inhibits the stimulation of secretion by all
these agents referred to above (Silva et al. 1985) and have claimed that this suggests
sites of somatostatin action both proximal and distal to the production of cAMP.
Clearly, full elucidation of the action of somatostatin in the rectal gland must
await further studies and, in particular, its action in relation to the likely natural sti-
mulator of secretory activity in vivo (i.e. rectin) should be investigated.
6.6.5.2 Hormonal Control - Steroid Hormones

It seems probable that hormones interacting with the adenylate cyclase-cAMP
system will modulate secretion rates over only relatively short time periods. The
possibility of a more prolonged regulation of secretory activity by steroid hormones
from the interrenal gland has been investigated by Holt and Idler (1975). These
authors found that removal of the interrenal gland in Raja ocellata resulted in a
decrease in secretion by the rectal gland which could be reversed by injections of
lel-hydroxycorticosterone, a major adrenocorticosteroid of elasmobranchs. In contrast
to this, Chan et al. (1967) reported preliminary findings that injections of cortisol and
deoxycorticosterone decreased rectal gland secretion in Hemiscyllium. Whether this
discrepancy is related to the use of different corticosteroids, species differences, or to
the fact that Chan et al. used intact rather than interrenalectomized animals is
unknown. Clearly further work is required to clarify the influence of interrenal
steroids on rectal gland function.
Evidence also suggests that other components of the general osmoregulatory
physiology of elasmobranchs, particularly urea balance and water permeability, may
be influenced by interrenal steroids, as well as possibly by prolactin and thyroid
hormones (payan and Maetz 1971; de Vlaming and Sage 1972; de Vlaming et al.
1975).
6.6.5.3 Gland Blood Flow and Secretion

In the rectal gland, as with other exocrine glands, it appears that important
relationships exist between secretory activity and the rate of blood flow to the gland.
Studies have shown that the vasculature within the rectal gland is markedly constricted
by physiological concentrations of circulating catecholamines operating at alpha-
adrenoceptors (Shuttleworth 1983 b). Since stress has been shown to elevate plasma
catecholamine titres (Butler et al. 1978), this probably explains the high variability
seen in measurements of rectal gland blood flow made in vivo (Kent and Olson
1982). Furthermore, at the low flow rates produced by such a vasoconstriction,
secretion rate by the gland is severely impaired (Shuttleworth and Thompson 1986),
although contrary to earlier suggestions (e.g. Solomon et al. 1984 b), this does not
appear to be the result of any limitation in the supply of oxygen. The conclusion
is that, under normal conditions in vivo, the gland may frequently be in a partially
vasoconstricted state and that for maximum rates of secretion, stimulation of the
gland must be associated with a simultaneous local vasodilation (Shuttleworth
1983 b), as is commonly seen in other exocrine glands. Some indication that such a
vasodilation in the rectal gland does indeed occur in vivo has been reported by
Solomon et al. (1984b). However, evidence suggests that this vasodilation does not
simply result from the increased metabolic activity of the secretory tissue, but is
6.7 Euryhaline and Freshwater Elasmobranchs 189
independently controlled in a manner synchronized to the stimulation of secretory

activity in the gland (Shuttleworth 1983 b). One possibility, as yet untested, is that this
secretion-related vasodilation is mediated via the same peptide hormone that regulates
secretory activity (i.e. rectin - see above) operating independently on receptors
located on the vascular smooth muscle of the gland.
6.6.5.4 Initiation of the Secretory Response In Vivo

As to the specific hydromineral parameter that is responsible for triggering the stimu-
lation of secretion rate in vivo, the early studies of Burger (1965) had shown that the
gland was highly active in animals exposed to diluted seawater. As such animals had
reduced blood ion concentrations, it seems likely that blood or body fluid volume,
rather than its concentration, is the principal factor responsible for the stimulation
of rectal gland secretion. This has been supported by more recent studies involving
direct volume loading (Solomon et al. 1985b; Erlij and Rubio 1986). Further
evidence suggests that there are receptors in the region of the heart which respond to
elevations in the vencus pressure by activating a pathway resulting in an increase
in secretory activity in the gland (Erlij and Rubio 1986). It has been suggested
(Solomon et al. 1985a) that this pathway may involve the release of a peptide
from cardiac cells, possibly related to the atrial natriuretic peptide or atriopeptin of
higher vertebrates, although current evidence is somewhat contradictory and it seems
unlikely that this' peptide has any direct effect on the secretory cells of the rectal
gland.
6.7 Euryhaline and Freshwater Elasmobranchs
Although it is clear that the elasmobranchs as a group are predominantly marine,

there are species that are known to frequent freshwater environments. However,
review of the published literature on this aspect of elasmobranch hydromineral
physiology reveals much debate and not insignificant confusion. Thus, although
Smith (1936) provides an extensive list of species that have been recorded from such
environments, it is clear that he includes many examples that represent only rare or
chance excursions into dilute media. In a similar way, much of the earlier work on
species of elasmobranch that were claimed to be euryhaline and/or freshwater was
bedevilled by confusion over the length of time they had been resident in reduced
salinities, and their degree of access to seawater. For example, the species of the
genus Carcharhinus, found in Lake Nicaragua some 200 km from the sea, was original-
ly named C. nicaraguensis and was thought to represent a truely freshwater species,
being completely land-locked by the presence of rapids in the river draining
the lake, the Rio San Juan. Subsequent studies (Bigelow and Schroeder 1966)
revealed that this species was indistinguishable from the marine bull shark,
C. leucas, and that specimens were capable of moving freely in both directions along
the Rio San Juan between the lake and the sea (Thorson 1971). As such, it is
better considered as a euryhaline elasmobranch rather than a truly freshwater species.
Similar arguments apply to the sawfish Pristis perotteti from the same locality and also
studied by Thorson (1967, 1982), as well as to the group of elasmobranch species

(P. microdon, C. melanopterus, Dasyatis uarnak and Hypolophus sephen) from the
Perak River of Malaysia studied by Smith (1931 a).
It now seems clear that there is only one group of elasmobranchs that can
genuinely be considered as being permanently resident in freshwater. These are the
members of the ray family Potamotrygonidae (Potamotrygon, Elipesurus, and Dis-
ceus) which are found particularly in the Amazon and Orinoco river systems as much
as 4000-4500 km from the sea (Thorson et al. 1967).
This distinction between the true freshwater elasmobranch species and those better
described as euryhaline is not made simply for the sake of ecological accuracy, as it is
now clear that these two groups demonstrate quite markedly different hydromineral
characteristics (Table 6.5). In fact, this distinction in the hydromineral characteristics
has been used as an argument to reassign a West African species from the genus
Potamotrygon to the genus Dasyatis (Thorson and Watson 1975). Thus, the euryhaline
species, when resident in freshwater, show only a relatively small reduction in
plasma ion concentrations and still retain significant, albeit reduced, levels of urea in
their body. Similar effects are seen in those marine species that are tolerant of
gradual dilution of the external medium (Goldstein and Forster 1971 a; de Vlaming
and Sage 1973). In the true1y freshwater species Potamotrygon, plasma sodium and
chloride concentrations are very much lower and urea concentrations are only
around 1.0 mmoll- 1 (Thorson et al. 1967), a concentration in fact somewhat lower
than that seen in most other ureotelic vertebrates. The net result is that the
euryhaline elasmobranch species are far more hyper osmotic when in freshwater than
is Potamotrygon, which has body fluids whose total osmotic concentration is similar
to those of freshwater teleosts.
table 6.5. Comparison between the body fluid composition in representative euryhaline and true
freshwater elasmobranch species
Species Na CI Urea Osm Reference
(mmoll- I ) (mOsmoll- l )
Euryhaline
Carcharhinus nicaragensis (= leucas) 200 180.5 132 Urist (1962)
245 219 169 Thorson et al. (1973)
Pristis perrotteti 217 193 Thorson (1967)
Freshwater
Potamotrygon spp. 150 150 1.25 308 Thorson et al. (1967)
164 152 1.08 282 Griffith et al. (1973)
It appears that these marked differences in plasma urea concentrations between

the two groups result, at least in part, from a fundamental difference in their
abilities to synthesize this compound. Thus, in the euryhaline species, the reduced
urea levels seen in dilute salinities result, in most cases, from a reduction in the
rate of synthesis, but this can be reversed on exposure to normal seawater (Gold-
stein and Forster 1971 a; Wong and Chan 1977) - see Chapter 9, this Volume, for
6.7 Euryhaline and Freshwater Elasmobranchs 191
details. In addition, exposure to dilute environments results in a marked increase

in urine flow rate which, coupled with a decreased tubular reabsorption (Goldstein
and Forster 1971 a; Forster et al. 1972), produces an elevated urinary loss of
urea. However, at the same time, branchial losses decline with the fall in plasma
urea levels (Payan et al. 1973).
The situation in the permanently freshwater species Potamotrygon is somewhat
different, in that exposure to increasing external salinities fails to result in any
osmotically significant elevation of plasma urea concentrations (Thorson 1970;
Griffith et al. 1973). For example, Bittner and Lang (1980) showed that, even at the
maximum salinity tolerated (525 mOsmoll- 1 ), a salinity in which the animals were
essentially isosmotic, plasma urea had risen to only 6 mmoll- 1 . The evidence suggests
that, compared to its marine ancestors, this species has evolved a very much reduced
rate of ure1:\ synthesis, as well as reduced urea reabsorption by the kidney tubule,
(Goldstein and Forster 1971 b). It further appears that these characteristic features
cannot be reversed on exposure to increased salinities (Goldstein and Forster 1971 b;
Gerst and Thorson 1977).
Euryhaline species, as well as the more genuinely freshwater Potamotrygon,
possess the relatively low permeability to ions and high permeability to water
typical of normal marine elasmobranchs (Carrier and Evans 1973). Whilst the former
is at an advantage in a freshwater environment, the latter is not, as it presumably
results in a large .osmotic flux of water into the animal which must, in turn, be
eliminated by a correspondingly high urine flow. Although this has not been
directly measured, Goldstein and Forster (1971 b) report average glomerular filtration
rates in Potamotrygon as high as 830 111100 g-l h -1. The high urine flows predicted
would inevitably result in significant renal losses of ions, although evidence from
euryhaline species suggests that elasmobranchs in freshwater are capable of reducing
ionic concentrations in the urine to very low levels, thereby minimizing such losses.
To these must be added diffusional losses which will occur despite the apparent
low permeability to ions. The mechanism, or indeed the location, of the ion uptake
processes necessary to compensate for these losses in elasmobranchs living in fresh-
water are unknown. Pang et al. (1972) recorded a unidirectional influx of sodium
ions from freshwater in Potamotrygon that showed saturation kinetics, but there was
no indication of whether this resulted in any net uptake of ions. It is tempting to
suggest that the branchial uptake mechanisms (at least for sodium) that have been
shown to exist in marine elasmobranchs, where their presumed role is in acid-base
balance (see Sect. 6.5 above), could form the basis of an uptake system essential
for ionic regulation.in freshwater species. However, in the absence of any detailed
studies on the true freshwater species, such suggestions must remain speCUlative.
Of course, in the absence of any requirement for the elimination of excess ions,
the rectal gland should play no part in osmoregulation in freshwater. It is interesting
therefore that, despite some earlier reports to the contrary, Potamotrygon appears
to possess a vestigial rectal gland (Thorson et al. 1978). What function it has, if any,
is not known, but it does perhaps support the notion of a typical marine origin
for these truly freshwater rays. In the euryhaline species, such as Carcharhinus
leucas, rectal glands of normal or somewhat reduced size are present and contain
fewer secretory tubules (Oguri 1964; Thorson et al. 1978).
6.8 Osmoregulation During Development
A particularly intriguing question arising from the unique pattern of osmoregulation

characteristic of elasmobranchs relates to exactly when and how, during ontogeny,
this pattern arises. For example, are the developing embryos isolated from the
osmotic and ionic stresses of the environment, during which time there is a gradual
accumulation of urea in the body fluids? If not, how is urea retained, and how
are excess sodium and chloride eliminated during development, particularly before
the rectal gland is fully developed? The problem is complicated, although, it may
be argued, made more interesting by the fact that elasmobranchs as a group show
a variety of patterns of development ranging from oviparity (e.g. Scyliorhinus
canicula, Raja erinacea), through ovoviviparity (e.g. Squalus acanthias), to various
forms of aplacental and placental viviparity (e.g. Mustelis canis). It has, in fact,
been claimed that there is a link between the osmoregulatory stresses experienced
during development and the evolution of viviparity in elasmobranchs, although
this now seems unlikely (see below).
For oviparous species, the opinion was long expressed in the literature that
relatively high levels of urea were supplied initially to the embryo and were retained
until hatching by the presence of an impermeable egg case (Smith 1936; Price and
Daiber 1967). Indeed, it had been claimed that the inability of the early embryo to
regulate urea was a major selection pressure leading elasmobranchs toward a vivi-
parous mode of reproduction (price and Daiber 1967). However, as early as 1930,
Needham and Needham had demonstrated that the oviparous elasmobranch egg case
was highly permeable to urea, a fact confirmed by several more recent studies
(Hornsey 1978; Foulley et al. 1981). Despite this, Read (1968) clearly showed that
the embryos of the oviparous Raja binoculata maintained high concentrations of both
urea and TMAO, just as in the adult, and that these remained essentially constant
throughout development. He further showed that even the undeveloped eggs were
capable of retaining urea against large diffusion gradients, and that early embryos
removed from their egg cases were perfectly able to survive in seawater. Indeed, it
is very easily demonstrated in Scyliorhinus that the fluid inside the oviparous egg
case surrounding the developing embryo is osmotically and ionically identical to
seawater and that the egg case provides no significant barrier to the movement of
ions, water or urea (Shuttleworth, unpubl. data). Clearly, the diffusion barrier to
urea must reside in the embryonic epithelia (e.g. filamental external gills) and in the
yolk sac, and the developing embryo must possess all the osmoregulatory and iono-
regulatory powers of the adult.
In those species showing ovoviviparity, such as Squalus acanthias, the developing
embryos are initially enclosed as a group within a thin membrane (the "candle").
In Squalus this stage lasts approximately 6 months. Subsequently, the embryos
(now called pups) with their large yolk sacs attached hatch, but are retained in the
maternal uterus for a further 18 months. The osmoregulatory abilities of the
embryos at these different stages, together with the nature of the environment to
which they are exposed, have been studied by Price and Daiber (1967) and, in more
detail, by Evans et al. (1982). At the candle stage, the embryo plasma, the fluid
contained within the candle membrane, and the surrounding uterine fluids are all
6.8 Osmoregulation During Development 193
'candle' stage ' pup' stage

500 sw adult
II I
r-
~ 400
5
g 300
r-
- -
o
o
r- -
~ 200
:;)
oro 100
z
-'- '- - ....
M u c E u E
Fig. 6.6. Concentrations of urea and sodium in the embryos and embryonic environment at
different stages of development in Squa/us acanthias, compared to those in the maternal plasma
and external seawater. Open bars represent sodium, solid bars urea. M maternal plasma; U uterine
fluids; C "candle" fluids; E embryo plasma. (Evans et al. 1982)
similar to each other and, apart from slightly higher ion concentrations and lower
urea concentrations, do not differ markedly from the maternal plasma (Fig. 6.6).
At the pup stage, the embryo plasma is virtually the same as the maternal plasma,
including the concentration of TMAO (Goldstein et al. 1967), but the uterine fluid
is osmotically and ionically equivalent to seawater (Fig. 6.6). This is to be anticipated,
as Burger (1967) had shown that , during this time, the mother periodically flushes the
uterus with seawater - although subsequent studies have shown that the uterine
fluid possesses characteristics of pH and ammonia levels that are quite distinct
from normal seawater (Kormanik and Evans 1986). Clearly, the pups are capable of
osmoregulating in a similar manner to the adult and can easily be maintained
extra-utero for extended periods in seawater. The fact that embryos at the candle stage
are surrounded by an environment that is identical to their own plasma and similar
to that of the mother was taken to imply that the embryos at this stage are
incapable of osmotic or ionic regulation (Price and Daiber 1967). However, as Evans
et al. (1982) showed, exposure of the candles to seawater for 4-6 days resulted in the
candle fluid surrounding the embryos coming into virtual ionic equilibrium with
the seawater, but the embryos themselves were still capable of maintaining their
plasma with normal high urea and low sodium chloride concentrations. It is clear
therefore, that osmo~ic and ionic regulatory mechanisms equivalent to those of the
adult exist even in these very early embryos.
Available information on the osmoregulatory stresses faced by the developing
embryos in viviparous species is very limited. Price and Daiber (1967) report that ,
in Mustelis canis, the uterine environment in which the embryos are retained for some
18 months, is similar, osmotically and ionically, to the maternal plasma. In this case
the young would not experience any osmotic or ionic problems until birth, when
presumably osmoregulatory capacities are fully developed.
In summary, it would seem that the combination of unique osmoregulatory
characteristics and a wide range of developmental patterns seen in elasmobranch
fish would make the study of the ontogeny of osmoregulation in this group
particularly interesting. Even though the early claims linking the evolution of vivi-
parity in the group with the presumed inability of the early embryos of oviparous
species to regulate adequately have been shown to be unjustified, the demonstration
that even the earliest embryos of both oviparous and ovoviviparous species apparently
po~sess full regulatory capabilities of urea retention and ion elimination, equivalent
to those seen in the adults, is particularly fascinating, and it is only a pity that more
detailed studies have not been performed.
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Chapter 7 I. W. HENDERSON!, L. B. O'TOOLE2 and N. HAZON2
Kidney Function
7.1 Gross Morphology

Elasmobranch kidneys are elongate paired structures lying along the dorsal wall of the
body cavity. In sharks they appear anteriorly as thread-like structures midway
along the abdominal cavity and gradually widen posteriorly on either side of the
dorsal aorta, to fuse and overlap at the level of the cloaca. In skates they are
lobulate bodies on either side of the cloaca, running cephalad, parallel to the mid-
dorsal line in the males, but often anteriorlyo.degenerate in the females (Hyman 1942;
Hickman and Trump 1969).
7.2 Blood Supply

The renal blood supplies of the dogfish, Squalus acanthias, and the little skate, Raja
erinacea, have been described in detail. In dogfish, the renal arterial supply starts
from ventIal branches ofthe segmental arteries (Fig. 7.1). There are 18 to 20 of these,
the majority being spread along the broader caudal portion of the kidney. Renal
branches are linked sequentially by a horizontal vessel to form a longitudinal chain
within the organ itself. Each renal artery gives rise to four further vessels, two
dorsal, one horizontal (Fig. 7.1), (Ghouse et al. 1968). The vascular pattern of the
skate differs in that there is only one renal artery per kidney. This branches
from the divided dorsal aorta, enters the kidney and runs anteriorly just below the
surface, giving off vessels to each lobe (Deetjen and Antkowiak 1970).
As in most non-mammalian vertebrates, elasmobranch fish possess a renal portal
system. The large caudal vein from the tail bifurcates to form the portal veins which
enter the kidney, divide and anastomose to form a matrix of small vessels. Blood
from these then mixes freely with blood from the glomerular vasa efferentia before
leaving the kidney via the renal veins. There is also evidence (Green 1986, J. A. Brown
1986, pers. commun.) of a glomerular bypass vessel wherein blood may pass from
the afferent to the efferent vessel, therby avoiding filtration (Fig. 7.2).
1 Department of Zoology, University of Sheffield, Sheffield SIO 2TN U.K.

2 Department of Physiology and Pharmacology, University ofSt. Andrews, Gatty Marine Laboratory,
St. Andrews KY16 8LB U.K.
202 Chapter 7. Kidney Function
DORSAL
VENTRAL
F
Fig. 7.1. Schema of arterial supply to the kidney. A" A2 renal arteries; B intrarenal arterial chain;
C" C2 ventral arcuates; D" D 2 , D3 dorsal arcuates; E horizontal artery; F archinephric Wolffian
duct; G ureter. (Ghouse et aL 1968)
Fig. 7.2. Electromicrograph of Mercox cast of a dogfish glomerulus showing preglomerular shunt.
(1. A. Brown and C. Green 1987, unpubL observ.)
7.3 Microanatomy of the Nephron 203
7.3 Microanatomy of the Nephron
The kidney of Raja erinacea has been reported to have a fixed glomerular population
of approximately 2260 glomeruli evenly distributed throughout each kidney, (Ant-
kowiak and Boylan 1974). The same authors comment on marked variation in
both number and distribution of glomeruli in the dogfish Squalus acanthias, giving
a wide spread of values with a mean of 10,350 per kidney for six female fish.
Green (1986), recorded 1140 and 1474 glomeruli per kidney for male and female
Scyliorhinus canicula respectively, and showed that these were not obviously related
to body weight. These studies also show that, in the female at least, the vast
majority of glomeruli are confined to the caudal 50% of the kidney. The nephron
itself is very long, Ghouse et al. (1968) reporting mean lengths of 3.3 cm for
Squalus acanthias, while Nash (1931) gives a value of 9 cm for Raja stabulaforis.
This great length, the labyrinthine meanderings of the tubule and an apparent
regional heterogeneity in its structure, alongside seasonal, sexual and species varia-
tions, clearly hamper an easy understanding of the three-dimensional structure and
basic function . However, a much clearer picture is now emerging, mainly through
work on the little skate, Raja erinacea, in which renal tubular micropuncture is
Collecting Duct
Blood Sinus
Fig. 7.3. Schematic drawing of the pathway of the skate nephron in the bundle zone (dorsal) and
in the sinus zone (ventral) showing some of the nephron complexity. (Lacy et al. 1985)
possible (Deetjen and Antkowiak 1970 ; Stolte et al. 1977; Lacy et al. 1985; Lacy
and Reale 1985a, b).
An area consisting of parallel limbs of the tubule had been reported in many studies,
as had a "special segment" which was striated and separated from the rest of the
tubule (Haller 1902; Borcea 1906; Marshall 1934; Smith 1931 ; Kempton 1939,
1956). Their relationship with the rest of the nephron , however, remained uncertain.
Lacy et al. (1985 ; Lacy and Reale 1985 a, b), in elegant accounts, have demonstrated
the presence of an elaborate counter-current flow system. Thus, starting from the
glomerulus, the nephron is divisible into four discrete loops (Loops I, II, III, IV),
a distal tubule and a collecting duct. Among the characteristics that vary within these
loops are overall tubular dimensions, type of epithelium, presence of a brush border,
w -
, - - - -,- -
z
OW
N...J ConVOluted
wO
...J3
~CD
':I
al~
~
~
Straight
~I--NECK
Q n SEGMENT
C3 1
_nz
EJ II
~m
PROXIMAL
SEGMENT
w IDJ I
z
o mn
N G m INTERMEDIATE
(f) E!iil til SEGMENT
:::>
z n: rDlZ:
en nnm
aD I DISTAL
CJ II SEGMENT
~ I COLLECTING
ell DUCT
Fig. 7.4. Schematic drawing of the course of the two dorsal (Loops I and III) and two ventral
(Loops II and IV) loops as well as of the distal tubule segment and collecting duct of the skate
nephron. Loops I and III and the distal tubule (early distal) form the tubular bundle (counter-
current system) and are wrapped by the peritubular sheath. RC renal corpuscle. The subdivisions
of each tuh!le part, based on the structure of the epithelium, are indicated by symbols. (Lacy and
Reale 1985a)
7.4 Kidney Function 205
flagella and cilia and mitochondrial density. The tubule can be thought of as folded
back upon itself twice, with two of the loops contained within a peritubular sheath
in the dorsal bundle zone of the kidney and two emerging into the ventral sinus zone.
The loops enter and leave the peritubular sheath at the same point, close to the urinary
pole of the renal corpuscle, then run parallel for a distance before becoming
highly convoluted. A counter-current system of five tubular segments is thus formed,
consisting of the ascending and descending limbs of the two peritubular loops and
the distal tubule, (Fig. 7.3 and 7.4). The glomerulus lies outside the peritubular sheath
in the bundle zone, while the remaining two loops lie in the sinus zone entwined with
the loops of adjacent nephrons. It is these latter two loops that may have been the
special segments described by early workers. The sheath is made up of squamous
epithelia linked by tight junctions and separates each bundle from the next. Apart
from the point where the five parallel tubular segments enter and the distal tubule
leaves to join the collecting duct, the sheath is broken only by an afferl!nt and an
efferent capillary. The~e vessels enter and leave near the tubules and anastomose
around them throughout the sheath (Lacy et al. 1985). The descriptions of both Stolte
et al. (1977) and Deetjeen and Antkowiak (1970) largely agree, but the terminology
differs with respect to the terms "distal" and "proximal" and some further sub-
divisions of the segments. The structural homologies of these many segments in the
various species are not absolutely clear. However, the descriptions of Lacy and
colleagues (1985) of the 16 divisions, defining the relative positions of each within
the framework of their three-dimensional model, may now be used to relate renal
structure to function in the elasmobranch.
7.4 Kidney Function
Table 7.1 summarizes some general characteristics of renal function in five species
of elasmobranchs. Elasmobranch plasma is hyperosmotic to seawater but contains
concentrations of sodium and chloride ions which are less than those of the
environment. Thus water will enter elasmobranchs osmotically and sodium and
chloride will enter by diffusion across permeable membranes (see Chap. 6). The
maintenance of normal body fluid and electrolyte balance requires an integrated
homeostatic response between the kidney and extrarenal structures such as the gill,
gut and rectal gland; this chapter focusses on the kidney.
Marine elasmobri1.11ch glomerular filtration rates (GFR) vary from 0.2 to 12 ml
kg -1 h -1, but seem to average about 4.0 mg kg -1 h -1 (Hickman and Trump 1969).
Thus GFR is significantly higher than in seawater teleosts (Hickman and Trump
1969). Changes in GFR appear to be mediated by changes in the number of
filtering glomeruli rather than by a change in single nephron filtration rates (Shannon
1940; Kempton 1953; Henderson et al. 1978). However, the sites and the neuroendo-
crine control of GFR are poorly understood. Thus, pharmacological doses of adrena-
line mayor may not raise GFR (Deetjen and Boylan 1968; Forster et al. 1972).
In a recent more detailed study (Brown and Green 1987), adrenaline induced a clear
glomerular diuresis, reflecting increased filtration rates in individual nephrons.
Table 7.1. General characteristics of renal function IV
0
'"
Squalus acanthias Scyliorhinus canicula Hemiscyllium plagiosum Raja erinacea Pristis microdon
Uosm 800 960 797 ± 34 966.9 ± 18.7 550 (")

::r
(mOsmoll- 1 ) .§
U pH 5.8 CD
....
U vo1 0.5 0.19 0.36 ± 0.06 10.4 ~
(ml kg-I h- 1 ) US" ~
p.
GFR (ml kg-I h- I ) 3.5" 1.13
Uurea 100 124 248 ± 21 14 ~
U TMAO 10 'Tj
UNa + 240 238 249 ± 18 179.8 ± 16.4 ::s

'g."
U C1 - 240 361 225 ± 16 208.9 ± 15.9 6.3 0
UK + 2 3.7 3.57 ± 0.37 31.6 ±8.8 n ::s
U ca2+ 3 3.29 ± 0.65 10.4 ± 1.6 1.7
U Mg2+ 40 50.8 ± 15.4 154 ± 29.1 1.3
Upol- 30 100.4 ± 22.5 6.9
Usoi- 70 3.29 ± 0.65 60.8 ± 10.9 0.3
Reference Burger (1967) Haywood, Brown and Wong and Chan (1977) Stolte et a!. (1977) Smith (1931)
aShannon (1940) Henderson
(unpub!. observ.)
a Urine concentrations in mmoll- I

7.4 Kidney Function 207
Indeed, fewer nephrons were filtering in these studies. About 75-85 % of filtered
fluid is reabsorbed in the kidney and, together with solute reabsorption, this renders
urine hypo-osmotic to plasma (Kempton 1953; Schmidt-Nielsen and Rabinowitz
1964).
7.4.1 Urea Reabsorption
One of the most impressive features of the elasmobranch kidney is its ability to
reabsorb urea (Marshall 1930; Smith 1931, 1936; Kempton 1953). The sites and
mechanisms of this reabsorption are uncertain, with conflicting and incomplete
evidence for active and/or passive urea reabsorption.
Evidence suggesting an active mode of reabsorption includes:
a) Fractional excretion of urea is only 0.5 % under normal conditions, a figure
remarkably similar to that found for the active reabsorption of glucose (Kempton
1953).
b) Urea reabsorption is iso-osmotic (Kempton 1953).
c) Elasmobranch nephrons will absorb 95 % of filtered urea but only 35 % thiourea,
suggesting a urea-specific transport mechanism (Boylan 1967).
d) Both phloretin and chromate inhibit urea reabsorption in the dogfish (Hays et al.
1977).
This has led to the proposal of on active urea reabsorption mechanism (Smith 1931;
Kempton 1953; Forster 1970). Micropuncture studies have implicated Loop II as a
possible site for active sodium-linked urea reabsorption (Stolte et al. 1977). However,
other investigations have demonstrated characteristics of passive transport mecha-
nisms. For example, Kempton (1953) failed to detect a transport maxima for
urea despite markedly raising plasma urea concentrations. Also, although the urea
analogues methyl urea and acetamide are partially reabsorbed by elasmobranch
nephrons, at high plasma concentrations they do not saturate the urea carrier mecha-
nism (Schmidt-Nielsen and Rabinowitz 1964). Finally, probenicid, a substance which
blocks active urea secretion in the frog, does not affect urea reabsorption in the
dogfish (Forster and Berglund 1957).
An alternative passive model for urea reabsorption has been proposed (Boylan
1972). This involves iso-osmotic active reabsorption of sodium and water, together
with tubular impermeability to urea in either or both loops II and III of the
nephron. This woul~ result in a low urea environment into which urea would diffuse
into the distal segments which are enveloped by the foldings of loops I and III.
The model assumes that the distal segment is both very permeable to urea and
impermeable to water. Some evidence in support of this theory is that the ultra-
structure of Loop III (but not Loop I) tubular cells has the intracellular characteristics
of cells known to actively transport sodium in other tissues (Lacy et al. 1975; Endo
1984). In addition, micropuncture analyses have suggested the distal tubule to be a
site for urea reabsorption (Thurau and Acquisto 1969), although this is not in
agreement with the data of Stolte and colleagues (1977). However, all studies agree
that there is no urea reabsorption in the collecting ducts (Thurau and Acquisto
1969; Deetjen et al. 1972; Stolte et al. 1977).
Definitive evidence for or against active or passive urea reabsorption is thus

lacking. What is evident is that this process in elasmobranchs differs from the urea
transport mechanisms described for all other vertebrates with the possible exception
of Rana cancrivora (Gordon et al. 1961). Given the structural complexity of the
elasmobranch nephron, this is perhaps not surprising, and future studies must con-
centrate on these novel properties of elasmobranchs rather than comparing them
with other vertebrates.
The kidney is also of great importance in reabsorbing another important osmotic
constituent, trimethylamine oxide (TMAO). Urinary concentrations of TMAO are
10 %those of plasma and plasma concentrations are maintained within a narrow range
of 60-80 mmoll- 1 (Norris and Benoit 1945; Cohen et al. 1958). Furthermore,
administering structural analogues such as basic trimethylamine or dimethylamine
inhibited tubular reabsorption of TMAO, whereas methylamine had no effect. This
was taken to indicate competition for a specific carrier (Cohen et al. 1959). The
kidney has therefore been implicated in TMAO conservation in a similar manner to
urea.
7.4.2 Reabsorption and Secretion of Ions
Sodium is actively reabsorbed in elasmobranch nephrons (Burger 1967; Stolte et al.

1977). Renal tubular micropuncture investigations have shown Loop II of the skate
nephron to be a site of both sodium and chloride reabsorption (Stolte et al.
1977), although on ultrastructural criteria this section of the nephron does not
appear to have the intracellular characteristics that one would associate with active
transport (Lacy et al. 1975; Endo 1984). The exact mechanisms of sodium reab-
sorption are not known, but it appears to be insensitive to ouabain but sensitive
to furosemide and ethacrynic acid (Hayslett et al. 1972). Histochemically Na + -K +-
ATPase activity has only been shown in the distal tubule and collecting duct
(Endo 1984).
Recently, single isolated perfused tubules from Squalus acanthias corresponding
to Loop II have been reported to be capable of actively secreting chloride and
sodium ions (Beyenbach and Fromter 1985). There also appears to be net fluid
secretion driven by active chloride transport coupled to sodium secretion CD. B. Sawyer
et al. 1985 a, b). Considering that sodium and chloride ions continually enter
elasmobranchs through the gills (Boylan 1967), this could be interpreted as a renal
mechanism supporting the rectal gland. At least this could explain that when the
rectal gland is removed, elasmobranchs still osmoregulate and maintain normal plasma
concentrations of sodium and chloride (Burger 1965; Chan et al. 1967). Urea and
sodium reabsorption have been linked, as they are reabsorbed in a fixed ratio of
1.6 mol urea: 1 mol sodium (Schmidt-Nielsen et al. 1972). However, chromate caused
a large irreversible inhibition of urea reabsorption with a much smaller effect on
sodium reabsorption (Hays et al. 1977). This suggests that there may be some
degree of independence in the reabsorptive mechanisms for sodium and urea.
Tubular secretion in elasmobranchs was first demonstrated in Squalus acanthias
(Smith 1939) and confirmed in the smooth dogfish, Mustelus canis, by Kempton
(1966). Net tubular secretion of divalent ions such as magnesium and phosphate,
7.5 Control of Kidney Function 209
and to a lesser extent calcium and sulphate has been demonstrated (Burger 1967;
Stolte et al. 1977; Henderson et al. 1978). Micropuncture studies have once again
suggested Loop II of the nephron as a site of divalent ion excretion (Stolte et al. 1977).
The ultrastructural studies used as evidence for active tubular reabsorption in Loop II
could just as easily be applied in support of active tubular secretion (Lacy et al. 1975;
Endo 1984).
As mentioned, active Cr-secretion has also been demonstrated in Loop II.
However, the excretory patterns for chloride ions are influenced by plasma levels
of other electrolytes such as magnesium, phosphate and calcium, as well as by urea,
and presumably these interactions take place at other sites along the nephron.
For example, anions such as phosphate and sulphate apparently reduce urinary
chloride excretion, whilst magnesium promotes its excretion in an obligatorily paired
fashion (Burger 1967).
7.5 Control of Kidney Function
The endocrine control of kidney function has been investigated to a very limited
degree (Table 7.2). Elasmobranchs possess a range ofnove1 hormones, some of which
may be involved in the control of kidney function. Perhaps the most unusual aspect
of elasmobranch endocrinology is the range of neurophysial peptides. To date, all
elasmobranchs investigated possess arginine vasotocin (AVT), rays possessing in
addition glumitocin (Acher et al. 1965, 1967; Chauvet et al. 1965; W. H. Sawyer
et al. 1969) and sharks both aspartocin and valitocin CW. H. Sawyer et al. 1969;
Acher et al. 1972). The physiological function of these hormones is unknown, but the
fact that elasmobranchs, unlike other fish (W. H. Sawyer 1972), may alter their
renal tubular water permeability (Henderson et al. 1978), suggests that a genuine
tubular-acting antidiuretic-hormone-like principle may be present. Only further study
will determine which, if any, of the elasmobranch neurohypophysial peptides are
involved.
Elasmobranchs have been reported to lack a renin-angiotensin system. In one
investigation, renin-like activity was not found (Nishimura et al. 1970) and, histologi-
cally, juxtaglomerular cells have not been identified (Oguri et al. 1970; Crockett
et al. 1973), although holocephalan ratfish and rabbitfish possess both juxtaglomerular
cells (Oguri 1978) and renin-like activity (Nishimura et al. 1973). However, in some
preliminary investigations, dogfish renal extracts incubated with relatively crude
preparations of rat and dogfish renin substrate generated angiotensin-like pressor
material as determined in the standard nephrectomized rat bioassay and in the free-
swimming dogfish (Henderson et al. 1980; O'Toole, Hazon, Uva, Ghiani and Masoni,
unpubl. observ.). Furthermore, relatively crude renal extracts were also pressor in
the same bioassay suggesting the presence of renin-like activity. Elasmobranchs also
possess an angiotensin I (AI)-converting enzyme, since mammalian AI was pressor
in sharks and the effect was blocked by the kininase II inhibitor SQ 20,881 (Opdyke
and Holcombe 1976). Thus elasmobranchs may possess the prerequisite enzymes for
a functional renin-angiotensin system. However, only future research will reveal
the systemic or intrarenal role for such a system in elasmobranchs.
Table 7.2. Endocrine control of kidney function ~
0
Hormone Species Method Dose Effect References
Adrenaline S. canicula Infusion 1 Ilg kg- 1 min- 1 GFR-UF diuresis Brown and Green (1987) (j
::r'
Noradrenaline S. acanthias Injection 1 mg kg- 1 GFR-constant excretion Forster et al. (1972) ~
of urea/TMAO up 1>
...,
Arginine Vasotocin S. canicula Injection 20 ng kg- 1 UF-constant Maetz and Lahlou (1974) :-'
Hypophysectomy S. canicula UF-down corrected by Payan and Maetz (1970) ~
ACTH Po
Calcitonin S. acanthias Injection 200 MRC units Ca2 + /urea excretion Hayslett et al. (1972)
ii
'<
constant ."
s::=:s
Thyroxine Ginglymostoma In vitro Incubation Reduced renal Na-K- Honn and Chavin (1977)
cirratum ATPase, cAMP and cGMP g.
=:s
UF = urine flow
GFR = glomerular filtration rate
7.5 Control of Kidney Function 211
A unique corticosteroid lex-hydroxycorticosterone (1ex-OH-B), has been isolated

from the elasmobranch interrenal gland along with its associated glycoprotein recep-
tors (Idler and Kane 1980). 1ex-OH-B is the major corticosteroid, at least in dogfish
(Kime 1977; Hazon and Henderson 1984, 1985a) and although a precise role has not
been identified, its function has been related to electrolyte and/or urea metabolism
in dogfish adapted to dilute seawater (Hazon and Henderson 1984, 1985b). In the
skate, renal tubular micropuncture showed that during adaptation of fish to a dilute
environment (75 % SW) there was an increase in sodium reabsorption which
required between 24 to 48 h adaptation to become apparent (Stolte et al. 1977).
These workers suggested that 1ex-OH-B may be responsible for this effect. Certainly,
the recent finding that dogfish renal extracts stimulate lot-OH-B production (Hazon
and Henderson 1985a, b) suggest that some form 'of renal-interrenal control
mechanism may exist analogous to the renin-angiotensin-corticosteroid system of
other vertebrates (Nishimura 1980).
Studies on the actions of other hormones on kidney function are essentially
preliminary. Cytologically and immunologically, lactotrophs and prolactin-like
activity have been identified in the rostral lobe of the pituitary gland in a number
of elasmobranchs (Della Corte and Chieffi 1961; Mellinger 1962). However, White
and Nicholl (1980) failed to detect any binding of I25I-labelled ovine prolactin with
renal membrane preparations from three species of chondrichthyeans. Hence, although
several reports implicate prolactin in .osmoregulation (De Vlaming and Sage 1973,
De Vlaming et aL 1975; Payan and Maetz 1970), its renal role is undefined
(Bern 1975).
Similarly, the control of renal calcium regulation is unknown. Salmon calcitonin
(200 MRC units) appeared to have little effect on the urinary excretion of calcium
or urea in Squalus acanthias, although the fractional sodium and potassium excretory
rates were reduced (Hayslett et al. 1972). Of other calcium-regulating hormones,
some preliminary studies have failed to identify a vitamin D3 metabolising system;
incubation of labelled 25-hydroxycholecalciferol with extracts of the kidney, rectal
gland, gills and liver failed to reveal hydroxylation of this substrate. Moreover,
HPLC analysis of dogfish plasma failed to show the presence of known hydroxylated
or dihydroxylated derivatives of cholecalciferol (Guilland-Cumming et al. 1982).
The only other hormones implicated in control of renal function are thyroxine,
noradrenaline and, 'as already mentioned, adrenaline. In vitro, thyroxine reduced
renal N a + - K + -ATPase and Mi + -ATPase activity with parallel reductions in the
levels of cyclic AMP and cyclic GMP (Honn and Chavin 1977). Exogenous
noradrenaline (1 mg kg-I) has been shown to increase excretion of urea 15 times,
chloride 2.6 times and TMAO 3.8 times with no concomitant effects on GFR
(Forster et al. 1972).
It is very difficult to produce an integrated theory of hormonal control of the
elasmobranch kidney at this stage. The physiological significance of some reports,
largely using pharmacological does or heterologous hormones, makes interpretation
almost impossible. Clearly only further investigation will reveal the physiological
role of hormones involved in renal control in elasmobranch fish.
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drichthyes and cyclostomes. Bull Jpn Soc Sci Fish 36: 881-884
Opdyke DF, Holcombe R (1976) Response to angiotensin I and II and to angiotensin I-converting
enzyme inhibitor in a shark. Am J Physiol 231: 1750-1753
Payan P, Maetz J (1970) Balance hydrique et mineralechez les elasmobranches: arguments en faveur
d'un contr61e endocrinien. Bull Inf Sci Tech Commt Energ Atom 146: 77-96
Sawyer DB, CliffWH, Wilhelm MM, Fromter RO, Beyenbach KW (1985a) Proximal tubules of the
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Sawyer DB, Cliff WH, Wilhelm MM, Fromter RO, Beyenbach KW (1985b) Mechanism of fluid
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Sawyer WH, Manning M, Heinicke E, Perks AM (1969) Elasmobranch oxytocin-like principles:
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Squaills acanthias. J Cell Comp Physiol 16: 285-291
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200: 85-96
Chapter 8 N. HEISLER
Acid-Base Regulation
Regulation of the acid-base status is an imperative task for maintenance of homeostasis

in living animals. Deviations of pH from the predetermined setpoint values may
result in reduced metabolic performance as a result of the often pronounced optima
in the overall pH/activity relationships of metabolic pathways. The mechanisms
involved in acid-base regulation do not differ among and within vertebrate classes,
but the extent to which certain processes are, or can be, utilized may be vastly
different. Accordingly, buffering, changes in Pe02 , and transmembrane and trans-
epithelial acid-base-relevant ion transfer, which are the main regulatory processes
in other vertebrates, are also valid mechanisms for elasmobranch fishes (for closer
analysis of relative importance and theoretical limitations of these mechanisms in
fishes, see Heisler 1986d, e).
Some of the general principles of elasmo branch acid-base regulation were described
decades ago in the pioneering studies of Smith (1929 a, b, 1931, 1939), Dill et al. 1932,
Hodler et al. (1955) and Robin et al. (1964a, b, 1966) on elasmobranch body
fluid composition and excretory mechanisms and sites, and analyzed by Murdaugh
and Robin (1967) in their classical chapter on Squalus acanthias. Closer understanding
of the interrelationships among various body fluid compartments and the environ-
mental water, and the interaction of the involved mechanisms was, however,
facilitated only recently by development of more sensitive techniques for the
determination of acid-base-relevant ion transfer mechanisms, and for practicable
and accurate determinations of intracellular buffering properties and pH. These
techniques have allowed closer insight irito the less accessible intracellular body fluid
compartments and have led to a more complete comprehension of the processes
involved in whole-body acid-base regulation (for a detailed description of metho dolo-
gies, see Heisler 1984 b).
The aim of this ·chapter is to review some of the relevant literature accumulated
during the last two decades, to closer characterize and quantify the mechanisms
involved, and to describe in more detail the correlations among regulatory processes.
Data collected in teleost species will also often be referred to when differences are
not evident and not to be expected, and relevant literature on elasmobranch fishes
is not available, or when comparison yields additional insight. Since the majority of
recent data on elasmobranchs have been collected in single species, and generally
Author's address: Max-Pianck-Institut fUr Experimentelle Medizin, Abteiiung Physioiogie, Hermann-

Rein-Strasse 3, 3400 Gi:ittingen, F.R.G
216 Chapter 8. Acid-Base Regulation
very few elasmobranch species have been studied in any detail, our knowledge is
still quite selective and fragmentary. Accordingly, this treatise must remain incomplete
and tentative.
8.1 Steady-State Acid-Base Regulation
8.1.1 Release of Acid-Base-Relevant Substances
Energy production by metabolic processes is generally accompanied by the production

of acid-base-relevant substances which, for maintenance of a steady state, have to be
balanced by an equivalent removal from the body fluids. Quantitatively, the most
important metabolic endproduct relevant to acid-base balance is CO2 which, during
aero bic metabolic processes, is produced in a relative amount of 0.7-1.0 of the oxygen
consumption, depending on the type of metabolic substrate utilized. CO2 is primarily
eliminated from the organism by non-ionic diffusion via the gill epithelium, supported
by convective transport in the blood from the tissues to the gills, and by the gill
water ventilation (see Piiper 1986).
A characteristic feature of elasmobranch protein catabolism is the production
of urea as the main nitrogenous waste product (ureotelism). Depending on species
and conditions, 70% to more than 90% of the nitrogen-containing metabolic end-
products is urea (e.g. Smith 1929b). This acid-base-irrelevant and relatively non-
toxic substance is utilized in elasmobranchs to balance the osmotic equilibrium, and
represents about one half of their total plasma osmotic activity (see Chapter 6 this
Vol.). Ammonia is produced in a much smaller fraction of the nitrogenous waste
than urea (less than 10 % up to 30 %, compared to 60-90 % in teleost fish species;
Smith 1929a; J. D. Wood 1958; Fromm 1963), but has a much larger impact for
acid-base regulation. Upon production from deamination of (X-amino acids mainly
in the liver, but also in gills and kidneys (see Goldstein et al. 1964; Pequin 1962;
Pequin and Serfaty 1963; Cameron and Heisler 1983), ammonia is almost immediately
ionized according to its high pK' value « 9.6, see Emerson et al. 1975; Bower and
Bidwell 1978; Cameron and Heisler 1983, 1985). This ionization of the highly
toxic NH3 (for references on toxicity see Heisler 1984 b, 1986 b) to NH: has an
alkalinizing effect, neutralizing an equivalent fraction of the concomitant CO2
production.
The production of bicarbonate from CO2 concomitant to, and equivalent with,
the ammonia ionization has to be eliminated together with the ammonium ions
(NHt) in order to prevent progressive alkalinization. This may be performed by
ionic transfer processes for both ions, or by non-ionic ammonia elimination
(NH 3-diffusion), which reverses the alkalinizing effect of the original NH3 ionization
before final elimination from the body fluids (for details Heisler 1984b, 1986b;
Claiborne and Heisler 1986). Both elimination mechanisms have an identical effect
on changes in bicarbonate and total ammonia concentrations in the environmental
water (see Heisler 1984b, 1986b; Claiborne and Heisler 1986), which accordingly
cannot provide any evidence for or against ionic or non-ionic elimination mecha-
nIsms.
Table 8.1. Net steady-state release to the environmental water of ammonium ions (~NH:w), bicarbonate (~HC03w)' and net H+ equivalent excretion (~H:~w)
in relation to oxygen consumption (V0 )a
2
Species Series ~NH: ~HC03W ~H:~w V02 ~H:~w' 100 Reference

(1) (2) (1)-(2) ----v
Elasmobranchii
Scyliorhinus Hypercapnia 0.83 0.57 0.26 34.8 b 0.74 Heisler et aI. 1976
stellaris Temperature 0.33 -0.09 0.42 34.8 b 1.20 Heisler 1978
change
Muscular 0.64 0.44 0.20 34.8b 0.57 Holeton atld:
activity Heisler 1983
Hyperoxia- 0.96 0.62 0.34 34.8 b 0.97 Heisler, Toews and
induced Holeton 1988
. hypercapnia
Squalus 1.4 Smith 1939
acanthias
Estimate 0.66 Smith 1929a ?O
to 0.88
Estimate 0.35 Murdaugh and
to 1.25 Robin 1967
Teleostei J
(Range) 1.48 0.19 -0.62 -1.12 Heisler 1984b, 1986b
to 6.83 to 5.58 to 3.29 to 5.94 ~
Man Normal man 0.49 0.37 0.85 220 0.39 Pitts 1945 a, .b ~
~
a Values expressed as /lmol kg- 1 body water min- 1
b Data of Randall et a1. 1976.
f
~
f
~
-.l
Ammonium and bicarbonate are produced during steady state in elasmobranchs

in a relative amount of up to about 4 % of the oxygen consumption (Table 8.1), which
is much less than the maximal values found in ammoniotelic fish species (up to
about 12% of the oxygen consumption, see Table 8.1 and Heisler 1986b). The
amount of bicarbonate-equivalent ions released from the fish is regularly lower
than that of ammonia, reflecting the net metabolic production of H+ ions. This
represents the difference of the production of fixed acids from break-down of
sulphur- and phosphorus-containing organic compounds and from incomplete
oxidation of fatty acids, amino acids or carbohydrates, minus the production of
fixed bases from oxidation of carbohydrate alkali metal salts or incomplete oxi-
dation of organic bases. The net H+ production in elasmobranchs (L'lH:~ w) is
comparable to that in ammoniotelic fish species (teleosts) and other vertebrates,
including man, when related to the respective specific oxygen consumption (Table 8.1).
8.1.2 Steady-State Acid-Base Status
Arterial pH in elasmobranch fishes is not significantly different from that found

in teleost fish species at the same temperature (Fig. 8.1). The body fluid pH is strictly
correlated with temperature, with pH falling when temperature rises. Robin (1962)
wai> the first to demonstrate in turtles that the reported larger variability of pH in
heterothermal. as compared to homoothermal animals (e.g. Austin et al. 1927;
Dill and Edwards 1935; Edwards and Dill 1935; Dill et al. 1935), was correlated
to different environmental temperatures. Rahn and Baumgardner (1972) showed that
the pattern of falling pH with rising temperature also applied to fish, although the
proposition that plasma pH varied with temperature in parallel with the pH of
neutral water (i.e. with a constant relative alkalinity, or a constant ratio of
[OH-]/[H+]; Rahn 1966, 1967) could not be confirmed by systematic studies. When
water-breathing fishes were acclimated to various body temperatures, plasma pH
was found to change with temperature generally much less (L'lpH/L'lt from -0.010
to -0.014, average -0.012 u;oe, Fig. 8.1) than expected on the basis of a constant
relative alkalinity (L'lpN/L'lt = -0.019 ure, range 5-20 De; L'lpN/L'lt = -0.017 Ure,
range 20-35 °e; Weast 1975, see Heisler 1986c).
The impressive uniformity of arterial plasma pH in fishes suggests that this
parameter is tightly regulated during steady-state conditions (Fig. 8.1). In contrast,
p C02 and bicarbonate concentrations are largely variable. Pcoo always rises with
increasing temperature, although the magnitude of change may vary considerably
between the two extremes of almost no change at all (Fig. 8.1, jJ.lvenile Scyliorhinus,
Sallno gairdneri) to a change larger than that required for the observed change in
pH at constant bicarbonate concentration (Fig. 8.1, adult Scyliorhinus). This variability
in Pco 2 may be attributed to limitations in gas exchange (Heisler et al. 1976 b). Pco2
in water-breathing species cannot be freely adjusted, and therefore modulation
of the arterial bicarbonate concentration may be the predominant and final regula-
tory mechanism to achieve the arterial pH setpoint value. This could also explain the
large variability seen in this parameter. This hypothesis is supported by experiments
in Scyliorhinus, reversing the normally observed rise in PC02 with increasing tem-
8.1 Steady-State Acid-Base Regulation 219
-0.08
-®~--~_ -0.012 l1pH/l1t
8.0
-o-;,f@:::-:. ®----
'-o.m1 2 . . . ~::_""'_'""":-.::::- __
~~~::-:?-=-==~s-i~~~~--':1:1: ............ ....

. . _- ................. __ <15°.013
)1
......... - ~--.. ..... .... _- ....

- _ ...
-- --
-0.013
--- - ....
..................... ®-O.017
9 _
....
7.4
PC0 2
2 _-~:==~--~~t;~::~~;~ii:~H~i~ ~ ! !l l li
immHg!
~ ..: :~~m0i ! !i ! ! ! ! ! ! !mi:imi im:imi!i i i lmi l ;
.::::r:::::,;::,;;:::::;::: :: ;::::::::: :: :::::::::: ::::::::: ::::::::::,;:::::
o .::::~;(~im~i:m1~:m: lim::mm:::m:m:!~1m:::m::m::mlm~::1:m::~:
15
10
[HCO;lpl
Immol]
5
o I I
10 20
Temperature
CD Scyliorhin.us ijuvenile) © Salmo (J) Ictalurus
Q) Scyliorhinus iadult) ® Cyprinus @ Anguilla
Q) Scyliorhinus ® Cynoscion ® Synbranchus
Fig. 8.1. Arterial plasma acid-base parameters of an elasmobranch fish species ( Scyliorhinus stellaris,
solid lines) compared to those of various water-breathing and one air-breathing ( Synbranchus
marmoratus) teleost fish species ( dashed lines). Values associated with the pH/temperature regression
lines are the respective temperature coefficients (~pH /~t). Data from Heisler et a!. 1980 ( 1 to 3);
Randall and Cameron 1973 (4) ; N. A. Andersen, M . L. Glass and N . Heisler, unpub!. (5);
Cameron 1978 (6); Cameron and Kormanik 1982 (7); Walsh and Moon 1982 (8); Heisler 1980,
1984a (9)
perature by moderate manipulation of the inspired P C02. The resulting atypical fall
of arterial P C02 with rising temperature was completely compensated by appropriate
adjustment of bicarbonate such that absolute pH and pH/temperature coefficients
(~pH/~t) in plasma (# 3, Fig. 8.1) as well as in white, red and heart muscle
remained unaffected (# 5, Fig. 8.2) (Heisler et al. 1980).
-0.016
7.1. White Muscle
7.2
--- ® :2:0~
--- --- ---
6.B
--- --
7.5
Red Muscle
pHi
7.3
7.1
7.6
--- --- --- Heart Muscle
pHi
...... ®- 0.020
--- -- ®~.~
---
7.4
.......... ~ ~~fO.007
> ...... ...~~-0 009
-.....-""-0...,- - - ___ ____
-0.005 .
----------
7.2 ® -0.003
--- - - - ___ ,~
----
®-~~5 _
7.0
10 20 30
Temperature (OC)
Fig. 8.2. Intracellular pH as a function of temperature in the elasmobranch Scyliorhinus stellaris

(solid lines, (l) and (3): juvenile, (2) and (4): adult specimens acclimated for 24 h or 3 weeks,
respectively. (5): adult specimens, acclimated for 3 weeks, 6.P co2 reversed, see text; Heisler et al.
1976 b, 1980) and in various teleost fish species (dashed lines). (6) Ictalurus punctatus; (7) Anguilla
rostrata; (8) Synbranchus marmoratus; (9) Cyprinus carpio; references as for Fig. 8.1
8.1 Steady-State Acid-Base Regulation 221
In contrast to plasma pH, the intracellular pH and pH/temperature coefficients

(~pH/~t) in tissues of fishes are quite variable with species and type of tissue.
As a general pattern, ~pH/~t in red muscle is highest, followed by that of white muscle,
and heart muscle (Fig. 8.2). This less uniform pattern (as compared to the extra-
cellular space) could be interpreted as a loose regulation of intracellular pH, or the
differences could be simply disqualified as experimental error. The more attractive
and likely alternative, however, that optimal cell performance is achieved at individual
tissue-specific and temperature-dependent pH values, is supported by the repeatabi-
*
lity of determinations, and the small effect of external disturbances (such as hyper-
capnia) on the adjustment of intracellular pH (Fig. 8.2, 5; see Heisler et al. 1976a,
1980; Heisler 1980).
8.1.3 Imidazole Alphastat Regulation
On the basis of some experiments in air-breathing species, Reeves (1972) propounded

that pH in heterothermic animals was regulated when temperature changed by
adjustment of ventilation and consequent changes in PC02 such that the fractional
dissociation of biological histidine-imidazole moieties was kept constant. If imidazole
were the predominant non-bicarbonate buffer in a fluid system, proper adjustment
of PC02 would result in no titration of non-bicarbonate buffers, and accordingly
no change in bicarbonate concentration. This type of buffer composition is prevalent
in the extracellular space (haemoglobin and plasma proteins), and similar conditions
were also suggested for the intracellular body compartments (Reeves and Malan
1976). Reeves claimed accordingly that constant imidazole ionization could be achiev-
ed simultaneously in all body fluid compartments by semi-closed system buffering
(except for CO2 ) without any changes in bicarbonate concentration, and without
any transmembrane and transepithelial ion transfer, exclusively via adjustment of
P C02 by changes in ventilation (Reeves 1972, 1977; Reeves and Malan 1976).
The pK/temperature coefficients (~pK/~t) of imidazole compounds may range
from -0.018 to -0.024 Ute, depending on ligands and steric arrangement
(Edsall and Wyman 1958; see Heisler 1986c), values' not met in the plasma of any
fish species (Fig. 8.1). The bicarbonate concentration, which is expected on the basis
of Reeves' model to be constant, is generally affected to a great extent by changes
in temperature. These data indicate that the criteria of the alphastat model do not
apply to extracellular fish acid-base regulation. In addition, with some exceptions,
the behaviour of intracellular pH does not support Reeves' model (Fig. 8.2).
In this context it should be emphasized that even if PC02 could be adjusted in
fish to arrive at a constant ionization of imidazole compounds in one of various
body compartments (e.g. the extracellular space) in spite of the physical limitations
of water as a gas exchange medium, alphastat regulation could not be expected in
the other, intracellular fluid compartments. The temperature coefficient of an ionically
closed buffer system, especially at low bicarbonate concentrations such as found in
the intracellular space of water-breathing species, is primarily determined by the
ratio of imidazole-like (~pK/~t '" -0.021) to phosphate-like (~pK/~t '" -0.002)
buffer values, and only to a lesser extent by changes in PC02 (Glass et al. 1985;
Heisler 1986c; cf. Heisler 1986a). Since this ratio is rather variable among tissues
(1 to 4, Heisler et al. 1980; Heisler 1984a, 1986c; Reeves and Malan 1976),
constant imidazole dissociation could be achieved only by extremely different PC02
values (which is physically impossible), or by transmembrane transfer of acid-base
relevant ions. These considerations and data suggest that the alphastat model is
incompatible with fish acid-base regulation (for more details on this topic, see
Heisler 1986a, b, c).
8.2 Acid-Base Stress Conditions
The intimate contact of fishes with their aqueous environment offers a number of
advantages for acid-base regulation (such as a much more effective transepithelial
ion transfer than is possible for terrestrial species, cf. Heisler 1986c, d), but also
provides additional environmental and endogenous challenges. Fishes, and in parti-
cular elasmobranchs, are more subject than terrestrial animals to changes in the
composition of their enviroment, of which changes in water Pco ,temperature and oxy~
gen content are the most frequent. Acid-base disturbancei may also arise from
internal production of acid-base-relevant ions over and above the steady state
production rate. The following description of acid-base challenges and the reactions
of regulatory mechanisms will focus on these specific perturbations, and attempt
to delineate generalized regulatory patterns.
In most cases the description will be limited to a single species, either because
other elasmobranchs have not been studied, or the described reaction appears to be
representative. The reaction of elasmobranchs is often not principally different
from that of teleost fishes, so that the reader is also referrred to a number of review
chapters, which include and emphasize teleost rather than elasmobanch fish species,
and on certain specific aspects such as the limiting factors for acid-base regulation
in fishes and the comparison with higher vertebrate species (Heisler 1984b, 1985,
1986 b, c, d, 1988). Due to space limitations, methodological aspects cannot be treated
in detail and the reader is referred to the original publications or to the more
extensive treatise of Heisler (1984 b).
S.2.1 Temperature Changes
Elasmobranchs may not only be subject to slow seasonal changes in water tem-
perature of up to 20°C or more (e.g. Kato and Carvallo 1967), but may also
experience large changes in temperature in a diurnal rhythm and during migratory
and predatory activities. Sharks may move from close to the surface down to
water depths of more than 600 m (e.g. Bullis 1967), which may be equivalent to a
temperature difference of 20°C or more (e.g. Harvey 1974). Small changes in depth
around a thermocline result in abrupt and large temperature changes which, although
normally avoided by the animals, may be imposed during certain predatory activities
and emergency situations. Such changes in water temperature are rapidly transferred
to the body fluids, facilitated by the large branchial interface area, and the high caloric
8.2 Acid-Base Stress Conditions 223
capacity of the environmental water. The following section will describe transients
of pH, P COz and [HCO;] resulting from experimental step changes in temperature
in the elasmobranch Scyliorhinus stellaris, and evaluate the contributions of physico-
chemical buffering and of bicarbonate-equivalent ion transfer processes to the read-
justment of the acid-base status after temperature changes.
8.2.1.1 Transients of Extracellular Acid-Base Regulation
Adjustment of the acid-base status after changes in temperature is a rather time-

consuming process. When temperature is elevated from 10°C to 20 DC, plasma pH
in Scyliorhinus changes initially more than three times (i1pH/i1t ~ -0.039 U;oC;
Fig. 8.3) the finally attained steady-state value (i1pH/M ~ -0.012 U;oq (Heisler
1978, 1984a). This undershoot in plasma pH is, at least during the first hours
after the change in temperature, mainly attributable to a more than threefold rise in
P coz ' whereas later adjustment of pH is mainly a function of the slow regulation
of the bicarbonate concentration, which requires more than 12 h to attain a new steady
state (Fig. 8.3). The response to a reverse temperature change from 20 to 10°C is
qualitatively similar with less overshoot in pH (i1pH/M ~ -0.019; Fig. 8.3). The
bicarbonate concentration presents almost a mirror image of the changes observed
during the equivalent rise in temperature (Heisler 1978, 1984a).
Slow adjustment of the bicarbonate concentration is evidently responsible for
the delayed adjustment kinetics of extracellular pH. This process is certainly little
affected by passive non-bicarbonate buffering in a semi-closed buffer system (i.e.
according to the alphastat model). The non-bicarbonate buffer capacity of blood and
extravascular extracellular space of Scyliorhinus (Holeton and Heisler 1983) and the
effective pH changes (cf. Heisler 1984 b, 1986a) are negligible compared to the
changes in extracellular bicarbonate concentration. Accordingly, the process is likely
to be attributable to transmembrane and transepithelial bicarbonate-equivalent
ion transfer processes, which may limit rapid extracellular pH regulation. The
limitation of acid-base adjustment kinetics by bicarbonate-equivalent transfer pro-
cesses is similar to that in the only other fish species studied with respect to
pH/temperature transients, the tropical freshwater teleost fish, Synbranchus marmora-
tus (Heisler 1984a).
8.2.1.2 Bicarbonate-Equivalent Ion Transfer Processes
Transepithelial ion transfer processes upon temperature changes can be determined

directly from the ch·anges in ionic composition of the ambient water of the fish
(Heisler et al. 1976a; Heisler 1978). For a 10°C increase in temperature Scyliorhinus
releases about 0.4 mmol of bicarbonate-equivalent ions per kg body water, to the
environment, an amount which is in turn taken up when an equivalent reverse
temperature change is imposed (Fig. 8.3; Heisler 1978). A slighly larger amount is
shifted across the epithelium in the teleost fish Ictalurus punctatus (0.55 mmol kg- 1
body water, Table 8.2, Cameron and Kormanik 1982), whereas in the tropical
freshwater teleost Synbranchus marmoratus only an insignificant amount (0.08 mmol
kg- 1 body water) is taken up for a 10°C temperature rise (Table 8.2 Heisler
1984a).
--10°C--j .. 20°C - - - - - - - - - - 20 oC--14 10oC-------- tv
~
8.0 I 8.0
(j
pH pl ~ I'lPHht=-0.~12 ::r
llpHfl'lt =-0.012 ~
t g
7.8 7.8 co
l=::= · ~to
e;
7.6 7.6 ":;0
"
~
~
o·
;:l
+1.0 o
to HCOj" I'l HCO;i_e
(mmollkg I'lHCO; e
body water) ....
II HCO;e_sw
...................................................
+ 0.5 I'l HC0 3e _ sw -0.5
........................................
I'l HC0:i" e
I'l HCO; i-e
o -1.0
o 10 20 30 o m w ~
Time (h) Time (h)
Fig. 8.3. Changes in plasma pH and total amount of extraceJlular bicarbonate (!l.HCO;c)' and the quantities of bicarbonate transferred between intraceJlular and
extraceJlular body compartments (!l.HCO;; _ c) and between extraceJlular space and ambient water (!l.HCO;c _ w) upon 10°C temperature changes. (Data from
Heisler 1978)
While transepithelial bicarbonate-equivalent ion transfer can be determined

directly, the only access to information on similar acid-base-relevant processes be-
tween extracellular and intracellular body compartments is provided by model
calculations based on experimental data. Two main approaches can be taken to
determine transmembrane acid-base-relevant ion transfer.
Approach 1 is based on the amount of bicarbonate equivalents produced by intra-
cellular non-bicarbonate buffering (Fig. 8.4). This amount can be determined from
Approach
I!. pHe
I!. [HCOjle
I!. [HCOjlNBe I!. HCOje+env
Temgerature constant
I!. [HCOjlNBi = f3lal·-1!. pH I!. HCOji+e = (f3lar-1!. pH -I!. [HCO;li) Vi
Temgerature variable.
I!. [HCOjlNBi = f3rm (I!. pKlm - I!. pH;! + f3Ph (I!. pKph - I!. pHi)
I!. HCOji+e ={l3Jm(l!.pK1m-l!.pHi)+ f3Ph(l!.pKPh-l!.pHi) -1!.[HCOjliJ ,Vi (for each individual lissue)
I!. HCOje+env = t
n
I!. HCOj i+e - I!. [HCO;le • Ve + I!. HCOj" NBe
Approach 2
ICS 1-n
I!.pHe
I!. [HCOjlenv
{
I!. HCOj env.. -;--
V.e Venv
I!. HCO; env-e = - I!. [HCOj lenv • Venv
I!. HCOj env-e -I!. [HCOjle • Ve + I!. HCOj' NBe
I!. HCOj' NBi I!. HCOj e-i + t I!. HCOj i

n
Fig. 8.4. Methodolological approaches to indirectly determine various parameters of the acid-base
regulation by model calculations based on experimental data~ ft buffer value; V volume. Indices 1m
and Ph designate imidazole- or phosphate-like buffer values, i and e intracellular and extracellular,
env and NB enviromental and by non-bicarbonate buffering, respectively. For details see text.
(Heisler 1984b, 1986a)
Table 8.2. Bicarbonate production by intracellular non-bicarbonate buffering (L'.[HC0 31NB ;), changes in intracellular bicarbonate (L'.[HC0 31;), and bicarbonate
equivalent transfer per kg intracellular tissue water, estimated total transmembrane bicarbonate-equivalent ion transfer (tlHC03i_e) determined by approach I
(see text), compared to the directly determined bicarbonate equivalent ion transfer between environmental water and extracellular space (tlHC03-e_ w), and to N
N
0'<
the overall bicarbonate-equivalent transfer between extracellular and intracellular space (tlHC03i_eo approach 2, see text) upon an increase in body temperature
by 10°C in the elasmobranch Scyliorhinus stellaris and in two freshwater teleost fish species'
Species Tissue Icsa Approach I Approach 2 Reference n

::r
Po>
volume
~
L'.[HCO;lNB tlHCO;NB. tl[HCD;l;-, tlHCO;; - e tlHCO;; _ e tlHCOie_w
(1) (2) (3) , (5) (6) (7)
"..,
?"
(4)
(2)-(3) (4) . (1) ()
>
0.:
Scyliorhinus White 357 0.53 0.19 0.34 0.32 Heisler and ~
Po>
V>
stellaris muscle Neumann 1980
Red 35 7.24 -0.25 7.49 0.27 Heisler et aJ. 1980 "::0
muscle "
~
Heart 0.1 -3.27 0.52 -3.79 -0.0004 fro·
muscle ::l
Other 340 0.53 b 0.19 b 0.34b 0.12b
tissues
732 0.51
0.77 0.41 Heisler 1978
Ictalurus 0.36 0.55 Cameron and

punctatus Kormanik 1982
Synbranchus White 600 -3.69 -0.64 -3.05 -1.83 Heisler 1984a

marmoratus muscle
Heart 0.3 -4.39 0.18 -4.57 -0.0014
muscle
Other 201 -3.69 b -0.64b -3.05 b -0.6Ib
tissues
Total 801 -2.44
-2.01 -0.08 Heisler 1984a
a ICS, intracellular space, ml kg body water-I.

b Other tissues assumed to behave like white muscle.
, Values expressed as mmol kg body water - [
the change in pH and the temperature-dependent buffer value matrix (Heisler and
Neumann 1980; Heisler 1984a, 1986a). The amount of bicarbonate equivalents
transferred from the specific intracellular compartment to the extracellular space
(MHCO;]i ~ e) is determined as the difference between produced bicarbonate and
the change in intracellular bicarbonate concentration. The overall transmembrane
transfer, as the sum of the individual bicarbonate-equivalent transfers for all intra-
cellular body compartments, can be utilized, together with the amount of bicarbonate
produced in the extracellular space and the extracellular [HCO;] difference, to
estimate the transepithelial ion transfer (~[HCO;]e ~ w)' although the extent of extra-
polation may probably be too large to yield significant information on the latter
parameter (Heisler 1986a).
Approach 2 is based on the bicarbonate-equivalent transfer at various epithelial
sites, determined directly as changes of the ionic composition of the ambient water
(Fig. 8.4). The overall transmembrane transfer is then the difference between the
extracellular HCO; production, the extracellular [HCO;] change, and the trans-
epithelial transfer. Any further extrapolation as to the amount of bicarbonate
produced by non-bicarbonate buffering cannot be expected to yield satisfying results
because of numerous uncertainties introduced by the impossibility of determining
[HCO;] and pH in all intracellular body compartments (Heisler 1986a).
Model calculations based on these approaches are very much dependent on,
and limited by, the quality of the involved experimental data. Also the obtained
results have to be regarded with discretion with respect to the extent of extra-
polation. A further complicating factor for the application of approach 1 is deviations
from steady-state conditions, whereas approach 2 is completely independent of this
factor. Appropriate application, however, can yield good quality estimates for
further analysis, as indicated by the similarity of values obtained for the trans-
membrane transfer (~HCO;i ~ e) determined by approach 1, and those determined
more directly by approach 2 (Table 8.2). The estimate based on approach 1 deviates
by only 33 % in Scyliorhinus and 18 % in the teleost Synbranchus from that obtained
from approach 2, differences which have to be considered as small compared to the
number of experimentally determined parameters involved.
The bicarbonate-equivalent ion transfer between intracellular body compartments
and extracellular space in Scyliorhinus (determined by approach I) is qualitatively and
quantitatively quite variable with the type of specific tissue (Table 8.2). The transfer
per unit volume of white muscle is relatively small, but contributes significantly to the
overall process because of the large relative fraction of the body mass represented
by this tissue. The opposite holds for red muscle. In spite of its small relative body
mass, the amount of bicarbonate-equivalent ions transferred upon changes in tem-
perature is fairly large, a fact attributed to the considerable mismatch between the
semi-closed system buffering characteristics and the in vivo change in intracellular
pH.
The transmembrane transfer of Synbranchus white and heart muscle is comparable
to that in heart muscle of Scyliorhinus on a per volume basis (Table 8.2). In
contrast to the elasmobranch, however, the overall transfer is much larger and in
the opposite direction. These differences are certainly not related to specific features
of e1asmobranch versus teleost acid-base regulation, as indicated by the similarity
between the transepithelial transfer in Scyliorhinus and Jctalurus, which are both in
contrast to that of Synbranchus (see above). More likely, the extent and direction of
transepithelial and transmembrane bicarbonate-equivalent transfer is species-specific
and may be related to differences in temperature-dependent intracellular buffering
characteristics and pH changes.
8.2.1.3 Contribution of Buffering and Ion Transfer to the Acid-Base Regulation
The negative temperature coefficients of the two main groups of physiological

non-bicarbonate buffers (imidazole-like, ~pK/M ~ -0.021; phosphate-like, ~pK/~t
~ -0.002) support adjustment of pH to lower values when temperature rises.
The same effect is expected on the basis of the Henderson-Hasselba1ch equation
from the general rise in Peo2' which is, however, counteracted by the fall in CO 2
solubility with increasing temperature (e.g. by a factor of about 1.4 for 10 to 20°C;
ref. for pK and cteo2 : Heisler 1984 b i , 1986 a). This fall in CO 2 solubility would tend
to increase pH with rising temperature (at least for those species in which PC02
rises by a smaller factor than CO2 solubility increases, e.g. juvenile Scyliorhinus,
Salrno gairdneri, Cyprinus carpio, Cynoscion arenarius, cf. Fig. 8.1). The effect of the
overall buffering processes for pH adjustment after changes in temperature can be
Table 8.3. pH changes induced by changes in temperature (ApH/tH) in extracellular and intracellular
body fluid compartments of an elasmobranch fish (Scyliorhinus stellar is) and various teleost fish
species in vivo (iv), and modelled as semi-closed buffer systems (cl)
ApH/Atcl
Species Body ApH/Ativ ApH/Atcl Reference
compartment ApH/Ativ
(1 ) (2) (3)
Scyliorhinus White muscle Heisler et a!. 1980

stellaris rcs -0.018 -0.017 0.95
Red muscle rcs -0.031 -0.013 0.41
Heart muscle Heisler and
res -0.007 -0.016 2.28 Neumann 1980
ECS -0.011 -0.021 1.90
Synbranchlls White muscle Heisler 1984a
marmoratus rcs -0.009 -0.017 1.89
Heart muscle
rcs -0.003 -0.021 7.00
ECS -0.017 -0.003 0.17
Sall110 gairdneri ECS -0.013 -0.001 0.07 Randall and
Cameron 1973
Cynoscion ECS -0.013 -0.002 0.15 Cameron 1978
arenarius
Cypnnus carpio ECS -0.012 +0.002 -0.16 Anderson, Glass and
Heisler, unpub!.
Ictalurus ECS -0.013 -0.007 0.53 Cameron and
punctatus Kormanik 1982
Based on model calculations according to Heisler and Neumann (1980) and Heisler (1984a, 1986a).
1 Note: the sign of the last line term of the crC0 2 -formula in Heisler (l984b) is misprinted and should
read '+'.
delineated by modelling intracellular and extracellular body compartments as semi-

closed systems by setting the transmembrane and transepithelial bicarbonate-equi-
valent ion transfers to zero (approach 1; Heisler and Neumann 1980; Heisler 1984a,
1986a; cf.Fig. 8.4).
pH in intracellular compartments modelled as semi-closed buffer systems generally
falls with rising temperature, due to the less important role of bicarbonate as
compared to the non-bicarbonate buffers in the total buffer capacity of the
intracellular space [Table 8.3, (2)]. The pattern in the extracellular fluid compart-
ments is diverse. The majority of species would exhibit hardly any negative change
(in one case even positive) in pH with rising temperature, when ionic transfers were
excluded. Only if the relative rise in PC02 is larger than the relative reduction in CX C02
(adult Scyliorhinus and Ictalurus, cf. Fig. 8.1), can an appreciable negative pH/tem-
perature coefficient be expected. This analysis modelling body compartments as semi-
closed buffer systems results in pH changes, which are (with one exception)
considerably different from those found in vivo [Table 8.3, (3) versus (1)]. This
comparison underlines the relative importance of ion transfer processes for the
adjustment of pH after changes in temperature. The amount of bicarbonate-equi-
valent ions transferred during temperature changes may appear small in absolute
terms (especially when compared to the amounts transferred during other acid-base
stress conditions), but referred to the compartment volume (L1[HCO;]i _ e)' they are
severalfold larger than the changes in intracellular bicarbonate concentration after
changes in temperature (Table 8.2).
8.2.1.4 Adjustment Kinetics ofIntracellular pH
Direct monitoring of intracellular pH kinetics after changes in temperature has been

impossible to date, so that information on this point can be obtained only by
appropriate model calculations on the basis of transmembrane bicarbonate-equiva-
lent transfer rates and tissue-specific buffer characteristics (Heisler and Neumann
1980; Heisler 1984a, 1986a). Such estimates are limited by a number of uncertainties
introduced by the required assumptions, of which the most important is homo-
geneous distribution of ion transfer rates across the cell membranes of various
tissues (cf. Heisler 1984 a, 1986 b).
The time course of intracellular pH regulation of Scyliorhinus white muscle
modelled on this basis is relatively fast, and the adjustment is almost complete after
about 30 min of exposure to the new temperature. This rapid adjustment is due to
the fact that the relative amount of bicarbonate equivalent ions to be transferred
across the cell membrane is relatively small. Assuming that the same specific
transfer rates apply for white muscle, red muscle, and heart muscle, establishment
of a new steady state would be related to the amount to be transferred per unit volume
(MHCO;t _ e' Table 8.2). A new steady intracellular pH in heart muscle would then
not be attained before about 2 h, and in red muscle not before 8 h after the
change in temperature.
The assumption that the ion transfer rates are the same for all three muscle
species represents a much larger factor of uncertainty for model calculations in
the elasmobranch Scyliorhinus than for the teleost fish Synbranchus marmoratus.
In the elasmobranch a much smaller fraction (~54 %) of the tissue-individual
transmembrane ion transfer, as well as of the tissue-specific buffering characteristics,

can be accounted for, whereas the equivalent fraction is much larger in Synbranchus
(~75 %) (Table 8.2). Furthermore the behaviour of the accounted fraction in the
transmembrane acid-base-re1evant ion transfer is rather homogeneous, whereas in
the elasmobranch a large tissue proportion (white muscle) contributes less to the
transmembrane transfer than red muscle, which represents a ten times smaller tissue
fraction (Table 8.2). Model calculations as to the intracellular pH adjustment
kinetics are accordingly much more secure for Synbranchus. Estimates on pH
adjustment kinetics in Synbranchus white muscle intracellular space yield similar
results to elasmobranch red muscle tissue, a new steady state being attained only
after about 7-8 h (Heisler 1984a).
The delayed rate of pH adjustment is evidently due to the fact that the observed
ion transfer rates are at least one order of magnitude smaller than those achieved
during the peak perturbations of other acid-base stress conditions. They are com-
parable to the transfer rates observed during extremely mild disturbances, or when
control values are approached. Thus the disturbances induced by temperature changes
may not be large enough to significantly affect cell functions, and to stimulate the
appropriate mechanisms to their maximal transfer rate. This hypothesis is supported
by the relatively wide pH optima (in terms of the pH changes usually dealt with in
connection with temperature changes) of the energy-producing enzymes, which
suggest that adequate metabolic activity can be well maintained at moderately affected
pH values (cf. Heisler 1986c).
8.2.2 Hypercapnia
8.2.2.1 Environmental Hypercapnia
Pco in natural freshwaters may frequently rise to peak values of up to 60 mm Hg

(8 k1>a) usually as a result of hindrance of gas exchange between water and air by
dense surface vegetation (e.g. Heisler et al. 1982), thermo stratification, and/or release
of CO2 from bicarbonate by the H+ -producing anaerobic metabolism of micro-
organisms. In contrast, surface seawater P C02 is usually only slightly affected by
temperature and photosynthesis-related processes and varies in the range of 0.15
to 0.30 mm Hg (0.02-0.04 kPa). Only at greater depths of 200-500 m may P C02
rise to values of more than 5 mmHg (0· 67 kPa) (Harvey 1974) as a result of
thermo stratification combined with anaerobic metabolism of bacteria and settling
organic debris.
Accordingly, elasmobranchs (and also many teleost fish species, Heisler 1984b,
1986b) have been subjected to increased environmental P C02 in order to mimic
natural elevations of postbranchial P C02 during predatory and migratory activities,
but mainly for studies of acid-base regulatory mechanisms in response to this stress
factor. The response is typical and rather common for many fish species: arterial
P C02 in the elasmobranch Scyliorhinus is rapidly elevated after onset of environmental
hypercapnia via the large branchial gas exchange surface, accompanied by an equi-
valent reduction in plasma pH (Fig. 8.5). Soon after elevation of plasma PCO 2 however,
plasma pH recovers towards control values, and is almost completely restored

(~pH < -0.1 U) by the elevation of plasma bicarbonate concentration (Fig. 8.5),
similar to the observations of Cross et al. (1969) in Squalus acanthias.
The increase in plasma bicarbonate concentration is effected by several mech-
anisms. The main proportion is supplied by net gain of bicarbonate from the
environmental water, equivalent to the change in net H+ excretion seen as the diffe-
rence between ammonia and bicarbonate release relative to control conditions
(Fig. 8.6). In Scyliorhinus, the ammonia release remains unaffected during hyper-
capnia, such that the observed large rise in net H + excretion is exclusively the
result of a reversal of the control release of bicarbonate into a net uptake of
bicarbonate-equivalent ions (Fig. 8.5).
10
PaC0 2
(mmHg)
5
0 I
---'----'_L-l...........l--L---'---'----'---"----'I ____ 1---''--'--.L........L-.L.-..l-..l.1_
0 5 10 25 30
pHa
7.8
7.6
\. .../
/
./
./ -- -- --
7.4
I
0
20
[HC0 31p
(mmol)
10 - - Scyliorhinus stellaris
- - Conger conger
o o 5 10
__ .Ll- - - , - - - " - - - - ,_ _
25
~~~~I_
30
Time (h)
Fig. 8.5. Changes in arterial plasma pH, Pco and bicarbonate concentration in the elasmobranch
Scyliorhinus stellaris (solid lines) and the 2marine teleost Conger conger (dashed lines) upon
exposure to about 7.5 mmHg (1 kPa) Pco2 in the ambient water. (Data of Holeton and Heisler 1983;
Toews et al. 1983)
5
--- ------
---------------
------- ---
-----~
------ --
___ --- Control
o --- -- 10 20 30
~HC03 sw
(mmol/kg
-- -- --
body water! --------------~~~-=------
-5
o 10 20 30
o >'@!!:
.......... Q .......
! I I I
~NH~ sw
(mmol/kg
body water!
0 10 20 30
0 ------ ------
Control
~H;-sw
(mmol/kg
body water)
5
.... -.-- -------
--
I I I
0 10 20 30
Time (h)
Fig. 8.6. Changes in the amount of bicarbonate (~HCOisw) and ammonium (~NHtsw) in the ambient
water, and the release of H+ ions (~H:_sw) from the elasmobranch Scyliorhinus stellaris (thick
solid lines) and the marine teleost Conger conger (thick dashed line) compared to the cumulative
control rates (thin lines) after exposure to environmental·hypercapnia. Note the offset in ~HCO; and
~NH: curves by the higher ammonia production rate of the teleost fish. (Data of Holeton and
Heisler 1983; Toews et al. 1983)
Only during the initial phase of hypercapnia ( ~ 15 min) is bicarbonate not taken
up from the environmental water, but actually lost at an increased rate from the fish
(Fig. 8.6). In spite of this initial loss of bicarbonate-equivalent ions the amount of
extracellular bicarbonate rises considerably more than expected from blood nonbi-
carbonate buffering (Fig. 8.7). Analysis on the basis of approach 2 (see above, and
Heisler 1986 a) indicates that bicarbonate-equivalent ions are transferred from the
intracellular to the extracellular space (Fig. 8.7). After an initial delay of about 15 min
there is a net gain of bicarbonate from the water, providing further compensation
of extracellular pH. At about the same time the flux of bicarbonate-equivalents from
intracellular to extracellular space is reversed and HCO; originally supplied to the
extracellular space is transferred back into the intracellular fluid compartments. The
uptake of bicarbonate-equivalents continu~s for at least 8 h, during which bicarbonate
is accumulated in both extracellular and intracellular spaces (Fig. 8.7).
4-
(',
;:.,HC03 I "- "- - - - - - -
2 I - -t.HCOj
- - -NB;- -
(mmol/kg
body water) I
tr
I
o I I
6 8 10 22 24
t. HCO; e
/::, HCO;
2
[mmol/kg
body water)
;:.,HC0:i
2
[mmollkg
body water)
o
22 24
Fig. 8.7. Quantitative evaluation of the amounts of bicarbonate accumulated in intracellular and
extracellular body compartments from non-bicarbonate buffering (8HC03NBi and 8HC03NB "
dashed lines), and transferred from the environmental water (8HC03sw~, and 8HC03'~io solid
lines) during pH compensation of environmental hypercapnia in Scyliorhinus stellaris. (Based on data
of Heisler et al. 1976a; Heisler and Neumann 1980; Heisler 1980)
The amount of bicarbonate transferred to the intracellular space is much less

than that accumulated in the much smaller extracellular space. The compensatory
effect, however, which is a function of the relative rather than of the absolute rise in
bicarbonate concentration (cf. Heisler 1986c), is comparable, or even larger, in the
intracellular body compartments due to the much lower control bicarbonate con-
centration and much higher non-bicarbonate buffer value. Accordingly, an amount
of about 1.3 mmol kg- 1 body water transferred to the intracellular space (Fig. 8.7),
which represents about 75 % of the body water volume, is sufficient to compensate
intracellular pH to less than -0.05 units (Fig. 8.8), whereas about 3.5 mmol kg- 1
body water (Fig. 8.7) accumulated in the extracellular volume (~25 % of the body
water) are required to yi~ld an extracellular pH recovery to -0.1 units (Fig. 8.5).
8
/'
[HC03] /'
/'\
(mmoll
6
4
r:I
I"NB
Q9.
o
7.2 7.3 7.4
Fig. 8.8. Intracellular pH compensation in muscle tissues of the elasmobranch Scyliorhinus stellaris
and the teleost Ictalurus punctatus during environmental hypercapnia. Bicarbonate is accumulated
in surplus to that produced by non-bicarbonate buffering (~NB) by transmembrane transfer of
HCO; -equivalent ions (vertical double lines). (Based on data of Heisler 1980; Cameron 1985)
During the initial phase of hypercapnia, compensation is certainly supported

by intracellular non-bicarbonate buffering, as suggested as the main mechanism of
compensation by Cross et al. (1969). The extent of buffering however, is always a
function of changes in pH (cf. Heisler 1986a, e), and falls with pH recovery towards
control values during the course of compensati9n, such that the proportion of
bicarbonate produced by non-bicarbonate buffering compared to the amount gained
from the environment is reduced to approximately 25 % after about 24 h of hyper-
capnia (Fig. 8.7).
The time course of pH compensation following environmental hypercapnia is

relatively fast in elasmobranchs, and a new steady state is attained within about
8-10 h (Cross et al. 1969; Heisler et al. 1976a), which is similar to that found in
marine teleost fish species (e.g. Toews et al. 1983). In contrast, compensation of
hypercapnia in freshwater fishes may be delayed by several days or even longer (e.g.
Janssen and Randall 1975; Claiborne and Heisler 1984, 1986). Large differences in
time course between studies in the same species, but in water of different quality (cf.
Janssen and Randall 1975; Eddy et al. 1977; see also Heisler 1982) suggest that a
lack of bicarbonate or counter ions for acid-base-re1evant ion exchange with the
environmental water is a factor at least partially responsible for these discrepancies
(cf. Heisler 1982, 1984b, 1985, 1986b, d).
Similar variations in the time course of pH compensation during hypercapnia
can be produced in the elasmobranch Scyliorhinus stellaris by manipulation of
seawater pH via changes in bicarbonate concentration. When water pH was
elevated to 7.8 at Pcoz of about 8 mmHg (1.07 kPa) (normal seawater pH 6.9),
extracellular pH recovered towards control values (LlpH < 0.1 U) in about half
of the time (~4 h) seen at normal seawater pH (Fig. 8.9, insert). In contrast,
reduction of seawater pH to below 6.2 did not allow any pH compensation at all.
Closer analysis of the data indicated that the rate of bicarbonate-equivalent ion
transfer was closely correlated with the difference between plasma pH (pH p1 ) and
seawater pH (pH sw )' or, since P C02 was constant, with the ratio of plasma and seawater
[HCO;]. The transfer rate was virtually constant in a range of pHpcpHsw of
-0.5 to +0.6, and decreased in an apparently linear fashion at higher pHp1-pH sw
values, attaining zero at pHpcpHsw = 1.1 (Fig. 8.9). W~1en seawater pH was even
lower, bicarbonate equivalents were lost to the environmental water in spite of
continuing hypercapnia. Since all ions, except HCO;, remained unchanged during
these experiments (except for very small elevations in [Na+] and [Cl-] due to the
seawater titrations with NaHC0 3 and HCl), the environmental [HCO;] appears
to be the limiting factor for transepithelial ion transfer for acid-base regulation in
Scyliorhinus during hypercapnia (Heisler and Neumann 1977; Heisler 1985; Heisler
1986d).
8.2.2.2 Hyperoxia-Induced Hypercapnia
The oxygen content of natural waters is a result of the balance between oxygen-
producing and oxy~en-consuming processes, and diffusive gas exchange with the
atmosphere. In freshwater habitats, Po may be extremely variable with particularly
z
low values resulting from oxygen consumption by aerobic metabolism of various
aquatic animals and microorganisms during dark periods, and extremely high values
close to atmospheric pressure induced by photosynthesis-related processes (cf. Heisler
1984b, 1986b). The variability of POz in seawater, however, is much smaller,
ranging from about 20-60 mmHg (2.7-8 kPa) at depths of 400-500 m in some ocean
areas, up to values of 150-190 mmHg (20-25.3 kPa) in near-surface water layers
(Harvey 1974).
Although elasmobranch fishes hardly ever experience significant hyperoxia in
their natural habitat, increased water P Oz levels have been applied as an experimental
n 5 7 12 14 9 13 11 10 20 23 18 21 13 14 18 17 21 12 19 21 16
+10
( Ilmol \
min· k9 body water/ 7.8
78~-
7.6
o 6.7
7.4 '--------6.2----
~ Time {hi
-10
I
-0.5 o 0.5 pHpl - pHsw
03 0.5 235 10 20 30
[HCOilpl/[HCO;lsw
Fig. 8.9. Rate of net bicarbonate-equivalent transfer as a function of the difference between plasma
and seawater pH, or the bicarbonate concentration ratio (indices pI and sw, respectively) in
Scyliorhinus stellar is during environmental hypercapnia. Insert time course of pH compensation at
different seawater pH values. (Data of Heisler and Neumann 1977)
tool during studies of respiratory physiology. Due to the primarily oxygen-oriented

regulation of ventilation in fishes (cf. Dejours 1975, 1981), any elevation of the
inspired oxygen content regularly results in a fall in gill water ventilation and a
concomitant rise in expired and arterial P C02 with an associated fall in plasma pH
(e.g. Dejours 1972, 1973; Dejours et al. 1977; Truchot et al.. 1980; Wood and
Jackson 1980; Wilkes et al. 1981; Heisler et al. 1981, 1988; H6be et al. 1984). In a
number of species, this fall in pH is either not at all, or only partially compensated by
an elevation of plasma bicarbonate (e.g. Bornancin et al. 1977; Dejours 1983, 1975;
Truchot et al: 1080), whereas in other species plasma pH is almost completely restored
to control values (Wood and Jackson 1980; H6be et al. 1984; Heisler et al.
1981,1988; Heisler 1984b).
The pattern for elasmobranchs is diverse. Exposure of Scyliorhinus canicula to
an environmental Po2 of more than 700 mm Hg (93.3 kPa) effected a relatively
steep rise in arterial PC02 to ~ 3.2 mm Hg (0.43 kPa), which was only partially
compensated during 3 h of exposure, and resulted in a fall in plasma pH by 0.3 pH
units (Truchot et al. 1980). In contrast, elevation of inspired P O 2 to 500 mm Hg
(66.7 kPa) in Scyliorhinus stellaris led to a much more gradual rise in P C02
(Fig. 8.10). After an initial fall and recovery, plasma pH remained essentially constant
r---- -------- ----

400
Pa0 2
(mm Hg)
200
Vg
-
(ml/min·kg)
•I •I •I
-------
0 I
PC0 2
10
...... . • • .
~
(mm Hg]
7.8
~ ----------
---------
pHa
7.7
20
• • •
..--- - • •
lHC0 31pl
o
......-------
5
Net t::,.H e _ sw
(mmol/kg
~
water]
o o 5
__ I I
2·':-1-----"---L....-.-l-----::!25 6
(h] (h) (d)
Time
Fig. 8.10. Arterial plasma acid-base parameters, oxygen partial pressure (PO 2 )' gill ventilation CVg ),
and net H+ excretion during environmental hyperoxia in Scyliorhinus stellaris. (Data of Heisler,
Holeton and Toews, unpub\.)
for more than 24 h (A pH < -0.04) and started to deviate significantly from control
values only at the end of the experiment, after about 6 days of hyperoxia.
PC02 continued to rise during this time up to values of 11 mm Hg (1.47 kPa). As
indicated by the small deviation of pH from control values, plasma bicarbonate
was considerably elevated concomitant to the rise in PC02' The rate of bicarbonate-
equivalent uptake from the environment was comparable to that observed during
environmental hypercapnia only during the initial phase of hyperoxia (- 15 ~mol
kg- 1 min-I), when plasma pH was maximally depressed, but was reduced to about
5 ~mol kg- 1 min -1, when pH recovered to less than -0.03 units after about 3-5 h
of hyperoxia, and fell to about 2 ~mol kg- 1 min -1 after 21-25 h of hyperoxia.
Although the capacity of transepithelial bicarbonate-equivalent ion transfer
was exploited to only a small fraction by Scyliorhinus during hyperoxia-induced
hypercapnia, except for the very first time after elevation of environmental Po2 '
plasma pH was little affected. This was due to a regulation of gill ventilation
independent of blood oxygen levels, which remained largely elevated for several
days of hyperoxia (Fig. 8.10). Apparently, under these conditions, gill ventilation
did not need to be adjusted primarily for sufficient oxygen acquisition (as during
normoxia), but was regulated according to the requirements of the acid-base status,
matched to the rate of transepithelial bicarbonate uptake.
2.0
.
1.5
Vg act.
\;g contr.
1.0
0.5
o
7.9 7.7 7.5 7.3 7.1
pH pl
Fig. 8.11. Gill ventilation during hyperoxia (V,a"> of Scyliorhinus stellaris as a function of plasma
pH (pH p1 ) in relation to the normoxic control value (V,con,')' Each solid curve represents the
least-square exponential regression of an individual specimen; the dashed line is the general exponential
regression of the data pairs of all individuals. (Data of Heisler, Andersen, Iwama, Boutilier and
Claiborne, unpubl.)
Gill ventilation was only reduced (and P C02 increased accordingly) such that
the transepithelial bicarbonate-equivalent uptake was sufficient to compensate the
concurrent rise in Pco2 , When P CO 2 started to rise steeply as a result of the more than
60 %initial reduction in gill ventilation, and pH was reduced by 0.08 units, ventilation
was again increased by a factor of two over the next 2 h, presumably in order to
keep plasma pH close to the control value. This tight respiratory pH regulation was
abandoned only when the plasma [HCO;] approached a level presumably close to
the maximal threshold (cf. Heisler 1984a, 1986b, c, d). These data suggest that
during hyperoxia the oxygen-sensitive respiratory drive is subordinate to a mechanism
sensitive to changes in the acid-base status (pH, P C02 or/and [HCO;D similar to that
known for higher vertebrates.
This hypothesis was confirmed by exposure of specimens of Scyliorhinus stellar is
to hyperoxia, and simultaneous manipulation of the arterial acid-base status by appli-
cation of various combinations of inspired P C02 and intra-arterial infusion of
bicarbonate. The hyperoxia-reduced gill ventilation was strongly correlated to
arterial pH in an exponential fashion (Fig. 8.11), whereas PC02 ' [HC03-] and gill
ventilation were not correlated (Heisler, Andersen, Iwama, Boutilier and Clai-
borne, unpub!. data). These data indicate that when the oxygen demand of the
animal can be supplied without any limitations, the respiratory drive is mainly
provided by pH-sensitive structures in, or closely related to, the arterial blood stream.
A point of special interest for future studies will be to evaluate to what extent
this mechanism is limited to the "less developed" elasmobranchs, or whether it is
also available to more "advanced" fish species.
8.2.3 Lactacidosis
Lactic acid is produced in vertebrates as the main end product of anaerobic

metabolism during periods of environmental anoxia or, more frequently, during
functional anaerobiosis when the "oxygen transport chain" (Piiper 1986) fails to
supply the oxygen demand to tissues. In fish, normal locomotion with cruising and
positioning activities is performed by the relatively small amount (cf. Table 8.2)
of well-vascularized and perfused red musculature (e.g. Totland et a!. 1981)
involving aerobic pathways, a fact reflected in the low normal plasma lactate
concentration of < 1.5 mmoll- 1 (see Heisler 1984 b). Only when the large amount
of poorly perfused white musculature is activated during emergency conditions and
predacious activities is a large fraction of the energy demand supplied by anaerobic
metabolism. The resulting "oxygen debt" is repaid with respect to creatine phosphate
and ATP usually within a short time period after reestablishment of aerobic
conditions (e.g. Piiper and Spiller 1970), whereas the removal of lactic acid is
comparatively delayed. Up to 84 mmol kg- 1 tissue weight may then be accumulated
in white muscles of fish (Wardle 1978).
Immediately after production, lactic acid is dissociated according to its low pK'
value (~3.9) into H+ affects the acid-base status within the tissue cells and, after
gradual elimination to the extracellular space, also in other body fluid compartments.
The time course of lactic acid elimination is variable among fish species, although
some general features of the process seem to be common to the majority of species
studied to date (Heisler 1984b).
Peak values of extracellular acid-base deflections are attained generally much
earlier than peak plasma lactate concentrations. In the elasmobranch Scyliorhinus
stellaris, plasma pH has fallen to its lowest value, and plasma [HCO;] is maximally
reduced, 15-30 min after the termination of strenuous muscular activity (Fig. 8.12).
Recovery of acid-base control conditions is a function of the extent of the imposed
anaerobic activity (cf. Fig. 8.12), but is always performed much earlier than plasma
lactate levels have reattained control values. This is due to the fact that the
animals are capable of excreting surplus H + ions to the environmental water until the
original stress factor, lactic acid, has been removed by further aerobic metabolic
processing from the body fluids.
Similar to the pattern observed during environmental hypercapnia, the small
ammonia release of Scyliorhinus stellaris remains essentially unchanged during
lactacidosis, whereas the typical slight control bicarbonate release is reversed into
8.0
..................................
I
7.0 I I I !
PC025[~
(mmHg) ...................................
o [I! I I I I ! I I ! _____ _ ! ! I !
- ....................................
[HC0
(mmeL)
3J;! '\,--~
o -L!-L-L-L-L-L~~~~~ ------
20
10
[LacfJ pl
(mmel)
o i····t····1.. ··T····r····r····r.... ~
o 5 10
Fig. 8.12. Arterial plasma pH, Pco ' [HCO;l and lactate concentration of Scyliorhinus stellaris
(peak lactate concentrations higher aJd lower than 15 mmoll- 1 thick and thin solid lines, respectively)
and Conger conger (dashed line) after strenuous muscular activity. (Data of Holeton and Heisler
1983)
a large net uptake, such that the net H + excretion rate attains values of about
15 Ilmol kg -1 body water min -1 (Fig. 8.13). After normalization of plasma pH and
[HCO;], the net H+ flux is reversed, and the H+ ions originally transferred to the
environmental water are taken up again at the rate at which lactic acid is further
processed in the organism. This type of utilization of the ambient water as a
transient store for surplus H+ ions is similar to that found in teleost fishes (e.g.
Conger conger, Fig. 8.13), although the pattern there is overlaid by the much larger
basal production of ammonia.
Although the acid-base deflections in the plasma of Scyliorhinus stellar is
peak much earlier than the lactate concentration (due to the fact that the time
course of H+ ion efflux from muscle cells is much faster than that for lactate, e.g.
Benade and Heisler 1978; Holeton and Heisler 1983), the absolute amount of H +
ions buffered in the extracellular compartment is relatively small compared to that of
lactate. This apparent "H+ ion deficit" (Piiper et al. 1972) is the result of various
interacting mechanisms. At least part of the surplus H+ ions produced from the
dissociation of lactic acid are transferred to the environmental water (Fig. 8.13).
Taking this mechanism into account, the amount of H+ ions released from the
intracellular compartment is actually larger, for most of the recovery period, than
the amount of lactate transferred to the extracellular space (Holeton and Heisler
1983).
Another factor involved is the difference in steady-state distribution of H+
ions and lactate between intracellular and extracellular spaces. At equilibrium, lactate
is distributed across the cell membrane either according to the membrane potential
(main transfer form = lactate ions), or according to the intracellular/extracellular
pH difference (main transfer form = lactic acid). In both cases equilibrium is
attained when the majority of lactate has been transferred to the extracellular
space (ECS).
In contrast, the distribution of H+ions is governed mainly by the compartment
buffer capacities, which are higher in the intracellular space of muscle tissues than
in the interstitial and plasma ECS by a factor between 5 and 100 (cf. Heisler
1986d, 1988). Accordingly, transfer of only a small amount of H+ ions lowers
extracellular pH so much that a new equilibrium is attained between intracellular
and extracellular pH which eliminates the driving force for any further transfer of
H+ ions to the extracellular space (equilibrium limitation, Holeton and Heisler 1983;
see also Heisler 1986b). With this condition the main proportion of surplus H+
ions would be left buffered in the intracellular compartments until further aerobic
metabolism Qf lactic acid is performed. If not, a considerable proportion of the surplus
H + ions would be transferred to the ambient water via the extracellular space
(Fig. 8.13).
The process of handling surplus H+ ions during muscular activity-induced
lactacidosis is complex, depends on various factors, and is certainly not yet
completely understood. However, the large amount of experimental infomiation
available for the elasmobranch Scyliorhinus stellaris should allow, more than for any
other fish species, at least a general description of the interaction of underlying
mechanisms, and the general strategy of the regulatory system on the basis of
simple model calculations (approach 2, see above; cf. Heisler 1984b, 1986b).
At the end of muscular activity the total amount of about 16 mmol kg- 1 body
t:,HC03sw
(mmol/kg
body waterl
-5
10 20 30
o I I
~
t:,NHZ sw
(mmollkg
body water)
10 30
o
t:,H;-.sw
----
(mmollkg
body water)
o 10 20 30
Time (h)
water of lactic acid (a value extrapolated to zero from the linear portion of the
~He ... sw curve during the time period of normalized acid-base status and continuing
lactic acid metabolism from 15-22 h after muscular activity) is accumulated exclusively
in intracellular body compartments (Fig. 8.14). H+ ions are initially released from
the muscle cells at an extremely high rate, indicated by the slope of the tangent to the
first part of the ~Ht curve (Fig. 8.14, Sl, > 150 Ilmol kg- 1 body water min-i).
This slope (Sl) is larger by the rate of lactic acid metabolism (S4) and the rate of
transepithelial elimination of H+ ions (S5) than the initial rise of the curve of H+
ions buffered in the extracellular space (S3). The rate of initial H+ effiux (Sl)
appears to be extremely high, but since the process is likely to be perfusion-limited
(Neumann et al. 1983), it probably represents a considerable underestimate of the real
transfer rate. Also, lack of any relevant data makes it impossible to take into
account any transfer of H +-equivalent ions into non-excitable intracellular body
compartments facilitated by the lowered extracellular pH, which will further tend to
reduce the apparent transfer rate.
The initial high transmembrane H+ transfer rate is considerably reduced to about
25 Ilmol kg- 1 body water min- 1 (Fig. 8.14, S2), mainly due to "equilibrium limita-
tion". pH in the small volume of poorly buffered extracellular fluid is reduced by
transfer of only about 8 % of the originally produced H+ ions (cf. ~Ht and
~H: curves) such that equilibrium (though at a reduced level) between intracellular
and extracellular fluid is attained (cf. Fig. 8.14, insert), eliminating further H +-equiva-
lent transfer in excess to the rate of removal from the ECS by other mechanisms.
When intracellular/extracellular pH equilibrium is attained, the time course of pH
normalization is therefore only dependent on two factors - lactic acid metabolism
( '" 11 Ilmol kg -1 min -1, S4)' and the transfer rate of H + equivalents to the environ-
mental water ('" 141lmol kg- 1 body water min- 1 , S5). When all surplus H+ ions
are eliminated from the organism, the aerobic lactic acid metabolism continues
until control plasma levels are attained with H+ ions taken back up from the
environment at a rate determined exclusively by metabolism ('" 11 !lffiol kg -1 body
water min -1, S4)' which is smaller than the maximal rate of transepithelial transfer
( '" 14 Ilmol kg -1 body water min -1, S5).
This simple model may not suffice to quantitatively describe the real conditions,
but certainly provides a general overview of the interaction of mechanisms involved
in acid-base regulation during endogenous lactacidosis. Two important features
become evident - first, the acid-base status is normalized before the original
stress factor, lactic acid, is removed. Secondly, the intracellular pH deviates, except
for immediately aft~r activity, less from control values than does extracellular pH.
Accordingly, this ana1ysis corroborates data collected during environmental hyper-
capnia indicating a preferential regulation of intracellular pH .
...
Fig. 8.13. Changes in the amount of bicarbonate· (~HCO;sw) and ammonium (~NH:'w) in the
ambient water, and the release of H+ ions (~H:_sw) from the elasmobranch Scyliorhinus stellaris
(peak plasma lactate higher or lower than 15 mmoll- 1 thick solid lines, 1 or 2 respectively)
and the marine teleost Conger conger (thick dashed line) compared to the cumulative control rates
(thin lines) aft~r exhausting muscular activity. Note the offset in ~HC03 and ~NH: curves by
the higher ammonia production rate of the teleost fish. (Data of I-Ioleton et al. 1983; Toews et al.
1983)
-1
pH
I
arltr t... _ ... -'" ••••
7.'
ICS
15
,
1
\
I
\
10 \
I
II Hi \
I
Immol / kg \
body water} ,
\ 1.I~_.L..--;--1._~..J....-}1_,-I--:'~~\k" "
6 Time S (hi
·.. ··1r-
5 \
,, \
'l
I
15 1
I
o
5
!l He
+
,
,53
(mmol / kg
body water) , _ -_ _
o Lr____~~~~~____~____L __ _~L_
/
15 /
/
//5 5
/
/
/
/
/
/
/
10 /
/
'Yo
!l H;_sw
Immol / kg
body water)
o
o 5 20 25
t (h)
8.3 Site and Utilization of Transepithe1ial Ion Transfer Mechanisms 245
8.3 Site and Utilization of Transepithelial Ion Transfer Mechanisms
The preceding sections have indicated that elasmobranchs (and fish in general)
rely heavily on ion transfer processes for pH regulation. Transfer of acid-base-
relevant substances takes place not only between intracellular and extracellular
body compartments, but also, to a great extent, between fish and environmental
water. A number of different epithelia may be involved in this process. The gill
epithelium with its large interface between water and plasma is the most likely
candidate for this task, although excretion of acid-base-relevant ions may also be
performed via kidneys, urinary bladder and skin, and, in elasmobranchs, via the
rectal gland and abdominal pores.
The information on transepithelial acid-base-relevant ion transfer in elasmo-
branchs is generally scarce. The kidney and urinary bladder have received some
attention in a number of studies, indicating that the urine H + excretion could be
raised during certain acid-base stress conditions by elevation of the urine titratable
acidity at more or less constant urine pH, but the results have not been related
to the total amount of transferred acid-base-relevant ions (e.g. Smith 1929a,
1939; Hodler et al. 1955; Cohen 1959; Murdaugh and Robin 1967; Goldstein and
Forster 1971; Deetjen and Maren 1974; Holliday et al. 1979; King and Goldstein
1979). The rectal gland has been studied mainly with respect to its contribution to
osmoregulation (see Chap. 6, this Vol.), although the existence of carbonic anhydrase
possibly suggests an involvement in acid-base regulation as well (e.g. Lacy 1983).
Release of abdominal fluid, reported for Squalus acanthias to be quite acidic (Mur-
daugh and Robin 1967), via the abdominal pores (Gans and Parsons 1964) is
another possible mechanism for acid-base regulation.
Branchial and renal contributions to the elimination of surplus acid-base-relevant
ions have been studied in only two elasmobranch species, Squalus acanthias and
Scyliorhinus stellaris, and for the latter only, skin, rectal gland and abdominal
pores have also been monitored during acid-base disturbances. The contribution of
extrabranchial sites is generally negligible in these two species (Table 8.4), which is
in accordance with most of the data collected in teleost fishes (cf. Heisler 1984 b,
1986b).
The fact that the branchial epithelium is the predominant site of acid-base
relevant ion transfer is not surprising. The large water jplasma interface area is irrigated
during resting conditions by about 12000 ml kg- 1 body wt h- 1 (e.g. Randall et al.
1976; Heisler et al. 1988), whereas the urine flow rate in fishes is in the range of
1-10 ml kg- 1 body wt h- 1 (e.g. Cross et al. 1969; Hunn 1982). Even with extremely
....
Fig. 8.14. Distribution of H+ ions among intracellular (i) and extracellular (e) body compartments,
and the environmental water (e ..... sw) after strenuous muscular activity in Scyliorhinus stellaris.
Sl represents the initial intracellular to extracellular transfer rate of H+ -equivalent ions, which is
reduced due to equilibrium limitation to S2' which is the sum oflactic acid metabolism ( -S4)' and the
rate of net H+ transfer to the environment (S5)' The total amount of lactic acid produced is
extrapolated to time zero from the slope of the ~H:~sw curve between 15 and 22 h. Insert Plasma
and intracellular pH values as a function of time after strenuous muscular activity. (Based on
data of Holeton and Heisler 1983; Heisler and Neumann 1980)
Table 8.4. Extrabranchial sites in elasmobranch acid-base regulationa
Species Stress factor Kidneys + Skin Rectal gland + Reference

urinary bladder abdominal pores
Squalus Hypercapnia <I Cross et a!. 1969

acanthias Hypercapnia <1 Evans 1982
HCO; infusion <1 Hodler et a!.
1955
HCO; infusion <I Murdaugh and
Robin 1967
HCI infusion <1 Murdaugh and
Robin 1967
Scyliorhinus Hypercapnia <1 <1 <1 Heisler et a!.
stellaris 1976a
Exercise <1 <1 Holeton and
Heisler 1983
HCI infusion <1 <1 <1 N. Heisler and
P. Neumann
(unpub!.)
HCO; infusion <1 <I <I N. Heisler and
P. Neumann
(unpub!.)
Temperature
changes 3 Heisler 1978
aContributions given as percentage of tota!' See original references for an evaluation of different
methodologies.
high titratable acidity and ammonia concentrations, the buffer capacity (i.e. the pro-
duct of buffer value and volume) is smaller by several orders of magnitude and
negligible as compared to the buffer capacity of the seawater (~2.3 mmoll- 1
HCO;) flowing past the gill epithelium.
Although the gills are not the main site of ammonia production (e.g. Cameron
and Heisler 1983), the large water flow facilitates elimination of this highly toxic
endproduct of nitrogen metabolism, possibly by ionic exchange for Na +. It has,
however, recently been demonstrated in teleost fishes that, at low environmental
NH;- concentrations, this ionic exchange mechanism is only insignificantly involved
in ammonia elimination, and the largest fraction ( ~ 90 %) is released to the environ-
ment by non-ionic diffusion similar to the mechanism for CO 2 elimination (Cameron
and Heisler 1983, 1985; Evans 1985). Non-ionic release of ammonia reverses the
alkalinizing a'nd CO 2 -neutralizing effect due to ammonia ionization upon production
in the tissues, and has accordingly no net effect for acid-base regulation. The
NH;- INa + exchange mechanism comes into play only when the environmental
ammonia concentration rises beyond values incompatible with non-ionic elimination
(Cameron and Heisler 1983). Recently these data have been corroborated by
Evans (1985), demonstrating that ammonia is eliminated from the elasmobranch
Squalus acanthias mainly by non-ionic diffusion (75 %), and the remainder by ionic
diffusion of NH;-. These data indicate that, of the three major ion exchange
mechanisms discussed as being involved in acid-base regulation, NH;- INa+ can
probably be neglected. This is supported by the fact that the rate of ammonia
8.3 Site and Utilization of Transepithelial Ion Transfer Mechanisms 247
Table 8.5. Ammonia excretion as mechanism for elasmobranch acid-base regulation
Species Stress factor Ammonia Reference

excretion'
Scyliorhinus Temperature changes 0 Heisler 1978

stellaris Hypercapnia 0 Heisler et al. 1976a
Hyperoxiajhypercapnia 0 Heisler et al. 1981
Exercise 0 Holeton and Heisler 1983
HCI infusion 0 N. Heisler and P. Neumann
(unpubl.)
NaHC03 infusion 0 N. Heisler and P. Neumann
(unpubl.)
Acidic water 0 Heisler and Neumann 1977
Squalus Hypercapnia 0 Cross et al. 1969
acanthias Hypercapnia (+) Evans 1982
, Symbols: 0, no change; (+), moderately increased excretion
production and release is hardly ever affected by changes in the acid-base conditions
of elasmobranch (Table 8.5), as well as of teleost, fish species (cf. Heisler 1984 b,
1986b).
Accordingly, the acid-base-relevant transepithelial ion transfer seems to be
mainly performed by 1: 1 electroneutral HCO; /CI- and/or H+ iNa + ion exchange
processes (for review see Maetz 1974; Evans 1979, 1980, 1984, 1986), rather than
by co-transfer of oppositely charged ions which would be in conflict with the require-
ments of osmoregulation. Utilization of the H +/Na + exchange mechanism adds the
H +-equivalent amount of Na + to the normal Na + influx of marine species. This
additional load may be of significance, as indicated by a comparison of the normal
passive Na + influx in Scyliorhinus canicula of between 10 and 22 J..lmol kg- 1 min- 1
(Bentley et al. 1976), the unidirectional Na + flux of between 6 and 9 J..lIDol kg -1 min- 1
for the same species (Payan and Maetz 1970; Maetz and Lahlou 1966), and between
11 and 16 J..lmol kg- 1 min- 1 in Squalus acanthias (Burger and Tosteson 1966;
Horowicz and Burger 1968), with the H+ -equivalent efflux of more than 15 J..lmol
kg- 1 min- 1 during environmental hypercapnia, and during severe lactacidosis in
Scyliorhinus stellaris (see above). If acid-base-relevant ions are equivalently trans-
ferred via the H+ /Na+ ion exchange mechanism, this amount would roughly double
the ionic load to be handled by the appropriate mechanisms. Nevertheless,
substantial evidence. ("albeit, equivocal", Evans 1986) for the utilization of this
mechanism has been provided by numerous investigators (for review see Evans 1986).
In contrast, uptake of HCO; in exchange for Cl- counteracts the passive Cl-
influx, and furthermore is osmotically neutral. This aspect may be of high significance
in face of the above quantitative comparison of flux data. While the H+ /Na +
exchange tends to accumulate additional osmotically active molecules in the body
fluids which have to be eliminated, the HCO; /Cl- ion exchange is osmotically neutral.
In spite of all accumulated evidence (see reviews by Maetz 1974; Evans 1979, 1980,
1984, 1986), a final conclusion cannot yet be drawn as to the branchial mechanisms
mainly responsible for the transepithelial acid-base-relevant ion transfer in elasmo-
branch fishes.
8.4 Conclusion
Elasmobranch acid-base regulation does not appear to be principally different from

that in other fish species, as far as can be judged on the basis of the available
scarce information. The main difference resides in the much lower fractional pro-
duction of ammonia as a nitrogenous waste product in favour of urea. Excretion
of ammonium ions as an acid-base-relevant substance is hardly ever utilized in
either elasmobranchs or teleost fish species.
Elasmobranch (as well as teleost) acid-base regulation is characterized by the
aim of pH normalization before the original stress factor is removed. Intracellular
pH is always preferentially regulated as compared to extracellular pH. This
strategy is supported by the high non-bicarbonate buffer capacity, which is mainly
located in the intracellular body compartments. Non-bicarbonate buffering alleviates
pH changes induced by external or endogenous acid-base disturbances, and assists
reduction of pH with rising temperature due to the negative pH/temperature
coefficients of biological non-bicarbonate buffers. Changes in P C02' a common
mechanism to compensate for non-respiratory disturbances in terrestrial vertebrates,
is not available to fishes due to the physical limitations of water as a gas exchange
medium. Only during hyperoxia, at least in one elasmobranch species, is the
primarily oxygen-oriented regulation of ventilation supplemented by a pH-sensitive
component, limiting a developing respiratory acidosis. The extremely fast, but more
transient, regulation on the basis of buffering is generally replaced by the comparative-
ly effective, although rate-limited, ion transfer mechanisms among intracellular and
extracellular spaces and environmental water, and it is these that are responsible
for the final adjustment of steady state acid-base conditions.
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Chapter 9 DEBORAH F. PERLMAN and L. GOLDSTEIN
Nitrogen Metabolism
Nitrogen metabolism is often viewed only as a set of pathways for forming nitrogenous
endproducts from protein degradation. However, these nitrogenous endproducts,
because of their small size and relative inertness, are also used as osmolytes
for general osmoregulation. Various strategies have been utilized for this function.
Ureotelic vertebrates use urea as their major nitrogenous endproduct and major
osmolyte in their body fluids and tissues. Other vertebrates, mainly birds and reptiles,
utilize uric acid as a nitrogen endproduct and are thus uricotelic. Uric acid is so
insoluble that it precipitates out of the urine and therefore' is removed as an
osmolyte, allowing the accumulation of high concentrations in this body fluid.
Ammoniotelic animals form ammonia as their main nitrogen endproduct. Because
ammonia is so highly diffusible and rapidly excreted its contribution as an osmolyte is
not significant.
The use of urea and other nitrogenous endproducts for osmoregulation by elasmo-
branchs makes the study of nitrogen metabolism in this class of vertebrates a
fascinating example of adaptive strategies in comparative physiology. In elasmo-
branchs, approximately 40-50 % of the plasma osmolarity and 65-75 % of the
intracellular osmolarity is due to non-protein nitrogenous compounds, the remainder
being mostly inorganic ions (Robertson 1975). The regulation of the extracellular
and intracellular nitrogen compounds through excretion, metabolism and transport
in response to changes in environmental salinity is thus an important area of
study in elasmobranch physiology.
In this chapter we will review nitrogen metabolism in elasmobranchs with
special emphasis on the evolutionary and physiological adaptations for osmoregula-
tion. We will also discuss recent research on amino acid metabolism as it relates to
intracellular osmoregulation in elasmobranchs.
9.1 Nature and Routes of Excretion
Quantitatively, the most significant non-protein nitrogenous compounds in elasmo-

branch extracellular and intracellular fluids are urea, trimethylamine oxide, and
amino acids. Betaine, creatine, and creatinine also contribute significant amounts
Author's address: Division of Biology and Medicine, Brown University, Providence, RI 02912, USA
IV
Table 9.1. Major nitrogenous compounds in plasma and muscle of the dogfish, Squalus acanthias, and skate, Raja erinacea v.
.j:>
(mmoll- ' plasma or tissue water)
Urea TMAO Amino N Betaine Creatine Creatinine Ammonia Q

~
Squalus acanthiai' ..,fO"
Plasma 308 ± 11.8 72.4 ± 5.7 11.6 ± 2.0 9.1 ± 6.8 0.126 ± 0.05 0.046 ± 0.005 0.40 ± 0.26 ~
(7) (7) (4) (4) (3) (3) (3) ~
Muscle 333 ± 7.1 180 ± 11.9 108 ± 9.1 100 ± 12.6 68.2 ± 2.3 1.02 ± 0.47 4.7 ± 1.9 g
(7) (7) (7) (7) (13) (3) (4) g
Raja erinacea b
Plasma 361 ± 17.8 39.2 ± 2.7 10.9 ± 0.49 NM NM NM NM ~
~
(6) (6) (6) ~
Muscle 398 ± 18.2 63.9 ± 14.2 214 ± 21.8 NM NM NM NM ~
(6) (6) (6) 8
Values are means ± SE. Numbers of fish in parentheses. NM = not measured .
• From Robertson 1975.
b From Forster and Goldstein 1976.
9.1 Nature and Routes of Excretion 255
to the nitrogen profile of elasmobranch tissues. Ammonia, although quantitively

low in body fluids because it is so readily diffusible and is constantly eliminated
through the gills, is also of importance as a nitrogen endproduct. Table 9.1 lists the
plasma and muscle concentrations of the major nitrogenous compounds found in
the dogfish, Squalus acanthias, and the little skate, Raja erinacea, (Robertson 1975,
Forster and Goldstein 1976). These values are typical for elasmobranchs living in
seawater. It is the regulation of these concentrations in varying environmental sali-
nities and in different body compartments (e.g. extracellular vs. intracellular) that
yields interesting information about the adaptive physiology and the evolutionary
history of elasmobranchs.
9.1.1 Urea
All marine and euryhaline elasmobranchs are ureotelic: urea is the major nitrogen
endproduct and major osmolyte (approximately 35 %of the total osmolarity) of both
extracellular and intracellular fluids and its concentration is regulated in response
to the varying osmolarity of the environment.
High urea concentrations are maintained in elasmobranchs by both the relative
impermeability of the gill membranes to urea and the active reabsorption of urea
by the kidneys. Urea is a dipole with a low oil-water partition coefficient and thus
penetrates cell membranes through aqueous pores (Goldstein and Forster 1970).
The impermeability of the gill to urea appears to be the result of a tight physical
barrier to its diffusion through the membrane rather than from a urea-retaining
transport system. Various studies with urea analogs, temperature dependency,
saturation kinetics and transport inhibitors have given no evidence of a specific urea-
retaining active process in the elasmobranch gills (see Goldstein 1982). This low
branchial permeability is in sharp contrast to the high permeability of other cell
membranes to urea (Fenstermacher et al. 1972).
Urea is avidly retained by the elasmobranch kidney: approximately 87 % of the
filtered urea is reabsorbed by Mustelus canis and 95 % or more by Squalus
acanthias (see Goldstein and Forster 1970). The role of bacterial degradation of urea
in the intestine of elasmobranchs and its contribution to total urea excretion has
been briefly studied. Lloyd and Goldstein (1969) have shown that approximately
41lmol kg- 1 body wt h- 1 of urea are degraded to ammonia in the spiny dogfish
intestine. Although contributing only 1 % of the total amount of urea excreted, the
bacterial degradation of urea should be considered in urea balance studies.
9.1.2 Trimethylamine Oxide ({MAO)

Trimethylamine oxide (TMAO) is another nitrogenous end-product that serves as
an important osmolyte in elasmobranchs. It contributes 5-20 % to the osmolarity of
plasma and muscle. Recent research has suggested that, in addition to its role as an
osmolyte, it functions to counteract the toxicity of urea to proteins (Yancey and
Somero 1979). Like urea, TMAO is avidly reabsorbed by the kidneys of elasmo-
branchs. Approximately 95 % of the filtered load is reabsorbed, in contrast to its
active secretion by the kidneys of teleosts (Forster et al. 1958; Cohen et al. 1958).
256 Chapter 9. Nitrogen Metabolism
9.1.3 Ammonia
Most aquatic animals excrete the major portion of their nitrogenous end products
via the gills in the form of ammonia. Elasmobranchs eliminate approximately 85 %
of their total nitrogenous waste through the gills; the remainder is excreted via the
kidneys (Goldstein and Forster 1962). In marine e1asmobranchs, in contrast to
teleosts, only 25 % of this gill excretion of nitrogen is in the form of ammonia;
75 % is excreted as urea (calculated from Goldstein and Forster 1962). In freshwater
e1asmobranchs such as the sting ray, Potomotrygon spp., urea is not accumulated as
a significant osmolyte and the total nitrogen excretion is through the gills in the form
of ammonia (Gerst and Thorson 1977).
9.1.4 Amino Acids
Urea and TMAO have long been known to be important osmolytes in elasmobranchs
(Smith 1936). Recently, however, attention has turned to amino acids because of
their role in intracellular osmoregulation and cell volume regulation. Free amino
acids can make up more than 20 % of the osmotically active solutes in elasmobranch
cells. Intracellular amino acid concentrations are regulated, since they fall significantly
when the fish are acclimated to diluted seawater. It seems that the concentrations
of selected amino acids are individually regulated through synthesis and transport
to allow the cells to maintain osmotic equilibrium with the extracellular fluid
(Forster and Goldstein 1976; Goldstein 1981). Interestingly, amino acids in marine
teleosts contribute approximately the same percentage to total intracellular osmolarity
as they do in elasmobranchs. However, because of the high plasma osmolarity
in elasmobranchs (695 mOsmoll- 1 versus 364 mOsmoll- 1 marine teleosts), the
intracellular amino acid concentration is significantly higher in elasmobranchs
(214 mmoll- 1 versus 71 mmoll- 1 in marine teleosts) (King and Goldstein 1983a).
This topic will be reviewed more completely below.
9.2 Biochemical Pathways of Formation

9.2.1 Urea
Urea may arise in elasmobranchs via three pathways.

1. The synthesis de novo from carbon dioxide and ammonia via the ornithine-urea
cycle:
ornithine + NH3 + HCO; + 3 ATP + aspartate ~
arginine + fumarate
arginine ~ ornithine + urea.
2. The breakdown of purines:
uric acid ~ allantoin ~ allantoic acid ~ urea + glyoxylate.
3. The breakdown of dietary arginine by arginase to ornithine and urea.
Schooler et al. (1966) examined the question of which pathway was quantitatively
9.2 Biochemical Pathways of Formation 257
most significant for urea production in elasmobranchs. Using in vivo methods and
liver slices they demonstrated a much greater incorporation of 14C-labelled bicarbonate
into urea (the ornithine-urea cycle route) than from 14C-labelled uric acid (the purine
to uric acid route) in dogfish. The arginase pathway is probably of little quantitative
importance in hepatic urea formation because it is limited to the breakdown of
dietary arginine. However, this pathway may have some significance in salvaging
urea from arginine in extrahepatic tissues. Campbell (1961) found arginase activity
not only in liver but also in kidney and rectal gland of the smooth dogfish,
Mustelus canis. For many years investigators assumed that the ornithine-urea cycle
pathway was the most important for urea production, but were unable to document
the occurrence of carbamyl phosphate synthetase (a rate-limiting enzyme in the
cycle) in elasmobranchs (Baldwin 1960). Brown, in 1964, was finally able to demonstra-
te the presence of this enzyme in the liver of the bonnet head shark, Sphyrna
tiburo, and Watts and Watts, in 1966, were able to show its occurrence in several spe-
cies of elasmo branchs.
Since then it has been shown that there are three kinds of carbamyl phosphate
synthetase enzymes, distinguished by their nitrogen-donating substrate and cofactor
requirements. CPS I, located in liver mitochondria of ureogenic animals, uses only
ammonia as the nitrogen-donating source and requires n-acetyl glutamate (NAG)
as a cofactor. The properties of the enzyme relate to its function in ammonia
detoxification. CPS II, located i~ the cytosol, utilizes glutamine as its substrate
and NAG is not a required cofactor. CPS II is involved in pyrimidine nucleotide
biosynthesis. CPS III, like CPS II, utilizes glutamine as the nitrogen source, but
like CPS I it requires NAG as a cofactor. Anderson (1980) found very high
CPS III activity in the elasmobranchs he surveyed. With glutamine as the substrate,
the activity of the enzyme is about ten times greater than with ammonia, and the
presence of NAG stimulates the enzyme activity. Interestingly, Webb and Brown
(1980) have measured high levels of glutamine synthetase activity in the hepatic
and renal tissue of these same urea-retaining elasmobranchs. This correlation between
glutamine synthetase activity and CPS III suggests a direct metabolic relationship
between the production of glutamine and urea in elasmobranchs. Glutamine can
be produced in the kidney for transport to the liver and the liver itself can
produce glutamine for urea production. The freshwater stingray, Potamotrygon
eireularis, which does not retain high levels of urea, has very low levels of CPS III
and correspondingly low levels of liver glutamine synthetase (Anderson 1980;
Webb and Brown 1980).
Leech et al. (1979) have reported that blood glutamine is not detectable in
dogfish during starvation and it is in very low levels in fed dogfish. However,
there is a net release of ammonia into the blood from the tail muscle in both
freshly caught (and presumably fed) and starved (up to 22 days) dogfish. The
ammonia must therefore be transported to the liver and kidney where glutamine
synthetase can covert it to glutamine and it can enter the urea cycle via CPS III
activity.
Anderson (1981) has prepared CPS III to a high degree of purity from
Squalus aeanthias. He studied the general kinetic and structural properties of this
enzyme and made comparisons with CPS from sources other than elasmobranchs.
He suggests that elasmobranch CPS III has unique properties which are specifically
related to its function in the synthesis of urea for osmoregulatory purposes rather
than as a major route for disposal of ammonia from protein and amino acid catabolism.
Varying the urea concentration within the physiological range affects the catalytic
activity of CPS III and may act as a general feedback for maintaining urea at the
appropriate osmotic concentration. The Km for glutamine and n-acetyl glutamate
increases when urea is present at physiological concentrations (0.4 moll- 1). The
concentration of n-acetyl glutamate required for activation is dependent on the con-
centration of glutamine. The requirement of glutamine as a substrate, the effect of
interaction between glutamine and n-acetyl glutamate on the kinetic properties, and
the effect of urea on activity may be specific properties of the elasmobranch
CPS III and relate to its role in urea synthesis for osmoregulation.
Shankar and Anderson (1985) have recently purified liver glutamine synthetase
from Squalus acanthias and studied its structural and kinetic properties. They
found that it is similar to mammalian and avian glutamine synthetases except for
two unique properties - a very low apparent Km for ammonia and an activation by
halogen anions. The very low Km for ammonia may be specifically related to the
role of the enzyme in providing glutamine as the substrate for urea biosynthesis.
The authors suggest that the extra energy-requiring step of converting ammonia to
glutamine and then the use of glutamine as the substrate for CPS III may provide
a more efficient means of extracting ammonia from the blood, particularly at low
concentration. This step may be more efficient and better regulated than if ammonia
were assimilated directly to carbamyl phosphate by ammonia-dependent carbamyl
phosphate synthetase. They do not speculate on the significance of the activation
by relatively low concentrations of chloride, bromide or iodide.
Interestingly, a few studies have been done with urea biosynthesis in elasmo-
branch embryos. Read (1968 a, b) has studied the activity of two urea cycle enzymes
(ornithine carbamoyl transferase and arginase) during development of the dogfish,
Squalus suckleyi and the skate, Raja binoculata. He was able to correlate the
increased biosynthetic activity of the enzymes with the increase in total urea that
accumulated during embryonic development.
9.2.2 Trimethylamine Oxide (TMAO)
There has been a great deal of discussion concerning the source of TMAO in
elasmobranchs. The fairly constant plasma level found in marine elasmobranchs is
assumed to .be maintained by the active reabsorption of TMAO by the kidney,
the large pool of TMAO in the muscle and the slow loss of TMAO from the
muscle (see Goldstein and Forster 1970). The question is whether the small loss
to the environment that does occur is replaced solely by diet (which has a high
concentration of TMAO) or by endogenous synthesis of TMAO. Goldstein et al.
(1967) were unable to demonstrate any in vivo biosynthesis of TMAO from the
putative 14C-labelled precursors, methionine. choline or trimethylamine, in either
the spiny dogfish or the little skate. However, a low rate of synthesis was found
in vivo in two warmer water species: the nurse shark, Ginglymostoma cirratum,
and the lemon shark, Negaprion breviostris (Goldstein and Funkhouser 1972;
Goldstein and DeWitt-Harley 1973). In addition, both liver slices and liver homogen-
9.2 Biochemical Pathways of Formation 259
ates from these species were capable of synthesizing TMAO from 14C-labelled
precursors.
An interesting way to look at endogenous biosynthesis ofTMAO is in the nutrition-
ally closed system of the skate embryo. Read (1968 b) measured TMAO concentra-
tion, embryo and yolk volume and water content throughout the development of
Raja binoculata enclosed in egg cases. Although the increase in total volume of the
system (mostly from increase in water content) caused a measured decrease in
TMAO concentration over time, the total TMAO content increased. The develop-
mental increase in TMAO is strong evidence that this elasmobranch must be capable
of producing the compound at least during development.
Goldstein and DeWitt-Harley (1973) examined the properties of nurse shark
trimethylamine oxidase in liver homogenates and showed that this enzyme shares
many properties with mammalian mixed function oxidase, an enzyme involved in
several detoxification reactions. They propose that the original function of this
enzyme was the oxidation of trimethylamine to trimethylamine oxide (and possibly
the oxidation of other amines). Because of its low specificity, this mixed function
oxidase was maintained or adapted by mammalian systems for catalyzing the oxidation
of a variety of foreign tertiary amines, including drugs.
The puzzling question is why some elasmobranchs have TMAO biosynthetic
capability and some do not, even though the latter seem to maintain a high
TMAO concentrati.on in a marine environment. Goldstein and Pallatt (1974) hoped
to correlate the presence or absence of biosynthetic capacity with a TMAO loss
coefficient (rate of loss of 14C TMAO from the extracellular compartment to the
environment). In fact, however, the lemon and nurse sharks, which can synthesize
TMAO, have loss coefficients at either extreme from that in dogfish and skate,
which were unable to synthesize TMAO. Thus, no correlation was found. The authors
suggested that the sporadic occurrence of TMAO biosynthesis in elasmobranchs
may be due to random deletion of the requisite genes in this group.
9.2.3 Ammonia
In elasmobranchs it has been assumed that the formation of ammonia comes from
the deamination of most amino acids in the trans-deamination pathway of Braustein
and Bychkov in a manner similar to that of other animal tissues (See Goldstein and
Forster 1970). This pathway involves transamination of various amino acids with
(X-ketoglutarate to form glutamate which is then deaminated by glutamate dehydro-
genase:
(transaminase)
amino acid + (X-ketoglutarate ¢ glutamic acid
(glutamate dehydrogenase)
glutamic acid + NAD + -> (X-ketoglutarate + NH;- + NADH .
It is interesting to determine how much of the branchially excreted ammonia is

brought to the gills in the blood as ammonia and how much is deaminated from
amino acids in the gill cells themselves. Using the ammonia venous-arterial differences
and measured cardiac output in Squalus acanthis (from Cross et al. 1969), ammonia
clearance from the blood through the gill can be estimated as 0.11 mmol kg- 1 body
wt h- 1 (out of a total gill ammonia excretion of 0.15 mmol kg- 1 body wt h- 1
(Goldstein and Forster 1962). This leaves only 25 % (0.04 mmol kg- 1 body wt h- 1 )
to be derived from deamination of blood amino acids by the gill itself. Gill
glutaminase activity (0.3 mmol kg- 1 body wt h -1 of ammonia) was found to be more
than sufficient to account for this component (Goldstein and Forster 1962).
Significant glutamate dehydrogenase (GDH) activity has been demonstrated in the
livers of two elasmobranchs, Raja ocel/ata and Squalus acanthias (McBean et al.
1966), which suggests that the liver may be an important site for ammoniogenesis in
elasmobranchs as well as teleosts.
Because the urinary excretion of ammonia is so low in elasmobranchs, renal
ammoniogenesis in response to acidosis has not usually been considered in studies of
elasmobranch acid base metabolism (Cross et al. 1969). However, King and
Goldstein (1983 b) were able to show in the dogfish, Squalus acanthias, a doubling
in renal ammonia excretion following acid loading (infusion of HC!). In vitro
studies with kidney slices showed that the dogfish kidney has the capacity to
synthesize ammonia from several amino acids (glutamine, glutamate, alanine, aspar-
tate and glycine) with the greatest ammonia production being from glutamine. The
authors studied the kidney enzymes for both the deamidation (glutaminase) and
synthesis (glutamine synthetase) of glutamine, which are both localized in the
mitochondria. The Km for the two enzymes were found to be about equal
(4.48 mmoll- 1 glutamine and 4.33 mmoll- 1 glutamate) and Very close to the sub-
strate concentration in the kidney (3.69 mmoll- 1 glutamine, 3.36 mmoll- 1 gluta-
Urine Renal cell Blood
Mitochondrion
GS
NH3 + Glutomote ~eGlutomine
NH/ GDH 1l
---+---t-'7':I---- NH3 + a.KG- - - Glucose
Glucose
Fig. 9.1 Hypothetical scheme for participation of substrate cycle in regulation of ammoniagenesis in
dogfish kidney cell. GDH glutamate dehydrogenase; GS glutamine synthetase. (King and Goldstein
1983b)
9.3 Urea Toxicity and Counteraction by Trimethylamine oxide 261
mate). They suggest that these mitochondrial enzymes might provide a substrate
cycle as a mechanism to increase renal ammonia production during acidosis. This
scheme is presented in Fig. 9.1. Regulation could be achieved by a modulation of the
activity of glutaminase or glutamine synthetase' or both. Depending on the relative
activities of glutaminase and glutamine synthetase, there could be either net glutamine
synthesis or net deamidation and ammonia production.
9.3 Urea Toxicity and Counteraction by Trimethylamine oxide
Ever since high levels of urea were observed in marine elasmobranchs, the question
of how these fish cope with what in mammals would be considered a "pathological
uremia" has been of interest. Urea at concentrations present in marine elasmobranchs
(approximately 0.4 mol 1-1 ) has strong perturbing effects on macromolecular structure
and function. The perturbing effect of urea may interfere specifically with binding
of ligands (substrates, cofactors, modulators) and active sites, and therefore disrupt
enzyme function, or may alter the hydration, solubility and charge interactions of
various protein groups (backbones or side chains) (Yancey et al. 1982). The question
is, therefore, whether elasmobranch macromolecules have adapted to high urea
concentrations to avoid these perturbations or whether there is some other mechanism
to counteract these effects.
Early 20th century experiments erroneously suggested that urea was an essential
biochemical for cardiac activity in elasmobranchs. The heart appeared to beat
satisfactorily in solutions containing 2.2 % urea and 2 % sodium chloride, but failed
to maintain its physiological activity in solutions containing no urea. Smith (1936)
reviewed this literature, critizing these experiments, and suggested that an appropriate
balance of electrolytes and optimum concentrations of proteins, amino acids and
lipids may be more important to physiological activity than the presence or absence
of urea. He concluded that in elasmobranchs urea exerted a significant osmotic
effect relative to the external environment, but internally urea was biochemically
inert.
Recent research has shown that some kinds of elasmobranch proteins do appear
to require urea for proper structure and function. The shark eye lens protein has
been shown to require urea for proper conformation (Zigman et al. 1965). The
M4 lactic dehydrogenase isozyme in marine elasmobranchs requires urea for correct
pyruvate:binding ability and optimal catalytic activity (Yancey and Somero 1978).
Interestingly the M4-lactic dehydrogenase of the freshwater elasmobranch, Potamo
trygon sp, which does not accumulate urea, does not require the presence of urea for
optimal kinetic properties (Yancey and Somero 1978). These examples seem to be
adaptations of elasmobranch proteins to high concentrations of urea.
Another solution to the problem of high urea concentration appears to be provided
by the presence of a family of methylamine compounds, especially TMAO, but also
betaine and sarcosine. Yancey and colleagues have shown that these compounds
act as stabilizers of protein structure and function. They counteract the destabilizing
effects of urea especially when present as a 2: 1 molar ratio (urea: TMAO).
Urea and TMAO balanced at this ratio yield the best rate and extent of enzyme
renaturation after acid denaturation of white shark (Carcharodon carcharias) lactate

dehydrogenase (Yancey and Somero 1979). Similarly, this ratio optimizes the tension
generated in vitro by dogfish muscle fibers (Altringham et a!. 1982). This stabilizing
effect of methylamines can also be shown for certain mammalian enzymes that are
not normally subjected to high urea concentrations. The thermal stability of bovine
ribonuclease and the reactivity of thiol groups of bovine glutamate dehydrogenase
best approximate control conditions when urea and trimethylamine are present
at the 2: 1 ratio (Yancey and Somero 1979).
Yancey et a!. (1982) suggest that these methylamine compounds have been selected
(partially at least) as osmolytes because of their stabilizing effects on macromolecules.
They act in a manner similar to the stabilizing ions of the Hofmeister series.
Hofmeister ranked cations and anions according to their stabilizing and destabilizing
effects on macromolecular conformations (See Von Hippel and Schleich 1969). Clark
and Zounes (1977) pointed out the striking similarity between certain organic
osmolytes and ions in the Hofmeister series that favour native macromolecular
structure (e.g. methylamines resembling Quaternary ammonium ions). Therefore the
modulation of TMAO concentrations in varying environmental salinities may be
significant not only for osmoregulation but also to maintain a 2: 1 ratio with
respect to urea as it varies in concentration.
9.4 Physiological and Evolutionary Adaptations of Nitrogen

Metabolism for Osmoregulation
It has long been known that elasmobranchs use various products of nitrogen
metabolism as osmotically active solutes to maintain themselves hyperosmotic to
their environment (Smith 1936). However, the mechanisms by which they regulate
the concentrations of these compounds in response to varying salinity is only begin-
ning to be understood. The comparative approach of examining elasmobranchs from
different environments and the regulation of nitrogen compounds in response to
salinity changes allows interesting information to be gained regarding both the adap-
tive physiology and the evolutionary history of this group of fish.
9.4.1 Physiological Adaptations
Various studies in different species have been aimed at understanding the physio-
logical and biochemical processes used to regulate the concentrations of nitrogen
compounds that are used as osmolytes. There have been two different approaches
to this question. One method has been to experimentally change the external salinity
either acutely or gradually and study the fish's adaptive response. This has been
done both with osmotically tolerant stenohaline elasmobranchs, which live entirely
in salt water, and with euryhaline elasmobranchs, which venture into brackish and
even freshwater. The second approach has been to collect fish from different salinities
and compare the concentrations of nitrogen compounds and the mechanisms by which
9.4 Physiological and Evolutionary Adaptations of Nitrogen 263
they are regulated. An important consideration in both these approaches is the

time allowed for acclimation to the various salinities. Different experimental designs
have involved either an abrupt transfer of a fish from one salinity to another, or a
gradual dilution or concentration of the environmental water over a longer period of
time. It has been shown that it can take 2 to 6 days after transfer for the fish
to reach a new equilibrium (price and Creaser 1967, De Vlaming and Sage 1973).
This may also be true for animals collected from different salinities in the wild.
It may not be known how long the fish had resided in that salinity or whether
they were in osmotic equilibrium with their environment at the time of capture.
Price (1967) reported a considerable scatter of data points when he compared serum
osmotic pressure of the clearnose skate, Raja eg/anteria, to the osmotic pressure of
the water from which they were collected. The clearnose skate that he was studying
lives in the estuaries of the lower Chesapeake and Delaware Bays in salinities
varying from 13-33%0' The tidal cycle in this area may produce salinity changes of
more than 10%0 in a 6-hour period. Therefore these skates may not have equilibrated
with their environment, which would explain the poor correlation between serum and
medium osmolarities.
The regulatory mechanisms for modulating urea and TMAO in response to
salinity changes must occur either through changes in production and/or total
body clearance. To maintain a constant plasma urea concentration, urea production
should equal total urea excretion. Using this argument, Goldstein et at. (1968)
found that in the lemon shark, Negaprion brevirostris, urea biosynthesis (production)
was not changed in fish adapted to 50 % seawater compared to controls in 100 %
seawater. In contrast, both the little skate, Raja erinacea, and the lipshark,
Hemiscyllium p/agiosum, did show a significant decrease in urea excretion and there-
fore urea biosynthesis in 50% seawater (Goldstein and Forster 1971 a; Wong and
Chan 1977). In addition, the little skate showed an increase in urea excretion and
therefore urea biosynthesis upon readaptation from 50 % back to 100 % seawater.
The reason for the different mechanisms of response (excretion vs. synthesis) in the
lemon shark, lipshark and little skate is not clear.
Total body urea clearance can be modified by changes in a variety of physiological
parameters: gill permeability, urine flow, glomerular filtration rate and reabsorption
by the kidney. Research has been done to try to delineate the role of each of these
factors in urea osmoregulation. Both Payan et at. (1973) and Wong and Chan (1977)
working with skates, Raja erinacea and R. radiata, and the lipshark, Hemiscyllium
p/agiosum, respectively, have shown that gill permeability to urea does not change
in either acute or gradual transfer to diluted seawater. By cannulating the urogenital
papilla (and the rectal gland in the lipshark) the rates of excretion of 14C-Iabelled
urea could be determined for the various routes - gills, kidney and rectal gland. In
the skate, following acute transfer from 100 % to 75 % seawater, branchial excretion
rate did not change significantly, while renal excretion increased fourfold. In
experiments where the lipshark and skate had been gradually acclimated over 5 to
6 days to 50 % seawater branchial urea excretion had decreased by 54 % and 65 %
in direct relation to the reduction in plasma urea concentration. Therefore the branchial
clearance of urea was not significantly different in fish adapted to dilute seawater
compared to seawater controls. The minor role of the gills can best be described
by looking at the percentage of total urea excreted. In marine elasmobranchs
85-97 % of the small amount of the total excreted urea is lost via the gills
(payan et al. 1973; Wong and Chan 1977), confirming the efficient reabsorption of
urea by the kidney. This percentage changes drastically to approximately 40-50 %
in fish adapted to 50 % seawater. The clearance of urea by the gills does not
change, while the renal clearance is greatly increased.
The effect of changes in environmental salinity on renal function has been
examined in several elasmobranchs. Both urine flow and glomular filtration rate
(GFR) increased four to six times in the skate and lipshark adapted to 50 %
seawater (Goldstein and Forster 1971 a; Wong and Chan 1977). Renal urea clearance
rates similarly increased fourfold in these fish. The important question is whether
the reduction in body urea in diluted elasmobranchs is accomplished solely by the
increase in urine flow and GFR or whether peri tubular controls are involved.
The classical view offered by Smith (1953) was that regulation of urea concentration
in body fluids was achieved by changes in urine flow. In a dilute seawater, blood volume
would be expanded by an influx of water due to the fish being hyperosmotic to
its environment. "The mechanism appears to work with great simplicity: as the
water absorbed through the gills dilutes the blood, the urine flow increases,
increasing the excretion of urea and thereby reducing the blood urea concentration,
which in turn reduces the absorption of water through the gills, reduces urine flow,
and starts a new cycle of urea retention, so that animal is always supplied with
enough free water to meets its urinary requirements." The renal tubular reabsorption
of urea, however, does seem to be independently affected. Forster et al. (1972) used
the dogfish, Squalus acanthias, as a model to separate peri tubular forces acting on the
reabsorption of urea from changes in urine flow and glomerular filtration rate.
Injection of epinephrine caused a marked osmotic diuresis with an increase in urine
flow and renal clearance of urea without affecting GFR. The elimination of urea,
expressed as percentage of filtered urea that is excreted, was increased 15 times
while urine flow increased only twofold and GFR did not significantly change.
Expansion of the extracellular volume by 20 % by the infusion of balanced isotonic
medium into the dogfish again caused an increase in urine flow and greatly
reduced reabsorption of urea without a concomitant change in GFR. The nature
of the tubular urea reabsorption mechanism is still not well understood, but important
controls must be operating here in the regulation of urea concentrations in varying
salinities.
Urea degradation by intestinal bacteria does not appear to playa significant role
in the adaptation of urea metabolism to environmental dilution. Goldstein and
DeWitt-Harley (1972) administered an antibiotic treatment to the skate, Raja
erinacea, to effectively reduce intestinal urease activity. There were no significant
differences in plasma urea concentration or urea excretion rates in antibiotic-treated
or untreated fish in either 100 % or 50 % seawater.
The regulatory mechanisms controlling TMAO concentrations have been much
less well defined. In the elasmobranchs studied (lemon shark, little skate and spiny
dogfish) the plasma TMAO concentration decreased by approximately the same
percentage as urea upon acclimation to 50 % seawater (Goldstein et al. 1968;
Goldstein and Forster 1971a; Forster et al. 1972). The renal reabsorption of
TMAO, however, does not seem to be regulated independently of urine flow and
GFR. While the percentage of filtered urea that was excreted increased 6- and 15-
9.4 Physiological and Evolutionary Adaptations of Nitrogen 265
fold in the skate and dogfish respectively, the percentage of filtered TMAO that was
excreted did not significantly increase in the skate and increased only 3.8-fold in the
dogfish. These experiments suggest that TMAO clearance may be modified more
simply by changes in water influx and resultant changes in GFR and urine flow than
by tubular changes in reabsorption.
9.4.2 Evolutionary Adaptations
Elasmobranchs are presumed to have evolved as a group in a marine environment

during the Devonian period (Romer 1966). Those presently living in freshwater
presumably represent a more recent invasion of that environment. Therefore it is
interesting to examine the nitrogen metabolism of freshwater elasmobranchs in rela-
tion to osmoregulation, and make comparisons with related marine species. As is
apparent from the previous sections, a significant amount of work has been done
on nitrogen metabolism in marine elasmobranchs. Only recently have freshwater
representatives of this group of fishes been investigated in detail. Smith (1936)
compiled a list of elasmobranchs known to inhabit freshwater. Included in the
list are species that only occasionally migrate into freshwater, while others remain
indefinitely in freshwater. It is important to understand fully a species' environment
and how long it has resided there in order to interpret its regulation of nitrogen
compounds for osmoregulation. Urea concentrations in various species captured in
freshwater have been reported to be 25-35 %of those found in marine elasmobranchs
(Smith 1931; Urist 1962; Thorson 1967). The particular species studied (sawfish,
Pristis microdon; sharks, Carcharhinus melanopterus, C. leucas; and rays, Dasyatis
warnak, Hypolophius sephen) were all obtained from freshwater bodies that still
maintain reasonable access to the sea. Therefore it was not known for certain
whether these fish were euryhaline and passed back and forth from freshwater to
saltwater or if now they were totally resident in freshwater and how long, evolutionarily
speaking, they had actually been there. For example, the shark found in Lake
Nicaragua was long considered to be a separate landlocked freshwater species,
Carcharhinus nicaraguensis, but was shown to be morphologically identical with
C. leucas of the Caribbean (Bigelow and Schroeder 1961). Thorson (1971) was able
to show by tagging that this fish moved freely between lake and sea in both
directions and was thus one euryhaline species.
Thorson et al. (1967) obtained rays of the genus Potamotrygon from the Amazon
drainage basin that had been at least 4000 to 5000 km from the ocean for many
thousands of years and could thus be called a true freshwater elasmobranch.
In contrast to the previous freshwater e1asmobranchs examined, these rays had
extremely low urea concentrations (about 1 mmoll- 1 ) and undetectable TMAO.
They appeared to osmoregulate in a manner similar to freshwater teleosts in that the
ions in their plasma and other body fluids contributed nearly the entire amount of
their fluid osmolarity.
Further research with Potamotrygon by Goldstein and Forster (1971 b) showed
that their low urea content was attributable to both very low urea biosynthetic
ability and low urea retention (see Tables 9.2 and 9.3). Although the hepatic urea
cycle enzymes were present, their activity was 1/2 to 1/20 th of those found in
Table 9.2. Liver ornithine-urea cycle enzyme activities and liver slice incorporation rates of 14C_
bicarbonate into urea in freshwater (Potomotrygon) and marine (Dasyatis, Squalus) elasmobranchs
Enzyme activities Incorporation rates
CPS OCT ASS Arginase

(J.lffi0lgliver- 1 h- 1)
Potomotrygon
spp" 0.36 ± 0.05 1600 ± 210 9.4 ± 0.5 4310 ± 240 0.021
Dasyatis
americana" 6.5 ± 1.3 14,360 ± 1420 21.6 ± 2.1 34,880 ± 820
Squalus
acanthiasb 2.25
Values for enzyme activities are means ± S.E. of four to six fish. Values for incorporation rates
are means of two to three fish. CPS = carbamoyl phosphate synthetase: OCT = ornithine carbamoyl
transferase; ASS = arginine synthetase system.
" From Goldstein and Forster 1971 b.
b From Schooler et al. 1966.
Table 9.3. Rates of ammonia and urea excretion in freshwater

(Potomotrygon) and marine (Raja erinacea) elasmobranchs
Urea Ammonia
Potomotrygon" BLD «100) 981

(3) (3)
Raja erinaceab 239 ± 49 111 ± 15
(6) (6)
Values are means + S.E. Numbers of fish per group shown in

parentheses. BLD = below limits of detection.
" From Goldstein and Forster 1971 b.
b From Goldstein and Forster 1971 a.
marine elasmobranchs and the incorporation of 14C-Iabelled bicarbonate into urea by

liver slices was 1/l00th of the rate of the marine dogfish, Squalus acanthias.
Urea excretion by Potamotrygon was below detectable limits whereas ammonia ex-
cretion was !J.bout 1000 !lmol kg -1 body wt h -1. This contrasts with the urea to
ammonia excretion ratio of 2: 1 in the marine skate, Raja erinacea. Similarly, the
excretion rate of injected 14C-Iabelled urea by Potamotrygon was 45-50 % per day
versus 1-3 % by Squalus acanthias, indicating the inability of this freshwater species
to reabsorb urea effectively in the kidney.
The interesting question remained, however, of whether Potamotrygon was able
to turn on a long repressed capability for urea synthesis and retention when faced
with an osmotic stress of increasing salinity, or whether it had permanently lost
this ability. Several studies have shown that Potamotrygon can survive in approximate-
ly 1/2 strength seawater but that urea accumulation plays an insignificant role in
this adaptation (Thorson 1970; Griffith et al. 1973; Gerst and Thorson 1977, and
9.5 Amino Acids and Intracellular Osmoregulation 267
Bittner and Lang 1980). Urea concentration increased only to about 6 mmoll- 1 when
Potamotrygon was acclimated to a salinity of 525 mOsmoll- 1 for 3 days (Bittner and
Lang 1980). The fish appeared to osmoregulate mainly via electrolytes with the
combined serum sodium and chloride concentrations nearly equivalent to the measured
serum osmolarity. Even when Potamotrygon was slowly acclimated to increasing
salinity, the urea biosynthetic capability and urea retention abilities did not change.
Gerst and Thorson (1977) basically repeated the measurements made by Goldstein
and Forster (1971 b) on rays acclimated to 40% seawater over a minimum period
of 2 months. They found no significant effect of hyperosmotic stress on the rate of
urea biosynthesis in liver slices or homogenates or on urea excretion rates in saline-
adapted rays. 14C-urea total body clearance also did not change, indicating no renal
conservation of urea. Potamotrygon appears to have truly lost the ability to
osmoregulate with urea.
An interesting classification problem appears to have been resolved by discovering
that Potamotrygon does not accumulate urea. The Garoua stingray found in the Niger
and Benoue rivers of Nigeria, Africa, has been variously assigned to the family
Dasyatidae and Potamotrygonidae. Thorson and Watson (1975) were able to obtain
three fresh specimens from the area and found the pericardial fluid urea concentration
to be approximately 250 mmoll- 1. This significant urea concentration, along with
morphological data, places this fish in the euryhaline family of Dasyatidae, which
also has reported urea concentrations in this range, rather than in the Potamotrygo-
nidae family with its negligible urea concentration.
9.5 Amino Acids and Intracellular Osmoregulation
Until recently, studies on the role of nitrogen metabolism for osmoregulation in

elasmobranchs have been concerned with the modulation of the extracellular fluid
(ECF) composition in response to varying environmental salinities. In vertebrates,
most cells are isosmotic with their extracellular fluid. Therefore, changes in ECF
osmolality in response to external salinity changes in turn present an osmotic stress
to the cells. A corresponding change in intracellular fluid (ICF) osmolality is
necessary for maintenance of cell volume and composition and for the functional
integrity of the cell.
Intracellular osmolytes present in elasmobranchs include both inorganic and
organic solutes. Although inorganic ions are involved in intracellular control of cell
volume, the change in their concentration and proportions are critically limited
by the necessity to maintain a milieu compatible with the metabolic and electrical
processes of the cell. Enzymes, membrane potentials, etc. are all dependent on
certain ion concentrations. Therefore, although the inorganic solutes do play some
role in intracellular osmoregulation, it is the organic solutes, namely urea, TMAO,
and amino acids, that are regulated to balance the extracellular osmolality.
As discussed above (Sect. 9.4.1) elasmobranchs venture into waters of varying
salinites and act as incomplete osmoconformers. Their extracellular fluid osmolarity
changes in relation to the environmental salinity, but they remain hyperosmotic to
the external medium. Quantitative changes take place in the total intracellular
Extracellular Intracellular
osmolytes osmolytes
1100 r
TMAO
1000 c- Amino acids Unmeasured
::::: osmolytes
$ 900 l-
E TMAO
<-
0 800 f- Urea
TMAO
(5
E 700 - Amino Amino acids Unmeasured
E acids osmolytes
VI
c
600
Urea TMAO
.Q
- 500 -
c;<- Amino acids
c
Ql
U
400 - Urea
C
0
-
u 300 e- Na+, K+, Cl-
Ql
Urea
:::l Na+, K+,Cl-
(5 200 c-
If)
100 I- Na+, K+, Cl-

Na+,K+,Cl-
0
100% SW 50% SW 100% SW 50% SW
Fig. 9.2. Extracellular (plasma) and intracellular (muscle cell) osmolyte concentrations for the little
skate Raja erinacea, acclimated to 100 or 50% seawater (SW). (King and Goldstein 1983a)
osmolarity to accompany those changes in the extracellular fluid. However, the

contribution of individual osmolytes differs between the two compartments. Figure
9.2 shows the osmolyte composition of the plasma and muscle cells of the little
skate, Raja erinacea, in 100 % seawater and adapted to 50 % seawater (King and
Goldstein 1983 a). The ions, Na +, K +, and Cl- , contribute 60 % of the osmolarity
of the extracellular compartment (plasma) in both 100% and 50% seawater and
only approximately 35 % to the osmolarity of the intracellular compartment (muscle
cells). In contrast, the organic solutes contribute approximately 40% to the ECF
osmolarity and approximately 60% to the ICF. This difference between ECF and
ICF is mostly due to amino acids (1 % in ECF vs. 19% in ICF). Plasma membranes
are highly permeable to urea and reasonably permeable to TMAO, so these organic
solutes are e~sentially in equilibrium between the plasma and intracellular environ-
ment in the skate. The cells therefore maintain themselves isosmotic with the ECF
mainly through the regulation of amino acids.
On the cellular level this regulation may include alterations in membrane transport
(influx and effiux) and/or metabolism (synthesis and degradation). Although a effiux
Fig. 9.3. Twelve most common free amino acids in skate wing muscle, skate erythrocytes, skate
.
heart, and sting ray brain. Half SW values significantly different from SW values are indicated by
p values. (Boyd et al. 1977)
60 60
Skate wing muscle Skate hea r t
50 50
1.0
I SW o 112SW
1.0
.....
0"1 0"1
.....
(530 LI> LI> "030
E 0
0
N
0 E
:l ci ci :1-
v v
0. 0.
20 20
10 10 LfI
LfI 0
o <>
ci ci
v v
0. 0.
o SLAJLRWC~lcURL1E..J..A!'"-
1 p"'R::J...o~1:WG"LY~I'-"THI:lR....I!"-A...S"'-'p~1~ o TLA"ULJR~
' CURLLE-"'!AIf-'-p"'SE:LR~'BJ....A"L"""A'F'1~
A L~A-:IF-s-E"-R~I-
BALA TAUR ALA SER GLU LEU ETHA GLU ASP THR GLY LEU
80
Skate erythrocytes 80 Stingray brain
70 70
60 0 60
ci
v
0.
50 50 0
ci
v
0.
.....0"1 .....0"1
"0 1.0 (51.0
E E
:1- :l
0
0
30 ci 30
v
0.
;:;
20 20 ci
v
0.
0
10 ci
V
In
0 10 LI>
0. 0 C!
ci o
V V
0. 0.
o lL..A"'U.LR~1J..JG"LY~ISL..JAL1R'-"C!-1 At::...BA~I~S...E"'-R~I-T..
IG.... R~1'-
H...... OL..TA"UJ..JR~ICLJRLJEL..JA~IG....IIALLB""AI!-'-B""AL.1.LA..I\I.LE"TH.LA~I"-T"H'--R"P-
BALA GLU ALA BABA ETHA ASP GLU GLY ASP GLUNH2 SER SARC
of amino acids may result in a reduced intracellular osmolarity, it may cause a

large increase in plasma concentration as a result of the release of amino acids from
cells into the extracellular fluid. Therefore, in terms of the whole animal, changes
at the cellular level are eventually dependent on metabolism and/or excretion of
these solutes. Interestingly, it is not the entire amino acid pool of the body that is
modulated in response to environmental dilution. Boyd et al. (1977) determined the
amino acid profiles of different tissues (muscle, heart, erythrocytes, brain) of the
little skate, Raja erincea, and the sting ray, Dasyatis sabina, acclimated to 100 %
or 50 % seawater. They showed that only a few selected amino acids contribute to
the high intracellular concentration (about 40-80 mmoll- 1 each) and it is these
major amino acids that decrease in response to environmental dilution (see Fig. 9.3).
In control fish, skate wing muscle contained high levels of sarcosine and ~-alanine,
sting ray brain had high concentrations of taurine, and skate erythrocytes had
high levels of both taurine and ~-alanine. In fish gradually adapted to 50 %
seawater over 7 days, these same amino acids fell to approximately half their control
level. Skate heart had taurine in high intracellular concentrations, but in these dilu-
tion experiments, neither taurine nor any of the other amino acids decreased.
The authors suggest that solutes other than amino acids may be involved in the
osmoregulatory response of heart muscle cells. However, Forster and Hannafin
(l980a) have shown that ninhydrin-positive substances (NPS) (representative of the
total amino acid pool) decreased significantly in both skate atrium and ventricle.
They also showed in in vitro experiments that the NPS concentration in the skate
atrium but not ventricle was directly related to medium sodium concentration. An
amino acid profile of the atrium might therefore show a decrease in taurine in
diluted skates in contrast to the sample from the ventricle.
The reason these specific amino acids, ~-alanine, sarcosine and taurine, are
accumulated and regulated intracellularly as osmolytes is assumed to be because
they are relatively inert from a metabolic point of view (King and Goldstein
1983 a). They are not found in protein, so changes would not disrupt protein bio-
synthesis, nor do they contribute to the major metabolic pathways of the cell.
Therefore their unique metabolic properties may have led to their selective uses in
intracellular osmoregulation.
The mechanisms for modulating the concentrations of these amino acids intracel-
lularly have been studied with in vitro techniques. The accumulation of ~-alanine and
taurine by skate cells appears to be regulated by a Na + -dependent and osmotically
dependent system in the cell membrane (Goldstein and Boyd 1978; Forster and Gold-
stein 1976; ~orster and Hannafin 1980b). Taurine uptake by skate atrium is
directly related to sodium concentration at a constant osmolarity. This taurine trans-
port is strongly inhibited by ~-alanine, suggesting a similar transport system for these
two amino acids that both act as significant intracellular osmolytes (Forster and
Hannafin 1980 b). Both the influx and efflux of ~-alanine in skate erythrocytes are
directly dependent on the Na + concentration (Goldstein and Boyd 1978). Influx,
but not efflux, is almost completely inhibited by omission of sodium from the medium.
In addition, with a constant sodium concentration but reduced osmolarity, efflux is
markedly accelerated, but influx is not affected. Amino acid efflux from cells
during hypo-osmotic stress may be in part a general effect caused by stretching of
the cell membrane resulting in a leaky cell. However, the effects of sodium con-
centration and the decrease in certain amino acids rather than a general overall
reduction suggest that specific transport mechanisms are involved.
The net release of these amino acid osmolytes upon environmental dilution
allows for a decrease in intracellular osmolarity and regulation of cell volume.
However, it also results in an increase in plasma concentration of these amino acids.
The sting ray showed a large increase in plasma levels of taurine, ~-alanine and
sarcosine upon acute transfer to 50 % seawater (King and Goldstein 1983 a). (See
Fig. 9.4). The concentrations of several other amino acids (alanine, valine, leucine,
isoleucine and proline) are also increased in the stingray plasma, but the remainder
of the amino acids show no change. These findings indicate that this response is
specific and not just a general release of solutes from cells.
The plasma concentration of an amino acid is important in the dynamics of
its distribution between the intracellular and extracellular compartments. The
intracellular concentrations of most amino acids are higher than their concen-
trations in extracellular fluid. Therefore there is a favourable diffusion gradient
for efflux of amino acids from cells to ECF. An uncontrolled rise in plasma
level of amino acids during environmental dilution could decrease the gradient
favourable for efflux from the cells and therefore impede intracellular osmoregula-
1.4
1.3
1.2
1.1
1.0
0.9
o
~ 0.8
o
0::: 0.7
!21 100 % seawater
~0.6 o
o 50 % seawater
3.0.5
0.4
0.3
0.2
0.1
OL--U~~~LA~~~AJ~~~~~~~~~~~~~~LL~~LL~~__
TAU SAR LEU MET ARG I lLEU I PHEA I

ALA VAL GLY SER LYS PRO ASG BALA
Fig. 9.4. Sixteen most common amino acids in plasma of the euryhaline sting ray, Dasyatis sayi,
acclimated to 100% seawater or following acute transfer (6 h) to 50% seawater. The values are the
means of data collected from three fish. (King and Goldstein 1983a)
tion. A high extracellular amino acid concentration could also alter the dynamics
of influx into the cells.
It is apparent that some mechanism is needed to remove these amino acids
from the whole body. This could be achieved via metabolic degradation or
excretion. The mechanism for handling ~-alanine and sarcosine appears to be
through metabolism while taurine is relatively inert metabolically and is therefore
handled through excretion (King et al. 1980). No urinary excretion of ~-alanine
or sarcosine is observed upon acute hypo-osmotic stress of the skate, while taurine
excretion is increased significantly. Oxidation of ~-alanine by liver and kidney slices
increased by 68 % and 45 % respectively in skates acclimated to 50 % seawater.
Sarcosine oxidation appears to occur in the skate liver, but a rigorous assessment
of its rate changes is technically difficult because of the lack of radioactively labelled
sarcosine.
A more detailed in vitro examination of increased ~-alanine oxidation in the liver
of the little skate following adaptation to diluted seawater has been done to better
understand the regulatory mechanism. (Leech and Goldstein 1983; Shuttleworth
and Goldstein 1984). This increased oxidation could be achieved, theoretically at
least, by modification at the enzyme level and/or by changes in transport of ~-alanine
into the liver cells. Leech and Goldstein (1983) could detect no significant changes
in the ~-alanine oxidative enzyme activities in skates maintained in 50 % seawater.
In addition, Shuttleworth and Goldstein (1984) found no significant change in
~-alanine uptake rate in isolated skate hepatocytes in diluted skates. The authors
suggest that, because of the kinetics of the processes involved, a sensitive, self-
regulating system may be in effect. The Km for the uptake of ~-alanine' by
hepatocytes (0.17 mmoll- 1 ) is within the normal range of the plasma con-
centration (0.1-0.4 mmoll- 1 ) and the Km of the transminase in the liver
(l.ll mmoll- 1) is very close to the liver amino acid concentration (0.97 mmoll- 1 ).
These kinetics would allow small increases in plasma ~-alanine concentrations in
vivo to produce significant changes in hepatic ~-alanine uptake and in turn changes
in oxidation by the liver (Shuttleworth and Goldstein 1984).
Taurine is not oxidized by any skate tissue examined but it is readily excreted
by the kidneys (King et al. 1980). Both the dogfish and skate have been shown to
increase taurine excretion upon gradual adaptation (dogfish) or acute transfer
(skate) to diluted seawater (Schrock et al. 1982; King et al. 1980). In the dogfish
the increased taurine excretion during environmental dilution results from both
increased glomerular filtration rates of taurine as well as increased rates of tubular
secretion. Experiments with dogfish kidney slices showed that taurine uptake occurs
mainly across the peritubular membrane and is carrier-mediated. This uptake is
is dependent on metabolic energy and completely inhibited by ouabain or low sodium
in the medium (Schrock et al. 1982).
Further characterization of renal taurine transport on the cellular level has been
done on the flounder, Pseudopleuronectes americanus, because the teased tubule
preparation in the flounder is simpler than in the e1asmobranchs, requiring no
enzymatic digestion and giving high U /P ratios. In vitro studies in the flounder
give results similar to the dogfish, indicating a common mechanism for taurine
secretion in fish (King et al. 1980; Schrock et al. 1982). These experiments have
shown taurine transport to occur via a system shared with other ~-amino acids
References 273
and y-aminobutyrate, but independent of both the IX-amino acids and organic acid
transport system (King et al. 1982). The Km for taurine in flounder is well above
the plasma taurine level (Km 1.8 mmoll- 1 , plasma 0.1 mmol 1-1, which strongly
suggests that secretion can be modulated by plasma concentration changes.
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Anderson PM (1981) Purification and properties of the glutamine- and n-acetyl-L-glutamate-
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King PA, Beyenbach KW, Goldstein L (1982) Taurine transport by isolate\! flounder renal tubulus. J
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McBean RL, Neppel MJ, Goldstein L (1966) Glutamate dehydrogenase and ammonia production in
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Price KSJr (19~7) Fluctuations in two osmoregulatory components, urea and sodium chloride, of
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Price KSJr, Creaser Epjr (1967) Fluctuations in two osmoregulatory components, urea and sodium
chloride, of the clearnose skate, Raja eglanteria Bosc 1802-1. Upon laboratory modifications of
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Read LF (1968 a) Ornithine-urea cycle enzymes in early embryos of the dogfish, Squalus acanthias, and
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Read LF (1968b) Urea aI)d trimethylamine oxide levels in elasmobranch embryos. Bioi Bull 135:
537-547
Robertson JD (1975) Osmotic constituents of the blood plasma and parietal muscle of Squalus
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Romer AS (1966) Vertebrate Paleontology, 3rd edn. U of Chicago Press, Chicago
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Schrock H, Forster RP, Goldstein L (1982) Renal handling of taurine in marine fish. Am J Physiol242:
R64--R69
Shankar RA, Anderson PM (1985) Purification and properties of glutamine synthetase from liver of
Squalus acanthias. Arch Biochem Biophys 239: 248-259
Shuttleworth TJ, Goldstein L (1984) ~-alanine transport in the isolated hepatocytes of the elasmo-
branch, Raja erinacea. J Exp Zool 231 : 39-44
Smith HW (1931) The absorption and excretion of water and salts by the elasmobranch fishes I.
Fresh water elasmobranchs. Am J Physiol 98: 279-295
Smith HW (1936) The retention and physiological role of urea in the elasmobranchii. Bioi Rev II:
49-82
Smith HW (1953) From fish to philosopher. The Natural History Library Anchor Books, Doubleday
Garden City, pp 65-66
Thorson TB (1967) Osmoregulation in fresh-water elasmobranchs. In: Gilbert PW, Mathewson RF,
Rail DP (eds) Sharks, skates and rays. Johns Hopkins, Baltimore, pp 265-270
Thorson TB (1970) Freshwater stingrays, Potamotrygon spp.: failure to concentrate urea when exposed
to saline medium. Life Sci 9, Part II: 893-900
Thorson TB (1971) Movement of bull sharks, Carcharhinus leucas between Caribbean Sea and Lake
Nicaragua demonstrated by tagging. Copeia 1971: 336-338
Thorson TB, Watson DE (1975) Reassignment of the African freshwater stingray Potamotrygon
garouaensis to the genus Dasyatis on physiological and morphological grounds. Copeia 1975:
701-712
Thorson TB, Cowan CM, Watson DE (1967) Potamotrygon spp: elasmobranch with low urea content.
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Urist M (1962) Calcium and other ions in blood and skeleton of Nicaraguan fresh-water shark.
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Von Hippel PH, Schleich T (1969) The effects of neutral salts on the structure and conformational
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Chapter 10 I. P. CALLARD and L. KLOSTERMAN (Part A)
GLORIA V. CALLARD (Part B)
Reproductive Physiology
Part A: The Female

The reproductive biology of the chondrichthyan fishes is remarkably sophisticated.
Using both oviparous and viviparous reproductive modes, the group in general
has adapted the strategy of giving birth to relatively few young at one time, each
representing the investment of a great deal of maternal energy. The oviparous
species foreshadow the situation common in oviparous reptiles and universal in birds.
On the other hand, viviparous species range from internal incubators, in which large
yolked eggs are retained, to other species in which the complexity of placentation
and egg yolk reduction approaches the eutherian condition. Further, in certain
viviparous elasmobranchs, the phenomenon of histiotrophic nutrition attains an
importance and complexity not seen in any other vertebrate group. Associated
with the elasmobranch priority on these female reproductive adaptations, internal
fertilization, amniote patterns of reproductive tract development, sex differentiation
and a typically vertebrate type of reproductive endocrinology are seen for the first
time in the vertebrates.
Much has been written of these general aspects of elasmobranch reproductive
biology in recent reviews (see Wourms 1977; Dodd and Sumpter 1984). However,
progress of knowledge in the reproductive endocrinology of both male and female
elasmobranchs has been slow due to the relatively few laboratories contributing
to elasmobranch research. It is the main purpose of this chapter to place newer
endocrine and reproductive information of a physiologic nature in the context of the
well-established patterns of reproduction in elasmobranchs.
10.1 Gonadal Cycles
10.1.1 The Little Skate, Raja erinacea
Sexually mature females undergo cyclic periods of spawning activity during which
eggs are produced in pairs every 5 to 7 days (Richards et al. 1963), and according
Department of Biology, Boston University, Boston, MA 02215, U.S.A. and

Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672, U.S.A.
278 Chapter 10. Reproductive Physiology - Part A: The Female
to Hisaw and Hisaw (1959), corpora lute a form after ovulation. Few follicles
exceed 20 mm, and these are judged to be preovulatory and are found only in
ovaries exceeding 20 g. Measurements of plasma estradiol 17~ showed a good corre-
lation between estradiol levels and increasing follicular size.
A composite graph showing the plasma gonadal steroid hormone levels associated
with the events of ovulation, encapsulation and oviposition is shown in Fig. 10.1
(Koob et al. 1986). As can be seen, estradiol and testosterone are the principal
steroids during the preovulatory phase of the cycle, and progesterone is elevated
approximately 24-28 h prior to ovulation. However, the precise time of ovulation is
not known, and since corpora lutea from previous cycles are said to persist through
several subsequent cycles (Hisaw and Hisaw 1959), we cannot be sure of the source of
the progesterone at this time. The levels of circulating hormones in the skate are
comparable to those reported for other elasmobranchs (see Sumpter and Dodd
1979, for Scyliorhinus canicula; Fletcher et al. 1969, for Raja radiata).
Encapsulation
20
~ Egg
retention
E 16
~ ~ Oviposition
I
......
Ol
C
(/) 12
<-
2
:;::
'7-'-'7 E
1J 8 0----0 T
'0 ",--"p
<-
OJ
U;
4
o 2 4 6 8 10
Days
Fig. 10.1. Average daily titers of estradiol (E). testosterone (T) and progesterone (P) computed
from the values of 7 females producing egg capsules. The day of encapsulation was taken as the
reference day (Raia erinacea) (From Koob et al. 1986)
10.1.2 The Spiny Dogfish, Squalus acanthias
According to Bigelow and Schroeder (1948), spiny dogfish migrate northward from
wintering grounds in deeper waters offshore from New York, Delaware and North
Carolina in early spring, summer in the Gulf of Maine and return southward in the
Fall. In 1947, Hisaw and Albert detailed the stages of gestation and ovarian
development associated with this migration.
These workers demonstrated that the gestation period of this aplacental viviparous
species occupied almost two full migratory cycles and lasts approximately 20-22
months, parturition occurring in late fall-early winter during the southward migration.
Clearcut temporal variations in plasma steroid levels are observed during the 2-year
10.2 Hormones and the Ovary 279
6
b
5
b
~ 3
OJ
c
Steroid PTE PTE PTE PTE

Stage A B C o
Fig. 10.2. Plasma steroid levels during the reproductive cycle (Squalus acanthias). Plasma progesterone,
testosterone and estradiol-17~ levels are shown and expressed as mean ng ml- 1 ± SE. For proge-
sterone and testosterone, different lower case letters indicate significance at the p < 0.01 level. For
estradiol-17~, dissimilar lower case letters indicate significance at the p < 0.001 level
follicular/gestation cycle of Squalus (Tsang and Callard 1987 a; Fig. 10.2). Testosterone
and estradiol levels are low during early pregnancy when follicles are small (Stages
A and B of pregnancy), but increase in the second year of follicular development
(Stages C and D) and reach maximum at full term. Plasma estradiol 17~ is highly
correlated with follicular diameter, as observed by Sumpter and Dodd (1979) for
oviparous Scyliorhinus. The levels of plasma testosterone and estradiol 17~ are lower
than previously reported in rays and skates (Idler and Truscott 1966; Lupo di
Prisco et al. 1967; Fletcher et al. 1969; Koob et al. 1986) and in Scyliorhinus
canicula (Sumpter and Dodd 1979). Unlike testosterone and estradiol 17~, proge-
sterone is highest in early-mid pregnancy before the final involution of corpora lutea
in Stage D, suggesting the corpus luteum as the primary source of progesterone.
10.2 Hormones ~nd the Ovary
10.2.1 In Vitro Ovarian Steroidogenesis
10.2.1.1 Follicular Wall
Recently (Tsang and Callard 1987b), we have expanded earlier observations on in

vitro steroidogenesis by follicular and luteal components of the ovary of Squalus
acanthias (see I. P. Callard and Leathem 1965; Lance and Callard 1969). It is well
known that ovarian steroidogenesis is a co-operative function of granulosa and theca
cells (see Falck 1959; Short 1962; Dorrington et al. 1975). Avian (Huang et al.
1979) and teleost (Kagawa et al. 1982) studies indicate a similar tissue interaction.
In Squalus, enzymatically isolated granulosa cells in vitro synthesize increasing
amounts of testosterone and estradiol 17~ as follicles enlarge during gestation.
Estradiol 17~ is the principal product, and progesterone synthesis was below the
levels of detection. Granulosa cell estradiol synthesis is highly substrate-dependent,
as indicated by the effect of testosterone added as substrate (see Fig. 10.3). Thecal
12
~
Qj
u
0
8 ~
0
0
0
Lf)
N
--..
4
W
01
C
0 n
c -9 -8 -7 -6 -5
Test (M)
Fig. 10.3. Effect of testosterone substrate on granulosa cell estradiol-17~ production (Squalus
acanthias). Stage C granulosa cells were incubated in the presence or absence of graded doses of
testosterone. The content of estradiol-17~ in medium was determined by radioimmunoassay. Values
are expressed as ng per 250,000 cells ± SE
tissue alone, in the presence of testosterone substrate was also able to synthesize
estradiol 17~. Co-incubation of granulosa and theca minces indicates that, although
theca is steroidogenic, the granulosa alone is able to synthesize all three steroids,
and the amounts produced are not increased by co-incubation of theca with granulosa.
This is the first observation that isolated vertebrate ovarian granulosa cells have
the capacity to synthesize significant quantities of all three steroids without the
co-operation of thecal elements, a conclusion supported by studies using radiolabeled
precursors. Nevertheless, a theca-granulosa cell interaction is suggested by the co-
incubation studies (Fig. lOA) where progesterone and estradiol synthesis is less in
the presence of theca, possibly due to activity of metabolic pathways not assayed in
these experiments. Comparison of this picture of follicular steroidogenesis in Squalus
with that of other vertebrates suggests that Squalus is most like the domestic hen in
that thecal elements can synthesize androgens and estrogens from granulosa-derived
progesterone but differs in that, in the bird, theca is the primary source of estradiol
17~ and the avian granulosa does not synthesize testosterone or estradiol 17~
(Huang et al. 1979). Although more studies are necessary in elasmobranchs, as well
as in amphibia and reptiles, these studies suggest that the elasmobranch situation
may be closer to the ancestral vertebrate condition. It is possible that the differences
in steroidogenic capacity exhibited by theca and granulosa elements in different
vertebrates are related to substrate supply, which is in turn associated with processing
of vitellogenin and low-density lipoproteins by the follicle wall and oocyte.
p T E
Gran
20
3 15
2 10
o
,nd .~ C o
Theca
"
~3
20
15
0
cry
"CJ) 2 10
c
5
' nd , nd ,
0
Gran &
"
Theca 20
3 15
' nd
0
C Pit C Pit C Pit
Fig. 10.4. Effect of homologous pituitary extract on co-incubates of granulosa and theca (Squalus
acanthias). Granulosa alone, theca alone or both granulosa and theca were incubated in the presence
of homologous pituitary extract for 4 h. Afterwards, the progesterone, testosterone and estradiol-17~
content was determined by radioimmunoassay and epressed as ng ± SE per 30 mg. Note different
axis for estradiol-17~. ND = not detectable
10.2.1.2 Corpus Luteum
Although progesterone was first definitively identified as a product of Squalus

ovarian tissue in 1965 (I. P. Cal1ard and Leathem 1965), the contribution of the
corpus luteum has only recently been defined. We have provided definitive proof of
the identity of progesterone as a product of the shark corpus luteum (Tsang and
Cal1ard 1987b). In addition, using radio-immunoassay, we have shown that basal
progesterone synthesis by Squalus luteal minces increases from Stage A of gestation
to a maximum in Stage B, declining thereafter (Fig. 10.5). In contrast, luteal weight
is greatest in Stage A and declines steadily until ful1 term.
250 2500
200
o CL wt.
2000 ::c
~P
~
_ 150 1500 ....
(j)
01
E ::
01
-l E
U 100 1000 ~
0.
0..
50 500
dID
CS D ~
0 0 0 0
4.,9mm 17-20 mm 30-34 mm 36- 50mm
Stage A Stage B Stage C Stage 0
Year I ~~~-t--~~ Year n
Fig. 10.5. Luteal weight and basal progesterone synthesis during the cycle (Squalus acanthias).
Corpora lutea were removed from animals at each stage of pregnancy. Mean individual luteal
weights (mg ± SE, open bar) are shown. Tissues from similar stages were scissor minced and
incubated for 4 h. The progesterone content in medium was determined by radioimmunoassay and
expressed as pg ± SE mg- 1 corpus luteum per 4 h (hatched bar). Cartoon (bottom) is adapted
from Hisaw and Albert (1947) and indicates correlated stages of follicular size in mm and
embryonic fetal development during pregnancy
10.2.2 Regulation of Steroid Secretion and Synthesis in Vivo and in Vitro
It is generally agreed that the ventral lobe of the elasmobranch pituitary is the
principal source of gonadotropins (Dodd and Sumpter 1984; Holmes and Ball 1974).
Injections of aqueous ventral lobe extracts stimulated an increase in plasma
androgen levels in hypophysectomized male dogfish (Scy£iorhinus; Sumpter et al.
1978). In Squalus Stage C animals, significant changes in plasma gonadal steroids
have been demonstrated after intravenous injection of aqueous extracts equivalent
~
Fig. 10.6. Effect of in vivo injections of homologous pituitary extract of plasma steroids (Squalus
acanthias). Stage A and stage C animals (4 specimens each) received a single injection of
homologous pituitary ventral lobe extract (from 3 lobes), and blood was sampled over a 24-h period.
Plasma progesterone, testosterone and estradiol-17~ were determined and levels (ng/ml ± SE) at
0, I, 5, 10, and 24 h post-injection are shown
Estradiol-17 ~
3000 Stage A Stage C
2000
1000
1
500
Testosterone
Stage A Stage C *
1000
E
"-
OJ
Q.
500
Progesterone
Stage A Stage C
I
o Ventral lobe extract
*
6000 E5l Control
~
OJ
4000
Q.
2000
0
0 5
~10 24
~~
0 5 10
J 24
Hours Hours
to three homologous ventral lobes (Fig. 1O.6a, b, c). In contrast, in Stage A

animals, only plasma progesterone was stimulated by the same treatment. The latter
response may represent an in vivo response of the corpora lutea, although this was not
seen in vitro (see below). The response of Stage C animals could be due to both the
corpora lutea (progesterone secretion) and gonadotropin-sensitive large ovarian
follicles (testosterone and estradiol secretion).
In vitro studies using follicular and luteal components have confirmed a ventral
lobe gonadotropic role in steroidogenesis. Thus, both theca and granulosa responded
to pituitary extracts with an increase in steroid synthesis (Fig. lOA) although
granulosa cells were most sensitive to gonadotropin and most active in steroid
synthesis. Stage C but not Stage A luteal minces responded to homologous ventral
lobe (but not neurointermediate lobe extract) with a marked increase in progesterone
synthesis (Fig.
.
10.7). Similarly, enzymatically dispersed
. luteal cells are stimulated
3.0
2.5
.c
~ 2.0
0
....
. -.J'
'i 1.5
Qj
~
01
....01E 1.0
C
a.. 0.5
0
4 8 VL VL NIL NIL VL VL
4 8 4 8 + +
NIL NIL
4 8
Fig. 10.7. Effect of ventral lobe and neurointermediate lobe extract on lut~al progesterone
production (Squalus acanthias). Minced stage C luteal tissue~ were incubated in the presence or
absence of ventral lobe, neurointermediate lobe or combination of ventral lobe and neurointer-
mediate lobe for 4 h or 8 h. The content of progesterone in medium was determined by radio-
immunoassay and expressed as ng ± SE mg- 1 corpus luteum per 4 or 8 h
by ventral lobe extract in Stage C but not Stage A (Fig. 10.8, Klosterman and
Callard 1986). The availability of cholesterol or a substrate for progesterone synthesis
is critical in both stages, however, as demonstrated in vitro by a marked enhancement
of hormone production by cells incubated in the presence of 25-0H-cholesterol
(Fig. 10.8).
+ 2lJ.g 25 - OH -chal.
6 I
5 o -pit.
~ +pit.
E
"-
01
C
QI
" S = + serum
c
0
L.. 3
2
If)
QI
01
0
ct 1.0
Fig. 10.8. Progesterone production (X ± S.E.
0.5 of triplicate tubes) by stage C luteal cells in
the presence or absence of 25-0H-cholesterol,
pituitary extract, and homologous serum. In-
0 cubation time, 18 h
S S S s
10.2.3 Ovarian Peptides
10.2.3.1 Relaxin
In 1959, Steinetz et al. reported "relaxin-like" activity in the ovaries of a viviparous
sand-tiger shark (Odontaspis taurus) based on the guinea pig pubic symphysis relaxa-
tion bioassay. The active molecule was recently identified as a true relaxin on the
basis of its amino acid composition (Reinig et al. 1981). Peptide analyses demon-
strated that sand-tiger shark (Carcharius taurus) relaxin is more homologous with
porcine insulin than with porcine relaxin. Further, the location of the disulphide
bridges is identical in all three molecules. Recently, we (Bullesbach et al. 1986) have
isolated a similar molecule from the ovaries of pregnant spiny dogfish, Squalus
acanthias. This relaxin has 75 % homology with sand-tiger shark relaxin, and 45 %
homology with mammalian relaxins. As with sand-tiger shark relaxin, Squalus
relaxin has a small (3 %) but unequivocal effect in the mouse relaxin bioassay.
Most recently, Bullesbach et al. (1987) have reported the structure of relaxin
from an oviparous species (skate, Raja erinacea) for the first time. Although the
number of amino 'acids in the A-chain (24) is the same as in Odontaspis and
Squalus, the skate peptide is unique among vertebrates so far examined in having
an extended B chain with 40 amino acids, compared to 23 for other species.
Relaxin structures of Odontaspis and Squalus show 75 % homology, but Raja is only
42 and 48 % homologous, respectively, with each of these. This is only slightly more
than the homology with human (31, 35 ~~) and pig (31 %). Thus, based on a limited
number of species, it appears that there has been almost as much amino acid substi-
tution and sequence change within the elasmobranchs as between the elasmobranchs
and the mammals. Nevertheless, the position of the disulphide bridges appears to be
invariant, suggesting that the three-dimensional structure of the whole molecule has
been strictly conserved whilst allowing considerable flexibility of primary structure.

As indicated below, both mammalian and elasmobranch relaxins and mammalian
insulins have significant relaxin bioactivity in elasmobranch systems, while elasmo-
branch relaxins have only minor effects in mammals. This suggests that the elasmo-
branch receptor has been highly conserved and that the three-dimensional molecular
structure of relaxin is more important than the primary structure in hormone action.
10.3 Gametogenesis and Vitellogenesis
Elasmo branch fishes produce small quantities of large eggs, ranging in size from 1 mm
in Scoliodon to 100 mm in Ginglymostoma and Chlamydoselachus (Gudger 1940;
Wourms 1977). Egg size is a reflection of reproductive strategy (oviparous or
viviparous) with small eggs being correlated with an enhanced maternal nutritive
role during gestation (see below for discussion of viviparity). In contrast, large
eggs are the rule when the embryo is exclusively dependent on the yolk reserve or
maternal histiotroph for development.
The appearance of the ovaries of different elasmobranchs varies according to the
stage of the reproductive cycle. In the dogfish, Scyliorhinus canicula (Metten 1939;
Dodd 1977), a variable number of vitellogenic oocytes of different sizes and
previtellogenic oocytes are present in winter and early spring. During the summer
(May-September) large yolky follicles which were not ovulated become atretic and
the next set of growing oocytes can be identified. Unequal growth of these follicles
results in a hierarchy of pairs of follicles, about 30 in all, ranging in weight from
0.05-28 g. In an oviparous skate (Raja erinacea) , reproductively active animals
undergo cyclic periods of spawning activity during which eggs are produced in
pairs every 5-7 days (Richards et al. 1963). Ovarian weight in mature females varies
from 1-30 g, the smallest ovaries containing previtellogenic follicles less than 1.0 mm
in diameter. With the acquisition of yolk, ovarian weight and follicular diameter
increase. Usually not more than two follicles reach a diameter greater than 20 mm in
a pair of ovaries and decreasing numbers of follicles are found in each size class up to
9.0 mm, from which stage two per class was maintained. Follicles greater than 20 mm
were judged to be preovulatory and were found only in ovaries heavier than 20 g
(Koob et al. 1986). In the viviparous aplacental species Squalus acanthias, ovaries
grow during the 22-month gestation period, increasing from a gonadosomatic index
of 0.89 in Stage A to 6.83 in Stage D near term, preovulatory animals (Tsang and
Callard 1987 a). At each stage of gestation, follicles can be divided into five stages
according to diameter: I 1-5 mm; II 6-10 mm; III 11-20 mm; IV 21-30 mm; V
31-40 mm. The larger-size follicles (IV and V) are found only in later pregnancy,
while a pool of size I follicles is present in early and mid-pregnant animals (Tsang
and Callard 1987a). The data suggest that by the fall of the first year of gestation
(6-9 months p.ost-ovulatory) the set of follicles which will ovulate next has been
selected and appears as Class III. By the following spring, in Stage III animals, these
follicles are growing and distributed between classes IV and V; they continue to grow
through the fall when a number of follicles approximately equivalent to the prior
fall Class III and the combined spring classes IV and V appear as class V. These
10.4 The Reproductive Tract 287
follicles will ovulate after parturition and a new set of follicles destined to ovulate
next will be identified by the next fall. Since most (60~70 %) follicular growth
occurs in the spring and fall of the second year, it would seem that the process of
vitellogenesis is most active at this time. This correlates well with observations that
the large follicles most actively synthesize estrogen (Tsang and Callard 1983) and that
plasma estradiol-17~ increases to a maximum during this time of the cycle (Tsang
and Callard 1987 a). Although atresia was not determined for the small follicular
size classes (I and II), atresia of large follicles was rarely observed in this study.
This is in contrast to the oviparous dogfish, S. acanthias (Dodd 1972), in which
atresia is common. Although follicular dynamics have not been described in other
elasmobranchs, general observations on the heterogeneity of the follicular population
in a number of species have been made (e.g. basking shark, Matthews 1950;
DuBuit 1976; skate, Koob et al. 1986).
Although D. M. J. Woodhead (1969) first demonstrated an increase of plasma
calcium after estrogen treatment in S. canicula, only one detailed study of vitellogenesis
in elasmobranchs has been made (Craik 1978a, b, c, d) on S. canicula. This study
showed that a yolk-granule-like phosphoprotein is present in the blood of vitello genic
female dogfish. The amount is relatively low, possibly correlated with the extended
egg-laying period, and the annual cycle of plasma levels of phosphoprotein is less
marked than in vertebrates with a more defined reproductive period (see Ho et al.
1982). Levels between September and March (peak egg-laying season) are significantly
higher than between March and August (see Craik 1978c; Dodd et al. 1983).
Intramuscular injections of estradiol-17~ (3.0 mg/kg) caused a 17-fold increase in
plasma phosphoproteins, indicating inducibility of the vitellogenin gene in this
species as in other vertebrate groups.
In Squalus acanthias, vitellogenin-like proteins could not be identified using
SDS-gel electrophoresis in male or early-pregnancy females. However, in late-preg-
nancy females with large follicles, low levels of vitellogenin were detectable. Whereas
estradiol-17~ (4x 1.0mg) failed to induce a vitellogenin band in adult males and
Stage A females, in Stage C females the vitellogenin band increased progressively
up to 10 days post-estradiol injection, and progesterone (4 x 2.0 mg) reduced the
effect of estrogen. This suggests that the high progesterone levels in early gestation
may slow the process of vitellogenesis (see Callard et al. 1980).
lOA The Reproductive Tract
1004.1 Gross Morphological and Histological Changes
Elasmobranch oviducts are of pronephric (Mullerian) duct derivation. Eggs are shed
by the ovary into the body cavity, and are carried to the opening of the oviduct
by ciliary action. The oviduct can be subdivided into several specialized regions.
The most anterior region is the oviducal shell or nidamental gland, which is best
developed in oviparous species. Metten (1939) has described this structure for
Scyliorhinus canicula, in which three regions are described: (1) an anterior albumen-
secreting zone; (2) a short mucus-secreting zone; (3) a large posterior region which
secretes the shell. This structure also serves as a receptacle in which sperm
survival of up to 5-6 weeks has been reported in the skate, Raja brachydura by
Clark'(1922). Dodd et al. (1983) reported laying of fertile eggs over 2 years after
the last possible insemination in S. canicula. The fine structure of the gland and
secretion of shell components has been described by Rusaouen (1978) and Rusaouen
et al. (1976). The shell gland in viviparous species is smaller, and in Squalus, for
example, the shell is reduced to a membrane in which several eggs may be packaged
to form the "candle" seen in Stage A pregnancies. Teshima et al. (1971) have
shown in viviparous Japanese smooth dogfish that the shell membrane persists through
development and may form part of the placenta. An excellent summary of work on
the egg case and egg case formation in the oviducal gland is provided by W ourms
(1977).
The oviducal gland of Scyliorhinus canicula is a compound tubular structure (see
Metten 1939), and the studies of Rusaouen et al. (1976) suggest that two cell classes
are present in alternative positions. One cell type secretes a collagen-like material, the
principal component of the egg case, and the other secretes a protein rich in
tyrosine and sulphydryl groups that is possibly important in cross-linking and tanning
of collagen. Although no studies of hormone regulation of the oviducal gland and its
secretions have come to our attention, the correlation between oviducal gland size,
ovarian follicular size and plasma estradiol in the little skate (see below) suggest
that this would be an excellent model for hormonal regulation of protein synthesis
and secretion (see I. P. Callard et al. 1978).
The shell gland is connected to the lower "uterine" region of the oviduct by
a narrow region, which, in Squalus, terminates in a "closing mechanism" (VerschluB-
vorrichtung; Widakowich 1907), which prevents refluxing of uterine contents up the
oviduct. In viviparous species, the lower, uterine region of the oviduct accommodates
developing young, and the main lining becomes highly vascular and may develop a
complex folding pattern with glandular epithelia and long villi called trophonemata
producing embryotrophe upon which embryos feed (see section below). In oviparous
species, such as the little skate, this region is highly muscular and serves to accommo-
date the egg cases briefly as they pass through the tract. Distal to the uterine
region, in viviparous Squalus a narrow "cervical" zone restricts passage of embryos
to the exterior until full term.
10.4.2 Hormonal Regulation of the Reproductive Tract
As in all other" vertebrates, the reproductive tract is an ovarian-dependent structure,

developing at sexual maturity. Once fully developed, cyclic changes in the size of
the regions of the oviduct occur which can be correlated with periods of ovarian
activity/inactivity and ovarian follicular development and/or pregnancy. Estradiol
is implicated in the development and maintenance of elasmobranch reproductive
tracts, as shown by the effects of injections of the hormone (Dodd and Goddard
1961) and the correlation between plasma estradiol levels, ovarian follicular and shell
gland size (see Koob et al. 1986).
As described above, we (Koob et al. 1986) have demonstrated that plasma pro-
gesterone titers peak 48 h prior to ovulation and encapsulation by the oocyte in the
little skate, Raja erinacea. Progesterone titers then dropped rapidly, and following
capsule formation eggs were retained in utero for several days before oviposition.
By this time, progesterone levels had declined to base line. These data suggested
that progesterone may be involved in the events of ovulation, capsule formation
and oviposition in Raja erinacea. For this reason, we injected progesterone (2.0 mg),
estradiol (250 /lg) and a combination of the two daily into fish carrying egg cases
until oviposition was complete. The data demonstrated that eight to nine proge-
sterone-treated animals retained eggs for less than 24 h versus 36 h to 8 days in
controls and 3 days in estrogen-treated animals. Similarly, progesterone reduced the
duration of oviposition (12 h vs. 18 h for estradiol-treated animals and 24 h for
controls). Thus, progesterone treatment advanced oviposition and reduced the time
spent in the reproductive tract from 3-4 days to less than 1 day. The site of
action of progesterone (peripheral or central) is not known at this time (Koob and
Callard 1985).
Estradiol is also necessary for the expression of relaxin bioactivity in Squalus
acanthias, first demonstrated by Koob et a1. (1984). As in mammals, in which
the cervix and pubic symphysis are restrictive to the passage of fetuses, in elasmo-
branchs at least two regions of the reproductive tract must enlarge or adapt
significantly to allow egg or fetus passage (see I. P. Callard and Koob 1982).
These are a specialized region of the lower oviduct, the VerschluBvorrichtung
(closing mechanism) (Widakowich 1907) and a region between the "uterus" and
common urogenital sinus referred to as the "cervix" (although homology is not
implied, these structures may be considered functionally analogous). The effects
of porcine relaxin and its disulphide homolog, insulin, on the "cervix" were studied
in the presence and absence of estradiol. Cervical cross-sectional area was increased
by both peptides, an effect which was magnified by prior exposure to estrogen. Of
interest is the fact that bovine insulin has a similar effect to that of porcine relaxin, and
the effect of insulin is unrelated to its effect on blood sugar level (Koob et a1. 1984). In
contrast to the cervix, the treatment with insulin or relaxin did not change the diameter
of the VerschluBvorrichtung; however, estradiol alone significantly enlarged both this
region and the cervix. It is also clear that the effect of relaxin and insulin on
cervical cross-sectional area is estrogen-dependent. In contrast to Stage A animals,
treatment of Stage C females with estradiol, relaxin and insulin resulted in premature
loss of developing fetuses within 24 h of peptide injection (Koob et a1. 1984).
It is interesting to note that the pattern of changes in response to relaxin after estrogen-
priming in Squalus is very similar to that of several mammalian species in which
estradiol is required for a full relaxin response (Steinetz et a1. 1982). The overall
result of increased cervical size is premature fetal loss which taken together with
the identification of relaxin in Squalus ovarian tissue (see above), suggests that the
hormone has an important function associated with parturition in this species. This
conclusion is supported by the absence of an effect of relaxin on the VerschluB-
vorrichtung which would ensure efficiency of delivery at a time when the cervix is
dilated.
In addition to the effect described above, which we believe to be connective
tissue related, we have recently demonstrated that homologous relaxin influences
myometrial activity, in vitro and in vivo, in Squalus (Sorbera et a1. 1986). Addition
of Squalus relaxin in increasing amounts (10, 100 and 1000 ng ml- I ) resulted in a
dose-related decrease in contractile rate without significantly influencing amplitude

or duration of contractions (Fig. 10.9a). A similar decrease in spontaneous rhythmic
oviduct activity in vivo was observed after intravenous administration of both Squalus
and porcine relaxin (Fig. 10.9b). In contrast to the effect of relaxin, neurointer-
mediate lobe extracts (0.5 equivalents) administered intravenously provoked a marked
contractile response of the oviduct (Fig. 10.9c). Despite these biological effects of
Con Control
10 ng
0.1I-Lg/ml~
100 ng ~--------'~
Con
I I
a 1 min b 1 min
I I
c 1min
Fig. 10.9. a Response of'Stage C Squalus uterine strip (in vitro) to increasing amounts of homo-
logous relaxin. Bottom trace shows return to normal after removing relaxin solution; b response of
Stage C Squalus uterine wall to in vivo SQ-RLX. Contractions were monitored using an intrauterine
catheter connected to a Grass physiograph; c response of stage C Squalus uterine wall to in vivo
homologous neurointermediate lobe (NIL) extracts
relaxin, we have been unable to demonstrate binding of iodinated porcine relaxin

to "uterine" membrane preparations made from Squalus. However, iodinated porcine
insulin binds specifically and increases in the presence of unlabelled porcine relaxin
(Mottola, Czech and Callard, unpubl.). These effects demonstrate that the purified,
naturally occurring, homologous ovarian peptide relaxin and native extracts of pitui-
tary neurointermediate lobe have effects on the reproductive tract which are of
potential importance to the progress of gestation and parturition in this species.
The studies suggest that the actions of relaxin and neurointermediate lobe peptides
on the vertebrate reproductive tract have been conserved since the earliest era of
gnathostome evolution.
10.4.3 Viviparity
Chondrichthyan viviparity is common (present in 90/145 genera and 420/600 species)

and has been reviewed by Budker (1958), Amoroso (1960), Breder and Rosen (1966),
Hoar (1969), Hogarth (1976), Wourms (1977, 1981) and Dodd (1983). Budker
(1958) proposed that viviparous species can be divided into those that develop
placentae and those that do not. There is· a well-defined progression from oviparity
to viviparity associated with a reduction in oocyte yolk reserves and the complexity
and efficiency of either placental or pseudoplacental structures for nutrient supply
(see I. P. Callard and Ho 1987).
Aplacental viviparity takes several forms. (1) Large-yolked eggs may be retained
in utero without yolk reduction or placentation (e.g. Squalus acanthias). (2) In other
species, placental analogs develop and provicie almost all of the nourishment (histio-
trophe) by secretion from the maternal uterine epithelium. The extreme development
of this mode is found in the trophonemata of certain rays and torpedoes, and
trophonemata may extend through the gill slits of the developing young into the
upper position of the gastrointestinal tract from which nutrients are directly absorbed
(e.g., Dasyatis violacea (3) Oophagy was first observed in porbeagle sharks (Loh-
berger 1910). In these species, ovulation of 1.0 cm oocytes continues throughout
gestation, itself an unusual condition during gestation in which ovulation is normally
suppressed in vertebrates. Developing young ingest the eggs as they descend the
oviduct. This strategy may extend to intrauterine "hunting" and sibling cannibalism
(see Springer 1948). In the sand-tiger shark there is a progressive decline in the
number of intrauterine embryos as gestation progresses.
Placental viviparity in elasmobranchs involves the yolk-sac (chorio-vitelline)
placenta. This structure functions to absorb yolk supplied during oocyte growth,
but it also forms a significant fetal/maternal interface through which nutrients are
absorbed as egg-yolk supplies are reduced (see, for example, the smooth dogfish,
Mustelus canis, Ten Cate-Hoedemaker 1933). In structure, at least, the yolk-sac
placenta resembles the epithelio-chorial placenta of eutherian mammals. This structure
persists in viviparous sharks, and in certain requiem (Carcharinidae) and hammerhead
(Sphrynidae) sharks it differentiates morphologically and functionally to form a
placental organ for ·intrauterine embryos (see Hamlett and Wourms 1984; for review
of the ontogeny of nutrient absorptive structures).
Although numerous papers have reported on the structure of the elasmobranch
yolk sac placenta, and functions of the placenta are inferred from morphological
correlates of nutrition (absorption, secretion and transport) and respiration (see
Hamlett et al. 1985a, b, c, 1987 for review), few physiological data are available.
The most important physiological studies of comparative elasmobranch reproduction
and development are still those of Ranzi (1932, 1934), which have been extensively
summarized by Needham (1950). Although the importance and efficiency of various
modes of fetal nutrition can readily be seen, we know nothing about the endOl~rine
292 Chapter 10. Reproductive Physiology - Part B: The Male
regulation of fetal-placental association and function or the secretion of histiotrophe

by placental analogs. However, since various components of the repr"oductive tract
are under endocrine control, it is logical to suggest that the nutritive funct.ions of these
structures are also endocrine-related.
Part B: The Male

Male elasmobranch fishes offer an exceptional opportunity for studies of the
physiology and biochemistry of reproduction, due primarily to an advantageous
organization of the testis in which the different germ cell stages are topographically
separated. Nonetheless, only a few of the 600 species of squaliformes and rajiformes
have been investigated, and only rarely have modern methods of experimentation
and observation been applied. A detailed historical perspective and comprehensive
bibiliography of reproduction in male elasmobranchs may be found in articles by
Wourms (1977), Dodd (1983), and Dodd and Sumpter (1984). Also, the elasmobranch
testis is compared with those of other vertebrates in reviews by Roosen Runge (1977)
and Pilsworth and Setchell (1981). The intent of the present chapter is to summarize
and update available information with a view to identifying opportunities for future
study.
10.5 Gross Anatomy of the Adult Male Reproductive Tract
The reproductive tract of male elasmobranchs resembles that of amphibians and

amniotes in its basic organization and embryologic origins and is typified by that
in the spiny dogfish Squalus acanthias (Gilbert 1973; Fig. 10.10). Paired testes are
located anteriorly in the body cavity and embedded in the epigonal organs which
have no known reproductive function but are comprised of lymphomyeloid tissue
(Fange and Pulsford 1983; Mattisson and Fange 1982). In adult sharks, the testes
represent from 1-5 %of the body weight. Spermatozoa exit the testis to enter the male
accessory ducts, all derived embryologically from the mesonephric (Wolffian) duct.
These include the efferent ducts, epididymis and ductus deferens. In skates there
is a single efferent duct, whereas in Squalus there are four, but these are too small to
be seen in gr'oss dissection. The paired epididymides serve as storage organs for
spermatozoa and also receive a secretion from accessory glands on their dorsal
surface. In blue sharks, this forms a matrix of supporting tissue in which the sperm
are embedded (Pratt 1979). Leydig's gland, not to be confused with testicular
Leydig cells, is the modified anterior section of the mesonephric kidney, and lies
dorsal to the d. deferens. In immature males urine is formed in this part of the
kidney and conveyed to the d. deferens via numerous collecting tubules, but in mature
males the tubules of Leydig's gland transmit a milky secretion to the seminal
fluid. At this level, the function of the d. deferens changes from sperm storage to
spermatophore formation, in species in which this occurs. Posteriorly, the d. deferens
10.5 Gross Anatomy of the Adult Male Reproductive Tract 293 .
1 abdominal pore
2 accessory urinary duct
3 anterior mesenteric artery
4 clasper
5 cloaca
6 colon
7 dorsal aorta
8 ductus deferens
9 epid idym is
10 kidney (opisthonephros)
11 left posterior cardinal vein
12 left testis
13 leydig's gland
14 lienogast ric artery
15 mesorchium
16 opening of siphon into
clasper tube
17 pelvic fin
18 posterior mesenteric artery
19 puboischiac bar
20 right posterior cardinal
vein
21 right testis
22 semi nal vesicle
23 siphon
24 sperm sac
25 urogenital papilla
Fig. 10.10. Diagram showing gross anatomy

of the male reproductive tract in Squalus
acanthias. For full description, see' text.
(Gilbert 1973)
is enlarged to form the ampulla d. deferens (seminal vesicles) which receives

no tubules from Leydig's gland but appears to be mainly a repository for sperm.
The ductus is closed by a sphincter muscle at its point of entry into the sperm sac,
small paired receptacles which are homologues of the posterior end of the oviduct,
but these are not well-developed in all species (viz. the blue shark, Pratt 1979). The
urogenital sinus is the part of the sperm sac posterior to the openings of the ampulla
and it vents into the common cloaca by means of a single large papilla that projects
from the dorsal body wall.
Matthews (1950) describes the epithelial lining of the male reproductive tract as
being organized into folds and glands and including several different secretory cell
types as well as ciliated cells. Little is known of the nature of these secretions. In
1927, Marshall discovered that the sperm sac or "urinary bladder", which is parti-
cularly well-developed in males of various species of the genus Raja, contained neither
sperm nor urine but a unique fluid of high electrolyte content and alkalinity (Smith
1929). Subsequent investigators have characterized the epithelium and secretory
functions of this "alkaline gland" (Maren et al. 1963; Smith 1985). A highly folded,
simple columnar cell lining overlies a dense connective tissue layer and is capable of
maintaining about a 100-fold concentration gradient of OH- and a 50-fold gradient
of CO2 from plasma to gland lumen. The conductance properties of this epithelium
rese!llble other CI- -secreting tissues in its transport of this ion, but the exact mechanism
by which a pH of 9.0 is maintained is not completely understood. It has been
suggested that the alkaline secretion neutralizes the fixed acid urine in these species
and thus protects the sperm (Smith 1929); however, Raja has been reported to
have an acid semen (Wourms 1977). Alternatively, the secretions of the alkaline gland
may somehow be related to the copulatory plugs found in female skates.
The copulatory organs of adult male elasmobranchs are paired claspers, a cylin-
drical modification of the inner lobe of the pelvic fin. Sperm are transferred to the
vagina of the female during copUlation by a propUlsive force generated by a water
piston which, in turn, is driven by the muscular subdermal siphon sac associated
with each clasoer. The clasper of the blue shark is a heavily calcified scroll-shaped
appendange (Pratt 1979) similar in form to that of the basking shark (Matthews
1950) and tope (Galeorhinus galeus) (Leigh-Sharpe 1920) but lacks a distinct spur or
clasper hook. During copulation the sharpened edges of the terminal parts are
splayed open to secure the clasper in the vagina.
10.6 Organization of the Testis
The seminiferous elements of the elasmobranch testis are organized into spherical
spermatocysts (lobules, follicles, ampullae) situated at the terminae of a highly bran-
ched collecting duct system. These are embedded directly in a connective tissue
. matrix and therefore not strictly homologous to mammalian tubules. The elasmo-
branch cyst is composed of an isogenetic clone of synchronously developing germ
cells and their associated Sertoli cells (nurse, sustentacular or follicle cell). All cells
within a single cyst are in virtually the same stage of maturation. The number of
Sertoli cells and germ cells per cyst is strictly dependent on the stage of sperma-
10.7 Germ Cells and Spermatogenesis 295
togenesis and, for anyone species, is predictable. In elasmobranchs the production

of new cysts is not restricted to embryonic or neonatal stages. Rather, new cysts
are formed continuously in adults, originating from fixed germinal sites on the lateral
or dorsolateral aspect of the testis (Fig. 10.11). These move steadily away from the
germinal sites as they grow and mature, followed closely by successively younger
elements. As the cysts approach the ventral and mesoventral aspects of the testis,
located just beneath the epigonal organ, they contain the most advanced germ cell
stages. When spermatogenesis is completed, an opening is affected into the attached
terminal branch of the collecting ducts and the spermatozoa exit the testis via the
efferent ducts. Growth of the collecting duct system is thought to be the mechanism
by which the developing cysts migrate gradually away from the germinal sites. More-
over, each "column" of migrating cysts progresses at the same rate away from the
germinal zone. Therefore, in cross-section, cysts in the same stage of differentiation
are recognizable as roughly concentric "rows" around a germinal hilus. The end result
of this process is the spatial segregation of the different stages of spermatogenesis.
When sectioned transversely, the testis appears to be filled with spherical cysts and,
even without the aid of a dissecting microscope, several different zones of maturation
are readily discernable~ This orderly arrangement of the different stages of spermato-
genesis has been used to advantage to study spermatogenesis per se and stage-
dependent biochemical changes (see below).
This organization of the elasmobranch testis at first seems radically different
from that of mammals, but in fact these differences are superficial. Although there
are no discrete spermatogenic cysts in the amniote testis, isogenetic germ cell clones
are synchronized developmentally, retain intercellular bridges through successive
cell divisions and share an intimate relationship with the same Sertoli cell set
(Fawcett 1975). Additionally, although mammalian germ cells themselves do not
actually migrate through the testis, as occurs in elasmobranchs, a maturational
wave radiates along the length of each tubule away from the rete testis, resulting
in a pattern whereby germ cells a few millimeters more distal from the rete are
somewhat less advanced than those located more proximally (Fig. 10.11). Pilsworth
and Setchell (1981) have likened the mammalian spermatogenic wave to a string
of open-ended contiguous cysts, a hypothetical model which closely resembles the
actual condition in the elasmobranch testis.
10.7 Germ Cells and Spermatogenesis
Based on light microscope observations, good accounts of cyst development

during spermatogenesis have been given for the spotted dogfish (J. P. Mellinger
1966; Stanley 1966); the spiny dogfish (Holstein 1969); the skate (J. P. Mellinger
1966); the ray (Stanley 1966); the smooth dogfish (Teshima et aL 1971) and the basking
shark (Matthews 1950). In addition, there are scattered electron microscope obser-
vations of germ cells in different stages of differentiation and particular attention
has been given to events occurring during spermiogenesis and to mature spermatozoa
(Boisson et al. 1968; Stanley 1971 a, b; Dobson and Dodd 1977 b; Gusse and Chevail-
lier 1978; Collenot and Damas 1980; Pudney and Callard 1984a). As first noted in
efferent
stem cell zone
~"(.
~
................... .
-<: .......
Sertoli cells
germ cells
efferent duct
Fig. 10.11. a Diagram showing spermatocysts in successive stages of spermatogenesis and organization
of the testis in Squa/us acanthias. For full description, see text. (Hoar 1969)
a comparative study by Moore (1895), cytological changes during spermatogenesis in

elasmobranchs are basically similar to those in mammals. The account which follows
essentially summarizes these reports.
Beneath the coelomic epithelium in the germinal zone there are two stem cell
lines, one which gives rise to germ cells and a second which gives rise to Sertoli cells.
10.7 Germ 'Cells and Spermatogenesis 297
Rete Testis
of
Modulation
Fig. 10.11. b Diagrain of the spermatogenic wave in a seminiferous tubule of the rat testis
showing the resemblance to that in Squa/us Numbers indicate successive maturational stages which
at.e arranged, in general, in descending order from both ends of the rete to a "site of reversal".
(Pilsworth and Setchell 1981)
Spherical primordial germ cells or gonocytes (18 f.!m in diameter) are grouped into
clusters or cords embedded in a dense matrix of stromal cells and collagenous
connective tissue fibers. While some germ cells in the cord remain in place, others
segregate after mitosis, forming spermatogonial cell masses which enter spermato-
genesis. Presumptive Sertoli cells are recognizable as small cells with thin cytoplasmic
processes resembling mesenchymal cells. Some congregate between the clusters of
gonocytes to form the excretory duct system and others differentiate into true Sertoli
cells. Each spermatocyst arises as a single spermatogonium in association with
a single Sertoli cell. The Sertoli cells and spermatogonia then undergo repeated
mitotic divisions (13 in Squalus; Fig. 10.12). Proliferation of the two cell types is
apparently tightly coordinated, since the germ cell: Sertoli cell ratio is constant
(1 : 1) during early spermatogonial stages. At first, somatic and germ cells line the
interior of the cysts in no apparent order, but soon they segregate into two
concentric single layers, the Sertoli nuclei located adjacent to the lumen and the
spermatogonial nuclei next to the basal lamina. In this two-layered stage, the cysts
are about 100 f.!m in diameter, the germ cells about 12 f.!m in diameter. As develop-
ment proceeds, no further mitoses occur in Sertoli cells but spermatogonia undergo
four more mitotic divisions, resulting in spermatocysts with a germ cell: Sertoli cell
ratio of 16: 1. Daughter cells grow in size between divisions, so that the cysts as a
whole continues to enlarge and the germ cells transform from the larger "primary"
VI
III
Fig. 10.12. Diagram showing origin and development of a single Sertoli/germ cell unit (spermatoblast)
during the spermatogenic progression in Squalus acanthias. From Stanley (1966). Segments I-VIII
depict the orderly increase in Sertoli and germ cell numbers such that a spermatocyst at a given stage
has a predictable number of cells of each type. Arabic numerals indicate the number of germ cells per
Sertoli cell at each development stage. An actual cyst contains many spermatoblasts (see Fig. 10.11),
all in the same stage of development, and their number per cyst at each stage is predictable for a
given species. I formation of a new spermatoblast; II period of mitotic proliferation of
spermatogonia and Sertoli cells; III cessation of Sertoli mitoses and continued mitotic proliferation
of spermatogonia; IV occurrence of primary spermatocytes in various stages of prophase prior to the
first meiotic division ; V occurrence of secondary spermatocytes prior to second meiotic division ;
VI spermatids in various stages of spermiogenesis; VII period of spermiation; VIII degeneration of
cells remaining after sperm release. Onset of III is characterized by association of a single Sertoli
cell with a single spermatogonium, thus forming a definitive spermatocyst. Transition of sperma-
togonia to spermatocytes at interface of segments III and IV marks position of " zone of degeneration"
which appears seasonally in early spring or after hypophysectomy (see text). Note repositioning of
Sertoli nucleus fyom lumen to base at end of segment III
spermatogonia with light-staining cytoplasm and finely granular chromatin, to the

smaller " secondary" spermatogonia with coarse dark-staining chromatin in large
flakes or patches. One or two very large nucleoli also characterize the spermatogonial
nuclei. Thus, the primary and secondary spermatogonia described here appear
similar in their behavior and cytology to spermatogonia "A" and "B" described in
rodent testis. Toward the end of the spermatogonial period in Scyliorhinus, but some-
what earlier in Torpedo, the Sertoli nuclei migrate from a central to a peripheral
10.8 Spermatozoa, Spermatophores and Fertilization 299
position in the cyst. The functional significance of this event is unknown, but it is
evidently rapid, since only occasional cysts are seen in which the Sertoli nuclei
lie somewhere between the central and peripheral areas. At the end of the period of
mitotic proliferation, it is estimated that there are 400--500 Sertolicells per spermato-
cyst in Squalus and Scyliorhinus and 230--260 in Torpedo (Fig. 10.12).
At the onset of the long meiotic prophase, the cysts increase sharply in size
from 150 J..lm to 225 J..lm diameter in Scyliorhinus. This increase is due to the
increase in size of the germ cells. By the end of this stage, the primary spermatocytes
are characterized by a large nucleus with distinct, elongate synaptic chromosomes.
The secondary spermatocyte stage is short, few cysts being seen in this stage
compared to those immediately preceding and following. Early in the spermatid stage,
or slightly earlier, fluid spaces develop among the germ cells and the cyst as a whole
appears to enlarge. Some preparations show that each Sertoli cell has formed a single
large pocket containing a group of spermatids. It is at this stage that the relationship
between a single Sertoli cell and its synchronously developing cohort of germ cells
(spermatoblast) first becomes obvious. With further development, the spermatids
elongate and gradually form loose bundles, oriented so that heads are facing
the basal lamina and tails projecting toward the lumen. With condensation of
chromatin and reduction of cytoplasm, the bundles of spermatids become more
compact and the lobule as a whole decreases in size from 350 J..lm to 115 J..lm at the
time of sperm release in Scyliorhinus. Nuclear shape and the organization of nuclei
within the seminiferous lobules have been used to divide spermiogenesis in the long-
nose skate (Raja rhina) into eight stages and in the spiny dogfish (Squalus acanthias)
into seven stages (Bois et al. 1980). When actual counts have been made, it is
evident that most spermatoblasts contain 64 spermatids with little variation from this
number. This is very close to theoretical counts based on the number of cell divisions,
indicating that germ cell degeneration is not extensive following spermatogonial
stages. By multiplying the estimated number of Sertoli cells in mature cysts by the
number of germ cells per spermatoblast), it is calculated that about 32,000 sperma-
tozoa are produced per cyst in Squalus and Scyliorhinus and about 16,000 per
cyst in Torpedo (Fig. 10.12).
Preceding spermiation, an opening is formed through the cyst wall into the
collecting duct. Spermiation occurs as an apical sloughing of Sertoli cell cytoplasm
and release of its constituent sperm bundle. Basal Sertoli cytoplasm remaining after
spermiation degenerates and is resorbed. This zone is evident macroscopically in
cross-section as a brownish orange area in the testicular parenchyma immediately
beneath the epigonal tissue. Upon entering the lumen of the cyst and the collecting
duct, sperm bundles dissociate, liberating individual spermatozoa.
10.8 Spermatozoa, Spermatophores and Fertilization
At spermiation, sperm bundles dissociate, liberating individual spermatozoa into the

lumens of the cyst and collecting ducts. The mature spermatozoan of selachians
was first described by Retzius (1902) as 160 J..lm in length with a long, pointed,
spirally twisted head. Structural features common to the spermatozoa of all chon-
drychthian fishes thus far studied are an elongate, helical nucleus with a small conical
acrosome fitted over the pointednucelar tip (Stanley 1983). In Squalus, the acrosome
is straight and the component microtubules take a helical course (Stanley 1971 a, b).
Spermatozoa rotate around their long axes with little lateral bending of their tails
and rapid rotation of the corkscrew-shaped head contributes to progressive movement
in essentially straight lines.
In some, but not all species, spermatophores are formed (blue shark, basking
shark) (Matthews 1950; Pratt 1979). During this process, which occurs in the lower
epididymis and the anterior section of the d. deferens, spermatozoa again aggregate
into groups of 60-70 with heads aligned in parallel. Hundreds of these packets then
coalesce into spermatophores which are stored in the ampulla of the d. deferens.
Matthews (1950) speculated that spermatophores preserve the sperm from loss by
leakage to the surrounding water during copulation; however, this protective mecha-
nism would seem to be unnecessary in species without spermatophores. An alter-
native explanation is that the aggregation of spermatozoa in the male ducts somehow
minimizes their exposure to an unfavorable milieu and facilitates maintenance of
conditions necessary for their survival during long periods of storage. In this regard
it is interesting to note that electron micrographs of seminal plasma collected from
the ampulla d. deferens of Squalus acanthias reveal the presence of numerous
membrane-bound cytoplasmic elements without nuclei but containing lipid droplets,
mitochondria, areas of agranular cytoplasmic reticulum and remnants of spermatozoa
(Pudney and Callard 1986). These were judged to be the apical portions of Serto1i
cells pinched off during spermiation (Sertoli cytoplasts) and are probably responsible
for steroidogenic enzyme activity and endogenous steroids reported to be present in
the semen of this species (Simpson et aI., 1963; 1964b). Similar structures have been
described in the semen of the Port Jackson shark (Heterodontus portusjacksoni) by
Jones et al. (1984). In addition, the semen contains large proteinaceous bodies
undergoing dissolution. The latter presumably are the remains of the problematic
bodies of mature Sertoli cells and, together with steroids, may somehow affect sperm
survival and motility in the male or female tract. More than 25 years ago, 5-hydroxy-
tryptamine was identified in high concentrations in the siphon fluid of Squalus and
shown to influence uterine contractions (Mann 1960; Mann and Prosser 1963).
Although this substance is not uniformly high in other elasmobranch species, its
unique role in elasmobranch reproduction has not been pursued.
Fertilization is internal in all elasmobranchs, whether oviparous or viviparous,
and takes place in the oviducal or shell gland present at the posterior end of the
female duct, not in the upper oviduct, as previously supposed. It occurs at the same
time as secretion of the shell substance but follows deposition of albumen around the
ovum by the anterior region of the gland (Metten 1941). Also the oviducal gland
serves as a "receptaculum seminis", and sperm are found in all mature females examin-
ed whether or not the gland is in the secretory stage (Metten 1941). Sperm may be
stored here for considerable periods of time, a single insemination being sufficient to
fertilize 30 eggs over a period of 5-6 weeks in captivity (Raja brachyura) (Clark
1922).
10.9 Leyding Cells, Sertoli Cells and Steroidogenesis 301
10.9 Leydig Cells, Sertoli Cells and Steroidogenesis
10.9.1 Leydig Cells
Leydig cells located in the interstices of the seminiferous tubules are considered the
primary endocrine element of the mammalian testis (Christensen 1975). They secrete
large quantities of testosterone and smaller amounts of estradiol into the general
circulation for actions on distant targets and are responsible for maintaining the high
tubular concentrations of testosterone necessary for the completion of spermatogenesis
(Steinberger 1971). Although Leydig cells have been described in the testes of
representatives of every vertebrate class, there is controversy as to the identity of
typical Leydig cells in the interstitium of elasmo branchs. Investigations have variously
reported, even within the same species, that Leydig cells are absent , e.g. Scyliorhinus
stellaris, Scyliorhinus canicula (Stephan 1902), Squalus acanthias (Simpson and
Wardle 1967); rare, e.g. Cetorhinus maximus (Matthews 1950), Scyliorhil1us canicula
(Collenot 1970); or conclusively present, e.g. Scyliorhinus stellaris, Scyliorhinus
caniculus (Chieffi et al. 1961; Della Corte et al. 1961). All these studies were carried
out at the light microscope level, the poor resolving power of which often precludes
observation of structural features that could be used to positively identify Leydig cells.
Furthermore, many of these studies relied on the histochemical reaction for ~5_3~_
Fig. 10.13. Electron micrograph of Squa/us testis showing Leydig-like cell from same stage of
spermatogenesis as shown in Fig. 10.14. Smooth endoplasmic reticulum and other steroidogenic
organelles are poorly developed, and morphologically these cells resemble fibroblasts. Bar = 111M.
(Micrograph courtesy of Dr. 1. Pudney)
hydroxy steroid dehydrogenase (30 HSD) activity known to be present in mammalian

Leydig cells. In elasmobranchs, however, this activity is present in Sertoli cells
(Collenot and Ozon 1964; Simpson and Wardle 1967). Therefore, the use of this techni-
que alone as a criterion for identifying Leydig cells can lead to erroneous con-
clusions.
In an early study using the electron microscope, Holstein (1969) stated that Leydig
cells were absent in Squalus acanthias, although no micrographs supporting this
conclusion were presented. Reexamination of Squalus testis using perfusion fixation
to preserve fine structural details revealed cells within the interstitium with the
cytological characteristics of steroidogenesis: agranular reticulum, tubulovesicular
mitochondria and lipid droplets (Pudney and Callard 1984b; Fig. 10.13). However,
these organelles were sparse and the cells themselves were small, rare and mesenchymal
in shape, resembling the undifferentiated Leydig cells seen in fetal mammals rather
than the fully mature epithelioid form. Furthermore, regardless of the adjacent
spermatogenic stage, there was no apparent difference either in abundance or fine
structural appearance of the Leydig cells, indicating that this cell type, in contrast
to the Sertoli cell (see below), is unlikely to contribute significantly to the total
steroid output of the testis.
10.9.2 Sertoli Cells
Based on their developmental history, intimate association with germ cells throughout
spennatogenesis, and common cytological features, it is evident that Sertoli cells
of elasmobranchs and mammals are homologues (Roosen Runge 1977). They differ
in two important respects, however, The Sertoli cell of elasmobranchs is associated
with a single, synchronously developing germ cell generation, while in mammals a
single Sertoli cell nourishes three or four germ cell generations simultaneously.
Second, after a single spermatogenic cycle the elasmobranch Sertoli cell degenerates,
whereas the mammalian Sertoli cell functions throughout life, cycle after cycle.
Thus, Sertoli cell proliferation is continuous in elasmobranchs, whereas they rarely,
if ever, divide in adult mammals.
His generally accepted that the Sertoli cell has a pivotal role in regulating
spermatogenesis (Fawcett 1975). In mammals, the Sertoli cell is differentiated from
base to lumen, good evidence that structural and functional changes are coordinated
with the spermatogenic progression. In addition, several biochemical end points
known to be associated with Sertoli cells have been correlated with the spermatogenic
wave (Parvineli 1982). Although mammalian Sertoli cells synthesize and secrete a
variety of proteinaceous products, they are limited in their ability to metabolize ste-
roids, and in this respect differ markedly from elasmobranch Sertoli cells which
are the primary steroidogenic element of the testis.
Numerous early investigators using light microscope techniques have described
the morphological differentiation of elasmobranch Sertoli cells which is associated
with germ cell maturation (Stephan 1902; Stanley 1966; Collenot 1970; Holstein
1969). Prominent features include (1) a change in position of the nucleus from
adluminal to basal toward the end of the spermatogonial divisions; (2) development
of cytoplasmic processes engulfing proliferating germ cells; (3) a marked increase in
cytoplasmic volume which first becomes apparent during spermiogenesis as the germ
cells condense. In an early electron microscope study, Holstein (1969) first described
the accumulation of lipid droplets and development of agranular reticulum in
Sertoli cells of Squalus, and ascribed this to enhanced secretory functions. A recent
electron microscope study in the same species demonstrated that Sertoli cells contain
Fig. 10.14. Electron micrograph of Squalus testis showing crossection of sperm bundle encompassed
by Sertoli cell (spermatoblast). Sertoli cytoplasm at this stage of spermatogenesis is filled with an
extensive network of smooth endoplasmic reticulum. (Micrograph courtesy of Dr. J. Pudney)
a well-developed agranular reticulum, mitochondria with tubulovesicular cristae and

numerous lipid droplets, organelles characteristic of steroid production (Pudney and
Callard 1984a). Moreover, as germ cells mature, there is a dramatic increase in the
abundance of agranular reticulum in adjacent Sertoli cells such that, by the time of
spermatid elongation, it has reached extraordinary proportions and fills the Sertoli
cell as a mass of tubules (Fig. 10.14). This cytologic development corresponds
exactly to an increase in microsomal enzymes that regulate androgen and estrogen
biosynthesis (G. V. Callard et al. 1985). Also, a histochemical study in Scyliorhinus
showed a strong positive reaction for 3~HSD but only in regions with mature
spermatids and spermatozoa (Collenot and Ozon 1964). Using Squalus, Simpson
and Wardle (1967) described 3~HSD activity in Sertoli cell cytoplasm, the lumens of
mature germinal cysts, and also in semen. The latter observation is consistent
with the presence of Sertoli cell remnants (Pudney and Callard 1986), steroidogenic
activity (Simpson et al. 1964b) and steroids (Simpson et al. 1963) in the seminal
fluid of the same species.
Based on cytologic observations, elasmobranch Sertoli cells, like their mammalian
counterparts, appear to synthesize and secrete proteinaceous products and this acti-
vity increases during spermatogenesis. This is' marked by the development of a
prominent Golgi, an increase in rough endoplasmic reticulum and the accumulation
of secretory products in its cisternae (Collenot and Damas 1975, 1980). Also, electron-
dense granules are observed at the apical pole of the fully mature Sertoli cell and
seem to be secreted into the lumen (Collenot and Damas 1975). At the end of
spermiogenesis a large, proteinaceous structure appears, the "problematic body"
(Semper 1975; Collenot and Damas 1975, 1980; Moyne and Collenot 1982). The
latter is present in the cytoplasm at the apices of Sertoli cells and, when the
apical cytoplasm is sloughed at spermiation, the problematic body is carried away
with the spermatozoa into the efferent ducts. In Squalus it can be recovered in
semen collected from the ampulla d. deferens (Pudney and Callard 1986). Cyto-
chemical techniques show that the proteins of the problematic body are rich in
lysine, cysteine and tryptophan (Collenot and Damas 1975). The elasmobranch
Sertoli cell would seem to be ideal for studying stage-dependent changes in specific
protein synthesis and secretion, a topic currently of great interest in mammalian
systems (Shabanowitz et al. 1986). In this regard, a sex steroid-binding protein
(SBP) was recently characterized in Squalus testis (Mak and Callard 1987). It was
distinctly different in its physicochemical properties from testicular steroid receptors
(G. V. Callard and Mak 1985) and serum steroid-binding proteins (Ho et al. 19110)
identified in the same species, but resembled the androgen-binding protein (ABP)
found in mammalian testis. Tissue concentrations of this novel steroid-binding protein
increased as spermatogenesis progressed and was maximal at the time when protein
secretory activity appeared to be maximal in Sertoli cells. Like mammalian ABP,
Squalus SBP may be a product of Sertoli cells and serve to maintain high androgen
concentrations in the testis and exurrent ducts. This condition may somehow be
involved in sperm maintenance and would be especially important in species with
long-term storage.
Several other cytologic features of mature Sertoli cells are worthy of note. In
Scyliorhinus, gap junctions are seen between adjacent Sertoli cells in the basal regions
where the plasmalemmas of two cells appear to be fused (Collenot and Damas 1980).
These junctions appear to be reinforced by microfilaments and by parallel arrays of

tubules of the smooth reticulum. Junctions between adjacent Sertoli cells in the
mammalian testis are responsible for the blood-testis barrier and divide the semini-
ferous epithelium into basal and adluminal compartments, the latter containing all
the haploid germ cells (Fawcett 1975). Although the blood-testis barrier has not been
investigated experimentally in elasmobranchs, an extracellular marker (Evans blue dye)
does not penetrate cysts in mature testicular regions although immature regions
are homogeneously stained (G. V. Callard, unpubl. observations). Also the nucleus
changes dramatically during spermatogenesis (Moyne and Collenot 1982). While the
apical surface remains smooth, that facing the basal lamina develops finger-like
projections, and intranucleolar granules and lamellar bodies appear in association
with the nucleolus. These resemble structures of unknown significance in mammalian
Sertoli cells.
10.9.3 Steroidogenesis
Progesterone, androstenedione, testosterone and estradiol hav.e been reported in

crude organic extracts of Scyliarhinus testis (Chieffi and Lupo di Prisco 1961).
Incubation of testicular tissues with radio labeled progesterone permitted identification
of the following products: testosterone; ll-deoxycorticosterone; Reichstein's
Substance S; 20~-hydroxypregn-4-en-3-one; 20~,21-dihydroxypregn-4-en-3-one;
171X,20~,21-trihyroxypregn-4-en-3-one; 3~-hydroxy-5~-pregnan-20-one; and 171X,20~
dihydroxypregn-4-en-3-one (Simpson et al. 1964a). Other pathways of steroid bio-
synthesis identified by radiolabeled tracer analysis are: (1) conversion of pregnenolone
to testosterone and 171X,20~-dihyroxy-4-pregnen-3-one (Kime 1978); (2) conversion
of testosterone to 51X-androstan-3~, 17~-diol and their respective conjugates, tenta-
tively identified as sulfates (Kime 1978); and (3) aromatization of androstenedione to
estrone (G. V. Callard et al. 1978). Recently, a steroid sulfotransferase was
characterized in Squalus testis and found to utilize both androgen and estrogen
as substrates (Cuevas and Callard 1986).
Simpson et al. (1963) reported that Squalus semen contains high levels of II-deoxy-
corticosterone (500 Ilg/l00 g). Smaller amounts of pregnenolone, progesterone, de-
hydroepiandrosterone, androstenedione, androsterone and three unidentified com-
pounds, but no testosterone, were found. At least some of the steroids present in
semen must be synthesized in situ, since the enzymes necessary for conversion of
14C-Iabeled substrates from cholesterol to II-deoxycorticosterone via pregnenolone
and progesterone were present (Simpson et al. 1964 b). Sertoli cytoplasts may be
responsible for a portion of the measured steroids and steroidogenic activity in
Squalus semen (Pudney and Callard 1986).
Free testosterone (22-2080 ngjml) and an approximately equivalent amount of
its glucuronide were definitively identified in the peripheral plasma of skates
(Raja radiata and Raja acel/ata) (Idler and Truscott 1966). These values are 10-
to IOO-fold higher than in mature rats and humans, and appear to be due to a low
metabolic clearance rate which, in turn, reflects high levels of a serum testosterone-
binding protein (Fletcher et al. 1969; Freeman and Idler 1972). Measured production
rates, although not established with certainty, are very low, a finding consistent with
the observation that Leydig cells are sparse and undeveloped (pudney and Callard
1984b). Presumably, androgen and other testicular hormones in blood are products
of steroidogenically active Sertoli cells and reach the circulation after diffusion
into testicular capillaries or, alternatively, after entry into the seminal fluid and
reabsorption in the excurrent ducts. The former route is suggested by the observation
that testosterone glucuronide levels are higher in testicular effluent than in the peri-
pheral circulation of the skate Raja radiata (Darrow and Fletcher 1972).
Recently, we have taken advantage of the strict topographical segregation of
different spermatogenic stages in Squalus. testis to show that steroidogenic enzyme
activities are stage-dependent (G. V. Callard et al. 1985; Cuevas and Callard 1986).
Testes were dissected as follows: Zone I (stem ceUs and. spermatogonia); Zone II
(primary and secondary spermatocytes; round spermatids); Zone III (elongating
spermatids and mature spermatozoa). The enzymes of androgen biosynthesis (3~HSD,
17<x-hydroxylase, C-17, 20-lyase) increased progressively during germ cell maturation
(ZIII > U > I), corresponding exactly to the dramatic development of smooth
reticulum in Serto1i cells and pointing to this cell type as the site of biosynthesis
(Pudney and Callard 1984a; see above). These data are consistent with the ob-
servation that 3~HSD activity is restricted to Sertoli cells in the spermatid stage of
development (Collenot and Ozon 1964). By contrast, aromat~se, the enzyme regulating
androgen to estrogen conversion, was highest in Zone II where germ cells are
undergoing meiosis (G. V. Callard et al. 1985). Since arterial blood flows from
mature to less mature regions (G. V. Callard, unpublished), androgen synthesized in
Zone III would be available for aromatization in Zone II. In turn, estrogen
synthesized in Zone II would be transported to ~strogen receptor sites concentrated
in Zone I (G. V. Callard et al. 1985; Ruh et al. 1986). The functional significance
of estrogen action in immature testicular regions is unknown; however, one possibility
is that it has a role in signalling the next wave of spermatogonial proliferation.
10.10 The Brain-Pituitary-Testicular Axis
Several lines of evidence indicate that elasmobranchs have a functional-brain-pituitary-

testicular axis. Selective ablation of portions of the pituitary gland (Dodd et al. 1960)
and heterologous immunocytochemical (J. C. A. Mellinger and Dubois 1973) and
radioimmunoassay studies (Scanes et al. 1972) point to the anatomically discrete
ventral lobe as the sole source of gonadotrophins. Bioassay of the ventral lobe
using testosterone production by dispersed testicular cells of the turtle has dem~n
strated directly the presence of gonadotrophic activity; intermediate/median lobe
extracts were 10-100-fold less active (Lance and Callard 1978). Removal of the ventral
10 bes in Soyliorhinus causes regression of the testis with the appearance of a localized
"zone of degeneration" between the last generation of spermatogonia and the first
appearance of spermatocytes (Dobson and Dodd 1977a, b; Fig. 10.12), suggesting
that this transition is the hormone-sensitive step. Paradoxically, replacement with
testosterone, human chorionic gonadotrophin or pregnant mares serum gonadotro-
phin did not prevent the effects of hypophysectomy. In these same studies, ventral
lobectomy reduced 3H-thymidine incorporation into whole testis, but no attempt was
10.11 Targets of Testicular Hormone Action 307
made to determine whether this effect was stage-specific. It was, however, seasonal;
only fish collected in summer (April to September) displayed a zone of degeneration
after ventral lobectomy (Dobson and Dodd 1977 a, b). In a subsequent study, Dobson
and Dodd (1977c) showed that temperatures resembling those in nature in summer
(10-15 0c) are critical for division of spermatogonia and for demonstrating effects of
hypophysectomy. In Squalus, a zone of degeneration appears in the testis annually in
early spring (Simpson and Wardle 1967). Together these data can be interpreted
as an indication that gonadotropin levels, and consequent testicular actions, fluctuate
seasonally as part of the normal breeding cycle. Nonetheless, it is important to note
that animals surviving as long as 2 years after hypophysectomy, although having
testes markedly reduced in weight, have all spermatogenic stages present" (Dobson
and Dodd 1977 a). From these observations, it is reasonable to conclude that
spermatogenesis is not absolutely dependent on pituitary hormones but is rendered
more "efficient" by their presence, a concept also supported by evidence in mammals
(Vernon et al. 1975). Another indication that the testis is to some degree autonomous
is the finding that hypophysectomy induces little or no change in circulating testo-
sterone (Dobson and Dodd 1977 a). Although ventral lobe extract significantly
increases plasma androgen, mammalian gonadotropins are ineffective (Sumpter
et al. 1978). Preliminary attempts have been made to purify gonadotrophic activity
from the dogfish pituitary, and specific antibodies raised against this preparation
neutralized its actions in homologous and heterologous bioassays but did not neu-
tralize activity of mammalian LH, indicating a considerable difference in structural
homology (Sumpter et al. 1978).
10.11 Targets of Testicular Hormone Action
In mammals, androgens and estrogens secreted by the testis into the peripheral cir-
culation are responsible for the development and maintenance of the male sex
accessories, external genitalia and secondary sex characters. They also have important
feedback and behavioral actions at the level of the brain and pituitary. In
addition, the testis itself is a target of steroid action and, in this instance,
locally synthesized androgen and estrogen function as parahormones or autohor-
mones regulating spermatogenesis and steroidogenesis. Androgen-dependent targets
have not been systematically studied in male elasmobranchs nor have androgen
receptors been identified. Although developmental studies show that clasper growth
accelerates coincident with the onset of testicular growth (Collenot 1969), treatment
of animals with androgen has little or no effect (W ourms 1977; Dodd 1983; Dodd
and Sumpter 1984). Studies in which animals were hypophysectomized do not report
changes in epididymaL weight or clasper length (Dobson and Dodd 1977a, b).
From the occurrence of sex-dimorphic behaviors (see below), we can infer that
steroids have triggering actions on the brain. Jenkins et al. (1980), using autoradio-
graphic and biochemical methods, provided the first evidence for estrogen-binding
activity in the dogfish (Scyliorhinus) brain and pituitary. In autoradiograms, target
cells were localized in preoptic, habenular and hypothalamic tuberal nuclei and in
the ependyma of the third ventricle. Although nuclear extracts were not studied,
a high affinity, limited capacity estrogen-binding protein was detected in cytosols

(hypothalamus> optic tectum > pituitary).
Recently we described the physicochemical characteristics of an estrogen receptor
in Squalus testis (G. V. Callard and Mak 1985). It resembled mammalian receptors
in its high affinity (Kd = 10- 9 M), limited capacity « 350 fmol g-l tissue), and
steroid specificity (estradiol> moxestrol > estrone> estriol); however, it was
unusual in that both empty and filled binding sites were extracted from nuclear
subfractions whereas cytosolic subfractions were receptor-negative. This was ascribed
to an exceptionally high affinity of the Squalus receptor for chromatin and a conse-
quent resistance to redistribution artifacts characteristic of mammalian and avian
systems. We postulated that the high oiSmolarity of body fluids during the course
of evolution in Squalus may have selected for a receptor with unusual nucleophilic
properties, since a freshwater elamobranch (Potamotrygon) had an estrogen receptor
more like that of mammals in its relatively low affinity for DNA. A second study
was undertaken to determine whether nuclear acceptor sites reflected these unusual
binding properties of the estrogen-receptor complex. Although nucleoacidic protein
binding sites were of high affinity (Kd = 10- 10 M), low capacity and species- and
tissue-specific, they were fundamentally similar to those in mammals, indicating
that the receptor itself is modified but the nuclear binding mechanism has been
conserved (Ruh et al. 1986). Total estrogen receptors, their percentage occupancy and
chromatin acceptor sites were all concentrated in Zone I (G. V. Callard. et al.
1985; Ruh et al. 1986), good evidence that the region in which stem cells and
spermatogonia are undergoing proliferation is the site of estrogen action; however,
the exact cellular targets and consequences of estrogen actions remain to be
determined.
10.12 Seasonal Cycles
Based on reports in the literature, Wourms (1977) has categorized elasmobranch

cycles as follows: (1) reproquction throughout the year (e.g. Scyliorhinus canicula,
Heterodontus francisci); (2) a partially defined annual cycle with one or two peaks
(Raja erinacea); (3) a well-defined annual or biennial cycle (Mustelus canis, Squalus
acanthias, Ceterohinus maximus). It should be noted, however, that many reports
are based on inadequate sampling methods. In addition, differences in reproductive
cycles between two populations of the same species, as well as differences between
species, suggest a need for careful study and cautious generalization (Wourms
1977). It is generally agreed, however, that the viviparous dogfish Squalus acanthias
shows a striking periodicity. The Scottish-Norwegian stock makes an autumn
breeding migration from the region of the Orkney and Shetland Isles to the breeding
areas in the Minch off northwest Scotland (Holden 1965). During January and
February those females carrying embryos at the end of their second year of
development move inshore and give birth to young in shallow water. This is
followed by copulation, ovulation and the onset of another pregnancy. At all
times of the year, Squalus testis contains every stage of sperm formation; however,
the relative abundance of the different stages and hence the width of the different
10.13 Sex and Sex-Related Behaviours 309
testicular zones vary seasonally (Simpson and Wardle 1967). Similar observations
have been recorded for the Japanese dogfish Mustelus manazo and Mustelus grizeo
(Teshima et al. 1971). In Squalus maximal accumulation of semen in the ampulla oc-
curs at the time of breeding (January-February) and is followed by a period of
testicular inactivity. The appearance in early spring of a "zone of degeneration"
separating spermatogonial from primary spermatocyte stages mimicks that occur-
ring after hypophysectomy in Scyliorhinus (Dobson and Dodd 1977 a, b) and suggests
that a deficiency of pituitary hormones occurs naturally at this time of year. This
zone moves progressively through the testis during the summer as less advanced
germ cells proliferate and reaches the mature surface of the testis in December to
February of the following year. The movement of this zone serves as a marker,
indicating that the duration of a complete spermatogenic sequence requires 9 months
(Simpson and Wardle 1967). Only animals maintained at "summer" temperatures
(13-18 0c) have a degenerative zone following hypophysectomy, an index that normal
spermatogonial proliferation may be triggered by rising temperatures in spring
(Dobson and Dodd 1977 c). Other evidence that reproductive functions are seasonal
comes from studies showing that 17et.-hydroxylase and C-17,20-lyase activities are
greater in Squalus testis in May than in late summer, possibly reflecting changes in
the proportion of different spermatogenic stages (Simpson et al. 1964a). Also,
pituitary gonadotrophic activity and circulating androgens and estrogens exhibit
an annual cycle in female Scyliorhinus with lowest activity in summer (Dodd and
Sumpter 1984).
10.13 Sex and Sex-Related Behaviours
Migratory and breeding behaviour supports the view that reproduction is seasonal in
many elasmobranch species (Wourms 1977). Collection data for Squalus acanthias
from Frenchman's Bay in 1976 shows a differential movement between the sexes
(A. D. Woodhead et al. 1976). Large mature females approach the coast first,
in early June, and form the bulk of the inshore catch throughout June to late
August. Mature males move into coastal waters later but remain in separate shoals,
generally further offshore than the females. Migration in unisexual shoals appears
to be common in the spiny dogfish and has been reported for populations off the
eastern seaboard of Canada and for populations in Europe.
Copulation has been observed infrequently in elasmobranchs and varies somewhat
in different species (see Wourms 1977). In small sharks, the male coils himself
around the female, seizes the left pectoral fin in his mouth and inserts the right
clasper into the cloaca; copulation lasts about half an hour, during which the female
is passive. Small species of skates mate with the ventral surfaces apposed, while
males of large species make either a dorsal or ventral approach; copulation lasts
for more than 2 hours. From the presence of vaginal scars, Matthews (1950)
determined that the basking shark employs one clasper at a time in copulation, a
conclusion supported by Pratt (1979), who notes further that, due to the size of the
claspers, the use of both may be an option only for young males; however,
Leigh-Sharpe (1920) killed two tope "in copula" and observed both claspers inserted.
Vaginal wounds are found only in mature females, presumably caused by the male
claspers, and are more frequent in the summer (Matthews 1950; Pratt 1979).
Wounds (bite marks) resulting from mating in large sharks have been described
by several authors as occurring only in mature females (Pratt 1979). Fresh bite
marks are most numerous from June to August in blue sharks and only healed scars
are seen in pregnant· females. Bite marks are never seen in males. A noteable
adaptation of female sharks, presumably to accommodate the males' aggressive
mating behaviour, is a skin over most of the body more than twice as thick as that
of the male (Pratt 1979). Indeed, the skin is thicker than the males' teeth are
long and only occasionally do the wounds penetrate the dermis and involve the
musculature. Pratt (1979) remarks that resistance to infection and healing rates must
be high because these injuries seem very serious. Biting is thought to be a component
of courtship behavior, serving as a pre-copulatory release mechanism in females
(Wourms 1977).
10.14 Sex Differentiation, Growth and Maturation
In the viviparous dogfish Squalus acanthias, pups are born after a 22-month gestation
period with the yolk sacs still attached, and measure 20-29 cm in length (Hisaw and
Albert 1947). Free-swimming, immature animals measure 40-50 em in length.
Among sexually mature Squalus, the males are generally smaller and sleeker than the
females which are almost always pregnant. In a large study of Scyliorhinus
canicula incubated and hatched in the laboratory or captured at sea, Collenot
(1969) categorized males as follows: embryonic «9 cm); juvenile (12~0 cm);
maturing (40-58 cm); adult (> 58 cm) She noted that immature stages were very
rarely seen.
The availability of large, readily accessible embryos and a lengthy period
of embryonic development (e.g. 4-5 months in oviparous skates; 22 months in
viviparous dogfish) would seem to be ideal for studying the mechanism of sex
determination and sex differentiation; however, few such studies have been carried
out and none of these are recent (Picon 1962; Thiebold 1964; Chieffi 1967;
Collenot 1969). Chieffi (1967) likened gonadal differentiation in embryonic elasmo-
branchs to that occurring in amphibia and amniotes, reporting that the ovaries and
testes arise after migration of primordial germ cells from extraembryonic endoderm
into paired genital ridges comprised of two anlage, respectively the cortex and
medulla.
In contrast to the situation in teleosts, in which sequential or simultaneous
hermaphoriditism is common, sex appears to be more stable and hermaphroditism
rare in elasmobranchs. Pratt (1979) describes a single occurrence in a blue shark,
that had both male and female sex organs; however, the presence of recently
incurred dermal mating scars indicate it was viewed as a female by conspecifics.
The idea that sex is relatively stable in elasmobranchs is further supported by
experimental studies. Transplantation of gonads onto extra-embryonic tissues show
that they differentiate into ovary or testis according to their genetic sex and
independent of the sex of the host (Thiebold 1964). On the other hand, in Torpedo,
10.14 Sex Differentiation, Growth and Maturation 311
experiments in which steroids were injected into embryos through the egg membranes
led to the' conclusion that all steroids (testosterone, estradiol, progesterone and
deoxycorticosterone) are feminizing, producing a hypertrophy of the "cortex"
and degeneration of the "medulla" (Chieffi 1967). These results are consistent with
the view that the female is the heterogametic sex in elasmobranchs (Dodd 1983).
In a study of the testis of Scyliorhinus canicula, Collenot (1970) reported that
the first cysts are visible even before birth (~l 0 cm) but sexual maturity does not
occur until the animals are 55-60 cm. During the embryonic and juvenile stages,
germ cell maturation is arrested prior to the onset of meiosis and, although their
appearance is normal, cysts containing spermatogonia are sparse. Also, degenera-
ting cysts are common. Sertoli cells remaining after germ cells degenerate are
relatively unaffected, and clusters of these cells display a positive reaction for lipid
and 3~HSD. These may have been mistaken for Leydig cell clusters reported
to be more frequent or present only in immature animals (Chieffi and Lupo di
Prisco 1961). At the onset of sexual maturation, there is a marked increase in
spermatogenic activity, and this is reflected in an increase in testicular weight
(Collenot 1969). Although germ cell degeneration still occurs to some extent,
eventually it is limited to a single zone of degeneration marking the transition
between spermatogonia and primary spermatocytes. These data in juveniles are
consistent with observations after hypophysectomy of mature animals (Dobson and
Dodd 1977b) and support the view that a deficiency in some hormone serves as a
brake on sexual maturation.
Secondary sex characters appear in embryos coincident with differentiation of
the gonads, and there is evidence that they are steroid hormone-dependent (Chieffi
1967). In common with most vertebrate embryos, elasmobranchs develop two sets
of urogenital ducts: the Wolffian and Mullerian systems. In males the Wolffian
duct, derived from the mesonephros, gives rise to the efferent ducts, epididymis and
ductus deferens, whereas the Mullerian ducts, derived from the pronephros,
atrophy or survive as vestiges in immature specimens or as part of the sperm sac in
adults (Wourms 1977; Gilbert 1973). Male dogfish are recognizable in utero and
immediately after birth by the presence of claspers. Thereafter clasper length increases
with total body length up to sexual maturity when further growth of this organ
ceases (Teshima et al. 1971; Collenot 1969, 1970). There are, however, changes in
the rate at which this organ develops, the first increase occurring shortly after
hatching and a second, greater increase coincident with the onset of testicular
development in maturing animals (Collenot 1969). Whether or not this organ is
androgen-dependent. is debatable (Chieffi 1967; Dodd 1983; Dodd and Sumpter
1984). In male elasmobranchs, changes in relative size, hardness and development
of the claspers is the most frequently used method for determining sexual maturity;
however, a more reliable index is the presence of spermatophores, milt and semina1
fluid, these events signaling abruptly the final stage of maturation (pratt 1979).
In the blue shark, the smallest mature male (with spermatophores) was 153 cm;
at 183 cm 50 % were mature and 95 % of males above 205 cm were mature,
estimated to occur at 6 years of age in this species (Pratt 1979). Because testicular
length and epididymal width follow the same gradual size increase as the claspers,
these measurements alone are not reliable for determining sexual maturity (Pratt
1979). Other sex dimorphic features in elasmobranchs are alar spines on the
pectoral fins (skates), tooth structure, body size, thickness of the skin and aggressive-
ness (Wourms 1977; Dodd 1983; Dodd and Sumpter 1984).
Acknowledgements. Supported by NSF grants DCB 85-18296 and 86-06344 to l.P.c., and NSF
grant DCB 85-19737 and NIH grant HD 16715 to G.V.c.
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Subject Index
Abdominal Androgen
fluid 245 biosynthesis 305,306
pores 245 binding protein (ABP) 304
Acid-base Androstenedione 305
branchial mechanisms 191, 245, 247 Arginine vasotocin 209,210
extrabranchial sites 246 Aromatization 305, 306
hypercapnia 230-239 Aspartocin 209
nitrogenous metabolism 216, 246-247 Atrium 2,3,150
regulation 215-252 Axillary bodies 146, 148, 149, 150
relevant substances, release 216-218
steady-state 218-221 Bicarbonate
temperature changes 222-230 -chloride (RCO; /CI-) exchange 247
transepithelial ion transfer 245-248 equivalent ion transfer
transients 223 hypercapnia 231-234
transmembrane ion transfer 223, 225, 227, temperature 223-228
229 plasma 218,219,231
Acetylcholine 143, 153, 163 steady-state release 217
Adenosine 31,187 Blood
carbon dioxide transport 19;
Adenosine triphosphate (ATP) 19, 31, 121,
equilibrium curves 16
187,239
oxygen carrying capacity 17, 18
Adrenaline 31, 34, 143, 150, 151, 154, 163, pso 17,18
210,211 PC02 15,16, 19
Adrenocorticosteroids 188 P0 2 15-19
~-alanine 270-272 pressure 11, 12
Alkaline gland 294 Blood-brain barrier 51
Alkalinity Blood/water diffusion distance 6
relative 218 Bohr
Alphastat model (see also Imidazole) 221, 223 coefficient 17, 18
Amino acids 256 effect 17,19
intracellular osmoregulation 267-273 Bombesin 151, 155, 159
Ammonia 216,255,256 Brain
diffusion 216 basic organization 49-51
excretion 247,256,260 reticular formation 51,58-59
formation 259-261 sensory processing 66-73
renal 260 Branchial
non-ionic elimination 246 artery 5-8
pK' 216 blood pathways
steady-state release 217 non-respiratory 8-10
toxicity 216 respiratory 5-8
Ammonium circulation 5-10
ionic diffusion 246 control 32-34, 151
-sodium (NRt /Na +) exchange 246 epithelium 4, 245
Ampulla ductus deferens (seminal vesicle) 292 innervation 32, 151
Ampullae of Lorenzini 93-96 ion fluxes 178
Anaerobiosis 121,239 ion permeabilities 176,191
320 Subject Index
mechanoreceptors 35 Claspers 294, 307

shunts 24--26 Companion vessels 9
urea permeability 173-174 Cones 86
vein 6 Copulation 309
Buffer Corpus cavernosum 5, 6
capacity 223 Corpus luteum 278,279,281
non-bicarbonate (see also Imidazole) 221, steroidogenesis 281
225,228,233 Cortisol 188
phosphate 228 Counter-current flow
semi-closed system 223 branchial 21,22
temperature coefficients 221, 228 nephron 204--205
Cranial nerves 59,60, 144--146, 163
Creatine phosphate 239
Cajal, cells of 149 Cuverian duct 2
Calcitonin 210,211 Cyc1icAMP
Capacitance coefficient 15, 16, 21 rectal gland function 182, 185-186
Carbamyl phosphate synthetase 257-258 renal 210,211
Carbon dioxide transport 19
Carbonic anhydrase 19, 177, 184,245
Cardiac output 10 Density (of body) 123,125
exercise 37 Deoxycorticosterone 188
Fick equation 27 Dermal dentic1es 134--135
filling 3 Diffusing capacity II
temperature 36 Ductus deferens 292
Cardinal sinuses 3, 149
Catecholaminei'> 121,149
branchial vasculature 34, 38, 151 Efferent ducts 292
exercise 38 Egg case 288
gas transfer 41 urea permeability 192
heart 31,150 Electric organs 60
hypoxia 41 Electroretinogram 87
rectal gland 188 Endothermic sharks (see also Lamnid sharks) 2
spleen 153 Epididymis 292
systemic vasculature 151 Epigonal organs 292
Cell volume regulation 267-271 Epiphysis 73
Central venous sinus 9 Estradiol 278-280,289,301,305
Cerebellum 61-65 Estrogen
Cerebrospinal fluid 51, 116 binding 307,308
Chloride receptor 308
branchial fluxes 176, 178 Euryhaline elasmobranchs 189-192,262-265
plasma concentration 172, 190
renal secretion 208, 209
secondary active transport 184 Fertilization 300
unidirectional fluxes 176 Fick equation 20, 27
urine concentrqtion 206 F ollic1es 278, 279
Chloride cells 178 granulosa cells 279-280
Cholecystokinin (CCK) - see gastrin/CCK steroidogenesis 279-280
Chloroid plexuses 51 theca cells 279-280
Chromaffin cells 146, 149, 150, 151 Food detection
Chrysopsin 87 by olfaction 80-81
Circulation by electroreception 93
branchial 5-10,32-34 Freshwater elasmobranchs 189-192,265-267
coronary 2-3
heart 2-3
renal 201-202 Gametogenesis 286,295
single I Gastrin/CCK 151, 155, 159
systemic 10-13, 34--35 Geomagnetic orientation 91,93
Subject Index 321
Gill (see also Branchial) Hypoxia 36,38-42

ventilation 13-15, 25, 236, 238, 239 catecholamine 41, 151
Glomerular heart rate 39-41
filtration rate 191, 205, 206 oxygen uptake 40
control 205,2\0 ventilation 39
number 203
Glumitocin 209
Gonadial differentiation 310-311 Imidazole 221
Gonocytes 297 imidazole alphastat regulation 221-222
Gonadotropins 282-284, 306-307 pK/temperature coefficients 221, 228
Gut Interbranchial septum 8, 9
anatomy 147
innervation Jaw-jerk reflex 71
adrenergic 158 J-type receptors 37
cholinergic 158 Juxtaglomerular apparatus 209
peptidergic 159
serotonergic 159
splanchic 157-158 Ketone bodies 122
vagal 156-157 Klinotaxis 80
motility 155 Krogh's diffusion coefficient 20
neural control 147-149
Lactic acid
Haematocrit 16,18,154 pK' 239
Hair cells 89-93 plasma 240
control 60 steady-state distribution 241
Haldane effect 18, 19 muscular activity 121,240
Heart 1,2,150 Lactacidosis 239-245
beat, initiation 3, 28 Lamellae (secondary) 4-8,24-26
synchrony with respiration 26, 30-31, 60 Lamnid sharks
humoral control 31-32, 150, 165 gas exchange 37
innervation 28,59, 150, 165 muscle temperatures 136-137
nervous control 28-31, 150 vascular anatomy 12, 136
pacemaker activity 28 Lateral line canal 91-93
rate 11,36 Leydig cells 292,301-302
control 28-32,165 Leydig's gland 292-294
hypoxia 29,39-41 Lipids
Heterocercal tail 124, 129 changes during pregnancy 127
low density 123, 125-127
Hill's coefficient 16, 18
metabolism 122, 127
Hydrogen ion (H+)
branchial efflux 177
deficit 241
Macula neglecta 91, 92
equilibrium limitation 241,243
Methylamines 122, 174,254,261
production 218
Muscle fibres
-sodium (H +INa +) exchange 176, 247
action potentials 117-118
steady-state distribution 241
Ca2 + -activated ATPase 105-107, 11 0, 121
I ex-hydroxycorticosterone 211
capillary distribution 109, 110, 120
Hypercapnia contractile properties 118-120
environmental 230-235,243 innervation III-liS
thermostratification 230 metabolism 120-123
hyperoxia-induced 235-239 membrane properties 115-118
Hypercapnic acidosis mitochondrial content \05-107,110, III, 120
compensation 234 pH 220
Hyperoxia 239 power output 119
-induced hypercapnia 235-239 sensory endings 113-115
Hypothalamus 73 types 100, lOS-liS
322 Subject Index
Muscle spindles 113 PCO z I2, 15-19218,219

Myotomes PO z 12, 15-19
internal pressures 103 Parvalbumin 121
organization 100-102 Pectoral muscles
swimming 104, 105
Nephron Pericardium 3
number 203 pH
loops 204-205 arterial 12, 218
Nerves intracellular 220,221,225-230,234,243
adrenergic 143, 150, 151, 152, 153, 155, plasma 12,218,219
158-159 sodium influx 177
cholinergic 143, 150,158 temperature coefficients 218,221
cranial 59,60, 144-146, 163 urine 206,245
enteric 147-148 venous 12
peptidergic 151, 155, 159-162, 165 Pineal organ (see Epiphysis)
serotonergic 151,152,155,159,165 Plasma
spinal chord 53, 56-57 bicarbonate 218,219,231
Neurohypophysial peptides 209 chloride 172, 190
Neuromuscular junctions III ion concentrations 190, 268
Neuropeptides 148,155 during development 193
Noradrenaline 31,34, 143, 149, 150, 154,210, lactate 240
211 osmotic concentration 172,190
Normoxia 238 PCOz 12, 15-19, 218, 219, 231, 237, 240
pH 12,218,219
Oestradiol (see estradiol) trimethylamine oxide 172, 193, 254, 268
Olfacory epithe1ium 81-82 urea 172, 190, 191, 193,254,268
Optic chiasma 72 Pregnancy
Organic phosphates (see also ATP) lipid metabolism 127
erythrocyte 17 Progesterone 278,279,281,289,305
Ornithine-urea cycle 256 Prolactin 188,211
Ostium Purkinje cells 61-65,67
atrio-ventricular 2
, sino-atrial 2
Otolith organs 90-91 Rebound contraction 156, 157
Ovary Rectal gland 171,179-189,191,245
cycles 277-279 adenosine 187
gametogenesis 286-287 atrial natriuretic peptide 189
steroidogenesis 279-281 carbonic anhydrase 184
regulation 282-284 contribution 177-178,180-181
Oviduct cyclic AMP 182, 185-186
contractile activity effects of removal 181
neurointermediate lobe hormones 290 hormonal control 186-188
relaxin 289-290 peptides 186-188
hormonal regulation 288-291 steroids 188
structure 287;-288 perfusion 182-183
Oviparity 192 rectin 187,189
gametogenesis 286 secretory cells 179
Oviposition 289 somatostatin 187
Ovoviviparity 192-193 (see also - Viviparity, vasculature 179,187,188
aplacental) vasoactive intestinal peptide (VIP) 186
Oxygen 15-20 Rectin 187, 189
consumption 11, 36 Relaxin 285, 289
debt 239 myometrial activity 289-290
diffusion resistance 21-24 Renal
percent extraction (%E) 12,24,25 function
transfer chain 239 endocrine control 209-212
transfer factor 23 ion transport 208-209
Subject Index 323
tubular water permeability 209 Substance P 151,155,161

urea reabsorption 173, 191, 207-208, 255, Suprarenal bodies 149
264
vasculature 201-202
Renal portal system 201
Taurine 270-273
Renin-angiotensin system 209
excretion 272
Reproductive behaviour
Telencephalon 73
role of olfaction 81
Temperature coefficients
Respiratory frequency 11
non-bicarbonate buffers 228
Rete mirabile 12, 13, 136
pH 218,219,220,228
Retinal epithelium 85-86
relative alkalinity 218
Rheotaxis 81, 88
Testis 292
Righting reflexes 88
cycles 308-309
Rhodopsin 87
maturation 311
Rods 86
steroidogenesis 302, 304, 305-306
Testicular hormones 304--306
Sarcosine 270-272
sites of action 307-308
Semi-circular canals 90-91, 92
Testosterone 278-280,301,305
Sertoli cells 294, 302-305
Thermostratification 230
cytoplasts 300
Thyroid hormones 188,210,211
gap junctions 304
Transamination 259
"problematic body" 300, 304
Trimethylamine oxide (TMAO) 171, 172, 192,
Sex differentiation 310-312
255
Shark repellants 81
concentration
Sinus venosus 2, 3, 150
muscle 254
Skin
plasma 172,193,254
acid-base balance 245
urine 206
connective tissue fibres 102, 103
counteraction of urea effects 122-123, 174,
locomotion 103
261-262
Sodium
excretion rate 172
-ammonium (Na + /NHt) exchange 246
renal reabsorption 208, 255
branchial fluxes 176-178
sources 258
-hydrogen (Na + /H +) exchange 176--177,247
synthesis 259
passive influx 247
plasma concentration 172, 190
renal transport 208
unidirectional fluxes 176, 191, 247 Urea 171, 172, 192,216,255
urine concentration 206 branchial permeability 173-174,255,263
Sodium-potassium activated ATPase (Na- K- concentration
ATPase) 171,178,183,210,211 muscle 254
Somatostatin 151, 155, 159, 187 plasma 172,190,191,193,254
Spermatids 299 urine 206
Spermatocysts 294--295 egg case permeability 192
Spermatocytes 299 embryonic development 192,258
Spermatogenesis 295-299 efflux 172,173,263
regulation 302-305' enzyme effects 122, 174, 261-262
Spermatogonia 297 counteraction by TMAO 122,174,261-262
Spermatophore 300 renal reabsorption 173, 191, 207-208, 255,
Spermatozoa 292, 299-300 264
Spermiation 299 synthesis 172, 190, 191, 256--258
Spinal chord neurons 53, 56-57 Uterine fluid 193
Spiracular organ 91 Urine
Spreading depression 63 flow rate 191,206
Squalene 126 ion concentrations 179, 191,206
Steroidogenesis osmotic concentration 206
ovary 279-281,282-284 pH 206,245
testis 302, 304, 305-306 titratable acidity 245
324 Subject Index
Valitocin 209 Ventilatory

Vasoactive intestinal peptide (VIP) muscles, innervation 35, 60
gut motility 161 rhythm 36, 60
neurones lSI, ISS, 161 Ventricle 2, 150
rectal gland 186 Vitellogenesis 287
Venous sinuses 2,9, ISO, lSI Viviparity [92,193,291-292
aplacental 291
Ventilation
gametogenesis 286
control 35-36, 60
placental 291
gill water \3-15, 25, 236, 238
hyperoxia 236-238 Water
hypoxia 39 diffusional permeability 175, 191
oxygen-oriented regulation 236, 238 drinking rate 175
volume 10 fluxes 175
exercise 37 Wunderer's corpuscles 56

P. J. Butler, J. D. Metcalfe (Auth.), Dr. Trevor J. Shuttleworth (Eds.) - Physiology of Elasmobranch Fishes (1988, Springer-Verlag Berlin Heidelberg)

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P. J. Butler, J. D. Metcalfe (Auth.), Dr. Trevor J. Shuttleworth (Eds.) - Physiology of Elasmobranch Fishes (1988, Springer-Verlag Berlin Heidelberg)

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Trevor 1.

With 116 Figures

Springer-Verlag Berlin Heidelberg NewYork

ISBN -13: 978-3-642-73338-3 e- ISBN-13 :978-3-642-73336-9

Library of Congress Cataloging in Publication Data.

Professor R. F. H. "Roy" Freeman

Rochester, January 1988 Trevor J. Shuttleworth

Chapter 1 Cardiovascular and Respiratory Systems

1.1 Functional Morphology of the Cardiovascular and Respiratory Systems ......... .

Chapter 2 The Central Nervous System

2.2.2 Is There a "Central Pattern Generator" for Locomotion? . . . . . . . . . . . . . . . . . . . . .. 52

Chapter 3 Sensory Physiology

Chapter 4 Muscles and Locomotion

4.1 General Organization of the Locomotor System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 100

Chapter 5 The Autonomic Nervous System

5.5.3.2 Adrenergic Innervation ................................ . . . . . . . . . . . . . . . . . . .. 158

Chapter 6 Salt and Water Balance - Extrarenal Mechanisms

Chapter 7 Kidney Function

Chapter 8 Acid-Base Regulation

8.1.2 Steady-State Acid-Base Status .............................................. 218

Chapter 9 Nitrogen Metabolism

9. F N'\ture and Routes of Excretion ............................................ 253

Chapter 10 Reproductive Physiology

ParI A The Female. , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 277

10.3 Gametogenesis and Vitellogenesis 286

Subject Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 319

Cardiovascular and Respiratory Systems

1.1 Functional Morphology of the Cardiovascular

1 Department of Zoology and Comparative Physiology, University of Birmingham, Birmingham

1.1.1 The Heart and Coronary Circulation

Vent r a I a o::::r:..:t.::a_.::$.~~0'Vi,o.\1. C ompacta

1.1.2 Branchial Circulation

1.1.2.1 The "Respiratory" Blood Pathway

Fig. 1.3. A light micrograph of a

Fig. 104. A light micrograph of a

Fig. 1.5. A scanning electron micro-

1.1.2.2 The "Non-Respiratory" Blood Pathway

Fig. 1.6. A light micrograph of the

Fig. 1.7. A scanning electron micro-

1.1.3 The Systemic Circulation

Species and source Mass Temperature Yentilation Cardiac Vw

Butler and Taylor 1975 0.86 7 19

a Now thought to be the same as S. acanthias (Wells and Weber 1983)

G vcnt Oxygen Diffusing Respiratory Heart Mean blood

1.7 36 4.12 59 32 5.1 3.9

d Calculated by Piiper and Baumgarten-Schumann (1968 a)

Table 1.1 (continued)

Species and source Pi 0 2 Pe 0 2 % Extr Pa 0 2 Pv0 2 p.C0 2 PvC0 2 pHa pHv

This rete arrangement acts as a counter-current heat exchanger which functions

1.1.4 The Respiratory System

Fig. 1.8. A diagrammatic representation

1.2 Gas Exchange

1.2.1 Properties of Oxygen and Carbon Dioxide in Water and Blood

elasmobranch blood than in seawater (Fig. l.10b). The blood is halfcsaturated at a

The predominant organic phosphate in elasmobranch erythrocytes and the relative

Species Authors Pso Bohr Hill Oxygen Haldane Haematocrit

stellar is Schumann 1968 b 0.2 kPa) ~

a Now thought to be the same as S. acanthias (Wells and Weber 1983).

et al. 1978), whereas in the dogfishes, S. canicuia, S. acanthias, the shark,

1.2.2 Analysis of Gas Exchange in Relation to the Morphology of the Gills

where M = amount of gas diffusing per unit time (mmol s -I)

Pi and P e = partial pressure of gas in inspired and expired water (kPa)

1.2.2.1 Direction of Blood Flow