comparattve biology of s alinity discrimination in ... · comparattve biology of s alinity...

COMPARATTVE BIOLOGY OF S ALINITY DISCRIMINATION IN CROCODILIANS

AND THE POSSIBLE ROLE OF THE INTEGUMENT

Katherine Jackson

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Zoology University of Toronto

@ Katherine Jackson 1995

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TABLE OF CONTENTS

TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

LISTOFTABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LIST OF ABBREVIATIONS xi

CHAPTER 1 : General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Osmoregul atory S trategies of Marine Animais . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Marine Adaptation versus Estuarine Adaptation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Osmoregulation in Estuarine Crocodilians 9

Permeability of the Integument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The renalcloacal system 10

Lingual salt-secreting glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

The role of selective drinking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phylogenetic trends 15

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Objectives of the thesis 18

CHAPTER 2: Habitat and Phylogeny Influence Salinity Discrimination

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

. . . . . . . . . . . . . . . . . . . . Lack of Salinity Discrimination in Caimm crocodilus 24

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and Methods 24

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resuits and Discussion 25

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Influence of Habitat 31


. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and Discussion 34

Influence of Phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35


. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and Discussion 39

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Discussion 40

CHAPTER 3: Evidence for Integumenial Chemoreception in C . porosus

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results 53

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

CHAPTER 4: Morphology and Ultrastructure of a Putative Integumentary Sense Organ

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction 60

Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

SEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Light microscopy and TEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gross morphology 65

SEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Light rnicroscopy and TEM 67

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion 71

C'EbWER 5: Conclusions and Directions for Future Research . . . . . . . . . . . . . . . . . . 77

ABSTRACT

COMPARATIVE BIOLOGY OF SALINITY DISCRIMINATION IN CROCODILIANS AND

THE POSSIBLE ROLE OF THE INTEGUMENT

Master of Science, 1995

Katherine Jackson

Department of Zoology, University of Toronto

This study investigated the mechanisms underlying salinity discrimination in

crocodilians. The results are as follows:

( 1 ) In crocodilians, the capacity for salinity discrimination has a phylogenetic

component. A11 crocodylids tested discriminated between salinities and did not drink hyper-

osmotic sea water. In alligatorids, however, only individuals collected in an estuarine area

were capable of salinity discrimination. (2) The salinity discrimination organ of the estuarine

crocody 1 id, Crocodylus porosus, is not located in the mouth. Dehy drated crocodiles

discriminate very precisely between salinities and do not drink hyperosmotic sea water when

they are immersed in it. However, they drink strongly hyperosmotic sea water when it is

dripped directly ont0 their tongues. Crocodiles must therefore have a salinity discrimination

organ on another part of the body. (3) Crocodylids but not alligatorids have sensory organs

on their post-cranial scales. The morphoiogy and ultrastructure of pits on the scales of C.

porosus was studied using SEM, TEM, and light rnicroscopy. Their structure is consistent

with a sensory function. Physiological shidy will be required to determine whether their

function is mechanosensory or chemosensory .

It has been an exciting year. Now in this totally inadequate space 1 will attempt to

thank the many people who have helped me out dong the way.

First of ail 1 wish to thank my supervisor, Prof. D. G. Butler, for providing suppon

while keeping me on a long leash when it came to research. 1 am especially grateful to

him for sending me ail the way to Singapore to follow up what was really just a hunch,

and for tolerating the encroachment of dip-nets and crocodiles arnong the glassware and

eels in his lab.

As my project dnfted îùrther and further away from comparative endocrinology, 1

came to rely more and more on my three adjunct supervisors. 1 wish to thank Dr. D. R.

Brooks for an inspiring introduction to the field of comparative biology, Dr. J. J. B.

Smith for his insights into sensory biology and experirnental design, and Dr. J. H. Youson

for advice on the interpretation of TEMs and for playing the useful role of devil's

advocate in the chemoreceptor-mechanoreceptor debate.

Many experts --herpetologists, morphologists, and physiologists --took time from

their own work to offer advice, suggest techniques, send me offprints, and answer my

letters and e-mail. 1 am especially grateful to T. H. Fritts, C. Gans, G. C. Grigg, S.

Hillyard, H. Hong, J. Lang, P. Maderson, N. Mrosovsky, 1. Orchard, T. Parsons, C. A.

Ross, R. Wassersug, R, Stephenson, H. D. Sues, A. Summers and G. Zug.

Toronto is not the ideal location for a crocodile biologist, and 1 have been

fortunate in k i n g able to visit places where crocodilians can be found. In Singapore,

Prof. T. J. Lam and his students generously provided laboratory space at the National

University of Singapore. Mr. Yap Boon Chark, Pnmary Production Department, helped

me through the bureaucratie maze associated with exporting crocodiles. Most of d l , 1

would like to thank Mr. and Mrs. Lee Bak Kuan, owners of the Long Kuan Hung

Crocodile Farm, and their daughters Peilin and Peihui who welcomed me like one of the

family. Mr. Lee showed me more crocodiles, from eggs to nesting adults, than 1 would

have thought possible in a month. Mrs. LRe taught me the secret to rearing hatchlings

successfully, and Peilin and Peihui devoted most of their summer holiday to helping with

my experiments and showing me Singapore. Also a big thankyou to Peilin, Mabel. and

the girls who helped get me and the crocs past the daunting Mr. Kwon and ont0 Our flight

home.

At the St. Augustine Alligator Farm, in St. Augustine, Florida, Kent Vliet let me

experiment on valuable and exotic hatchlings. At Sape10 Isiand, Dr. J. Alberts, Director

of the University of Georgia Marine Institute, made an exception for me to his usuai rule

about herpetologists, Charles Durant and Mary Price provided a warm welcome to the

island, and Greg Balckom and the Wildlife Management Students from Archibaid Baldwin

Agricul tural College heIped me coilect alligators.

In spite of al1 this travelling, 1 acnially spent most of the ycar in Toronto.

Essential technical assistance was provided by N. White, S. Norwood, R. Villadiego, E.

Lin, and E. Knapp, often at very short notice. My labmates, Gavin, Donald, and Colin

were very good Company (even though they won? stop teasing me about chiggers). 1

want to thank al1 rny friends in the Deparment. 1 know I'm going to miss you ail next

year. 1 especially want to thank Tuhin, Stevie and Jose for coffee, beer. moral support,

ruthless proofreading, tiring runs through deep snow, and (a mal test of friendship)

reading my entire thesis.

This research was funded by an NSERC operating grant to D. G. Butler. Persona1

support was provided by an Ann Sheppard Memorial Scholarship in Biology, from

University College.

Finally, 1 wish to thank the crocodiles. We've been through a lot together this

year -Singapore customs, the baggage scanner in the Seoul airport, vaseline. minnows,

CoCl,, and bags and bags of Instant Ocean Sea Salt. 1 wish them long and happy lives in

their new home at the St. Augustine Alligator F m .

LIST OE FIG-

Fig. 1.1. Distribution of osmoreplatory strategies across taxa The three strategies are as follows: (A) osmoconfoming, so that the M y fluids have an ionic composition similar to that of sea water, (B) osmoregulating by concentrating organic osmons in the body fluids, and (C) osmoregulating by excreting NaCl and maintaining a plasma osmolality below that of sea water.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Fig. 1.2. Independent derivations of salt-secreting glands in marine and estuarine lineages of non-mamrnalian amniotes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Fig. 2.1. Mass of water ingested by Cairnan crocodilus (n=6) foiiowing dehydration. Values are mean +/- s.e.m. Differences in arnount dnink at different salinities are not significant (P>0.05, ANOVA, Scheffe test). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Fig. 2.2. Body mass in gram (mean +/- s.e.rn.) of Ca. crocodilus (n=9), and mass of 20 ppt sea water ingested as a percentage of initial body mass (mean +/- s.e.m.): initial mass (prior to dehydration), dehydrated mass (following dehydration), mass after 15 minutes exposure to sea water, and after 75 minutes exposure. ' hdicates significant increase in mass (P4.001, rrnANOVA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Fig. 23. Body mass and water ingested (mean +/- s.e.m.) by dehydrated ALLigator mississipiensis (n=10) from a captive, freshwater population before and after 15 min exposure to (a) 30 ppt sea water and (b) fresh water. ' Indicates a significant mass increase from the dehydrated condition (Pd.OO1, t-test). The amount drunk by the group exposed to 30 ppt sea water is not significantly different from the amount dmnk by the group exposed to fresh water (t-test, PzO.05). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Fig. 2.4. Body mass and water ingested (mean +/- s.e.m.) by dehydrated A. mississipiensis (n=3) from an estuarine population, before exposure to water, after 15 min exposure to 30 ppt sea water, and after 15 min exposure to fresh water. ' Indicates a significant mass increase relative to the mass increase in sea water (paired t-test, P<0.025). . . . . . . . . . . . . . . . . . . . . . . . 34

Fig. 2.5. Body mass and water ingested (mean +/- s.e.m.) by dehydrated Crocodylus porosus (n=6), before exposure to water, after 15 min exposure to 30 ppt sea water, and after 15 min exposure to fresh water. ' Indicates a significantly pater mass increase relative to the mass increase in sea water (paired t-test, P<0.005). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Fig. 2.6. Body mass and water ingested (mean +/- s.e.m.) by dehydrated CrocodyLus siamiensis (n=lO) from a captive, freshwater population (a) before and after 15 min exposure to 30 ppt sea water, and (b) before and after 15 min exposure to fresh water. ' Indicates a significantly greater mass increase in fresh water relative to the mass increase in 30 ppt sea water (t-test, P4.005). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

vii

Fig. 2.7. Body mass and water ingested (mean 4- s.e.m.) by dehydrated Osteolaemus tetraspis (n=3), before exposure to water, after 15 min exposure to 30 ppt sea water, and after 15 min exposure to fresh water. ' Indicates a significantly p a t e r mass increase relative to the mass increase in sea water (paired t-test, P4.05). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Fig. 2.8. The phylogenetic distribution of salinity preference among crocodilian species examined. (F/S) indicates animals which drank both fresh water and sea water, and (F/-) indicates those which drank fresh water but not sea water. . . . . . . . . . . . . . . . . . . . . . . 41

Fig. 2.9. Two equally parsimonious hypotheses for the evolutionary significance of salinity preference in crocodilians. In (a) lack of salinity discrimination is the plesiomorphic condition for crocodilians, in (b) it is the derived condition in freshwater alligatorids. (F/S) indicates drinking of both fresh water and sea water; (F/-) indicates drinking of frcsh water but not sea water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Fig. 3.1. Methodology for Experiment 1. F=fresh water, S=30 ppt sea water. Dehydrateci crocodiles drink when immersed in fresh water (a). do not dnnk when immersed in 30 ppt sea water (b), drink 30 ppt sea water from a bottle (c), drink 30 ppt sea water from a bottle while immersed in fresh water (d), and drink fresh water from a bottle when immersed in 30 ppt sea water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Fig. 3.2. A dehydrated C. porosus drinking hyperosmotic sea water from a wash bonle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Fig. 4.1. (A) Ventral scales of a crocodylid, C. porosus, showing the pits (arrow). . . . . . . . . . . . . (B) Ventral scales of an alligatorid, A. mississipiensis, which lacks pits. 62

Fig. 4.2. Touch papillae (sensu von During, 1973) from the cranid scales of C. porosus: (A) head of a crocodile with touch papillae indicated (arrow), (B, C, D) SEMs of touch papillae from the mandibular region. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Fig. 4.3. SEMs of the pst-cranial pits €rom the ventral scdes of C. porosus: (A) ventro-lateral view, (B, C, D) ventral view. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Fig. 4.4. Light micrographs of cross-sections a pst-cranial pit (A) at l4OX magnification, showing diffuse pit region of the dermis (pr) and collagen-rich non-pit region (nr), and (B) at 640X magnification, showing stratum germinativum (sg) and stratum comeum (SC) layers of the epidermis (ep). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Fig. 4.5. TEM (2400X) of the apex of pit region of the dermis, showing high concentration of cells (bl=basal lamina of the stratum germinativum of the epidermis, f=fibroblast cell, ir=iridocyte). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

viii

Fig. 4.6. TEM (7400X) of fibroblast cells frorn the pit region of the demis (c-collagen fibres). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Fig. 4.7. TEM (8900X) of nerve tenninals (nt) supported by a fibroblast ce11 (0. . . . . . . 69

Fig. 4.8. TEM (135ûûX) of an iridocyte from the pit region of the demis. . . . . . . . . . . 69

Fig. 4.9. TEM (2700X) of epidermis from (A) the pit region, and (B) from another area of the same scale (sc=stratum comeum, sg=stratum germinativum, ddermis). . . . . . . . . . . . . . 70

Fig. 4.10. Summary illustration of the integumentary sense organ (sc=stratum comeum, sg=straturn germinativum, pr=pit region of the dermis, m o n - p i t region of the demis. d=derrnis). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Fig. 4.11. Altemate interpretations of the hinction of the integumentary sense organ: (a) mec hanoreceptor , (b) chemoreceptor, (c) osrnoreceptor. . . . . . . . . . . . . . . . . . . . . . . . . 74

LIST OF TABLES

Table 1.1. Osmolality. Na'. CI.. and K+ concentrations of plasma and cloacal urine of C . porasirs and A . mississipiensis exposed to 20 ppt sea water . . . . . . . . . . . . . . . . . . . . . . I l

. . . . . . . . . . Table 1.2. Phylogenetic trends in osmoregulatory strategies of crocodiiians 17

Table 2.1. Osmolaiity. Na' and K+. and blood hematocrit in cairnan plasma before and after dehydration by IO% of body mass . ' Indicates a significant increase (P4.05, paired t.test. corrected for mu1 tiple comparisons) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

LIST OF ABBREVIATIONS

ANOVA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anaiysis of variance

OC .....................................................Deg reecelcius

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ca2' Calcium cation (divalent)

COCI, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cobaltchloride

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cl' Chloride anion (monovalent)

cm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Centimetre

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ECM Extracellular matrix

F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Freshwater

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g Gravitational force

g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gram

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hr Hour

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ISO Integumentary sense organ

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K' Potassium cation (monovalent)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KCl Potassium chloride

kg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kilogram

krn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kilometre

kV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kilovolt

min . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Minute

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mosm Milliosmole

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MC Magnesium cation (divalent)

. . . . . . . . . . . . . . . . . . . . . . . . . . . pi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - Microlitre

p m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Micrometre

p o l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Micromole

mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Millimetre

rnM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Millimolar

n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Samplesize

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NaCl Sodium chloride

OsO, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Osmiumtetroxide

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ppt Parts per thousand

. . . . . . . . . . . . . . . . . . . . . . . . . . . . rrnANOVA Repeated measures analysis of variance

S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Seawater

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s.e.m. Standard error of the mean

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SEM Scanning electron micrograph

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TEM Transmission electron micrograph

xii

CHAPTER ONE

General Introduction:

Osmoregulation in Marine and Estuarine Environments

. 1: Generai Introduction -- -- --

Osmoregulatory strategies of marine animais

Animals which live in marine environments face the challenge of maintaining

- physiologicd function in a highly saline extemal environment A wide variety of

osmoregulatory mechanisms are employed, but these fa11 into three basic categories: (A)

tolerating high concentrations of inorganic osmons in the body fluids, (B) eliminating the

osmotic gradient by concentrating organic osmons in the body fluids, and (C) actively

excreting inorganic osmons so as to maintain an interna1 osmolality below that of the

surrounding sea water.

The term "sea water" will be used throughout this thesis to indicate ionic

composition but not concentration. Full sea water has a concentration of approximately

30 ppt. Thus "15 ppt sea water" means brackish water. The term "salt water" is avoided

because it is ambiguous and could mean any sait in soiution (e.g. NaCi. KCI, CoCI,, etc.).

The more precise term "NaCI solution" is also avoided, on the grounds that it is

misleading. Although sea water is mostly an NaCl solution, it also contains traces of

other physiologically important ions (e.g. Ca2', K*, ~ g 2 ' , etc.).

The distribution of different osmoregulatory strategies across taxa is summarised in

figure 1.1. The first and simplest strategy (A) is simply to osmoconform, so that the body

fluids have an ionic composition similar to that of sea water. This strategy is employed

by the marine invertebrates, as well as by the presumably basal vertebrate group, the

hagfishes (Myxinoidea) (Bentley 1971). With the exception of the hagfishes, however, al1

marine vertebrates employ some sort of mechanism to keep the concentration of NaCl

1 : General Introduction

Fig. 1.1. Distribution of osmoregulatory strategies across taxa. The three strategies are as

follows: (A) osmoconforming, so that the body fluids have an ionic composition similar

to that of sea water, (B) osmoregulating by concentrating organic osmons in the body

fluids, and (C) osmoregulating by excreting NaCl and maintaining a plasma osmolality

below that of sea water.

1: G e n e d Introduction

in the body fluids lower than that of the surrounding sea water. The elasmobrmchs have

adopted strategy (B), and concentrate urea in their tissues. Their plasma osmolality is

. thus comparable to that of sea water while their plasma NaCl concentration is kept much

lower than that of their surroundings. They obtain water from sea water by drinking and

then excreting the NaCl by rneans of a gland ("digitifoxm" or "rectal" gland) which

concentrates NaCI fiom the bloodstream and empties it into the posterior intestine

(Bentley 197 1 , Kirschner 1979).

Teleost fishes maintain a low plasma osmolality relative to their surroundings

(strategy C), and excrete excess NaCl by actively transporting Cl- out across the giil

membranes. This system can work equally well as an osmoregulatory mechanism in fresh

water, by actively transporting Cl' into the body, thereby allowing the retention of NaCl.

The larnprey , Petromyzon, uses the same osmoregulatory mechanism (J. H. Youson; pers.

cornrn.). The coelocanth, Lutimeriu osmoregulates by hyperuraemia of the interstitial

fluids (Bentley 1970, Kirschner 1979).

The Amphibia have not colonised the sea as other vertebrate groups have. The

closest thing to a marine amphibian is the Crab-eating frog, Ranu cancrivora, which lives

in southeast Asian estuariss. Interestingly, this species adopts two different

osmoregulatory strategies, depending on its ontogenetic stage. Tadpoles maintain a low

plasma osmolality relative to their surroundings @unson 1977). presumably by actively

transporting Cl- out across the gills, as teleost fishes do. Adults osmoregulate like

elasmobranchs, using hyperuraernia of the interstitial fluids (Gordon et al. 1961).

- 1 : Gcncral Introduction

Regardless of habitat type (marine, aquatic. terresuial), al1 amniotes maintain a plasma

osmolality of about 300 mosm kg", and excrete excess NaCl under hyperosmotic

. conditions. Marine and terrestrial habitats have similar osmotic stresses (the need to

conserve water and excrete salt) and as a result, marine and desert amniotes often use the

same mechanisms. Mamrnals are able to use the kidney for osmoregulation, since the

mamrnalian nephron has lmps of Henle which allow the concentration of NaCl in the

urine (Romer and Parsons 1986). Marine mammals are therefore able to excrete excess

NaCl renaily.

Non-rnammalian amniotes excrete excess salt extra-renally by means of specialised

salt-secreting glands. These glands have k e n independently derived in several lineages

(Fig. 1.2). Among the Chelonia, the lachrymal gland has become modified for salt-

secretion in marine-adapted lineages (Peaker and Linzell 1975). The tnily marine

representatives of this order, the sea aides (Cheloniidae, Dermochelyidae) dnnk sea

water, consume osmoconforming prey, and have lachrymal glands capable of high

secretory rates (up to 134-950 pmoles of Na' 100g" hi1) (Mazzotti and Dunson 1989) for

the excretion of excess salt.

In the marine lizard, Amblyrhynchus, the nasai gland has k e n adapted for

secreting salt (Peaker and Linzell 1975). In marine and estuarine snakes salt glands have

evolved independently at l e s t three times, through the modification of either the

premaxillary or the posterior sublingual glands in the mouth (Heatwole and Guinea 1993).

Birds excrete NaCl through supra-orbital salt glands which drain into the nares (Peaker

- 1: General Introduction

and Linzell, 1975).

Marine adaptation versus estuarine adaptation

The physiological stresses of estuarine environments are often discussed as

though they were simply those of marine environments manifested to a lesser degree.

This view, however, is rnisleading. Salinity in marine environments is high and constant.

In an estuarine environment, by contrat, the salinity fluctuates, and may change in a few

hours from highly saline to almost fresh. Estuarine habitats therefore have two important

characteristics with respect to osmoregulatory physiology: (1) the stress of a constantly

changing environmental osmolality, and (2) the opportunity to make use of intermittently

available fresh or brackish water.

Many of the physiologicai adaptations of estuarine animals are the same as those

of marine animals, but there are other adaptations which are specific to estuarine habitats.

Osmoconforrning invertebrates require special modifications to ailow them to survive

fluctuating environmental salinities without becorning flooded with water. Protozoans

actively remove water by means of water excreting vesicles (Brusca and Brusca 1990).

Osmoconforming metazoan invertebratess use osmoregulatory organs such as nephridia,

and a variety of mechanisms such as manipulating the concentrations of other inorganic

osmons normally present in sea water in their body fluids (e.g. ca2+. M$) so as to

modify osmotic gradients (Nicol 196 1).


Fig. 1.2. Independent denvations of salt-secreting glands in marine and estuarine lineages

of non-mammalian amniotes.

1: Generai Introduction

For animals which maintain a plasma osmolality lower than that of sea water by

actively removing NaCl, adapting to estuarine life presents less of a chailenge than

adapting to a fully marine existence, since there is simply less NaCl to actively remove.

This is especially true of estuarine animals which take advantage of the fluctuating salinity

by selectively drinking when the water is hypo-osmotic and avoiding drinking when it is

hyper-osmotic.

The selective drinking of only hypo-osmotic sea water is an important

osmoregulatory mechanism for estuarine reptiles. Estuarine populations of normally

freshwater species such as the turtle, Chelydra, and the water snake Nerodia, tack salt

glands but are able to survive brief periods of exposure to hyperosmotic salinities by only

drinking when the salinity is hypo-osmotic. Individuals from freshwater populations of

these species lack this adaptation and will drink al1 salinities indiscrirninately (Dunson

1980, 1986). Other estuarine reptiles such as the turtle Mahclemys and the snake

Cerberrcs have sait glands of low secretory capacity (0.4-221 p o l e s of Na' i00g-' hi1)

and combine this adaptation with selectively avoiding drinking hyperosmotic sea water

(Dunson 1985, Mazzotti and Dunson 1989).

It is important to note that salinity discrimination and selective drinking of only

hypo-osmotic salinities are specialisations for estuarine life and are completely useless as

osmoregulatory mechanisms in marine environments when the salinity is hyperosmotic

and unchanging. Fully marine osmoregulating animals must thetefore dnnk hyperosmotic

sea water to obtain water, and have a good mechanism for excreting the salt.

8


Osmoregulatory strategies of estuarine crocodilians

The living crocodilians are primarily freshwater animais, some populations of

which survive in estuarine areas. Two species, Crocodylus acuzus and C. poroslcs are

largely estuarine, and large individuais are often seen far out at sea (Taplin 1988).

However, the frequently used cornmon name "Salt-water crocodile" for C. porosus is

misleading. None of the living crocodilians is as specialised for marine life as the tntiy

marine reptiles, the sea snakes and sea turtles (discussed above). Crocodilians do,

however, possess some morphological and physiologicai specialisations for estuarine life.

Penneabiliry of the integwnent

Reducing integumental pemeability so as to minimise osmotic water loss is

probably an important osmoregulatory mechanism for crocodilians living in hyperosmotic

water. Unfortunately, however, Our understanding of the importance of integumental

permeability as an osmoregulatory mechanism has suffered from a lack of comprehensive

studies and from differences in methodology. Methodological differences include in vivo

studies versus studies of isolated pieces of skin, and studies of restrained versus

unrestrained animals. Distinguishing between dnnking and osmotic uptake of water

through the buccal epithelium can be very difficult, and as a resuit, much confusion

surrounds integumental permeability. The integumental pemeabilities of Crocodylus and

of Alligator, for example, have been contrastingly descnbed as approximately equal

. 1 : General Introduction

(Dunson and Mazzotti 1988) and as differing by an order of magnitude (Taplin 1988).

Ellis and Evans (1984) measured integumentary Na' and water flux in Alligator

mississipiensis in fresh water, and noted that the rate of Na' efflux was 0.02 v o l cmJ hr-

' in the head region. compared with 0.01 p o 1 cm-2 hr-' for the ~ s t of the integument.

The buccal epithelium of crocodylids and gavialids has a smooth, keratinised surface,

while in alligatonds the buccal epitheliurn is characterised by the presence of mucous

zones and dense papillae. This difference is thought to represent an adaptation by

crocodylids to reduce osmotic water loss through the buccal epithelium (Taplin and Grigg

1989). It would be interesting to measure the difference in permeability of the buccal

epithelium between crocodylids and alligatonds so as to detennine exactly how much of

an osmotic advantage a keratinised buccal epithelium confers on a crocodilian in a

hyperosmotic habitat. No data are currently available.

The renal-cloaca1 sysrem

The non-mammalian nephron lacks loops of Henle (Romer and Parsons 1986). and

the crocodilian kidney is therefore an unlikely route for the excretion of excess salt.

Schmidt-Nieslen and Skadhauge (1967) confinned this experimentally in C. acutus by

comparing osmolality and electrolyte composition of ureteral urine with that of cloaca1

urine. As Na' load was increased, ureteral urine showed no significant change in

electrolyte composition. In the cloaca, resorption of Na' and Cr was found to occur

under hypo-osmotic conditions. However, increased Na' loading led to a decrease in

10

- 1 : General Introduction

Table 1.1. Osmolality, Na', Cld, and K+ concentrations of plasma and cloaca1 urine of C.

- porosus and A. mississippiensis exposed to 20 ppt sea water.

Osmolality (mosm kg")

Crocodylus porosud 1 Alligator mississippiensi~

from Grigg (1981)

from Lauren (1985)

Plasma

302

130

114

3.8

Urine

277

10.1

9.7

21.5

Plasma

393

182.8

187

4.3

. .

Urine

387

150.0

25.5

53.5

- 1: Generai Introduction

cloacal resorption of these ions.

Grigg (198 1) collected cloacai urine from C. porosus captured dong a saiinity

gradient in order to investigate the possible role of the renal-cloacai system as an

osmoregulatory mechanism. NaCl composition of cloaca1 urine remained constant as

environmental salinity increased. However, urine osmolality increased, largely as a result

of increasing K+ concentration. There was also an increase in the solid component (K4,

~ g " , and ca2' salis) of the urine. suggesting that cloacal water resorption was occumng.

Lauren (1985) measured plasma and cloacal urine osmolality and ion

concentrations in A. mississippiensis which had been kept in water of different salinities

ranging from fresh water to 20 ppt (no data were recorded from higher salinities because

mortalities started to occur at 20 ppt). Baxd on their own observations of C. porosus and

C. n iloticus and compari son w ith Lauren' s ( 1985) observations of A. mississippiensis,

Taplin (1988) and Taplin and Lovendge (1988) point out that the concentration of Na' in

the cloacal urine of Alligator is markedly higher than that of Crocodylus (Table 1.1).

This may indicate that Alligator but not Crocodylus uses the renalcloacal system for Na'

excretion.

- 1: General Introduction

Lingual salt-secreting glands

With the exception of the mammds, al1 marine amniotes studied to date excrete

excess Na+ extra-renally through specialised glands. Given this precedent, the presence of

some sort of gland capable of secreting a hyperosmotic NaCl solution seemed a likety

answer to the question of how crocodiles survive in hyperosrnotic sea water. The

osmolalities of nasal and lachrymal secretions were duly measured in search of a possible

route for Na' excretion (Peaker and Linzell 1975). The lingual salt-secreting glands were

identified by Taplin and Grigg (1981) in C. porosus. These consist of 28-40 highly

vascularised glands, each with a duct leading to the surface of the tongue. Each gland is

divided into 14-20 lobular subunits, and blood flow from capillaries of the lingual vein is

from the centre of the gland outward to the periphery, countercumnt to the flow of

secretion as a mechanism for producing a concentrated NaCl solution (Taplin and Grigg

198 1, Franklin and Grigg 1993). Intra-peritoneal injection of methacholine chloride

stimulates secretion by the lingual glands of a NaCl solution 3-5 times more concentrated

than the plasma (Taplin and Grigg 1981). The secretory rate is comparable to that of

estuarine reptiles (e.g. Malaclemys) rather than marine reptiles (e.g. Chelonia) (Mazzotti

and Dunson 1989). Lingual glands capable of secreting a hyperosmotic solution of NaCl

(ma'] = 365 to 740 mm01 La') have been identified in d l crocodylids studied (12 species)

including freshwater species, while alligatorids and gavialids appear to lack salt-

secreting glands (Taplin et al. 1985).

Surprisingly, the lingual salt glands do not secrete NaCl in response to Na+ loading

13

. 1 : Gencral Introduction

(Taplin and Grigg 1981). Al1 rneasurements of secretory rate must therefore be made by

stimulating secretion through intra-peritoneal injection of the parasympathetic nerve

stimulant methacholine chloride. The failure of the lingual salt-secreting glands to

respond to Na' loading has been interpreted as evidence that these glands are not involved

in osmoregulation in estuarine environments (Mazzotti and Dunson 1989). However, the

same phenornenon has recently been reported in ostriches (Gray and Brown 1995).

Further study will be required to determine why some sait-secreting glands do not respond

to Na4 loading.

Other experiments have demonstrated indirectly the function of crocodile salt

glands. Taplin and Loveridge (1988) observed that C. niloticus became hypernatraemic

more rapidly when kept in sea water with lingual glands sealed with glue than the control

goup in which the glands wen not sealed. Franklin and Grigg (1993) found that in C.

porosus the volume of the vasculature supplying the lingual tands increased when animals

were kept in hyperosmotic sea water. The lingual salt glands of crocodylids have

generally been interpreted as an osmoregulatory adaptation for estuarine life. The

presence of lingual glands in freshwater crocodylids has therefore k e n considered

vestigial and interpreted as evidence of a marine ancestry (Taplin et al. 1985. Taplin and

Grigg 1989). An alternative hypothes is is that the lingual glands of freshwater

crocodylids are adaptations allowing the animal to deal with the osmotic stress of

aestivation (Mazzotti and Dunson 1989). The osmoregulatory physiology of aestivating

crocodiles has yet to be studied, however.

. 1 : General Introduction

The role of selective drinking

Selective drinking of only hypo-osmotic sea water has been shown to be an

important osmoregulatory mechanism for many estuarine reptiles (Dunson 1980, 1985,

1986, Dunson and Mazzotti 1989). Taplin (1984) demonstrated that estuarine C. porosus

wiII not drink hyperosmotic sea water even when severely dehydrated. Mazzotti and

Dunson ( 1984) found that C actrtus dehydrated by 10% of body mass could distinguish

very precisely between sea water of hyper- and hypo-osmotic saiinities, and would only

drink salinities of 9 ppt or less (these k i n g hypo-osmotic). Moreover, when the plasma

osmoldity was increased by further dehydrating them (by 20% of body mass) the

crocodiles drank sea water of salinities up to 15 ppt.

With the exception of these two studies, the study of salinity discrimination and

selective dnnking has been much neglected. Although it has been studied as a possible

osrnoregulatory mechanism for two species of estuarine crocodylid, neither salinity

discrimination nor selective drinking has been considered in a phylogenetic context. Their

underlying mechanism has yet to be studied, and they have been virtually ignored as a

possible confounding factor in skin permeability experiments comparing different

crocodilian species (e.g. Lauren 1985, Mazzotti and Dunson 1984).

Phylogenetic trends

Perhaps the most surprising result of recent research into the osmoregulatory

adaptations of crocodilians is the extent of the differences between alligatorids and

15

crocodylids. Although the farnilies Alligatoridae and Croçodylidae both have estuarine

representatives, the crocodylids possess a suite of morphological and physiological

modifications which make them bener adapted to estuarine life than alligatorids. These

are summarised in Table 1.2.

The best-studied of these. especidly in t e m of phylogenetic trends, are the

lingual salt-secreting glands in crocodylids, which are absent in alligatorids (Taplin et al.

1985). Another modification is the keratinised buccal epithelium of crocodylids, which is

presumabiy less permeable to salt and water than that of alligatorids (Taplin and Grigg

1989). Less well-studied are the differences in the renal-cloacal system of alligatorids and

crocodylids (Table 1.1). Taplin (1988) and Taplin and Loveridge (1988) interpret the

difference in cloaca1 urine Na' concentration between A. mississippiensis and two species

of Crocodylus kept in 20 ppt sea water as a phylogenetic difference between alligatorids

and crocodylids. They propose that alligatorids (which lack lingual sait-secreting glands)

are able to excrete Na' in their urine when exposed to hyperosmotic saiinities.

The presence of estuarine adaptations (e.g. lingual sait-secreting glands) in

freshwater crocodylids is of paiticular interest since these adaptations appear to have no

function in freshwater habitats. These have been interpreted as evidence for a marine

phase in the recent evolutionary history of crocodylids (Taplin et al. 1985, Taplin 1988.

Taplin and Grigg 1989).

. 1 : G e n e d Introduction

Table 1.2. Phy logenetic trends in osmoregulatory strategies of crocodilians.

Integumental permeability

Sai t-secreting glands3

Buccal epithelium'

Selective drinking *

Mainiy freshwater, some estuarine populations

Americas (except for one Asian species)

Mucous zones, dense papillae

Some Na' excreted

Croeod y lidae

Some species primarily estuarine

Yes

Keratinised to reduce osmotic water loss

Not used for Na' excretion

SUBJECT OF THIS THESIS

'Taplin (1988), 'Taplin and Grigg (1989). qaplin et al. (1985). 'Lauren ( 1985). %rigg (198 1 )

- 1 : General Introduction

Objectives of the thesis

The objective of this thesis was to study the role of salinity discrimination and

selective drinking as an osmoregulatory mechanism used by estuarine crocodilians.

S pecificail y, 1 attempted to determine (1) whether crocody lids and alligatorids differ in

their capacity for saiinity discrimination, and (2) what mechanism crocodilians use to

discriminate between salinities. Chapter 2 documents a series of experiments undertaken

to test for salinity preference in a variety of crocodilian species from freshwater and

estuarine habitats, in search of phylogenetic and ecological trends. Chapters 3 and 4

explore possible mechanisms underlying the phylogenetic difference revealed by the

experirnents in chapter 2. Specificaliy, chapter 3 describes an expriment undertaken to

determine the role of the buccal epithelium in salinity discrimination. Chapter 4 is a

detailed morphological study of a possible salinity discrimination organ. Finally, chapter

5 is an attempt to integrate the phylogenetic, morphologicai, and physiological data, and

to discuss areas of particular interest for future research.

C-R TWO

Habitat and Phylogeny Influence Salinity Discrimination

(Adapted from: Jackson. K., Butler, D. G., and D. R. Brooks. Habitat and phylogeny

influence salinity discrimination in crocodilians: implications for osmoregulatory

physiology and historical biogeography. Biological Journal of the Linnaean Society (In

press))

2 Habitat and Phylogeny Influence Salinity Discrimination

Abstract

The ability to discriminate fresh water from hyperosmotic sea water, and to avoid

drinking the latter, is known to be an important osmoregulatory mechanism for estuarine

crocody 1 ids. The crocodilian families Crocody l idae and Alligatoridae both include species

in w hich some populations inhabit brackish or coastal areas. Estuarine crocodylids,

however, are more common than estuarine alligatorids, and members of the crocodylid

farnily possess morphological specialisations (e-g. lingual salt-secreting glands, keratinised

buccal epithelium) which confer an advantage to them over alligatorids in adapting to

hyperosmotic conditions. This study was undertaken to determine whether the ability to

discriminate between hyper- and hypo-osmotic salinities is determined by habitat, as it is

in other normally freshwater reptiles, or whether, like morphological adaptations

associated with estuarine life, it has a phylogenetic bais.

Two species of freshwater alligatorid were found to drink fresh water and

hyperosrnotic sea water indiscriminately, while an estuarine population of a normally

freshwater ailigatorid species drank only fresh water. This indicated that salinity

discrimination is determined by habitat. However, al1 three crocodylid species tested

drank fresh water but not hyperosmotic sea water, suggesting that, in crocodilians, the

ability to distinguish between fresh water and sea water is influenced by phylogeny as

well as by habitat.

2: Habitat and Phyiogeny Influence Salinity Discrimination

Introduction

Reptiles inhabiting marine and estuarine environments face the challenge of

maintaining a constant plasma osmolality while living in a hyperosmotic medium. They

employ a variety of osmoregulatory strategies, including behavioural modifications such as

avoiding drinking hyperosmotic sea water together with morphological adaptations

associated with osrnoregulation such as salt-secreting glands and reduced integumental

permeability. Dunson (1980, 1985, 1986) and Mazzotti and Dunson (1989) studied the

physiological b a i s of putative marine adaptations in reptiles using snakes and turtles as

models. Dunson and Mazzotti (1989) recognised a number of conditions which they

interpreted as a gradient of evolutionary specialisations. In their model, the presumed

plesiomorphic condition is represented by aquatic (freshwater) snakes and turtles (e.g.

Nerodia, Chelydra). The second stage is represented by estuarine populations of the same

species, which differ from freshwater populations in k i n g able to tolerate limited

exposure to hyperosmotic sea water by selectively drinking only hypo-osmotic water

(Dunson 1980, 1986). Reptilian nephrons lack loops of Henle, and are therefore not

capable of producing a hypertonic urine. Like marine birds, therefore, some reptiles

possess extrarend salt-secreting glands capable of secreting a hypertonic NaCl solution.

The third stage of marine adaptation is marked by the appearance of salt-secreting glands

of low secretory capaci ty (volume and concentration) (e.g. Acrochordus, Malaclemys),

which allow a constant plasma osmolality to be maintained when used in conjunction with

selective drinking of only hypo-osmotic water @unson 1985). The fourth and final stage

2; Habitat and Phylogeny Influence Salinity Discrimination

is represented by the tmly marine reptiles, the sea snakes (Hydrophiinae, Laticaudinae),

sea turtles (Cheloniidae, Derrnochelyidae), and the marine iguana (Amblyrhychus). In

these species the sait-secreting glands are well developed and ailow the maintenance of a

constant plasma osmolality even when hyperosmotic sea water and osmo-conforming prey

such as jellyfish are ingesteci. Sait glands have been independently derived several times

in reptiles. Salt glands are sub-lingual in snakes, lachrymai in turtles, and nasal in iizards

(Peaker and Linzell 1975). Crocodylids but not alligatorids have lingual salt glands of

low secretory capacity (Taplin and Grigg 198 1, Taplin et al. 1982, 1985).

Crocodilians include species in which some populations inhabit brackish or

es tuari ne habitats. Crocodylus acutrcs and C. porosus (Crocody 1 idae) are primari 1 y

estuarine, while several other normally freshwater croçodylid species (e.g. C.

cala ph ractus, C. johnstoni, C. more letii, C. niloticus, and C. palustris) have some

estuarine populatio~s. Estuarine populations of Alligator mississippiensis, Caiman

crocodilus and Ca. lutirostris (Alligatondae) are also known to exist (reviewed by Taplin,

1988). Estuarine crocodylids are more common than estuarine alligatorids, perhaps

because they possess morphological specialisations, independent of habitat, which confer

an advantage to them over alligatorids in adapting to hyperosmotic conditions. The

lingual glands of crocodylids, for example, secrete an hyperosmotic solution of NaCl in

response to stimulation with methacholine chloride, while those of alligatorids secrete an

iso-osmotic solution (Taplin and Grigg 198 1, Taplin et al. 1985). Additionally,

crocodylids possess a keratinised buccal epithelium, so that osmotic water loss under

2: Habitat and Phylogcny Influenee Sdinity Discrimination

hyperosmotoc conditions is less than for alligatorids, which lack protection for the highly

permeable buccal epithefium (Taplin and Gngg, 1989).

Opportunistic drinking of fresh water or hypo-osmotic sea water is thought to be

an important mechanism allowing crocodilians inhabiting estuarine areas to maintain a

constant plasma osmolality in a fluctuating hyperosmotic environment. The estuarine

crocodyiids, C. acutus and C. porosus, as well as estuarine populations of C. johnstoni,

will not drink hyperosmotic sea water even when severely dehydrated (Mazzotti and

Dunson 1984, Taplin 1984, 1988, Taplin et al. 1993). Moreover, these species can

distinguish very precisely between brackish water of hyper- and hypo-osmotic salinities

(Mazzotti and Dunson 1984, Taplin 1984).

No experimental data exist on the ability of alligatorids to selectively avoid drinking

hyperosmotic sea water. Lauren (1985) found that juvenile A. mississipiensis died after

three weeks of continuous exposure to salinities of 15 ppt or greater, and Mazzotti and

Dunson (1984) observed that the mortality rate for A. mississippiensis was higher than chat

of C. acutus when both species were exposed to the same regime of altemating hyper- and

hypo-osmotic salinities. Bentley and Schmidt-Nielsen (1965) observed in the course of an

experiment on skin permeability in the freshwater alligatorid Ca. crocodilur that 20% of

their experimental animals died 18-24 hours after k i n g placed in a 33 ppt NaCl solution.

apparently as a result of drinking the medium.

The following study was undertaken to obtain experimental data on drinking of

h y perosmotic sea water by a freshwater alligatorid, Cu. crocodilus, for cornparison w ith


existing data from estuarine crocodylids, and to obtain data from representative alligatorid

and crocodylid species from freshwater and estuarine habitats, to determine whether the

- reported difference in capacity for salinity discrimination represented (1) a difference

between freshwater and estuanne populations, similar to those observed in other normally

freshwater reptiles in which some populations are estuarine, or (2) a difference between

crocodylids and alligatorids anaiogous to morphological differences associated with marine

adaptation between these two families.

Lack of salinity discrimination in Caiman crocodilrrs

Materials and Methods

Captive-raised juvenile (100-300 g) Ca. crocodilus were imported from Venezuela,

housed in a tank of dechlorinated tap water with a land/water choice, at 30°C (air

temperature), and fed live minnows ad libirum. Blood was sampled in order to determine

which sea water dilutions were hyper- and hypo-osmotic to cairnan plasma. Blood

samples of 0.2 ml were withdrawn from the caudal vein and centrifuged (4000 g) at 5OC

for 10 minutes. Plasma was collected from caimans before and after dehydration (10% of

initial body mass), and stored at -80°C until analysis. Na' and K+ concentrations were

measured by flarne photometry (Instrument Laboratories, Model 9431, and osmolality by

freezing point depression (Advanced Instruments micro-osmorneter, Model 3MO).

Two experiments were conducted to determine whether dehydrated caimans would

drink sea water. The first experiment measured the amount of water ingested by

- 2: Habitat and Phylogcny Influence Salinity Preference

Table 2.1. Osmolality, Na' and K4, and blood hematocrit in caiman plasma before and after

dehydration by 10% of body m a s . ' Indicates a significant increase (P4.05, paired t-test,

corrected for multiple comparisons).

Plasma 1 ~ l o o c î I

Before dehydration

n -

6

After dehydration

% change

hematocrit (%)

16.2 +/- 0.7 ,

6 23.û+/- 1.3'

+42%

'

Osmolality (m0s-g)

289 +/- 1.6

Na' (mM)

145.7 +/- 1.4

327 +/- 2.4'

+13%

K' (mM)

4.10 +/- 0.25

167.1 +/- 1.6'

+15%

4.38 +/- 0.30

no change

2: Habitat and Phylogeny Influence Salinity Discrimination

Fig. 2.1. Mass of water ingested by Caiman crocodil~~ (n=6) following dehydration. Values

are mean +/- s.e.m. Differences in amount dmnk at different salinities are not significant

(b0.05, ANOVA, Scheffe test).

10 15

salinity (ppt)


Fig. 2.2. Body mass in grarns (mean +/- s.e.m.) of Ca. crocodilus (n=9), and mass of 20 ppt sea

water ingested as a percentage of initial body mass (mean +/- s.e.m.): initiai mass (prior to

dehydration), dehydrated mass (following dehydration), mass after 15 minutes exposure to sea

water, and after 75 minutes exposure. ' Indicates significant increase in mass (P4.001.

rmANOVA).

initial dehydrated 15 min 75 min

2; Habitat and Phylogeny Influence Sdinity Discrimination

dehydrated caimans at different sea water dilutions. Sea water solutions in both

experiments were made using Instant Ocean sea salt (Aquarium Systems, Mentor, Ohio

44060, USA). Six unfed caimans were seiected from a group of nine and weighed. They

were then dehydrated by 10% of their initial body m a s , in a current of air (30°C),

re-weighed and placed in a 50-gallon plastic tank containing fresh water 10 cm in depth.

After 15 minutes, they were re-weigned to determine, by difference, the amount of water

ingested. This procedure was repeated for 5, 10, 15, 20, and 30 ppt sea water to

determine whether or not increases in salinity would affect drinking by dehydrated

caimans. Integumentary osmotic uptake of water in this species is known to be 1 . l pl

h f l (Bentley and Schmidt-Nielsen 1965). In a 200g animal immersed in fresh water for

15 minutes this represents a gain of only 95 pl (Sudace ma= 1 1.7~assO-", Dunson and

Mazzotti 1988), so this was not an important factor in measuring drinking by increased

m a s . Between experiments, the test caimans were retumed to the fresh water holding tank

for a period of at least seven days to allow time for rehydration. The second experiment

was used to determine the amount of water ingested by dehydrated caimans during a

longer period of exposure to hyperosmotic sea water. Body mass of nine unfed caimans

was measured before and after dehydration, following a 15-minute exposure, and finally, a

75-minute exposure to 20 ppt sea water.

Results and Discussion

Following dehydration there was a significant increase in plasma Na+ concentration

2; Habitat and Phylogcny Influence Salinity Discrimination

(15%) and osmolality (13%) but no significant change in plasma K* (Table 2.1). Blood

hematocrit increased by 42%, indicating that the vascular cornpartment had become

smailer in response to dehydration. Cairnan plasma was found to be hyprosmotic to 10

ppt sea water and hypo-osmotic to 15 ppt sea water, both before and after dehydration.

Following dehydration, caimans were transferred to fresh water or to one of a

series of diluted sea waters for an observation period of 15 minutes. At dl sea water

concentrations tested, dehydrated caimans drank a significant volume of water, regaining

of 2-5% of their initial body mass (Fig. 2.1). No overall statistical difference was found

in the arnount drunk of the different sea water dilutions (ANOVA, PM.05). and a Scheffe

test showed no significant difference in arnount drunk between any two dilutions (PM.05).

When the 15-minute observation period was increased to 75 minutes (Fig. 2.2) caimans

transferred to 20 ppt sea water continued to drink water and to increase in mass so that

the final mass was 99.6% of the original. This indicated that the caimans continued to

drink hyperosmotic sea water when given access to it for more than 15 minutes.

These results (Figs. 2.1, 2.2) show that caimans drink water of ail salinities tested

when dehydrated by 10% of body mass. That the caimans regained 2 4 % of their initial

body mass during 15 minutes of exposure to water over the range of salinities, is

interpreted as an indication of drinking, as opposed to integumental uptake by dehydrated

animals, because (1) the exposure period was short (15 minutes) in order to minimise the

possible effects of osmotic uptake, and (2) the animals increased in mass during exposure

to water of hyperosmotic salinities in which the osmotic gradient should have led to a


decrease rather than an increase in mass if diffusion across the integument had been an

important factor.

Results from the first experiment (Fig. 2.1) indicated a slight. though statisticaily

nonsignificant. decrease in the amount drunk at the two highest salinities (20 ppt and 30

ppt). It seemed possible that this result rnight indicate that although the caimans were

drinking at al1 salinities, they stopped dnnking after an initial mouthful at strongly

hyperosmotic salinities. However. when the experiment was repeated using 20 ppt sea

water, rhis time weighing the caimans twice and extending the period of exposure to 75

minutes, the mean increase in mass after 15 minutes was 6.5% of initial body mass (Fig.

2.2). This is the largest mass increase recorded at any salinity, suppoxting the view that

the amount drunk does not decrease at the highest salinities. When re-weighed after 75

minutes, the caimans were found to have further increased their mass (Fig. 2.2). This

result is evidence against the idea that the caimans stopped drinking after an initiai

mouthfül at hyperosmotic salinities.

These data provide quantitative evidence of the inability of the freshwater alligatorid.

Ca. crocodilus to osmoregulate by selectively drinking only water of hypo-osmotic

salinities. These results are strikingly different from those obtained in studies of the

estuarine crocodylids. C. ocutus (Mazzotti and Dunson 1984) and C. porosus (Taplin

1984). These species will not drink sea water of hyperosmotic salinities and are capable

of distinguishing very precisely between hyper- and hypo-osmotic sea water. What Our

experimental data for a freshwater alligatorid, and those of other researchers from


Fig. 2.3. Body mass and water ingested (mean +/- s.e.m.) by dehydrated Alligator

mississippiensis (n= 10) from a captive, freshwater population before and after 15 min

exposure to (a) 30 ppt sea water and (b) fresh water. ' Indicates a significant mass

increase from the dehydrated condition (P<0.001, t-test). The amount dmnk by the group

exposed to 30 ppt sea water is not significantly different from the amount dnink by the

group exposed to fresh water (t-test, P>0.05).

(6) s s o u Kpoq

(6) s s o u Kpaa


Fig. 2.4. Body mass and water ingested (mean +/- s.e.m.) by dehydrated A.

rnississippiensis (n=3) from an estuarine population, M o r e exposure to water, after 15

min exposure to 30 ppt sea water, and after 15 min exposure to fresh water. ' indicates a

significant mass increase relative to the mass increase in sea water (paired t-test,

PcO.025).

dehydrated seo water fresh water


estuarine crocodylids fail to reveal, however, is whether this difference in capacity for

behavioural osmoregulation represents (1) a phylogenetic constraint on the capacity of

aliigatorids to adapt to estuarine conditions (e.g. inability of alligatorids to taste salt), (2) a

behavioural modification with the potential to evolve independently in any crocodilian

population exposed to fluctuating sa1 inities, or (3) a behaviour learned by individual

crocodilians exposed to fluctuating saiinities (Le. without a genetic basis).

Infïuence of Habitat

Materials and Meth&

This experiment assessed the effect of habitat in determining the capacity for

salinity discrimination in crocodilians, by comparing Alligator mississippiensis from

freshwater and estuarine populations. Experiments on freshwater A. mississippiensis were

perforrned on captive-bred hatchlings (52-68g) (n=20) at the St. Augustine Alligator Farm

in Florida. Estuarine A. mississippiensis juveniles (248-372g) (n=3) were collected from a

freshwater pond on Sapelo Island, one of a string of barrier islands off the Coast of

Georgia. Sapelo Island is approximately 20 km long by 6 km wide and is separated from

the shore by a salt marsh 10 km wide, although it formed part of the mainland as recently

as five to ten thousand years ago (Martof, 1963). Alligators from this population live in

the freshwater ponds and salt marshes of the island. Ailigators ranging in size from large

juveniles (>lkg) to adults have k e n observed on the beach facing the Atlantic Ocean, and

large individuals are often seen several kilometres from shore. Captured alligators were

2: Habitat and Phylogeny Influence Sdinity Discrimination

housed temporarily in fibreglass tanks (approx. M)cm3), one alligator per tank, in an

outbuilding where they were exposed to outdoor temperatures but protected frorn rain and

- direct sunlight.

In preparation for the expenment, alligators were dehydrated by 10% of initial

body mass, to stimulate thirst, by keeping them out of the water for 24-4û hours. Once

dehydrated, the animals were weighed and transferred to a tank containing either fresh

water or 30 ppt sea water 10 cm in depth. After 10- 15 minutes they were rernoved frorn

the water, blotted dry, and reweighed in order to determine, by difference, the amount of

water ingested. For the freshwater alligators, in which the sample size was large, the

animals were separated into two groups of ten. One group was exposed to sea water and

the other to fresh water. For the estuarine alligators, the sample size was much smaller.

Al1 the dehydrated animals were therefore exposed initially to 30 ppt sea water and then,

if they did not drink, transferred to fresh water, since response (dnnking versus not

drinking) to sea water would be more informative than response to fresh water.

Moreover, animals which did not dnnk 30 ppt sea water in the course of 15 minutes

exposure were still dehydrated by 10% of initial body mas, and could therefore be

transferred to fresh water afterward and used to test response to fresh water. Sea water

solutions were prepared as described above for Ca. crocodiius.


Freshwater A. mississippiensis increased in mass following exposure t o fresh water


Fig. 2.5. Body mass and water ingested (mean +/- s.e.m.) by dehydrated Crocodylus porosus

(n=6), before exposure to water, after 15 min exposure to 30 ppt sea water, and after 15 min

exposure to fresh water. ' Indicates a significantly greater mass increase relative to the mass

increase in sea water (paired t-test, P<0.005).

dehydrated seo w a t e r fresh water


Fig. 2.6. Body mass and water ingested (mean +/- s.e.m.) by dehydrated Crocodylus siamiensis

(n= 10) from a captive, freshwater population (a) before and after 15 min exposure to 30 ppt sea

water, and (b) before and after 15 min exposure to fresh water. ' Indicates a significantly greater

mass increase in fresh water relative to the mass increase in 30 ppt sea water (t-test, P4.005).

(ssouj Apoa Z) palsa6ui ~a10M

(6) ssow Aooc

(6) s s o u Apoa

2: Habitat and PhyIogeny Influence Sdinity Discrimination

Fig. 2.7. Body mass and water ingested (mean +/- s.e.m.) by dehydrated Osteolaemus tetmspis

(n=3), before exposure to water, after 15 min exposure to 30 ppt sea water, and after 15 min

exposure to fresh water. ' Indicates a significantly greater rnass increase relative to the m a s

increase in sea water (paired t-test, Pe0.05).

body mass b-1 water ingested

dehydrated seo wate r fresh woter


and to 30 ppt sea water (Fig. 2.3). There was no significant difference in amount of water

drunk in fresh water versus sea water. By contrast, the estuarine A. mississippiemis drank

fresh water but not sea water (Fig. 2.4). This result supports the hypothesis that the

ability to selectively drink only water of hypo-osmotic salinities is determined at least in

part by habitat. G. C. Grigg (pers. comm.) has observed that estuarine populations of Ca.

latirostris also avoid drinking hyperosmotic sea water.

Other estuarine populations of normally freshwater reptiles follow the same pattern.

In the turtle, Chelydra, and the aquatic snake, Nerodia, individuds from freshwater

populations drink al1 salinities, whereas those from estuarine populations selectively avoid

drinking hyperosmotic sea water (Dunson 1980, 1986).

Influence of Phylogeny

Materials and Methodr

In order to evaluate the possible role of phylogeny in determining the capacity for

salinity discrimination in crocodilians, an attempt was made to obtain salinity

discrimination data from a large number of crocodilian taxa so as to reveal phylogenetic

patterns. Experiments were therefore undertaken to test for salinity preference in an

addi tional t hree crocody lid species, C. porosus, C. siamiensis. and Osreolaernus tetraspis.

Of these, two were exclusively freshwater crocodylid species: C. siamiensis (Asian) and

0. tetraspis (African). Data from 0steolaemu.s were especially interesting from a

phylogenetic perspective, since this genus is the sister group of Crocodylus (Fig. 2.8).


Although it is known that estuarine C. porosus and C. acutus hatchlings are able to

distinguish very precisely between hyper- and hypo-osrnotic salinities (Taplin 1984,

- Mazzotti and Dunson 1984), we included C. porosus in our study and used captive-bom,

freshwater-raised hatchlings which had never previously been exposed to sea water, so as

to rule out the possibility that avoidance of drinking hyperosmotic sea water is a

behaviour tearned by individual crocodilians in response to exposure to sea water.

Experirnents with C. siamiensis (n=20), and 0. tetraspis (n=3) were carried out at

the S t. Augustine Alligator Farm, using captive-bom, freshwater-raised hatchlings (6 1 -78g.

0. tetraspis; 54-696, C. siamiensis). Freshwater-raised C. porosus hatchlings ( 127- 17 1 g)

(n=7) were obtained from the Long Kuan Hung Crocodile Farm in Singapore, and

experiments were performed under laboratory conditions at the University of Toronto.

The experimental procedure was the sarne as that described above for the estuarine and

freshwater alligatoriàs. For C. simiensis, in which the sample size was relatively large

(n=20), the animals were divided into two groups of ten. One group was exposed to fresh

water and the other to 30 ppt sea water. The sample sizes of 0. tetraspis (n=3) and C.

porosus (n=6) were smaller. Animals were therefore first exposed to 30 ppt sea water and

then, if they did not drink, transferred to fresh water, as described above for estuarine A.

mississippiensis.


Al1 three crocodylid species drank fksh water but not 30 ppt sea water. C.


Fig. 2.8. The phylogenetic distribution of salinity preference among crocodilian species

exarnined. ( W S ) indicates animals which drank both fresh water and sea water, and (FI-)

indicates those which drank fresh water but not sea water.

2: Habitat and Phylogcny Influence Salinity Discrimination

porosus (Fig. 2.5) and 0. tetrmpis (Fig. 2.7) increased in mass significantly more in fresh

water than in sea water. The mass increase in the freshwater group of C. siamiensis was

significantly greater than that for the sea water group (Fig. 2.6).

The cornparison of fresh water and estuarine populations of A. mississipiensis

indicated that for alligatonds at least, the capacity to discriminate between fresh water and

hyper-osmotic sea water, and to avoid drinking the latter, is an adaptation found only in

populations inhabiting areas where they are exposed to sea water. However, the results of

the experiments on the freshwater crocodylids, C. siamiensis and 0. terraspis. and on

freshwater-raised hatchlings of the estuarine species, C. porosus, suggest that phylogeny is

also involved. Al1 crocodylids, whether freshwater or estuarine md with or without

previous experience of hyperosmotic sea water, discriminate between fresh water and

hyperosmotic sea water and will not drink the latter. Although previous studies have not

tested freshwater crocodylids, it has previously been shown that estuarine C. acuzus

(Mazzotti and Dunson, 1984) as well as estuarine populations of the normaily freshwater

C. johnstoni (Taplin et al. 1993) selectively avoid drinlcing hyperosmotic sea water.

General Discussion

Al1 crocodylid species, whether estuarine or freshwater, drank only fresh water.

Among the alligatorids, however, only those from an estuarine population distinguished

between salinities. These results are presented in Fig. 2.8. superimposed on a

phylogenetic tree depicting the evolutionary relationships of the taxa involved (Norell, fide

2; Habiîat and Phylogcny Innuencc Saihity Discrimination - --

Benton and Clark 1988). In crocodilians. therefore, the capacity for salinity

discrimination has a strong phylogenetic component, analogous to morphological

- adaptations associated with estuarine life. such as lingual salt-secreting glands and a

keratinised buccal epithelium, which are present in crocodylids and absent from

alligatorids. A search for the physiological mechanism underlying salinity discrimination

in crocodylids may shed new light on the evolutionary significance of this adaptation. It

would be useful, for example. to know whether crofodylids and estuarine alligators use

the same mechanism to distinguish between fresh water and sea water.

Di fferences in osmoregulatory phy siology between crocody lid and alligatorid

crocodilians are of particular interest in the context of hwo conflicting hypotheses to

explain the cumnt global distribution of crocodilians. The transsceanic migration

hypothesis (Densmore 1983). explains the distribution of living cracodilian species as the

result of a post-Pliocene tram-oceanic migration on the part of a marine-adapted ancestral

crocodylid. This hypothesis depends on an upper Cretaceoudearly Tertiary divergence

between crocodylid and alligatorid lineages, which is more ment than indicated by the

fossil record (Buffetaut 1982, Si11 1973, Steel 1968), but supporteci by molecular clock

calculations based on haemoglobin sequence data (Densmore 1983). Lingual sait-secreting

glands have been interpreted as crocodylid synapomorphies associated with adaptation to

marine conditions on the part of a crocodylid ancestor, and consistent with the trans-

oceanic migration hypothesis (Taplin et al 1985, Taplin and Grigg 1989). as has the

keratinised (and consequently less permeable) buccal epithelium of crocodylids and

2: Habitat and Phylogcny Influence Salinity Discrimination

Fig. 2.9. Altemate hypootheses for the evolutionary significance of salinity preference in

crocodilians. In (a) lack of salinity discrimination is the plesiomorphic condition for

crocodilians, in (b) it is the derived condition in freshwater alligatorids. (F/S) indicates

drinking of both fresh water and sea water. (F/-) indicates dnnking of fresh water but not

sea water.


gavialids and the non-keratinised buccal epithelium of alligatorids. The presence of sait-

secreting glands in freshwater crocodiles is thus considered vestigiai.

An alternative hypothesis ex plains the distribution of living crocodilians as the

result of speciation and upstream migration by a widely distributed, estuarine ancestral

group. Systematic evidence from the cocvolving digenean parasites of crocodilians

indicates a cosmopolitan distribution in the early Cretaceous, which supports the fossil

data suggesting an ancient origin (Brooks 1978, Brooks and O'Grady 1989, Brooks and

MacLennan 1993). However, the parasite data also show a mixture of freshwater (e.g.

digeneans of the family Proterodiplostomidae) and estuarine-derived parasite groups (e-g.

digeneans of the subfamily Acanthostominae), and could therefore be consistent with

either a freshwater or an estuarine origin.

The phyiogenetic significance of salinity discrimination in crocodilians can be

interpreted in four different ways, depending on whether the capacity for salinity

discrimination is assumed to be the plesiomorphic or the derived condition, and on

whether the Crocodilia had a cosmopoliîan distribution in the Cretaceous (as suggested by

the parasite data) or diverged more recently (as suggested by the molecular data). The

first possibility (Fig. 2.9.a.) is that lack of salinity preference is plesiomorphic, and that

the ability to distinguish between salinities has evolved independently in alligatorid

populations exposed to sea water, and in an ancestral crocodylid. This interpretation is

consistent with both the trans-oceanic migration hypothesis and the estuarine origin

hypothesis depending on the time scaie involved. If the marine adaptation by the


ancestral crocodylid is recent (after the separation of the continents), the tram-oceanic

migration hypothesis is supported. However, if the adaptation at the base of the

- crocodylid lineage occurred prior to the break-up of Pangaea, it could also be interpreted

as consistent with the hypothesis that modem crocodilians arose from widely distributed

estuarine ancestors and that upstrearn migration and Ereshwater adaptation occurred

secondarily .

An alternative interpretation (Fig. 2.9.b.), is that the ability to distinguish between

salinities is plesiomorphic and has been secondarily lost in fieshwater alligatorids. This

view is consistent with the estuarine origin hypothesis, although it still does not

necessarily require a marine or estuarine ancestor. At this point, each of these trees

requires exactly thme steps. Obtaining data from more taxa, especially from Gavialis and

from basal alligatorids (e.g. Palaeosuchur) should help to determine which of these two

trees more accurately reflects evolutionary history.

Although these two alkrnatives each require an equal number of evolutionary

events, it is not known whether or not the derivation of salinity preference from lack of

salinity preference oçcurs more readily than the opposite, lack of salinity preference from

salinity preference. It is also not known whether it is possible for a population to

secondarily lose its capacity to avoid drinlcing hyperosmotic sea water. In contrast to the

reverse situation, in which lack of salinity preference confers an obvious selective

disadvantage on an individual inhabiting an estuarine are& there is no immediately

obvious disadvantage associated with the latent capacity for sea water avoidance in an

2; Habitat and Phy logtny Influence Salinity Discrimination - - -

individual living in fresh water. The same question applies to morphological

speciaiisations associated with marine adaptation. The presence of lingual salt-secreting

- glands in crocody lids, for exarnple. has been interpreted as a crocodylid synapomorphy

consistent with the trans-oceanic migration hypothesis (Taplin and Grigg 1989). An

alternative interpretation is that the presence of lingual salt-secreting glands is

plesiomorphic, reflecting the estuarine ancestry of the group, and that their absence from

alligatorids represents a secondary loss associated with upstrearn migration and speciation.

Once again, it is not known whether Ioss of salt-secreting glands is a likely result of

freshwater adaptation by estuarine reptiles. Part of the problern is that models of marine

adaptation in reptiles are based on studies of snakes and turtles, and assume that the

direction of the evolutionary trend is aiways from fresh water to sea water (e.g. Dunson

and Mazzotti 1 989). Such models are therefore not necessari1 y transferable to

crocodilians, for which b t h historical biogeogqhical sceniuios (trans-oceanic migration

and estuarine origin hypotheses) involve estuarine to fresh water adaptation at some point.

Further study of physiological adaptation to fresh water by marine or estuarine reptiles

may provide answers to some of these questions and a better ba i s from which to evaiuate

alternative reconstructions of the historical biogeography of the Crocodilia

CWU"ER THREE

Evidence for Integumental Chemoreception in C. porosus

3: Evidence for Intcgumental Chemoreccption - - -

Abstract

Crocodylids possess a suite of morphological and physiological specialisations

- which make them better-adapted than alligatorids to the osmotic stresses of estuarine

habitats. One of these modifications is a coating of keratin on the buccal epithelium to

reduce osmotic water loss. An expected side-effect of a coating of this sort might be

reduced chemosensory capacity on the buccal epithelium. However, another phylogenetic

di fference associated w ith osmoregulation is that al1 crocody lids are able to distinguish

very precisely between sea water of hyper- and hyposmotic saiinities, whereas in

alligatorids this adaptation is only found in individuals from estuarine populations.

This study investigated the role of the buccal epithelium in salinity discrimination

in the Estuarine crocodile, Crocodylus porosus. When immersed in water, dehydrated

crocodiles drink fresh water but not hyperosmotic sea water. However, crocodiles do

drink hyperosmotic sea water when it is dripped directly into their mouths. Crocodiles

drink hyperosmotic sea water dripped into their mouths while their bodies are immersed in

fresh water, as well as fresh water dripped into their mouths while they are immersed in

sea water.

These results suggest that C. porosus does not use the buccal epithelium for

salinity discrimination, and that one or more chemosensory organs must be located on

another part of the body.

3: Evidenct for Intcgumental Chemottception - - -- . . - - - - ~ - - --

In traduction

Salinity discrimination and selective drinking are important osmoreplatory

- mechanisms for esniarine crocodilians (chaptcr 1). This mechanism has a phylogenetic

component, as al1 crocodylids, regardless of habitat (freshwater or estuarine) discriminate

between fresh water and hyperosmotic sea water and will not drink the latter.

Ailigatorids, however, are only capable of salinity discrimination if they belong to an

estuarine population (chapter 2).

Crocodylids possess a suite of estuarine adaptations which are absent in alligatorids

(chapter 1). The ability to discriminate between salinities (regardless of habitat or

experience) is one of these (chapter 2). Paradoxically, another of these adaptations is a

modified buccal epithelium. In crocodylids the buccal epithelium is smooth and covered

by a yellowish layer of keratin, presumably to reduce osmotic water loss under

hyperosmotic conditions (Taplin and Grigg 1989). In alligatorids, by contrast, the buccal

epithelium is characterised by the presence of mucous zones and dense papillae. Taplin

and Grigg (1989) have speculated that the keratin coating on the buccal epithelium of

crocodylids may interfere with sensory transduction. It seems surprising, therefore, that

crocodylids are better at tasting salinity differences than alligatorids.

Given these phylogenetic differences between crocodylids and alligatorids in

salinity discrimination capacity and in buccal morphology, it seems possible that

crocodylids and alligatorids may use different mechanisms to distinguish between sea

water of hyper- and hypo-osmotic salinities. The following study was undertaken to

3: Evidcncc for Intcgumental Chcmomxption -

investigate the mechanism used to distinguish between salinities by the Estuarine

crocodile, Crocodylus porosus, especially the role of the keratinised buccal epi thelium in

saiinity discrimination and selective drinking.

Although the capacity for salinity discrimination is well-donimented in C. porosus

(Chapter 2, Taplin 1984, Mazzotti and Dunson 1984). al1 studies of selective dnnking

have used animals immersed in tanks of water, so that al1 parts of the crocodile are

exposed to the sarne salinity. The purpose of the following study was to study selective

drinking in animals in which the buccal epithelium and the post-cranial integument were

exposed to different salinities, in order to determine what part of the crocodile tastes salt.


JuveniIe C. porosus ( n 4 ) (250g) were obtained from the Long Kuan Hung

Crocodile Fam in Singapore. Crocodiles were housed in a tank of dechlonnated tap

water with a l adwate r choice, at 30°C (air temperature), and fed live rninnows ad

libitum. In preparation for the expriment, crocodiles were dehydrated by 10% of their

body mass, in order to stimulate thirst. This was achieved by preventing access to water

for 48 to 72 hours. Crocodiles were weighed every 1-6 hours until 10% dehydration was

reached.

Two plastic tanks (approx. 20cm X 60cm) were filled to a depth of approx. 5 cm,

one with fresh water (dechlonnated tap water), the other with 30 ppt sea water. Two

wash bottles were also filled, one with frcsh water, the other with 30 ppt sea water. The

3: Evidence for Integumcntal Chemorcception .

Fig. 3.1. Methodology for Expriment 1 . F=fresh water, S=30 ppt sea water.

Dehydrated crocodiles drink when immersed in fresh water (a), do not drink when

immersed in 30 ppt sea water (b), drink 30 ppt sea water from a bottle (c), drink 30 ppt

sea water from a bottle while irnmersed in fresh water (d), and drink fresh water from a

bottle when immersed in 30 ppt sea water.

DRINKS

DOES NOT DRINK

DRINKS

DRINKS

3: Evidcncc for Intcgumental Chcmorcception

30 ppt sea water solution was made using the rnethod described in chapter 2. Following

dehydration, crocodiles were allowed access to drinking water (either fresh water or 30

ppt sea water) in the form of water dripped into their mouths with a wash bottle. To test

whether the crocodiles would drink when the salinity of the water in contact with their

buccal epithelium differed from that in contact with the rest of the integument, crocodiles

were offered drinking water from the wash bottle while sitting in one of the tanks of

water. Crocodiles were not restrained during these experiments. They showed little

inclination to submerge their heads in the water however, perhaps because of the

shallowness of the water, the small size of the tanks, and the presence of the researcher.

Three permutations were tested: (1) 30 ppt sca water from a wash bottle while out of the

water (n=6) (Fig. 3. lx ) , (2) 30 ppt s t a watcr from a wash bottle while sitting in fresh

water (n=3) (Fig. 3.l.d). and (3) fresh water from a wash bonle while sitting in 30 ppt sea

water (n=3) (Fig. 3.1 .e).

Following the 30 min period of exposure, crocodiles were removed from the water

and blotted dry. As in chapter 2, an increasc in body mass following exposure to water

was considered evidence of drinking. The methodology of this experiment differed from

chapter 2 in that the crocodiles had water dripped into their mouths from a wash bottle

rather than k i n g immersed in it. Since drinking from a wash bonle requires more time,

the tirne Iimit was increased to 30 min. This introduced the problem of allowing for

different rates of osmotic and evaporative water loss for crocodiles in fresh water, in sea

water, and out of the water. Because of these problems, and the problems of comparing

3: Evidence for Integumcntal Chcmorcception -

Fig. 3.2. A dehydrated C. porosus drinking hyperosmotic sea water from a wash bottle.

3: Evidcnct for Intcgurncntal Chemorcccption

amounts drunk from this experiment with amounts from prcvious experiments (chapter 2)

in which the animals were immened in the water, this expriment tested only wheiher the

crocodiles drank or did not drink, rather than comparing amounts of water ingested. An

increase in body mass of 2g in 30 min indicated that water had been ingested. This a

conservative estimate. as Mavotti and Dunson (1984) used an increase of Ig in one hour

as indicating drinking in C. acurus, even though these were largcr animals (20e70g) and

immersed in water rather than having it dripped into their mouths.

Resuits

The results are surnmarised in figure 3.1. Revious experiments (chapter 2) have

shown that dehydrated C. porosur drink when immersed in fresh water (Fig. 3.1 .a) and do

not drink when immersed in 30 ppt sea water (Fig. 3.1.b).

However, in this study, dehydrated crocodiles (n=6) did drink 30 ppt sea water

when it was dripped directly into their mouths (Fig. 3.1 .c). Crocodiles (n=3) also drank

30 ppt sea water when it was dripped into their mouths while they were irnmersed in a

tank of fresh water (Fig. 3.l.d). The dehydrated crocodiles (n=3) also drank fresh water

when it was dripped into their mouths while they were immersed in a tank of sea water

(Fig. 3.1 .e).

Discussion

Previous experiments (chapter 2) showed that dehydrated crocodiles drink when

3: Evidence for Intcgumcntal Chcmoreccpùon

immersed in fresh water and do not drink when immersed in hyperosmotic sea water. In

this expenment, however, it was found that dehydrated crocodiles do drink hyperosmotic

- sea water when it is dripped directly ont0 the buccal epithelium.

The fact that crocodiles immersed in water drink fresh water but do not drink

hyperosmotic sea water indicates that crocodiles have a mtchanism for distinguishing

between salinities. This presumably allows hem to avoid drinking when it is osmotically

disadvan tageous to do so. Although dehydrated crocodiles do not drink hyperosmotic sea

water when irnmersed in it, they do drink it when it is dripped dirrcdy into their mouths

(Fig. 3.2). This suggests that the salinity discrimination mechanism must be located

somewhere other than on the buccal epithelium. The apparent lack of chemosensation in

the mouth may be the rtsult of the keratin coating of the buccal epitheiium interfenng

w i th sensory transduction.

To test the hypothesis that the salinity discrimination organ of crocodiles is located

on the integument of the body rather than in the mouth, two further experiments were

undertaken in which crocodiles had water of one salinity dripped into their mouths while

imrnersed in waîer of another salinity. Dehydrateci crocodiles drank hyperosmotic sea

water dnpped directly into their mouths while immersed in fresh water (Fig. 3.l.d),

supporting the hypothesis that crocodiles rely on scnsory information h m their skin

rather than from their mouths to detennine salinity. In this case, the crocodiles were

presumably drinking in response to the prcsencc of fresh watcr in contact with their skin.

in spite of the fact that the water k i n g drippcà into their mouths was strongly hyperosmotic.

3: Evidcnce for Intcgumcntal Chcmorcccption

Dehydrated crocodiles do dnnk f ~ s h water dripped into their mouths when they

are imrnersed in sea water (Fig. 3.1.e). Following the same reasoning as above, if the

- salinity of available drinking water is detennined by an organ on the skin rather than in

the mouth, a crocodile ought not to drink if the water in contact with its skin is

hyperosmotic, even if the water actually k i n g dripped into its mouth is fmsh.

One possible explanation for this .apparently anornalous result is that the crocodiles

recognise that the contents of the wash bottle belongs to a different body of water than the

one they are imrnersed in. The faft that the crocodiles drank hyperosmotic sea water

ciripped into their mouths indicates that they are unable to discriminate salinities using

only the buccal epithelium. It may be thercfore, that since the crocodiles are unable to

taste the saiinity of water drïpped into their mouths from above, they assume that it is

fresh water. Although under laboratory conditions this may ôe a dangerous assumption to

make, in an ecologicai context it makes a certain amount of sense, as rainwater is never

hyperosmotic.

This idea could be ttsted by rcpeating the experiments outlined above, in which

the salinity of the water in contact with the integument differs from the water in contact

with the buccal epithelium. However, instead of having the two saiinities divided into

two separate bodies of watcr (the wash bottle and the tank), the experiments should be

carried out under conditions in which the intcgumcnt and the buccal epithelium can be

ex posed to di fferent salinities, w ithou t the crocodiles perceiving them as separate bodies

of water. One possible way of acheiving this might be in a flow tank in which waters of

3: Evidtnce for Inwgumcntal Ckrnoreception --- - p~

different salinities could bc separated by cumnu.

One important expcrimcnt is notably absent from this chapter What mechanism

do estuarine alligatorids use to discriminate ktwecn salinities? My hypothesis is that

crocodylids have adapted to estuarine habitats by devcloping a keratinised buccal

epithelium, and then made up for the loss of chemosensory function in the mouth by

developing integumentai chemorcctption. Aiiigatorids lack a keratinised buccal

epithelium. and ought therefore to be able to use the buccal epitheliurn for sensoy

transduction. Although freshwatcr alligatorids drink sea water of al1 salinities

i ndiscriminatel y (Chapter 2). alligatorids from estuaxine populations arc able to

discriminate between salinities, like crocodylids. If my hypothesis is correct, alligatorids

and crocody 1 ids use dif fertnt mechanisms to discriminate between salinities: estuarine

alligatorids taste sea water using the buccal epithelium, while crocodyüds use the

integument. If this expriment were repeated using estuarine aiiigatorids, therefore, the

alligatorids should not drinlc hyperosmotic sea water when it is dripped directiy into their

mouths.

CHAPTER FOUR

Morphology and Ultrastmcture of a Putative Integumentary Sense Organ

Abstract

The structure of darkiy pigmented pits which arc prrsent, one per scale, on the

. post-cranial scales of crocodylids was studied using light microxopy and scanning and

transmission electron microscopy. The stratum comeum of the epidermis in the area of

the pit is thinner than on the rcst of the scale. The outer surface of the seanirn comeum

covering the pit is continuous with a dcepr layer of the straRIm comeum covering the

rest of the scaie, and is rcvealed through a circular opening in the outer surface of the

stratum comeum proper. Widel y disperscd fibroc ytes, ne rve terminais and

chromatophores are found throughout the pit region of the dermis, but these elements are

concentrated in the area immediateiy beneath the stratum germinativum. The stratum

germinativum of the epidennis is thicker in the area of the pit than in other regions of the

integument, and bcneath the epidermai layer the pit region is fret of collagen fibres

relative to the rest of the demis.

The morphology of the pits suggests that they are sensory organs of some sort.

Sensory organs on the amniote integument have traditionally been assumed to be

mechanosensory. In this study, however, alternate interpretations of the stnicture

(mechanosensory, chemosensory, osmosensory) are discussed.

Introduction

Integumental pits an present, one per scale, on the post-cranial scales of some

. crocodilians, and have been used extensivcly as taxonomic characters in the identification

of crocodilian skins (King and Brazaitis 1971, Brazaitis 1987, Wennuth and Fuchs 1978).

They are pksent on the post-cranial scales of crarodylids and gavialids (Fig. 4.1.a). but

absent from the post-cranial scales of alligatorids (Fig. 4.1.b). The prcsence or absence of

pits can therefore be useû to distinguish ailigatorids from the other two crocodilian

families (even when the specimen involved is a smdl skin fragment such those used in a

wallet or a belt). The pits have been rcfcrrcd to in the taxonomic Iiterature as "follicle

pores" (King and Brazaitis 1971), "Porcn" (Wermuth and Fuchs 1978). "follicle glands".

"follicle pits" (BraLaitis 1987). and "integumcntary sense organs" or "ISOs" (Brazaitis

1987).

Although the pits have been well studied as taxonornic characters, their structure

and function are not known. Brazaitis (1987) says that they are thought to be

mechanosensory, while Grigg and Gans (1993) speculate that they may be either sensory

structures or secretory porcs. An extensive search of the literahire reveals no detailed

study of their structure or function. In contrast, detailed morphological and ultrastructural

studies of mechanoreceptors or "touch papillae" on the cranial scales of the alligatorid,

Cairnan crocodilus, have ken done (von During 1973, 1974, von During and Miller

1979). These "touch papillae" are confined to the cranial scales and are found in al1

crocodilians (Fig. 4.2). Alligatorids lack pits on the pst-cranid scales. The touch

Fig. 4.1. (A) Ventral scales of a crocodylid, C. porosus. showing the pits (arrow).

(B) Ventral scales of an alligatorid, Ca. crocodilus, which lacks pits.

Fig. 4.2. Touch papillae (sensu von hiring, 1973) from the cranial scales of C. porosur:

(A) head of a crocodile with touch papillae indicated (arrow), (B. C, D) SEMs of touch

papillae from the mandibular region.

- - -

papillae on the cranial wales of Ca. crocodiIus are obviousiy not the pst-cranial pits

referred to in the taxonomie literature, therefore. Von During's work may nonetheless be

the source of the idea that the pits have a mechanosensory function, however. Guibe

(1970) reports that the abundance of pits decreases as the animal ages. The source of this

information, however, is a study which compared juvenile Crocodylus with adul t Alligator

(Hulanicka, 1913), and it therefore seems Iikely that this difference may have more to do

with phylogeny than with ontogeny.

Evidence from previous experiments (chapter 3) indicates that the Crocodylid,

Crocodylus porosus discriminates between sea water salinities using an organ located

sornewhere other than in the mouth. The objective of this study was to investigate the

general morphology and the ultrastructure of the post-cranial pits of C. porosus, in order

to determine whether these might be salinity discrimination organs.


Animals

Captive-bred juvenile (400-700 g ) crocodiles (Crocodylus porosus) were obtained

from the Long Kuan Hung Crocodile F m in Singapore, where they had been housed in

fresh water in an outdoor enclosure. A total of five specimens were obtained, of which

four were frozen specimens for gross morphological snidies. in addition, one live

specimen was euthanised by cervical dislocation in order to fix tissues for electron

microscopie examination.

4: Intcgumentary Sense Organs

Scanning electron microscopy

Integument was dissected from the ventral surface of the freshly killed crocodile

. and transferred immediately to Bouin solution. FolIowing 24 h fixation, tissue was

transferred to 70% ethanol. Cubes of tissue (lmm3), each with a pit at its centre, were cut

out of the ventral integument. Specirnens were dehydrated in a graded series of ethanol,

dried in a Sowall 49300 critical point drying system, mounted on metai stubs, sputter-

coated with gold, and observed in a Hitachi H-2500 scanning electron microscope at an

acceleration voltage of 1 5 kV.

Light microscopy and transmission electron microscopy

Integument was dissected from the ventral surface of the freshly killed crocodile

and transferred immediately to a solution of ice-cold 2.5% giutaraldehyde in 0.1 M

Millonig's phosphate buffer at pH 7.3. The tissue was cut into lrnm cubes, each with a

pit at its centre, and fixed in the a b v e fixative for 3 hours. The tissue cubes were then

rinsed in the buffer, stored in the buffer for 3 days, and postfixed for 2 hours in 1% OsO,

in the same buffer. Tissues were dehydrated in ethanol and propylene oxide, and

embedded in Spurr's resin. Tissue blocks were sectioned with glass knives, using a

Sorvall MT2 ultra-microtome. Semithin (0.5 um thickness) sections were piaced on glass

slides and stained with 1% toluidine blue in saturated sodium tetraborate. Thin (silver)

sections were cut using a diamond knife, and mounted on copper grids. The specirnens

were stained with saturated uranyl acetate and lead citrate, and examined using a Hitachi

4: Integumentary Sense Organs

Fig. 4.3. SEMs of the posi-cranial pits from the ventral scales of C. porosus: (A) ventro-

lateral view, (B, C, D) ventral view.

H-7000 transmission electron microscope. Ail fixation and staining procedures were those

of Bozzola and Russell (1992).

Resul ts

Gross morphology

Pits are present on al1 the post-cranial scales. The pits arc darkly pigmented and

are therefore most clearly visible on the large and relatively unpigrnented ventral scales

(Fig. 4.1 .a). However, they are also present on the darkly pigmented dorsal scales and on

the very small scales surrounding the proximal ends of the limbs. There is usually one pit

on each scale, although the number occasionally varies from zero to t h e . The pit is

usually centred (sagitally) in the caudal third of the scale. When more than one pit is

present on a single scale, the two (or three) pits are positioned in line dong the same

transverse plane.

Scanning electron microscopy

In SEM, the pit is revealed frorn the outer surface as a roughly circular opening,

approx. 300 um in diameter, in the stiff outer layer of the stratum comeum that fonns a

protective coating over the scales. A slightiy convex surface of the epidermis of the pit is

revealed through the opening (Fig. 4.3.a,b). At the edges of the circular opening, the

outer layer of keratin is seen to be flaking off in stiff sheets. The convex surface of the

pit, when viewed at high magnification (Fig. 4.3.c), shows the margins between adjacent

Fig. 4.4. Light micrographs of cross-sections a pst-cranid pit (A) at 140X

magnification, showing diffuse pit region of the demis (pr) and collagen-rich non-pit

region (nr), and (B) at 640X magnification, showing (coUectively) stratum germinativum,

stratum spinosum, and straturn granulosum (sg) and stratum comeum (SC) layers of the

epidermis (ep).

Fig. 4.5. TEM (2400X) of the apex of pit region of the demis, showing high

concentration of cells (bl=basal lamina of the stranim germinativum of the epidexmis,

f=fibroblast ce11 , ir=iridocyte).

Fig. 4.6. TEM (7400X) of fibroblast cells from the pit region of the dermis (c=collagen

fibres).

Fig. 4.7. TEM (8900X) of nerve terminais (nt) supported by a fibroblast ce11 (0.

Fig. 4.8. TEM (135ûûX) of an indocyte from the pit region of the dermis.


Fig. 4.9. TEM (27ûûX) of epidennis from (A) the pif region, and (B) from another area

of the sarne scde (sc=stratum corneum, sg=collectively: stratum germinativurn, straturn

s pinosum, stratum granulosum, d=dennis).

- -

epidermal cells. The surface of these cells is pitted and possibly porous (Fig. 4.3.d).

Light microscopy and transmission electron microscopy

In cross-section and at low magnification (Fig. 4.4) the pit is revealed as a diffuse,

lightly stained p k e t in the darkly stained. collagen-rich surrounding dermal tissue. The

pit occupies an approximately spherical space in the dermis imrnediaîely underlying the

circular opening in the stratum corneum proper of the epidennis. Celis in the dermal

portion of the pit are widely separated, and there are very few collagen fibres compared

with the surrounding dermis. Those cells that are present are usually densely concentrated

at the apex of the demal sphere, in the area immediately underlying the stratum

germinativum. The stratum corneum appears to have two distinct layers: an outer layer

through which the underlying second layer protrudes through a circular opening. The

outer layer tended to separate from the underlying layer and to break off the tissue block

during sectioning.

The cells of the pit beneath the epidermal layer are widely dispersed among a few

collagen fibres and extensive ground substance of extracellular matrix. Three types of

cells were observed in the dermal region of the pit, and al1 of these are most numerous at

the apex of the dermal ngion of the pit. in the ana irnmediately underlying the stratum

germinativum of the epidecmis (Figs. 4.44 4.5). Fibroblast cells (Fig. 4.6) are the most

abundant ce11 type, but melanocytes are also cornmon. Al1 cells are often found close to

nerve terminais (Fig. 4.7). but structural support for the nerve terminais may be provided

by the attenuated processes of fibroblasts (Fig. 4.7). There are two types of

chromatophores: melanocytes and iridocytes (Fig. 4.8). The former contain many

melanosornes, and the cytoplasm of the latter has many indophores o r guanine crystals.

- The epidemis in the pit region (Fig. 4.9.a) differs from the epidermis of non-pit regions

(Fig. 4.9.b). Although the total thickness of the epidermis is equal in both regions, the pit

region has a thimer stratum corneum than the non-pit region. Hence, the germinativa,

spinosa, and granulosa strata are more prominent in the pit region.

Discussion

The location and general morphology of the poskrania.1 pits in C. porosus suggest

that they are sensory structures of some sort. The pit is an opening in the stiff outer layer

of the stratum corneum, through which a thinner, underlying layer of the epidermis is

revealed. Immediately beneath this exposed region of the epidemiis is a fluid-filled area

in the demis. Nerve terrninals are found in the fluid-filled region, immediately beneath

the epidermis.

The post-cranial pits of C. porosus differ from known cranial touch papillae of Ca.

crocodilus (von During 1973, 1974) in several ways. The pits are present on only the

post-cranial scales of crocodylids and gavialids, while the touch papillae are present on

the cranial but not the post-cranial scales of d l crocodilians. There is usuaily only one pit

on each post-cranial scale (with occasionai exceptions) and the pi& are not concentrated in

any one particular region of the skin. The number of touch papillae on the cranial scales,

by contrast, is much more variable (4-16). Papillae are most numerous on the scales

4: Integumentary Stnx Organs

Fig. 4.10. Summary illustration of the integumentary sense organ (sc=stratum corneum,

sg=collectively: stratum germinativum, stratum spinosum, stratum granulosum, p v i t

region of the dermis, nr=non-pit region of the demis, ddennis).


Fig. 4.11. Altemate interpretations of the function of the integumentary sense organ: (a)

rnechanoreceptor, (b) chemoreceptor, (c ) osmoreceptor.

(a) mechanoreceptor

resting state phy sical pressure

Physical pressure from outside changes the shape of the convex surface of the sensory organ. Nerve terminals are stimulated by the change in shape.

(b) chemoreceptor

sea water

(c) osmoreceptor

A fresh water Hz0 hyperosrnotic sea water

t t t

The epidennis is Grmeable to water. Osmotic changes in theextemal medium cause changes in the volume of the fluid-filled region, changing the shape of the convex surfrice. Nerve terrninals are stimulated by the change in shape

> A *

surrounding the nares and the mouth, and least numemus on the scales between the eyes.

Whereas each pit is centred in the caudal third of the scale, the touch papillae are

- randomly distributed. The pits are also larger than the touch papillae (300 um diameter

versus 200 um diameter). Although the outer surface of the pits is slightly convex, it is

not raised to the degree described by von During (1973, 1974) for cranial touch papillae.

In spite of these differences, the overall morphology of the pits is similar to that of

the touch papillae. In both structures the surface of the stratum comeum covering the

organ is exposed by a circular opening in the stiffer outer layer of the stratum comeum

which covers the rest of the scaIe. Baden and Maderson (1970) have determined by x-ray

diffraction that the stratum corneum of lepidosaur reptiles has an inner layer of alpha-

keratin and an outer layer of beta-keratin, while that of Alligator has a single layer of

beta-keratin. with alpha-keratin only at the hinge regions of the scales. We observed,

using SEM and TEM, what appeared to be two distinct layers in the stratum comeum of

Crocodylus: a stiff outer layer which peeled off in flakes, and a pitted and apparently

more pliable inner layer. The outer layer tended to separate from the inner layer during

sectioning, perhaps suggesting the presence of a mesos layer (Lillywhite and Maderson

1982). X-ray diffraction analysis of these two layers has the potentid to reveal whether

these two layers are indeed both beta-keraîin, or whether the skin of Crocodyfus has an

inner layer of alpha-keratin and an outer layer of beta-keratin, like that of Lepidosaurs.

Although we identified nerve terminais in the pits immediately beneath the

epidermal layer and observed that al1 ce11 types were most densely concentrateci in that

area of the dermis. we did not observe al1 the receptor types described by von During in

the touch papillae (intraepidermal nerve endings. Merkel ce11 neurite cornplex, lamellated

receptors). However. it is possible that this apparent difference rnay reflect the fact that

we used semi-thin sections and iight microscopy for serial reconstruction and TJZM only

for fine detail, whereas von During (1973) used TEM of thin sections for the entire seriai

reconstruction.

The presence of nerve tenninals immediately beneath the epidermal layer is

consistent with the hypothesis that the pits are sensory organs of some son. and we

therefore favour the term "integumentary sense organs" (ISO) over other ternis which have

k e n used in the taxonomie literature (e.g. "follicle glands", "follicle pores", etc.).

The touch papillae have been described as mechanosensory on the basis of their

structure (von During 1973, 1974), and have been described as elevated relative to the

surrounding integument as though under pressure from fluid inside (von During, 1974).

Figure 4.10 summarises Our observations of the morphology and ultrastructure of the ISO.

We observed that the dermal region of the pst-cranid pit has a high component of

ground substance and little collagen and cells. It is possible that these cells and fibres of

the extracellular matrix (ECM) have been dispersed by a unique gel or fluid-like

component of the ECM. This ground substance could be interpreted as an important

element of the mechanoreception systern which when stimulated by extemal pressure

stimulates the nerve tenninals near the epidemal-dermal junction (Fig. 4.1 1 .a).

Alternatively, the morphology of the pits could be interpreted as consistent with a

4: Intcgumenmy Sense Organs

chemosensory function (Fig. 4.1 1.b). The stratum comeum of the epidermis of the pit

region is thinner than that of the rest of the integument, with a surface that is pitted and

- possibly porous rather than flaking off in flat sheets like the surface surrounding it. if the

epidermis of the pit region is indeed porous and allows the passage of fluid from the

outside environment, then the diffuse, possibly fluid-filled area of the dermis in the pit

region could be interpreted as a sampling ce11 in which the nerve terminals of the pit

region are bathed in fluid from outside, and are stimulatecl by the chemical characteristics

of this fluid. There is strong evidence for the importance of salinity discrimination as an

osrnoregulatory mechanism for estuarine crocodylids, as well as evidence for a

phylogenetic difference between crocodylids and alligatorids in capacity for salinity

discrimination (chapter two).

Another possible way that the pits could be acting as salinity discrimination organs

is as osmoreceptors. This interpretation (Fig. 4.11.c) combines characteristics of both the

mechanosensory and chemosensory interpretations, in that chemical changes in the

external medium could cause a mechanical change in the shape of the fluid-filled region.

If the epidermis in the pit region is permeable to water but not to salt, then water will be

drawn into the fluid-filled region by osmosis when the animal is in hypo-osmotic sea

water, and out of the fluid-filled region when the animai is in hyperosmotic sea water.

The resulting change in the shape of the epidermis may stimulate the nerve terminals as in

the mechanoreceptor interpretation.

To date only one study of integumental mechanoreception in reptiles has k e n

4: Integumenwy Scnse Organs

undertaken in which nerve action potentials were recoded as the integument was

mechanically stimulated (Necker 1974). Because of the technical difficulty of recording

, electrical activity in the small efferent nerve fibres of individual mechanoreceptors,

recordings are made from larger nerve fibres far dowmtrearn, so that it is difficult to

attribute the observed response to the stimulation of any particuiar proposeci sensory

structure. Morphological study of the pst-cranial pits reveals a structure which is

potentially consistent with either a mechanosensory or a chemosensory function. If the

function of the post-cranial pits is to be determined, physiological study will be required.

Such studies have the potential to reveal whether the sensory function of the pits is

rnechanoreception, or whether morphologicai differences between the pst-cranial pits and

the cranial touch papillae reflect a functional difference between these organs.

CHAPTERFIVE

Conclusions and Directions for Future Research

5: Conclusions

The results of the research descnbed in this thesis provide circumstantial evidence that

the integumentary sense organs of crocodylids are chemosensory organs used to distinguish

between hyper- and hypo-osmotic sea water salinities. In summary, the evidence is as

follows: In chapter 2 the capacity for saiinity discrimination was found to have a

phylogenetic component. All crocodylids discriminate between salinities and drink only those

that are hypo-osmotic, whereas only alligatorids from estuarine areas possess this adaptation.

Chapter 3 provides evidence that crocodylids have a chemosensory organ on some part of the

body other than the buccal epithelium. Although the crocodylid, C. porosus is capable of

very precise salinity discrimination, it apparently does not use its mouth for chernosensation

and drinks fresh water and hyperosmotic sea water indiscriminately when these are dnpped

directly into its mouth. Chapter 4 is a detailed study of the morphology and ultrastructure

of a possible integemental chemoreceptor. The integurnentary a n s e organ is a sùucture of

previously unknown function on the pst-cranial scaies of crocodylids but not of alligatorids.

This phylogenetic difference may reflect the crocodylid-alligatorid difference in capacity for

salinity discrimination, and in the keratinised versus non-keratinised buccal epithelium. The

morphology and ultrastructure of the ISO were found to be consistent with either a

chemosensory or a mechanosensory function.

in many ways this thesis has produced more questions than answers, and there are

many possible directions for further research which it was not feasible to follow up in a one-

year study. One that should be given high priority is the physiological study of the ISOs, to

evaluate the different possible functions of this organ (chapter 4).

80

5: Conclusions

Chapter 4 explains two possible mechanisms by which the ISO could respond to

chemical stimuli. The dermal region of the ISO is free of collagen fibres relative to the

surrounding tissue, and is apparently filled with fluid. Nerve tcnninals are present

immediately beneath the epidermis. The mechanosensory interpretation of this structure is

that the fluid in the ISO is maintained under pressure from within. Extemal pressure on the

epidermis causes pressure on the fluid and stimulates the nerve endings beneath the

epidermis. The chemosensory interpretation is that the fluid in the ISO cornes from outside

rather than inside, so that the fluid-filled region of the ISO acts as a sampling cell, allowing

the nerve terminais protected beneaîh the epidermis to be exposed to sea water which cornes

into contact with the skin. Another possibility is that the ISO acts as an osmoreceptor in

response to chexnical changes in the external medium, by osmotic pressure on the fluid-filled

region causing a mechanicd change (chapter 4)-

If the ISO is a chemoreceptor which functions as a sea water sampling cell, the

epidennis covering it must be permeable to sea water. Morphologically it seems likely that

the epidermis would be more permeable above the ISO, because the stratum corneum in this

area is much thinner than in other paru of the scale. However, this could also be interpnted

as consistent with a mechanosensory or osmosensory fûnction, since the thin stratum corneum

might ailow the epidermis to change shape in response to pressure, or might be penneable

to water but not to salts.

The chemoreceptor hypothesis depends o n the epidennis of the ISO king permeable

to sea water (chapter 4). An attempt was made to test the pemeability hypothesis using a

8 1

5: Conclusions

30 ppt CoCI, solution applied to the outside of the ISO in place of sea water. After a 10-

minute exposure period, the ISO was excised and solubilised. and x-ray micro-analysis was

used to detect the presence or absence of Co2+. This sample was compared with a tissue

sample excised from a CoC1,-exposed area on another part of the scaie, and another ISO was

wiped briefly with the CoCI, solution and then excised, as a control for surface

contamination. Unfortunately, this project was beset with technical problems such as finding

a substance which would solubilise the sarnples and then dry on the SEM stub without

leaving so much residue as to interfere with co2' detection. No data have yet been obtained.

therefore, on the permeability of the ISO epidennis relative to the rest of the crocodile. A

similar experirnent using radio-isotopes might answer the question.

An experiment is currently in progress to test the osmoreceptor hypothesis. If the ISO

functions as an osmoreceptor, the shape of the expoad. convex area of the epidermis

covenng the ISO must change shape when the animal moves from fresh water to

hyperosmotic sea water. However, these changes are likely to be very slight. as

mechanosensory organs respond to very slight pressure. To measure these changes, high

resolution casts will be made, using dental cernent. of the same individual ISOs on a

crocodile following exposure to fresh water, and then to 30 ppt sea water. These casts will

be filled with tesin to produce a replica of the ISO, and these replicas will be examined using

SEM.

A more direct way to approach the question would be to use nerve recordings while

stimulating the ISOs with sea waters of different concentrations. Initial inquiries and

82

5: Conclusions

dissections indicated that this project was going to k vcry tcchnicaily demanding. This

perhaps explains why nerve recording studies of integumental sensation in mptiles are so rare

(chapter 4).

An important experiment which remains to bc done is the estuarine alligator

experiment suggested in chapter 3. Estuarine alligatorids distinguish saiinities but lack ISOs.

They also lack a keratin coating on the buccal epithclium, however. if estuarine alligators

continue to distinguish between salinities even when the water is dripped directly into their

mouths, it will provide further circurnstantial support for the idea that the crocodylids

developed a keratinised buccal epithelium as an estuarine adaptation to reduce osmotic water

loss, and then developed integumental chemoreceptors to make up for the reduced sensory

capacity in the buccal epithelium.

There is some precedent for the idea of an integumental chemoreceptor to allow an

animal to selectively avoid osmotically stressfui areas. Von Seckendorff Hoff and Hillyard

(1993) have shown that the toad, Bufo punctatus, tastes sodium with its skin. Like selective

drinking of hypo-osmotic sea water by estuarine crocodilians, this mechanism allows the toad

to osmoregulate behaviourally by moving away from concentrated NaCl solutions.

Amphibians obtain water by osmotic uptake through the skin rather than by ingesting it, and

the chemosensory mechanism in this case is a Na' channel in the slcin modified for sensory

transduction.

If the ISO is indeed the organ tesponsible for salinity discrimination in crocodylids,

then this is the first report of an integumental chemo- or osmoreceptor in an amniote

83

5: Conclusions

vertebrate. integumental chemoreceptors axe found exclusively in water-living animals such

as fishes and amphibians (Reutter 1986). Sensory organs on the integument of amniotes are

therefore usually described as mechanorecepton by default, even in the absence of any

physiologicd evidence for a mechanosensory function (e.g. von During and Miller 1979).

Crocodilians are amniotes which are secondarily aquatic. The morphological similarities of

the mechanosensory "touch papillae" on the cranial scaies of crocodilians and the possibly

chemosensory ISOs on the postcranial scales of crocodylids, may reflect the derivation of

an integumental chemo- or osmoreceptor from a mechanoreceptor after the return to the

aquatic habitat.

Baden, H. P. and P. F. A. Maderson. 1970. Morphological and biophysical identification

of fibrous proteins in the amniote epidermis. J. Exp. Zool. 174: 225-232.

Bentley, P. J. and K. Schmidt-Nielsen. 1966. Perrneability to water and sodium of the

crocodilian, Cairnan sclerops. J. CeU and Comp. Physiol. 66: 303-3 10.

Bentley, P. J. 197 1. Endocrines and Osmortplation: A Comparative Account of the

Remdation of Water and Salt in Vertebrates. Springer-Verlag.

Benton, M. J. and J. M. Clark. 1988. Archosaur phylogeny and the relationships of the

Crocodylia Benton. M. J. (ed.) The Phvloeenv and Classification of the Tetrawds.

Clarendon Press, Oxford.

Benton, M. J. 1990. Vertebrate Palaeontoloav. Unwin Hyman Ltd., London.

Bozzola, J. J., and L. D. Russell. 1992. Electron Microsco~v: Princi~les and Techniques

for Biologists. Jones and Bartlett, Boston.

Brazaitis, P. 1987. Identification of crocodilian skins and products. Pp. 373-386. In:

Webb, G. J., Manolis, S. C., and P. J. Whitehead (eds) Wildlife Management: Crocodiles

and Alligators. Surrey Beatty & Sons, Australia.

LitCram Citcd - - -

Brooks, D. R. 1979. Testing hypotheses of evolutionary relationships among parasites:

The digeneans of crocodilians. Amer. Zool. 19: 1225- 1238.

Brooks. D. R. and R. T. O'Grady. 1989. Crocodilians and their helminth parasites:

Macroevolutionary considerations. Amer. Zoolm 29:873-883.

Brooks, D. R. and D. MacLennan. 1993. Parascript: Parasites and the lanauaae of

evolution. Srnithsonian Institution Press.

Brusca, R. C. and G. J. Brusca 1990. Invertebrates. Sinauer Associates Inc.

Buffetaut, E. 1978. The evolution of the crocodilians. Sci. Amer. 241 (4): 130- 14-4.

Densmore, L. D. 1983. Biochernical and immunological systematics of the order

Crocodilia. Hecht, M. K., Wallace, B.. Prance, G. T. (eds.) Evolutionary Biology,

Vol. 16. Plenum Press, New York.

Dueilman, W. E. and L. Tmeb. 1994. Biolom of Amhibians. Johns Hopkins

University Press.

Dunson, W. A. 1970. Some aspects of electrolyte and water balance in three estuarine

reptiles, the Diamondback terrapin. Amencan and "Salt water" crocodiles. Comp.

- Biochem. Physiol. 32: 16 1 - 174.

Dunson, W. A. 1979. Control mechanisms in reptiles. In: Gilles, R. (ed.) Mechanisms

of Osmoregulation in Animals: Maintenance of CeIl Volume. John Wiley & Sons, Ltd.

Dunson, W. A. 1980. The relation of sodium and water balance to survival in sea water

of estuarine and freshwater races of the snakes Nerodia fasciafa, N. sipedon, and N.

valida. Copeia 2: 268-280.

Dunson, W. A. 1985. Effect of water salinity and food salt content on growth and

sodium efflux of hatchling Diarnondback terrapins (Malaclemys). Physiol. Zool. 58(6):

736-747.

Dunson, W. A. 1986. Estuarine populations of the Snapping turtle (Chelydra) as a mode1

for the evolution of marine adaptation in reptiles. Copeia 3: 741-756.

Dunson, W. A. and F. J. Mazzotti. 1988. Some aspects of water and sodium exchange of

freshwater crocodilians in frcsh water and sea-water: Role of the integument. Comp.

Biochern. Physiol. 90A: 39 1-396.

Dunson, W. A. and F. J. Mazzotti. 1989. Salinity as a limiting factor in the distribution

of reptiles in the monda Bay: A theory for the estuarine origin of marine snakes and

turtles. Bull. Marine Sci. 44( 1 ): 229-244.

Ellis, T. M. and D. H. Evans. 1984. Sodium balance in the Amencan alligator. J. Exp.

2001. 23 I : 325-329.

Evans. D. E. and T. M. Ellis. 1977. Sodium balance in the hatchling Arnerican

crocodile, Crocodylus a c u m Comp. Biocbem. Physiol. 58A: 1 59- 1 62.

Franklin, C. E. and G. C. Grigg. 1993. Increased vascularity of the lingual salt glands of

the estuarine crocodile, Crocodylus porosus, kept in hyprosmotic salinity . J. Morphol.

218: 143-151.

Gray, D. A. and C. R. Brown. 1995. Saline-inhision-induced increases in plasma

osmoiality do not stimulate nasal gland secretion in the ostrich (Stmthio camelus).

Physiol. Zool. 68(1): 164- 175.

Gordon, M. S., Schmidt-Nielsen, K, and H. M. Kelly. 1961. Osmotic rcgulation in the

Crab-eating frog (Rana cmcrivora). J. Exp. Biol. 38: 659-678.

Grigg, G. C., Taplin, L. E., Harlow, P., and J. Wright. 1980. Survival and growth of

- hatchling Crocodylus porosus in saltwater without access to fresh drïnking water.

Oecologia 47: 264-266.

Grigg, G. C. 198 1. Plasma homeostasis and cloaca1 urine composition in Crocodylus

porosus caught dong a salinity gradient. J. Comp. Physiol. 144: 261-270.

Grigg, G. C., Taplin, L. E., Green, B., and P. Harlow. 1986. Sodium and water fluxes in

free-living Crocodyfus porosus in marine and brackish conditions. Physiol. Zool. 59(2):

240-253-

Grigg, G. C. and C. Gans. 1993. 40: Morphology and physiology of the crocodilia. Pp.

326-336 In. Glasby, C. J., Ross, G. J. B., and P. L. Beesley (eds.) Fauna of Australia

Vol. 2A: Am~hibia and Reptilia Austraiian Govemment Publishing Service: Canberra.

Guibe, I. 1970. La peau et les productions cutanees. & Grasse, P. (ed.): Traite de

Zoolo~ie: Anatomie. Svstematiaue. Biolo~ie. X l V Reptiles: Caracteres Genereaux et

Anatomie. Masson et ci', Paris, pp. 6-32.

Heatwole, H. and M. L. Guinea. 1993. 37: Farnily Laîicaudidae. Glasby, C. J.,

Ross, G. J. B., and P. L. Beesley (eds.) Fauna of Australia. Vol. 2A: Amvhibia and

- Reptilia. Austraiian Govemment Publishing Service: Canberra.

Hulanicka, R. 19 13. Recherches sur les terminaisons nerveuses dans la langue, le palais,

et la peau du crocodile. Arch. de Zool. Exp. et Gen. 53: 1 - 14.

King, F. W. and P. Brazaitis. 1971. Species identification of commercial croçodilian

skins. New York Zoological Society: Zoologica.

Kirschner, L. B. 1979 Control mechanisms in crustaceans and fishes. Gilles, R.

(ed.) Mechanisms of Osmoremlation in Animals: Maintenance of Ce11 Volume. John

Wiley & Sons, Ltd.

Lauren, D. J. 1985. The efTect of chronic saline exposure on the electrolyte balance,

nitrogen metaboiism, and corticosterone titer in the American alligator, Alligator

rnissippiensis. Comp. Biochem. Physiol. 8 1 A(2): 2 17-223.

Lillywhite, H. B. and P. F. A. Maderson. 1982. Skin structure and permeability.

Gans, C. and P. H. Pough (eds) Bioloev of the Reptilia - 12. Phvsiolow C:

Phvsiolonical Ecoloav. Academic Press, pp. 397-442.

Martof, B. S. 1963. Some observations on the herpetofauna of Sapelo Island, Georgia

Herpetologica 19( 1 ): 70-72.

Mazzotti, F. J. and W. A. Dunson. 1984. Adaptations of Crocodylus acutus and Alligator

for life in saline water. Comp. Biochem. Physiol. 79A(4): 641-646.

Mazzotti, F. J. and W. A. Dunson. 1989. Osrnoregulation in crocodilians. Amer. Zool.

29: 903-920.

Necker, R. 1974. Dependence of mechanoreceptor activity on skin temperature in

sauropsids. 1. Cairnan. J. Comp. PhysioL 92: 65-73.

Nicol, J. A. C. 1961. The Bioloav of Marine Animals. Sir Isaac Pitrnan and Sons Ltd.,

London.

Peaker, M. and i. L. Linzell. 1975. Mononraohs of the Phvsioloaical Societv No. 32:

Salt Glands of Birds and Re~tiles. Cambridge University Press.

Reutter. K. 1986. Chemoreceptors. In: Bereiter-Hahn, J., Matoltsy, A. G., and K. S.

Richards (eds.) Biolom of the Intemiment. 2: Vertebrates. Springer-Verlag.

Romer, A. S. and T. S. Parsons. 1986. The Vertebrate Body. Sixth Edition. Saunders

College Publishing.

Schmidt-Nielsen, B. and E. Skadhauge. 1967. Function of the excretory system of the

crocodile (Crocoàylus acutus). Am. J. Physiol. 2 l2(5): 973-980.

Sill, WD. 1968. Zoogeography of the Crocdilia. Copeia 1:76-88.

Steel, L. 1973. Crocodylia. In O. Kuhn (ed.), Encvclo~edia of Paleoher~etolonv. 16: 1-

1 l S. Gustav Fisher Verlag, Stuttgart.

Taplin, L. E. and G. C. Grigg. 198 1. Salt glands in the tongue of the estuarine crocodile,

Crocodylus porosus. Science 2 12: 1045-1047.

Tapiin, L. E., Grigg, G. C., Harlow, P., Ellis, T. M., and W. A. Dunson. 1982. Lingual

salt glands in Crocodylus acutta and C. johnstoni and Caïman crocoàilus. J . Comp.

Physiol. 149: 43-47.

Taplin, L. E. 1984. Drinking of fresh water but not seawater by the estuarine crocodile,

Crocodylus porosus. Com p. Biochem. Physiol. 77A(4): 763-767.

-

Taplin, L. E., Grigg, G. C., and L. Beard. 1985. Sait gland function in fresh water

crocodiles: evidence for a marine phase in eusuchian evofution? 4. 403410 & Grigg,

. G. C., Shine, R., and H. Ehmann (eds.) Biolonv of Australasian Froas and Reptiles.

Royal Zoological Society of New South Wales.

Taplin, L. E. and J. P. Loveridge. 1988. Nile crocodiles, Crocodylus nilotieus and

estuarine crocodiles, Croeodylus porosus, show similar osmoregulatory responses on

exposure to seawater. Comp. Biochem. Physiol. 89A(3): 443-448.

Taplin, L. E. 1988. Osmoregulation in crocodilians. Biol. Rev. Caxnb. Phil. Soc. 63:

333-377

Taplin, L. E. and G. C. Grigg. 1989. Historical zoogeography of the eusuchian

crocodilians: a physiological perspective. Amer. Zool. 29: 885-90 1.

Taplin, L. E., Gngg, G. C., and L. Beard. 1993. Osmoregulation of the Australian

freshwater crocodile, Crocodylus johnstoni, in fresh and saline waters. J. Comp. Physiol.

B. 163: 70-77

Von During, M. 1973. The ultrastnicture of lamellated mechanoreceptors in the skin of

reptiles. 2. Anat. Entwicki.-Gesch. 143: 8 1-94.

Von During, M. 1974. The ultrastructure of the cutaneous receptors in the skin of Ca.

crocodilus. Abhandlungen rheiin.-westfai. Alrad. 53: 123- 134.

Von During, M. and M. R. Miller. 1979. Sensory nerve endings of the skin and deeper

structures. In: Gans, C., Northcutt, R. G., and P. Ulinski (eds) Biolom of the Reptiiia 9.

Neurologv A. Acadernic Press, pp. 407-442.

Von Seckendorff Hoff, K. and S. D. Hillyard. 1993. Toads taste sodium with their skin:

sensory function in a transporting epithelium. J. Exp Biol. 183: 347-35 1.

Werrnuth, H. and K. Fuchs. 1978. Bestimmen von Krokodilen und ihrer Haute: Eine

Anleitun~ m m Identifizieren der Art- und Rassen-Zuaehoriakeit der Krokodile. Gustav

Fischer Verlag, Stuttgart.

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