Camp. Biochem. Physiol., 1974, Vol. 48A, pp. 489 to 500. Pergamon Press. Printed in Great Britain

OBSERVATIONS ON EXTRARENAL EXCRETION BY ORBITAL GLANDS AND OSMOREGULATION IN

MALACLEMYS TERRAPIN

F. B. M. COWAN

Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada

(Received 2 July 1973)

Abstract-l. Body weight in Malaclemys terrapin progressively declined during extended stays in sea water, whereas in fresh water body weight remained stable.

2. Lachrymal “salt” gland weight did not increase in sea water acclimated animals in terms of absolute weight, but did increase in terms of relative weight. This increase was small but significant, P < 0.01.

3. Initially, plasma sodium, and osmotic pressure increased in animals acclimated to sea water, but after this phase remained stable for periods up to 6 months in sea water.

4. Animals acclimated to sea water and sodium loaded with 517 m-equiv. kg-l have a cranial loss of sodium of 18 CL-equiv. 100 g-l hr-l. Those loaded with an equivalent volume of distilled water had cranial sodium losses of less than 1 I*-equiv. 100 g-l hr-l.

INTRODUCTION

SCHMIDT-NIELSEN & F&GE (1958) made the original observation that a hyper- tonic sodium chloride solution could be collected from the orbital glands in Malaclemys terrapin. They gave no details as to the source of the secretion, the method of fluid collection or of the concentration of sodium in the collected fluid. Dunson & Taub (1967) made reference to work they had done on MaZacZemys which agreed with earlier work. However, Bentley et al. (1967) in a more extensive study of osmoregulation in MaZacZemys made no direct statement concerning the involvement of salt glands in osmoregulation. The relatively vague anatomical information accompanying the earlier work reflected the general lack of know- ledge of the gross anatomy of the orbital region in emydine turtles. This was the starting point for the work reported in this paper. Cowan (1967,1969) studied the gross anatomy and histology of the orbital glands in Mahclemys and in several closely related emydines. These studies showed that a lachrymal gland is present in all species studied as is an Harderian gland. Morphological evidence suggested it is the former which might be involved in some function related to salinity. This evidence included the size of the gland in the euryhaline Malademys as compared to the stenohaline species, an accumulated sulfated polyanion in Malaclemys adapted to sea water, and the lack of obvious organic secretory function in the

490 F. B. M. COWAN

lachrymal gland of Malaclemys as compared to the homologue of all other species studied (Cowan, 1969). Further studies with the electron microscope (Cowan, 1971) showed that the lachrymal gland of MaZac2emy.s has many structural charac- teristics found in other cells or tissues involved in active transport; notably the

lachrymal gland of Chelonia mydas (Abel & Ellis, 1966), the nasal “salt” glands in birds (Doyle, 1960; Ernst & Ellis, 1969), the human kidney (Rhodin, 1958) and the reptilian kidney (Schmidt-Nielsen & Davis, 1968). The electron micrographs

of the lachrymal gland in Malaclemys also showed little evidence of organic secretion These anatomical data were essentially in agreement with the earlier work indi-

cating the presence of an orbital gland with a structure and histochemical profile consistent with the function of electrolyte secretion.

However, in the study of the gross anatomy of the lachrymal gland (Cowan,

1969) suggested caution was necessary in interpreting the earlier work. The lachrymal gland in Malaclemys and other emydines is drained by a series of ducts which open through small apertures over a considerable expanse of the lateral

surface of the nictitating membrane. It is also thought that salt gland activity is

accompanied by frequent blinking movements. Thus the small amount of fluid secreted is spread in a thin layer over the cornea and surface of the eyelid and

nictitating membrane. This seems to make the possibility of evaporative water

loss likely; this would cause an overestimation of sodium concentration in fluid collected from these surfaces. With this possibility of error, it seemed timely to

repeat some of the physiological experiments to clarify the function of the lachrymal gland in Malaclemys. This work was undertaken almost simultaneously by Dunson (1970, 1971) and myself; however, this paper will report observations not covered

by him, with a minimum of repetition.

MATERIALS AND METHODS

Animals and their maintenance

The animals used in this study were an intergrade of Malaclemys terrapin terrapin and Malaclemys t. centrata, collected off Chincoteaque, Virginia, by Miles Hancock. The animals were kept in fresh water (defined as 1.7 mM sodium chloride and 0.87 mM calcium chloride per kg of distilled water at 22°C) or sea water. The sea water was artificial, called Instant Ocean, manufactured by Aquarium Systems Inc., Ohio. The full strength pre- paration with a specific gravity of 1.025 at 25°C is designated 100% or full strength sea water in this paper. Fifty per cent sea water is a 1 : 1 dilution with distilled water of the full strength preparation described in the preceding sentence. The animals were fed every 3 days fresh shrimp or salt water fish.

The Malaclemys were routinely divided into three groups upon arrival at the laboratory, and acclimated to fresh water. At the beginning of the experiment one group was placed in 1OO0/0 sea water, another in 50% sea water and one was left in fresh water. Groups were kept in each of these salinities for periods up to 52 weeks. In another experiment the animals were kept in either fresh water or 100% sea water. Half of those kept in 100 per cent sea water were given a drink of fresh water every 3 days at feeding times. This experiment was designed to see if some of the effects, especially morphological variations, which had been found in Malaclemys acclimated to sea water for long periods, would occur in animals kept in a saline environment, but not undergoing prolonged and continued osmotic stress.

EXTRARENAL EXCRETION AND OSMO~~ULATION IN hfALACLEh4YS TERFSHN 491

In another experiment ten animals were salt loaded by subcutaneous injection of 1 ml of a 30 per cent solution of sodium chloride per kg body weight. This load of 5.17 m- equiv. kg-* body weight is similar to that used by Holmes & M&lean (1964) working with CheZoniu. Five of the salt loaded Malaclemys were given a drink of fresh water 12 hr before killing. The other five were not allowed access to fresh water.

An additional ten animals were used for the collection of sodium secreted cranially. The same salt load, 5.17 m-equiv. kg-’ was used, and the collection period began 1 hr after salt loading. Controls were loaded with an equal volume of water.

Orbital fluid collection

Method 1. In this method the animals were restrained as shown in Fig. 1. The animal was able to put his head under water voluntarily into the forward compartment which

FIG. 1. F.C., Forward compartment; C.F., collecting fluid (0 litre distilled water); R.C., rear compartment for isolation of cloacal discharge; M.C., middle compartment, sealed off to separate renal excretory products from cranially

derived secretions.

contained 1 1. of triple glass distilled water. As a rule the animals kept their heads sub- merged most of the time. The horizontal bars kept water from leaving the forward com- partment, and also prevented the flow of cloacal fluid forward from the posterior compart- ment. This apparatus was used as described for five salt loaded animals, and five water loaded animals.

Method 2. This method was similar to the one above, using the same chamber. The head was rinsed at hourly intervals with a known volume of distilled water, and the water was immediately drained out of the compartment. Samples of 4 ml were set aside at hourly intervals and quickly frozen. These were later measured for osmolarity and sodium con- centration and accumulative values for total sodium loss from the cranial region were calculated.

Measurements

For osmotic pressure and ion concentrations blood was collected at decapitation in centrifuge tubes rinsed with ammonium heparin. After centrifugation the plasma was pipetted off and frozen in lo-ml snap-capped plastic vials. Plasma sodium and potassium concentrations were measured on the thawed samples with a Zeiss PF-5 flame photometer. Plasma was diluted 1 : 250 for sodium analysis, allowing a sample concentration to be determined from a standard concentration curve ranging from 0 to 1.5 m-equiv. 1-r. A

492 F. B. M. COWAN

plasma dilution of 1 : 50 was used to determine potassium concentration from a standard curve ranging from 0 to 0.2 m-equiv. 1-r. The osmotic concentration of plasma samples and water-bath samples were measured with a Fiske osmometer using undiluted plasma. The weights of animals were measured on an Ohaus top-loading balance. For determination of gland weights, the glands were dissected from the right orbit. Adherent connective tissue was removed, and the glands were weighed immediately to obtain the wet weight. Weights were taken to the nearest mg, and are expressed in the following figures as absolute weight, and weight 100 g-l body weight. The Student’s t-test with appropriate corrections for small samples, or unequal samples was used to test the statistical significance.

RESULTS

The weights of the animals kept in the various salinities were taken every

3 days, prior to feeding and tank cleaning. There was much fluctuation from day to day in each animal. In general, the trend was for Malaclemys kept in fresh water to gain weight, but the difference between initial weights and weights at killing was not significant. The same trend was true for animals kept in 50% sea

water. Animals kept in lOOo/o sea water lost weight progressively, and the dif- ference between initial weight and weight at 21 days in 100% sea water was significant (PC 0.01). The difference increased with time. Table 1 shows the

TABLE ~-BODY WEIGHTS OF M. terrapin ACCLIMATED TO A REGIME OF SEA WATER WITH DRINKING WATER SUPPLIED EVERY 3 days

Trial 1 Trial 2

Acclimated to regimen Acclimated to regimen 3 months 5 months

Before After Before After

560 564 557 627 532 560 521 575 468 504 475 475 524 555 508 546 587 644 575 607 540 555 453 470

Average increase 1OO-n’ body weight 5.15 7.61

Trials 1 and 2 show body weights before and 30 min after receiving a drink of fresh water

immediate weight change which occurred in a group of M. terrapin when they were transferred from lOOo/o sea water to their fresh drinking water tanks, for a period of 30 min. In both trials the animals had been in sea water several months, but had been receiving a supply of fresh drinking water every 3 days. The figure

EXTRARENAL EXCRETION AND OSMOREGULATION IN MALACLEMYS TERRAPIN 493

indicates that the turtles drank an amount of fresh water equivalent to approxi- mately 5 and 7 per cent of their body weight respectively in the two trials, even though in both cases it had only been 3 days since their last access to fresh water. Table 2 shows that the animals kept in fresh water, or given access to fresh water every 3 days, weighed significantly more than animals confined to sea water over extended periods of time. With regard to the weight of the lachrymal gland

TABLE ~-BODY WEIGHT OF M. terrafiin KEPT IN MEDIA OF VARIOUS SALINITIES FOR VARIOUS

EXPOSURE TIMES

Condition of exposure Sample size Mean body weight Standard error Significance

Experiment 1

Fresh water 6 months 6 866 81.2 P<O*l >o-05

100% sea water 6 months 10 748 53.8

Experiment 2

100°/O sea water 4 months, 9 575 18.9 with fresh drinking water every 3 days P<O*Ol

100% sea water 4 months 9 431 24.2

from these same animals, it is seen that the absolute glandular weight remains stable; there is no significant difference between the means in either of the paired groups. However, when glandular weight is expressed on a relative basis (per 100 g body weight), the relative weight of the glands from animals confined to sea water was significantly greater than from those kept in, or given access to, fresh water. The same was true for dry weights of the lachrymal gland. The abso- lute dry weights of the lachrymal glands from either of the two groups were not significantly different. However, on a relative basis the dry weight of the glands from Muhdemys kept in sea water was significantly greater (PC 0.01) than that from animals with access to fresh water. With respect to the other orbital gland, the Harderian gland, wet or dry weights were no different between either of the two groups shown in Tables 2 and 3 on an absolute or relative basis. These data seem to indicate that the size of the lachrymal gland does not vary directly as the body weight in a linear fashion, whereas the weight of the Harderian gland and body weight do vary in a linear fashion irrespective of the salinity of the environ- ment.

Figure 2 presents data with regard to plasma sodium concentration from four groups kept under the following conditions. Group A was confined to sea water, while group B was kept in sea water, but given fresh water to drink every 3 days. Group C was kept in sea water, and given a salt load of 5.17 m-equiv. sodium chloride kg-l body weight, 24 hr before killing. Group D was treated the same

494 F. B. M. COWAN

TABLE %-WET WEIGHT OF LACHRYMAL GLAND FROM M. terrapin EXPOSED TO VARIOUS SALINITIES FOR VARIOUS EXPOSURE TIMES

Absolute weight Relative weight

Sample Mean * Mean* Exposure size (mg) Significance (mg/lOO-r) Significance

Experiment 1

Fresh water 6 months 6 142+15 16+1-2 P<O.2>0.1 P<O*Ol

Sea water 6 months 10 172+12 21 f 1.0

Experiment 2

100% sea water fresh drink- 9 116+11 21 f 0.9 ing water every 3 days. 4 months P<O-5 P<O*Ol

100% sea water 4 months 9 119f6.0 28 + 0.8

Iv=9

IV=6

r I

B

1 -

N-9

r I-

i - C 1 -

Iv=9

1 D

FIG. 2. Effect of environmental salinity on plasma sodium levels.

way but received a drink of fresh water after salt loading, 12 hr before killing. Groups A and B differ significantly (PC OaOl), as do groups C and D. The figures for the salt loaded animals in group C had a great deal of variation; however, the mean was very high and significantly different from the salt loaded animals in group D which had access to fresh water. This indicates that the elevated sodium was either not sufficient to stimulate salt secretion or the maximal rate of secretion was insufficient to excrete the entire load in the 24-hr period. This is hard to

EXTRARENAL EXCRETION AND OSMOREGULATION IN MALACLEMYS TERRAPIN 495

reconcile with later results of cranial sodium loss, and with the following facts from another experiment. In the latter, Maladmys were kept in fresh water, 50% sea water or lOOo/o sea water. In this experiment the plasma sodium values were 125.3 + 10.4, 135.3 _t 7.3 and 159.2 -+ 11.8 m-equiv. l-l, respectively for the three groups, after 2 months’ exposure. However, subsequent sampling for the three groups, at various intervals up to 6 months, show that there were no further changes. Thus in animals kept in lOOo/o sea water, plasma sodium seems to rise initially to a higher but more stable level. This rise differs significantly (PC 0.01) from initial values, and from all the values obtained for the groups kept in fresh water or 50% sea water.

Plasma osmolarity figures for the same animals are shown in Fig. 3 and indicate the same trend. The sampling periods illustrated are typical of a much larger series, and show that in lOOo/o sea water the plasma osmolarity (initially) rises

u)

5 ‘I- v Se0 water (100%) E :

x-x Sea water(50%) .-A Fresh woter

k

_ 400- T

), C ,‘T I

1

i h E”

1’ / 6 300- ,’

z ,/

d -ii

T tl I P

0 200 I I 1

o-5 2 5

Time, months

FIG. 3. Effect of long term adaptation to varying environmental salinities on plasma osmotic pressure.

significantly (PC 0.01) above the plasma osmolarity of animals kept in fresh water of 50% sea water, within the first 2 weeks. Thereafter the plasma osmolarity rises no further, becoming stabilized at 400 mosmols. Experiments are now under way to measure changes in plasma sodium and osmolarity during the early stages of acclimation to various salinities, but some of this work has been done by others (Bentley et al., 1967). In another experiment, using animals described under experiment 2 in Tables 1 and 2, similar plasma osmolarity values were found. The animals kept in sea water without access to fresh water had plasma osmolarities of 400 mosmols 5 16.2 after 4 months’ exposure, while those kept in sea water but given access to fresh water every 3 days maintained plasma osmolarities of 285 f 13.5 mosmols. The difference between these two groups was significant (P” 0.01).

Figure 4 shows data collected from experiments on cranial loss of sodium with fresh water available during the collection period. This graph shows that with

496 F. B. M. COWAN

I 2 3 4 5 6

Tme. hr

FIG. 4. Cumulative cranial sodium secretion in unloaded and salt loaded animals.

animals adapted to sea water, and salt loaded with 5.17 m-equiv. kg-l sodium chloride there was a cranial loss of sodium of 17 p-equiv. 100 m-l hr-r. The control animals also adapted to sea water for 4 months but loaded with an equal volume of distilled water had a cranial loss of sodium of O-8 p-equiv. 100 g-l hr-l f 0.9. The difference between the two groups was significant (IQ 0.01). Using

the second variation of the collection method, in which the animal was not allowed access to fresh water during the collection period, similar values were found, with a total cranial loss of sodium equal to 19 _+ 4.6 p-equiv. 100 g-l hr-l for salt loaded animals, and 1.3 CL-equiv. 100 g-1 hr-1 in distilled water controls. The

difference here was also significant to the P-c O-01 probability level. Finally, the histochemical observations reported in an earlier paper (Cowan,

1969) were confirmed and extended. The lachrymal glands of animals kept in fresh water showed a weak reaction for sulfated polyanions. This was also true for animals kept in sea water, but given access to fresh water at 3-day intervals. The animals restricted to sea water, however, showed a very strong reaction for sulfate groups.

DISCUSSION

The observations reported here could be taken to illustrate the following points concerning osmoregulation in the euryhaline M. terrapin.

The animals are in fact capable of surviving extended periods in sea water or fresh water. There seems no necessity for supplying fresh water to animals kept in sea water for 1 year (cf. Coker, 1951). Behaviour, appetite and general condition do not appear different in the two groups, and there is no significant difference in the incidence of mortality.

EXTRARENAL FXCFU3TION AND OSMOReGULATION IN MALACLEMYS TERRAPIN 497

One detected change in the condition of the animals was in total body weight. Matactemys restricted to sea water appear to progressively lose weight at a slow rate, while animals in fresh water and SOo/0 sea water seem to maintain stable body weights. This drop in weight might indicate a progressive dehydration (Bentley et al., 1967).

This suggestion is partially borne out by the fact that plasma osmolarity is increased in animals kept in sea water. However, this increase in plasma osmolarity is not linear with time. The data show that there is a rapid increase in plasma osmolarity with the first 2 weeks exposure to sea water, but thereafter the plasma osmotic pressure remains constant, with no further increase up to 6 months exposure to sea water.

The bulk of the increase in plasma osmotic concentration can be accounted for on the basis of increased sodium concentration. This increase in sodium values appears to follow quite closely the increase in osmotic pressure, with a rapid initial raise after the animals are put in sea water, followed by a stabilization, throughout the subsequent exposure. This initial rise of plasma sodium and osmolarity might represent the time necessary for induction of euryhaline mecha- nisms, for example see Bornancin & de Renzis (1972). However, even if this is true the assumption must be made that Malachzys tolerates relatively high plasma sodium and osmotic concentrations, because even after sufficient time for the induction of euryhaline mechanisms, these plasma concentrations remain high compared to the animals kept in fresh water. This would indicate a certain degree of osmoconformity, which is perhaps not unusual in the reptilian class (Bradshaw & Shoemaker, 1967).

The results with respect to changes in plasma osmolarity and sodium concen- tration in animals kept in 50% sea water vary somewhat with those reported by Gilles-Baillien (1970). He found animals kept in 50% sea water, for at least 15 days, had significant increases in both parameters. This slight difference in results might be due to the past history of the animals, and the degree of activation of excretory mechanisms. The present results on plasma osmolarity and sodium concentration in animals kept in fresh water or lOOy& sea water are in essential agreement with his results.

Whether induction of salt gland tissue is part of this acclimation to sea water remains unclear. There is no great enlargement of the salt gland in animals kept in sea water. It could be debated that the relative gland weight in animals kept in sea water is greater, while in the Harderian gland no changes are seen; but any increase in size is not anywhere near the same magnitude seen in other species. However, the lachrymal gland in M~~c~~~s is large in all animals studied with the relative weight similar to that of salt glands in other marine species. The lachrymal gland of Malaclemys is also much larger than the homologous gland in several closely related, but stenohaline emydines (Cowan, 1969). So it could be that the size of the gland is determined early in the life of the animals, when it is almost certain that they were exposed to sea water (Dunson, 1970). Unfo~unately, hatchlings have not been available to test this theory. The lachrymal gland from

498 F. B. M. COWAN

Malaclemys kept in sea water but given fresh drinking water was also significantly

smaller when compared with the gland taken from animals kept in sea water without fresh drinking water. This indicated that the increase in size, if function- ally significant, is not related to exposure to sea water per se. The same conclusion

is suggested by the fact that the increase in sulfate staining is found in animals exposed to sea water but without fresh drinking water, whereas in the animals exposed to sea water but given fresh drinking water there is no increase in sulfate

staining. The presence of sulfate in secretory cells therefore apparently is not related to exposure of the eye and related structures to sea water. A material of similar histochemical staining properties has been found in a number of osmo- regulatory organs undergoing functional changes (Morard & Poirier, 1968 ; van

Hegan, 1967). However, this circumstantial evidence does not prove whether the lachrymal

gland of Malaclemys is primarily an osmoregulatory organ. The fact that Malac- lemys alone of all the emydines studied is able to survive in sea water or distilled water for extended periods of time indicates that it does have mechanisms to

retard dehydration in sea water, but the progressive loss of weight indicates that these mechanisms have relatively low efficiency. Dunson (1970), in his review of reptilian salt glands, has shown that there is wide variation in the osmolarity of

secreted fluid and maximal secretory rates of the various extrarenal excretory organs found within the class. The figures for cranial loss of sodium in this paper agree very closely with those of Dunson, who used similar techniques. The present work shows that 2.16 m-equiv. of sodium would be excreted cranially in a

500-g animal over a 24-hr period. Thus it would take approximately 1 day to excrete cranially the relatively small injected sodium load. In the present experi-

ments this rate of excretion appeared to remain the same whether fresh water was available during the collection period (method 1) or not as in method 2. This

excretory capability is modest; well below that of Chelonia mydas (Holmes &

McBean, 1964). Without direct cannulation, however, it cannot be said whether this cranial sodium loss occurs as isotonic loss, concomitant with the secretion of

organic material, or hypertonic secretion from an organ primarily subserving the function of osmoregulation. Direct cannulation of the lachrymal gland from Malaclemys is difficult for anatomical reasons, but studies on isolated gland tubules are under way in this laboratory. Until these data are available the following

hypothesis seems worthy of consideration. Malaclemys in natural surroundings can tolerate sea water for extended periods, but fresh water is normally available. The animal, when it has access to fresh water, fully hydrates and stores relatively large amounts of water interstitially, or perhaps in definitive organs. Upon return to sea water this stored water is available initially to counteract dehydration, and salt influx. During this period other mechanisms for retarding dehydration are activated. This process takes several days before the animal is able to retard hydration by these more active mechanisms, and the progressive weight loss would indicate that even then these mechanisms of osmoregulation are not fully

effective. One such mechanism might be sodium secretion by the lachrymal

EXTRARENAL EXCRETION AND OSMOREGULATION IN MALACLEMYS TERRAPIN 499

gland, perhaps by a secretion of a fluid hypertonic to plasma, but not necessarily hypertonic to sea water. Direct cannulation is probably necessary to decide this point.

Acknowledgements-I gratefully acknowledge the help of Dr. T. S. Parsons and Dr. V. E. Engelbert in doing a part of this work. This work was supported by a National Research Council operating grant, No. A-6365, to the author.

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Key Word Index-Turtles ; osmoregulation ; salt glands ; extra-renal excretion; Mulac- lemys terrqpin; euryhalinity.