STUDIES ON OSMOREGULATION AND ENDOCRINE CONTROL OF OSMOREGULATION IN THE ATLANTIC STINGRAY, DASYATIS SABINA

STUDIES ON OSMOREGULATION AND ENDOCRINE CONTROL OF OSMOREGULATION IN THE ATLANTIC STINGRAY, DASYATIS SABINA

by

BARRY EDWARD BEITZ, B.S.

THESIS

Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of MASTER OF ARTS

THE UNIVERSITY OF TEXAS AT AUSTIN

August, 1974

ACKNOWLEDGMENTS

I would like to thank Dr. Martin Sage for his guidance and concern, Mike Litwin and Dr. Vic deVlaming for their technical assistance and helpful discussion, and Boat Captain Elgie Wingfield for providing animals. My thanks also to Welder Wildlife Foundation for their financial assistance.

May 20, 1974

ABSTRACT

Osmoregulation and endocrine control of osmoregulation were studied in the euryhaline stingray, Dasyatis sabina. In hypersaline sea water (above 35ppt), the ability to regulate plasma solutes was reduced relative to regulation in the optimum, hyposaline (12-28ppt) range. Survival in high salinities was dependent on slow acclimation, while rapid change to low salinities was tolerated. These results appear consistent with adaptations necessary for salinity changes occurring in a bay environment.

Natural, uninduced rectal gland secretion was obtained in 2* sabina. The average chloride concentration of the rectal gland fluid was 583.3 m-moles/1. The average flow rate was 0.13 ml/kg/hr. Occurrence and amount of rectal gland secretion were erratic.

Thyroidectomy, in sea water of 23ppt salinity, caused a significant rise in plasma urea concentrations in D. sabina. Replacement injections of thyroxine lowered plasma urea levels in thyroidectomized animals.

Removal of the rostal pars distalis caused plasma urea to increase. Replacement injections of prolactin lowered plasma urea levels, as did replacement ACTH injections. Replacement of both

prolactin and ACTH caused variable results concerning urea. Problems of dosage, stress, and possible seasonal influences, most likely affected the results of the endocrine study.

TABLE OF CONTENTS

Page

General Introduction 1

Materials and Methods 5

Introduction —Osmoregulation 11

Part I —Results 15

Osmoregulation in hypersaline media 15

Rectal gland studies 21

Discussion 21

Introduction —Endocrine control of osmoregulation in elasmobranchs 30

Part II —Results 37

Thyroidectomy 37

Rostralobectomy 37

Discussion ..... 42

References 49

LIST OF TABLES

Page

Table

I Plasma solute concentrations in ID. sabina after six days in hypersaline media 16

II Rectal gland fluid (RGF) electrolyte concentrations and flow rate in D. sabina 22

III Plasma solutes in D. sabina exposed to dilute media expressed as percentage of plasma concentrations in sea water 25

IV Effects of thyroidectomy and replacement therapy with thyroxine on osmoregulation in ID. sabina 38

V(a) Effects of rostralobectomy and hormone replacement therapy on osmoregulation in D. sabina. —May, 1973 ... 40

V(b) Effects of rostralobectomy and hormone replacement therapy on osmoregulation in D. sabina. —March, 1974 . . 41

VI Effects of rostralobectomy and prolactin replacement therapy on osmoregulation in ID. sabina 46

LIST OF FIGURES

Figure

Page

1. Change in plasma sodium and potassium concentrations with salinity in D. sabina 18

2. Change in plasma urea concentration with salinity in D. sabina 20

3. Diagrammatic median longitudinal section of the pituitary gland of D. sabina, indicating zonation and location of hormones 32

GENERAL INTRODUCTION

Osmoregulation is a homeostatic mechanism whereby both the volume of water and the concentration of electrolytes of the internal body fluids are controlled within narrow limits (Conte, 1969). The cartilaginous fishes use such a mechanism in order to exist in the sea water environment.

Elasmobranchs maintain their plasma slightly hyperosmotic to sea water (for review see Bernard, Wynn and Wynn, 1966; Holmes and Donaldson, 1969). The inorganic plasma solutes, mainly Na + and Cl , account for approximately two-thirds of plasma osmolarity (for review see Holmes and Donaldson, 1969), their concentration in the blood being about the same as or a little greater than that of marine teleosts.

The high osmolarity of elasmobranch blood is due to the active conservation of the nitrogenous end products produced in the liver, namely urea and trimethylamine oxide (TMAO) (Goldstein, 1967; Goldstein and Funkhouser, 1972). These organic solutes are responsible for onethird to 55% of plasma osmolarity (Cohen, Krupp and Chidsey, 1958; Holmes and Donaldson, 1969; deVlaming and Sage, 1973).

The hyperosmotic condition allows for a homeostasis in water balance achieved through opposing osmotic and hydrostatic forces. Water entering osmotically would tend to dilute the blood leading to an increased excretion of urea due to an increased rate of glomerular filtration and urine flow. A lowered blood concentration of urea would lower the absorption rate of water through the gills which would decrease urine flow. In actuality, these processes are normally in balance. The result of this simple mechanism is that the fish receives water without expending any free energy and in sufficient quantity to meet urinary demands and maintain fluid equilibrium (Smith, 1953; Forster, 1967). Other aspects of osmoregulation do require energy expenditure, such as active transport of plasma solutes to and across various membranes.

Presumably elasmobranchs do not drink sea water, in contrast to the hypoosmotic marine teleosts which must drink to maintain osmotic balance (Burger, 1967; Hickman and Trump, 1969). This theory is supported by the fact that sodium flux in the dogfish, Squalus acanthias, can be balanced without considering sea water ingestion (Horowicz and Burger, 1968).

Maintenance of internal equilibrium depends on balancing chemical influx and efflux. Since the concentration of salts in elasmobranch blood is less than that of sea water, electrolytes tend to enter by diffusion. Sites of invasion include the gills and skin. Also, eating is a source of chemical uptake in the gastro-intestinal tract.

Burger (1967) suggests that SL acanthias can control electrolyte influx so that excretory mechanisms are not swamped. The elasmobranch gill is relatively impermeable to sodium and urea (Smith, 1953; Boylan, 1967). Also, low epithelial permeability to electrolytes is suggested by low turnover rates of sodium and chloride (Carrier and Evans, 1972).

The role of the kidneys is control of water, excretion of multivalent ions and conservation of urea and TMAO (Burger, 1965). Most of the filtered water is reabsorbed, with loss of 15-30% of the filtrate. The urine produced is hypoosmotic to the blood, indicating solute is more effectively reabsorbed than water. Variations in water influx can be compensated for by adjustment of tubule permeability and filtration rate (Burger, 1967; Hickman and Trump, 1969).

Since the elasmobranch kidney is a water excretory device, the efficient retention of plasma solutes is necessary. This problem is alleviated by the diffusion of salts into the animal from sea water due to the osmotic gradient, and the result is the ability to critically regulate electrolytes. Each blood solute is independently regulated. Sodium, chloride, potassium and calcium are reabsorbed from the filtrate, while magnesium, sulfate, and phosphate are actively excreted (for review see Hickman and Trump, 1969).

The organic solutes urea and TMAO are freely filterable at the glomerulus, but are actively reabsorbed to maintain the proper osmotic pressure of the blood for water acquisition. 90-95% of urea is reabsorbed in the renal tubules, while 95-99% of TMAO is retained (Cohen et al., 1958; Forster, 1967). The per cent of filtered urea reabsorbed depends on the plasma urea concentration (Kempton, 1953).

The rectal gland secretes a sodium chloride solution at concentrations twice those of the plasma concentration (Burger and Hess, 1960). Burger (1967) notes this gland is instrumental in holding plasma electrolyte levels to a narrow range through sensitive response to blood salt concentration.

Our knowledge of elasmobranch osmoregulation is rather meager. Information on endocrine control of osmoregulation is especially limited. Using the euryhaline elasmobranch, Dasyatis sabina, this study surveys various aspects of osmoregulation. Change in concentration of plasma solutes with high salinity, as well as rectal gland function, was investigated. Special attention was given to indications of hormonal control of osmoregulation.

MATERIALS AND METHODS

Animals

The Atlantic stingray, Dasyatis sabina (Lesueur), is a bottom dwelling elasmobranch, living generally on sand flats in bays or other shallows (Bigelow and Schroeder, 1953; Bohlke and Chaplin, 1968). Those rays studied inhabited the bays near Port Aransas, Texas, from the late spring to fall. During the winter, the stingrays migrate to the shallow Gulf of Mexico. This seaward movement is mainly prompted by low temperatures (Gunter, 1945; Sage, Jackson, Klesch and deVlaming, 1972).

D. sabina is euryhaline. Gunter (1945) found the stingray in salinities ranging from 2.2-36.7 ppt. It was most commonly obtained above 25 ppt. Simmons (1957) and Hoese (1960) have found D. sabina in water of 45 ppt salinity. Gunter (1945) also noted D. sabina was found from 13.7-30.5°C.

Collections were made, in both the bays and shallow Gulf, using an otter trawl similar to those used for shrimp. Animals were kept in live boxes with circulating sea water on the boat, and immediately transferred to covered concrete holding ponds upon return to port. The holding ponds used aerated, circulating natural sea water at ambient temperatures. The stingrays were allowed to acclimate to these conditions for at least four days prior to experiments. The rays were usually not fed during holding, and never during experiments.

PART I: METHODS

Osmoregulation

Animals for the hypersaline studies were transferred from the holding ponds to 227 1 polyethylene tanks containing aerated high salinity water, (salinities specified in the results section). After six days the rays were killed. Blood samples were obtained with syringe by cardiac puncture. Plasma samples and sea water samples from test tanks were frozen (-4°C) until used for analysis.

Urea was determined colorimetrically by the diacetyl monoxime method (see Sigma Technical Bulletin No. 535), chloride titrimetrically according to its reaction with mercuric nitrate (see Sigma Technical Bulletin, No. 830). Sodium, potassium and calcium were determined photometrically using a Beckman Flame Photometer (Model 105).

Rectal gland

Animals for rectal gland studies were placed in the 227 1 tanks for 2-3 hours acclimation to the test salinity (see results). Operations, as described below, were then performed and rectal gland fluid collected four hours later.

Collection of rectal gland fluid in various shark studies was made with a catheter inserted into the duct of the rectal gland (Burger and Hess, 1960; Chan, Phillips and Chester Jones, 1967). However, in Dasyatis sabina this method of collection is almost impossible (Burger, 1972). The method employed here consisted of opening the body wall just anterior to the pelvic girdle and tying off the intestine on either side of the rectal gland duct. The intestine was washed out with distilled water prior to tying off the posterior constricture. The body wall was then sutured. Shams had the rectal gland duct tied off as well. Animals were anesthetized prior to operation by immersion in a MS-222 (tricaine methanesulphonate) solution (1 g/4000 ml sea water) neutralized with 0.1 N sodium hydroxide.

Upon completion of the test period, the sac formed by the intestine was removed. Any fluid present was obtained by direct puncture with a 1 cc syringe. This fluid, blood and test water samples were frozen until analyzed.

PART II: METHODS

Rostralobectomy

Animals were anesthetized in 1:4000 MS-222. The pituitary was approached through the mouth from the ventral side of the braincase. A hinged flap was cut in the roof of the mouth just anterior to the carotid anastomosis. The anastomosis lies on a line between two small

protuberances, one on either side of the roof of the mouth near the internal spiracular openings. The flap exposed the rostral lobe of the pars distalis, which was then removed. In sham-operated animals, the pituitary was exposed, but left intact. Upon completion of the operation, the flap was closed and the buccal mucosa joined by adhesive (Eastman, 910). (Operations were performed by Dr. V. L. deVlaming and the author).

Aminals were then placed in the test tanks of aerated, filtered sea water. Dilutions were made by the addition of dechlorinated tap water. Replacement therapy consisted of daily (five total) injections of 1 I.U. prolactin (Sigma, ovine) for one group, 1 I.U. ACTH (Sigma, porcine) for another, and 1 I.U. of both for another. The injections were given intramuscularly. After six days, the animals were sacrificed, and blood samples taken as above.

Thyroidectomy

Animals were anesthetized in 1:4000 MS-222. The thyroid in D. sabina is encapsulated, and removed easily. In sham-operated animals, the thyroid was exposed, but the gland left intact. (Operations were performed by Mike Litwin.) The incision was closed by suturing.

Animals were placed in aerated, filtered test tanks for six days. Daily injections (five total) of 6.5-7.2 pg/day of L-thyroxine (Sigma, sodium salt) were given intramuscularly to half the shams and half the fish with the thyroid removed. The total iodine release rate in D. sabina averages 3-5 pg/day in a 700 g animal (Litwin, unpublished). Blood and tank water samples were taken on the sixth day

Note

The experiment length of six days was previously determined (deVlaming and Sage, 1973) as the period necessary for D. sabina to equilibrate its blood ion levels after a change in the external medium.

The survival of animals in experimental conditions was poor until the methods of water changes twice in six days, and constant filtering of the water with were employed. All animals thereafter were in excellent condition at the end of an experiment.

Statistics

All comparisons were a. priori planned comparisons for which the t-test called ’’least significant difference" was used (Sokal and Rohlf, 1969). The F -test was employed to test for homogeneity of max variance. For the groups whose variances were heterogeneous (thyroidectomy experiment-potassium; rostralobectomy May experiment-potassium), the approximate t-test was used (Sokal and Rohlf, 1969).

PART I

INTRODUCTION—OSMOREGULATION

Elasmobranch plasma solutes change in concentration as external salinity varies. The direction of change follows that of the sea water’s change in osmotic pressure (Chaisson, 1930; Price and Creaser, 1967; Goldstein, Oppelt, and Maren, 1968; deVlaming and Sage, 1973; Jones and Price, 1974). The blood is maintained hyperosmotic to the external medium, although the degree of regulation varies with the species, concentration of the sea water and duration of exposure.

In previous studies, changes in plasma urea concentration were the major factor in adjustment of blood osmotic pressure. The method of urea regulation appears to vary with species and salinity tolerance. In the euryhaline lemon shark, Negaprion brevirostris, exposure to 50% sea water caused a 55% decrease in plasma urea concentration, due mainly to a threefold increase in renal clearance of urea (Goldstein et al., 1968). In the stenohaline little skate, Raja erinacea, exposure to 50% sea water caused a 45% decrease in plasma urea concentration, caused by a twenty-twofold increase in renal clearance in urea, and a decrease in urea biosynthesis (Goldstein and Forster, 1971). Forster, Goldstein and Rosen (1972) suggest that in addition to changes in glomerular activity and hepatic biosynthesis of urea, peritubular forces can help

control osmotic adjustments via tubular reabsorption of urea and other osmotically important solutes.

Of the blood electrolytes, sodium chloride is the most important as a component of plasma osmotic pressure. Sodium chloride and urea are responsible for over 90% of plasma osmolarity, with each contributing a corresponding fraction.

Electrolytes tend to enter the body by diffusion. In the spiny dogfish, S>. acanthias, sodium influx through the head is balanced by efflux through the kidney, rectal gland, and the head (gills) (Horowicz and Burger, 1968). However, the rectal gland is the specific regulator of sodium chloride (Burger, 1965). Hickman and Trump (1969) postulate that sodium chloride appears in the urine only as an "osmotic ballast," which when combined with the divalent ions, urea and other minor urinary osmolytes, permits the kidney to dispense with forming an exceedingly dilute urine.

The rectal gland is a cylindrical gland with a central canal. This canal continues as a duct which empties into the intestine behind the spiral valve. A single rectal artery arising from the posterior mesenteric artery enters the capsule dorsally and gives rise to many branches which eventually form a single ventral rectal vein. The blood flows in the same direction as the fluid is secreted, thus no countercurrent mechanism is operating (Burger, 1962; Chan and Phillips, 1967).

Various histological studies of the rectal gland indicate its glandular tubules actively extract sodium via some sort of sodium pump (Doyle, 1962; Bulger, 1963; Chan and Phillips, 1967). Since the resulting rectal gland fluid is isosmotic with the plasma, no osmotic work is required to secrete the solution (Bulger, 1963).

The salt solution concentrated by the rectal gland is mainly sodium chloride at a concentration twice that of the plasma (Burger and Hess, 1960). The gland tends to secrete on a continuous, yet variable, basis indicating a persistent influx of salt from the external medium (Burger and Hess, 1960). The volume of flow is quite sufficient to significantly affect plasma salt levels (Burger and Hess, 1960; Burger, 1962, 1965). The secretory mechanisms are capable of fairly exact evaluation. Following three successive injections of equal amounts of saline, the increase in rectal gland output in acanthias was approximately the same each time (Burger, 1962, 1967).

To discern the relative importance of the rectal gland in the salt economy of 8. acanthias, Burger (1965) removed the gland. An increase in urine flow was observed in glandless fish, resulting in higher urinary chloride loss than in intact fish, and stable plasma concentrations. However, no specific renal response to concentrate chloride above plasma levels was observed in the face of a salt load. Thus, the kidney cannot act as a substitute rectal gland. The response to the absence of a rectal gland is reduced uptake of salt so influxing

solutions are nearly equal to or below plasma concentrations (Burger, 1965).

Three components are suggested in the induction of secretion in the rectal galnd: an osmotic, a volume and a sodium chloride response. The nature of the receptor is unknown. Nervous control of the gland is not evident, therefore humoral factors are postulated (Burger, 1962). Chan, Phillips, and Chester Jones (1967) suggest corticosteroidtype hormones can affect rectal gland function, although conclusive results were not obtained.

PART I—RESULTS

Osmoregulation in hypersaline media

In experiment I (see Table I) of the hypersaline study, rays were placed in aerated tanks of 36, 38, 39 and 41 ppt salinity sea water. Prior to the experiment, the animals were held in 33 ppt salinity, at ambient temperature 25.8°C. The rays in tanks 39 and 41 ppt died within one or two days of transfer, while only one animal was lost in 38 ppt and none in 36 ppt. Following a six day acclimation period, the stingrays were removed and plasma obtained.

Since in experiment I animals transferred directly to high salinity died, an alternative experimental procedure was adapted. Rays in experiment II were placed in test tanks of 26 ppt salinity sea water The water was allowed to evaporate over a period of 14 days, reaching approximately 40 ppt, with a sodium concentration of 705.6 m-equiv/1, chloride concentration of 822.5 m-equiv/1 and a potassium concentration of 16.2 m-equiv/1. The electrolyte values reported in Table I for the sea water in experiment II are the approximate concentrations the sting rays were acclimated to by the end of the 14 day period.

Values for the plasma electrolyte concentrations were graphed (see figures 1 and 2) along with values obtained in a previous study in

this laboratory of D. sabina osmoregulation in hypoosmotic media (deVlaming and Sage, 1973). The values reported here extend the graphs, and show a gradual loss in regulating ability as the extreme end of the salinity tolerance in I), sabina is approached.



l. Change in plasma sodium and potassium with salinity in D■ sabina. (° = data from deVlaming and Sage, 1973; ■ = data from this study.)



Figure 2. Change in plasma urea concentration with salinity in D. sabina. (° = data from deVlaming and Sage, 1973; ■ = data from this study.)

Sample Plasma and Sea Water Electrolyte Concentrations* Fluid Size Na"*~ Cl -i ++ K Ca Urea (N) (m-moles/1) (m-equiv/1) EXPERIMENT I sw a — — 562.4 595.0 13.6 23.0 plasma (2) 426 ±11 408.0 ± 78.0 343.2 ± 6.8 7.0 ± 1.6 5.1 ± 1.0 sw a — — 571.6 655.0 13.9 21.0 plasma (2) 434 ± 41 369.2 ± 3.2 381.2 ± 18.8 6.3 ± 0.6 6.2 ± 1.1 EXPERIMENT II sw b — — 635.0 731.0 15.0 20.5 plasma (4) 377 ± 14 378.0 ± 12.0 386.2 ± 9.4 9.6 ± 0.3 4.8 ± 0.4 * Values (x ± S.E.) a = sea water, 22°C, animals prior acclimation to 33 ppt. b = 24.6°C, sea water allowed to evaporate from 26 ppt to 40 ppt over a 14 day period (see text).

TABLE I PLASMA SOLUTE CONCENTRATIONS IN D. SABINA AFTER SIX DAYS IN HYPERSALINE MEDIA

Rectal gland studies

Animals for the rectal gland experiments were held prior to the tests in water of 31.9 ppt salinity and 13°C, except for the ray weighing 1011 grams (see Table II), which was in 31.9 ppt and 18°C. Rays were put in two groups, each containing a control, sham and expermentals. One group was put in water of 29-32 ppt, the other in high saline water ranging from 37-40 ppt. Temperature was 17°C, except for the ray mentioned above, which was in 20°C water. Rectal gland secretion observed during these experiments proved erratic. Salinity did not seem to influence the rectal gland’s secretion (three samples were obtained in high salinity, two in normal salinity). Also, rectal gland fluid was obtained in only 5 of 11 test animals (see Table II).

The chloride concentration of the rectal gland fluid (RGF) ranged from 1.7 to 2.3 times as great as the plasma values. The RGF volume varied tenfold, and the flow rate sixfold among samples obtained.

Discussion

The stingray, I). sabina, is a euryhaline elasmobranch, migrating between marine and brackish waters. The previous study by deVlaming

and Sage (1973) shows efficient ionic regulation by _D. sabina over a range of approximately 12-28 ppt salinity sea water. Thus it appears D. sabina is best adapted to dilute sea water.

Since the stingray has been found in salinities of up to 45 ppt, the present experiments were carried out to determine its osmoregulating ability in high salinities. The resulting data extends the graphs (see figures 1 and 2) of deVlaming and Sage (1973). Above 28 ppt salinity, the concentration of plasma electrolytes increases markedly. However, plasma potassium appears to vary freely with the external potassium concentration until about 21 ppt, whereupon it is somewhat regulated until 37 ppt. Above that salinity, it again starts to increase sharply. Also, urea differs in that it is regulated fairly well over a wide range, approximately 12-35 ppt salinity.

Since Dasyatis spends most of its time in brackish water bays and the shallow ocean, it regulates best in lower than normal sea water salinities. DeVlaming and Sage (1973) note that in D. sabina, with increasing dilution of the medium, the gradient between plasma and sea water osmotic pressure increases. This general trend is also observed in Raja eglanteria (Price, 1967), R. erinacea (Goldstein and Forster, 1971) and Negaprion brevirostris (Goldstein et al., 1968). All the above mentioned elasmobranchs are known to enter brackish waters except R. erinacea, which is strictly marine (Bigelow and Schroeder, 1953).

In adapting to low salinities, D_. sabina lowers Na and Cl more than urea until about 11-12 ppt (see Table III). In changes from 100% to 35% sea water, urea is reduced by 20.1%, chloride by 25.5%. In R. eglanteria, however, changes from 86% to 46% sea water reduced urea by 44% and chloride by only 25% (Price and Creaser, 1967). In N. brevirostris, change from 100% to 50% sea water reduced urea by 55% and chloride by only 20% (Goldstein et al., 1968). Also, in S_. acanthias, a change from 74% to 46% sea water reduced urea by 18%, and chloride by 11% (Jones and Price, 1974). The differences between species may be due to different ranges of tolerance to salinity, different acclimation times or just different physiologies. More data is needed to elucidate the problem.

Data on survival in high and low salinity studies with D. sabina can be related to environmental adaptation to salinity changes. Upon transfer to high salinity water, survival depended on the difference in salinities. Animals acclimated to 33 ppt, survived direct transfer to 36-38 ppt, while there was total failure upon abrupt transfer to 39-41 ppt. Rays, allowed a slower acclimation by letting the tank water evaporate, survived fairly well to high (40 ppt) salinities. Animals transferred directly from 24 ppt to 12 ppt sea water all survived.

This difference in tolerance to rapid change in salinity appears to reflect an adaptation to environmental situations. Rapid

increases in salinity rarely occur naturally in the environment. Often a long drought will raise the salinity of a bay above 40 ppt, but the change is slow, allowing time for adaptation or escape. However, a rapid drop in salinity is more commonly encountered in the natural environment. A heavy rainfall, whether local or inland, can result in greatly increased fresh water runoff and rapid salinity drops in surrounding bays. The ability to tolerate rapid decreases in salinity would obviously be advantageous to a common resident of the bays. D. sabina seems to possess this adaptation to environmental stress.

The rectal gland in elasmobranchs is a specific regulator of sodium and chloride. The great majority of research on this gland has been done with the spiny dogfish, acanthias. This study on D. sabina was carried out in an attempt to broaden the knowledge of rectal gland function in cartilaginous fishes.

One previous study has been done on the rectal gland in D. sabina (Burger, 1972). However, no uninduced, natural secretion was obtained. Injections of NaCl did result in three samples of rectal gland fluid in that experiment. The average chloride value of the rectal gland fluid was 770 m-moles/1, with plasma values of 305-340 m-moles/1 chloride for injected fish. Maximum observed secretion rate of RGF was 0.6 ml/kg/hr.

In this study on D. sabina, six samples of natural, uninduced rectal gland fluid were obtained. Average RGF chloride values were

583.3 m-moles/1, with plasma values ranging from 287.5-340 m-moles/1. Maximum observed secretion rate was 0.26 ml/kg/hr, with an average flow rate of 0.13 ml/kg/hr.

The fairly large discrepance in RGF average chloride concentration between studies could be due to many factors. According to Burger’s (1962) work with acanthias, an injection of NaCl raises the concentration of the RGF. However, since Burger (1972) does not mention the amount or concentration of the NaCl injections given D. sabina in his experiment, no exact analysis can be made. Also, a NaCl injection causes a low rate of secretion to increase tremendously (Burger, 1962). This may be responsible for the difference in maximum observed secretion rate in D. sabina between studies.

The variation seen in this study of natural, uninduced rectal gland secretion can probably be attributed to inherent problems with the collection technique (see Material and Methods), or more important, to matters of threshold. Burger (1967) postulates that a persistent influx in salt must build to a threshold level, which then induces rectal gland secretion. The secretion would lower the plasma’s concentration and volume, and another build-up period would begin. The resulting variation in output has been observed in JS. acanthias (Burger, 1965), and most likely is present in I), sabina.

In S_. acanthias, the average chloride concentration of unin- duced RGF is 490-499 m-moles/1. The maximum observed secretion rate is about 4 ml/kg/hr. The average flow rate is 0.47 ml/kg/hr, varying from 0.11-0.82 ml/kg/hr for individual fish averages (Burger, 1962).

The rectal gland in elasmobranchs is obviously important in maintaining internal chemical homeostasis via specific regulation of sodium chloride. The function of this gland is no doubt especially valuable to the euryhaline stingray in adjusting to varying salinities. The specific receptors and controlling mechanisms of the rectal gland are, however, unknown at present.

Electrolyte Concentrations Fish no. Weight (g) Fluid urea + Na Cl K + Volume (ml) RGF flow rate (ml/ kg / hr) (m-moles/1) (m-equiv/1) 1 a RGF 59 575.0 575.0 12.0 0.200 — plasma 334 318.0 340.0 5.7 7 1011 RGF 31 624.0 687.5 10.0 1.000 0.257 plasma 344 309.0 300.0 6.8 17 1756 RGF * — 568.8 — 1.000 0.142 plasma 269 — 302.5 — 19 839 RGF — — 525.0 — 0.100 0.041 plasma 215 — 317.5 — 26 946 RGF — — ; — 531.2 — 0.245 0.089 plasma 279 — 287.5 — 29 853 RGF — — 612.5 — 0.430 0.138 plasma 311 — 302.5 — Averages (x ± S of RGF • E.) 583. 3± 24.6 11.0 ± 1.0 0.50± 0.17 0.1334± 0.0360 a = results of a preliminary experiment, not included in text. * = dashes (— —) mean the value was not determined.

TABLE II RECTAL GLAND FLUID (RGF) ELECTROLYTE CONCENTRATIONS AND FLOW RATE IN D. SABINA

Urea + Na Cl % sea water plasma concentration in dilute sea water/ /plasma concentration in normal sea water 80 91.4 82.7 82.2 61 93.2 84.3 85.8 55 87.5 75.1 70.0 35 79.9 77.1 74.5 26 54.8 63.7 69.3

TABLE III PLASMA SOLUTES IN D. SABINA EXPOSED TO DILUTE MEDIA EXPRESSED AS PERCENTAGE OF PLASMA CONCENTRATIONS IN SEA WATER. (FROM DEVLAMING AND SAGE, 1973).

PART II

INTRODUCTION—ENDOCRINE CONTROL OF OSMOREGULATION IN ELASMOBRANCHS

Osmoregulation in fishes is under the control of various hormones released by endocrine organs into the blood stream. Teleost endocrinology has been studied fairly extensively, while information concerning hormone responses and actions on other fishes, especially elasmobranchs, is lacking. In general, the anatomy and histophysiology of the endocrine system of elasmobranchs has been described (see Hoar and Randall, 1969).

The pituitary gland in elasmobranchs has been investigated. Although the functional identity of the various cell regions is still somewhat uncertain, the general location of the pituitary hormones has been determined (see figure 3).

Prolactin has been found in the rostral pars distalis of elasmobranch pituitaries (Grant and Waterman, 1962; Grant, 1962). This prolactin is of the "fish-type" that gives no response in the pigeon crop and mammary gland tests (Nicoll and Bern, 1964; 1968).

Considerable information is known about the effect of fish prolactin in teleosts (for review see Ensor and Ball, 1972). Prolactin controls osmoregulation in hypotonic media, having a high secretion rate

Hormone references: prolactin (Grant, 1962; Grant and Waterman, 1962); ACTH (deßoos and deßoos, 1967); TSH (Dent and Dodd, 1962; Dodd et al., 1962; Jackson and Sage, 1973); gonadotropin (Dodd, Evennett and Goddard, 1960); MSH (Lowry and Chadwick, 1970); neurohypophyseal principles (Perks, Dodd and Dodd, 1960; Sawyer, 1965).

in fresh water, and a very low secretion rate in sea water. In those teleosts in which hypophysectomy causes death in fresh water, prolactin injections maintain survival. Prolactin also affects osmoregulation in fish not needing the pituitary to survive in fresh water. Prolactin maintains normal plasma electrolyte levels. Prolactin affects sodium fluxes at the gill, kidney, urinary bladder and possibly the skin. Water movement is also controlled at these sites. Secretion of prolactin is responsive to osmotic changes in the blood, and may also be under hypothalamic control.

Prolactin’s role in elasmobranch osmoregulation is unknown. The published information states that prolactin increases diffusional branchial water permeability in Scyliorhinus canicula (Payan and Maetz, 1971), and reduces water retention in hypoosmotic sea water in Raja erinacea (Grant and Banks, 1968). DeVlaming and Sage (1972) found that rostralobectomy in Dasyatis sabina caused signigicant increases in plasma urea and electrolyte concentrations. Mammalian prolactin injections lowered urea levels significantly below the rostralobectomy values. Sage and deVlaming (unpublished) found a transient increase in prolactin content in the pituitary of D. sabina upon transfer to dilute sea water.

Adrenocorticotrophic hormone (ACTH) is found in elasmobranch pituitaries in the rostral pars distalis and the neurointermediate lobe (deßoos and deßoos, 1967). However, the ACTH activity detected in the

neurointermediate region is less than that of the rostral pars distalis, and is thought to be due to the chemically similar melanophore stimulating hormone (MSH) principle located in the neurointermediate lobe (deßoos and deßoos, 1967; Klesch and Sage, 1973). In D. sabina, the rostral pars distalis showed the greatest ACTH activity in vitro, and was the only pituitary region whose removal caused atrophy of the interrenal gland (Klesch and Sage, 1973). This latter result further suggests a possible pituitary-adrenocortical axis. Klesch and Sage (1973) showed that production of la-hydroxycorticosterone (lOt-OH-B) by interrenal tissue of D. sabina was significantly increased by incubation with homogenates of Dasyatis rostral pars distalis. lot-OH-B is the major steroid in elasmobranch blood (Idler and Truscott, 1967; 1969).

It should be noted that the pituitary-adrenocortical axis may differ physiologically among the elasmobranchs. The observation of interrenal atrophy following hypophysectomy has been seen in rajaform elasmobranchs only (Dittus, 1941; Klesch and Sage, 1973), while Dodd (1961) failed to find this result in a squaliform (dogfish) elasmobranch.

ACTH, when released by the pituitary of higher vertebrates, augments steroidogenesis and release of adrenocorticosteroids into the circulation. The circulating levels of corticosteroid control ACTH secretion by a negative feedback system. This mechanism may be operative in teleosts, while in cyclostomes and elasmobranchs, very little

information is available. The release of ACTH can be drastically increased in response to stress (Chester Jones, Chan, Henderson and Ball, 1969; Butler, 1973).

Adrenocorticosteroids play an important role in the maintenance of water and electrolyte homeostasis in all vertebrates. In teleosts (for review see Butler, 1973), the main mineralocorticoid, cortisol, is a salt-excreting factor for fish in a hypertonic media, and a salt-absorbing factor for fish in fresh water (Maetz, 1969). In elasmobranchs, the role of ACTH and the interrenal in osmoregulation is not known. Idler and Szeplaki (1968) reported no change in blood electrolyte or urea concentrations, or total osmolality, in Raja radiata in 100% or 75% sea water after interrenalectomy. DeVlaming and Sage (1972) found lowered plasma osmotic pressure and urea levels following interrenalectomy in sabina in 80% sea water. It should be noted that R. radiata is stenohaline, rarely found in water less than 31.2 ppt salinity (Bigelow and Schroeder, 1953), while D. sabina is euryhaline. This obvious difference in physiology cound easily account for the contradictory results observed in the two elasmobranchs.

Little research has been done on the function of the thyroid gland in elasmobranchs. Changes in the thyroid’s activity have been correlated to seasonal migration in SL acanthias (Woodhead, 1966). In this lab, cyclic activity in the thyroid of D. sabina has been related to reproductive development and the reproductive cycle (Sage, Litwin,

and Jackson, unpublished). The relationship of the thyroid to osmoregulation is unclear. In teleosts, thyroxine has been frequently claimed an influence in salt and water movements. However, the importance of thyroxine’s action is uncertain, with more potent hormones possibly acting simultaneously (for review see Gorbman, 1969; Sage, 1973). In elasmobranchs, deVlaming and Sage (1972), report a decrease in plasma osmotic pressure, due to a fall in the urea concentration, in I), sabina in 80% sea water following thyroidectomy. Previously, Dodd and Matty (1964) suggested that the thyroid may affect plasma urea levels because of the thyroid hormone’s implication in nitrogen metabolism. Further study is obviously needed.

Thyroid stimulating hormone (TSH) activity has been found in the elasmobranch pituitary, specifically in the ventral lobe (Dent and Dobb, 1961; Dodd, Ferguson, Dodd and Hunter, 1963; Jackson and Sage, 1973). However, the functional significance of this activity is undetermined.



Figure 3. Diagrammatic median longitudinal section of the pituitary gland of D. sabina, indicating zonation and location of hormones (from Klesch and Sage, 1973).

PART II — RESULTS

Thyroidectomy

Final results of the thyoidectomy experiment consisted of pooled data from individual experiments performed from August, 1973 to January, 1974. Sea water in the experiments was approximately 23 ppt, with temperature ranging from 19-25°C. The results (See Table IV) show that thyroidectomy in D. sabina caused a significant increase in plasma urea concentration, compared to normal animals. However, when thyroidec tomized animals received replacement therapy injections of thyroxine, plasma urea levels returned to normal.

Thyroxine injections also had an effect on the electrolyte levels of normal and thyroidectomized stingrays. Chloride and calcium plasma concentrations were significantly raised in both groups, compared to normal animals with no injections. Also, plasma potassium was lowered in both groups mentioned above, significantly in the thyroidectomized animals (see Table IV).

Rostralobectomy

Two identical experiments involving rostralobectomy and hormone replacement therapy were performed. Since the results of the two experiments differ significantly, the data is reported separately.

The first experiment (see Table Va) was performed in May, 1973. The sea water contained 12 ppt salinity. Stingrays were acclimated to 24 ppt sea water prior to the experiment. It should be noted that some deaths were occuring towards the end of the experiment.

Removal of the rostral pars distalis resulted in increased plasma urea concentrations compared to control animals. Replacement injections of prolactin decreased plasma urea levels significantly below rostralobectomy. Sodium was also lowered significantly. Replacement injections of ACTH also reduced plasma urea and sodium concentrations below rostralobectomy. A rise in muscle lipid content over rostralobectomized rays was significant.

Injections of prolactin and ACTH to rostralobectomized animals resulted in plasma urea levels slightly higher than rostralobectomy values, but not statistically different. This result is hard to explain, and is the main difference between experiments. Hematocrit values were significantly higher compared to rostralobectomy. This lowered blood volume may explain the more concentrated plasma urea levels. Muscle lipid content was also increased, while plasma sodium was lowered.

The second rostralobectomy experiment was identical in procedure to the first, except that it was performed in March, 1974 (see Table Vb). Water temperature during the experiment was 21°C. Animals were in excellent condition at the end of the experiment.

Rostralobectomy caused a significant rise in muscle lipid content over controls. Muscle water was lowered. Plasma urea was somewhat higher than controls, although not statistically different. Replacement injections of prolactin raised muscle water content significantly over rostralobectomy values. Plasma urea was lowered compared to rostralobectomy, although not significantly. Replacement injections of ACTH slightly lowered plasma urea concentrations compared to rostralobectomy. Replacement injections of prolactin and ACTH also slightly lowered urea levels below rostralobectomy values. Muscle lipid content was significantly lowered compared to rostralobectomy levels.

An attempt was made to find the ultimobranchial gland in I). sabina. The area around the last gill pouch was carefully dissected numerous times. However, no tissue appearing to be a gland was seen. For a description of the location of the ultimobranchial gland in jS. acanthias, see Camp (1917) or Copp (1969).

Treatment Sample Size (N) Plasma Electrolyte Concentrations 3 Muscle Water (%) Hematocrit (%) Body Weight Loss (%) Urea (m-moles/1 Na + Cl" K + Ca (m-equi v/1) Sham 11 311 ± 10 b 253.0 + 4.4 259.1 ± 2.3 6.9 + 0.7 2.9 + 0.1 75.8 ± 0.5 22.2 ± 1.3 6.98 Sham + thyroxine 10 309 ± 15 247.2 + 2.7 268.3 ± 1.5** 5.2 + 0.7 3.6 + 0.2* 76.4 ± 0.6 18.7 ± 0.9* IO.78 Thyroidectomy 11 351 ± 20* 248.7 + 3.8 262.3 ± 1.9 + 0.5 3.2 + 0.2 76.2 ± 0.4 21.2 ± 1.1 7.31 Thyroidectomy thyrox ine + 10 315 ± 10 248.7 + 6.5 267.O ± 2.1** 4.0 + 0.1** 4.1 + 0.3*** 75.4 ± 0.3 21.0 ± 0.7 9.81 * * * * * * o' co- il ii ii ii ii values (x ± S. E. ) for sham urea, N = 10 significantly different (p significantly different (p significantly different (p AAA 0.05) 0. 01) 0.001 from shams from shams ) from shams

TABLE IV EFFECTS OF THYROIDECTOMY AND REPLACEMENT THERAPY WITH THYROXINE ON OSMOREGULATION IN D. SABINA

a Plasma Electrolyte Concentrations fl CD s co CD N w Urea CD Na + Cl" K .+ c ++ !a (D O w 2 fl 0 i> — W -H fl ft fl 0 0 fl Eh ft § (m-moles/1) CO (m-equiv/1) Is CO g CD Sham + no hormones 3 170 ± 9 206.4 ± 8.7 197.1 ± 4.6 3.2 + 0.0 1.9 + 0.4 79.8 + 0.2 5.8 ± 0.6 21.3 + 1.2 Rostralo- bectomyb + no hormones 3 201 ± 3** 215.7 ± 4.6 212.9 ± 4.o 4.3 ± 0.7 2.4 + 0.1 78.8 + 0.4 5.6 ± 0.6 24.0 + 0.6* Rostralo- bectomy 0 + prolactin 3 125 ± 9*** 186.5 ± 2.6* 202.5 ± 13.8 5.2 + 0.4 2.9 + 0.2 79.8 + 0.7 6.4 ± 0.3 23.7 + 0.3 Rostralo- bectomy c + ACTH 5 161 ± 3*** 187.3 ± 6.5* 213.2 ± 6.1 3.2 + 0.2 2.4 + 0.1 79.3 + 0.2 7.0 ± 0.2* 25.6 ± 0.4 Rostralo- bectomy + prolactin + ACTH 3 208 ± 4 175.5 ± 11.7** 217.8 ± 10.2 4.2 + 0.8 2.1 + 0.3 78.8 + 0.1 7.3 ± 0.4* 31.7 + 0.9*** a b c * ** *** = values (x ± S. = statistically = statistically = significantly = significantly = significantly E. ) compared to sham compared to rostralobectomy + different (p < 0.05) different (p < 0.01) different (p < 0.001) no hormones

TABLE V(a) EFFECTS OF ROSTRALOBECTOMY AND HORMONE REPLACEMENT THERAPY ON OSMOREGULATION IN D. SABINA. (DATA OBTAINED IN COLLABORATION WITH DR. V. L. DEVLAMING) --MAY, 1973

a -p 3 Plasma Electrolyte Concentrations x-~ £ 0) (D N £ 'H -p m Urea «) <D <D Na + Cl" K .+ ++ Ca Muscle Water C O T? W 'H £ ft 0 0 Sh rd Eh ft § (m-moles/1) co (m- ■equiv/1) S J CO £ (D K Sham + no , hormones 4 270 ± 10 227.4 ± 1.6 243.4 ± 8.2 4.2 ± 0.4 1.7 + 0.1 78.9 ± 0.2 11.7 ± o.8 b 19.0 ± 1.2 Rostralo- bectomy 0 + 4- 280 ± 17 no hormones 226.0 + 3.0 229.3 ± 2.5 3.5 ± 0.2 2.6 + 0.4 77.1 ± 0.2*** 17.4 ± 0.7*** 18.8 + 1.3 Rostralo- + 4 253 ± 8 prolactin 219.4 ± 5.4 226.9 ± 2.7 4.2 ± 0.4 3.2 + 0.1 78.2 ± 0.3** 17.6 ± 0.5 17.4 + 1.0 Rostralo- bectomy 4 269 ± 14° + ACTH 217.7 4 3.4 226.4 ± 3.4 3.6 ± 0.3 2.4 + 0.6 76.8 ± 0.3 18.6 ± 0.3 20.2 + 1.1 Rostralo- + prolactin 4 265 ± 10 + ACTH 229.6 ± 5.8 241.3 ± 6.7 3.7 + 0.5 2.6 + 0.2 77.4 ± 0.2 13.2 ± 0.5*** 21.0 + 1.2 a = values (x ± S. b = (N = 3) c = statistically d = statistically ** = significantly *** = significantly E. ) compared to compared to different (p different (p sham rostralobectomy < 0.01) < 0.001) + no hormones

TABLE V(b) EFFECTS OF ROSTRALOBECTOMY AND HORMONE REPLACEMENT THERAPY ON OSMOREGULATION IN D. SABINA--MARCH, 197*+

Discussion

The relationship of the thyroid gland to osmoregulation in fishes is not clear. The many and varied effects of thyroid hormone, plus possible interactions of other hormones, make interpretation of experimental data difficult. Also, little data is available, as lower vertebrates’ thyroid function has received little attention. From the existing studies, some generalizations may be made. In teleosts, the thyroid gland has been implicated in the control of salt and water balance (Dodd and Matty, 1964; Gorbman, 1969). An increase in thyroid activity is seen in many diadromous fish at migration time (for review see Baggerman, 1960). A decrease in thyroid activity occurs at the end of migration. The changes in thyroid activity are seen prior to the beginning and end of migration. In juvenile salmon, and in sticklebacks, the level of thyroid hormone in the blood can induce changes in salinity tolerance and preference (Baggerman, 1960).

In elasmobranchs, the thyroid’s activity has been related to migration also (Woodhead, 1966). In acanthias, the thyroid’s activity increases prior to or at migration time, and decreases prior to the end of migration. The causal relationship of thyroid and migration is further suggested by the fact that immature dogfish do not migrate, and their thyroid activity correspondingly remains constant throughout the year.

DeVlaming and Sage (1972) reported a rise in plasma urea concentration, following thyroidectomy, in D. sabina. This same effect was observed in this study. However, no other plasma electrolytes measured (see Table IV) were significantly affected. Since hematocrit, muscle water, and body weight loss do not differ between controls and thyroidectomized stingrays, water balance was maintained. Thus, the results are not due to a simple change in plasma volume.

The rise in urea concentration following thyroidectomy in I). sabina suggests that thyroxine helps control urea levels. The control could result from thyroxine’s influence on protein synthesis, protein storage site shifts, or nitrogenous end-product excretion rates. Thyroxine’s effect on osmoregulation in elasmobranchs is essentially unknown. These effects are better known in higher vertebrates, and are only speculative here. Thyroxine has been implicated in affecting nitrogen metabolism in teleosts (Gorbman, 1969), birds and mammals (for reviews see Dodd and Matty, 1964; Wolff and Wolff, 1964). Replacement therapy of thyroxine injections in I), sabina repaired urea levels to normal. This confirms the influence of the thyroid on urea levels.

Apparently thyroxine has some additional effects (see Table IV). A general diuresis seems evident in controls and thyroidectomized animals injected with thyroxine, as shown by greater body weight loss than noninjected animals. Hematocrit shows plasma volume is elevated. Plasma sodium and potassium concentrations were lowered, most likely by increased urinary loss (see Turner and Bagnara, 1971). However, plasma chloride and calcium concentrations were increased significantly. Further experiments would be necessary to elucidate the matter.

The rostral pars distalis of the elasmobranch pituitary is reported to contain prolactin (Grant and Waterman, 1962; Grant, 1962) and ACTH (deßoos and deßood, 1967; Klesch and Sage, 1973). The effects of prolactin and ACTH on osmoregulation in teleosts and higher

vertebrates have been documented. However, very little information exists concerning endocrine control of osmoregulation in cartilaginous fishes.

The results obtained in the May, 1973 rostralobectomy experiment (see Table Va) are similar to those found in a previous experiment (see Table VI) done in various months (deVlaming and Sage, unpublished). The rostralobectomy raised plasma urea concentrations above controls in each experiment, while replacement injections of prolactin resulted in urea levels below rostralobectomy values. This same trend is followed in the March, 1974 experiment (see Table Vb) , except that the differences in urea levels are not statistically significant. This variation may be due to animal condition during the experiment (stress), or to seasonal-migrational influences. In March, the stingrays are usually in the inshore Gulf waters, while in May, they reside in the bays. The more marine conditions may not require the same degree of endocrine control to maintain internal homeostasis as brackish water conditions. Thus, although the experiments were both performed at low salinity, the previous history of the animals may influence experimental results.

The rostralobectomy with prolactin replacement therapy resulted in lowered plasma urea concentrations compared to rostralobectomy. The rostralobectomy with ACTH replacement therapy also resulted in lower levels of plasma urea. This suggests that both these hormones are influential in controlling plasma urea concentrations. Prolactin

may affect urea through nitrogen metabolism or gill permeability. ACTH may affect protein (nitrogen) metabolism through its influence over steroidogenesis, or it may also influence urea conservation at the gills and kidneys. These effects have been documented in teleosts (for reviews see Ball 1969 a; Chester Jones et al., 1969).

The role of prolactin in osmoregulation is well known in teleosts (Ball, 1969 b). However, in elasmobranchs, information is lacking concerning prolactin and osmoregulation. The results obtained in this and other recent studies strongly suggest a role for prolactin in the osmoregulation of D. sabina. The action of lowered plasma urea concentrations following prolactin injections of rostralobectomized animals was seen in all three experiments. Also, trends in sodium and water movements were consistent throughout. The fact that transient increases in prolactin content of D. sabina pituitaries occur upon transfer to dilute sea water (Sage and deVlaming, unpublished) further suggests prolactin is involved in elasmobranch osmoregulation.

The results obtained from replacement injections of prolactin and ACTH differ between the two experiments performed. Plasma urea was somewhat lowered below rostralobectomy values in the March experiment, whereas urea was slightly higher than rostralobectomy levels in the May experiment. Since prolactin and ACTH both occur in the rostral pars distalis, replacement of these hormones in a rostralobectomized animal should approximate normal conditions. Although there are no doubt

dosage problems involved in these experiments, plasma urea levels should be reduced below rostralobectomy values based on the other treatments’ results. The higher urea values obtained in the May experiment may be questionable, since hematocrit was 49% higher than controls, and 32% higher than the rostralobectomy group. These differences may indicate excessively stressed animals (diuresis), possibly affecting plasma volume and solute concentrations. Since the muscle lipid content of the prolactin + ACTH group is higher than the rostralobectomy group, possibly nitrogen metabolism was affected also.

Also of note was the comparative muscle lipid data. The March shams had muscle lipid content 100% higher than the May shams. The higher storage of fat may be in response to impending copulation and/or migration (see Sage et al., 1972). Also, in the March experiment, rostralobectomy caused a significant rise in muscle lipid content that could only be repaired by replacement of both prolactin and ACTH. Thus, both of these hormones may influence fat metabolism and storage in D. sabina.

Treatment Sample Size (N) Plasma Electrolyte Concentrations Muscle Urea (m-moles/1) K + (m-equiv/1) Ca ++ Water Hematocrit Na + Cl" Sham 6 296 ± 7 Sea Water 30.2 272 ±1 293 ± 7 ppt— July, 1972 5.6 ± 0.1 3.3 ± 0.1 76.6 ± 0.2 26.5 ± 0.4 Rostralobectomy 5 329 ± 8** 286 ± 4* 311 ± 10 6.5 ± 0.1 2.8 ± 0.1** 75.4 ± 0.1* 25.2 ± 0.2* Sham 5 264 + 9 Sea Water 10. 5 ppt--November, 197 2 192 ± 3 230 ± 6 4.5 ± 0.5 1.5 ± 0.1 — — Rostralobectomy 5 309 ± 6** 201 ± 2* 226 ± 13 4.7 ± 0.4 1.5 ± 0.1 — — Sham 5 253 ± 9 Sea Water 10.8 192 ±3 205 ± 7 ppt--June, 1972 3.5 * 0.1 2.8 ± 0.1 79.7 ± 1.2 25.5 ± 0.7 Rostralobectomy 13 5 282 ± 4 196 ±1 212 ± 9 4.5 ± 0.2 2.5 ± 0.1 77.2 ± 0.3 22.2 ± 0.8 Rostralobectomy 0 + Prolactin 5 228 ± 4** 186 ± 3* 207 ± 4 2.9 ± 0.2** 1.4 ± 0.1** 81.6 ± 0.5** 20.5 ± 0.4* a = values (x ± S.E.) b = not compared to sham c = statistically compared to rostralobectomy * = significantly different (p < O.O5) ** = significantly different (p < 0.01)

TABLE VI EFFECTS OF ROSTRALOBECTOMY AND PROLACTIN REPLACEMENT THERAPY ON OSMOREGULATION IN D. SABINA. (FROM DEVLAMING AND SAGE, UNPUBLISHED)

REFERENCES

Baggerman, B. 1960. Factors in the diadromous migrations of fish. Symp. Zool. Soc. London 1: 33-60.

Ball, J. N. 1969 a. Prolactin (fish prolactin or paralactin) and growth hormone. In Fish Physiology (Edited by Hoar, W. S. and D. J. Randall), Vol. 11, Chap. 3, pp. 207-240. Academic Press, New York.

Ball, J. N. 1969 b. Prolactin and osmoregulation in teleost fishes: A review. Gen. Comp. Endocrinol. Suppl. 2: 10-25.

Bernard, G. R. , R. A. Wynn and G. G. Wynn 1966. Chemical anatomy of the pericardial and perivisceral fluids of the stingray, Dasyatis americana. Biol. Bull. 130: 18-26.

Bigelow, H. B. and W. C. Schroeder 1953. Fishes of the Western North Atlantic. Part II: Sawfishes, Guitarfishes, Skates, Rays and Chimaeroids. Yale University Press, New Haven.

Bbhlke, J. E. and C. Chaplin 1968. Fishes of the Bahamas and Adjacent Waters. Livingston Pub. Co., Wynnewood, Pa.

Boylan, J. W. 1967. Gill permeability in Squalus acanthias. In Sharks, Skates, and Rays (Edited by Gilbert, P. W. , R. F. Mathewson and D. P. Rall), Chap. 12, pp. 197-206. Johns Hopkins Press, Baltimore.

Bulger, R. E. 1963. Fine structure of the rectal (salt-secreting) gland of the spiny dogfish, Squalus acanthias. Anat. Rec. 147: 95-127.

Burger, J. W. 1962. Further studies on the function of the rectal gland in the spiny dogfish. Physiol. Zool. 35: 205-217.

Burger, J. W. 1965. Roles of the rectal gland and the kidneys in salt and water excretion in the spiny dogfish. Physiol. Zool. 38: 191-196.

Burger, J. W. 1967. Problems in the electrolyte economy of the spiny dogfish, Squalus acanthias. In Sharks, Skates, and Rays (Edited by Gilbert, P. W., R. F. Mathewson and D. P. Rall), Chap. 10, pp. 177-185. Johns Hopkins Press, Baltimore.

Burger, J. W. 1972. Rectal gland secretion in the stingray, Dasyatis sabina. Comp. Biochem. Physiol. 42A: 31-32.

Burger, J. W. and W. N. Hess 1960. Function of the rectal gland in the spiny dogfish. Science 131: 670-671.

Butler, D. G. 1973. Structure and function of the adrenal gland of fishes. Amer. Zool. 13: 839-879.

Camp, W. E. 1917. The development of the suprapericardial (postbranchial, ultimobranchial) body in Squalus acanthias. J. Morphol. 28: 369-415.

Carrier, J. C. and D. H. Evans 1972. lon, water and urea turnover rates in the nurse shark, Ginglymostoma cirratum. Comp. Biochem. Physiol. 41A: 761-764.

Chaisson, A. F. 1930. The changes in the blood concentration of Raja erinacea produced by modification of the salinity of the external medium. Contr. Can. Biol. Fish. 5: 477-484.

Chan, D. K. 0. and J. G. Phillips 1967. The anatomy, histology and histochemistry of the rectal gland in the lip-shark, Hemiscyllium plagiosum (Bennett). J. Anat. 101: 137-157.

Chan, D. K. 0., J. G. Phillips and I. Chester Jones 1967. Studies on electrolyte changes in the lip-shark, Hemiscyllium plagiosum (Bennett), with special reference to hormonal influence on the rectal gland. Comp. Biochem. Physiol. 23: 185-198.

Chester Jones, 1., D. K. 0. Chan, I. W. Henderson, and J. N. Ball 1969 The adrenocortical steroids, adrenocorticotropin and the corpuscles of Stannius. In Fish Physiology (Edited by Hoar, W. S. and D. J. Randall), Vol. 11, Chap. 6, pp. 321-376. Academic Press, New York.

Cohen, J. J., M. A. Krupp, and C. A. Chidsey 111 1958. Renal conserve tion of trimethyamine oxide by the spiny dogfish, Squalus acanthias. Am. J. Physiol. 194: 229-235.

Conte, F. P. 1969. Salt secretion. In Fish Physiology (Edited by Hoar, W. S. and D. J. Randall), Vol. I, Chap. 3, pp. 241-292. Academic Press, New York.

Copp, D. H. 1969. The ultimobranchial glands and calcium regulation. In Fish Physiology (Edited by Hoar, W. S. and D. J. Randall), Vol. 11, Chap. 7, pp. 377-398. Academic Press, New York.

Dent, J. N. and J. M. Dodd 1962. Some effects of mammalian thyroid stimulating hormone, elasmobranch pituitary gland extracts and temperature on thyroidal activity in newly hatched dogfish (Scyliorhinus caniculus). J. Endocrinol. 22: 395-402.

deßoos, R. and C. deßoos 1967. Presence of corticotropin activity in the pituitary gland of chondrichthyean fish. Gen. Comp. Endocrinol. 9: 267-275.

deVlaming, V. L. and M. Sage 1972. Some aspects of endocrine control of osmoregulation in the euryhaline elasmobranch, Dasyatis sabina. Amer. Zool. 12: 676.

deVlaming, V. L. and M. Sage 1973. Osmoregulation in the euryhaline elasmobranch, Dasyatis sabina. Comp. Biochem. Physiol. 45A: 31-44.

Dittus, P. 1941. Histologie und Cytologie des Interrenal-organs der Selachier unter normalen und experimentellen Bedingungen. Ein Beitrag zum Kennti der Wirkungsweise des kortikotropen Hormons und des Verhaltnisses von Kern zu Plasma. Z. Wiss. Zool. A 154: 40-124.

Dodd, J. M. 1961. Adenohypophyseal hormones of fishes. Abstracts, Symposium Papers, 10th Pacific Sci. Congress, Honolulu, p. 167.

Dodd, J. M., P. J. Evennett and C. K. Goddard 1960. Reproductive endocrinology in cyclostomes and elasmobranchs. Symp. Zool. Soc. London 1: 77-103.

Dodd, J. M., K. M. Ferguson, M. H. I. Dodd, and R. B. Hunter 1963. The comparative biology of thyrotropin secretion. In Thyrotropin (Edited by S. C. Werner). Charles Thomas, Springfield.

Dodd, J. M. and A. J. Matty 1964. Comparative aspects of thyroid function. In The Thyroid Gland (Edited by Pitt-Rivers, R. and W. R. Trotter), Vol. I, Chap. 13, pp. 303-356. Butterworths, London.

Doyle, W. L. 1962. Tubule cells of the rectal salt-gland of Urolophus Am. J. Anat. Ill: 223-237.

Ensor, D. M. and J. N. Ball 1972. Prolactin and osmoregulation in fishes. Fed. Proc. 31: 1615-1623.

Forster, R. P. 1967. Osmoregulatory role of the kidney in cartilaginous fishes (Chondrichthyes). In Sharks, Skates, and Rays (Edited by Gilbert, P. W., R. F. Mathewson and D. P. Rall), Chap. 11, pp. 187-195. Johns Hopkins Press, Baltimore.

Forster, R. P., L. Goldstein and J. K. Rosen 1972. Intrarenal control of urea reabsorption by renal tubules of the marine elasmobranch, Squalus acanthias. Comp. Biochem. Physiol. 42A: 3-12.

Goldstein, L. , 1967. Urea biosynthesis in elasmobranchs. In Sharks, Skates, and Rays (Edited by Gilbert, P. W., R. F. Mathewson and D. P. Rall), Chap. 13, pp. 207-214. Johns Hopkins Press, Baltimore.

Goldstein, L. and R. P. Forster 1971. Osmoregulation and urea metabolism in the little skate Raja erinacea. Am. J. Physiol. 220: 742-746.

Goldstein, L. and D. Funkhouser 1972. Biosynthesis of trimethylamine oxide in the nurse shark, Ginglymostoma cirratum. Comp. Biochem. Physiol. 42A: 51-58.

Goldstein, L., W. W. Oppelt and T. H. Maren 1968. Osmotic regulations and urea metabolism in the lemon shark Negaprion brevirostris. Am. J. Physiol 215: 1493-1497.

Gorbman, A. 1969. Thyroid function and its control in fishes. In Fish Physiology (Edited by Hoar, W. S. and D. J. Randall), Vol. 11, Chap. 4, pp. 241-274. Academic Press, New York.

Grant, W. C. 1962. Studies on the endocrinology of the skate pituitary. Bull. Mt. Desert Island Biol. Lab. 4: 37.

Grant, W. C. and P. M. Banks 1968. Immunologic investigations of elas mobranch pituitary hormones. Bull. Mt. Desert Island Biol. Lab. 8: 31-32.

Grant, W. C. and A. J. Waterman 1962. The elasmobranch thyroidpituitary system. Bull. Mt. Desert Island Biol. Lab. 4: 36-37.

Gunter, G. 1945. Studies on marine fishes of Texas. Publ. Inst. Mar. Sci. Univ. Texas, 1: 1-190.

Hichman, C. P. and B. F. Trump 1969. The kidney. In Fish Physiology (Edited by Hoar, W. S. and D. J. Randall), Vol. I, Chap. 2, pp. 91-239. Academic Press, New York.

Hoar, W. S. and D. J. Randall (eds.) 1969. Fish Physiology, Vols. I-111. Academic Press, New York.

Hoese, H. D. 1960. Biotic changes in a bay associated with the end of a drought. Limnol. and Oceanogr. 5: 326-336.

Holmes, W. N. and E. M. Donaldson 1969. The body compartments and the distribution of electrolytes. In Fish Physiology (Edited by Hoar, W. S. and D. J. Randall), Vol. I, Chap. 1, pp. 1-89. Academic Press, New York.

Horowicz, P. and Burger, J. W. 1968. Unidirectional fluxes of Na in the spiny dogfish, Squalus acanthias. Am. J. Physiol. 214: 635-642.

Idler, D. R. and B. J. Szeplaki 1968. Interrenalectomy and stress in relation to some blood components of an elasmobranch (Raja radiata). J. Fish. Res. Bd. Canada 25(12): 2549-2560.

Idler, D. R. and B. Truscott 1967. la-Hydroxycorticosterone: synthesis in vitro and properties of an interrenal steroid in the blood of cartilaginous fish (Raja). Steroids 9: 457-477.

Idler, D. R. and B. Truscott 1969. Production of Icx-hydroxycorticosterone in vivo and in vitro by elasmobranchs. Gen. Comp. Endocrinol. Suppl. 2: 325-330.

Jackson, R. G. and M. Sage 1973. Regional distribution of thyroid stimulating hormone activity in the pituitary gland of the Atlantic stingray, Dasyatis sabina. Fish. Bull. 71: 93-97.

Jones, R. T. and K. S. Price 1974. Osmotic responses of spiny dogfish (Squalus acantias L.) embryos to temperature and salinity stress. Comp. Biochem. Physiol. 47A: 971-979.

Kempton, R. T. 1953. Studies on the elasmobranch kidney-11. Reabsorp tion of urea by the smooth dogfish, Mustelus canis. Biol. Bull., Woods Hole. 104: 45-56.

Klesch, W. L. and M. Sage 1973. The control of the interrenal by the pituitary in the elasmobranch, Dasyatis sabina. Comp. Biochem. Physiol. 45A: 961-967.

Lowry, P. J. and A. Chadwick 1970. Interrelations of some pituitary hormones. Nature 226: 219-222.

Maetz, J. 1969. Observations on the role of the pituitary-interrenal axis in the ion regulation of the eel and other teleosts. Gen. Comp. Endocrinol. Suppl. 2: 299-316.

Nicoll, C. S. and H. A. Bern 1964. ’’Prolactin” and the pituitary glands of fishes. Gen. Comp. Endocrinol. 4: 457-471.

Nicoll, C. S. and H. A. Bern 1968. Further analysis of the occurrence of pigeon crop sac-stimulating activity (prolactin) in the vertebrate adenohypophysis. Gen. Comp. Endocrinol. 11: 5-20.

Payan, P. and J. Maetz 1971. Balance hydroque chez les elasmobranches: agruments en faveur d’un controle endrocinien. Gen. Comp. Endocrinol. 16: 535-554.

Perks, A. M., M. H. I. Dodd and J. M. Dodd 1960. A neurohypophyseal principle in the elasmobranch pituitary. Nature 185: 850-851.

Price, K. S. 1967. Fluctuations in two osmoregulatory components, urea and sodium chloride, of the clearnose skate, Raja eglanteria BOSC 1802-11. Upon natural variation of the salinity of the external medium. Comp. Biochem. Physiol. 23: 77-82.

Price, K. S. and E. P. Creaser 1967. Fluctuations in two osmoregulatory components, urea and sodium chloride, of the clearnose skate, Raja eglanteria, BOSC 1802-1. Upon laboratory modification of external salinities. Comp. Biochem. Physiol. 23: 65-76.

Sage, M. 1973. The evolution of thyroidal function in fishes. Amer. Zool. 13: 899-905.

Sage, M., R. G. Jackson, W. L. Klesch and V. L. deVlaming 1972. Growth and seasonal distribution of the elasmobranch Dasyatis sabina. Contrib. Mar. Sci. 16: 71-74.

Sawyer, W. H. 1965. Active neurohypophyseal principles from a cyclostome (Petromyzon marinus) and two cartilaginous fishes (Squalus acanthias and Hydrolagus collei). Gen. Comp. Endocrinol. 5: 427-439.

Simmons, E. G. 1957. An ecological survey of the Upper Laguna Madre of Texas. Publ. Inst. Mar. Sci., Univ, of Texas. 4(2): 156-200.

Smith, H. W. 1953. From Fish to Philosopher. Little, Brown and Co., Boston, 304 p.

Sokal, R. R. and F. J. Rohlf 1969. Biometry. W. H. Freeman and Co., San Francisco. 776 p.

Turner, C. D. and J. T. Bagnara 1971. General Endocrinology. p. 210. W. B. Saunders Co., Philadelphia.

Wolff, E. C. and J. Wolff 1964. The mechanism of action of the thyroid hormones. In The Thyroid Gland (Edited by Pitt-Rivers, R. and W. R. Trotter), Vol. I, Chap. 11, pp. 237-282. Butterworths, London.

Woodhead, A. D. 1966. Thyroid activity in the ovo-viviparous elasmobranch Squalus acanthias. J. Zool. 148: 238-275.

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