Body Fluid Solutes of Juveniles and Adults of the Euryhaline Bull Shark Carcharhinus Leucas from Freshwater and Saline Environments

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Investigations of the Ichthyofauna of Nicaraguan Lakes Papers in the Biological Sciences

1976

Body Fluid Solutes of Juveniles and Adults of the Euryhaline Bull Shark Carcharhinus Leucas from Freshwater and Saline Environments

Thomas B. Thorson University of Nebraska-Lincoln

C. Michael Cowan Associated Environmental Services Corp.

Donald E. Watson University of Lagos

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Thorson, Thomas B.; Cowan, C. Michael; and Watson, Donald E., "Body Fluid Solutes of Juveniles and Adults of the Euryhaline Bull Shark Carcharhinus Leucas from Freshwater and Saline Environments" (1976). Investigations of the Ichthyofauna of Nicaraguan Lakes. 47. https://digitalcommons.unl.edu/ichthynicar/47

This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Investigations of the Ichthyofauna of Nicaraguan Lakes by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Published in INVESTIGATIONS OF THE ICHTHYOFAUNA OF NICARAGUAN LAKES, ed. Thomas B. Thorson (University of Nebraska-Lincoln, 1976). Copyright © 1976 School of Life Sciences, University of Nebraska-Lincoln.

BODY FLUID SOLUTES OF JUVENILES AND ADULTS OF THE EURYHALINE BULL SHARK CARCHARHINUS LEUCAS FROM FRESHWATER AND SALINE ENVIRONl\1ENTSl

THOMAS B. THORSON, C. MICHAEL COWAN, AND DONALD E. WATSON Depar~ment of Zoology, University of Nebraska, Lincoln, Nebraska 68508' Department of BIology, N~bras~a We~leyan University, Lincoln, Nebraska 68504; and School of BIOlogIcal SCIences, University of Lagos, Yaba, Lagos, Nigeria

INTRODUCTION Krogh ( 1939) , Prosser and Brown (1961), Potts and Parry (1964), sev Since the discovery of the phenome eral authors in Gilbert, Mathewson, and nally high urea content of the body flu RaIl (1967), and Hoar and Randall ids of cartilaginous fishes by Staedeler (1969). and Frerichs (1858), the unique osmo regulatory system of this vertebrate However, chondrichthians have also class has been studied by many inves been reported in brackish or fresh water tigators. In broad outline, the mecha i? many places around the world, par nisms are essentially similar in all three tIcularly in the tropics and subtropics. of the major subtaxa: the selachians, Known occurrences have been tabu the batoids, and the holocephalans. A lated by Engelhardt ( 1913 ) , Smith single exception is the genus P otamo (1936), and Boeseman (1964). These trygon (fresh-water stingrays of South s?ecies are, to various extents, euryha America and Africa), which has appar lme and should provide excellent sub ently lost the ability to concentrate urea jects for studies on osmoregulation. Yet even when transferred to salt water in spite of the ready availability of eu~ (Thorson, Cowan, and Watson 1967; ryhaline forms in certain places very Thorson 1970; Goldstein and Forster little work has been done on any ~spect 1971b). of the physiology of elasmobranchs in Since the Chondrichthyes are primar fresh water until the past decade. The ily marine animals, research on them only exception was the classical inves has centered mainly upon marine spe tigation of Smith (1931a) who studied cies, either in their natural environment the blood and certain other body fluids or following transfer to various dilu of four species of elasmobranchs taken tions of seawater. These studies have in the Perak River in the Federated been reviewed by Smith (1931b, 1936), Malay States (now Malaysia). These included a sawfish, Pristis microdon; 1 We are indebted to Colegio Centro America, a shark identified as Carcharhinus mel Managua, Nicaragua, for facilities provided. Some anopterus; and two rays, Dasyatis of the laboratory work was done by Jeffery W. Gerst. Appreciation is expressed to him and to warnak and H ypolophus sephen. For others too numerous to name for a vrrriety of more than 30 years Smith's results pro help. The study was supported by NIH grant vided the only available basis for sub HE-09075, by National Science Foundation funds for operation of the RjV Rhincodon at the Mote sequent discussions of osmoregulation Marine Laboratory, and by the University Re of elasmobranchs in fresh water. In search Council of the University of Nebraska, through Biomedical Sciences Support Grant RT- general, Smith demonstrated that (1) 07055. the osmotic pressure of the serum is 29

599 30 T. B. THOI{SON, C. M. COWAN, AND D. E. WATSON about 45 % lower in fresh-water elas fresh water, the only sharks that com mobranchs than in marine ones but is monly undertake extended excursions still higher than that of either fresh into fresh water in appreciable numbers water or marine teleosts; (2) in fresh are Carcharhinus leucas and its close water, chloride content is reduced by relatives included in the C. leucas-gan about 25 %, to approximately the range geticus group of Garrick and Schultz of teleosts; ( 3) the urea level is lower (1963). Carcharhinus leucas is the spe by about 70%, but still much higher cies that forms a sizable population in than that of teleosts; and (4) urine ex Lake Nicaragua and its drainage into hibits a marked increase in flow accom the Caribbean Sea, the Rio San Juan. panied by a manyfold decrease in os Contrary to an earlier and still common motic concentration. popular belief, they do not constitute a Values reported by Smith (1931a) separate, land-locked species but are for urea, as well as other body fluid identical with the bull sharks of the parameters, varied widely both among Atlantic and are able to move freely and within species. This has also been back and forth between the Caribbean true for marine elasmobranchs reported Sea and Lake Nicaragua (Thorson, by other authors. These variations no Watson, and Cowan 1966; Thorson doubt represent, in part, genuine spe 1971). They are also known to remain cific differences, and in some cases in both fresh and salt water for ex might reflect technical problems. How tended periods (unpublished data). In ever, in many cases (Smith excepted), spite of its potential usefulness and the environmental history of the sub availability, C. leucas remained unex ject animals was unknown, or at least ploited for physiological studies until not indicated, and the variations in urea 1962. Since that time, studies on vari levels reported may to some extent re ous aspects of the osmoregulation of flect differences in the salinity of the this species have been reported by environment from which the experimen Thorson (1962a, 1962b, 1967), Urist tal animals were taken. (1962), Oguri (1964), Gerzelli, Ger The ideal experimental subject would vaso, and De Stefano (1969), and be one that could be taken from both Thorson and Gerst (1972). fresh and salt water, in situations where In this paper, we present data on it was reasonably certain that the in the chemical anatomy of serum, peri dividuals had been in one environment visceral fluid, pericardial fluid, and cra or the other for an appreciable length nial fluid of (1) adult sharks from of time. Such a species is Carcharhinus marine waters off the west coast of leucas (family, Carcharhinidae), the Florida; (2) adults from the fresh bull shark, which occurs along the water of Lake Nicaragua; (3) juveniles coasts of tropical and SUbtropical land from the fresh water of the lowest masses all around the world. This spe reaches of the Rio San Juan; and (4) cies occurs in full-strength seawater adults from water of undetermined but and also congregates around the mouths varied salinities around the mouth of of rivers and is known to travel up the Rio San Juan. completely fresh-water rivers and into lakes in a number of places. Although MATERIAL AND METHODS the listings mentioned above include The sharks were obtained by local several species of sharks reported in fishermen in Lake Nicaragua and at

600 BODY FLUIDS OF BULL SHARKS 31

I I I I I 10 20 30 .«J 50 Km

1 N

w co

«'" u

II< « "l:

COSTA

FIG. I.-Map of Lake Nicaragua, Rio San Juan, and Rio Colorado (drawn by Norman H. Jensen)

Barra del Colorado, Costa Rica (fig. 1). cluded a group of specimens taken at They were caught almost exclusively on each end of the lake (near Granada handlines with large shark hooks baited and at San Carlos). They were, there with chunks of various local fishes. A fore, about 180-345 km from the sea, few in the vicinity of Granada were distances that assure that they had been taken on sport gear with caiman tail as in fresh water for an appreciable, al bait. The Florida sharks were taken on though undetermined, length of time. longlines, also baited with chunks of Since the major fluid parameters showed fish. no differences between the two groups, Here, adults include both sexuaIly they have been combined in a single mature and immature individuals, in series. most cases more than 110 em long, The juvenile series was taken almost while juveniles are 72 em or less in exclusively a kilometer or more inside length. Very few sharks between 80 and the river mouth, where the water was 110 em in length are taken in the re completely fresh, as it is throughout the search area. length of the Rio San Juan and Rio The adult series from Florida was Colorado. taken in fuIl-strength seawater of ap After leaving the river mouth, the proximately 34 ppt total salinity, within fresh water extends 2 or 3 km out to a few miles of the Mote Marine Labo sea and is washed down the coast by ratory at Sarasota. the prevailing current and graduaIly The water of Lake Nicaragua is com dissipates. Therefore, the surface water pletely fresh. The lake is about 165 km immediately outside the mouth is fresh, long, while the Rio San Juan-Rio Colo although there may at times be an in rado system is somewhat longer. The trusion of brackish water underneath. Lake Nicaragua series of sharks in- In any case, all adult sharks taken near

601 32 T. B. THORSON, C. M. COWAN, AND D. E. WATSON the river mouth were probably caught in the case of blood, from a caudal ves in fresh water, since fishing could only sel or by cardiac puncture. Blood was be done from the small dugouts em allowed to clot, and serum was removed ployed, in relatively calm water, and following centrifugation. the fishermen rarely ventured as far as Sodium, potassium, and calcium were the salt-water intrusion. However, it is determined by flame photometry; mag conceivable that a few may have been nesium by the titan yellow method of taken from the deeper, brackish-water Heagy (1948); chloride by mercuric underlayer. Preliminary tagging and te nitrate titration, modified from Schales lemetry results (Thorson 1971; unpub and Schales (1941); bicarbonate by the lished data) indicate free movement of method of Scribner and Caillouette sharks up and down the coast, in and ( 1954) ; inorganic phosphorus by a out the river mouth, and up and down method based on Fiske and Subbarow the river. Thus, the environmental (1925); total protein by the biuret background of the series of sharks method (after Weichselbaum 1946); taken at the river mouth is undoubtedly urea by the Nessler reaction, the car heterogeneous, and even though caught bamido diacetyl reaction, modified from in fresh water they may very recently Fearon (1939), or by the microdiffu have come from salt water, or, in some sion method of Conway and O'Malley cases, possibly from the lake. (1942); trimethylamine oxide by a Field collection of body fluids was method based on Dyer, Dyer, and Snow carried out in the summers of 1965- (1952). The pH was read on an In 1970. In 1965, a complete field labo strumentation Laboratory Delta-matic ratory was set up at Colegio Centro model 145 pH/mV electrometer. America, Granada, Nicaragua, and all laboratory analyses were completed as DISCUSSION OF RESULTS the samples became available. In sub sequent years, more limited facilities Table 1 summarizes data for the four were set up at Barra del Colorado, series of sharks. It should be borne in Costa Rica, where hematocrit, pH, and mind when interpreting comparisons bicarbonate were determined. Fluid that the sample size of the marine series samples were then frozen and shipped is smaller than that of the other series on dry ice to our Lincoln, Nebraska, and that ranges of some parameters are laboratory, where most of the analyses appreciable. were performed. This procedure was Solute concentrations are expressed also followed with the marine specimens in mM/liter, except total protein which at the Mote Marine Laboratory. Fluids is recorded in grams percent. Partial from all but two fresh-water specimens data for the adult fresh-water series were analyzed in our field laboratory from Lake Nicaragua were previously in Nicaragua. All others were done in presented by Thorson (1967). Lincoln. Essentially the same equip ment and methods were employed in SERUM SOLUTES both places, and no appreciable differ The total concentration of all mea ences were noted between analyses sured serum solutes of the marine series made in the two situations. is a little more than 1,000 mM/liter, All fluids were drawn from their re approximately what would be expected spective sources by needle and syringe, of elasmobranchs in seawater, as previ-

602 BODY FLUIDS OF BULL SHARKS 33 ously reported. In the fresh-water series unknown but probably mixed environ it is approximately two-thirds of the mental history. Their fresh-water speci marine level. mens had 198 mM/liter sodium, 234 Of the individual parameters, potas mM/liter chloride, and 231 mM/liter sium, calcium, bicarbonate, phosphorus, urea. The sodium and chloride were, in and total protein show very little dif general, similar to ours, but the urea ference between the fresh-water and content was higher than that of any of marine series, whereas the fresh-water our specimens from the lake. The magnesium level is about 37% of the "marine" (estuarine) specimens had marine level, the total of sodium and 213 mM/liter sodium, 360 mM/liter chloride is about 80% and urea about chloride, and 435 mM/liter urea. In this 47%. case, the urea was at the high end of Differences are somewhat less pro our marine range, but the sodium was nounced than those reported between far below any individual sodium value fresh-water and marine elasmobranchs found in our marine series and at the by Smith (1931a, 1931b, 1936). How low extreme for all others. The chloride ever, his comparisons were between dif was well above any individual in any of ferent species, and it is well established our series. that there are appreciable species dif Our estuarine series predictably is ferences completely unrelated to habi highly variable. Ranges of the inorganic tat. ions so broadly overlap those of the In Urist's (1962) comparison of Car other series that little confidence can be charhinus leucas nicaraguensis (from placed in the apparent differences. The fresh water) with marine C. l. leucas, individual urea levels range from well mean figures for sodium, chloride, and below the fresh-water average (157 urea suggested somewhat lower levels compared with 169 mM/liter to ap than ours for fresh-water sharks. How proximately the marine average (357 ever, he used only four specimens, and compared with 356 mM/liter). A simi his figures in most cases fall wi thin the lar range of urea levels (134-336 mM/ low part of our ranges. No important liter) was found in a series of eight differences are evident. U rist's figures pregnant females taken from the same for marine sharks, based on a small but collecting grounds at Barra del Colo unspecified number of specimens, are rado (Thorson and Gerst 1972). From likewise largely within our ranges. How tagging and tracking records (unpub ever, his values for sodium (223.4 mM/ lished data) it is known that these liter) and chloride (236) are well below sharks move freely in and out of the our ranges (276-300 and 267-309, re river mouth and from fresh water to spectively) as well as below most values salt water and vice versa. They are given by other investigators for elasmo highly tolerant of either fresh or salt branchs (summarized by Bernard, water for extended periods of time. Wynn, and Wynn 1966). Since they are strong swimmers and are Gerzelli et al. (1969) reported data constantly moving, it is a reasonable on three C. leucas from Lake Nicaragua assumption that they are not confined and two from "the sea." The latter to a narrow range of salinity, even for were taken at the mouth of the Rio short periods. It is impossible to know, Colorado and are therefore comparable even if the exact spot of capture is with our estuarine series, which were of known, where they have been in the im-

603 34 T. B. THORSON, C. M. COWAN, AND D. E. V·,1ATSON

TABLE 1

SUMMARY 01' DATA ON BODY-FLUID PARAMETERS OF "CARCHARHINUS LEUCAS"

Body Fluid Hematocrit pH Na Ca ~Ig Marine adults (Florida): Serum ...... 21.6 ( 6) 7.00 ( 4) 288 ( 7) 6.1 ( 6) 5.7 ( 7) 3.8 ( 7) 18-27 6.81-7.15 276-300 5.0-8.0 4.1-6.3 1.7-6.0 1.31 0.08 3.54 0.47 0.29 0.59 Perivisceral fluid ...... 5.36 ( 2) 313 ( 5) 6.7 ( 5) 1.7 ( 4) 3.3 ( 4) 5.32-5.39 290-328 4.2-10.6 1.2-2.6 2.5-5.3 8.21 1.11 0.32 0.70 Pericardial fluid ...... 5.05 ( 2) 336 ( 5) 11.6 ( 5) 2.4 ( 5) 1.8 ( 5) 5.00-5.10 308-370 9.6-16.9 2.2-2.8 1.4-2.6 11.89 1.41 0.11 0.22 Cranial fluid ...... 7.26 ( 3) 266 ( 4) 3.7 ( 4) 4.6 ( 4) 3.0 ( 4) 7.02-7.42 231-278 2.1-4.6 4.2-5.1 1.9-4.0 0.12 11.59 0.57 0.18 0.47 Fresh-water adults (Lake Nicaragua) : Serum ...... 24.9 ( 8) 6.83 (15) 245 (17) 6.4 (12) 4.5 (15) 1.4(16) 19-29 6.28-7.15 214-280 5.0-8.2 3.5-5.3 0.9-2.3 1.30 0.06 4.14 0.29 0.15 0.09 Perivisceral fluid ...... 5.50 ( 7) 213 ( 7) 5.8 ( 7) 0.8 ( 6) 1.8 ( 6) 5.26-5.85 186-236 4.8-6.4 0.5-1.6 1.0-2.3 0.07 6.64 0.22 0.18 0.18 Pericardial fluid ...... 5.57 (11) 249 (11) 11.1 ( 8) 1.4(11) 1.5 (13) 5.04-5.95 198-276 5.5-15.6 0.6-1.9 1.0-2.2 0.08 6.23 1.05 0.13 0.11 Cranial fluid ...... 7.49 (13) 247 (11) 4.8 (10) 3.1 (10) 1.6 (11) 7.01-7.97 206-268 4.2-5.4 2.2-3.8 1.2-2.3 0.08 5.96 0.12 0.19 0.10 Fresh-water juveniles (lower Rio Colorado): Serum ...... 21.8 ( 9) 228 (12) 6.3 (12) 4.3 ( 5) 2.1 ( 5) 18-25 204-262 4.4-9.8 2.7-5.3 1.6-2.5 0.76 4.45 0.43 0.46 0.17 Estuarine adults (mouth of Rio Colorado): Serum ...... 22.5 (19) 6.71 (13) 233 (29) 5.9 (29) 4.2 (27) 2.0 (28) 13-31 6.45-7.20 196-270 3.9-13.2 2.0-5.4 1.4-2.9 1.20 0.06 2.97 0.37 0.17 0.03 Perivisceral fluid ...... 5.41 ( 8) 234 (11) 5.3 (11) 0.7 ( 8) 2.9 ( 8) 5.10-5.84 198-278 3.0-8.6 0.4-1.6 1.6-4.7 0.10 8.64 0.42 0.14 0.35 Pericardial fluid ...... 5.41 ( 9) 255 ( 9) 8.4 ( 9) 1.1 (11) 2.0 (11) 5.12-5.73 185-319 4.2-12.4 0.4-1.7 1.1-3.0 0.08 12.33 1.07 0.14 0.21 Cranial fluid ...... 7.39 ( 9) 238 (12) 4.7 (12) 3.0 (10) 1.5 (13) 7.20-7.70 209-254 2.9-10.2 1.3-4.3 1.1-1.9 0.05 3.54 0.66 0.26 0.06 NOTE.-All fluid concentrations expressed in mM lliter except total protein in g/IOO. The means, in italic, are fol. lowed by number of specimens in parentheses. Ranges are on the second line, followed by standard errors on the third.

mediate past. Price and Creaser (1967) to changed environmental salinity ob found that the skate, Raja eglanteria, viously are slow and will not accurately required more than 48 hr to reach reflect short-term salinity changes. osmotic equilibrium when the salinity However, there can be no doubt that of its medium was changed by only 2.4 the animal's urea level is at least a ppt. Changes in urea level in response crude indication of its more extended

604 BODY FLUIDS OF BULL SHARKS 35

TABLE 1 (Continued)

Total Body Fluid Cl HC03 Pi Protein Urea TMAO

Marine adults (Florida): Serum ...... 288 ( 7) 8.1 ( 5) 2.3 ( 4) 3.3 ( 7) 356 ( 7) 46.6 (4) 267-309 6.0-10.0 1.8-3.5 2.4-3.9 289--450 30.7-53.6 5.96 0.71 0.41 0.22 20.90 5.34 Perivisceral fluid ...... 382 ( 5) 5.0 ( 2) 0.3 ( 5) Trace 380 ( 4) 366--400 3.5-6.5 Trace-I. 1 328--443 6.07 0.22 25.78 Pericardial fluid ...... 372 ( 5) 6.5 ( 2) 0.1 ( 5) Trace 418 ( 4) 350--400 6.0-7.0 Trace-O.3 393--464 8.33 0.06 15.77 Cranial fluid ...... 279 ( 4) 6.8 ( 3) 1.5 ( 4) 429 ( 3) 275-284 4.5-8.5 1.4-1.7 410--450 2.46 1.20 0.07 11.57 Fresh-water adults (Lake Nicaragua) : Serum ...... 219 (17) 7.0 (13) 2.6 (10) 3.1 (16) 169 (16) 179-247 5.0-9.0 2.0-3.0 2.5-3.6 121-194 4.91 0.38 0.10 0.07 5.40 Perivisceral fluid ...... 217 ( 7) Trace Trace 179 ( 3) 200-228 129-208 3.75 24.97 Pericardial fluid ...... 258 (13) 6.0 ( 1) Trace Trace 161 ( 3) 221-293 121-187 6.59 20.42 Cranial fluid ...... 224 ( 7) 6.6 ( 7) 1.2 ( 5) 1.2 (11) 172 ( 4) 192-250 4.0-9.0 0.8-1.6 Trace-1.9 136-207 7.06 0.39 0.13 0.17 14.77 Fresh-water juveniles (lower Rio Colorado): Serum ...... 207 (12) 2.0 ( 5) 138 (12) 179-233 1.8-2.4 114-171 5.15 0.10 6.01 Estuarine adults (mouth of Rio Colorado): Serum ...... 233 (29) 5.8 (11) 2.7 (18) 2.6 (26) 220 (28) 132 (7) 183-293 4.0-8.0 1.6--4.8 1.2-3.8 152-357 8.9-23.3 4.06 0.36 0.17 0.12 9.82 2.14 Perivisceral fluid ...... 243 (13) 5.9 ( 6) Trace Trace 182 ( 6) 196-270 4.5-7.0 160-209 6.28 0.35 8.12 Pericardial fluid ...... 287 (12) 5.9 ( 8) Trace Trace 220 ( 7) 208-375 3.5-7.5 157-286 13.10 0.45 18.04 Cranial fluid ...... 236 (13) 8.8 ( 9) 1.7 (11) 1.1 (11) 213 ( 9) 16.2 (2) 198-270 7.0-11.5 1.2-2.2 0.6-1.9 147-264 13.1-19.3 5.02 0.48 0.11 0.11 14.86 environmental history. Since each ani series (46.6 mM/liter) and the estuarine mal of the estuarine series had its own series (13.2 mM/liter). Although our peculiar but unknown history, the figures are lower than those frequently grouping is actually an artificial one, attributed to elasmobranchs, they agree but it did not seem warranted to list the with the findings of Goldstein, Oppelt, data for 29 sharks individually. and Maren (1968) who found that, in Trimethylamine oxide levels can be the lemon shark (Negaprion breviros compared only between the marine tris), TMAO concentration dropped by

605 36 T. B. THORSON, C. M. COWAN, AND D. E. WATSON about 60 % when the fish were trans Dulce the juveniles are at least loosely ferred from full-strength to half segregated from the adults. strength seawater. A lower protein content was noted in The 12 juvenile specimens range from juvenile serum than in adult serum from newly born individuals with the umbili either fresh or salt water. The same ob cal scar still unhealed to those possibly servation was reported in ex utero pups several months of age. Their size range by Cowan (1971) and by Thorson and (56-72 cm) is entirely within the Gerst (1972). range of unborn pups of this species (Jensen 1972). Although they move PERIVISCERAL, PERICARDIAL, AND CRANIAL FLUIDS freely about the fresh-water channels Both perivisceral and pericardia1 of the Rio San J uan-Rio Colorado sys cavities are parts of the coelomic sys tem, they are concentrated in Laguna tem. Their fluids are regarded basically Agua Dulce, a shallow channel about 12 as secretions of their bounding mem km long, which runs parallel to the branes, the peritoneum and pericar Caribbean Coast toward the north from dium. The two cavities do not the mouth of the Rio Colorado. Most of communicate, at least not freely and the 12 specimens were taken from this continuously (Smith 1929; Maren channel. 1967), and each fluid has its own Most serum parameters of the unique chemical anatomy. In all three juvenile series, including urea, were adult series, both fluids had reduced similar to, or slightly below, those of calcium (especially in the perivisceral the adult fresh-water series, suggesting fluid) and only a trace of inorganic a freshwater habitat as the preferred phosphorus and protein. As has been environment of the neonatal sharks. noted in other elasmobranchs by Smith This is borne out by 5 years' fishing (1929), Hartman et al. (1941), Rod records, which indicated that over 99 % nan, Robin, and Andrus (1962), and of more than 1,000 neonatal sharks were Bernard et al. (1966), chloride levels of taken at points inside the river mouth, both coelomic fluids were elevated in strictly fresh water (unpublished above those of serum and cranial fluid. data). This was particularly true in the marine The ability to tolerate marine levels series. In fresh water, the chloride of of urea is clearly present in this species perivisceral fluid dropped to the serum before birth. Ex utero pups taken from level, while pericardial chloride re females with urea levels ranging from mained somewhat elevated. Sodium of 134 to 336 mM/liter, usually contained both fluids in marine sharks was also similar urea levels in their own serum slightly elevated but dropped to near the (Thorson and Gerst 1972). It would levels found in serum in fresh-water appear that they should be able to live sharks. In all cases, sodium was some in either fresh or marine water at birth. what higher in pericardial than in peri The actual habitat selection is probably visceral fluid. The pericardial fluid had less a physiologic requirement than a a potassium content greater than that of matter of survival from predation. The the other fluids by about a factor of 2. heaviest concentration of adults, includ This condition was also reported by ing a few other species of sharks, is Smith (1929) and by Maren (1967), around the river mouth, while in the but Bernard et al. (1966) reported high "nursery grounds" of Laguna Agua potassium in all fluids tested, with the

606 BODY FLUIDS OF BULL SHARKS 37 least in pericardial fluid. We agree with marine series similar to those shown by Bernard et aI. (1966) in that we did not the serum. observe the elevated magnesium in perivisceral fluid reported by Smith HYDROGEN ION CONCENTRATION (1929) and by Hartman et al. (1941). The serum pH of the fresh-water, In marine sharks, magnesium of peri estuarine, and marine series of adults visceral fluid was approximately the showed broadly overlapping ranges and same as in serum. However, it was were very little different from one an higher than that of pericardial fluid. other (6.83, 6.71, and 7.00, respec The cranial fluid of elasmobranchs tively). Values reported in the litera lies between the brain and the cranium ture for a variety of elasmobranchs and is distinct from the cerebrospinal (cited by Bernard et al. 1966; Berger fluid, with which it has sometimes been 1967; Maren 1967; Murdaugh and confused. It was regarded by Smith as Robin 1967) largely fall between 7.2 a protein-free dialysate of the plasma. and 7.6, although Bernard et al. (1966) We found it to be considerably more gave figures of 6.0 for Dasyatis say and similar to serum than to the coelomic 6.3 and 6.9 for D. americana. Differ fluids, but it nevertheless had its own ences may be real and/or may depend distinct chemical composition. \Ve did on differences in procedures. Changes not find that it was protein free but that in pH occur slowly. Samples of cranial it had approximately 40% of the serum fluid tested 1, 3, and 5 hr after being protein concentration in both the fresh drawn had pH readings of 7.3, 7.4, and water and estuarine series. Oppelt et al. 7.5 in one instance and 7.2, 7.3, and (1964) also reported approximately 7.35 in another. Serum with a pH of 1-2 g/100 protein in this fluid. 6.79 had a reading of 6.96 on the fol Our data do not appear either to sup lowing day. Readings in the present port or disprove Smith's (1936) specu study were taken on serum as soon as lation that the perivisceral fluid provides possible after clot separation and in an avenue of excretion. We occasionally other fluids as soon as possible after noted the escape of coelomic fluid from samples were collected. the abdominal pores when sharks were Both coelomic fluids (perivisceral brought on land and no longer had the and pericardial) had appreciably higher pressure of the water supporting their hydrogen ion concentrations (ca. 5.0- bodies. However, we have no evidence 5.5) than the serum. They were essen that such a discharge takes place when tially the same in all three environ the animals are in water. mental series. This greater acidity has In the fluids in general we did not also been reported by others who have find a reciprocal relation between con made observations on coelomic fluids of centration of inorganic substances and various elasmobranchs (Smith 1929; urea, as reported by Smith (1929). Rodnan et aI. 1962; Bernard et aI. Urea content in the four fluids tested 1966; Maren 1967). was, in general, similar. In marine The cranial fluid in all three series sharks the minor fluids showed higher exhibited a hydrogen ion concentration mean values for urea than did serum, lower than that of the serum (approxi but the ranges overlapped broadly. The mately 7.25-7.50), although in most minor fluids in general exhibited differ respects this fluid is similar to the ences between the fresh-water and serum. Smith (1929) reported pH

607 38 T. B. THORSON, C. M. COWAN, AND D. E. WATSON values for the cranial fluid of several reported here and by various other in species which were about the same as vestigators emphasize the need for con ours. However, his cranial fluid pH sidering the subject species' degree of values were nearly the same as those of euryhalini ty. the plasma, which were somewhat lower To illustrate, Alexander et a1. (1968), (less acid) than our serum values. studying Scyliorhinus canicula, placed these dogfish in 60 % seawater and HEMATOCRIT noted a rapid increase in weight which When the Lake Nicaragua series is was attributed to retention of water. combined with the juvenile series from They calculated that the decrease in the fresh-water nursery area, the aver urea concentration observed in dogfish age fresh-water hematocrit value is 23.3 kept in diluted seawater could be ex compared with 21.6 for the marine plained entirely by dilution of the body series. The mixed estuarine population fluids as water was taken up. They is intermediate, with 22.5. This rela found no support for Smith's (1936) tionship is the reverse of what might assumption that elasmobranchs respond have been anticipated between hydrat to a diuretic stress by increased urea ing and dehydrating environments. excretion. Furthermore, they (as well However, these figures, for a single as Watts and Watts 1966) reported an species, are in close agreement with increase in activity of carbamoyl phos figures of Thorson (1962 b), comparing phate synthetase (and presumably urea fresh-water Carcharhinus nicaraguensis synthesis) in S. canicula placed in (== leucas) , with a hematocrit of 22.8, diluted seawater. These observations with three basically marine species of are undoubtedly true for the species in sharks with a hematocrit of 18.9. The question. However, one must keep in same relationship was also found by mind that S. canicula is not a markedly Thorson (1961) in a comparison of the euryhaline shark. It is possible that it hematocrits of three species of fresh may make excursions into somewhat water teleosts with seven marine teleost brackish water, but its performance in species (31.8 and 30.4, respectively). diluted seawater clearly marks it as In none of these comparisons are the relatively stenohaline. These fish sur differences great enough to be statisti vived in 60 % seawater only up to 10 hr cally valid, but they demonstrate that (Alexander et a1. 1968). Although they the fresh-water environment does not survived for 2 or 3 days when trans elicit dilution of the plasma in species ferred gradually to 80% seawater that can normally inhabit fresh-water. (Watts and Watts 1966), even then they showed signs of distress, in stiff ness of the tail, difficulty of tail flexion, CONCL UDING REMARKS and imbibition of large amounts of sea The wide range of urea concentra water. tions in our estuarine series and the im Somewhat different observations were plied diversity of salinity from which reported by Goldstein and Forster the individual animals were taken un (1971a) who successfully transferred derscore the importance of knowing the Raja erinacea, over a period of 4 days, environmental history of the subjects to approximately 50% seawater, kept before interpretations are made. Also, them in this medium for 4 days, and re differences in physiological responses turned them during an additional 4 days

608 BODY FLUIDS OF BULL SHARKS to full strength seawater. They reported cas from Lake Nicaragua was shown to that, as salinity of the medium was be virtually identical (72.1 % of body reduced, plasma urea concentration weight) with the average of three dropped, because of increased renal marine species, N egaprion brevirostris, clearance; and urea synthesis decreased Ginglymostoma cirratum, and Squalus rather than increased. It is obvious from acanthias (71.5 %). Also in that study, the survival of the subjects that this the pattern of water partitioning among species is somewhat more tolerant of the major body-fluid compartments was salinity changes than S. canicula. How found to be similar in the fresh-water ever, it was apparently approaching its and marine sharks. The homeostatic limit of toler ace to dilution of its mechanisms appear to be highly effec medium as suggested by an 18% in tive as regards water content and parti crease in body weight and a 20% drop tioning in spite of the radically different in hematocrit. No calculations were external medium and the resultant dif made to determine whether or not the ference in osmoregulatory requirements. drop in urea concentration could be ex Most of the findings concerning plained on the basis of body-fluid dilu fresh-water elasmobranchs by Smith tion. (1931a, 1936) were made on the saw Goldstein et al. (1968) also trans fish, Pristis microdon, a species either ferred lemon sharks (N egaprion bre identical with or closely related to P. virostris) to 50% seawater over a perotteti, which occurs in Lake Nicara period of 7 days and maintained them gua. Although it is also found in coastal in that salinity for an additional 7 days. salt and brackish water, it appears to They ascribed an observed drop in urea spend major portions of its life in fresh concentration in the plasma to a nearly water, probably reproducing there, and threefold increase in urea clearance is, if anything, even more thoroughly from the body fluids and reported no adapted to fresh water than C. leucas significant change in the rate of urea (unpublished data) . Smith's results synthesis. There was likewise no signifi must be interpreted in light of these cant change in hematocrit, indicating facts. His assumption that elasmo that water balance was being main branchs respond to a diuretic stress by tained. These sharks are among the increased urea excretion (supported by more euryhaline species and are known Goldstein et al. [1968] in Negaprion to occur in brackish and even fresh brevirostris) is undoubtedly valid for water (Bigelow and Schroeder 1948), elasmobranchs equipped to deal with al though there are no known popula this stress ( euryhaline species) , but tions that regularly occur in fresh water quite possibly not for stenohaline for extended periods. species. The assumption is not even The fully euryhaline shark, Carchar pertinent to the fresh-water stingrays hinus leucas, as reported above, occurs (Potamotrygonidae) which have ap regularly in fresh water as well as in parently abandoned the mechanisms for seawater for extended periods, and re urea retention (Thorson et al. 1967; ported hematocrit values indicate that Thorson 1970; Goldstein and Forster there is no dilution of body fluids when 1971b). the animals enter fresh water. Further It is clear from these and other ap more, in a study by Thorson (1962a, parently conflicting reports concerning 1962b), the total body water of C. leu- various aspects of the osmoregulation

609 40 T. B. THORSON, C. M. COWAN, AND D. E. WATSON of a number of elasmobranchs that the sery grounds," probably because they range of environmental salinity toler are segregated there from predatory ance of the species studied is very im adults rather than for physiologic rea portant. It must be considered in inter sons. preting results and caution must be ex 5. In comparison with serum, the ercised in extrapolating findings to coelomic fluids (perivisceral and peri elasmobranchs in general. cardial) had elevated chloride levels, reduced calcium, and only a trace of SUMMARY inorganic phosphorus and protein. Sodium was somewhat higher in peri 1. Comparisons were made between cardial than in perivisceral fluid, and various chemical parameters of bull pericardial potassium concentration was shark serum, perivisceral fluid, peri greater by a factor of about 2 than in cardial fluid, and cranial fluid from other fluids. adults taken from marine water, adults 6. Cranial fluid was more similar to from fresh water, juveniles from fresh serum than to coelomic fluids, but its water, and adults from a river mouth protein content was intermediate be where environmental salinities of the tween them. subjects were unknown but probably 7. The minor fluids, in general, ex heterogeneous. hibited differences between the fresh 2. Serum of fresh-water adults con water and marine series similar to those tained reduced levels of magnesium, shown by the serum. sodium, chloride, and urea compared 8. Hydrogen ion concentration was with marine adults, while potassium, virtually the same in the three environ calcium, bicarbonate, inorganic phos ments. However, the coelomic fluids phorus, and total protein were approxi were distinctly more acid than the mately equal. serum, and the cranial fluid was less 3. The estuarine series was highly acid. variable. The serum urea content 9. Mean hematocrit values were vir ranged from levels found in fresh-water tually equal in the fresh-water and specimens to those of marine ones. The marine series, indicating that osmotic urea concentration was interpreted as dilution of fluids does not occur in the an index of the shark's recent environ fresh-water environment. mental history. 10. The environmental history of ex 4. Except for a reduced protein con perimental elasmobranchs and the range centration, the juveniles' serum solute of environmental salinity tolerance of concentrations were similar to those of the subject species must be considered fresh-water adults. Even before birth, in interpreting experimental results, and they have the urea tolerance necessary great caution must be exercised in ex for life in a marine environment, but trapolating results from one species to they occur mainly in fresh-water "nur- elasmobranchs in general.

LITERATURE CITED ALEXANDER, M. D., E. S. HASLEWOOD, G. A. D. BERNARD, G. R., R. A. WYNN, and G. G. WYNN. HASLEWOOD, D. C. WATTS, and R. L. WATTS. 1966. Chemical anatomy of the pericardial and 1968. Osmotic control and urea biosynthesis perivisceral fluids of the stingray, Dasyatis in selachians. Compo Biochem. Physio!. 26: americana. BioI. Bull. 130: 18-27. 971-978. BIGELOW, H. B., and W. C. SCHROEDER. 1948.

610 BODY FLUIDS OF BULL SHARKS 41

Sharks. In Fishes of the western North Atlan physiology. Vol. 1. Excretion, ionic regulation, tic. Mem. Sears Found. Marine Res. Vol. 1, and metabolism. Academic Press, New York. no. 2. Yale Univ., New Haven, Conn. 465 pp. BOESEMAN, M. 1964. Notes on the fishes of west JENSEN, N. H. 1972. Reproduction and develop ern New Guinea. III. The fresh water shark ment of the bull shark, Carcharhinus leucas, of Jamoer Lake. ZooI. Mededelingen 40:9-22. in the Lake Nicaragua-Rio San Juan system. BURGER, J. W. 1967. Problems in the electrolyte Unpublished Ph.D. thesis. University of Ne economy of the spiny dogfish, Squalus acan braska, Lincoln, Nebraska. thias. In P. W. GILBERT, R. F. MATHEWSON, KROGH, A. 1939. Osmotic regulation in aquatic and D. P. RALL (eds.) , Sharks, skates, and animals. Cambridge Univ. Press, Cambridge. rays. Johns Hopkins Press, Baltimore. 242 pp. CONWAY, E. J., and E. O'MALLEY. 1942. Micro MAREN, T. H. 1967. Special body fluids of the diffusion methods: ammonia and urea using elasmobranch. In P. W. GILBERT, R. F. buffered absorbents. Biochem. J. 36:655-661. MATHEWSON, and D. P. RALL (eds.) , Sharks, COWAN, C. M. 1971. Serum protein variation in skates, and rays. Johns Hopkins Press, Balti the bull shark, Carcharhinus leucas MUller and more. Henle, 1841. Int. J. Biochem. 2:691-696. MURDAUGH, H. V., and E. D. ROBIN. 1967. Acid DYER, W. J., F. E. DYER, and J. M. SNOW. 1952. base metabolism in the dogfish shark. In P. W. Amines in fish muscle. V. Trimethylamine ox GILBERT, R. F. MATHEWSON, and D. P. RALL ide estimation. J. Fisheries Res. Board Can. (eds.) , Sharks, skates, and rays. Johns Hop 8(5):309-313. kins Press, Baltimore. ENGELHARDT, R. 1913. Monographie der Selachier OGURI, M. 1964. Rectal glands of marine and der MUnchener zoologischen Staatssammlung. fresh-water sharks: comparative histology. I. Tiergeographie der Selachier. AbhandI. Bay Science 144: 1151-1152. erischen Akad. Wiss. 4(3): 1-110. OPPELT, W. W., C. S. PATLAK, C. G. ZUBROD, and FEARON, W. 1939. The carbamido diacetyl reac D. P. RALL. 1964. Ventricular fluid production tion: a test for citrulline. Biochem. J. 33: 902- rates and turnover in elasmobranchii. Compo 907. Biochem. PhysioI. 12: 171-177. FISKE, C. H., and Y. SUBBAROW. 1925. The POTTS, W. T. W., and G. PARRY. 1964. Osmotic colorimetric determination of phosphorus. J. and ionic regulation in animals. Macmillan, BioI. Chem. 66:375-400. New York. 423 pp. GARRICK, J. A. F., and L. P. SCHULTZ. 1963. A PRICE, K. S., and E. P. CREASER, JR. 1967. Fluc guide to the kinds of potentially dangerous tuations in two osmoregulatory components, sharks. In P. W. GILBERT (ed.), Sharks and urea and sodium chloride, of the clearnose survival. Heath, Lexington, Mass. skate, Raja eglanteria Bose 1802-1. Upon lab GERZELLI, G., M. V. GERVASO, and G. F. DE oratory modification of external salinities. STEFANO. 1969. Aspetti della ghiandola rettale Compo Biochem. Physiol. 23:65-76. e della regolazione osmotica in Selaci manm PROSSER, C. L., and F. A. BROWN, JR. 1961. e d'acqua dolce. Atti 38 Convegno U.Z.I., Comparative animal physiology. Saunders, Senigallia, Boll. Zool. 36. Philadelphia. 688 pp. GILBERT, P. W., R. F. MATHEWSON, and D. P. RODNAN, G. P., E. D. ROBIN, and M. H. ANDRUS. RALL. 1967. Sharks, skates, and rays. Johns 1962. Dogfish coelomic fluid. I. Chemical anat Hopkins Press, Baltimore. 624 pp. omy. Bull. Mount Desert Island BioI. Lab. GOLDSTEIN, L., and R. P. FORSTER. 1971a. Osmo 4(4) :69. regulation and urea metabolism in the little SCHALES, 0., and S. S. SCHALEs. 1941. A simple skate Raja erinacea. Amer. J. Physiol. 220: and accurate method for the determination of 742-746. chloride in biological fluids. J. BioI. Chern. ---. 197Ib. Urea biosynthesis and excretion in 140:879-884. freshwater and marine elasmobranchs. Compo SCRIBNER, B. H., and J. CAILLOUETTE. 1954. Serum Biochem. Physiol. 39B :415-421. bicarbonate: bedside determinant. J. Amer. GOLDSTEIN, L., W. W. OPPELT, and T. H. MAREN. Med. Ass. 155: 644. 1968. Osmotic regulation and urea metabolism Sl.fITH, H. W. 1929. The composition of the body in the lemon shark Negaprion brevirostris. fluids of elasmobranchs. J. BioI. Chem. 81: Amer. J. PhysioI. 215: 1493-1497. 407-419. HARTMAN, F. A., L. A. LEWIS, K. A. BROWNELL, ---. 1931a. The absorption and excretion of F. F. SHELDON, and R. F. WALTHER. 1941. water and salts by the elasmobranch fishes. I. Some blood constituents of the normal skate. Fresh water elasmobranchs. Amer. J. PhysioI. PhysioI. Zoo1. 14:476-486. 98:279-295. HEAGY, F. C. 1948. Determination of magnesium ---. 1931b. The absorption and excretion of in body fluids by the use of Titan Yellow. water and salts by the elasmobranch fishes. II. Can. J. Res. 26E:295. Marine elasmobranchs. Amer. J. Physiol. 98: HOAR, W. S., and D. J. RANDALL. 1969. Fish 296-310.

611 42 T. B. THORSON, C. M. COWAN, AND D. E. WATSON

---. 1936. The retention and physiological role charhinus leucas, between Caribbean Sea and of urea in the elasmobranchii. BioI. Rev. 11: Lake Nicaragua demonstrated by tagging. 49-82. Copeia 1971:336-338. STAEDELER, G., and F. T. FRERICHS. 1858. tiber THORSON, T. B., C. M. COWAN, and D. E. WAT das Vorkommen von Harnstoff, Taurin und SON. 1967. Potamotrygon spp.: elasmobranchs Scyllit in den Organen der Plagiostomen. J. with low urea content. Science 158:375-377. Prakt. Chem. 73:48-55. THORSON, T. B., and J. W. GERST. 1972. Compari THORSON, T. B. 1961. The partitioning of body son of some parameters of serum and uterine water in Osteichthyes: phylogenetic and eco fluid of pregnant, viviparous sharks (C ar logical implications in aquatic vertebrates. BioI. charhinus leucas) and serum of their near-term Bull. 120:238-254. young. Compo Biochem. Physioi. 42A:33-40. ---. 1962a. Body water partitioning of the THORSON, T. B., D. E. WATSON, and C. M. fresh water shark, Carcharhinus nicaraguensis, COWAN. 1966. The status of the freshwater compared with that of marine selachians. shark of Lake Nicaragua. Copeia 1966:385- Amer. Zooi. 2(3) :452-453. 402. ---. 1962b. Partitioning of body fluids in the URIST, M. R. 1962. Calcium and other ions in Lake Nicaragua shark and three marine sharks. blood and skeleton of Nicaraguan fresh-water Science 138: 688-690. shark. Science 137: 984-986. ---. 1967. Osmoregulation in fresh water WATTS, D. C., and R. L. WATTS. 1966. Carbamoyl elasmobranchs. In P. W. GILBERT, R. F. phosphate synthetase in the elasmobranchii: MATHEWSON, and D. P. RALL (eds.), Sharks, osmoregulatory function and evolutionary im skates, and rays. Johns Hopkins Press, Balti plications. Compo Biochem. Physioi. 17: 785- more. 798. ---. 1970. Freshwater stingrays, Potamotrygon \VEICHSELBAUM, T. E. 1946. An accurate and spp.: failure to concentrate urea when exposed rapid method for the determination of proteins to saline medium. Life Sci. 9:893-900. in small amounts of blood serum and plasma. --_. 1971. Movement of bull sharks, Car- Amer. J. Clin. Path. 7:40-49.

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