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Osmotic accomodation in Callinectes sapidus Rathbun
Comp. Biochem. Physiol., 1969, Vol. 29, pp. 671 to 687. PergamonPress. Printed in Great Britain
O S M O T I C A C C O M M O D A T I O N IN C A L L I N E C T E S S A P I D US RATHBUN* B U E N A S. B A L L A R D t and W A L T E R A B B O T T Gulf Coast Research Laboratory, Ocean Springs, Mississippi, U.S.A.; Department of Zoology, Mississippi State University, State College, Mississippi, U.S.A. (Received 16 September 1968)
Abstract--1. Salinity and temperature influence blood osmoconcentration of adult Callinectes sapidus Rathbun. Osmoregulation is possible over a wide salinity range, hut hyporegulation is exhibited only at high temperatures. 2. An inverse relationship exists between temperature and blood concentration. A seasonal effect persists under experimental conditions of constant temperature. 3. Male crabs have lower blood concentrations than females at salinities below 20 ~ooexcept at extreme dilutions. 4. Both sexes exhibit hysteresis-like response at salinities above 15 ~oo. Blood concentration after acclimation is higher when a given salinity is approached from a higher salinity than when approached from a lower one. 5. Total organic substances and ninhydrin reactive substances in the blood vary without apparent relation to blood osmoconcentration. Inorganic substances change with blood concentration. INTRODUCTION CALLINECTES SAPIDUS Rathbun, although essentially an estuarine species, occurs in completely fresh water (Gunter, 1938) and in hypersaline waters of 60~oo or more (Simmons, 1957). Its life cycle involves movement across salinity gradients, with differential, apparently salinity-related, age, sexual and seasonal distribution patterns. W h e t h e r or not such patterns are related to variations in osmoregulatory ability is not established. Larvae of C. sapidus do not develop normally in low salinities (Sandoz & Rogers, 1944; Costlow & Bookhout, 1959) but reasons for this p h e n o m e n o n are not known. T a n & Van Engel (1966) noted a differential ability for blood sodium regulation in adult males and females and concluded that this difference is one of the major factors involved in their preferences for different salinities. However, * This study is part of a thesis submitted to the Graduate School, Mississippi State University, in partial fulfillment of the requirements for the degree Doctor of Philosophy. These investigations were supported by Research and Service Grants from Mississippi State College for Women, National Science Foundation Grant No. GB-3452, National Science Foundation Graduate Traineeship (Mississippi State University) and Gulf Coast Research Laboratory research fund. t Present address: Southwestern State College, Weatherford, Oklahoma. 671
672
BUENA S. BALLARD AND WALTER ABBOTT
Rees (1966) reported that the regulatory ability of the males and females was about the same in eight of nine salinity-temperature combinations. Only at the lowest tested combination, 10°C and 1"75~oo sea water, did females show less regulatory ability than males. T e m p e r a t u r e is known to affect blood osmoconcentration of a n u m b e r of animals (Broekema, 1941; Kinne, 1952; Dehnel, 1962). An inverse relationship between these factors has been postulated to favor invasion of low salinity waters at high temperatures (Pannikar, 1940; Pearse, 1950). C. sapidus shows m a x i m u m invasion of brackish waters in s u m m e r (Hay, 1904; Pearson, 1948) b u t t e m p e r a t u r e effect on osmoregulatory ability has not been established unequivocally as a contributing factor. Rees (1966) reported that in full-strength sea water C. sapidus had about the same blood concentration regardless of temperature, b u t in dilute sea water, blood concentration generally increased with decreases in temperature. S o m e animals are known to exhibit hysteresis effects in osmotic adjustment (Kinne & Rotthauwe, 1953 ; Bryan, 1960), b u t the relationship of this p h e n o m e n o n to behavior of animals such as C. sapidus in fluctuating osmotic situations or during migration across salinity gradients is not established. T h i s p a p e r reports experimental studies on several aspects of osmoregulation in C. sapidus including responses to changes in salinity and temperature, hysteresis effects and sexual differences. T h e work was carried on at G u l f Coast Research Laboratory, Ocean Springs, Mississippi, located on Mississippi Sound in the northern G u l f of Mexico.
M A T E R I A L S AND M E T H O D S
Collection and maintenance of crabs Adult male and female crabs were collected from various locations in Mississippi Sound and from bayous with access to the Sound. Surface salinities ranged from 0"7%o to 30"2~oo and surface temperatures from 12.0 to 34.0°C. Although bottom salinities and temperatures undoubtedly varied somewhat from values obtained at the surface, the latter were taken to be sufficient indicators of the field environment for the purposes of the present laboratory study (cf. Mulkena, 1968). The maximum depth of collection areas was approximately 12 ft (3.7 m). Gunter (1945) found that in two Texas bays with depths of 4-12 feet (1"2-3"7 m) the average differences in surface and bottom salinities were 0.5 and 1"7%o. Crabs were transported in boxes in which they were covered with Spanish moss or burlap wet with sea water from the collection area. In the laboratory, crabs were immersed in aerated water of field salinity in glass aquaria, two crabs per aquarium. An acclimation time of 48-72 hr was allowed before experiments were begun. Crabs were fed daily at first, but feeding was later extended to 3-day intervals and then discontinued when preliminary experiments showed that starvation had no significant effect on blood concentration (Ballard, 1967). Water was changed dally in all cases. Temperature varied from experiment to experiment but was held relatively constant for any one experiment. Natural waters were used for all salinities between 3"8 and 33'4~oo and for some experiments at fresh-water concentration levels. For salinities above 33"4%0 an artificial sea-water preparation or reagent grade NaCI was added to 33"0%o sea water. Dehnel (1962) concluded that the ion balance was not altered when sea water was concentrated to 150% with NaCI and then diluted to 100% with distilled water.
OSMOTIC ACCOMMODATIONIN CALLINECTES SAPIDU,S RATHBUN
673
Three different methods were used to obtain very low salinities. Natural waters were taken at increasing distances upstream in the Pascagoula River which empties into Mississippi Sound; 5"0Yoosea water was mixed with increasing amounts of fresh water taken from Bluff Creek, Vancleave, Mississippi, 12 miles from the Sound; and 3"8~oo sea water was mixed with increasing amounts of distilled water. Ionic ratios were assumed to differ in waters obtained by the three methods. Salinities were determined with a G M hydrometer set, by silver nitrate titration of chlorinity or with a freezing point osmometer calibrated against titrated standards. Results were equated b y testing a series of waters of various salinities by all three methods and adjusting to titration. Copenhagen Standard Sea Water with a chlorinity of 19"373~oo was used as a standard for titration. T h e precision of salinity determinations using the osmometer was evaluated by carrying out replicate sets of five determinations each on each of eight sea waters ranging from salinities of 9"2 to 26"0~oo (standard deviation after correction for regression: + 0"08~oo) (cf. Abbott, 1967). B l o o d concentration determinations
Total osmoconcentration of the blood was determined either b y comparison of melting time with that of known concentrations of sodium chloride, using a modification of the method described b y Gross (1954), or with the freezing point osmometer. T h e concentrations of ten blood samples were determined comparatively b y the two methods and, although exactly equivalent values were not obtained, the average difference was not statistically significant (P > 0"1). F o r the melting point comparisons, blood samples were collected b y inserting a melting point capillary tube (1.3-1.5 m m diameter) through the arthroidal membrane at the base of any leg and allowing blood to flow directly into the tube. T h e tube was then sealed with vaseline and quick frozen on dry ice. F o r the osmometer determinations, a 2.5-ml disposable syringe with 21-gauge needle was used for sampling. T h e needle was inserted at the base of a leg; 0.25 ml of blood was drawn into the syringe and transferred to the osmometer cuvette. B l o o d analysis
Determinations were made of total water content, organic content and inorganic residue of blood taken from adult female crabs in ten salinities. After blood osmoconcentration was determined, a 5-ml blood sample was tzken by syringe from each crab and frozen until it could be weighed and analyzed. Approximately 3 g of frozen blood were placed in a 100-ml beaker that had been ignited to constant weight. T h e beaker plus blood was carefully weighed, dried to constant weight at 100°C and weighed again to determine water content. Water-content values were verified independently b y the Chemistry Section, G u l f Coast Research Laboratory, using separate aliquots of the same blood samples. Organic material in the dried samples was removed with concentrated nitric acid and an oxidizing reagent composed of a 2 : 1 : 1 mixture of nitric, sulfuric and perchlorie acids. T h e organic material in the blood proved very resistant to oxidation. Therefore, after the sample was evaporated to dryness, high localized heat was applied with a Bunsen flame. After fuming had ceased, the sample was ignited at 450°C for 2 hr in a muffle furnage, allowed to cool in a desiccator and reweighed. Loss of weight was regarded as total organic content and weight of residue as total metallic cation content as oxides. Estimations were made of free amino acids as leucine and of total reducing substances as glucose. Extraction was carried out b y the method of Awapara (1948) using precisely weighed blood samples (approximately 1 g). Total free amino acids, as leucine equivalents, were determined on 1-ml aliquots using the procedure of Landua & Awapara (1949). Total reducing substances as glucose were determined on 2-ml aliquots b y a modification of the F o l i n - W u procedure (U.S. Army, 1951).
BUENA S. BALLARDAND WALTER ABBOTT
674
R E S U L T S AND D I S C U S S I O N
Preliminary experiments Results of preliminary investigations of acclimation requirements, weight changes with changes in salinity and effects of starvation were in general agreement with those obtained b y other workers for other brackish water decapods (Hukuda, 1932; Krogh, 1939; Gross, 1957; Dehnel, 1962). I n m o s t salinities equilibration was attained within 24 hr after transfer (Table 1). Even with sudden transfer between 15.5 and 33"4~oo, 48 hr was sufficient. However, in extremely low salinities blood concentration was still dropping at the end of 48 hr. I n early experiments 24-hr acclimation was allowed but the time was extended to 48 hr to be within safe
TABLE 1--ACCLIMATION OF C.
sapidusIN VARIOUS SALINITIES
Blood concentration in equivalent sea water (%0) Salinity (~o)
24 hr
48 hr
72 hr
96 hr
30"2-33"4 30"2-33"4 30"2-33"4 30"2-33"4 15"5-33"4 30"2-33"4 30"2-33"4 30"2-33"4 30'2-33"4 30"2-33"4 30"2-33"4 30"2-33"4 30"2-33"4 25 "7-30"2 20"7-25"7 15"5-20"7 3"8- 3"0 33"4---15"5 15"5-20"1 10"0- 5"3 10"0- 5"3 3"0- 2"0 3"0- 2"0 2"0- 1"0 2"0- 1"0 2"0- 1"0 25"7-30"1 25"7-30"1 15"5-20"7 15"5-20"7 15"5-20"7
32"3 32"0 31"5 31"0 31"7 33"0 33"0 32'8 33"1 33"0 31"6 31"5 32"2 30"5 28"5 28"4 24'9 28"5 26'4 26"4 25'1 27"4 21"4 19"9 20"4 23"9 30"5 31"2 29"0 27"9 28"0
33"9 33"6 33"1 33"2 33"4 32"1 32"1 32"2 34"2 32"9 33'2 33"3 33"6 30'0 28"3 27"7 23"8 27"9 26"4 25"8 25"2 25"2 19"8 19"8 19"0 22"9
33"4 32"8 32"0 32"1 32"9 33"1 32"3 32"2 33 "7 32"4 33"4 33"5 33"4 30"5 28"2 28"0 24"2
33"2 32"8 32"0 32"2 33"0
9 days
11 days
30"6 31"4 28"7 28"5 28"8
OSMOTIC ACCOMMODATION I N CALLINECTES SA[P1DUS RATHBUN
675
limits for most salinities. Blood concentrations recorded for osmotic extremes probably do not represent full equilibration but as all crabs were treated alike, trends and sexual comparisons should be valid. When animals were transferred through a series of salinities ranging from 20.7 to 1.0~oo, weight remained constant within 1 per cent of the mean except in some cases in extremely dilute water (Ballard, 1967). Results agreed with the accepted premise that osmoregulation is accomplished by net changes in blood salt content rather than water content (Hukuda, 1932; Schwabe, 1933; Gross, 1957).
Bleeding and starvation effects An experiment to test the effect of frequent bleeding on total blood osmoconcentration was combined with a test for starvation effect. Twenty-eight animals, fourteen fed and fourteen starved, were maintained for 7 days in a constant salinity of 29"7~oo. On the first day, blood concentrations were determined for two fed and two unfed crabs. On each successive day two additional fed and two unfed crabs were sampled, along with those bled on previous days. In this manner four crabs were bled for 7 consecutive days, four for 6 days, four for 5 days and so on. Fed and unfed crabs were otherwise treated alike. Water temperature was not controlled and fluctuated over a range of 19-23°C. Crabs that died during the course of the experiment were excluded from the data (Table 2). Although frequent bleeding reduces the mean serum protein level of the blood (Horn & Kerr, 1963) and starvation may cause a decrease in reducing substances (Florkin, 1960), analysis of variance showed that daily bleeding and starvation for as long as 7 days had no significant effect on blood osmoconcentration.
Response to salinity changes When crabs were passed through a series of salinities in approximately 5~oo intervals, osmotic responses showed the same general pattern described for various other brackish water decapods (Shaw, 1960; Dehnel, 1962). The crabs demonstrated good hyperregulatory ability at low salinities, were nearly homoiosmotic at medium salinities and approached iso-osmoticity at high salinities (Figs. 1, 2, 3 and 5). The results varied somewhat in different experiments. The slopes and positions of the blood concentration vs. salinity curves were affected by temperature, season, sex and direction of movement across a salinity gradient. When late spring crabs were moved gradually from a field salinity of 17"0%o either toward a low salinity of 3"8~oo or toward a high salinity of 30"2~oo sea water, at experimental temperatures of 21.0-21"5°C, the results agreed very well with those obtained by Mantel (1967) for changes in blood sodium with changes in salinity (Fig. 1). Her use of summer crabs from Woods Hole, Massachusetts, and experimental temperatures of 19.0-21"0°C makes the two experiments reasonably comparable. Summer field temperatures at Woods Hole are no higher than late spring temperatures of the north central Gulf of Mexico. Surface temperatures near Woods Hole average 19-21°C during June, July and August (Schroeder, 1966). For purposes of comparison, Mantel's sea water values in percentage were
676
BUENA S. BALLARD AND WALTER ABBOTT
TABLE 2~STARVATION
AND BLEEDING EFFECT ON BLOOD CONCENTRATION OF C.
sapidus
Bleeding No. Diet
1
2
3
4
5
6
7
Fed
30"8 31.6 30.8 31"6 30.2 30-6 30"6 30.5 30"9 30"3 30"4 29"9
30'5 31.0 31.4 30"1 30.0 30.4 30"0 30.6 30.3 29-4
30"5 30.8 30.8 30.7 29-9 30.0 29.9 30.2
30"5 31 "2 31"3 30"3 30.1 30"2
30"6 30.1 30.3 30"2
29-8 30"6 31.0
29.5
Starved
30"8 31"1 30"7 30.5 30.6 30"8 30"8 30.3 30.4 29.8 29.9 29.8 29.7
30.8 30.6 30"5 30.6 30.0 30.8 30"6 29.6 29"7 29"8 30.1
31.2 30"8 30.8 30"4 30.4 30.6 29.7 29.3 29.9
30.4 30"4 30"0 29.4 30"1 30"3 29.5
30"2 29.8 29.4 30.3 29-7
29.5 29.9 29.5
29"7
Blood value in equivalent sea water (~oo); salinity 29"7~o. converted to sea-water sodium in m-equiv./1 based on a sodium content of 470.2 m-equiv./1 for a salinity of 34"33~oo (Barnes, 1954). Mantel's blood sodium value at 1 0 0 ~ sea water indicated a higher total blood concentration than that found b y other workers (Odum, 1953; Rees, 1966).
Hysteresis-like phenomena T h e general experimental protocol was to begin each experiment with crabs in a salinity approximating field conditions from which they were collected. As most collections were made from m e d i u m salinities, transfers were often through a series of salinities from m e d i u m to high and from m e d i u m to low as shown in Figs. 1 and 3. T o determine if the equilibrated blood concentration at a given salinity was dependent on whether the salinity was reached from a lower or a higher level, the experiment represented in Fig. 1 was extended to include a reversal procedure in which crabs in 3"8~oo and in 30"2~oo sea water were moved through the same series of salinities they had previously encountered but in reverse order (Fig. 2). In
OSMOTICACCOMMODATIONIN CA.LLINECTES S~PIDUS RATHBUN
677
salinities below approximately 15~oo direction of movement had little effect on blood concentration in either sex, but in higher salinities blood concentration at a 500
.35= Osmoconcentrotion 21.0°-21.5"C (Ballord) // ..... 400 "5
No+concentration 1 9 , 0 e - Z I . O ' C (Mantel)
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. ~ - • . f / j
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E
g
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200
,'5
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'/fo
2'5
~'o
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4~0 '
340
Sea water Na~meq.lt,
Fio. 1. Changes in osmoconcentration and sodium concentration of blood of C. sapidus with changes in salinity. 31
• Up
30
o Down
.
j
•
--.
/ ~ i////
....
2B
27 >_
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25
23 2ZO
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5
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Sea water,
20
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%=
FIG. 2. Changes in blood osmoconcentrations of both sexes of C. sapldus moved up and down a salinity gradient.
given salinity was higher when the crab arrived there from a higher salinity. T h e response of the two sexes was very much alike. Although the lines are shown as meeting at the extreme ends of the curves, they merely follow the transfer pathway
678
BUENA S. BALLARD AND WALTER ABBOTT
of the crabs. Actually, 3"8~o0was reached only from a higher salinity, and 30"3~o0 only from a lower salinity. All other salinities were approached from both directions. The reason for this differential behavior at concentrations above and below 15"0~0 is not known. Perhaps a higher-lower-higher sequence of transfer is not the same as a lower-higher-lower sequence. However, the procedure, as followed, represents a natural sequence because during their life cycle, these adult crabs must, of necessity, encounter low salinities before moving to high salinities. Further, the probability is that they move down to the very low salinity of 3"8~o rather than up from even lower salinities. Figure 1 shows a change in degree of accommodation to the external medium at a salinity of approximately 15~oo in the present study and similar blood sodium changes at a comparable salinity in Mantel's study (1967). This change seems to coincide with the point at which the hysteresis-like effect disappeared (Fig. 2). A similar effect is indicated in Fig. 3. If the differences in blood concentration with direction of movement above a salinity of 15"0~oo simply represented a lag in the active uptake mechanism, then 31
~ pmearliemSentaltemperature 21.0o-21.5oC
30 29
March field temp. 20.0°-2i.0°C May field temp. 2 6 . 0 ° - 2 8 , ~ e
.....
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2B 27
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26
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25 24
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31 30 m El
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~ :
/'//
29 28 27 26 25 24 23 22
7...........:¢" ,'o
,'~ Sea water,
FIQ. 3.
2'o
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2'5
~o
%o
Blood osmoconcentrations of male and female C. sapidus at the experimental salinities and temperatures b u t at different seasons.
same
OSMOTIC ACCOMMODATION IN C A L L I N E C T E S S A P I D U S RATHBUN
679
equilibration would eventually be achieved. However, preliminary experiments indicated that at the saiinities involved in this experiment full equilibration is reached in the 48-hr acclimation period. Therefore, the observed differences presumably would be maintained over an indefinite period of time if conditions remained the same, and so do not represent true hysteresis or lag effect. For this reason the phenomenon is referred to here as hysteresis-like. Too little is known at present about the mechanism controlling the rate of active osmotic accommodation to postulate an explanation.
Sexual comparisons When equal numbers of male and female crabs were subjected to the same experimental conditions there was little sexual difference in blood concentration between approximately 20.0 and 30"0%0 sea water. At lower salinities, however, the males showed consistently lower blood concentrations than the females (Figs. 2, 3). These results do not agree with Rees' observations (1966) of no sexual differences within the salinity range covered by the present experiment or with Tan & Van Engel's findings (1966) of higher blood osmoconcentration for males in 10 and 20%0 water and lower for those in 30%0. Tan & Van Engel's results are puzzling. Although total blood concentration was as indicated above, their blood sodium values showed an opposite trend. At 10 and 20%o, males had lower sodium concentrations than females, but at 30%o females had lower sodium values. A tolerance to lowered blood concentration would, presumably, favor the observed preference of males for low salinity waters (Churchill, 1919; Darnell, 1959; Gunter, 1967). Osmotic extremes A few animals were subjected to extreme osmotic stress. In general, females maintained higher blood concentrations than males except at extremely low salinities (Fig. 4a). A tendency for the situation to reverse at a certain point was evident. This reversal point may vary with the ionic composition of the medium and may represent an earlier breakdown of osmoregulation for the females. However, no generalizations can be made on the basis of these limited experiments. More individual variation was noted at the osmotic extremes than at other salinities. In addition, medium differences, seasonal and temperature differences, and technique variations could have had an effect on results represented as the three experiments were conducted at different times. The melting point comparison technique for determining blood concentration was employed in the experiment using Bluff Creek water for dilution and a 24-hr acclimation time was allowed. An osmometer and a 48-hr acclimation were used for the other two experiments. With these reservations, some observations can be made. Although the curves are displaced, their shapes are similar, except at extreme dilutions. The displacement could be due in part to sexual differences, but may also represent temperature and seasonal effects. Bluff Creek dilutions were made in late summer when field temperatures were 30-0-32.0°C and experimental
680
BUENA S. BALLARD AND WALTER ABBOTT
temperatures were 23.0-24.0°C. Experimental temperatures were 21.0-21.5°C when Pascagoula River water and distilled water dilutions were used, but field temperatures were 28.0-30-0°C and 20.0-21.0°C, respectively. Dehnel (1962) noted that winter Hemigrapsus nudus (Dana) and H. oregonensis (Dana) had higher blood concentrations than summer crabs at the same experimental temperature and salinity, and Mantel (1967) obtained a blood sodium value of 513 m-equiv./1 for winter C. sapidus at the same salinity and temperature as summer crabs with blood sodium of 466 m-equiv./l. 60
(b)
25
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........
Poscagoula river water
3O m./" .....
13
l
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3o
3'5
23.0°-24.0°C 28.O°-30.O°C
4'~
25
conc. with a r t { f i c i a l sea water conc. with N a C t
;o
;5
%o
o f m a l e a n d f e m a l e C. sapidus in l o w - a n d high-s~inity media.
FIG. 4. B l o o d o s m o c o n c e n t r a t i o n s
The concentrations of waters composed of mixtures of Bluff Creek water and 5"0~oo sea water were established by calculation, using a value of 0-06~oo as the salinity of Bluff Creek water at Vancleave, Mississippi (Price & Gunter, 1964). Other salinities were determined with the osmometer calibrated against sea water standards. Individual variation was too great to permit estimation of tolerance ranges and sexual differences at low salinities. The only crab to tolerate fresh water for more than 12 hr was a male which became moribund after 36 hr but survived for 63 hr. All males died immediately in distilled water, but four females survived for approximately 4 hr. Blood concentrations of the latter at the time of transfer from 1"0~oo water were equivalent to 20.8, 19.8, 21.2 and 22"7~oo sea water. Although males generally tended to have lower blood concentrations than females at low salinities, females had the lowest concentrations at extreme dilutions. Such low blood concentrations probably reflect osmoregulatory breakdown and intolerable
OSMOTIC ACCOMMODATION IN CALL1NECTES SAP1DUS RATHBUN
681
dilution of body fluids. However, the lowest recorded blood concentration at which a crab appeared active and normal was obtained with a female in a salinity of 0"2~oo. Blood concentration was equivalent to 13"8~oo. Tan & Van Engel (1966) reported no survival of females in fresh water beyond 36 hr, but 40 per cent survival of males beyond 48 hr. They did not report blood osmoconcentrations in fresh water. At sea-water concentrations above approximately 35~oo males tended to have higher blood concentrations than females (Fig. 4b). Both sexes were nearly isoosmotic to salinities above 30~ooat temperatures of 23-0-24.0°C, but at 28.0-30.0°C both could hyporegulate to some degree. This ability appeared to be better developed in females. At a salinity of approximately 40~oo, the blood of the males had reached iso-osmoticity and that of females was approaching it. The lag at 37~oo is unexplained but presumably could be related to the addition of NaC1 to the medium. Three males and one female tolerated extreme environmental salinities up to 70"8~oo at 23.0-24.0°C but no blood concentration determinations were made at this salinity. The length of time for which the crabs could withstand these conditions was not determined, but exceeded 7½ hr, the termination time for the experiment. All crabs were sluggish and probably were near their upper salinity limit. The female was somewhat more active than the male, but whether this behavior was attributable to a differential tolerance to the osmotic stress was not determined. The population of crabs represented in the present study is not usually subjected to salinities above normal sea water but is well represented in very low salinities. The highest salinity at which the writers have personally observed crabs in the field is 31.6~oo. Several adult females were observed swimming at the surface. As these crabs were moving from the sea, they may have encountered even higher salinities. However, surface salinities in this part of the Gulf of Mexico seldom exceed 35~oo even as far as 80 miles (130 km) offshore (Gulf Coast Research Laboratory, unpublished data). Only a few adult females were taken in low-salinity waters. The minimum salinity from which a female was recorded was 8"8~oo. This situation is to be expected since much of the life of a mature female is spent in spawning activity, which requires high-salinity water. Adult males and juveniles of both sexes were quite plentiful at 4"0~oo and were observed on one occasion at an extreme low of 0"7~oo. The latter value is very close to the 0-5%o upper limit set for fresh water by the Venice Symposium (1959), although Price & Gunter (1964) consider this level to be oligohaline.
Temperature and seasonal effect Results of this study indicate an inverse relationship between blood concentration and temperature as found by Rees (1966). When gradual transfer experiments were conducted using crabs from the same field temperatures of 28-030-0°C but different experimental temperatures, blood concentrations were higher at lower temperatures (Fig. 5). Results differed from those of Rees in that the
682
Btrv~A S. BALLARDANDW.~a'ma ABBOTT
inverse relationship persisted even in full-strength sea water. High experimental temperatures caused shifting of the iso-osmotic point and increasing hyporegulation. On the basis of the previously described experiment (Fig. 4b), the points at which crabs would be approximately iso-osmotic at both temperature ranges are between 40 and 45~oo, a considerably higher concentration than that at which Rees observed
36
Temp.25.0"-24.0"C ......
/;
Temp. 2 8 . 0 * - 3 0 . 0 * C
g
/"
z8 g
z6
J
;
,;
" /;7
jl/"1""
,;
Z/,~o
~'s
3'o
~5
Sea water, %. Fro. 5. Blood osmoconcentrations of C. sapidus in same experimental salinities but at different experimental temperatures. Crabs for both experiments were collected from field temperatures of 28"0-30"0°C.
lack of temperature effect. Gifford (1962) reported that in high salinities in the Laguna Madre of Texas, high temperatures resulted in increased hyporegulation for some C. sapidus but had no effect on blood concentrations of others. Dehnel (1962) noted that for H. nudus and H. oregonensis temperature effect decreased with increasing salinity, with a minimum between 100 and 125% sea water. Temperature effect could account, in part, for conflicting results obtained by various workers. Mantel (1967) observed hypertonicity for sodium at full sea water, but Odum (1953) reported hypotonicity. Mantel's experimental temperature range was 19.0-21.0°C. While Odum did not report temperature, he indicated that his experiments were conducted in August when atmospheric temperatures were high. His animals were approximately iso-osmotic at a salinity of 30~o, but Tan (1962) observed considerable hyperosmoticity at that salinity with 20.0°C experimental temperature. In both experiments represented in Fig. 5, blood concentration declined at a faster rate than in previous experiments using crabs from lower field temperatures (21.0-21.5°C). The curves are steeper at both experimental temperature ranges, although 23-0-24.0°C is fairly close to the range used previously. Odum (1953) showed a similar curve for his experiments which presumably utilized crabs from high field temperatures. Such a fall in blood concentration should favor greater
OSMOTIC ACCOMMODATION IN CALL1NECTES SAPIDUS RATHBUN
683
tolerance of low salinities in summer, the season of maximum invasion of very low salinities by C. sapid~. Although an attempt was made to equate results obtained by different techniques as much as possible, such differences could affect results and must be noted. For the experiments represented in Fig. 5, melting point comparison technique was employed to determine blood concentration and 24-hr acclimation was allowed. For other experiments at comparable salinities, osmometer determinations and 48-hr acclimations were used. In the 24-hr acclimation experiments, the direction of transfer was not recorded and varied from salinity to salinity. The hysteresis-like effect, previously noted, could be the cause of the differences in shapes of these curves when compared to curves from other experiments. Seasonal effect such as observed by Dehnel (1962) for Hemi~,'ap~us was noted for C. sapid~. Crabs collected from colder waters showed higher blood concentrations than those collected from warmer waters, even when kept at equivalent temperature in the laboratory (Fig. 2). As already mentioned, Mantel (1967) also obtained higher blood sodium values for winter C. sapidus than for summer crabs at the same experimental temperature and salinity. The present study verifies this result, but does not explain it.
Blood analysis Aliquots of blood samples were analyzed for water, total organic content, inorganic content, amino acids as leucine and reducing substances as glucose (Table 3). Results were erratic for water and organic substances except at the osmotic extreme of 1"0%o, where water :increased and total solids decreased, possibly because of breakdown in osmoregulation. Horn & Kerr (1963) observed that individual variations in hemolymph protein content of C. sapidus showed a tenfold range and were not related to salinity. Their observed range of 15.2-119-2 mg/100 ml for non-sponge females is not unreasonable in view of the 38-4--140 mg/g range for total organic substances found in the present study. Concentration of inorganic substances (total metallic cations as oxides) paralleled total blood concentration. Presumably the major inorganic component was sodium, since this ion forms 85.5 per cent of the total cations of the blood of C. sapidus (Florkin, 1960). Mantel's (1967) sodium curve, shown in Fig. 1, indicates a rather sudden drop in concentration at approximately 15~oo sea water, whereas the inorganic concentration in the present study drops at 10%o. However, the total osmoconeentration of the blood also drops in this experiment at a salinity of 10~oo, whereas it dropped at approximately 15~oo in most of the experiments. Ninhydrin-reactive substances in the blood varied erratically, although they showed some tendency to be more concentrated at higher salinities. Jeffries (1966) obtained a range in non-protein nitrogen concentration of 8.6--102.9 mg/100 ml in C. sapidus blood and concluded that composition was independent of salinity. Variations in reducing substances tended to reflect variations in total blood concentration. Presumably, such substances are not involved in osmoregulation,
684
BUENA S. BALLASDAND W A L ~ ABBOTT
8~ 0 e~ 0
~NN~ 0
0
0 0 ° ~ .~
,...1
~'~ s~ ©
N~N
NNN
~
OSMOTIC ACCOMMODATIONIN CALL1NECTES SAPIDUS RATHBUN
685
a view supported by the data of Jeffries (1966). In the present study the drop in concentration of reducing substances may have been related to starvation effect rather than to changes in blood concentration. Blood samples were taken at 48-hr intervals in each successive salinity, beginning with 20"7~oo. The crabs were not fed during the course of the experiment and those bled at a salinity of 1.0~oo had fasted considerably longer than those at 20"7~oo. Although starvation does not affect total blood osmoconcentration, according to Florkin (1960) a decrease of concentration of fermentable reducing substances takes place in some species of crabs but not in others. Callinectes sapidus may be in the former group. Morgulis (1922, fide Florkin, 1960) obtained blood glucose values of 175-182 mg/100 ml for freshly caught C. sapidus and values of 9-6-19-0 and 12.5-13.8 mg/100 ml after 1 and 2 days, respectively, of fasting. CONCLUSION
C. sapid~ is a euryhaline species demonstrating good hyperregulatory ability and limited hyporegulatory ability. Sexual differences in osmoregulatory responses may account, in part, for differential salinity preferences of the sexes. Because of the steeper gradient between medium and blood, low salinities demand more osmotic work by females than by males. At very high salinities the gradient between blood and medium is slightly higher for males. However, the sexassociated differences in blood-medium gradients do not appear sufficient to account for the observed differences in sexual ratios. The differential distribution of the sexes may be behavioral rather than physiological. Temperature effect on distribution is not clear. High temperatures may favor tolerance of low salinities by lowering blood concentration and reducing osmotic work. High temperatures may also favor tolerance of hypersalinity by increasing hyporegulatory ability. Such a relationship would cause crabs to move out of both low salinity and hypersaline areas in winter, and such migration does, in fact, occur (Jeffries, 1966; Gifford, 1962). REFERENCES
ABBOTT W. (1967) Salinity determination by freezing-point osmometry. Simposio Internacional sobre Lagunas Costeras, Mexico, D.F., Mexico, 28-30 November, 1967. AWAPARAJ. (1948) Application of paper chromatography to the estimation of free amino acids in tissues. Archs Biochem. 19, 172-173. BALLARD B. S. (1967) Osmotic accommodation in Callinectes sapidus Rathbun. Ph.D. Thesis, Mississippi State University, State College, Mississippi. BARNES H. (1954) Some tables for the ionic composition of sea water. 07. exp. Biol. 31, 582-
588. BROEKEMAM. M. M. (1941) Seasonal movements and the osmotic behavior of the shrimp Crangon crangon L. Archs neerl, zool. 6, 1-100. BRYAN G. W. (1960) Sodium regulation in the crayfish Astacusfluviatilis--II. Experiments with sodium depleted animals. 07. exp. Biol. 37, 100-112. CHURCHILLE. P. (1919) Life history of the blue crab. Bull. U.S. Bur. Fish. 36, 95-128. COSTLOWJ. D., JR. & BOOKHOUTC. G. (1959) The larval development of Callinectes sapidus Rathbun reared in the laboratory. Biol. Bull., Woods Hole 116, 373-396. 23
686
BUENA S. BALLARD AND WALTER ABBOTT
DARNELLR. M. (1959) Studies of the life history of the blue crab, Callinectes sapidus Rathbun, in Louisiana waters. Trans. Am. Fish. Soc. 88, 294--304. DEHNELP. A. (1962) Aspects of osmoregulation in two species of intertidal crabs. Biol. Bull., Woods Hole 122, 208-227. FLORKINM. (1960) Blood chemistry. In The Physiology of Crustacea (Edited by WATERMAN T. H.), Vol. I, 670 pp. Academic Press, New York. GIFFOROC. A. (1962) Some aspects of osmotic and ionic regulation in the blue crab, Callinectes sapidus, and the ghost crab, Ocypode albicans. Publ. Inst. Marine Sci. (Univ. Texas) 8, 97-125. GRoss W. J. (1954) Osmotic responses in the sipunculid Dendrostomum zostericolum, ft. exp. Biol. 31,402--423. GROSSW. J. (1957) An analysis of response to osmotic stress in selected decapod Crustacea. Biol. Bull., Woods Hole 112, 43-62. GImTER G. (1938) The common blue crab in fresh water (Callinectes sapidus). Science 87, 87-88. GUNTERG. (1945) Studies on marine fishes of Texas. Publ. Inst. Marine Sci. (Univ. Texas) 1, 1-190. GUNTER G. (1967) Some relationships of estuaries to the fisheries of the Gulf of Mexico. In Estuaries (Edited by LAUn~G. H.), 757 pp. Am. Inst. Biol. Sci. Pub. 83, Washington, D.C. HAY W. P. (1904) The life history of the blue crab (Callinectes sapidus). Rep. U.S. Comm. Fish. 347-413. Hom~ E. C. & KERR M. S. (1963) Hemolymph protein and copper concentration of adult blue crabs (Callinectes sapidus Rathbun). Biol. Bull., Woods Hole 125, 499-507. HUKUDA K. (1932) Change of weight of marine animals in diluted media, ft. exp. Biol. 9, 61-68. Jm~FmEs H. P. (1966) Internal conditions of a diminishing blue crab population (Callinectes sapidus). Chesapeake S t . 7, 164--170. K I m ~ O. (1952) Zur Biologic und Physiologie yon Gammarus duebeni LilIj--V. Untersuchungen fiber Blutkonzentration, Herzfrequenz und Atrnung. Kid. Meeresforsch. 9, 134-150.
KINNE O. & ROTTrlAUWEH. W. (1952) Biologisehe Beobachtung und Untersuchungen fiber die Blutkonzentration an Heteropenope tridentatus Maitland (Decapoda). K i d Meeresforsch. 8, 212-217. KROCH A. (1939) Osmotic Regulation in Aquatic Animals, 242 pp. Cambridge University Press, London. LANDUA A. J. & AWAPARAJ. (1949) Use of modified ninhydrin reagent in quantitative determination of amino acids by paper chromatography. Science 109, 385. MANTEL L. H. (1967) Asymmetry potentials, metabolism and sodium fluxes in gills of the blue crab, Callinectes sapidus. Comp. Biochem. Physiol. 20, 743-753. MOROULISS. (1922) As cited by Florkin (1960). MULKANA M. S. (1968) Seasonal changes in the nutritional components of the standing plankton biomass in Mississippi Sound. Ph.D. Thesis, Mississippi State University, State College, Mississippi. ODUM H. T. (1953) Factors controlling marine invasion into Florida freshwaters. Bull. mar. Sci. Gulf Carib. 3, 134-156. PANNIKAR N. K. (1940) Osmotic properties of the common prawn. Nature, Lond. 145, 108. PEA~E A. S. (1950) The Emigrations of Animals from the Sea, 210 pp. Sherwood Press, Dryden, N.Y. PEARSONJ. C. (1948) Fluctuations in the abundance of the blue crab in Chesapeake Bay. Res. Rep. U.S. Fish. Serv. No. 14, 26 pp.
OSMOTIC ACCOMMODATION I N CALLINECTES SAP1DUS RATHBUN
687
PRICE J. B. & GUN~R G. (1964) Studies on the chemistry of fresh and low salinity waters in Mississippi and the boundary between fresh and brackish water. Int. Rev. ges. Hydrobiol. 49, 629-636. REIn G. H. (1966) Blue crab studies. Informal Progress Report. U.S. Bur. Comm. Fish. Beaufort, N.C. SANDOZM. & ROBERTSR. (1944) The effect of environmental factors on hatching, moulting, and survival of zoea larvae of the blue crab, Callinectes sapidus Rathbun. Ecology, 52 216-228. SCHROEDER E. H. (1966) Average surface temperatures of the Western North Atlantic. Bull. mar. Sci. 16, 302-323. SCmVABE E. (1933) Ober die Osmoregulation verschiedener Krebse (Malacoetracen). Z. vergl. Physiol. 19, 183-236. SHAW J. (1960) The mechanism of osmoregulation. In Comparative Biochemistry (Edited by FLORKINM. & MASONH. S.), Vol. II, 685 pp. Academic Press, New York. SIMMONS E. G. (1957) An ecological survey of the upper Languna Madre of Texas. Publ. Inst. mar. Sci. (Univ. Texas) 4, 156-200. TAN ENc-CHow (1962) Studies on osmotic and ionic regulation in the blue crab, Callinectes sapidus Rathbun. M.A. Thesis, College of William and Mary, Williamsburg, Virginia, 66 pp. TAN ENo-CHow & VAN ENO~ W. A. (1966) Osmoregulation in the adult blue crab, CaUinectes sapidus Rathbun. Chesapeake So/. 7, 30-35. U.S. Depart~,ent of the Army (1951) Methods for Medical Laboratory Technicians, Technical Manual TM-227, 746 pp. Venice Symposium on the classification of brackish waters. Final resolution (1959) Arch. Oceanogr. Limnol. Suppl. 11, 243-245.
Key Word Index--Osmoregulation; crab; Callinectes sapidus; temperature on osmoregulation; salt regulation; amino acids in osmoregulation.
O S M O T I C A C C O M M O D A T I O N IN C A L L I N E C T E S S A P I D US RATHBUN* B U E N A S. B A L L A R D t and W A L T E R A B B O T T Gulf Coast Research Laboratory, Ocean Springs, Mississippi, U.S.A.; Department of Zoology, Mississippi State University, State College, Mississippi, U.S.A. (Received 16 September 1968)
Abstract--1. Salinity and temperature influence blood osmoconcentration of adult Callinectes sapidus Rathbun. Osmoregulation is possible over a wide salinity range, hut hyporegulation is exhibited only at high temperatures. 2. An inverse relationship exists between temperature and blood concentration. A seasonal effect persists under experimental conditions of constant temperature. 3. Male crabs have lower blood concentrations than females at salinities below 20 ~ooexcept at extreme dilutions. 4. Both sexes exhibit hysteresis-like response at salinities above 15 ~oo. Blood concentration after acclimation is higher when a given salinity is approached from a higher salinity than when approached from a lower one. 5. Total organic substances and ninhydrin reactive substances in the blood vary without apparent relation to blood osmoconcentration. Inorganic substances change with blood concentration. INTRODUCTION CALLINECTES SAPIDUS Rathbun, although essentially an estuarine species, occurs in completely fresh water (Gunter, 1938) and in hypersaline waters of 60~oo or more (Simmons, 1957). Its life cycle involves movement across salinity gradients, with differential, apparently salinity-related, age, sexual and seasonal distribution patterns. W h e t h e r or not such patterns are related to variations in osmoregulatory ability is not established. Larvae of C. sapidus do not develop normally in low salinities (Sandoz & Rogers, 1944; Costlow & Bookhout, 1959) but reasons for this p h e n o m e n o n are not known. T a n & Van Engel (1966) noted a differential ability for blood sodium regulation in adult males and females and concluded that this difference is one of the major factors involved in their preferences for different salinities. However, * This study is part of a thesis submitted to the Graduate School, Mississippi State University, in partial fulfillment of the requirements for the degree Doctor of Philosophy. These investigations were supported by Research and Service Grants from Mississippi State College for Women, National Science Foundation Grant No. GB-3452, National Science Foundation Graduate Traineeship (Mississippi State University) and Gulf Coast Research Laboratory research fund. t Present address: Southwestern State College, Weatherford, Oklahoma. 671
672
BUENA S. BALLARD AND WALTER ABBOTT
Rees (1966) reported that the regulatory ability of the males and females was about the same in eight of nine salinity-temperature combinations. Only at the lowest tested combination, 10°C and 1"75~oo sea water, did females show less regulatory ability than males. T e m p e r a t u r e is known to affect blood osmoconcentration of a n u m b e r of animals (Broekema, 1941; Kinne, 1952; Dehnel, 1962). An inverse relationship between these factors has been postulated to favor invasion of low salinity waters at high temperatures (Pannikar, 1940; Pearse, 1950). C. sapidus shows m a x i m u m invasion of brackish waters in s u m m e r (Hay, 1904; Pearson, 1948) b u t t e m p e r a t u r e effect on osmoregulatory ability has not been established unequivocally as a contributing factor. Rees (1966) reported that in full-strength sea water C. sapidus had about the same blood concentration regardless of temperature, b u t in dilute sea water, blood concentration generally increased with decreases in temperature. S o m e animals are known to exhibit hysteresis effects in osmotic adjustment (Kinne & Rotthauwe, 1953 ; Bryan, 1960), b u t the relationship of this p h e n o m e n o n to behavior of animals such as C. sapidus in fluctuating osmotic situations or during migration across salinity gradients is not established. T h i s p a p e r reports experimental studies on several aspects of osmoregulation in C. sapidus including responses to changes in salinity and temperature, hysteresis effects and sexual differences. T h e work was carried on at G u l f Coast Research Laboratory, Ocean Springs, Mississippi, located on Mississippi Sound in the northern G u l f of Mexico.
M A T E R I A L S AND M E T H O D S
Collection and maintenance of crabs Adult male and female crabs were collected from various locations in Mississippi Sound and from bayous with access to the Sound. Surface salinities ranged from 0"7%o to 30"2~oo and surface temperatures from 12.0 to 34.0°C. Although bottom salinities and temperatures undoubtedly varied somewhat from values obtained at the surface, the latter were taken to be sufficient indicators of the field environment for the purposes of the present laboratory study (cf. Mulkena, 1968). The maximum depth of collection areas was approximately 12 ft (3.7 m). Gunter (1945) found that in two Texas bays with depths of 4-12 feet (1"2-3"7 m) the average differences in surface and bottom salinities were 0.5 and 1"7%o. Crabs were transported in boxes in which they were covered with Spanish moss or burlap wet with sea water from the collection area. In the laboratory, crabs were immersed in aerated water of field salinity in glass aquaria, two crabs per aquarium. An acclimation time of 48-72 hr was allowed before experiments were begun. Crabs were fed daily at first, but feeding was later extended to 3-day intervals and then discontinued when preliminary experiments showed that starvation had no significant effect on blood concentration (Ballard, 1967). Water was changed dally in all cases. Temperature varied from experiment to experiment but was held relatively constant for any one experiment. Natural waters were used for all salinities between 3"8 and 33'4~oo and for some experiments at fresh-water concentration levels. For salinities above 33"4%0 an artificial sea-water preparation or reagent grade NaCI was added to 33"0%o sea water. Dehnel (1962) concluded that the ion balance was not altered when sea water was concentrated to 150% with NaCI and then diluted to 100% with distilled water.
OSMOTIC ACCOMMODATIONIN CALLINECTES SAPIDU,S RATHBUN
673
Three different methods were used to obtain very low salinities. Natural waters were taken at increasing distances upstream in the Pascagoula River which empties into Mississippi Sound; 5"0Yoosea water was mixed with increasing amounts of fresh water taken from Bluff Creek, Vancleave, Mississippi, 12 miles from the Sound; and 3"8~oo sea water was mixed with increasing amounts of distilled water. Ionic ratios were assumed to differ in waters obtained by the three methods. Salinities were determined with a G M hydrometer set, by silver nitrate titration of chlorinity or with a freezing point osmometer calibrated against titrated standards. Results were equated b y testing a series of waters of various salinities by all three methods and adjusting to titration. Copenhagen Standard Sea Water with a chlorinity of 19"373~oo was used as a standard for titration. T h e precision of salinity determinations using the osmometer was evaluated by carrying out replicate sets of five determinations each on each of eight sea waters ranging from salinities of 9"2 to 26"0~oo (standard deviation after correction for regression: + 0"08~oo) (cf. Abbott, 1967). B l o o d concentration determinations
Total osmoconcentration of the blood was determined either b y comparison of melting time with that of known concentrations of sodium chloride, using a modification of the method described b y Gross (1954), or with the freezing point osmometer. T h e concentrations of ten blood samples were determined comparatively b y the two methods and, although exactly equivalent values were not obtained, the average difference was not statistically significant (P > 0"1). F o r the melting point comparisons, blood samples were collected b y inserting a melting point capillary tube (1.3-1.5 m m diameter) through the arthroidal membrane at the base of any leg and allowing blood to flow directly into the tube. T h e tube was then sealed with vaseline and quick frozen on dry ice. F o r the osmometer determinations, a 2.5-ml disposable syringe with 21-gauge needle was used for sampling. T h e needle was inserted at the base of a leg; 0.25 ml of blood was drawn into the syringe and transferred to the osmometer cuvette. B l o o d analysis
Determinations were made of total water content, organic content and inorganic residue of blood taken from adult female crabs in ten salinities. After blood osmoconcentration was determined, a 5-ml blood sample was tzken by syringe from each crab and frozen until it could be weighed and analyzed. Approximately 3 g of frozen blood were placed in a 100-ml beaker that had been ignited to constant weight. T h e beaker plus blood was carefully weighed, dried to constant weight at 100°C and weighed again to determine water content. Water-content values were verified independently b y the Chemistry Section, G u l f Coast Research Laboratory, using separate aliquots of the same blood samples. Organic material in the dried samples was removed with concentrated nitric acid and an oxidizing reagent composed of a 2 : 1 : 1 mixture of nitric, sulfuric and perchlorie acids. T h e organic material in the blood proved very resistant to oxidation. Therefore, after the sample was evaporated to dryness, high localized heat was applied with a Bunsen flame. After fuming had ceased, the sample was ignited at 450°C for 2 hr in a muffle furnage, allowed to cool in a desiccator and reweighed. Loss of weight was regarded as total organic content and weight of residue as total metallic cation content as oxides. Estimations were made of free amino acids as leucine and of total reducing substances as glucose. Extraction was carried out b y the method of Awapara (1948) using precisely weighed blood samples (approximately 1 g). Total free amino acids, as leucine equivalents, were determined on 1-ml aliquots using the procedure of Landua & Awapara (1949). Total reducing substances as glucose were determined on 2-ml aliquots b y a modification of the F o l i n - W u procedure (U.S. Army, 1951).
BUENA S. BALLARDAND WALTER ABBOTT
674
R E S U L T S AND D I S C U S S I O N
Preliminary experiments Results of preliminary investigations of acclimation requirements, weight changes with changes in salinity and effects of starvation were in general agreement with those obtained b y other workers for other brackish water decapods (Hukuda, 1932; Krogh, 1939; Gross, 1957; Dehnel, 1962). I n m o s t salinities equilibration was attained within 24 hr after transfer (Table 1). Even with sudden transfer between 15.5 and 33"4~oo, 48 hr was sufficient. However, in extremely low salinities blood concentration was still dropping at the end of 48 hr. I n early experiments 24-hr acclimation was allowed but the time was extended to 48 hr to be within safe
TABLE 1--ACCLIMATION OF C.
sapidusIN VARIOUS SALINITIES
Blood concentration in equivalent sea water (%0) Salinity (~o)
24 hr
48 hr
72 hr
96 hr
30"2-33"4 30"2-33"4 30"2-33"4 30"2-33"4 15"5-33"4 30"2-33"4 30"2-33"4 30"2-33"4 30'2-33"4 30"2-33"4 30"2-33"4 30"2-33"4 30"2-33"4 25 "7-30"2 20"7-25"7 15"5-20"7 3"8- 3"0 33"4---15"5 15"5-20"1 10"0- 5"3 10"0- 5"3 3"0- 2"0 3"0- 2"0 2"0- 1"0 2"0- 1"0 2"0- 1"0 25"7-30"1 25"7-30"1 15"5-20"7 15"5-20"7 15"5-20"7
32"3 32"0 31"5 31"0 31"7 33"0 33"0 32'8 33"1 33"0 31"6 31"5 32"2 30"5 28"5 28"4 24'9 28"5 26'4 26"4 25'1 27"4 21"4 19"9 20"4 23"9 30"5 31"2 29"0 27"9 28"0
33"9 33"6 33"1 33"2 33"4 32"1 32"1 32"2 34"2 32"9 33'2 33"3 33"6 30'0 28"3 27"7 23"8 27"9 26"4 25"8 25"2 25"2 19"8 19"8 19"0 22"9
33"4 32"8 32"0 32"1 32"9 33"1 32"3 32"2 33 "7 32"4 33"4 33"5 33"4 30"5 28"2 28"0 24"2
33"2 32"8 32"0 32"2 33"0
9 days
11 days
30"6 31"4 28"7 28"5 28"8
OSMOTIC ACCOMMODATION I N CALLINECTES SA[P1DUS RATHBUN
675
limits for most salinities. Blood concentrations recorded for osmotic extremes probably do not represent full equilibration but as all crabs were treated alike, trends and sexual comparisons should be valid. When animals were transferred through a series of salinities ranging from 20.7 to 1.0~oo, weight remained constant within 1 per cent of the mean except in some cases in extremely dilute water (Ballard, 1967). Results agreed with the accepted premise that osmoregulation is accomplished by net changes in blood salt content rather than water content (Hukuda, 1932; Schwabe, 1933; Gross, 1957).
Bleeding and starvation effects An experiment to test the effect of frequent bleeding on total blood osmoconcentration was combined with a test for starvation effect. Twenty-eight animals, fourteen fed and fourteen starved, were maintained for 7 days in a constant salinity of 29"7~oo. On the first day, blood concentrations were determined for two fed and two unfed crabs. On each successive day two additional fed and two unfed crabs were sampled, along with those bled on previous days. In this manner four crabs were bled for 7 consecutive days, four for 6 days, four for 5 days and so on. Fed and unfed crabs were otherwise treated alike. Water temperature was not controlled and fluctuated over a range of 19-23°C. Crabs that died during the course of the experiment were excluded from the data (Table 2). Although frequent bleeding reduces the mean serum protein level of the blood (Horn & Kerr, 1963) and starvation may cause a decrease in reducing substances (Florkin, 1960), analysis of variance showed that daily bleeding and starvation for as long as 7 days had no significant effect on blood osmoconcentration.
Response to salinity changes When crabs were passed through a series of salinities in approximately 5~oo intervals, osmotic responses showed the same general pattern described for various other brackish water decapods (Shaw, 1960; Dehnel, 1962). The crabs demonstrated good hyperregulatory ability at low salinities, were nearly homoiosmotic at medium salinities and approached iso-osmoticity at high salinities (Figs. 1, 2, 3 and 5). The results varied somewhat in different experiments. The slopes and positions of the blood concentration vs. salinity curves were affected by temperature, season, sex and direction of movement across a salinity gradient. When late spring crabs were moved gradually from a field salinity of 17"0%o either toward a low salinity of 3"8~oo or toward a high salinity of 30"2~oo sea water, at experimental temperatures of 21.0-21"5°C, the results agreed very well with those obtained by Mantel (1967) for changes in blood sodium with changes in salinity (Fig. 1). Her use of summer crabs from Woods Hole, Massachusetts, and experimental temperatures of 19.0-21"0°C makes the two experiments reasonably comparable. Summer field temperatures at Woods Hole are no higher than late spring temperatures of the north central Gulf of Mexico. Surface temperatures near Woods Hole average 19-21°C during June, July and August (Schroeder, 1966). For purposes of comparison, Mantel's sea water values in percentage were
676
BUENA S. BALLARD AND WALTER ABBOTT
TABLE 2~STARVATION
AND BLEEDING EFFECT ON BLOOD CONCENTRATION OF C.
sapidus
Bleeding No. Diet
1
2
3
4
5
6
7
Fed
30"8 31.6 30.8 31"6 30.2 30-6 30"6 30.5 30"9 30"3 30"4 29"9
30'5 31.0 31.4 30"1 30.0 30.4 30"0 30.6 30.3 29-4
30"5 30.8 30.8 30.7 29-9 30.0 29.9 30.2
30"5 31 "2 31"3 30"3 30.1 30"2
30"6 30.1 30.3 30"2
29-8 30"6 31.0
29.5
Starved
30"8 31"1 30"7 30.5 30.6 30"8 30"8 30.3 30.4 29.8 29.9 29.8 29.7
30.8 30.6 30"5 30.6 30.0 30.8 30"6 29.6 29"7 29"8 30.1
31.2 30"8 30.8 30"4 30.4 30.6 29.7 29.3 29.9
30.4 30"4 30"0 29.4 30"1 30"3 29.5
30"2 29.8 29.4 30.3 29-7
29.5 29.9 29.5
29"7
Blood value in equivalent sea water (~oo); salinity 29"7~o. converted to sea-water sodium in m-equiv./1 based on a sodium content of 470.2 m-equiv./1 for a salinity of 34"33~oo (Barnes, 1954). Mantel's blood sodium value at 1 0 0 ~ sea water indicated a higher total blood concentration than that found b y other workers (Odum, 1953; Rees, 1966).
Hysteresis-like phenomena T h e general experimental protocol was to begin each experiment with crabs in a salinity approximating field conditions from which they were collected. As most collections were made from m e d i u m salinities, transfers were often through a series of salinities from m e d i u m to high and from m e d i u m to low as shown in Figs. 1 and 3. T o determine if the equilibrated blood concentration at a given salinity was dependent on whether the salinity was reached from a lower or a higher level, the experiment represented in Fig. 1 was extended to include a reversal procedure in which crabs in 3"8~oo and in 30"2~oo sea water were moved through the same series of salinities they had previously encountered but in reverse order (Fig. 2). In
OSMOTICACCOMMODATIONIN CA.LLINECTES S~PIDUS RATHBUN
677
salinities below approximately 15~oo direction of movement had little effect on blood concentration in either sex, but in higher salinities blood concentration at a 500
.35= Osmoconcentrotion 21.0°-21.5"C (Ballord) // ..... 400 "5
No+concentration 1 9 , 0 e - Z I . O ' C (Mantel)
/•
/
/
~o
g
.B
E
+o z
25
. ~ - • . f / j
t
/"
g
tn
300
E
g
~ z• ,'o
0 ,(~0
200
,'5
Sea water, 200
'/fo
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Sea water Na~meq.lt,
Fio. 1. Changes in osmoconcentration and sodium concentration of blood of C. sapidus with changes in salinity. 31
• Up
30
o Down
.
j
•
--.
/ ~ i////
....
2B
27 >_
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5
I
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' 15
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20
I
I
25
I
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%=
FIG. 2. Changes in blood osmoconcentrations of both sexes of C. sapldus moved up and down a salinity gradient.
given salinity was higher when the crab arrived there from a higher salinity. T h e response of the two sexes was very much alike. Although the lines are shown as meeting at the extreme ends of the curves, they merely follow the transfer pathway
678
BUENA S. BALLARD AND WALTER ABBOTT
of the crabs. Actually, 3"8~o0was reached only from a higher salinity, and 30"3~o0 only from a lower salinity. All other salinities were approached from both directions. The reason for this differential behavior at concentrations above and below 15"0~0 is not known. Perhaps a higher-lower-higher sequence of transfer is not the same as a lower-higher-lower sequence. However, the procedure, as followed, represents a natural sequence because during their life cycle, these adult crabs must, of necessity, encounter low salinities before moving to high salinities. Further, the probability is that they move down to the very low salinity of 3"8~o rather than up from even lower salinities. Figure 1 shows a change in degree of accommodation to the external medium at a salinity of approximately 15~oo in the present study and similar blood sodium changes at a comparable salinity in Mantel's study (1967). This change seems to coincide with the point at which the hysteresis-like effect disappeared (Fig. 2). A similar effect is indicated in Fig. 3. If the differences in blood concentration with direction of movement above a salinity of 15"0~oo simply represented a lag in the active uptake mechanism, then 31
~ pmearliemSentaltemperature 21.0o-21.5oC
30 29
March field temp. 20.0°-2i.0°C May field temp. 2 6 . 0 ° - 2 8 , ~ e
.....
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Blood osmoconcentrations of male and female C. sapidus at the experimental salinities and temperatures b u t at different seasons.
same
OSMOTIC ACCOMMODATION IN C A L L I N E C T E S S A P I D U S RATHBUN
679
equilibration would eventually be achieved. However, preliminary experiments indicated that at the saiinities involved in this experiment full equilibration is reached in the 48-hr acclimation period. Therefore, the observed differences presumably would be maintained over an indefinite period of time if conditions remained the same, and so do not represent true hysteresis or lag effect. For this reason the phenomenon is referred to here as hysteresis-like. Too little is known at present about the mechanism controlling the rate of active osmotic accommodation to postulate an explanation.
Sexual comparisons When equal numbers of male and female crabs were subjected to the same experimental conditions there was little sexual difference in blood concentration between approximately 20.0 and 30"0%0 sea water. At lower salinities, however, the males showed consistently lower blood concentrations than the females (Figs. 2, 3). These results do not agree with Rees' observations (1966) of no sexual differences within the salinity range covered by the present experiment or with Tan & Van Engel's findings (1966) of higher blood osmoconcentration for males in 10 and 20%0 water and lower for those in 30%0. Tan & Van Engel's results are puzzling. Although total blood concentration was as indicated above, their blood sodium values showed an opposite trend. At 10 and 20%o, males had lower sodium concentrations than females, but at 30%o females had lower sodium values. A tolerance to lowered blood concentration would, presumably, favor the observed preference of males for low salinity waters (Churchill, 1919; Darnell, 1959; Gunter, 1967). Osmotic extremes A few animals were subjected to extreme osmotic stress. In general, females maintained higher blood concentrations than males except at extremely low salinities (Fig. 4a). A tendency for the situation to reverse at a certain point was evident. This reversal point may vary with the ionic composition of the medium and may represent an earlier breakdown of osmoregulation for the females. However, no generalizations can be made on the basis of these limited experiments. More individual variation was noted at the osmotic extremes than at other salinities. In addition, medium differences, seasonal and temperature differences, and technique variations could have had an effect on results represented as the three experiments were conducted at different times. The melting point comparison technique for determining blood concentration was employed in the experiment using Bluff Creek water for dilution and a 24-hr acclimation time was allowed. An osmometer and a 48-hr acclimation were used for the other two experiments. With these reservations, some observations can be made. Although the curves are displaced, their shapes are similar, except at extreme dilutions. The displacement could be due in part to sexual differences, but may also represent temperature and seasonal effects. Bluff Creek dilutions were made in late summer when field temperatures were 30-0-32.0°C and experimental
680
BUENA S. BALLARD AND WALTER ABBOTT
temperatures were 23.0-24.0°C. Experimental temperatures were 21.0-21.5°C when Pascagoula River water and distilled water dilutions were used, but field temperatures were 28.0-30-0°C and 20.0-21.0°C, respectively. Dehnel (1962) noted that winter Hemigrapsus nudus (Dana) and H. oregonensis (Dana) had higher blood concentrations than summer crabs at the same experimental temperature and salinity, and Mantel (1967) obtained a blood sodium value of 513 m-equiv./1 for winter C. sapidus at the same salinity and temperature as summer crabs with blood sodium of 466 m-equiv./l. 60
(b)
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........
Poscagoula river water
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13
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~ Water,
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4'~
25
conc. with a r t { f i c i a l sea water conc. with N a C t
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o f m a l e a n d f e m a l e C. sapidus in l o w - a n d high-s~inity media.
FIG. 4. B l o o d o s m o c o n c e n t r a t i o n s
The concentrations of waters composed of mixtures of Bluff Creek water and 5"0~oo sea water were established by calculation, using a value of 0-06~oo as the salinity of Bluff Creek water at Vancleave, Mississippi (Price & Gunter, 1964). Other salinities were determined with the osmometer calibrated against sea water standards. Individual variation was too great to permit estimation of tolerance ranges and sexual differences at low salinities. The only crab to tolerate fresh water for more than 12 hr was a male which became moribund after 36 hr but survived for 63 hr. All males died immediately in distilled water, but four females survived for approximately 4 hr. Blood concentrations of the latter at the time of transfer from 1"0~oo water were equivalent to 20.8, 19.8, 21.2 and 22"7~oo sea water. Although males generally tended to have lower blood concentrations than females at low salinities, females had the lowest concentrations at extreme dilutions. Such low blood concentrations probably reflect osmoregulatory breakdown and intolerable
OSMOTIC ACCOMMODATION IN CALL1NECTES SAP1DUS RATHBUN
681
dilution of body fluids. However, the lowest recorded blood concentration at which a crab appeared active and normal was obtained with a female in a salinity of 0"2~oo. Blood concentration was equivalent to 13"8~oo. Tan & Van Engel (1966) reported no survival of females in fresh water beyond 36 hr, but 40 per cent survival of males beyond 48 hr. They did not report blood osmoconcentrations in fresh water. At sea-water concentrations above approximately 35~oo males tended to have higher blood concentrations than females (Fig. 4b). Both sexes were nearly isoosmotic to salinities above 30~ooat temperatures of 23-0-24.0°C, but at 28.0-30.0°C both could hyporegulate to some degree. This ability appeared to be better developed in females. At a salinity of approximately 40~oo, the blood of the males had reached iso-osmoticity and that of females was approaching it. The lag at 37~oo is unexplained but presumably could be related to the addition of NaC1 to the medium. Three males and one female tolerated extreme environmental salinities up to 70"8~oo at 23.0-24.0°C but no blood concentration determinations were made at this salinity. The length of time for which the crabs could withstand these conditions was not determined, but exceeded 7½ hr, the termination time for the experiment. All crabs were sluggish and probably were near their upper salinity limit. The female was somewhat more active than the male, but whether this behavior was attributable to a differential tolerance to the osmotic stress was not determined. The population of crabs represented in the present study is not usually subjected to salinities above normal sea water but is well represented in very low salinities. The highest salinity at which the writers have personally observed crabs in the field is 31.6~oo. Several adult females were observed swimming at the surface. As these crabs were moving from the sea, they may have encountered even higher salinities. However, surface salinities in this part of the Gulf of Mexico seldom exceed 35~oo even as far as 80 miles (130 km) offshore (Gulf Coast Research Laboratory, unpublished data). Only a few adult females were taken in low-salinity waters. The minimum salinity from which a female was recorded was 8"8~oo. This situation is to be expected since much of the life of a mature female is spent in spawning activity, which requires high-salinity water. Adult males and juveniles of both sexes were quite plentiful at 4"0~oo and were observed on one occasion at an extreme low of 0"7~oo. The latter value is very close to the 0-5%o upper limit set for fresh water by the Venice Symposium (1959), although Price & Gunter (1964) consider this level to be oligohaline.
Temperature and seasonal effect Results of this study indicate an inverse relationship between blood concentration and temperature as found by Rees (1966). When gradual transfer experiments were conducted using crabs from the same field temperatures of 28-030-0°C but different experimental temperatures, blood concentrations were higher at lower temperatures (Fig. 5). Results differed from those of Rees in that the
682
Btrv~A S. BALLARDANDW.~a'ma ABBOTT
inverse relationship persisted even in full-strength sea water. High experimental temperatures caused shifting of the iso-osmotic point and increasing hyporegulation. On the basis of the previously described experiment (Fig. 4b), the points at which crabs would be approximately iso-osmotic at both temperature ranges are between 40 and 45~oo, a considerably higher concentration than that at which Rees observed
36
Temp.25.0"-24.0"C ......
/;
Temp. 2 8 . 0 * - 3 0 . 0 * C
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;
,;
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lack of temperature effect. Gifford (1962) reported that in high salinities in the Laguna Madre of Texas, high temperatures resulted in increased hyporegulation for some C. sapidus but had no effect on blood concentrations of others. Dehnel (1962) noted that for H. nudus and H. oregonensis temperature effect decreased with increasing salinity, with a minimum between 100 and 125% sea water. Temperature effect could account, in part, for conflicting results obtained by various workers. Mantel (1967) observed hypertonicity for sodium at full sea water, but Odum (1953) reported hypotonicity. Mantel's experimental temperature range was 19.0-21.0°C. While Odum did not report temperature, he indicated that his experiments were conducted in August when atmospheric temperatures were high. His animals were approximately iso-osmotic at a salinity of 30~o, but Tan (1962) observed considerable hyperosmoticity at that salinity with 20.0°C experimental temperature. In both experiments represented in Fig. 5, blood concentration declined at a faster rate than in previous experiments using crabs from lower field temperatures (21.0-21.5°C). The curves are steeper at both experimental temperature ranges, although 23-0-24.0°C is fairly close to the range used previously. Odum (1953) showed a similar curve for his experiments which presumably utilized crabs from high field temperatures. Such a fall in blood concentration should favor greater
OSMOTIC ACCOMMODATION IN CALL1NECTES SAPIDUS RATHBUN
683
tolerance of low salinities in summer, the season of maximum invasion of very low salinities by C. sapid~. Although an attempt was made to equate results obtained by different techniques as much as possible, such differences could affect results and must be noted. For the experiments represented in Fig. 5, melting point comparison technique was employed to determine blood concentration and 24-hr acclimation was allowed. For other experiments at comparable salinities, osmometer determinations and 48-hr acclimations were used. In the 24-hr acclimation experiments, the direction of transfer was not recorded and varied from salinity to salinity. The hysteresis-like effect, previously noted, could be the cause of the differences in shapes of these curves when compared to curves from other experiments. Seasonal effect such as observed by Dehnel (1962) for Hemi~,'ap~us was noted for C. sapid~. Crabs collected from colder waters showed higher blood concentrations than those collected from warmer waters, even when kept at equivalent temperature in the laboratory (Fig. 2). As already mentioned, Mantel (1967) also obtained higher blood sodium values for winter C. sapidus than for summer crabs at the same experimental temperature and salinity. The present study verifies this result, but does not explain it.
Blood analysis Aliquots of blood samples were analyzed for water, total organic content, inorganic content, amino acids as leucine and reducing substances as glucose (Table 3). Results were erratic for water and organic substances except at the osmotic extreme of 1"0%o, where water :increased and total solids decreased, possibly because of breakdown in osmoregulation. Horn & Kerr (1963) observed that individual variations in hemolymph protein content of C. sapidus showed a tenfold range and were not related to salinity. Their observed range of 15.2-119-2 mg/100 ml for non-sponge females is not unreasonable in view of the 38-4--140 mg/g range for total organic substances found in the present study. Concentration of inorganic substances (total metallic cations as oxides) paralleled total blood concentration. Presumably the major inorganic component was sodium, since this ion forms 85.5 per cent of the total cations of the blood of C. sapidus (Florkin, 1960). Mantel's (1967) sodium curve, shown in Fig. 1, indicates a rather sudden drop in concentration at approximately 15~oo sea water, whereas the inorganic concentration in the present study drops at 10%o. However, the total osmoconeentration of the blood also drops in this experiment at a salinity of 10~oo, whereas it dropped at approximately 15~oo in most of the experiments. Ninhydrin-reactive substances in the blood varied erratically, although they showed some tendency to be more concentrated at higher salinities. Jeffries (1966) obtained a range in non-protein nitrogen concentration of 8.6--102.9 mg/100 ml in C. sapidus blood and concluded that composition was independent of salinity. Variations in reducing substances tended to reflect variations in total blood concentration. Presumably, such substances are not involved in osmoregulation,
684
BUENA S. BALLASDAND W A L ~ ABBOTT
8~ 0 e~ 0
~NN~ 0
0
0 0 ° ~ .~
,...1
~'~ s~ ©
N~N
NNN
~
OSMOTIC ACCOMMODATIONIN CALL1NECTES SAPIDUS RATHBUN
685
a view supported by the data of Jeffries (1966). In the present study the drop in concentration of reducing substances may have been related to starvation effect rather than to changes in blood concentration. Blood samples were taken at 48-hr intervals in each successive salinity, beginning with 20"7~oo. The crabs were not fed during the course of the experiment and those bled at a salinity of 1.0~oo had fasted considerably longer than those at 20"7~oo. Although starvation does not affect total blood osmoconcentration, according to Florkin (1960) a decrease of concentration of fermentable reducing substances takes place in some species of crabs but not in others. Callinectes sapidus may be in the former group. Morgulis (1922, fide Florkin, 1960) obtained blood glucose values of 175-182 mg/100 ml for freshly caught C. sapidus and values of 9-6-19-0 and 12.5-13.8 mg/100 ml after 1 and 2 days, respectively, of fasting. CONCLUSION
C. sapid~ is a euryhaline species demonstrating good hyperregulatory ability and limited hyporegulatory ability. Sexual differences in osmoregulatory responses may account, in part, for differential salinity preferences of the sexes. Because of the steeper gradient between medium and blood, low salinities demand more osmotic work by females than by males. At very high salinities the gradient between blood and medium is slightly higher for males. However, the sexassociated differences in blood-medium gradients do not appear sufficient to account for the observed differences in sexual ratios. The differential distribution of the sexes may be behavioral rather than physiological. Temperature effect on distribution is not clear. High temperatures may favor tolerance of low salinities by lowering blood concentration and reducing osmotic work. High temperatures may also favor tolerance of hypersalinity by increasing hyporegulatory ability. Such a relationship would cause crabs to move out of both low salinity and hypersaline areas in winter, and such migration does, in fact, occur (Jeffries, 1966; Gifford, 1962). REFERENCES
ABBOTT W. (1967) Salinity determination by freezing-point osmometry. Simposio Internacional sobre Lagunas Costeras, Mexico, D.F., Mexico, 28-30 November, 1967. AWAPARAJ. (1948) Application of paper chromatography to the estimation of free amino acids in tissues. Archs Biochem. 19, 172-173. BALLARD B. S. (1967) Osmotic accommodation in Callinectes sapidus Rathbun. Ph.D. Thesis, Mississippi State University, State College, Mississippi. BARNES H. (1954) Some tables for the ionic composition of sea water. 07. exp. Biol. 31, 582-
588. BROEKEMAM. M. M. (1941) Seasonal movements and the osmotic behavior of the shrimp Crangon crangon L. Archs neerl, zool. 6, 1-100. BRYAN G. W. (1960) Sodium regulation in the crayfish Astacusfluviatilis--II. Experiments with sodium depleted animals. 07. exp. Biol. 37, 100-112. CHURCHILLE. P. (1919) Life history of the blue crab. Bull. U.S. Bur. Fish. 36, 95-128. COSTLOWJ. D., JR. & BOOKHOUTC. G. (1959) The larval development of Callinectes sapidus Rathbun reared in the laboratory. Biol. Bull., Woods Hole 116, 373-396. 23
686
BUENA S. BALLARD AND WALTER ABBOTT
DARNELLR. M. (1959) Studies of the life history of the blue crab, Callinectes sapidus Rathbun, in Louisiana waters. Trans. Am. Fish. Soc. 88, 294--304. DEHNELP. A. (1962) Aspects of osmoregulation in two species of intertidal crabs. Biol. Bull., Woods Hole 122, 208-227. FLORKINM. (1960) Blood chemistry. In The Physiology of Crustacea (Edited by WATERMAN T. H.), Vol. I, 670 pp. Academic Press, New York. GIFFOROC. A. (1962) Some aspects of osmotic and ionic regulation in the blue crab, Callinectes sapidus, and the ghost crab, Ocypode albicans. Publ. Inst. Marine Sci. (Univ. Texas) 8, 97-125. GRoss W. J. (1954) Osmotic responses in the sipunculid Dendrostomum zostericolum, ft. exp. Biol. 31,402--423. GROSSW. J. (1957) An analysis of response to osmotic stress in selected decapod Crustacea. Biol. Bull., Woods Hole 112, 43-62. GImTER G. (1938) The common blue crab in fresh water (Callinectes sapidus). Science 87, 87-88. GUNTERG. (1945) Studies on marine fishes of Texas. Publ. Inst. Marine Sci. (Univ. Texas) 1, 1-190. GUNTER G. (1967) Some relationships of estuaries to the fisheries of the Gulf of Mexico. In Estuaries (Edited by LAUn~G. H.), 757 pp. Am. Inst. Biol. Sci. Pub. 83, Washington, D.C. HAY W. P. (1904) The life history of the blue crab (Callinectes sapidus). Rep. U.S. Comm. Fish. 347-413. Hom~ E. C. & KERR M. S. (1963) Hemolymph protein and copper concentration of adult blue crabs (Callinectes sapidus Rathbun). Biol. Bull., Woods Hole 125, 499-507. HUKUDA K. (1932) Change of weight of marine animals in diluted media, ft. exp. Biol. 9, 61-68. Jm~FmEs H. P. (1966) Internal conditions of a diminishing blue crab population (Callinectes sapidus). Chesapeake S t . 7, 164--170. K I m ~ O. (1952) Zur Biologic und Physiologie yon Gammarus duebeni LilIj--V. Untersuchungen fiber Blutkonzentration, Herzfrequenz und Atrnung. Kid. Meeresforsch. 9, 134-150.
KINNE O. & ROTTrlAUWEH. W. (1952) Biologisehe Beobachtung und Untersuchungen fiber die Blutkonzentration an Heteropenope tridentatus Maitland (Decapoda). K i d Meeresforsch. 8, 212-217. KROCH A. (1939) Osmotic Regulation in Aquatic Animals, 242 pp. Cambridge University Press, London. LANDUA A. J. & AWAPARAJ. (1949) Use of modified ninhydrin reagent in quantitative determination of amino acids by paper chromatography. Science 109, 385. MANTEL L. H. (1967) Asymmetry potentials, metabolism and sodium fluxes in gills of the blue crab, Callinectes sapidus. Comp. Biochem. Physiol. 20, 743-753. MOROULISS. (1922) As cited by Florkin (1960). MULKANA M. S. (1968) Seasonal changes in the nutritional components of the standing plankton biomass in Mississippi Sound. Ph.D. Thesis, Mississippi State University, State College, Mississippi. ODUM H. T. (1953) Factors controlling marine invasion into Florida freshwaters. Bull. mar. Sci. Gulf Carib. 3, 134-156. PANNIKAR N. K. (1940) Osmotic properties of the common prawn. Nature, Lond. 145, 108. PEA~E A. S. (1950) The Emigrations of Animals from the Sea, 210 pp. Sherwood Press, Dryden, N.Y. PEARSONJ. C. (1948) Fluctuations in the abundance of the blue crab in Chesapeake Bay. Res. Rep. U.S. Fish. Serv. No. 14, 26 pp.
OSMOTIC ACCOMMODATION I N CALLINECTES SAP1DUS RATHBUN
687
PRICE J. B. & GUN~R G. (1964) Studies on the chemistry of fresh and low salinity waters in Mississippi and the boundary between fresh and brackish water. Int. Rev. ges. Hydrobiol. 49, 629-636. REIn G. H. (1966) Blue crab studies. Informal Progress Report. U.S. Bur. Comm. Fish. Beaufort, N.C. SANDOZM. & ROBERTSR. (1944) The effect of environmental factors on hatching, moulting, and survival of zoea larvae of the blue crab, Callinectes sapidus Rathbun. Ecology, 52 216-228. SCHROEDER E. H. (1966) Average surface temperatures of the Western North Atlantic. Bull. mar. Sci. 16, 302-323. SCmVABE E. (1933) Ober die Osmoregulation verschiedener Krebse (Malacoetracen). Z. vergl. Physiol. 19, 183-236. SHAW J. (1960) The mechanism of osmoregulation. In Comparative Biochemistry (Edited by FLORKINM. & MASONH. S.), Vol. II, 685 pp. Academic Press, New York. SIMMONS E. G. (1957) An ecological survey of the upper Languna Madre of Texas. Publ. Inst. mar. Sci. (Univ. Texas) 4, 156-200. TAN ENc-CHow (1962) Studies on osmotic and ionic regulation in the blue crab, Callinectes sapidus Rathbun. M.A. Thesis, College of William and Mary, Williamsburg, Virginia, 66 pp. TAN ENo-CHow & VAN ENO~ W. A. (1966) Osmoregulation in the adult blue crab, CaUinectes sapidus Rathbun. Chesapeake So/. 7, 30-35. U.S. Depart~,ent of the Army (1951) Methods for Medical Laboratory Technicians, Technical Manual TM-227, 746 pp. Venice Symposium on the classification of brackish waters. Final resolution (1959) Arch. Oceanogr. Limnol. Suppl. 11, 243-245.
Key Word Index--Osmoregulation; crab; Callinectes sapidus; temperature on osmoregulation; salt regulation; amino acids in osmoregulation.