Variations in serum constituents of the blue crab, Callinectes sapidus: Free amino acids and total ninhydrin positive substances

Comp. Biochem. PhysioL, 1973, Vol. 4513, pp. 407 to 418. Pergamon Press. Printed in Great Britain

VARIATIONS IN SERUM C O N S T I T U E N T S OF T H E BLUE CRAB, C A L L I N E C T E S S A P I D U S : FREE A M I N O ACIDS AND T O T A L N I N H Y D R I N POSITIVE SUBSTANCES* M. P. LYNCH and K. L. WEBB Department of Environmental Physiology, Virginia Institute of Marine Science, Gloucester Point, Virginia 23062, U.S.A.

(Received 31 October 1972) Abstract--1. Five amino acids, glycine, taurine, alanine, proline and arginine, constitute 72-90 per cent of the total free amino acids and 39-70 per cent of the total ninhydrin positive substances (TNPS) in the serum of mature blue crabs. 2. Serum TNPS is positively correlated with salinity for female crabs taken along a salinity gradient. No significant correlation was found between serum TNPS and salinity in male crabs. 3. The positive correlation in female crabs is attributed to increased synthesis of intracellular amino acids during the migration to higher salinity waters for spawning. The increased production of amino acids is considered an acclimation phenomenon rather than a permanent response to higher salinity. 4. A positive correlation exists between serum TNPS and glucose and serum TNPS and serum chloride in female crabs. 5. Serum TNPS was highest during months with the lowest temperatures. INTRODUCTION Pm~vlovs reports from this laboratory have discussed variations in serum chloride and osmotic concentration (Lynch et al., 1973), serum total protein (Lynch & Webb, 1973a) and serum glucose (Lynch & Webb, 1973b) in mature blue crabs, Callinectes sapidus Rathbun. This report discusses serum free amino acids and total ninhydrin positive substances (TNPS) in the same organisms. Serum non-protein nitrogen in decapod crustaceans has been reported to vary from 8 to 37.5 mg/100 ml, with amino-nitrogen ranging from 2.2 to 12.0 mg/100 ml (Florkin, 1960). Serum free amino acids have been determined in several species; total serum amino acids range from 26 to 80 mg/100 ml with glycine, alanine, glutamic acid, proline, serine, ornithine and taurine making up the greater portion of the free amino acid pool with different amino acids being the most concentrated in different crustaceans (Camien et al., 1951 ; Duch~teau-Bosson & Florkin, 1961 ; Stevens et al., 1961 ; Stewart et al., 1966; Vincent-Marique & Gilles, 1970). Little information is available on the variability of blood or serum non-protein nitrogen, free amino acids or TNPS in a single species. Delaunay (1931) reported * Contribution No. 500 from the Virginia Institute of Marine Science. 407

408

M . P . LYNCH AND K. L. WEBB

blood amino nitrogen ranged f r o m 1.6 to 8.4 rag/100 ml in Ma]a squinado. Stewart et al. (1955) reported a twofold variation of serum amino nitrogen in Homarus americanus sampled at different times during the year (35.8 rag/100 ml in M a y ; 74.7 mg/100 ml in January). Glycine showed the greatest change (0.45/,moles/ml in M a y ; 2.40/,moles/ml in January). Vincent-Marique & Gilles (1970) found up to a 600 per cent increase in serum proline in Eriocheir sinens~ 4 days after transfer f r o m sea water to fresh water. After 8 days in fresh water, blood free amino acids (including proline) were below the levels found in sea-water acclimated animals. I n the blue crab, blood non-protein nitrogen was reported to vary f r o m 24.7 rag/100 ml in freshly caught animals to 9.0 rag/100 ml in animals held 2 days in the laboratory, indicating the serum non-protein nitrogen is dependent u p o n the nutritional state of the animal (Morgulis, 1922). Jeffries (1966) reported plasma non-protein nitrogen ranged f r o m 8-6 to 102.9 mg/100 ml in male and 12-842-9 mg/100 ml in female blue crabs. H e reported an inverse correlation between s e r u m non-protein nitrogen and s e r u m chloride. MATERIALS AND M E T H O D S Collection of crabs, environmental data, morphometric and life stage data and serum are described in Lynch et al. (1973). All crabs with the exception of a few used to determine the relation between TNPS and free amino acids were mature, intermolt individuals. Those crabs used in seasonal studies were supplied by commercial crabbers and were out of water several hours before bleeding. The crabs used in salinity gradient studies were bled immediately upon removal from the water. Short-term serum TNPS variation was determined in the same crabs used to determine short-term glucose variation (Lynch & Webb, 1973b). Serum T N P S Fresh serum or serum which had been stored, frozen and thawed once was deproteinized by adding four parts of absolute ethanol (100%) to one part of serum (the usual volumes used were 0"2 ml serum and 0"8 ml ethanol) and centrifuging at 2000g for 10 rain. An aliquot of the deproteinized serum (usually 0-5 ml) was mixed with 1.0 ml of ninhydrin reagent (Moore & Stein, 1954), brought to 2"0 ml of solution with distilled water, heated in a boiling water-bath for 20 rain in a capped test-tube, cooled to room temperature, diluted with 5"0 ml 50% ethanol and thoroughly mixed. A standard (0"5 ml, 1"0 millimolar leucine) and blank (2"0 ml distilled water) were treated the same way. Absorbance was determined at 570 nm. In practice the ninhydrin reagent used was drawn from the reservoir used with a Technicon (Ardsley, N.Y.) amino acid analyzer. T N P S was calculated as/.moles leucine equivalents/ml original serum.

Serum free amino acids A qualitative and quantitative analysis of the free amino acids in serum was made with a semi-automatic ion exchange analyzer (Technicon Auto-Analyzer). A volume of serum (0-2-1"0 ml) was mixed with 4 vol. of 100% ethanol and centrifuged as above. The entire supematant was decanted and treated as described by DuPaul & Webb (1970). RESULTS Serum T N P S D u r i n g the seasonal study, mean s e r u m T N P S ranged f r o m 0.8 to 16.1 t~moles/ ml in male crabs and 1.0-30.1 #molcs/ml in female crabs (Table 1). T N P S were

409

V A R I A T I O N S I N S E R U M C O N S T I T U E N T S O F T H E B L U E CRAB

066666~

~

666h~6

6~6~

v

v

v

•

•

~o ~D

®v

~..~ v

v

v

~,... v

®~

v

( ~

.

,

°

°

.

.

v

~

.

.

.

.

.

v

.

°

v

.

v

.

v

,

v

.

O xx~

~;g:g

0)

l

L

~'

~. ~

l.w

~

410

M . P. LYNCH AND K . L . W ~ B

found in the serum of all the crabs sampled. Variability was high within each sample (the mean C was 52 per cent). Month to month variation was also high (Fig. 1). The highest serum T N P S values appeared in January 1970, September 1970, February 1971 and April 1971. Mean serum T N P S was higher in males in

3O

3O

L

'-- ~ - ~ ' ~ ~.

Salinity

. P -- - - ~ . . .

Temperature

//

20 10 o o

1

zo

IO

~

28

-:.

- -

~ 26

~ 24

::k 22 20 _--

•. /,~ I I,t I P,|

.

g

o. c

c

~

/1',1

--

- 1968 ----Ma,e ........ 1969 • 1967 .... 1'70

....

Female Female Female Fem°le

[ . ~t1~

'!"

/11 8

"

-_

...... --.i

\":i .... \\

4

2 A

$

O N O~d

F

r

I M A M d

d

A

S O

I

r d I I I I [ I N D F M A M d

I

FIG. 1. Seasonal variation in mean serum total ninhydrin positive substances in mature blue crabs, C. sapidus, from the York Spit area of Chesapeake Bay, Virginia. 2 months and in females in 5 months. In the other 13 months in which comparisons were possible, no differences were found. Overall, there was probably no difference between serum T N P S levels in male and female blue crabs. Differences in serum T N P S between year classes of female crabs were found in 2 months (August 1970 and July 1971). In these months old year class females had significantly higher T N P S than new year class females. In the other 9 months in which 2 year classes of female crabs were sampled, no differences were found. Overall there was probably no difference between serum T N P S levels in older and younger year class crabs taken at the same time. The higher level of T N P S in the older year class in August 1970 was due primarily to higher serum T N P S in two egg bearing (sponge) crabs. In June and July 1970, the sponge crabs and non-egg bearing (clean) crabs had no significant differences in serum TNPS. In August, however, the serum T N P S levels were significantly higher in the sponge females (Table 2). In the salinity gradient study, serum T N P S was found to range from about 1.0 to 19.8 #moles/ml in males and 1.9-32.1/zmoles/ml in females. Serum T N P S was detected in all crabs sampled. Mean serum T N P S ranged from 4.3 to

VARIATIONSIN S~UM CONSTITUENTSOF TH~ BLUECSAB TABLE 2~CoMPARISONOF SERUMTNPS n~ sc~ B ~ I N o (SPONGE) ~ (CLEAN) 1968 ~

411

NON-~3G BEARING

CLASS C. sap/dus

Mean and S.E. Sample date (1970)

State (N)

Serum TNPS (pmoles/ml)

June

Sponge (12) Clean (5)

8.7 :[: 1"1 8"2 __2"0

July

Sponge (8) Clean (13)

14.9 + 2"8 14.6 + 2-0

August

Sponge (2) Clean (4)

13"9 + 0.8 5.9 + 1"0"

* Significant difference (P<0'05).

16.3 pmoles/ml in males and 4.1-21.4 pmoles/ml in females (Table 3). Variability was essentially the same for both sexes (mean C = 23.4 per cent for females, 27.3 per cent for males). Serum T N P S levels were significantly higher in male compared to female crabs in three of eighteen possible comparisons. No differences were found in the other fifteen comparisons. Serum T N P S in female crabs was positively correlated with salinity (r = 0.56, N = 152). The correlation between serum T N P S and salinity in male crabs was not significant (r -- 0.18, N = 131). TABLE 3 - - M e ~

SFJ~UM T N P S CONCENTRATION IN MATUI~, HARD BLUE CRABS, C. $~J~df/$, COLLECTED FROM VARIOUS SALINITIES IN VIRGINIA

Serum TNPS _+S.E. (pmoles) Area

Salinity (%o)

Temperature (°C)

1969 19 August 19 August 19 August 19 August 19 August 19 August 21 August 21 August

RR RR RR RR RR RR ES ES

19.00 17.50 14.00 11.50 6.40 1.30 27.50 18.90

27.5 27.6 28.0 28.2 28.0 28.0 22.0 22.0

5.97 9.72 (1) 8.10 6.85 7.49 6-36 5.01 5.24

1970 14 July 14 July 14 July 14 July

RR RR RR RR

19.11 16.50 13.82 10.08

24.0 24-6 24.9 25-6

2.55 (1) 2.85 (1) 8-00 (1) 5.70 0.24 (5)

Sample date

Males (N)

Females (N)

0.76 (5)

4.51

0-43 (12)

0.47 0.42 0.33 0.92 0.35 0.21

7.03 5.06 6.03

0-46 (11) 0.40 (7)* 0.50 (12)*

5.11 4.09

0-55 (4) 0-22 (4)

(12) (10)* (12)* (2) (12) (69)

[Continued overleay

412

M. P. LYNCH AND K. L. W,V.BB TABLE 3 (cont.) Serum T N P S _+S.E. (#moles)

Sample date

Area

Salinity (~oo)

1970 14 July 16 July 16 July 16 July 16 July 3 August 3 August 3 August 3 August 7 August 7 August 7 August 11 August 11 August 11 August 11 August 11 August 11 August 13 August 13 August 13 August 13 August 13 August 14 September 14 September 14 September 17 September 17 September 17 September 17 September 17 September 17 September 17 September 8 October 8 October 12 October 12 October

RR YR MB MB MB JR JR JR JR CB YR CB RR RR RR RR RR RR YR YR YR YR PR CB CB YR YR YR YR YR PR PR PR PR PR CB YR

4.75 24.00 21.69 19.07 18.71 21.25 10.71 4.62 2.84 30"38 27"01 26.82 15-34 15-02 14,66 13-90 11.49 9,68 19.28 18.49 16.00 13'90 7"13 30.65 27.18 24"06 21.93 20.42 18.21 17.00 12.94 8"97 0"89 12.41 4.26 29.74 23.50

Temperature (°C) 26.4 26.0 25.6 26.8 25-8 27.2 28.4 28.8 29.0 22.2 24.1 24.0 25.7 25.8 26"4 25.6 25"8 25"7 26,6 26.5 25"8 25"8 26"3 22.5 23.9 24.1 25.4 25.7 26.1 26"0 26-0 26.3 26.8 21.6 21.4 21.1 21-6

Males (N) 5.30 (1) 13.57 (1) 1.10 (1) 9.38 6.52 7-00 11.61 (1) 6.96 (1) 10-88

1.44 (9) 1-72 (2) 0-67 (2)

16"33

2"33 (3)*

9.04 7.85 7.74 5.44 9.02 6"55 9~20 (1) 10"96

1.12 0.67 1.45 0.55 1.28 1.89

9.18 (1)

(4) (8)* (5) (4) (7) (2)

2"20 (3)*

0.66 (2) 0.80 (2)

7.13 (1) 7.49 0.65 (4) 2.73 (1) 4.31 1"52 (4) 5-52

8.58 (1)

0-18 (2)

9"36 1.38 (2) 5-84 (1)

5"92 7-80

Females (N)

0.96 (7)

21.43 1.40 5.64 0.74 11"34 2.06 6.31 (1) 4.56 0"17 7"18 4'78 9"74 (1) 6.00 9"09 7-91

(3) (2)* (3) (2)*

0"58 (2) 0"81 (4) 0.43 (5)* 0.67 (13) 0.70 (9)

11"84 0.75 (15) 11-58 (1) 7.58 0"65 (8) 6-39 1.25 (5) 5.60 0.87 (4) 8.60 (1) 6.13 0.74 (8) 8.30 (1)

2.96 0"45 (2) 6"95 0"86 (8) 5.64 (1)

Area code--RR, Rappahannock River; YR, York River; MB, Mobjack Bay; JR, James River; CB, Chesapeake Bay; PR, Pamunkey River; ES, Eastern Shore (sea side). * Significant difference. I n the s h o r t - t e r m variability study, serum T N P S in male crabs ranged f r o m 0"9 to 14.8/zmoles/ml in males and 4-6-6.2/~moles/ml in females. M e a n T N P S varied f r o m 4.2 to 8.5 tzmoles/ml during this period. D a y to day variation was high.

413

VARIATIONS I N SERUM CONSTITUENTS OF THE BLUE CRAB

In one 3-day period (15-17 July 1969) serum T N P S varied from 4"9 to 8"5 #moles/ ml (Fig. 2). Mean C = 37"4 per cent for these samples. Serum T N P S and serum chloride (Lynch et al., 1973) are positively correlated in female crabs of the salinity gradient studies (r = 0.44, N = 152). Serum TNPS and serum glucose (Lynch & Webb, 1973) are positively correlated in females of both the salinity gradient (r = 0.60, N = 100) and seasonal studies (r = 0.16, N - - 429). All of these correlation coefficients were significant at the 99 per cent confidence level ( P < 0.01).

5

I0

$

9

g

s

Mole

~-Mean } +-S.E.

Femole

×

18

,IS E'6 6 ~e ~:t 5 -6

4

3

I

I

I0 July

I

20

I

31

I

I0

I

20 August

I 31

Fxo. 2. Meat; serum total ninhydrin positive substances in mature blue crabs, C. sapidus, from the North River, Mathews County, Virginia. No significant correlation (P>0.05) was found between serum TNPS and serum total protein (Lynch & Webb, 1973a) in either females or males or between serum TNPS and serum chloride or glucose in males in either salinity gradient or seasonal samples. Neither was a significant correlation found between serum T N P S and serum chloride in females of the seasonal samples. Serum free amino acids were determined in seven adult hard crabs. Glycine was the most abundant amino acid, followed by taurine, alanine, arginine and proline (Table 4). These five amino acids accounted for 72-90 per cent of the total free amino acids measured and 39-70 per cent of the total ninhydrin positive substances. A total of fourteen serum free amino acid determinations (including serum free immature crabs and crabs from other stages of the molt cycle) were made (Fig. 3). The linear correlation between serum T N P S and the sum of the serum free amino acids was very high (r = 0.96). DISCUSSION Both Morgulis (1922) and Jeffries (1966) have reported values for serum nonprotein nitrogen in the blue crab. An approximate conversion of non-protein 14

414

M . P . LYNCH AND K. L. WEBB

T A B L E ~----CoNCENTRATION OF INDIVIDUAL FREE AMINO ACIDS (FAA) IN THE SERUM OF THE BLUE CRAB, C. sapidus, FROM V I R G I N I A

M

F

F

F

F

F

F

Salinity (~o) Temperature (°C)

Sex

16-5 24-6

19"5 24.0

19"5 24.0

21"0 23"5

21"0 23"5

21"0 23.5

21"0 23"5

Amino acids Cysteic acid Taurine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Tryptophan Histidine Arginine Sum FAA Total NPS

-0"37 0"03 0-09 0-08 0.05 0"16 0"43 0"28 0"05 Tr 0"02 Tr 0"04 Tr 0"03 0"05 0-04 -0-04 0.12 1"88 2"85

0-01 0"24 0"01 0-05 0"03 0.03 0"13 0"32 0"15 0"01 -Tr 0"01 0"01 0"01 0-01 0"02 0"01 -0"01 0"19 1-25 2"62

0"01 0"29 0-02 0-04 0-04 0-05 0"06 0-51 0"24 0-02 --0'01 0"01 0-01 0"02 0"02 0"01 -0"02 0.16 1-54 1"89

0zmoles) Tr 0-52 0-02 0-23 0-15 0-05 0"75 1"04 0"69 0"06 0-01 0"03 0"03 0"05 0"01 0"05 0"01 0"11 0"02 0-04 0.35 4"22 7"27

0"01 0"78 0-02 0"07 0"09 0"06 0"29 1"11 0"58 0"07 -0"04 0-03 0"05 0"04 0"04 0"02 0"07 -0"15 0.94 4"46 8"54

0"01 1-32 0-11 0"13 0"18 0.07 0"62 5.20 0-90 0"10 Tr 0"02 0"04 0"07 0"04 0"07 0-02 0-08 -0"11 1-96 11"05 14"18

0"03 0"39 0"01 0.05 0.04 0.02 0"18 0"47 0"27 0"03 -0"02 0"02 0"03 0"01 0"04 0"01 0-02 0.02 0"02 0.18 1"86 3'55

Tr, trace,

nitrogen to T N P S was made by using a 30 per cent amino nitrogen fraction in non-protein nitrogen (Florkin, 1960) and assuming amino nitrogen accounts for 70 per cent of the T N P S (Table 4). T h e converted values of Morgulis (1922) ranged from 7.4/zmoles/ml in a freshly caught crab to 2.7/zmoles/ml in a crab held for 2 days in an aquarium. Jefferies' (1966) values converted to a range of 2.630.8/~moles/ml for males and 3.8-12.9/zmoles/ml for females. These values are within the ranges found in this study. Jeffries (1966) reported that non-protein nitrogen in blue crab serum is inversely correlated with serum chloride. Unfortunately he combined data from both male and female crabs in various stages of the molt cycle to determine the correlation. I n the present study distinct differences are found between the relationship of serum T N P S and serum chloride in male and female crabs. N o significant correlation between serum T N P S and serum chloride is found in male crabs. I n female crabs, however, a significant positive correlation is found in data

VARIATIONS I N SERUM CONSTITUENTS OF THE BLUE CRAB

ZO u

-

/7.'"

r8

14

=~

I0

"6

4 2

415

¢t /e

I

2

I

4

I

6

I

8

r

I0

I

12

Total free amino acids,

J

14

I

16

~moles/mt.

FIG. 3. Relationship between the free amino acids and total ninhydrin positive substances (NPS) in serum of the blue crab, C. sapidus. taken during the salinity gradient studies. This correlation is not found in data from the seasonal studies. Serum T N P S and salinity were not correlated for either male or female crabs in the seasonal studies, but in the salinity gradient study significant positive correlation is found between serum T N P S and salinity in crabs of both sex. Analysis of covariance indicated the correlation was significantly higher in females. Because of the lack of correlation between serum T N P S and salinity during the seasonal studies and the lack of correlation between serum T N P S and serum chloride in males in either phase of the present study, the relationship between salinity and serum T N P S in females found in the salinity gradient study is probably due to some factor other than different osmotic conditions. Free amino acids are known to be intracellular osmotic effectors in euryhaline marine invertebrates (Lange, 1968; Schoffeniels & GlUes, 1970). Gilles & Schoffeniels (1964, 1968, 1969) have shown that the increase in tissue free amino acids occurs primarily by de no'oo synthesis within the cells. Several investigators have shown, however, that intracellular amino acids can be passed (or are leaked) to the extracellular spaces (Gilles & Schoffeniels, 1969; Vincent-Marique & Gilles, 1970), particularly during volume regulation (Fugelli, 1967). When isolated blue crab muscle fiber are exposed to a hypertonic medium, there is an immediate decrease in fiber volume (Lang & Gainer, 1969). This volume reduction is due to an efflux of water from the cell to the surrounding fluid (Lange & Mostad, 1967). Volume readjustment is accomplished by increasing the intracellular concentration of organic solutes, primarily free amino acids. Gilles & Schoffeniels (1969) have demonstrated two mechanisms to accomplish this increase in intracellular free amino acids, modifications of the permeability of membranes and modifications of pathways responsible for biosynthesis and degradation of these compounds.

416

M.P.

LYNCH AND K. L. WEBB

Female blue crabs begin migrating towards higher salinity waters upon reaching maturity, whereas male crabs do not have such a migratory pattern (Van Engel, 1958). Female crabs captured along a salinity gradient during the periods of migration, therefore, are most likely in a state of active osmotic readjustment that involves increased amino acid biosynthesis. Upon reaching higher salinity waters, an equilibrium is reached, and the serum TNPS concentrations no longer reflect the higher rate of metabolic activity required during passage along the salinity gradient. The secondary peak of TNPS found in the fall of 1970 (Fig. 1) occurs at about the same time of year as the peak of the migration of mature females to higher salinity waters. This peak might be a reflection of active osmotic adjustment. The correlation between TNPS and salinity in females is not thought to be related to ovogenesis or ovary development as is the correlation between total serum protein and salinity (Lynch & Webb, 1973a) since no significant correlation was found between serum protein and serum TNPS, and essentially no differences were found between serum TNPS levels in different year classes. The cause for the differences in the relationship between serum TNPS and serum glucose in males and females is not readily apparent. Since there is no significant correlation between serum glucose and salinity (Lynch & Webb, 1973b), the significant correlation between serum T N P S and serum glucose in females taken along the salinity gradient cannot be explained by the migratory behavior of the mature females, particularly as a significant correlation between these constituents is also found in the seasonal samples. The seasonal variation in serum TNPS (Fig. 1) is marked by a high value in each winter (1970 and 1971) of the study. These high values coincide relatively closely with the highest serum chloride values found during the year. A distinct difference is seen, however, in that serum chloride varies continuously during the year, closely related to the environmental temperature (Lynch et al., 1973). Serum TNPS, however, increase sharply during one month and decrease just as sharply the following month. Duch~teau & Florkin (1955) have demonstrated lower concentrations of tissue free amino acid in E. sinews at 1-3°C than at 10-11°C. One possible explanation for the sharp increase in TNPS in blue crab serum is that at some low temperature, approximately 5°C, the permeability of cell membranes to free amino acids changes drastically allowing intracdlular free amino acids to leak out to the surrounding medium. As is the case with the sudden extrusion of intracellular free amino acids to the serum of E. sinensis transferred from sea water to fresh water (VincentMarique & Gilles, 1970), the higher amino acid levels are detectable in the serum for several days after the imposed stress. Free amino acid distribution in blue crab serum is generally similar to the free amino acid distribution reported in other crustaceans (Camien et al., 1951; Duch~teau-Bosson & Florkin, 1951). The major difference appears to be that serine, which is among the five most concentrated amino acids in Cancer irroratus, E. sinensis and H. americanus (Stevens et al., 1961; Stewart et al., 1966; VincentMarique et al., 1970), is not a major portion of the free amino acids pool in C. sapidus.

VARIATIONS IN ~.RUM CONSTITUENT.q OF THE BLUECRAB

417

Acknowledgements--We wish to acknowledge the several persons who assisted in the serum analysis, particularly Mrs. Rebecca Goldstein, Miss Carolyn Watson and Miss Rosemary Green. Thanks must also go to Mr. W. A. Van Engel and Dr. P. A. Haefner, Jr., who assisted in the processing of the monthly samples and to Dr. C. P. Mangum for her very helpful review of the manuscript. This study was supported b y funds provided by the Commonwealth of Virginia and by the Virginia Institute of Marine Science Sea Grant Program (P.L. 89-688) in 1969 b y Grant No. G H 67 administered by the National Science Foundation, in 1970 by Grant No. G H 37 administered by the National Oceanic and Atmospheric Administration (NOAA) of the U.S. Department of Commerce and in 1971 by Grant No. 1-36032 administered by NOAA. In addition, animals supplied by commercial crabbers were purchased from funds provided by the Bureau of Commercial Fisheries, U.S. Department of the Interior, now the National Marine Fisheries Service ( N M F S ) , NOAA, under the co-operative crab disease study sponsored by the N M F S Biological Laboratory, Oxford, Maryland, for the period February 1969 to December 1970. REFERENCES CAMIEN, M. N., SARLET H., DUCH~TEAU G. L. ~ FLORKIN M. (1951) Non-proteinamino

acids in muscle and blood of marine and fresh water crustacea. `7. biol. Chem. 193, 881885. DELAUNAY H. (1931) L'excr~tion azot~e des invert~br~s. Biol. Rev. 6, 265-301. DUCH~,TEAU G. L. & FLORKIN M. (1955) Influence de la temperature sur l'~tat stationnaire du pool des acides aminSs non prot6iques des muscles d'Eriocheir sinensis Milne Edwards. Archs int. Physiol. Biochim. 63, 213-221. DUCH~,TEAU-BosSON G. L. & FLORKIN M. (1961) Change in intracellular concentration of free amino acids as a factor of euryhalinity in the crayfish Astacus astacus. Comp. Biochem. Physiol. 3, 245-249. DuPAuL W. D. & WPBB K. L. (1970) T h e effect of temperature on salinity-induced changes in the free amino acid pool of Mya arenaria. Comp. Biochem. Physiol. 32, 785-801. FLORKIN M. (1960) Blood chemistry. In The Physiology of Crustacea (Edited by WATERMAN W. H.), Vol. 1, pp. 141-159. Academic Press, New York. FUGELLI K. (1967) Regulation of cell volume in flounder (Pleuronectes flesus) erythrocytes accompanying a decrease in plasma osmolarity. Comp. Biochem. Physiol. 22, 253-250. GILLES R. & SCHOFFENIELSE. (1964) Action de la v~ratrine de la cocaine et de la stimulation 61ectrique sur la synth~se et sur le pool des acides amin6s de la chalne nerveuse ventrale du homard. Biochim. biophys. Acta 82, 525-537. GILLES R. & SCHOFF~.NIELSE. (1958) Fixation de 14CO2 par les acidds amines de la chalne nerveuse ventrale du crustac~ Homarus vulgaris M. Edw. Archs int. Physiol. Biochim. 76, 441-451. GILLES R. & SCHOFFENIELS E. (1969) Isosmotic regulation in isolated surviving nerves of Eriocheir sinensis Milne Edwards. Comp. Biochem. Physiol. 31, 927-939. J'~FFRI~SH. P. (1966) International condition of a diminishing blue crab population (Callinectes sapidus). Chesapeake Sci. 7, 164-170. LANO M. & GAINER H. (1969) Volume control by muscle fibers of the blue crab. Volume readjustment in hypotonic salines..7, genet. Physiol. 53, 323-341. LANO~. R. (1968) Isosmotic intracellular regulation. Y y t t Mag. Zool. 16, 1-13. LANG-. R. & MOSTAD A. (1967) Cell volume regulation in osmotically adjusting marine animals. `7. exp. mar. Biol. Ecol. 1, 209-219. LYNCH M. P. & WPBB K. L. (1973a) Variations in serum constituents of the blue crab, Callinectes sapidus: total serum protein. Comp. Biochem. Physiol. 44A, 1237-1249. LYNCH M. P. & WEBB K. L. (1973b) Variations in serum constituents of the blue crab, Callinectes sapidus: glucose. Comp. Biochem. Physiol. 45A, 127-139.

418

M. P. LYNCH AND K. L. WEBB

LYNCH M. P., WEBB K. L. & VAN ENOEL W. A. (1973) Variations in serum constituents of the blue crab, Calcinectes sapidus: chloride and osmotic concentration. Comp. Biochem. Physiol. 44A, 719-734. MORGULtS S. (1922) A study of the non-protein constituents in blood of some marine invertebrates, ft. biol. Chem. 50, lii-liv. MOORE S. & STEIN W. H. (1954) A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. biol. Chem. 211, 907-913. SCHOFFENmLS E. & GmLES R. (1970) Osmoregulation in aquatic arthropods. In Chemical Zoology (Edited by FLORKINM. & SCHEERB. T.), Vol. 5, Arthropoda, Part A, pp. 255286. Academic Press, New York. STmVART J. E., DINGLE J. R. & ODENSE P. H. (1966) Constituents of the hemolymph of the lobster, Homarus americanus Milne Edward. Can. ft. Biochem. 44, 1447-1459. STEVENS T. M., HOWARDC. E. & SCHtaZSXNOERR. W. (1961) Free amino acids in sera of the marine invertebrates, Cancer irroratus, Limulus polyphemus and Homarus americanus. Comp. Biochem. Physiol. 3, 310-314. VAN ENGEL W. A. (1958) The blue crab and its fishery in Chesapeake Bay.--I. Reproduction, early development, growth and migration. Comml Fish. Rev. 20, 6-17. VINCENT-MARIQUE C. ~; GILLES R. (1970) Modification of the amino acid pool in blood and muscle of Eriocheir sinensis during osmotic stress. Comp. Biochem. Physiol. 35, 479-485.

Key Word Index---Callinectes sapidus; crab amino acids; amino acids; serum amino acids; osmoregulation; ninbydrin positive substances.