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Variations in serum constituents of the blue crab, callinectes sapidus: Copper and zinc
Comp. Blochem. Physiol., 1975, Vol. 50A, pp. 135 to 139. J~erganwn Press..Printed in Great Britain
VARIATIONS IN SERUM CONSTITUENTS OF THE BLUE CRAB, CALLINECTES SAPIDUS: COPPER AND ZINC J. A. COLVOCORESSES AND M. P. LYNCH* Virginia Institute of Marine Science, Gloucester Point, Virginia 23062, U.S.A. (Received 15 October 1973)
Abstraet--l. Serum copper and serum zinc concentrations in mature blue crabs, Callinectes sapidus, range from 35-150ppm and 6-22 ppm respectively. Variation in serum metal concentrations followed the general seasonal pattern of total serum protein variation. No significant correlation was established between the serum metal ,~ariation and variation in environmental temperature and salinity. 2. Significant positive correlations were found between serum copper and serum zinc and both metals and total serum protein and serum calcium. 3. Serum copper was significantly more positively correlated with total serum protein than serum zinc while serum zinc was significantly more correlated with serum calcium than serum copper. 4. Using atomlc absorption spectroscopy, no detectable quantities of cadmium or lead (0.3 ppm or greater) were found in blue crab serum.
INTRODUCTION
Bryan (1968) found that while most decapods maintained similar copper to blood solid ratios, IN PREVIOUS reports we have attempted to establish the zinc to blood solid ratio varied from species to "normal" or baseline values of serum chloride and species. Hemocyanin and other blood proteins serum osmotic concentration (Lynch et aL, 1973), have been found to comprise nearly all of the total total serum protein (Lynch & Webb, 1973a), serum organic content of most crustacean sera (Florkin, glucose (Lynch & Webb, 1973b), serum total nirthy1960). drin positive substances (Lynch & Webb, 1973c) Lynch & Webb (1973) reported differences in and major serum cations (Colvocoresses et al., 1974) serum protein levels with sex and year class as well in the blue crab, Calllnectes sapldus, Rathbun. This as seasonal differences in the serum protein of female report considers the variation in serum copper and blue crabs. These differences should also apply to zinc in the same organisms. serum levels of copper and zinc if the aforementioned Copper and zinc are both known to be of conrelationship to serum protein is sufficiently strong. siderable metabolic importance in decapod crustacea. Copper and zinc have been found to be accumCopper is the metallic oxygen carrier bound with ulated by crustacea to concentrations many times hemocyanin, the respiratory pigment of the blue higher than those found in normal sea water. Serum crab, and zinc is a known co-factor of carbonic anhydrase, the principal enzyme involved in calci- concentrations of these metals have been reported to be well regulated and relatively independent of fication (Florkin, 1960). Serum copper and zinc environmental concentrations under normal cirlevels have been reported for a substantial number of decapod species (Bryan, 1968) and considerable cumstances (Bryan, 1966, 1967; Djanghmah, 1970). work has been reported concerning the metabolism Under conditions of extremely high environmental of these metals in higher crustacea (Florkin, 1960; concentrations, these metals may inhibit respiration Bryan, 1964, 1966, 1967; Djanghmah, 1970; (Corner & Sparrow, 1956; Kerkut & Munday, 1962; Hubschmalm, 1966) but these effects occur at Djanghmah & Grove, 1970). concentrations so high (about 20--30 mg/1) that they Serum copper in the blue crab has been studied by Horn & Kerr (1963, 1969) but no information is could not reasonably be expected in an estuary available concerning zinc in this animal. Serum except in the immediate vicinity of a severe pollution levels of both metals have been found to be strongly source of these metals. related to serum protein concentrations in the other decapod species thus far studied, a fact verified for MATERIALS AND METHODS copper in the blue crab by Horn & Kerr (1963). Collection of crabs, environmental data, morpho* Contribution No. 578 from the Virginia Institute of metric and life stage data and serum are described in Lynch et al. (1973) and Colvoeoresses et al. (1974). Marine Science. 135
136
J.A. COLVOCORE~ESAND M. P. LYNCH
All crabs used in this study were mature, intermolt individuals. All of the serum samples used had been previously analyzed for the constituents reported in the previous papers. Serum levels of copper and zinc were determined by atomic absorption spectroscopy. Preparation of samples for analysis is described in Colvocoresses et al. (1974). Both metals were analyzed on dilutions of prepared serum approximately matched to the optimum concentration range of the most sensitive line. RESULTS The data on total serum protein and serum calcium used here for correlative purposes have been reported in Lynch & Webb (1973a) and Colvocoresses et aL (1974) respectively. None of the other constituents thus far studied were found to be related to the serum levels of either zinc or copper. M e a n serum copper ranged from 41 to 101 parts per million (ppm) in the seasonal study (Table 1) a n d 35 to 150ppm in the salinity gradient study (Table 2). A significant difference was found between different year class females in September a n d between males and females in November,
Mean serum zinc ranged from 6'7 to 21.8 ppm in the seasonal study (Table 1) and from 6.2 to 20.7 ppm in the salinity gradient study (Table 2). A significant difference was found between different year class females in August and between males and females in April. There was no indication of any relationship between serum levels of either of these metals and environmental temperatures or salinities. The relationship between serum levels of these metah and the levels of some of the other serum constituents was, however, significant. Serum levels of copper and zinc were found to be positively correlated (r = 0.77), but copper was significantly more d o s d y related ( r = 0.91) to total serum protein levels than was zinc (r = 0'81). Serum levels of these metals are also related to serum calcium levels, with copper showing a significantly weaker relationship (r = 0'59) than zinc (r = 0.76). The serum samples used in this study were also analyzed for cadmium and lead, but n o detectable amounts (0.3 ppm or greater in undiluted serum) of cadmium or lead were found in a n y serum sample, although about one-third of the samples,
Table 1. Seasonal variation of mean serum copper and zinc concentrations in mature, hard blue crabs, C. sapidus, from the York Spit area, Chesapeake Bay, Virginia Serum constituent + S.E. Sample date
Salinity (~o)
Temperature CC)
18 August
27.01
24.1
23 September
24.06
24.1
Sex
Year c/ass
N
Copper
Zinc
2 11 1 4 8 3 10 2 11 1 12
87.1-+3 5 . 3 54.7+_ 5.3 72"9 82"6+_12.2" 53'2+_ 5"7* 81"3+27.7 54"65:3.8 41"1 _+ 4"0* 93"5+10.7" 38"8 65"6+- 3.4
18.6~8.5" 7.5+_0'7* 11.7 10'5 +_2"4 6'7+ 1"1 12"55:3'2 8'8_+ 1'3 9.5 5:8"8 14'4+2.1 20.8 13.2+- 1.3
1 2 10 2 11 13 5 3 1 10 1 8 3
38.5 101'3+ 9.5* 73.9+_ 7.8* 57"8+_1 4 . 9 59"1+_4.1 94-9+-11.3 79.7+_15.3 70.7+_11.4 15.7 100.2+_ 8.7 52.2 72.95:8.1 84.65:21.4
14.2 15.0+4.3 15.3+2.4 13"4+ i'0 11'7+0'9 15.2+- 1.5 21.8+4.8" 12.1 +_2.8* 7.3 14.9+1.3 9.3 13.85:3.8 7.3+_2'1
1970
20 October
23.50
21.6
12 November
22.48
14.9
8 December
22.02
7.6
F F M F F F F M F M F
1968 1969 1968 1969 1968 1969 1969 1969
1971 21 January
19.94
4.8
19 February
22.34
0.5
17 March 13 April
17.28 21.22
6.2 8.8
2 June
24.26
19.7
21 July
21.31
* Significant difference.
24.1
M F
1968
F
1969
F F F M F M F F M F
1968 1969 1969 1969 1969 1970 1970
Serum constituents of the blue crab including at least one sample from each collection, were analyzed for these metals. DISCUSSION Serum levels of copper and zinc have been found to be extremely well regulated in crustacea (Kerkut et al., 1961; Bryan, 1964, 1966, 1967; Djangmah, 1970). Excesses are stored in the hepatopancreas or excreted, while low environmental concentrations are overcome by the binding of these metals with serum protein, thus maintaining an inward diffusion gradient. U n d e r conditions of normal environmental concentrations, crustaceans maintain serum levels of these metals on the order of 103 to 104 times greater than that o f the environment. Because these metals are being concentrated to such an extent, there is p r o p o r t i o n a l l y little unbound zinc or copper in the sera o f decapods (Bryan, 1966).
137
The relationship between serum copper a n d serum protein shown in Fig. 1 is essentially the same as that found by Horn & Kerr (1963) for the blue crab. Bryan (1968) found very similar ratios between serum copper and blood solid content in eighteen other species of decapods. This is n o t surprising inasmuch as hemocyanins frequently account for most of the organic content of crustacean blood (Manwell & Baker, 1963). H e m & Kerr (1969) found that the copper to protein ratio was useful in predicting the relative amounts o f hemocyanin and apohemocyanin, with a copper to protein ratio o f 0,002 representing pure hemocyanin and smaller ratios indicating an apohemocyanin contribution. Those values which fall to the left o f the dotted line in Fig. 1 stand in contrast to H o r n & Kerr's (1969) consideration of 0.002 as the probable upper limit for the copper/protein ratio. Because this only occurs at low protein concentrations it
Table 2, Mean serum copper and zinc concentrations in mature, hard blue crabs, C. sapidus, collected from various salinities in Virginia Serum constituent_+ S.E. (ppm) Sample date
Area*
Salinity (%0)
Temperature (~
1971 3 June 3 June
YR YR
14,42 12"69
20.7 20.5
3 June
YR
9,05
21.1
3 June
YR
5.09
21"7
7 June
YR
24.26
19"7
7 June 8 July 8 July
CB YR YR
26.34 19.60 16.72
19.5 24"6 25"6
8 July
YR
13.52
26.5
8 July 8 July 12 July 12 July
PR PR CB CB
6.42 0.15 20.88 28.97
28.2 28.3 24.1 21.5
4 August
YR
21.63
26.1
4 August 4 August
YR YR
19.90 17.40
27.5 27.6
4 August
YR
14.29
28"7
4 August 4 August 9 August
PR PR YR
9"30 3.72 21.46
28.9 29"3 26.5
9 August
CB
24.85
25.7
Sex F M F M F M F M F F F M F M F F M F M F M F M M F M F M M M F M F
N 1 5 1 4 2 4 4 4 3 3 3 1 4 2 3 2 1 1 1 2 4 2 1 4 4 4 4 4 2 5 2 2 3
* YR, York River; CB, Chesapeake Bay; PR, Pamunkey River,
Copper 150.2 92.0 _+24,6 66,9 62'7+_20,1 50.3+_ 0'1 79'7+21'7 55.6 + 5.0 49.5+ 5,5 98"1+39.5 76.9+- 9.6 48.54- 8'4 35.3 60.6+11.3 85.9_+ 1.6 79.4_+16.2 71.2-1-20.6 81.2 95.9 113.9 107.0+- 0.5 60,4+ 1 5 . 3 47.7_+ 0.7 39.6 42.1_+ 7.3 56'8+_11'7 52'9_+11,9 78'44-30,8 85"9_+27,4 95"34-22.2 59.8 +_ 8.4 76.4+-32.2 69.0_+27.9 93.4-+ 4.0
Zinc 20.7 12.8 + 2.4 9.6 9.7+_2.8 9.8 • 9.9-4-1.3 9.2 + 1'2 7"9+0'8 14.4-1-3.2 9.9+-3.0 5.7+-0.9 5.5 6.8+0.5 10.9_+1.9 9.3_+0.8 8.34-1.8 8-4 14-9 17.1 17.4-+1.5 10-25:2.2 6.2-+1.5 6.5 8.6_+0'8 10.6+-1'6 9"6• 1'8 10.04-2"6 11"0+_3'0 12'1 -+2'9 7.5 4-1'2 10.4_+3.3 11.24-4.3 12.0+2.0
138
J.A. COLVOCOR~.SSmAND M, P. LYNCH j A*
~2o~
.002
Cu / Pr
/ / /
9
= r
//0
80" /
6o-
9
/o
/So
/t.
/o" 9
9
o
/ 9 "0
9 O0 9
9 d
9 9
9
9
9
40--
I
20
I
I
30
I
I
I
I
I
1'
I
,u
40 50 60 70 PROTEIN (mgl ml)
I
g
80
I
I
90
Fig. 1. Relationship of mean serum copper and mean serum zinc to mean total serum protein in mature, hard blue crabs, C. sapidus, taken in Virginia waters. could indicate that the animals in question were under some kind of stress. Bryan's extensive work (1964, 1966, 1967, 1968) with zinc regulation in decapods has indicated that there are. species specific ratios of zinc to blood solid content. The reason for this is unclear, b u t likely dei~ends on both the binding properties of the blood proteins and o n the efficiency of the mechanisms for removing zinc from the blood. The ratio shown by this study for the blue crab is about intermediate when compared to the other species studied. Working with Aastropotamobhts pallipes, Bryan (1967) found that serum zinc was chiefly associated with the non-bemocyanin proteins while the converse held true for copper. Horn & Kerr (1969) found that sponge female blue crabs had a lower copper/protein ratio than non-sponge females or males due to a decrease in the per cent contribution of hemocyanin to total protein. It would be interesting to know if there was a corresp o n d i n g increase in the zinc/protein ratio. Serum zinc and calcium levels were found in this study to be as strongly correlated with each other as they were with total serum protein. This indicates that both may be bound in common to a certain protein or group of proteins. The preceding discussion suggests that any such protein or proteins are non-hem 9 Zinc is a co-factor of carbonic anhydrase, the chief enzyme involved in calcification, b u t a study by Bryan (1964) indicates that serum levels of carbonic anhydrase and zinc may be only loosely related. He found that a ten- to thirty-fold increase in carbonic anhydrase activity was associated with only a two- to three-fold increase in zinc
concentration in Homarus vulgaris. Therefore, while some of the relationship between serum levels of zinc and calcium may bc considered to be a result of corresponding metabolic responses to carbonic anhydrase activity, it is probable that common binding with other serum proteins is more important. The correlation between serum levels of copper and calcium is probably not a result of common complexing with protein but rather a reflection of concomitant increases in the hemocyanin and non-hemocyanin protein fractions as total serum protein increases. Because of the strong correlations between serum zinc and copper and total serum protein, somewhat the same pattern of seasonal variation is in evidence as was found earlier for protein, with new females having increasingly higher concentrations during the summer and early fall a n d then leveling off during late fall and winter. Acknowledgements--We wish to acknowledge the assistance of Dr. K. L. Webb throughout this study and the several persons who assisted in the serum analysis, particularly Miss Rosemary Green. The analytical work done in this study was supported by funds provided by the University of Virginia/USPHS Biomedical Sciences Support Sub-Grant No. 72-279-L. Collecting and processing of samples prior to January 1971 were funded by the National Marine Fisheries Service (NMFS), NOAA, under the co-operative crab disease study sponsored by the NMFS Biological Laboratory, Oxford, Maryland. After that time collection and processing were supported throughout the Virginia Institute of Marine Science Sea Grant Program. REFERENCES
BRYAN G. W. (1964) Zinc regulation in the lobster Homarus vulgaris I. Tissue zinc and copper concentrations. I. mar. Biol. Ass. U.K. 44, 549-563. BRYAN G. W. (1966) The metabolism of Zn and eBZn in crabs, lobsters and freshwater crayfish. I n Symposium on _~adioecological Concentration Processes, Stockholm, Sweden, pp. 1005-1016. Pergamon Press, Oxford. BRYAN G. W. (1967) Zinc regulation in the freshwater crayfish (including some comparative copper analyses). J. exp. Biol. 46, 281-296. BRYAN G. W. (1968) Concentrations of zinc and copper in the tissues of decapod crustaceans. J'. mar. Biol. Ass. U.K. 48, 303-321. COLVOCOgESSESJ. A., LYNCHM. P. & W~BSK. L (1974) Variations in serum constituents of the blue crab, Callinectes sapidus: major serum cations. Comp. Biochem. Physiol. 49A, 787-803. CORr,mR E. D. S. & SPARROWB. W. (1956) The modes of action of toxic agents--I. Observations on the poisoning of certain crustaceans by copper and mercury. J. mar. Biol. Ass. U.K. 35, 531-548. DJANGMAH J. S. (1970) The effects of feeding and starvation on copper in the blood and hcpatopancreas and on blood proteins of Crangon vulgarls (Fab.). Comp. Biochem. Physiol. 32, 709-731.
Serum constituents of the blue crab DJANGMAFIJ. S. • GROVE D, J. (1970) Blood and hepatopancreas copper in Crangon vulgaris (Fab.). Comp. IJioehern. Physiol. 32, 733-745. FgORKr~ M. (1960) Blood chemistry. In The Physiology of Crustacea (Edited by WATE~N T. H.), Yol. I, pp. 141-159. Academic Press, New York. HORN E. C. & KERR M. S. (1963) Hemolymph protein and copper concentrations of adult blue crabs (Callinectes sapidus, Rathbun). BioL Bull. mar. biol. Lab., Woods Hole 125, 499-507. HORN E. C. & KERR M. S. (1969) The hemolymph proteins of the blue crab, Callinectes sapidus--I. Hemocyanins and certain other major protein constituents. Comp. Bioehem. Physiol. :29, 493-508. HtmSCHMANNJ. H. (1966) Effects of copper on the crayfish Orconectes rttsticus (Girard)--I. Acute toxicity. Crustaceana 12, 33--42. I~Rv:trr G. A., MORITZP. M. & MUrrAY K. A. (1961) Variations of copper concentrations in Carcinus maenas. Cah. BioL Mar. 2, 399-408. KERKUT G. A. & MUNDAY K. A. (1962) The effect of copper on the tissue respiration of the crab Carcinus maenas. Cah. BioL Mar. 3, 27-35.
139
LYNCrI M. P. & WEBBK. L. (1973a) Variations in serum constituents of the blue crab, Callinectes sapidus: total serum protein. Comp. Bioehem..PhysioL 44A, 1237-1249. LYNCHM. P. & WBBBK. L. (1973b) Variations in serum constituents of the blue crab, Callinectes sapidus: glucose. Comp. Bioehem. PhyaioL 45A, 127-139. LYNCRM. P. & WEan K. L. (1973c) Variations in serum constituents of the blue crab, Callinectes sapidus: free amino acids and total ninhydrin positive substances. Cornp. Bioehem. PhysioL 4513, 407--418. LYNCI~ M. P., WEan K. L. & VAN ENO~.LW. A. (1973) Variations in serum constituents of the blue crab, Callinectes sapfdus: chloride and osmotic concentration. Comp. Biochem. Physiol. 44A, 719-734. MANW~LLC. & BAKERC. M. A. (1963) Starch gel electrophoresis of sera from marine arthropods: studies on the heterogenicity of hemocyanin and on a "ceruloplasmin-like protein". Comp. Biochem. PhysioL 8, 193-268.
Key Word Index--Blue crab; Calltnectea saptdus; copper; protein; seasonal variation; serum; zinc.
VARIATIONS IN SERUM CONSTITUENTS OF THE BLUE CRAB, CALLINECTES SAPIDUS: COPPER AND ZINC J. A. COLVOCORESSES AND M. P. LYNCH* Virginia Institute of Marine Science, Gloucester Point, Virginia 23062, U.S.A. (Received 15 October 1973)
Abstraet--l. Serum copper and serum zinc concentrations in mature blue crabs, Callinectes sapidus, range from 35-150ppm and 6-22 ppm respectively. Variation in serum metal concentrations followed the general seasonal pattern of total serum protein variation. No significant correlation was established between the serum metal ,~ariation and variation in environmental temperature and salinity. 2. Significant positive correlations were found between serum copper and serum zinc and both metals and total serum protein and serum calcium. 3. Serum copper was significantly more positively correlated with total serum protein than serum zinc while serum zinc was significantly more correlated with serum calcium than serum copper. 4. Using atomlc absorption spectroscopy, no detectable quantities of cadmium or lead (0.3 ppm or greater) were found in blue crab serum.
INTRODUCTION
Bryan (1968) found that while most decapods maintained similar copper to blood solid ratios, IN PREVIOUS reports we have attempted to establish the zinc to blood solid ratio varied from species to "normal" or baseline values of serum chloride and species. Hemocyanin and other blood proteins serum osmotic concentration (Lynch et aL, 1973), have been found to comprise nearly all of the total total serum protein (Lynch & Webb, 1973a), serum organic content of most crustacean sera (Florkin, glucose (Lynch & Webb, 1973b), serum total nirthy1960). drin positive substances (Lynch & Webb, 1973c) Lynch & Webb (1973) reported differences in and major serum cations (Colvocoresses et al., 1974) serum protein levels with sex and year class as well in the blue crab, Calllnectes sapldus, Rathbun. This as seasonal differences in the serum protein of female report considers the variation in serum copper and blue crabs. These differences should also apply to zinc in the same organisms. serum levels of copper and zinc if the aforementioned Copper and zinc are both known to be of conrelationship to serum protein is sufficiently strong. siderable metabolic importance in decapod crustacea. Copper and zinc have been found to be accumCopper is the metallic oxygen carrier bound with ulated by crustacea to concentrations many times hemocyanin, the respiratory pigment of the blue higher than those found in normal sea water. Serum crab, and zinc is a known co-factor of carbonic anhydrase, the principal enzyme involved in calci- concentrations of these metals have been reported to be well regulated and relatively independent of fication (Florkin, 1960). Serum copper and zinc environmental concentrations under normal cirlevels have been reported for a substantial number of decapod species (Bryan, 1968) and considerable cumstances (Bryan, 1966, 1967; Djanghmah, 1970). work has been reported concerning the metabolism Under conditions of extremely high environmental of these metals in higher crustacea (Florkin, 1960; concentrations, these metals may inhibit respiration Bryan, 1964, 1966, 1967; Djanghmah, 1970; (Corner & Sparrow, 1956; Kerkut & Munday, 1962; Hubschmalm, 1966) but these effects occur at Djanghmah & Grove, 1970). concentrations so high (about 20--30 mg/1) that they Serum copper in the blue crab has been studied by Horn & Kerr (1963, 1969) but no information is could not reasonably be expected in an estuary available concerning zinc in this animal. Serum except in the immediate vicinity of a severe pollution levels of both metals have been found to be strongly source of these metals. related to serum protein concentrations in the other decapod species thus far studied, a fact verified for MATERIALS AND METHODS copper in the blue crab by Horn & Kerr (1963). Collection of crabs, environmental data, morpho* Contribution No. 578 from the Virginia Institute of metric and life stage data and serum are described in Lynch et al. (1973) and Colvoeoresses et al. (1974). Marine Science. 135
136
J.A. COLVOCORE~ESAND M. P. LYNCH
All crabs used in this study were mature, intermolt individuals. All of the serum samples used had been previously analyzed for the constituents reported in the previous papers. Serum levels of copper and zinc were determined by atomic absorption spectroscopy. Preparation of samples for analysis is described in Colvocoresses et al. (1974). Both metals were analyzed on dilutions of prepared serum approximately matched to the optimum concentration range of the most sensitive line. RESULTS The data on total serum protein and serum calcium used here for correlative purposes have been reported in Lynch & Webb (1973a) and Colvocoresses et aL (1974) respectively. None of the other constituents thus far studied were found to be related to the serum levels of either zinc or copper. M e a n serum copper ranged from 41 to 101 parts per million (ppm) in the seasonal study (Table 1) a n d 35 to 150ppm in the salinity gradient study (Table 2). A significant difference was found between different year class females in September a n d between males and females in November,
Mean serum zinc ranged from 6'7 to 21.8 ppm in the seasonal study (Table 1) and from 6.2 to 20.7 ppm in the salinity gradient study (Table 2). A significant difference was found between different year class females in August and between males and females in April. There was no indication of any relationship between serum levels of either of these metals and environmental temperatures or salinities. The relationship between serum levels of these metah and the levels of some of the other serum constituents was, however, significant. Serum levels of copper and zinc were found to be positively correlated (r = 0.77), but copper was significantly more d o s d y related ( r = 0.91) to total serum protein levels than was zinc (r = 0'81). Serum levels of these metals are also related to serum calcium levels, with copper showing a significantly weaker relationship (r = 0'59) than zinc (r = 0.76). The serum samples used in this study were also analyzed for cadmium and lead, but n o detectable amounts (0.3 ppm or greater in undiluted serum) of cadmium or lead were found in a n y serum sample, although about one-third of the samples,
Table 1. Seasonal variation of mean serum copper and zinc concentrations in mature, hard blue crabs, C. sapidus, from the York Spit area, Chesapeake Bay, Virginia Serum constituent + S.E. Sample date
Salinity (~o)
Temperature CC)
18 August
27.01
24.1
23 September
24.06
24.1
Sex
Year c/ass
N
Copper
Zinc
2 11 1 4 8 3 10 2 11 1 12
87.1-+3 5 . 3 54.7+_ 5.3 72"9 82"6+_12.2" 53'2+_ 5"7* 81"3+27.7 54"65:3.8 41"1 _+ 4"0* 93"5+10.7" 38"8 65"6+- 3.4
18.6~8.5" 7.5+_0'7* 11.7 10'5 +_2"4 6'7+ 1"1 12"55:3'2 8'8_+ 1'3 9.5 5:8"8 14'4+2.1 20.8 13.2+- 1.3
1 2 10 2 11 13 5 3 1 10 1 8 3
38.5 101'3+ 9.5* 73.9+_ 7.8* 57"8+_1 4 . 9 59"1+_4.1 94-9+-11.3 79.7+_15.3 70.7+_11.4 15.7 100.2+_ 8.7 52.2 72.95:8.1 84.65:21.4
14.2 15.0+4.3 15.3+2.4 13"4+ i'0 11'7+0'9 15.2+- 1.5 21.8+4.8" 12.1 +_2.8* 7.3 14.9+1.3 9.3 13.85:3.8 7.3+_2'1
1970
20 October
23.50
21.6
12 November
22.48
14.9
8 December
22.02
7.6
F F M F F F F M F M F
1968 1969 1968 1969 1968 1969 1969 1969
1971 21 January
19.94
4.8
19 February
22.34
0.5
17 March 13 April
17.28 21.22
6.2 8.8
2 June
24.26
19.7
21 July
21.31
* Significant difference.
24.1
M F
1968
F
1969
F F F M F M F F M F
1968 1969 1969 1969 1969 1970 1970
Serum constituents of the blue crab including at least one sample from each collection, were analyzed for these metals. DISCUSSION Serum levels of copper and zinc have been found to be extremely well regulated in crustacea (Kerkut et al., 1961; Bryan, 1964, 1966, 1967; Djangmah, 1970). Excesses are stored in the hepatopancreas or excreted, while low environmental concentrations are overcome by the binding of these metals with serum protein, thus maintaining an inward diffusion gradient. U n d e r conditions of normal environmental concentrations, crustaceans maintain serum levels of these metals on the order of 103 to 104 times greater than that o f the environment. Because these metals are being concentrated to such an extent, there is p r o p o r t i o n a l l y little unbound zinc or copper in the sera o f decapods (Bryan, 1966).
137
The relationship between serum copper a n d serum protein shown in Fig. 1 is essentially the same as that found by Horn & Kerr (1963) for the blue crab. Bryan (1968) found very similar ratios between serum copper and blood solid content in eighteen other species of decapods. This is n o t surprising inasmuch as hemocyanins frequently account for most of the organic content of crustacean blood (Manwell & Baker, 1963). H e m & Kerr (1969) found that the copper to protein ratio was useful in predicting the relative amounts o f hemocyanin and apohemocyanin, with a copper to protein ratio o f 0,002 representing pure hemocyanin and smaller ratios indicating an apohemocyanin contribution. Those values which fall to the left o f the dotted line in Fig. 1 stand in contrast to H o r n & Kerr's (1969) consideration of 0.002 as the probable upper limit for the copper/protein ratio. Because this only occurs at low protein concentrations it
Table 2, Mean serum copper and zinc concentrations in mature, hard blue crabs, C. sapidus, collected from various salinities in Virginia Serum constituent_+ S.E. (ppm) Sample date
Area*
Salinity (%0)
Temperature (~
1971 3 June 3 June
YR YR
14,42 12"69
20.7 20.5
3 June
YR
9,05
21.1
3 June
YR
5.09
21"7
7 June
YR
24.26
19"7
7 June 8 July 8 July
CB YR YR
26.34 19.60 16.72
19.5 24"6 25"6
8 July
YR
13.52
26.5
8 July 8 July 12 July 12 July
PR PR CB CB
6.42 0.15 20.88 28.97
28.2 28.3 24.1 21.5
4 August
YR
21.63
26.1
4 August 4 August
YR YR
19.90 17.40
27.5 27.6
4 August
YR
14.29
28"7
4 August 4 August 9 August
PR PR YR
9"30 3.72 21.46
28.9 29"3 26.5
9 August
CB
24.85
25.7
Sex F M F M F M F M F F F M F M F F M F M F M F M M F M F M M M F M F
N 1 5 1 4 2 4 4 4 3 3 3 1 4 2 3 2 1 1 1 2 4 2 1 4 4 4 4 4 2 5 2 2 3
* YR, York River; CB, Chesapeake Bay; PR, Pamunkey River,
Copper 150.2 92.0 _+24,6 66,9 62'7+_20,1 50.3+_ 0'1 79'7+21'7 55.6 + 5.0 49.5+ 5,5 98"1+39.5 76.9+- 9.6 48.54- 8'4 35.3 60.6+11.3 85.9_+ 1.6 79.4_+16.2 71.2-1-20.6 81.2 95.9 113.9 107.0+- 0.5 60,4+ 1 5 . 3 47.7_+ 0.7 39.6 42.1_+ 7.3 56'8+_11'7 52'9_+11,9 78'44-30,8 85"9_+27,4 95"34-22.2 59.8 +_ 8.4 76.4+-32.2 69.0_+27.9 93.4-+ 4.0
Zinc 20.7 12.8 + 2.4 9.6 9.7+_2.8 9.8 • 9.9-4-1.3 9.2 + 1'2 7"9+0'8 14.4-1-3.2 9.9+-3.0 5.7+-0.9 5.5 6.8+0.5 10.9_+1.9 9.3_+0.8 8.34-1.8 8-4 14-9 17.1 17.4-+1.5 10-25:2.2 6.2-+1.5 6.5 8.6_+0'8 10.6+-1'6 9"6• 1'8 10.04-2"6 11"0+_3'0 12'1 -+2'9 7.5 4-1'2 10.4_+3.3 11.24-4.3 12.0+2.0
138
J.A. COLVOCOR~.SSmAND M, P. LYNCH j A*
~2o~
.002
Cu / Pr
/ / /
9
= r
//0
80" /
6o-
9
/o
/So
/t.
/o" 9
9
o
/ 9 "0
9 O0 9
9 d
9 9
9
9
9
40--
I
20
I
I
30
I
I
I
I
I
1'
I
,u
40 50 60 70 PROTEIN (mgl ml)
I
g
80
I
I
90
Fig. 1. Relationship of mean serum copper and mean serum zinc to mean total serum protein in mature, hard blue crabs, C. sapidus, taken in Virginia waters. could indicate that the animals in question were under some kind of stress. Bryan's extensive work (1964, 1966, 1967, 1968) with zinc regulation in decapods has indicated that there are. species specific ratios of zinc to blood solid content. The reason for this is unclear, b u t likely dei~ends on both the binding properties of the blood proteins and o n the efficiency of the mechanisms for removing zinc from the blood. The ratio shown by this study for the blue crab is about intermediate when compared to the other species studied. Working with Aastropotamobhts pallipes, Bryan (1967) found that serum zinc was chiefly associated with the non-bemocyanin proteins while the converse held true for copper. Horn & Kerr (1969) found that sponge female blue crabs had a lower copper/protein ratio than non-sponge females or males due to a decrease in the per cent contribution of hemocyanin to total protein. It would be interesting to know if there was a corresp o n d i n g increase in the zinc/protein ratio. Serum zinc and calcium levels were found in this study to be as strongly correlated with each other as they were with total serum protein. This indicates that both may be bound in common to a certain protein or group of proteins. The preceding discussion suggests that any such protein or proteins are non-hem 9 Zinc is a co-factor of carbonic anhydrase, the chief enzyme involved in calcification, b u t a study by Bryan (1964) indicates that serum levels of carbonic anhydrase and zinc may be only loosely related. He found that a ten- to thirty-fold increase in carbonic anhydrase activity was associated with only a two- to three-fold increase in zinc
concentration in Homarus vulgaris. Therefore, while some of the relationship between serum levels of zinc and calcium may bc considered to be a result of corresponding metabolic responses to carbonic anhydrase activity, it is probable that common binding with other serum proteins is more important. The correlation between serum levels of copper and calcium is probably not a result of common complexing with protein but rather a reflection of concomitant increases in the hemocyanin and non-hemocyanin protein fractions as total serum protein increases. Because of the strong correlations between serum zinc and copper and total serum protein, somewhat the same pattern of seasonal variation is in evidence as was found earlier for protein, with new females having increasingly higher concentrations during the summer and early fall a n d then leveling off during late fall and winter. Acknowledgements--We wish to acknowledge the assistance of Dr. K. L. Webb throughout this study and the several persons who assisted in the serum analysis, particularly Miss Rosemary Green. The analytical work done in this study was supported by funds provided by the University of Virginia/USPHS Biomedical Sciences Support Sub-Grant No. 72-279-L. Collecting and processing of samples prior to January 1971 were funded by the National Marine Fisheries Service (NMFS), NOAA, under the co-operative crab disease study sponsored by the NMFS Biological Laboratory, Oxford, Maryland. After that time collection and processing were supported throughout the Virginia Institute of Marine Science Sea Grant Program. REFERENCES
BRYAN G. W. (1964) Zinc regulation in the lobster Homarus vulgaris I. Tissue zinc and copper concentrations. I. mar. Biol. Ass. U.K. 44, 549-563. BRYAN G. W. (1966) The metabolism of Zn and eBZn in crabs, lobsters and freshwater crayfish. I n Symposium on _~adioecological Concentration Processes, Stockholm, Sweden, pp. 1005-1016. Pergamon Press, Oxford. BRYAN G. W. (1967) Zinc regulation in the freshwater crayfish (including some comparative copper analyses). J. exp. Biol. 46, 281-296. BRYAN G. W. (1968) Concentrations of zinc and copper in the tissues of decapod crustaceans. J'. mar. Biol. Ass. U.K. 48, 303-321. COLVOCOgESSESJ. A., LYNCHM. P. & W~BSK. L (1974) Variations in serum constituents of the blue crab, Callinectes sapidus: major serum cations. Comp. Biochem. Physiol. 49A, 787-803. CORr,mR E. D. S. & SPARROWB. W. (1956) The modes of action of toxic agents--I. Observations on the poisoning of certain crustaceans by copper and mercury. J. mar. Biol. Ass. U.K. 35, 531-548. DJANGMAH J. S. (1970) The effects of feeding and starvation on copper in the blood and hcpatopancreas and on blood proteins of Crangon vulgarls (Fab.). Comp. Biochem. Physiol. 32, 709-731.
Serum constituents of the blue crab DJANGMAFIJ. S. • GROVE D, J. (1970) Blood and hepatopancreas copper in Crangon vulgaris (Fab.). Comp. IJioehern. Physiol. 32, 733-745. FgORKr~ M. (1960) Blood chemistry. In The Physiology of Crustacea (Edited by WATE~N T. H.), Yol. I, pp. 141-159. Academic Press, New York. HORN E. C. & KERR M. S. (1963) Hemolymph protein and copper concentrations of adult blue crabs (Callinectes sapidus, Rathbun). BioL Bull. mar. biol. Lab., Woods Hole 125, 499-507. HORN E. C. & KERR M. S. (1969) The hemolymph proteins of the blue crab, Callinectes sapidus--I. Hemocyanins and certain other major protein constituents. Comp. Bioehem. Physiol. :29, 493-508. HtmSCHMANNJ. H. (1966) Effects of copper on the crayfish Orconectes rttsticus (Girard)--I. Acute toxicity. Crustaceana 12, 33--42. I~Rv:trr G. A., MORITZP. M. & MUrrAY K. A. (1961) Variations of copper concentrations in Carcinus maenas. Cah. BioL Mar. 2, 399-408. KERKUT G. A. & MUNDAY K. A. (1962) The effect of copper on the tissue respiration of the crab Carcinus maenas. Cah. BioL Mar. 3, 27-35.
139
LYNCrI M. P. & WEBBK. L. (1973a) Variations in serum constituents of the blue crab, Callinectes sapidus: total serum protein. Comp. Bioehem..PhysioL 44A, 1237-1249. LYNCHM. P. & WBBBK. L. (1973b) Variations in serum constituents of the blue crab, Callinectes sapidus: glucose. Comp. Bioehem. PhyaioL 45A, 127-139. LYNCRM. P. & WEan K. L. (1973c) Variations in serum constituents of the blue crab, Callinectes sapidus: free amino acids and total ninhydrin positive substances. Cornp. Bioehem. PhysioL 4513, 407--418. LYNCI~ M. P., WEan K. L. & VAN ENO~.LW. A. (1973) Variations in serum constituents of the blue crab, Callinectes sapfdus: chloride and osmotic concentration. Comp. Biochem. Physiol. 44A, 719-734. MANW~LLC. & BAKERC. M. A. (1963) Starch gel electrophoresis of sera from marine arthropods: studies on the heterogenicity of hemocyanin and on a "ceruloplasmin-like protein". Comp. Biochem. PhysioL 8, 193-268.
Key Word Index--Blue crab; Calltnectea saptdus; copper; protein; seasonal variation; serum; zinc.