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Muscle, eye and serum proteins in the blue crab, Callinectes sapidus, rathbun
Comp. Biochem. Physiol., t978, Vol. 59B, pp. 25 to 26. Pergamon Press. Printed in Great Britain
MUSCLE, EYE AND SERUM PROTEINS IN THE BLUE CRAB, CALLINECTES SAPIDUS, RATHBUN MARTHE A. COLE1 AND RAYMONDP. MORGAN 112 1Chesapeake Biological Laboratory, Center for Environmental and Estuarine Studies, University of Maryland, Solomons, MD 20688; and 2Batelle Industries, William F. Clapp Laboratories, Inc., Duxbury, MA 02332, U.S.A. (Received 2 April 1977) Abstract--1. Eye, muscle and serum of the blue crab, Callinectes sapidus were examined through discon-
tinuous polyacrylamide techniques. 2. Low polymorphism was found in eye and serum proteins from 3 salinity ranges. 3. Muscle proteins were identical for all samples. INTRODUCTION The objective of this study was to characterize eye, muscle and serum protein patterns of the blue crab, Callinectes sapidus, Rathbun. Crabs from two proximate estuaries, Chincoteague and Chesapeake Bays, were used. Whereas the Chesapeake Bay exhibits a continuous salinity gradient, Chincoteague Bay is highly saline throughout. Crabs were taken from three salinities to examine possible differences in eye, muscle and serum protein configuration. Except for an early paper on serum proteins (Woods et al., 1958) little work has been done on the biochemical genetics of Callinectes sapidus. Most studies (Horn & Kerr, 1963, 1969) have been concerned with concentrations of hemocyanin and other proteins in blue crab serum. Additional information on 20 soluble enzyme systems from other tissues of Callinectes sapidus is presented elsewhere (Cole & Morgan, 1977, in press).
d.c. current was applied to the system; 30 mA for the initial 10 min and 45 mA until the end of the run, approximately 45 min. Gels were removed from glass tubes and cut at the front edge of dye band, allowing measurement of relative mobility of bands. Gels were stained for protein for a minimum of 1 hr in a 0.1% Buffalo Black NBR in 7% acetic acid. Destaining of gels was done in a Canalco Quick Destainer with 7% acetic acid. After destaining gels were scored, drawn and stored in acetic acid in 10 x 75 mm culture tubes. RESULTS AND DISCUSSION
Figure 1 shows the electropherograms of muscle, eye and serum proteins of the blue crab. Muscle pro-
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MATERIALSAND METHODS
Illll
Specimens of Callinectes sapidus were obtained commerMuscle proteins cially from three sources; from water of 8-12 ppt Choptank River, MD, from water of 17-20 ppt Backbay, VA and specimens from water of 35 ppt, Chincoteague Bay, VA. Only adult males were used. Sample size ranged from 12 to 48. Live crabs were cooled on ice, transported and dissected on the same day for muscle and eye tissue, as well as being sampled for hemolymph. Whole tissues were frozen immediately in an equal volume of Poulick's grinding buffer (Poulick, 1957) (0.08M Tris-citrate, pH 8.6) and stored at -20°C. Hemolymph was allowed to clot prior to freezing. At the time of assay, tissues were thawed, Eye proteins macerated and homogenized for approximately 1 rain and centrifuged at 1600g at 5-10°C for 7 min. Samples were kept cool by storing in a Kryorack prior to usage. Soluble muscle, eye and serum proteins were separated on 7% acrylamide gels with a Canalco Model 1200 bath and Beckman Duostat power supply. Protein content, determined by an A/O TS refractometer was maintained between 512 and 820 #g. The acrylamide gels were formed in glass tubes (5 mm i.d., 7 mm o.d., 67 mm long). The 10 #! sample was separated at room temperature with a Tris (0.005 M)-glycine (0.039 M) buffer pH 8.3. The buffer was Serum proteins made fresh for each run. Several drops of bromophenol blue were added to the buffer prior to etectrophoresis as Fig. 1. Electropherograms of protein systems of blue crab, a dye. Once this dye band had migrated to within 5 mm Callinectes sapidus, Rathbun. Arrows indicate variable of the gel end, electrophoresis was terminated. A constant bands. --
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MARTHE A. COLEAND RAYMONDP. MORGANII
tein patterns for all salinities were identical in electrophoretic migration based on 12-24 samples from each salinity range. Twelve proteins were present. The most prominent bands were the first three near anodal end of gel and the band in position 6 (as counted from anodal end) near middle of gel. The close proximity of bands 1 and 2 occasionally results in slight fusion producing an intense band. The cathodal end in these gels is generally fuzzy throughout, although six bands are usually discernible. Very little polymorphism was noted among muscle proteins. The pattern for eye proteins was identical among Choptank (8-12 ppt)and Back Bay (17-20 ppt) crabs, and very similar to Chincoteague samples, except for occasional absences in the first clearly visible band near middle of gel (band no. 5 counting from anodal end). This protein band was variable in intensity, ranging from moderately intense in Chesapeake crabs, to relatively faint in Back Bay crabs, to almost indiscernible or absent in Chincoteague crabs. Bands nos. 1, 2 and 4 showed the greatest intensity among all salinities. However, in all eye tissue gels (sample size was at least 12 in each salinity), bands appear somewhat undeveloped or unclear, owing perhaps to protein degradation during the freezing and thawing process, Serum proteins were also similar among crabs from all three salinities (based on 12-24 samples for each salinity) except for variation in band no. 6 (as counted from anodal end). This band varied in intensity, sometimes occurring as a distinct band, other times as a faint blur, and occasionally not at all. The pattern for serum proteins exhibits 10 fractions. Anodally migrating bands nos. l, 2 and 4 were well developed and thick. Bands no. 3 and 9 were almost as intense, but much narrower. Bands no. 5 and 7 were of a uniform, moderate intensity throughout all gels. The cathodal end of the gel was dense and smeared, therefore individual bands were indiscernible. Using starch gel electrophoresis, Horn & Kerr (1969) were able to resolve 7-9 serum protein fractions of Callinectes depending upon sex of sample. Ghidalia et al. (1970) point out that the number of serum protein fractions is a function of gel and buffer systems employed, therefore it appears that the acrylamide Tris--glycine system offers better resolution for analyzing blue crab sera. The low variability observed in eye and serum proteins may result from natural variation within the population, or perhaps from physiological changes associated with molt cycle. Working with lobster serum,
Barlow & Ridgway (1969) found that serum proteins build up prior to molt, are diluted at molt and are nearly constant during intermolt period. Through electrophoretic and other techniques they found shifts in both serum components and protein concentration during molt cycle. To insure against just such variability due to physiological changes, only adult male crabs were used in this study. Presumably then, these crabs were in an intermolt (or post molt) stage in which serum proteins were likely to be constant, producing electrophoretic bands of uniform intensity. Thus the variation in protein intensity observed in band no. 6 of serum proteins and band no. 5 of eye proteins is probably not a result of ontological-physiological changes. Further sampling of adult male crabs, as well as crabs from various stages of the molt cycle would be necessary to confirm this hypothesis. Just as it is difficult to ascribe the observed variation to physiological changes, sampling was inadequate to evoke ecological correlates, such as salinity differences, of this genetic variability. Acknowledgements--Mr Phil Jones assisted in procurring crabs. This is contribution No. 764, Center for Environmental and Estuarine studies of the University of Maryland. Ms Cole was supported in part by a National Science Foundation Undergraduate Research Participation grant no. SMI 76-03130 to the Chesapeake Biological Laboratory. REFERENCES
BARLOW J. 8,~ RIDGWAYG. J. (1969) Changes in serum protein during molt and reproductive cycles of the American lobster (Homarus americanus). J. Fish. Res. Bd Can. 26(8), 2101-2109. COLE M. A. • MORGANR. P. lI (1977) Genetic variation in the blue crab, Callinectes sapidus, Rathbun. Chesapeake Sci. In press. GHIDALIAW., VENTRELY R. 8/, COIRAULTY. 0970) Use of different filtering media for the electrophoretic analysis of crustacean sera, Comp. Biochem. Physiol. 35, 597-606. HORN E, C. & KERR M, S. (1963) Hemolymph protein and copper concentrations of adult blue crabs (Callinectes sapidus, Rathbun). Biol. Bull. 125, 499-507. HORN E. C. 8¢. KERR M. S. 0969) The hemolymph proteins of the blue crab, Callinectes sapidus. Comp. Biochem. Physiol. 29, 493-508. POULICK M. D. (1957) Starch gel electrophoresis in a discontinuous system of buffers. Nature, Lond. 180, 1477-1479. WOODS R. K., PAULSEN E. C., ENGLE R L. & PERT J. H. 0958)Starch gel electrophoresis of some invertebrate sera. Science 12'7, 519-520.
MUSCLE, EYE AND SERUM PROTEINS IN THE BLUE CRAB, CALLINECTES SAPIDUS, RATHBUN MARTHE A. COLE1 AND RAYMONDP. MORGAN 112 1Chesapeake Biological Laboratory, Center for Environmental and Estuarine Studies, University of Maryland, Solomons, MD 20688; and 2Batelle Industries, William F. Clapp Laboratories, Inc., Duxbury, MA 02332, U.S.A. (Received 2 April 1977) Abstract--1. Eye, muscle and serum of the blue crab, Callinectes sapidus were examined through discon-
tinuous polyacrylamide techniques. 2. Low polymorphism was found in eye and serum proteins from 3 salinity ranges. 3. Muscle proteins were identical for all samples. INTRODUCTION The objective of this study was to characterize eye, muscle and serum protein patterns of the blue crab, Callinectes sapidus, Rathbun. Crabs from two proximate estuaries, Chincoteague and Chesapeake Bays, were used. Whereas the Chesapeake Bay exhibits a continuous salinity gradient, Chincoteague Bay is highly saline throughout. Crabs were taken from three salinities to examine possible differences in eye, muscle and serum protein configuration. Except for an early paper on serum proteins (Woods et al., 1958) little work has been done on the biochemical genetics of Callinectes sapidus. Most studies (Horn & Kerr, 1963, 1969) have been concerned with concentrations of hemocyanin and other proteins in blue crab serum. Additional information on 20 soluble enzyme systems from other tissues of Callinectes sapidus is presented elsewhere (Cole & Morgan, 1977, in press).
d.c. current was applied to the system; 30 mA for the initial 10 min and 45 mA until the end of the run, approximately 45 min. Gels were removed from glass tubes and cut at the front edge of dye band, allowing measurement of relative mobility of bands. Gels were stained for protein for a minimum of 1 hr in a 0.1% Buffalo Black NBR in 7% acetic acid. Destaining of gels was done in a Canalco Quick Destainer with 7% acetic acid. After destaining gels were scored, drawn and stored in acetic acid in 10 x 75 mm culture tubes. RESULTS AND DISCUSSION
Figure 1 shows the electropherograms of muscle, eye and serum proteins of the blue crab. Muscle pro-
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MATERIALSAND METHODS
Illll
Specimens of Callinectes sapidus were obtained commerMuscle proteins cially from three sources; from water of 8-12 ppt Choptank River, MD, from water of 17-20 ppt Backbay, VA and specimens from water of 35 ppt, Chincoteague Bay, VA. Only adult males were used. Sample size ranged from 12 to 48. Live crabs were cooled on ice, transported and dissected on the same day for muscle and eye tissue, as well as being sampled for hemolymph. Whole tissues were frozen immediately in an equal volume of Poulick's grinding buffer (Poulick, 1957) (0.08M Tris-citrate, pH 8.6) and stored at -20°C. Hemolymph was allowed to clot prior to freezing. At the time of assay, tissues were thawed, Eye proteins macerated and homogenized for approximately 1 rain and centrifuged at 1600g at 5-10°C for 7 min. Samples were kept cool by storing in a Kryorack prior to usage. Soluble muscle, eye and serum proteins were separated on 7% acrylamide gels with a Canalco Model 1200 bath and Beckman Duostat power supply. Protein content, determined by an A/O TS refractometer was maintained between 512 and 820 #g. The acrylamide gels were formed in glass tubes (5 mm i.d., 7 mm o.d., 67 mm long). The 10 #! sample was separated at room temperature with a Tris (0.005 M)-glycine (0.039 M) buffer pH 8.3. The buffer was Serum proteins made fresh for each run. Several drops of bromophenol blue were added to the buffer prior to etectrophoresis as Fig. 1. Electropherograms of protein systems of blue crab, a dye. Once this dye band had migrated to within 5 mm Callinectes sapidus, Rathbun. Arrows indicate variable of the gel end, electrophoresis was terminated. A constant bands. --
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26
MARTHE A. COLEAND RAYMONDP. MORGANII
tein patterns for all salinities were identical in electrophoretic migration based on 12-24 samples from each salinity range. Twelve proteins were present. The most prominent bands were the first three near anodal end of gel and the band in position 6 (as counted from anodal end) near middle of gel. The close proximity of bands 1 and 2 occasionally results in slight fusion producing an intense band. The cathodal end in these gels is generally fuzzy throughout, although six bands are usually discernible. Very little polymorphism was noted among muscle proteins. The pattern for eye proteins was identical among Choptank (8-12 ppt)and Back Bay (17-20 ppt) crabs, and very similar to Chincoteague samples, except for occasional absences in the first clearly visible band near middle of gel (band no. 5 counting from anodal end). This protein band was variable in intensity, ranging from moderately intense in Chesapeake crabs, to relatively faint in Back Bay crabs, to almost indiscernible or absent in Chincoteague crabs. Bands nos. 1, 2 and 4 showed the greatest intensity among all salinities. However, in all eye tissue gels (sample size was at least 12 in each salinity), bands appear somewhat undeveloped or unclear, owing perhaps to protein degradation during the freezing and thawing process, Serum proteins were also similar among crabs from all three salinities (based on 12-24 samples for each salinity) except for variation in band no. 6 (as counted from anodal end). This band varied in intensity, sometimes occurring as a distinct band, other times as a faint blur, and occasionally not at all. The pattern for serum proteins exhibits 10 fractions. Anodally migrating bands nos. l, 2 and 4 were well developed and thick. Bands no. 3 and 9 were almost as intense, but much narrower. Bands no. 5 and 7 were of a uniform, moderate intensity throughout all gels. The cathodal end of the gel was dense and smeared, therefore individual bands were indiscernible. Using starch gel electrophoresis, Horn & Kerr (1969) were able to resolve 7-9 serum protein fractions of Callinectes depending upon sex of sample. Ghidalia et al. (1970) point out that the number of serum protein fractions is a function of gel and buffer systems employed, therefore it appears that the acrylamide Tris--glycine system offers better resolution for analyzing blue crab sera. The low variability observed in eye and serum proteins may result from natural variation within the population, or perhaps from physiological changes associated with molt cycle. Working with lobster serum,
Barlow & Ridgway (1969) found that serum proteins build up prior to molt, are diluted at molt and are nearly constant during intermolt period. Through electrophoretic and other techniques they found shifts in both serum components and protein concentration during molt cycle. To insure against just such variability due to physiological changes, only adult male crabs were used in this study. Presumably then, these crabs were in an intermolt (or post molt) stage in which serum proteins were likely to be constant, producing electrophoretic bands of uniform intensity. Thus the variation in protein intensity observed in band no. 6 of serum proteins and band no. 5 of eye proteins is probably not a result of ontological-physiological changes. Further sampling of adult male crabs, as well as crabs from various stages of the molt cycle would be necessary to confirm this hypothesis. Just as it is difficult to ascribe the observed variation to physiological changes, sampling was inadequate to evoke ecological correlates, such as salinity differences, of this genetic variability. Acknowledgements--Mr Phil Jones assisted in procurring crabs. This is contribution No. 764, Center for Environmental and Estuarine studies of the University of Maryland. Ms Cole was supported in part by a National Science Foundation Undergraduate Research Participation grant no. SMI 76-03130 to the Chesapeake Biological Laboratory. REFERENCES
BARLOW J. 8,~ RIDGWAYG. J. (1969) Changes in serum protein during molt and reproductive cycles of the American lobster (Homarus americanus). J. Fish. Res. Bd Can. 26(8), 2101-2109. COLE M. A. • MORGANR. P. lI (1977) Genetic variation in the blue crab, Callinectes sapidus, Rathbun. Chesapeake Sci. In press. GHIDALIAW., VENTRELY R. 8/, COIRAULTY. 0970) Use of different filtering media for the electrophoretic analysis of crustacean sera, Comp. Biochem. Physiol. 35, 597-606. HORN E, C. & KERR M, S. (1963) Hemolymph protein and copper concentrations of adult blue crabs (Callinectes sapidus, Rathbun). Biol. Bull. 125, 499-507. HORN E. C. 8¢. KERR M. S. 0969) The hemolymph proteins of the blue crab, Callinectes sapidus. Comp. Biochem. Physiol. 29, 493-508. POULICK M. D. (1957) Starch gel electrophoresis in a discontinuous system of buffers. Nature, Lond. 180, 1477-1479. WOODS R. K., PAULSEN E. C., ENGLE R L. & PERT J. H. 0958)Starch gel electrophoresis of some invertebrate sera. Science 12'7, 519-520.