Comp. Biochem. Physiol.Vol. 102B,No. 1, pp. 175-185, 1992 Printed in Great Britain
0305-0491/92 $5.00 + 0.00 © 1992Pergamon Press Ltd
HIGH TAURINE LEVELS IN THE S O L E M Y A V E L U M SYMBIOSIS NOELLETTE M. CONWAY* and JUDITH E. McDOWELL CAPUZZO Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, U.S.A. (Received 25 July 1991) Abstraet--l. To compare biochemical differences between bivalves with and without endosymbiotic
chemoautotrophic bacteria, specimens of Solemya velum, a bivalve species known to contain bacterial endosymbionts, and the symbiont-free soft-shelled clam Mya arenaria, were collected from the same subtidal reducing sediments during October and November 1988. 2. Total and free amino acid compositions were determined for both species. Protein-bound amino acids were calculated as the difference between total and free amino acids. In addition, stable isotope ratios of the total and free amino acids of each species were measured to determine potential sources for these molecules. 3. Both species had similar total hydrolyzable- and protein-bound amino acid compositions; approximately 50% of the protein-bound amino acids were essential amino acids. In S. velum, the small size of the digestive system suggests that these amino acids are probably synthesized by the endosymbiotic bacteria and translocated to the animal tissue. The 6 ~3C and 6~5N ratios of the amino acids are very similar to the isotope ratios previously found in both the endosymbionts and whole tissues of S. velum. The relative and absolute amounts of free amino acids are very different in the two species. In S. velum, the absolute concentrations of taurine, a sulfur-containing amino acid, were greater than the total free amino acid concentrations found in other bivalves. 4. The ~34S ratios of the free amino acids of S. velum, which were predominantly composed of taurine, were extremely negative ( - 17.29'oo)suggesting that taurine is synthesized using sulfur originally derived from external reduced sulfur sources, such as pore water sulfides. The possible roles for taurine in this animal-bacteria symbiosis are discussed.
INTRODUCTION Chemoautotrophic bacteria occur as symbionts in at least four marine invertebrate phyla, including molluscs, annelids, pogonophoran and vestimentiferan worms (Cavanaugh et al., 1981; Feibeck et al., 1981; Giere et al., 1984; Southward et al., 1981). In most cases, the symbiotic bacteria derive their cellular carbon from the fixation of CO2 and their energy from the oxidation of reduced sulfur compounds, such as H2S, S°, and $2O2- (Anderson et aL, 1987; Chen et al., 1987; Hand, 1987; Giere et al., 1988); however, species capable of growth on methane have also been reported (Childress et al., 1986; Cavanaugh et al., 1987; Schmaljohann and Fliigel, 1987). Many of the host species are characterized by reduced digestive capabilities (Cavanaugh, 1983) while some are completely gutless (Reid, 1980) suggesting a trophic function for the bacterial symbionts. In this context, recent studies (Anderson et al., 1987; Conway et al., 1989; Conway and McDowell Capuzzo, 1991) suggest that intact invertebratechemoautotroph symbioses may obtain their entire carbon budgets and much of their nitrogen requirements through bacterial autotrophy. The endosymbiotic bacteria, in turn, appear to be provided with both a sheltered environment and the inorganic compounds necessary for chemoautotrophy (CO2, 02 and reduced sulfur compounds). *Author to whom correspondence should be addressed. Present address: 515 Hastings St, Pittsburgh, PA 15206, U.S.A.
The invertebrate--chemoautotroph symbioses discovered to date generally occur in either inaccessible environments, such as hydrothermal vents, or involve relatively obscure species. Consequently, little information is available regarding the basic biology of the host species, and the design and interpretation of experiments to elucidate the interactions between bacteria and host is difficult. In this laboratory, we have been investigating the trophic interactions within animal-bacterial symbioses using the shallowwater protobranch bivalve Solemya velum as a general model of this type of animal-bacterial symbioses (Conway et al., 1989; Conway, 1990; Conway and McDowell Capuzzo, 1990, 1991). In order to determine basic differences in physiology between the S. velum symbiosis and symbiont-free clams occupying similar habitats, and to provide background data regarding the biology of this species, we have conducted a detailed examination of the biochemical composition and stable isotope ratios of S. velum and the soft-shelled clam Mya arenaria, a bivalve lacking symbionts. We report here the results of a comparative study of the amino acid composition of S. velum and the symbiont-free species Mya arenaria collected from the same subtidal reducing sediments, in order to characterize basic differences in the biochemical composition of S. velum which may be attributed to endosymbiont activity. In addition we compare the stable isotope ratios of the amino acid pools of both species. Stable isotope ratios are important biochemical markers which have been used to examine trophic exchanges in marine ecosystems and to
175
176
NOELLETTE M. CONWAY and JUDITH E. McDOWELL CAPUZZO
estimate the c o n t r i b u t i o n o f bacterial symbionts to host n u t r i t i o n (Fry a n d Sherr, 1984; Rau, 1985; Spiro et al., 1986; C o n w a y et al., 1989). D e t e r m i n i n g the stable isotope ratios o f the a m i n o acids of S. velum should provide i n f o r m a t i o n o n the c a r b o n a n d nitrogen sources of these molecules. MATERIALS AND METHODS Animal collection Specimens of Solemya velum and Mya arenaria were collected from shallow subtidal areas in Little Buttermilk Bay, Cape Cod, MA during October and November 1988. This site is characterized by organically-rich (0.5-8% organic carbon, dry wt) reducing sediments. Both species may be found within close proximity of each other. Animals were divided into gills, foot (gonads removed in S. velum) and visceral mass (defined here as all remaining soft parts excluding adductors) within 24 hr of collection and immediately frozen in liquid nitrogen. Tissues were stored at - 7 0 ° C until analysis. Phenylisothioeyanate derivitization o f amino acids The formation of a phenycarbamyl derivative of amino acids, through the use of phenylisothiocyanate (PITC, the Edman reagent) was first demonstrated by Koop et al. (1982) for the analysis of amino acids liberated by carboxypeptidase digestion of peptides from cytochrome P-450. Modifications of the technique for the derivatization of free amino acids released by the acid hydrolysis of pure proteins, and subsequent high pressure liquid chromatography (HPLC) analysis are outlined by Bidlingmeyer et al. (1984), Henrickson and Meredith (1984) and Yang and Sepulveda (1985). Advantages of this technique over traditional ionexchange and o-phthalaldehyde derivitization methods induce the formation of stable reaction products with all primary and secondary amino acids, and the presence of a linear relationship between the concentration of the amino acid derivative and the electrical signal of the detector. Detection limits are in the picomolar range. Amino acid standards, PITC, triethylamine (TEA) and glacial acetic acid were purchased from Sigma; HPLC grade solvents and water were obtained from Burdick and Jackson. All glassware was washed in a strong dichromic acid solution, rinsed I0 times in water and sonically cleaned with distilled water, methanol and acetone prior to use. Total hydrolyzable amino acids (THAAs). For THAA analysis, subsamples of each tissue fraction (1 I0 mg) were placed in tared 10 ml ampoules and the wet wt was determined. Two milliliters of 6 N HCI were added to each ampoule along with known amounts of the internal standards norieucine and gamma aminobutyric acid (GABA). Each ampoule was flushed successively with nitrogen and evacuated under vacuum to remove oxygen. Ampoules were sealed, kept at 110°C for 36 hr and subsequently frozen at -20°C prior to PITC derivatization. Conversion of asparagine and glutamine to aspartic and glutamic acids occurs during hydrolysis, and yields of serine, tryptophan and threonine are reduced. Free amino acids (FAAs). Subsamples (5-30 mg) of each tissue fraction were placed in tared 5 ml homogenization tubes, and 2ml of 5% trichloroacetic acid (TCA) in ethanol:water (50:50, v:v) was added. The samples were homogenized after the addition of internal standards and sealed under nitrogen. The free amino acids were extracted for 36 hr; each sample was then filtered through 0.45/~m PTFE filters and stored at - 2 0 ° C until derivatization. For PITC derivatization, THAA and FAA samples were transferred to 5 ml derivatization vials and the HCI and TCA removed under vacuum. One hundred microliters of ethanolic solution (ethanol:water:TEA = 1:1 : 1) were added to each vial and removed under vacuum, to ensure com-
plete removal of HCI and other reagents. Derivatization to PITC amino acids was achieved by adding 330-550/.tl of PITC solution to the sample (ethanol:water:TEA:PITC = 7:1:2:1, made freshly daily to prevent the formation of PITC degradation products; PITC was stored at - 2 0 ° C under nitrogen). After 15min, the PITC solution was removed under vacuum; the sample was then diluted with I-2 ml of eluent A (0.03% sodium acetate and 0.005% TEA in 6% acetonitrile in water), filtered through a 0.22 #m PFTE syringe filter and analyzed for PITC amino acids. PITC amino acids were separated on a Beckman high resolution C~8 reverse-phase column at 48°C and analyzed at 254 nm using a flow rate of 0.8 ml/min and the gradient program described below. Eluent A--0.03% sodium acetate and 0.005% TEA in 6% acetonitrile in water. The pH of this eluent significantly affects the retention time of many peaks. Optimum pH was found to be in the range 6.15~.25. Eluent B--50% acetonitrile in water. The gradient programmer was programmed to deliver the eluents to the Varian 2010 pump in the following program at a rate of 0.8 ml/min. Eluent A (%)
Eluent B (%)
100 65 20 0 0 100 100
0 35 80 100 100 0 0
Time Start In 20 min In 10 min In 1 min Hold for 10 min In 5 min Hold for 20 min (to re-equilibrate column)
Amino acids were identified and quantified by comparison with the retention times and response factors determined for amino acid standards and the internal standards. Blanks were run of each derivatization, and any amino acid concentrations in the blanks were subtracted from the sample concentrations. Typically the blank values accounted for less than 1% of the sample values. Total protein and carbohydrate determination Gills, foot, and visceral mass tissue fractions of Solemya velum and Mya arenaria were dissected and dried to constant weight at 60°C. Five to I0 mg of tissue were ground to a fine powder and homogenized with 2 ml of HPLC-grade distilled water; 500#1 aliquots of the homogenate were used for each total protein and carbohydrate determination. Total protein was estimated using the biuret assay, with bovine serum albumin as the standard; carbohydrate was determined using the H2SO4/phenol technique, with D-glucose as the standard (Raymont, 1964). Stable isotope analyses For 613C and 615N measurements, filtered, TCAextracted or HCl-hydrolyzed samples from each species were evaporated under nitrogen and combusted at 900°C, followed by cryogenic combustion of CO2 and N2 as described by Conway et al. (1989). For 634S determination of the free amino acids, TCA extracts of five specimens of S. velum were combined, passed through an activated copper column to remove any elemental sulfur, and concentrated by evaporation of solvents under nitrogen; HPLC analyses of this eluent demonstrated that taurine was not removed by this procedure. Samples were combusted to sulfate at 590°C for 12 hr. The sulfate was precipitated with barium and subsequently decomposed to yield pure SO2 for isotopic analysis (Yanigusawa and Sakai, 1983). Isotope measurements were determined using a Finnigan 251 ratio mass spectrometer in the laboratory of Dr Brian Fry, MBL Ecosystems Center, Woods Hole, MA. Standards were high purity gases from commercial cylinders calibrated
H i g h t a u r i n e levels in Solemya velum a g a i n s t N a t i o n a l B u r e a u o f S t a n d a r d s reference materials. T h e 613C, 6*~N a n d 6~4S r a t i o s are reported relative to Pee Dee Belemnite (PDB), n i t r o g e n in air, a n d C a n y o n D i a b l o Troilite ( C D T ) , respectively, using the s t a n d a r d delta notation: ¢~X ---- [(Rsample/Rstandard ) - -
1] x 10~,
where X = t3C, ~SN, or ~4S a n d R = ~3C/~2C, ~N/~4N, or ~2S/3~ S.
177
Table 1. Comparative aspects of the biochemical composition of S. velum and M. arenaria; means + SD, N = 5 Parameter
Gill
Foot
Visceral mass
75 + 6.9 40.9_+2.0 11.5 _+0.9 2.3 76.0 _+ I 1 8.1 + 1.2
72 + 6.9 ND ND ND 73.0 _+ 8.0 8.1 + 2.2
78 + 2.6 39.9* 10.5" ND 65.3 + 0.2 11.8 _+0.2
79 + 0.4
Solemya velum % Water 78 + 4.7 % C 41.8+2.0 % N 9.6 5- 0.5 % S 1.9 % Protein 85 + 3.0 % Carbohydrate 9.0 + 3.0
Mya arenaria
RESULTS
Overall there were few discernible differences between the gross biochemical composition of the two species (Table 1). Although samples of the foot and viscera of Mya arenaria appeared to have slightly greater carbohydrate levels than those of Solemya velum, these differences were not significant (Student's t-test). The protein levels of the foot and visceral mass of M. arenaria were greater than those of the gills. This is not surprising considering the 20-
% Water 82 _+ 2.5 % C % N % S % Protein 53.3 +_4.0 % Carbohydrate 6.1 + 0.7
70.0 + 2.8 13.3 _ 2.0
*Combined values for all tissues.
greater lipid levels found in the gills of this species. The high gill protein levels found in S. velum are in all probability artifacts caused by the interference of
a) Solemya velum •
15
Gill
[] Foot
.<
0305-0491/92 $5.00 + 0.00 © 1992Pergamon Press Ltd
HIGH TAURINE LEVELS IN THE S O L E M Y A V E L U M SYMBIOSIS NOELLETTE M. CONWAY* and JUDITH E. McDOWELL CAPUZZO Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, U.S.A. (Received 25 July 1991) Abstraet--l. To compare biochemical differences between bivalves with and without endosymbiotic
chemoautotrophic bacteria, specimens of Solemya velum, a bivalve species known to contain bacterial endosymbionts, and the symbiont-free soft-shelled clam Mya arenaria, were collected from the same subtidal reducing sediments during October and November 1988. 2. Total and free amino acid compositions were determined for both species. Protein-bound amino acids were calculated as the difference between total and free amino acids. In addition, stable isotope ratios of the total and free amino acids of each species were measured to determine potential sources for these molecules. 3. Both species had similar total hydrolyzable- and protein-bound amino acid compositions; approximately 50% of the protein-bound amino acids were essential amino acids. In S. velum, the small size of the digestive system suggests that these amino acids are probably synthesized by the endosymbiotic bacteria and translocated to the animal tissue. The 6 ~3C and 6~5N ratios of the amino acids are very similar to the isotope ratios previously found in both the endosymbionts and whole tissues of S. velum. The relative and absolute amounts of free amino acids are very different in the two species. In S. velum, the absolute concentrations of taurine, a sulfur-containing amino acid, were greater than the total free amino acid concentrations found in other bivalves. 4. The ~34S ratios of the free amino acids of S. velum, which were predominantly composed of taurine, were extremely negative ( - 17.29'oo)suggesting that taurine is synthesized using sulfur originally derived from external reduced sulfur sources, such as pore water sulfides. The possible roles for taurine in this animal-bacteria symbiosis are discussed.
INTRODUCTION Chemoautotrophic bacteria occur as symbionts in at least four marine invertebrate phyla, including molluscs, annelids, pogonophoran and vestimentiferan worms (Cavanaugh et al., 1981; Feibeck et al., 1981; Giere et al., 1984; Southward et al., 1981). In most cases, the symbiotic bacteria derive their cellular carbon from the fixation of CO2 and their energy from the oxidation of reduced sulfur compounds, such as H2S, S°, and $2O2- (Anderson et aL, 1987; Chen et al., 1987; Hand, 1987; Giere et al., 1988); however, species capable of growth on methane have also been reported (Childress et al., 1986; Cavanaugh et al., 1987; Schmaljohann and Fliigel, 1987). Many of the host species are characterized by reduced digestive capabilities (Cavanaugh, 1983) while some are completely gutless (Reid, 1980) suggesting a trophic function for the bacterial symbionts. In this context, recent studies (Anderson et al., 1987; Conway et al., 1989; Conway and McDowell Capuzzo, 1991) suggest that intact invertebratechemoautotroph symbioses may obtain their entire carbon budgets and much of their nitrogen requirements through bacterial autotrophy. The endosymbiotic bacteria, in turn, appear to be provided with both a sheltered environment and the inorganic compounds necessary for chemoautotrophy (CO2, 02 and reduced sulfur compounds). *Author to whom correspondence should be addressed. Present address: 515 Hastings St, Pittsburgh, PA 15206, U.S.A.
The invertebrate--chemoautotroph symbioses discovered to date generally occur in either inaccessible environments, such as hydrothermal vents, or involve relatively obscure species. Consequently, little information is available regarding the basic biology of the host species, and the design and interpretation of experiments to elucidate the interactions between bacteria and host is difficult. In this laboratory, we have been investigating the trophic interactions within animal-bacterial symbioses using the shallowwater protobranch bivalve Solemya velum as a general model of this type of animal-bacterial symbioses (Conway et al., 1989; Conway, 1990; Conway and McDowell Capuzzo, 1990, 1991). In order to determine basic differences in physiology between the S. velum symbiosis and symbiont-free clams occupying similar habitats, and to provide background data regarding the biology of this species, we have conducted a detailed examination of the biochemical composition and stable isotope ratios of S. velum and the soft-shelled clam Mya arenaria, a bivalve lacking symbionts. We report here the results of a comparative study of the amino acid composition of S. velum and the symbiont-free species Mya arenaria collected from the same subtidal reducing sediments, in order to characterize basic differences in the biochemical composition of S. velum which may be attributed to endosymbiont activity. In addition we compare the stable isotope ratios of the amino acid pools of both species. Stable isotope ratios are important biochemical markers which have been used to examine trophic exchanges in marine ecosystems and to
175
176
NOELLETTE M. CONWAY and JUDITH E. McDOWELL CAPUZZO
estimate the c o n t r i b u t i o n o f bacterial symbionts to host n u t r i t i o n (Fry a n d Sherr, 1984; Rau, 1985; Spiro et al., 1986; C o n w a y et al., 1989). D e t e r m i n i n g the stable isotope ratios o f the a m i n o acids of S. velum should provide i n f o r m a t i o n o n the c a r b o n a n d nitrogen sources of these molecules. MATERIALS AND METHODS Animal collection Specimens of Solemya velum and Mya arenaria were collected from shallow subtidal areas in Little Buttermilk Bay, Cape Cod, MA during October and November 1988. This site is characterized by organically-rich (0.5-8% organic carbon, dry wt) reducing sediments. Both species may be found within close proximity of each other. Animals were divided into gills, foot (gonads removed in S. velum) and visceral mass (defined here as all remaining soft parts excluding adductors) within 24 hr of collection and immediately frozen in liquid nitrogen. Tissues were stored at - 7 0 ° C until analysis. Phenylisothioeyanate derivitization o f amino acids The formation of a phenycarbamyl derivative of amino acids, through the use of phenylisothiocyanate (PITC, the Edman reagent) was first demonstrated by Koop et al. (1982) for the analysis of amino acids liberated by carboxypeptidase digestion of peptides from cytochrome P-450. Modifications of the technique for the derivatization of free amino acids released by the acid hydrolysis of pure proteins, and subsequent high pressure liquid chromatography (HPLC) analysis are outlined by Bidlingmeyer et al. (1984), Henrickson and Meredith (1984) and Yang and Sepulveda (1985). Advantages of this technique over traditional ionexchange and o-phthalaldehyde derivitization methods induce the formation of stable reaction products with all primary and secondary amino acids, and the presence of a linear relationship between the concentration of the amino acid derivative and the electrical signal of the detector. Detection limits are in the picomolar range. Amino acid standards, PITC, triethylamine (TEA) and glacial acetic acid were purchased from Sigma; HPLC grade solvents and water were obtained from Burdick and Jackson. All glassware was washed in a strong dichromic acid solution, rinsed I0 times in water and sonically cleaned with distilled water, methanol and acetone prior to use. Total hydrolyzable amino acids (THAAs). For THAA analysis, subsamples of each tissue fraction (1 I0 mg) were placed in tared 10 ml ampoules and the wet wt was determined. Two milliliters of 6 N HCI were added to each ampoule along with known amounts of the internal standards norieucine and gamma aminobutyric acid (GABA). Each ampoule was flushed successively with nitrogen and evacuated under vacuum to remove oxygen. Ampoules were sealed, kept at 110°C for 36 hr and subsequently frozen at -20°C prior to PITC derivatization. Conversion of asparagine and glutamine to aspartic and glutamic acids occurs during hydrolysis, and yields of serine, tryptophan and threonine are reduced. Free amino acids (FAAs). Subsamples (5-30 mg) of each tissue fraction were placed in tared 5 ml homogenization tubes, and 2ml of 5% trichloroacetic acid (TCA) in ethanol:water (50:50, v:v) was added. The samples were homogenized after the addition of internal standards and sealed under nitrogen. The free amino acids were extracted for 36 hr; each sample was then filtered through 0.45/~m PTFE filters and stored at - 2 0 ° C until derivatization. For PITC derivatization, THAA and FAA samples were transferred to 5 ml derivatization vials and the HCI and TCA removed under vacuum. One hundred microliters of ethanolic solution (ethanol:water:TEA = 1:1 : 1) were added to each vial and removed under vacuum, to ensure com-
plete removal of HCI and other reagents. Derivatization to PITC amino acids was achieved by adding 330-550/.tl of PITC solution to the sample (ethanol:water:TEA:PITC = 7:1:2:1, made freshly daily to prevent the formation of PITC degradation products; PITC was stored at - 2 0 ° C under nitrogen). After 15min, the PITC solution was removed under vacuum; the sample was then diluted with I-2 ml of eluent A (0.03% sodium acetate and 0.005% TEA in 6% acetonitrile in water), filtered through a 0.22 #m PFTE syringe filter and analyzed for PITC amino acids. PITC amino acids were separated on a Beckman high resolution C~8 reverse-phase column at 48°C and analyzed at 254 nm using a flow rate of 0.8 ml/min and the gradient program described below. Eluent A--0.03% sodium acetate and 0.005% TEA in 6% acetonitrile in water. The pH of this eluent significantly affects the retention time of many peaks. Optimum pH was found to be in the range 6.15~.25. Eluent B--50% acetonitrile in water. The gradient programmer was programmed to deliver the eluents to the Varian 2010 pump in the following program at a rate of 0.8 ml/min. Eluent A (%)
Eluent B (%)
100 65 20 0 0 100 100
0 35 80 100 100 0 0
Time Start In 20 min In 10 min In 1 min Hold for 10 min In 5 min Hold for 20 min (to re-equilibrate column)
Amino acids were identified and quantified by comparison with the retention times and response factors determined for amino acid standards and the internal standards. Blanks were run of each derivatization, and any amino acid concentrations in the blanks were subtracted from the sample concentrations. Typically the blank values accounted for less than 1% of the sample values. Total protein and carbohydrate determination Gills, foot, and visceral mass tissue fractions of Solemya velum and Mya arenaria were dissected and dried to constant weight at 60°C. Five to I0 mg of tissue were ground to a fine powder and homogenized with 2 ml of HPLC-grade distilled water; 500#1 aliquots of the homogenate were used for each total protein and carbohydrate determination. Total protein was estimated using the biuret assay, with bovine serum albumin as the standard; carbohydrate was determined using the H2SO4/phenol technique, with D-glucose as the standard (Raymont, 1964). Stable isotope analyses For 613C and 615N measurements, filtered, TCAextracted or HCl-hydrolyzed samples from each species were evaporated under nitrogen and combusted at 900°C, followed by cryogenic combustion of CO2 and N2 as described by Conway et al. (1989). For 634S determination of the free amino acids, TCA extracts of five specimens of S. velum were combined, passed through an activated copper column to remove any elemental sulfur, and concentrated by evaporation of solvents under nitrogen; HPLC analyses of this eluent demonstrated that taurine was not removed by this procedure. Samples were combusted to sulfate at 590°C for 12 hr. The sulfate was precipitated with barium and subsequently decomposed to yield pure SO2 for isotopic analysis (Yanigusawa and Sakai, 1983). Isotope measurements were determined using a Finnigan 251 ratio mass spectrometer in the laboratory of Dr Brian Fry, MBL Ecosystems Center, Woods Hole, MA. Standards were high purity gases from commercial cylinders calibrated
H i g h t a u r i n e levels in Solemya velum a g a i n s t N a t i o n a l B u r e a u o f S t a n d a r d s reference materials. T h e 613C, 6*~N a n d 6~4S r a t i o s are reported relative to Pee Dee Belemnite (PDB), n i t r o g e n in air, a n d C a n y o n D i a b l o Troilite ( C D T ) , respectively, using the s t a n d a r d delta notation: ¢~X ---- [(Rsample/Rstandard ) - -
1] x 10~,
where X = t3C, ~SN, or ~4S a n d R = ~3C/~2C, ~N/~4N, or ~2S/3~ S.
177
Table 1. Comparative aspects of the biochemical composition of S. velum and M. arenaria; means + SD, N = 5 Parameter
Gill
Foot
Visceral mass
75 + 6.9 40.9_+2.0 11.5 _+0.9 2.3 76.0 _+ I 1 8.1 + 1.2
72 + 6.9 ND ND ND 73.0 _+ 8.0 8.1 + 2.2
78 + 2.6 39.9* 10.5" ND 65.3 + 0.2 11.8 _+0.2
79 + 0.4
Solemya velum % Water 78 + 4.7 % C 41.8+2.0 % N 9.6 5- 0.5 % S 1.9 % Protein 85 + 3.0 % Carbohydrate 9.0 + 3.0
Mya arenaria
RESULTS
Overall there were few discernible differences between the gross biochemical composition of the two species (Table 1). Although samples of the foot and viscera of Mya arenaria appeared to have slightly greater carbohydrate levels than those of Solemya velum, these differences were not significant (Student's t-test). The protein levels of the foot and visceral mass of M. arenaria were greater than those of the gills. This is not surprising considering the 20-
% Water 82 _+ 2.5 % C % N % S % Protein 53.3 +_4.0 % Carbohydrate 6.1 + 0.7
70.0 + 2.8 13.3 _ 2.0
*Combined values for all tissues.
greater lipid levels found in the gills of this species. The high gill protein levels found in S. velum are in all probability artifacts caused by the interference of
a) Solemya velum •
15
Gill
[] Foot
.<
