Development of a Saxidomus purpuratus (mollusca: bivalvia) egg-specific antibody for the quantification of eggs using an enzyme-linked immunosorbent assay.

ABSTRACT We developed a polyclonal antibody from egg proteins of the butter clam, Saxidomus purpuratus, to quantify eggs using an enzyme-linked immunosorbent assay (ELISA). SDS-PAGE showed that the egg protein was composed of several peptides with molecular weights of 247, 200, 99, 91, 54 and 47 kDa. An immunoblotting assay indicated that the egg-specific antibodies were developed from the 200 and 99 kDa peptides. The rabbit anticlam egg IgG was able to detect as little as 0.078 [micro]g/mL of S. purpuratus egg protein by ELISA. A weight-normalized gonadosomatic index (GSI, mg dry egg/mg dry tissue) was determined to follow the monthly changes in egg production of the clams. Clam-egg protein could be detected by ELISA during the entire sampling period (December to July), and GSI ranged from 0.035 (December) to 0.156 (February). The fecundity of the clams was measured from two spawning peaks in February and May and was estimated to be 22.6 million eggs (GSI of 0.16) in February and 16 million eggs in May (GSI of 0.15). The indirect ELISA used in this study was rapid, affordable and sensitive enough to assess minute amounts of egg protein, which is difficult to measure using traditional methods such as induced spawning or histology.

KEY WORDS: Saxidomus purpuratus, immunoassay, reproduction, egg, ELISA, butter clam

INTRODUCTION

The butter clam, Saxidomus purpuratus Sowerby 1852, is a highly valued shellfish species commonly found on subtidal, silty-sand substrates along the southern and western coasts of Korea. S. purpuratus is commercially harvested in small bays on the southeast coast of Korea and supports the local shellfish industry. By 1999, annual landings of this clam in Korea had reached approximately 9,000 metric tons (Kim et al. 2001). Over the past several years, however, clam landings have been declining, and intensive clam fishing is considered to be one of the reasons for the current decline.

To protect wild shellfish populations from the effects of heavy fishing, marine spats are often artificially produced in hatcheries and released into suitable habitats to enhance natural populations (Loosanoff & Davis 1963, South Sea Fisheries Institute 2002). Baseline reproductive information such as timing of spawning or quantity of eggs produced during spawning is crucial for the development of an artificial breeding program (Galsoff 1964, Pillay 1990, Massapina et al. 1999). In particular, an understanding of the quantitative aspect of reproduction (i.e., reproductive output or fecundity), and an understanding of the life history of the target species, are critical to hatchery operations (Choi et al. 1993, Powell et al. 1994, Hyun et al. 2001, MOMAF 2001).

The reproductive effort of marine bivalves has been assessed using various methods. The number of gametes or the area occupied by the gametes is often measured from histologic slides (Tirado & Salas 1998, Ceballos-Vazquez et al. 2000). The reproductive effort has been estimated from the difference between the body weight of an individual before and after spawning (Deslous-Paoli & Heral 1988, Tirado & Salas 1998). Gravid females are often induced to spawn by various spawning stimulants, and the number of eggs released from each individual is directly counted or weighed (Galinou-Mitsoudi & Sinis 1994, Chung et al. 2001). However, these quantitative methods may underestimate the true quantity of eggs because spawning is incomplete in most cases and occurs several times with different intensities during the reproductive season (Kang et al. 2003, Lucas 1982, Ahn 2001).

Immunologic techniques have also been used to quantify the reproductive output of marine bivalves. Choi et al. (1993, 1994) developed an egg-specific polyclonal antibody to the American oyster, Crassostrea virginica, and the quantity of egg protein per oyster was then assessed using this egg-specific antibody as a primary antibody in indirect ELISA. The technique was sensitive enough to measure the reproductive output from a very small quantity of egg protein remaining in some oysters collected in winter (Choi et al. 1993). Kang et al. (2003) developed antibodies to the Pacific oyster (C. gigas) to study the reproductive effort during an annual reproductive cycle. Recently, Park & Choi (2004) used ELISA to measure the reproductive output of the Manila clam, Ruditapes philippinarum, with a polyclonal antibody derived from the clam eggs. Because of its rapidity and high sensitivity, this immunoassay is considered to be the method of choice for the quantification of bivalve reproductive output (Kang et al. 2003, Park & Choi 2004).

In the present study, we developed a polyclonal antibody to eggs of the butter clam to measure the quantity of eggs in individual clams. Using this antibody and ELISA, we assessed the reproductive effort of clams collected from a major clam bed. Here we report the seasonal changes in the reproductive effort of clams and the immunologic methods used to evaluate the reproductive effort.

MATERIALS AND METHODS

Sampling Effort

Butter clams, S. purpuratus, were collected from Geoje-Do Island on the southern coast of Korea on a monthly basis by SCUBA from December 2001 to June 2002 (Fig. 1). After recording shell length (SL) and wet body weight (TWWT), individual clam bodies containing gonads were freeze-dried, weighed and kept at -70[degrees]C until further use.

[FIGURE 1 OMITTED]

Purification of Eggs

During the spawning season in May 2002, the visceral masses containing ripe eggs were excised from the collected clams and gently squeezed to release eggs onto a Petri dish. The crude egg extracts were diluted in filtered seawater containing 0.02 mM N[H.sub.4] OH to prevent aggregation of the eggs. The extracts were then filtered successively through 100- and 63-[micro]m mesh screens to remove impurities. The filtered eggs were washed several times by centrifuging the eggs at x 100g for 10 min, and the number of eggs in the filtrate was then counted under a microscope using a hemocytometer. A known quantity of eggs was freeze-dried and weighed to determine the dry weight of an individual egg.

Proximate Composition of the Eggs

The protein concentration of the purified egg extracts was determined using a BCA Protein Assay Kit with bovine serum albumin (BSA) as the standard. The total carbohydrate in the extract was determined according to Taylor (1995) using dextrose anhydrose as the standard. The total lipid concentration of the eggs was determined by the sulfuric acid-charring technique of Marsh & Weinstein (1966) using tripalmitin as the standard after extracting lipids according to the methods of Bligh and Dyer (1959).

Development of a Clam Egg-specific Antibody

A New Zealand white rabbit was selected as the host animal to raise the antibody to S. purpuratus egg protein. The rabbit was injected subcutaneously with 0.5 mL of 500-[micro]g/mL egg protein mixed in an equal volume of Freund complete adjuvant (FCA). After the initial injection, the rabbit received 0.5 mL of a 250 [micro]g/mL egg protein emulsification in 0.5 mL Freund incomplete adjuvant (FIA) on a biweekly basis over 6 wk. Ten milliliters of blood were withdrawn from the rabbit in the fourth week to examine the specificity and titer of the antiserum. Twenty milliliters of rabbit blood were withdrawn from an ear vein 1 wk after the final injection when the titer of the antiserum was high enough for use in this study. Rabbit anticlam egg IgG was isolated by ammonium sulfate precipitation and dialysis against PBS.

Specificity of the rabbit antibody was investigated first by using a double immunodiffusion test according to Ouchterlony & Nilsson (1978). For this test, we prepared 1.5% agarose in PBS and 4.5 mL of the gel were cast on a 75 x 25-mm glass slide. After solidifying the gel at 4[degrees]C for 20 min, a hexagonal pattern of 2-mm diameter wells was arranged on the plate. A 20-[micro]L aliquot of the rabbit antiserum was added to the central well, and 20-[micro]L aliquots of positive and negative controls were placed in the wells around the central well. Purified clam egg protein was used as a positive control, and protein extracts of somatic tissues such as gills, mantle, labial palps and adductor muscle were used as negative controls. The immunodiffusion slide was then incubated for 24 h in a humidified chamber at 4[degrees]C, dried, stained with 0.25% Coomassie Brilliant Blue and de-stained in 40% methanol containing 7% acetic acid. A positive antibody-antigen reaction was identified as precipitin lines formed between the serum and the protein preparations.

The rabbit antiserum initially exhibited a weak but recognizable cross-reaction to the somatic tissues in the double immuno-diffusion test. The cross-reacting antibodies in the serum were then removed using an affinity column prepared according to Fuchs & Sela (1979). Immuno-adsorbents packed in the column were prepared by cross-linking clam somatic tissue extracts with glutaraldehyde. To remove the cross-reacting antibodies, the antiserum was added onto the column packed with the immuno-adsorbent and incubated at room temperature for several hours. The column was then centrifuged to harvest the egg-specific antibody in the supernatant. The egg-specific antibody was then precipitated using saturated ammonium sulfate. Specificity of the antibody was tested again by indirect ELISA using alkaline phosphatase-labeled goat antirabbit IgG as a secondary antibody. No cross-reaction to the somatic protein was demonstrated from the egg-specific antibody by ELISA.

SDS-PAGE and Immunoblotting

Egg protein homogenates were precipitated with acetone, and the quantity was measured using a BCA Protein Assay Kit (Pierce, USA). Egg protein was loaded into a 10% SDS-polyacrylamide gel and electrophoresed (SDS-PAGE) according to LaemmLi (1970). For SDS-PAGE, the egg protein was treated with 5% [beta]-mercaptoethanol by boiling (i.e., under reducing conditions). After electrophoresis, protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane at 100 V for 60 min using a Mini TransBlot Transfer Cell. The membrane was blocked in 1% BSA in PBST and incubated in 1/200 diluted rabbit anticlam egg IgG for 1 h. The blots were then incubated with goat antirabbit IgG as the second antibody conjugated to horseradish peroxidase (Sigma, 1:1000 dilution) for 1 h at room temperature. The blots were developed using FAST DAB enzyme substrate to visualize the antibody-antigen reaction.

Immunofluorescence Assay

Fully mature female clam gonadal tissue was fixed in Davidson fixative, embedded in paraffin, sliced to a thickness of 6 [micro]m and mounted onto a glass slide. The tissue was rehydrated through a graded ethanol series and equilibrated in 0.15 M PBS containing 0.05% Triton X-100 (PBST). After blocking the slide with 1% BSA dissolved in PBST for 30 min at room temperature, the rabbit anticlam egg IgG (10 [micro]/mL) diluted with the blocking buffer as a primary antibody was reacted with the tissue for 30 min. The slide was then washed with PBST for 30 min and reacted with FITC conjugated goat antirabbit IgG as a secondary antibody. The slides were finally washed with PBST for 30 min and mounted in a glycerol-PBS solution (1:1). The antibody-stained tissue was then observed under a fluorescence microscope (Olympus).

Quantification of Clam Eggs Using ELISA

Indirect ELISA was used for the quantification of clam eggs. The rabbit anticlam egg IgG developed in this study was used as the primary antibody, and alkaline phosphatase-labeled goat anti-rabbit IgG (Sigma) was used as the secondary antibody in ELISA. To quantify the eggs, triplicates of 100 [micro]L of each tissue extract dissolved in 0.15 M PBS (pH 7.5) and duplicates of the standard solution (i.e., S. purpuratus egg extract at 0.156-5 [micro]g/mL in 0.15 M PBS, pH 7.5) were included in a 96-well ELISA microplate. The optical density (OD) of the antibody-antigen complex developed in ELISA was read at 405 nm using a microplate spectrophotometer. A standard regression curve was plotted based on the OD of the standards and their protein concentration. The concentration of egg protein in each clam was then estimated from the curve. Finally, a weight-based gonado-somatic index (GSI) and fecundity (i.e., number of eggs per clam) were estimated from the following relationships: (1) GSI = quantity of egg protein estimated from ELISA x 2.67/total dry weight (TDWT), where the constant 2.67 is the conversion factor of egg protein to egg dry weight; (2) Fecundity = quantity of eggs estimated from ELISA/ weight of a single egg (95 ng).

RESULTS

Biometric and Biochemical Characteristics of Butter Clam Eggs

The eggs initially aggregated because of the gelatinous outer membrane; however, aggregation was successfully dissociated by the application of 0.02 mM N[H.sub.4]OH in filtered seawater. Impurities in the extract were removed by successively passing the egg extract through 100- and 63-[micro]m mesh screens followed by several washings by low speed centrifugation. Table 1 summarizes the biometric and biochemical features of the eggs estimated from the purified eggs. Sizes of the eggs varied from 70.81 [+ or -] 7.52 [micro]m in histologic preparations to 88.56 [+ or -] 11.31 [micro]m in an intact condition. The dry weight of an individual egg was estimated to be 95 ng, which was somewhat heavier than the egg weights reported for other venerid clams (Table 1). Protein was a major component of the eggs and accounted for 37.4% of the dry weight, whereas lipid and carbohydrate comprised 11.4 and 9.6% of the dry weight, respectively.

SDS-PAGE and Western Blotting of S. purpuratus Egg Protein

Figure 2 shows the results of electrophoresing the egg protein in a reduced condition and the subsequent results of Western blotting. The electrophoretic analysis revealed that S. purpuratus egg protein is composed of six major peptides with molecular masses of 247, 200, 99, 91, 54 and 47 kDa. Immunoblotting of the egg protein using S. purpuratus egg-specific antibody developed in this study revealed that the 200 and 99 kDa peptides were the eggspecific proteins that induced antibody production.

[FIGURE 2 OMITTED]

Specificity of the Antibody

Table 2 shows the specificity test results for the clam egg-specific antibody developed in this study. After eliminating cross-reactivity using the immuno-adsorbent, the rabbit anticlam egg IgG did not exhibit any cross-reaction to the somatic tissues. In ELISA, the rabbit IgG detected <78 ng - 20 [micro]g/mL of S. purpuratus egg protein. The immunofluorescence assay conducted on histologic sections of mature eggs also confirmed the specificity of the IgG; only eggs were stained with the antibody, indicating that the antibody was specific to eggs (Fig. 3).

[FIGURE 3 OMITTED]

Reproductive Output Estimated by ELISA

The rabbit anticlam egg IgG detected as little as 0.078 [micro]g/mL of S. purpuratus egg protein by ELISA. Table 3 and Figure 4 summarize the monthly changes in the reproductive output of S. purpuratus as measured by ELISA from December 2001 to June 2002. Most of the clams used in this analysis were >80 mm in size and were estimated to be between 3 and 5 y old (i.e., old enough to reproduce sexually). The quantity of clam eggs measured with ELISA was weight-normalized and expressed as the GSI. The monthly mean GSI varied from 0.035 (December 2001) to 0.156 (February 2002). The maximum GSI observed in this study was 0.268, which was recorded in May, suggesting that butter clams in the study area produced as much as 26.8% of their body weight in eggs. Fecundity of the clams was also estimated by dividing the number of eggs measured with ELISA by the weight of a single egg (95 ng) estimated in this study (Table 3). Our data indicate that clams may produce 0.9-22 million eggs (February 2002) during spawning.

[FIGURE 4 OMITTED]

DISCUSSION

Because of their high specificity and sensitivity, immunologic techniques have been widely applied in various fields of marine biology, including pathology (Bernoth 1990, Choi et al. 1991, Sitja-Bobadilla & Woo 1994, Parker & Lewis 1995, Anguilera et al. 1996, Montes et al. 1996), predator-prey analysis (Fichter & Stephen 1981, Theilacker et al. 1986, Pierce et al. 1990), identification of larval organisms (Demers et al. 1993, Paugam et al. 2000), and reproductive biology (Quackenbush 1989a, Quackenbush 1989b, Suzuki et al. 1992). In particular, ELISA has been adapted as the method of choice for quantification of oyster and clam eggs (Choi et al. 1993, Choi et al. 1994, Kang et al. 2003, Park & Choi 2004). From a practical aspect, ELISA is rapid and sensitive enough to measure the very small quantity of egg protein remaining in clams or oysters collected even in winter.

The rabbit antiS, purpuratus egg protein antibody developed in this study was able to detect as little as 0.156 [micro]g/mL of egg-specific protein by ELISA, which indicated that ELISA is sensitive enough to measure the minute quantities of egg protein present in clam tissues. Table 3 shows that the lowest GSI estimated in this study was 0.002, implying that ELISA, in conjunction with the antibody developed in this study, can provide measurements even when eggs represent <1% of the body weight. Other studies using egg-specific antibodies in ELISA also reported similar detection ranges of antibodies: rabbit antiC, virginica egg IgG detected 0.2-10 [micro]g/mL of egg protein with ELISA (Choi et al. 1993), rabbit antiC, gigas egg IgG detected 0.01-0.3 [micro]g/mL of the egg protein (Kang et al. 2003), and rabbit antiR, philippinarum egg IgG detected 0.23-15 [micro]g/mL of the clam egg protein by ELISA (Park & Choi 2004). These studies also reported that ELISA detected as little as 1% of egg protein relative to the total body weight, as was observed in this study.

Western blot analysis suggests that the antigenic determinants inducing the egg-specific antibody in this study are large molecular weight peptides with molecular sizes of 200 and 99 kDa. These large molecules in animal eggs, called vitellins, are a major constituent of yolk granules and provide nutrition during development (Shafir et al. 1992). Vitellin-like high-molecular egg-specific peptides have been reported from other marine bivalves. Lee & Heffernan (1991) reported that egg proteins of M. mercenaria are composed of several peptides with molecular weights of 98, 87, 68, 60, 56, 36 and 19 kDa. Park & Choi (2004) reported that egg proteins of R. philippinarum consist of several peptides with molecular masses of 330, 96, 64, 50 and 31 kDa. Accordingly, electrophoretic profiles of vitellins may vary among species.

The rabbit antibutter clam egg antibody developed in this study initially exhibited cross-reactions to nongonadal tissues such as gill, mantle and adductor muscles. Such cross-reactions were also observed in polyclonal antibodies raised from other marine bivalve eggs. Polyclonal antibodies derived from Pacific oyster eggs initially showed a minor but recognizable positive reaction to proteins extracted from nongonadal tissues such as gills, mantle and adductor muscle (Kang et al. 2003). Park & Choi (2004) also reported weak but noticeable cross-reactions of rabbit antiManila clam egg antibody to nongonadal protein extracted from gills and mantles. The antigenic determinants of cross-reacting antibodies observed in these studies may be peptides of smaller molecular sizes commonly occurring in both somatic tissues and eggs, as observed in oyster eggs (Choi et al. 1993, Kang et al. 2003). Indeed, Renwrantz et al. (1998) reported the occurrence of similar sized peptides in egg and serum samples of Mytilus edulis by electropboresis. Suzuki et al. (1992), Gagne et al. (2001) and Matozzo & Marin (2005) also reported common peptides present both in the eggs and hemolymph or somatic tissues of marine bivalves. The occurrence of the common peptides in the eggs as well as nongonadal tissues can be attributed to RNA synthesis in oocytes during gametogenesis as well as transportation of nutritive substances to eggs from somatic tissues by hemolymph (Dohmen 1983, Gagne et al. 2001).

Microscopic examination of gonadal tissues showed that S. purpuratus carries sexually mature eggs as early as February, indicating that this species is capable of spawning during the winter season. Figure 4 shows that the GSI gradually increased from December, reached a peak in February and dropped in April. Another GSI peak that was observed in late May dropped in late June, suggesting that this clam spawns at least twice between December and June. By comparing the GSI between the peak and postpeak periods, we estimated that the egg mass released during spawning represented 7.5% to 13.7% of the body weight of these clams. This value is two to three times lower than the values reported for other marine bivalves. In the case of the mussel M. edulis, the eggs released during spawning account for 38% to 52% (Kautsky 1982), 50% to 59% (Thompson 1979) or 50% to 60% (Griffiths 1977) of the body weight. By calculating the weight loss before and after spawning, Deslous-Paoli and He (1988) estimated that the quantity of eggs that C. gigas releases during spawning represented 43.1% to 61.9% of the body weight. In contrast, Park & Choi (2004) reported that the quantity of eggs released from spawning R. philippinarum comprised 6.5% to 14.3% of the body weight by calculating the difference between the GSI before and after spawning. Although this estimation was much lower than the values reported from oysters or mussels, it is similar to the value estimated in our study. The wide-ranging values among the reported amount of eggs released during spawning for different bivalves can be partially explained by different methods used in the estimation and different reproductive traits of the various bivalve species (Strathmann & Strathmann 1982, Choi et al. 1993, Park & Choi 2004).

Relatively few studies have investigated the measurement of fecundity in venerid clams, including the butter clam (Table 4). Dietrich et al. (2002) reported that the hard clam M. mercenaria releases approximately 30,000,000 eggs during the spawning season. The fecundity of R. philippinarum, a small venerid clam, has been reported to be 0.20-1.79 (Chung et al. 2001) or 0.94-11.0 million eggs (Park & Choi 2004). The number of eggs present in a sexually mature butter clam was estimated by directly counting the eggs excised from the gonads (Y.-S. Choi personal, communication). According to Choi, S. purpuratus produces as many as 20,000,000 eggs during a spawning season. The fecundity of the clam as estimated by Choi is comparable to the fecundity estimated by ELISA in this study: 3.1-9.3 million eggs per clam, with a mean of 1.6 million. The fecundity of the butter clam estimated by direct counting is considered to be a minimum estimate, because the entire egg mass could not be separated from the body and not all of the eggs were released from the excised tissue. However, the fecundity estimated from direct counting is within the range of the fecundity estimated from ELISA in this study, which suggests that our ELISA method is reliable for measuring fecundity. ELISA, as applied to estimates of clam fecundity in this study, is considered to yield a maximum estimate because it measures all eggs in an individual clam, which may release eggs into the water several times during a spawning period (Davis & Chanley 1956, Peterson 1983, Chung & Kim 1994, Ahn 2001).

The size and weight of S. purpuratus eggs estimated in this study were larger and heavier than those of other marine bivalves. The dry weight of oyster eggs ranges from 12-13 ng (Choi et al. 1993, Kang et al. 2003). Park & Choi (2004) reported that the dry weight of a R. philippinarum egg is 22 ng. Compared with oyster and other clam eggs, S. purpuratus eggs are approximately four to eight times heavier. Lee and Heffernan (1991) reported that the dry weight of eggs of Mercenaria mercenaria, a clam species taxonomically close to S. purpuratus, is 51 ng. Our data suggest that hard clams such as S. purpuratus and M. mercenaria produce larger and heavier eggs, which may sink easily when discharged into the water column during spawning. The proximate composition of S. purpuratus eggs is similar to that of other marine bivalves; a major constituent of oyster and clam eggs is protein, which accounts for 38% to 50% of the egg weight, followed by lipids and carbohydrates.

CONCLUSION

We developed an S. purpuratus egg-specific antibody and applied it to quantify the reproductive effort of clams collected between December 2001 and July 2002. The amount of egg protein in an individual clam was assessed by ELISA in conjunction with the polyclonal antibody. The weight-based GSI and fecundity of the clam was estimated based on ELISA data. The GSI ranged from 0.035 (December) to 0.156 (February), and fecundity varied from 16 million (May) to 22.6 million (February) eggs. The indirect ELISA analysis used in this study was rapid, affordable and sensitive enough to assess minute amounts of egg protein; it is therefore considered to be the method of choice for quantifying clam eggs.

ACKNOWLEDGMENTS

The authors thank the staff of the Shellfish Research Laboratory at Cheju National University for their help in data acquisition. This study was supported by the Korea Research Council of Public Science and Technology (KORP) under the project "Eco-environmental studies for the restocking and enhancement of bivalve resources on the southern coast of Korea" (BSPG 337-00-1447-3).

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KYUNG-IL PARK, (1) JIN-WOO CHOI (2) AND KWANG-SIK CHOI (1) *

(1) School of Applied Marine Science, Cheju National University, 1 Ara 1-Dong, Jeju 690-756, Korea; (2) Environmental Science Laboratory, South Sea Institute, KORDI, 391 Jangmok, Geoje 656-830, Korea

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Biometric data and biochemical composition of marine bivalve eggs.
(histology indicates that cells were fixed and stained).

                                                            Egg
                               Egg Diameter ([micro]m)   Dry-Weight
Species                              [+ or -] SD         in ng/egg

C. virginica                            --                   12
M. mercenaria                           --                   51
C. gigas (Portuguese strain)            --                   --
C. gigas                                --                   13
R. philippinarum               60 (histology)                22
S. purpuratus                  70.81 [+ or -] 7.52           95
                                 (histology), 88.56
                                 [+ or -] 11.31
                                 (suspension)

                                Protein in       Lipids in
Species                         ng/egg, (%)     ng/egg, (%)

C. virginica                       6 (50)          2.5 (21)
M. mercenaria                     20 (40)          7 (14)
C. gigas (Portuguese strain)     (44-74)         (16-38)
C. gigas                         5.3 (41)        3.3 (25.5)
R. philippinarum                   9 (41)          --
S. purpuratus                  35.57 (37.44)   10.83 (11.40)

                               Carbohydrate
                               in ng/egg,
Species                        (%)                   Source

C. virginica                   1.1 (9)        Lee and Heffernan (1991)
M. mercenaria                    4 (8)        Lee and Heffernan (1991)
C. gigas (Portuguese strain)    (7-12)        Massapina et al. (1999)
C. gigas                       1.5 (11.7)     Kang et al. (2003)
R. philippinarum                  --          Park and Choi (2004)
S. purpuratus                  9.2 (10.83)    Present study

SD = standard deviation.

TABLE 2.
Dilution and specificity test by ELISA of rabbit anti-egg
IgG against purified egg and somatic tissues of S. purpuratus.

                        Optical Density (405 nm)
Protein Concentration
    ([micro]g/ml)        Egg     Somatic Tissue

       20.000           0.778         0.010
       10.000           0.657        -0.006
        5.000           0.452        -0.003
        2.500           0.296        -0.002
        1.250           0.191        -0.005
        0.625           0.137        -0.001
        0.313           0.086        -0.002
        0.156           0.024         0.001
        0.078           0.014        -0.004

TABLE 3.
Reproductive output (GSI, g dry egg/g dry clam) of S. purpuratus
measured by ELISA.

                      Mean Shell
                        Length
  Period     N     (mm) [+ or -] SD       Mean Fecundity [+ or -] SD

Dec 23 '01    6   89.12 [+ or -] 2.08    4,393,201 [+ or -] 4,129,279
Jan 21 '02   13   83.68 [+ or -] 4.17    8,564,612 [+ or -] 6,202,413
Feb 20 '02   10   90.20 [+ or -] 5.59   22,634,503 [+ or -] 10,355,859
Apr 02 '02   17   84.59 [+ or -] 5.82   17,191,137 [+ or -] 8,360,012
Apr 19 '02    9   90.17 [+ or -] 8.37   13,816,468 [+ or -] 6,098,517
May 20 '02   13   88.77 [+ or -] 3.91   16,931,893 [+ or -] 6,253,074
Jun 27 '02    6   67.70 [+ or -] 1.40      945,876 [+ or -] 274,291

              Highest      Lowest             Mean
  Period     Fecundity    Fecundity     GSI [+ or -] SD

Dec 23 '01   10,934,767   282,401     0.035 [+ or -] 0.030
Jan 21 '02   22,120,843   2,178,444   0.072 [+ or -] 0.042
Feb 20 '02   35,457,185   3,121,846   0.156 [+ or -] 0.076
Apr 02 '02   33,010,372   6,731,357   0.136 [+ or -] 0.063
Apr 19 '02   25,794,873   6,137,485   0.081 [+ or -] 0.036
May 20 '02   31,156,333   9,307,309   0.154 [+ or -] 0.060
Jun 27 '02   1,429,542    669,868     0.017 [+ or -] 0.006

              Highest      Lowest
  Period        GSI          GSI

Dec 23 '01     0.078        0.002
Jan 21 '02     0.147        0.033
Feb 20 '02     0.243        0.016
Apr 02 '02     0.232        0.051
Apr 19 '02     0.138        0.039
May 20 '02     0.268        0.077
Jun 27 '02     0.026        0.011

SD = standard deviation.

TABLE 4.
Reproductive output of marine venerid clams reported from
various studies.

                                       Shell
Species            Location         Length (mm)

R. philippinarum   Gomso Bay,       21.11-43.50
                     Korea

S. purpuratus      Taean, Korea         --

M. mercenaria            --             --

S. purpuratus      Geoje-Do Isl.,      86.94
                     Korea

Species                    Egg Diameter (pm)

R. philippinarum   89.57 [+ or -] 8.66 wm (suspended)
                   61.19 [+ or -] 5.66 (histology)

S. purpuratus                    --

M. mercenaria               60-85 [micro]m

S. purpuratus      88.56 [+ or -] 11.31 p.m (suspended)
                   70.81 [+ or -] 7.52 (histology)

                                           Highest
                   Estimation              Monthly
Species            Method                 Mean GS1

R. philippinarum   Immunological            0.250
                     method (ELISA)

S. purpuratus      Direct egg counting       --
                     after chopping off
                     clam body

M. mercenaria             --                 --

S. purpuratus      Immunological            0.154
                     method (ELISA)

Species                  Fecundity           Author

R. philippinarum   0.94-11 x [10.sup.6]   Park and Choi
                                            (2004)

S. purpuratus           20 x [10.sup.6]   Choi (personal
                                            communication)

M. mercenaria           30 x [10.sup.6]   Dietrich et al.
                                            (2002)

S. purpuratus         9-31 x [10.sup.6]   Present study