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Development of an immunological probe to quantify reproductive effort in the Suminoe oyster, Crassostrea ariakensis (Gould 1861).

ABSTRACT A polyclonal antibody specific to an egg protein of the Suminoe oyster, Crassostrea ariakensis, was developed to assess reproductive effort. After 2 mo of immunization, rabbit antiserum showed strong specificity to the egg protein in an enzyme-linked immunosorbent assay (ELISA). The rabbit antioyster egg immunoglobulin G detected as little as 0.2 [micro]g/mL of Suminoe oyster egg protein by ELISA. The quantity of eggs present in an oyster was estimated using ELISA and was expressed as a gonadosomatic index (GSI). Gonadosomatic index values of Suminoe oysters were assessed monthly from January to July 2007 at the Seomjin River estuary off the south coast of Korea. Histology indicated that most oysters were mature and ready to spawn by the middle of July. Mean GSI values for oysters collected in April, when most female oysters are in early developmental stages, varied between 0.6% and 14.0%. In July, most oysters were ready to spawn and GSI values ranged from 17.5-67.0%, with a mean of 47.7%. The potential fecundity of ripe oysters was determined by dividing the number of eggs, which was estimated by ELISA, by the mean dry weight of a single egg (14 ng); fecundity ranged from 162-910 million eggs. The immunological technique used in this study was affordable and sensitive enough to measure variation in the number of eggs present among gametogenic stages.

KEY WORDS: Crassostrea ariakensis, Suminoe oyster, ELISA, reproductive effort, gametogenesis, Seomjin River estuary, Korea

INTRODUCTION

The Suminoe oyster, Crassostrea ariakensis (Fujita 1913), is an estuarine bivalve that occurs commonly along the coasts of northern (Yellow River) and southern China (Zhejiang, Yangjiang, and Zhuhai), Japan (western Kyushu), and Korea (west and south coasts), from low intertidal to shallow subtidal areas (Okutani 2000, Zhou & Allen 2003, Lam & Morton 2004, Yoo et al. 2004, Zhang et al. 2005, Yoon et al. 2008). As an estuarine species, the Suminoe oyster occurs in habitats with wide temperature and salinity ranges (Zhou & Allen 2003, An et al. 2006, Joo 2006, Park 2007). Because of its fast growth and unique flavor, the Suminoe oyster has been considered a candidate for aquaculture in Korea (Yoo et al. 2004, An et al. 2006). The Suminoe oyster is also relatively resistant to the protozoan parasites Haplosporidium nelsoni and Perkinsus marinus (Calvo et al. 2001, Paynter et al. 2008), and therefore was being considered for introduction from Asian waters to the Chesapeake Bay (Allen 2000, Calvo et al. 2001, Grabowski et al. 2003). Accordingly, numerous studies have been conducted to understand the genetics, ecology, and reproduction of the Suminoe oyster in China and the United States (Perdue & Erickson 1984, Langdon & Robinson 1996, Zhang et al. 2005, Bushek et al. 2008, Tamburri et al. 2008, Wang & Guo 2008, Wang et al. 2008, Xiao et al. 2010, Xiao et al. 2011). In contrast, few studies have investigated the reproduction and ecology of the Suminoe oyster in Korean waters (Yoo et al. 2004, Joo 2006, Park 2007, Yoon et al. 2008).

Understanding the reproductive patterns of marine bivalves, such as annual reproductive cycles and the quantity of gametes produced during spawning, is important for the management of natural populations and the development of the aquaculture industry. In particular, measurement of reproductive effort (i.e., the proportion of energy or materials allocated to reproduction) is crucial for the management of broodstocks, either in natural populations or in the hatchery (see Gosling 2003). In oysters, reproductive effort is often estimated by measuring the difference in body weight prior to and after spawning or by counting the number of gametes using histology (Lucas 1982, Deslous-Paoli & Heral 1988, Choi et al. 1993, Pouvereau et al. 2000, Kang et al. 2003a, Royer et al. 2008). Immunological methods also have been applied to the quantification of marine bivalve reproductive effort. For immunological measurements, polyclonal antibodies are raised from egg proteins and reproductive effort is estimated using an enzyme-linked immunosorbent assay (ELISA) with the polyclonal antibodies (Choi et al. 1993, Kang et al. 2003b, Park & Choi 2004, Park et al. 2005, Long et al. 2008, Royer et al. 2008). Unlike histology, ELISA is a true quantitative technique that measures mass of eggs in an individual oyster or clam. Enzyme-linked immunosorbent assay is also rapid and sensitive enough to detect small quantities of egg protein remaining in some oysters during early development and during the spent stage (Choi et al. 1993, Kang et al. 2003b). Because of these advantages, ELISA is considered the method of choice in quantitative measurement of marine bivalve reproductive effort (Kang et al. 2003b, Park & Choi 2004; Park et al. 2005).

The objective of this study was to develop an immunological probe to measure the reproductive effort of Suminoe oysters during their annual reproductive cycle. The current study also estimated fecundities of Suminoe oysters during the spawning period, which is valuable for appropriate management of wild oyster populations and for facilitating hatchery operations.

MATERIALS AND METHODS

Sampling Effort

Thirty to 40 adult Suminoe oysters were collected monthly from an estuarine area of the Seomjin River on the southern coast of Korea from January to July 2007 (Table 1, Fig. 1). On arrival at the laboratory, biometric parameters of individual oysters were recorded, including shell length (SL, longest axis) and wet tissue weight. The internal shell cavity volume was determined by the water displacement method, according to Galtsoff (1964) to determine the condition index (CI). The CI was calculated according to the following equation: CI = Dry tissue weight (DTWT)/Internal shell cavity volume. Dry tissue weight was determined from the freeze-dried wet tissue (Table 1).

Histological Analysis

To examine gonadal maturation of oysters histologically, a thin longitudinal section was cut in the middle of each oyster; the section included the gonad, digestive gland, gills, and mantle (Fig. 2). The remaining tissues were lyophilized on each sampling date and were kept at -70[degrees]C. The cross-section accounted for 5-8% of an individual's total tissue weight. For histology, each cross-section was fixed in Davidson's solution (1 part of glycerin, 2 parts of formaldehyde (35%), 3 parts of ethanol (95%), 3 parts of filtered seawater, and 1 part of glacial acetic acid) for 48 h. After fixation, the tissue was dehydrated and embedded in paraffin. The paraffin block was sliced to 6 [micro]m and stained with Harris' hematoxylin and eosin Y (Sigma). The level of gonadal development was then analyzed quantitatively using the planimetric technique (Heffernan & Walker 1989, Kang et al. 2003). The image of each embedded and mounted cross-section was digitized using a scanner (Epson) connected to a personal computer. After defining the boundary of the gonad in the digitized image manually, the percent gonad area (PGA) on each slide was measured using image analysis software (Image Pro Plus). Percent gonad area was calculated as the gonadal area divided by the total cross-sectional area, excluding the gill (Fig. 2).

Purification of Eggs

Ripe eggs were harvested from fully mature market-size female oysters (n = 20, 14-23 cm in SL). Female gonadal tissues containing ripe eggs were excised using scissors and were placed on a Petri dish. The tissues were squeezed gently using a syringe piston to release the eggs from the connective tissues. The crude mature egg extract was then filtered through a 100-[micro]m mesh screen to remove tissue debris. The filtered egg suspension was transferred into a 50-mL conical tube at 4[degrees]C for 1 h, and the supernatant containing egg debris was decanted. This cleaning procedure was repeated 5 times. The purified oyster eggs were stored at -70[degrees]C until use.

Biochemical Analysis of Egg

To estimate the weight of a single egg, a known volume of the purified egg suspension was taken and the number of eggs in the suspension was counted using a hemocytometer. The same volume of eggs was then lyophilized and weighed. The total weight of the pooled eggs was divided by the estimated number of eggs to determine the approximate weight of a single egg.

Protein concentrations in purified eggs and in oyster tissues were determined using the method of Lowry et al. (1951) after alkaline hydrolysis with 0.1 M sodium hydroxide at 37[degrees]C for 2 h, using bovine serum albumin (Pierce) as the standard. Total carbohydrate values were quantified photometrically by the phenol-sulfuric acid extraction method of Taylor (1995), with dextrose anhydrase (Sigma) as a standard. Total lipids were extracted using a mixture of chloroform and methanol (Bligh & Dyer 1959) and were measured gravimetrically. Ash content was obtained by igniting a subsample (100 [mg]) of homogenized tissue in a muffle furnace at 450[degrees]C for 24 h. The estimates of total protein, lipid, carbohydrate and ash content were expressed as milligrams divided by the DTWT.

Development and Purification of Antibody

The development of the rabbit antioyster egg immunoglobulin G (IgG) followed the protocol outlined by Kang et al. (2003b) and Park and Choi (2004). In brief, a New Zealand white rabbit was selected to produce the antibody. The rabbit was injected subcutaneously with 0.5 mL 500 [micro]g/mL egg protein mixed in an equal volume of Freund's complete adjuvant. After the first injection, the rabbit received 0.5 mL of a 250-[micro]g/mL egg protein emulsification in 0.5 mL Freund's incomplete adjuvant biweekly for 6 wk. Twenty milliliters of blood was withdrawn from an ear vein 2 wk after the final injection.

Enzyme-Linked Immunosorbent Assay Analysis

Specificity of the rabbit antibody was tested using a double immunodiffusion test (Ouchterlony & Nilsson 1973) and ELISA. In the tests, Suminoe oyster egg protein was used as a positive control, and protein extracts of somatic tissues such as gills, mantle, labial palp, and adductor muscle were used as negative controls. Enzyme-linked immunosorbent assay indicated that the antibody developed in this study exhibited a very weak but detectable cross-reaction to nongonadal tissue proteins. To remove the nonspecific antibodies, an immunoadsorbent was prepared by polymerizing sexually undifferentiated oyster tissue using glutaric dialdehyde according to Fuchs and Sela (1973). Twenty milliliters of the rabbit antiserum was mixed with an equal volume of the immunoadsorbent and incubated for 3 h at room temperature. Nonspecific antibodies in the antiserum bound to the surface of the immunoadsorbent particles during incubation whereas the egg-specific antibody remained free. The oyster egg-specific antibody was then isolated from the unbound fraction of the antiserum by centrifugation. The rabbit antioyster egg IgG in the supernatant was precipitated using 50% saturated ammonium sulfate, and the precipitate was dissolved and dialyzed in phosphate-buffered saline (PBS; 0.15 M NaCl, 0.01 M Na[H.sub.2]P[O.sub.4], pH 7.5). The specificity of the rabbit antioyster egg IgG was tested again using ELISA, and a cross-reaction with somatic proteins was no longer demonstrated.

Immunofluorescence Assay

An immunofluorescence assay was carried out to visualize and localize the rabbit antioyster egg IgG and oyster egg protein interaction. A series of histological blocks containing ripe eggs were sliced to 6 [micro]m, deparaffinized, and rehydrated. Five ripe oysters collected in June were used in the assay. The sections were incubated with 5 % bovine serum albumin in Triton-X 100 in PBS (PBST-100) as a blocking agent. After blocking, the tissue sections were incubated with the rabbit antioyster egg IgG (1:100 dilution) at room temperature for 1 h. The tissues were then washed 5 times in PBST-100, and incubated with fluorescein isothiocyanate-conjugated goat antirabbit IgG (1:400 dilution; Sigma) as a secondary antibody for 1 h. The presence of the antibody-antigen reaction in the slides was observed under a fluorescence microscope (Olympus).

Suminoe Oyster Egg Protein Analysis

Oyster egg proteins and whole tissue homogenates of ripe, late-, and early-developing female oysters were separated by protein size using 10% sodium dodecylsulfate polyacrylamide gel electrophoresis. Separated proteins were transferred to a polyvinylidene difluoride membrane (Pierce) and blocked with 5% skim milk in 0.15 M Tris-buffered saline with 1% Tween 20 (TBS-T; Yakuri) for 3 h. The membrane was then incubated overnight at 4[degrees]C with diluted (1:1,000) antioyster egg IgG as a primary antibody in a blocking buffer. After washing with TBS-T, goat antirabbit IgG horseradish peroxidase (Koma), conjugated (1:3,000 diluted with TBS-T), was added onto the membrane as the secondary antibody and incubated for 1 h at room temperature. The final antibody-antigen complexes in the blotted membrane were visualized with luminescent reagents (Thermo Scientific).

Quantification of the Reproductive Effort and Potential Fecundity Using ELISA

An indirect ELISA was used to determine the quantity of oyster egg protein in individual oysters collected from January to July 2007 (Table 1), following the protocol developed by Kang et al. (2003b). As positive controls, Suminoe oyster egg protein was prepared in 0.1-10 [micro]g/mL PBS and added to a polystyrene 96-well microplate. Ten micrograms per milliliter of oyster somatic protein extracted from a sexually undifferentiated oyster collected in January was also included in the plate as a negative control. The microplate containing the controls and oyster samples was incubated at 4[degrees]C overnight or at 37[degrees]C for 3 h. After incubation, the wells were washed 4 times with washing solution containing PBST-100, and 150 [micro]L 1% bovine serum albumin was added to each well as a blocking agent. After incubation, the oyster egg-specific antibody developed in this study was applied as the primary antibody (6.8 [micro]g/mL) and was given 1 h of reaction time. After incubation, goat antirabbit IgG alkaline phosphatase conjugate (1:1,000 dilution; Sigma) was added to the plate as the secondary antibody and the plate was incubated again for 1 h. Finally, 9-nitrophenylphosphate (Fluca) substrate dissolved in 0.1 M glycine buffer was added as a coloring agent and the optical density of each well was measured at 405 nm using a microplate reader (BioTek). The quantity of egg protein in an oyster sample was then estimated from a titration curve constructed using a regression between the optical density of the oyster egg protein in the plate and the concentration in the egg preparation. The quantity of egg was then estimated by multiplying the quantity of egg protein measured by ELISA by 1.96, the ratio of total egg to egg protein weight estimated in this study. Finally, a weight-based gonadosomatic index (GSI), the ratio of total egg mass to total tissue weight, was established.

Potential fecundity was estimated for oysters collected during June and July that were fully ripe or ready to spawn. To estimate fecundity, the quantity of eggs estimated by ELISA was divided by the weight of a single egg (i.e., 14 ng). A total of 39 ripe oysters collected in June and July were included in the analysis.

RESULTS

Reproductive Stages and CI

In January 2007, most of the oysters (59%) were in early developmental stages, exhibiting early vitellogenic oocytes and spermatogonia. Late developmental stages were observed from May to July, and ripe oysters could be observed as early as May (25%). Spawning by both sexes commenced in July, when 6.7% of the oysters engaged in spawning. Most oysters were fully mature and ready to spawn in June (84.6%) and July (86.7%).

Figure 3 shows monthly changes in PGA values measured from January to July 2007. Percent gonad area was measurable in April, when most of the oysters were in the early and late developmental stages. Percent gonad area increased dramatically from April (5.1%) to June (51%) as the oysters became reproductively mature and ready to spawn.

As shown in Figure 4, the CI of the Suminoe oysters ranged from 0.04-0.22 during the study period. The CI increased gradually from February to April and peaked in June, when most of the oysters were ready to spawn.

Biochemical Composition of the Egg and the Somatic Tissues

Ripe eggs were purified successfully from the ovaries of Crassostrea ariakensis using different sizes of mesh screening and centrifugation. The dry weight of an average egg was estimated to be 14 ng, consisting of 51.1% protein, 5.3% carbohydrate, 24.1% lipid, and 9.4% ash.

Total protein, carbohydrate, lipid, and ash content in oyster tissues used in this study are plotted in Figure 5. The monthly mean total proteins ranged from 270.4-387.5 mg/g DTWT. Total protein was found to be lower in the winter (January and February: 270.4-245.6 mg/g DTWT) and higher from March to June (376.2-387.5 mg/g DTWT), when the oysters became sexually mature. Tissue carbohydrate levels increased from March to May then decreased in June. Carbohydrate values were stable from January to March, increased in April, and decreased dramatically from May (459.5 mg/g DTWT) to July (322.4 mg/g DTWT). The lipid level showed clear monthly changes during the study period. Lipid values gradually increased from January (39.2 mg/g DTWT) to May (86.2 mg/g DTWT), and then decreased from June to July, similar to the pattern observed for carbohydrates. Ash levels showed no marked differences during the study period, with values ranging from 61.5-72.0 mg/g DTWT.

Specificity of the Rabbit Anti-Crassostrea ariakensis Egg IgG

The rabbit antiserum raised from purified oyster eggs demonstrated initially a weak cross-reaction to nongonadal proteins on ELISA and Western blotting (Fig. 6A). After eliminating the cross-reacting antibody using an immunoadsorbent, a strong precipitin line was observed between the egg protein and the antibody in the double immunodiffusion. Western blotting and ELISA demonstrated that no cross-immunological reaction with somatic proteins isolated from sexually undifferentiated oyster was observed (Fig. 6B).

Figure 6 shows the immunologically active peptides in purified eggs and eggs from ripe, late- and early-developing, and sexually undifferentiated oysters. Oyster egg proteins are a complex mixture comprised of peptides approximately 150, 120, 95, 90, 82, and 55 kDa. The egg proteins isolated from late-developing and ripe oysters showed major bands with molecular weights of 150 kDa and 55 kDa, and very weak bands of 120, 95, 90, and 82 kDa. In contrast, egg proteins from oysters during early developmental stages and the undifferentiated stage exhibited no immunological reaction during Western blotting, suggesting that these oysters did not contain the larger molecular weight peptides present in ripe oysters.

Immunofluorescence Assay

An indirect immunofluorescence assay performed on mature female gonadal tissue revealed a highly specific interaction between the antibody and egg proteins. The oocytes were stained with the fluorescent antibody, but no positive staining was observed in the connective tissue, nucleus, mantle, or gills (Fig. 7). It was suggested that the rabbit antioyster egg IgG reacts with vitellins, egg-specific proteins in animal eggs. The titration curve indicated that the antioyster egg IgG in ELISA detected 0.2-8 [micro]g/mL oyster egg protein in the standard solution.

Reproductive Effort and Potential Fecundity of Suminoe Oyster

A total of 270 oysters ranging from 150-195 mm in SL and 40-51 g in wet tissue weight were collected from the Seomjin River estuary during January and July 2007 (Table 1) to assess reproductive effort and potential fecundity. The reproductive effort of female Suminoe oysters was assessed using the rabbit antioyster egg IgG in ELISA. Reproductive effort remained undetectable from January to March, when most oysters were sexually undifferentiated or in early developmental stages (Fig. 3). In April, reproductive effort was measurable for 13 of 25 oysters examined; the average effort was 3.62 [+ or -] 4.42%. The GSI increased dramatically between April and July, from 23.38 [+ or -] 12.05% in May to 42.95 [+ or -] 14.61% in June and to 47.69 [+ or -] 11.80% in July, when most oysters were fully mature. The highest GSI value (66.93%) was recorded in July.

Potential fecundity was estimated for 38 ripe oysters. Values ranged from 162-910 million eggs, with a mean of 446 million eggs.

DISCUSSION

Enzyme-linked immunosorbent assay has been applied in studies conducting quantitative assessments of reproductive effort in marine bivalves (Kang et al. 2003b, Park & Choi 2004, Royer et al. 2008). In the current study, we developed an immunological probe to measure the reproductive effort of Suminoe oysters. The rabbit anti-Crassostrea ariakensis egg IgG applied during an ELISA detected 0.2-8 [micro]g egg protein in oyster tissue homogenate. The detection range of the polyclonal antibody developed in this study is comparable with the sensitivity of other polyclonal antibodies used to quantify oyster egg proteins. Choi et al. (1993) first used ELISA in the quantification of egg mass in Crassostrea virginica and the rabbit anti-C, virginica egg IgG detected 0.2-10 [micro]g/mL egg protein. Kang et al. (2003b) also developed a polyclonal antibody for the eggs of the Pacific oyster, Crassostrea gigas, to assess reproductive effort using ELISA. According to Kang et al. (2003b), for Pacific oysters in Goseong Bay off the southern coast of Korea, up to 67% of the body weight was contained in eggs (i.e., GSI = 67%), although the mean GSI value recorded during the spawning period was 40%. Royer et al. (2008) estimated GSI values for Pacific oysters that were 1, 2, and 3 y old in Normandy, France, using the Pacific oyster egg-specific antibody in ELISA. In their study, reproductive effort was 36-60% and GSI increased with increasing oyster size and age. Figure 5 indicates that Suminoe oysters from the Seomjin River estuary contained 42-48% of their body weight as eggs during the spawning season. Reproductive effort of Suminoe oyster estimated in this study is somewhat comparable with, though slightly higher than, the reproductive effort of Pacific oysters reported by Kang et al. (2003b) and Royer et al. (2008).

As listed in Table 2, the potential fecundity of Suminoe oysters estimated from 38 ripe oysters ranged from 162-910 million eggs, with an average of 446 million. This estimate is somewhat higher than the fecundity of Crassostrea virginica reported by Choi et al. (1993), which ranged from 3.7-65.4 million eggs for individuals in Galveston Bay. Kang et al. (2003b) also estimated the fecundity of Crassostrea gigas as 4-196 million eggs in Goseong Bay, whereas Royer et al. (2008) reported C. gigas fecundity as 2.6-234 million eggs in Normandy. The relatively high fecundity of Suminoe oysters recorded in this study could be explained by the size of oysters used in the analysis. As shown in Table 2, the Suminoe oysters collected in June and July weighed 40-55 g in wet tissue weight, which is 2-3 times higher than 2 to 3-y-old C. gigas or C. virginica (Choi et al. 1993, Kang et al. 2003b). Because the size and weight of mature eggs is similar among C. virginica, C. gigas, and Crassostrea ariakensis, estimates of fecundity as the total number of eggs in an individual oyster should increase as body size increases. Such size-dependent fecundity also has been shown in other marine bivalves (Park & Choi 2004, Park et al. 2005).

Several studies have reported that marine bivalves store biochemical energy reserves in tissues, prior to gametogenesis, in the form of carbohydrates, lipids, and proteins. These reserves are then used during gametogenesis as building blocks of gonadal materials or to sustain metabolism during periods of low food supply and reduced feeding (Gabbott 1976, Thompson et al. 1996, Ahn et al. 2003, Ojea et al. 2004). Consequently, seasonal changes in the biochemical composition of marine bivalves are associated closely with environmental parameters and annual gametogenesis (Kang et al. 2000, Ren et al. 2000, Ngo et al. 2006, Delgado & Camacho 2007, Dridi et al. 2007).

It is notable that the total protein level during this study increased gradually from February to June as gonadal maturation progressed from early development to being ripe and ready for spawning. In contrast, the total carbohydrate level in the tissue decreased from April to July (Fig. 7). Such an inverse relationship between protein and carbohydrate levels in oyster tissues has been reported previously in species that undergo annual gametogenesis (Berthelin et al. 2000, Kang et al. 2000, Ren et al. 2000, Dridi et al. 2007). Increase in the total protein level during gametogenesis is attributable to increases in the size and number of oocytes as the oyster becomes ripe and ready to spawn (Li et al. 2000). According to Choi et al. (1993) and Kang et al. (2003b), proteins in ripe or partially spawning Crassostrea virginica and Crassostrea gigas account for almost 40% of the total dry weight. In this study, proteins comprised 51% of the dry weight of Suminoe oysters. Therefore, it is believed that, like other oysters, the Suminoe oyster maintains a biochemical nutrient reserve in the form of carbohydrates during early gametogenesis. The carbohydrates are stored in tissues, possibly in the mantle, and are converted subsequently to yolk proteins as oogenesis progresses, and as the size and number of oocytes in the ovary increases. Accordingly, Perdue and Erickson (1984) reported an inverse relationship between the percentage of carbohydrate and the extent of gonadal maturation in Suminoe oysters during an annual reproductive cycle in Washington state.

The dry weight of a single Suminoe oyster egg estimated in this study (i.e., 14 ng) was comparable with the dry weights of eggs from other oysters, including Crassostrea virginica (12 ng (Lee & Heffernan 1991), 13 ng (Choi et al. 1993)) and Crassostrea gigas (13 ng (Kang et al. 2003b)). In contrast, the dry weights of single eggs from clams such as Mercenaria mercenaria (Lee & Heffernan 1991), Saxidomus purpurdatus (Park et al. 2005), and Ruditapes philippinarum (Park & Choi 2004) are reported to be heavier than oyster eggs, ranging from 22-95 ng.

Western blotting revealed that Suminoe oyster egg proteins are a complex of proteins including various-size peptides of approximately 150, 120, 95, 90, 82, and 55 kDa (Fig. 3). Several studies have reported egg-specific peptides, with molecular weights of 105, 85, 66, 64, 60, 45, and 41 kDa in Pacific oysters (Suzuki et al. 1992); 76, 56, 50, 48, 18, and 17 kDa in Crassostrea virginiea (Lee & Heffernan 1991); 98, 87, 68, 60, 56, 36, and 19 kDa in Mercenaria mercenaria (Lee & Heffernan 1991); and 330, 96, 64, 50, and 31 kDa in Ruditapes philippinarum (Park & Choi 2004). These egg-specific peptides are so-called vitellins, a major component of invertebrate eggs, and are used as energy and nutrient sources during gonadal development and spawning (Lee & Heffernan 1991, Suzuki et al. 1992, Osada et al. 2003).

Western blot analysis demonstrated that the rabbit anti-Suminoe oyster egg IgG developed in this study showed a strong immune reaction to proteins present in late-developing and ripe oysters, whereas a similar reaction was absent in oysters that were sexually undifferentiated or in early development (Fig. 3). In addition, the Western blot analysis results suggested that the antigenic determinants raised in the oyster egg-specific antibody are vitellin-like proteins that occur commonly in mature marine bivalve eggs (Suzuki et al. 1992, Osada et al. 2003, Park & Choi 2004, Arcos et al. 2009). During the immunofluorescence assay, the rabbit anti-Suminoe oyster egg IgG responded strongly to yolk granules (Fig. 4), supporting the hypothesis that the rabbit antibody was raised from yolk proteins, which are vitellin or vitellinlike molecules (Suzuki et al. 1992, Kang et al. 2003b, Park & Choi 2004, Park et al. 2005).

In summary, we developed a polyclonal antibody to an egg protein of the Suminoe oyster to quantify reproductive effort. The polyclonal antibody was sensitive enough when used during ELISA to detect the minute amount of the egg protein present in the early developmental stage of the Suminoe oyster collected in April. Oysters collected in July 2007 were ready for spawning, and the reproductive effort ranged from 17.5-67.0%, with a mean of 47.7%, suggesting that the Suminoe oyster on the southern coast of Korea may release 162-910 million eggs during a spawning season.

ACKNOWLEDGMENT

We thank the staff of Shellfish Aquaculture and Research Laboratory of Jeju National University for sampling and laboratory works. This work was supported by Korea Science and Engineering Foundation (KOSEF) grant funded by the Ministry of Education, Science and Technology (grant code 20090074167) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant no. 2010-0009352).

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BONG-KYU KIM AND KWANG-SIK CHOI *

School of Marine Biomedical Science (Post BK 21) and Marine and Environmental Research Institute, Jeju National University, 66 Jejudaehakno, Jeju 690-756, Republic of Korea

* Corresponding author. E-mail: skchoi@jejunu.ac.kr

DOI: 10.2983/035.031.0415

TABLE 1.

Monthly mean values for shell length (SL), tissue wet weight
(TWWT), and tissue dry weight (DTWT; N = 270).

2007       n          SL (mm)               TWWT (g)

January    40   189.2 [+ or -] 14.6   44.62 [+ or -] 7.75
February   40   194.6 [+ or -] 13.9   51.04 [+ or -] 6.90
March      40   192.9 [+ or -] 14.7   49.97 [+ or -] 12.10
April      40   172.8 [+ or -] 16.7   42.65 [+ or -] 8.82
May        40   180.1 [+ or -] 16.3   51.48 [+ or -] 12.72
June       40   149.3 [+ or -] 12.4   40.87 [+ or -] 8.07
July       30   181.1 [+ or -] 21.3   55.38 [+ or -] 14.67

2007            DTWT (g)

January    10.24 [+ or -] 2.12
February   13.17 [+ or -] 1.94
March      12.81 [+ or -] 3.43
April      10.50 [+ or -] 2.23
May        13.63 [+ or -] 3.36
June       10.35 [+ or -] 2.19
July       12.71 [+ or -] 3.49

TABLE 2.

Fecundity of marine bivalves reported from various studies.

Oyster Species                Location          SL (mm)     TDWT (g)

Crassostrea virginica    Galveston Bay, US      70-120       0.7-1.9
Crassostrea gigas        Goseong Bay, Korea     74-91.6      0.9-2.9
C. gigas                 Normandy, France     55.2-88.6      0.7-5.9
Crassostrea ariakensis   Seomjin River        125.2-230.1   10.5-13.6
                           estuary, Korea

Oyster Species           EW (ng)         Fecundity

Crassostrea virginica      13      3.7-65.4 x [10.sup.6]
Crassostrea gigas          13        4-196 x [10.sup.6]
C. gigas                   22      2.6-234 x [10.sup.6]
Crassostrea ariakensis     14      162-910 x [10.sup.6]

Oyster Species                  Study

Crassostrea virginica    Choi et al. (1993)
Crassostrea gigas        Kang et al. (2003b)
C. gigas                 Royer et al. (2008)
Crassostrea ariakensis   Current study

In this study, 39 ripe oysters collected in June and July were
included exclusively in the analysis. EW, egg weight; SL, shell
length; TDWT, tissue dry weight.
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Author:Kim, Bong-kyu; Choi, Kwang-sik
Publication:Journal of Shellfish Research
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Date:Dec 1, 2012
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