Reproductive cycle and biochemical composition of ...

Aquaculture 261 (2006) 752 – 759 www.elsevier.com/locate/aqua-online

Reproductive cycle and biochemical composition of the Zhe oyster Crassostrea plicatula Gmelin in an eastern coastal bay of China Qi Li a,⁎, Wenguang Liu a , Kunio Shirasu b , Weimin Chen b , Shouxuan Jiang c b

a Fisheries College, Ocean University of China, Qingdao 266003, China Marine Biological Technology Center, Nippon Suisan Kaisha, Ltd., Saiki876-1204, Japan c Rushan Fishery Technique Promoting Station, Rushan 264500, China

Received 15 April 2006; received in revised form 15 August 2006; accepted 17 August 2006

Abstract The reproductive cycle and biochemical composition of the oyster Crassostrea plicatula in Rushan Bay, an eastern coastal bay of China, were investigated between March 2004 and February 2005 in relation to environmental factors (temperature, salinity and chlorophyll a). Histological analysis, combined with oocyte examination and measurements of protein, glycogen and lipid levels and RNA/DNA ratio from gonad, adductor muscle and mantle tissue of each sex were performed. The gametogenic cycle of C. plicatula comprised two phases: a resting phase (November–February) and reproductively active phase, including ripeness and spawning, during the rest of year. Gametogenesis in spring and summer was associated with an increase in protein and lipid contents in the ovaries, and took place at the expense of reserves (mainly glycogen) accumulated previously during winter. The RNA/DNA ratio is a good indicator of sexual maturity in the ovary; the increasing RNA/DNA ratio in the ovary appears to show the rising synthetic activity of vitellin within the ovary. Spawning occurred at the moment when water temperature and chlorophyll a levels were highest, and salinity lowest throughout the year. High variation in glycogen contents during storage and gametogenic development suggests that carbohydrates are the main respiratory substrate in this species. The results demonstrated that C. plicatula may be considered a conservative species in gametogenic pattern. © 2006 Elsevier B.V. All rights reserved. Keywords: Crassostrea plicatula; Biochemical composition; Reproductive cycle; Environment variables

1. Introduction The Zhe oyster Crassostrea plicatula Gmelin, which is small and thin-shelled as compared with the Pacific oyster C. gigas, naturally occurs along the entire coast of China, and is one of the dominant ⁎ Corresponding author. Tel.: +86 532 82031622; fax: +86 532 82894024. E-mail address: [email protected] (Q. Li). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.08.023

species of mollusc aquaculture (Wang et al., 2004). The production of this oyster reached 3.62 × 106 metric tons in 2002 in China, accounting for 37.6% of total marine molluscan yield (DOF, 2003). Official statistics do not distinguish individual oyster species, but it is estimated that the C. plicatula accounts for 50–60% of the total oyster production (Guo et al., 1999). Culture of C. plicatula depends entirely on natural seed. Often, seed is collected on stones, shells, bamboo, and cement blocks in one area and moved to another area for

Q. Li et al. / Aquaculture 261 (2006) 752–759

culture. C. plicatula is cultured primarily in Fujian province and other parts of the southern coast where seeds are usually collected in May and September (Guo et al., 1999). Recently, culture of C. plicatula has been done in the northern coast, but there is few information on its reproductive cycle. Detailed knowledge of reproductive strategy in a given area could have essential importance for collecting C. plicatula seed from nature. Up to now, biological studies on C. plicatula have been limited to artificial seed collection (Zhang, 1996; Li, 1996; Liu, 2005), culture techniques (Yuan et al., 1998; Bao and Li, 1995; Yuan et al., 2003), and genetic characteristics (Yang et al., 2000). Little attention has been paid to the physiological ecology and gametogenic cycle. The reproductive cycles of marine bivalves are strongly related to energy storageutilization cycles and environment parameters such as water temperature and food availability (Taylor and Venn, 1979; Zandee et al., 1980; Beninger and Lucas, 1984; Arellano-Martinez et al., 2004). In general, when food is abundant, reserves accumulate prior to gametogenesis in the form of glycogen, lipid and protein substrates, and subsequently are utilized in the production of gametes when metabolic demand is high (Mathieu and Lubet, 1993). The specifics of which substrates are important, and how the timing of their consumption is related to gametogenesis vary between species as well as between populations of the same species (Barber and Blake, 1981). In some species such as Chlamys opercularis (Taylor and Venn, 1979), Argopecten irradians concentricus (Barber and Blake, 1981) and A. purpuratus (Martinez, 1991), energy for gametogenesis comes from substrates stored in various organs and tissues (adductor muscle, digestive gland, or mantle) through feeding prior to its gametogenesis (conservative species) (Bayne et al., 1976); in some species such as Tellina tenuis and Abra alba, it comes from the recently ingested energy from the seston (opportunistic species) (Bayne et al., 1976); in others such as Placopecten magellanicus, it comes from a combination of both stored reserves and ingested food (Thompson, 1977). The seasonal cycles of gametogenesis and reserve storage in C. plicatula may reveal its reproductive strategy in relation to local environmental conditions. This study investigated the seasonal variation in biochemical composition and reproductive cycle of C. plicatula cultured in Rushan Bay, a predominant area for oyster production in eastern coast of China. The objectives of the study are to monitor gametogenesis, the breeding season, and the seasonal cycles of energy

753

storage and depletion in different organs in relation to the reproductive cycle. 2. Materials and methods 2.1. Sample collection Samples of C. plicatula (shell height, 8.3 ± 1.5 cm; shell length, 5.4 ± 0.8 cm; soft body dry weight, 12.0 ± 4.2 g) cultured on ropes suspended from a raft were collected monthly from March 2004 to February 2005 in Rushan Bay (36°43′–37°36′N and 121°28′–121°39′ E), Shandong Province, China. They were transported live to the laboratory, and were dissected to obtain gonads (digestive gland not included), mantles, and adductor muscles. The tissues were then frozen and stored at − 80 °C until used. Temperature and salinity of sea water were measured in situ during sampling, and chlorophyll a (from 0–1 m of depth) at the site of the raft was determined according to Parsons et al. (1984). 2.2. Histology Twenty individuals from each sample were used for histological examination. A transverse cut was made across the middle part of the soft body of the oyster and a 5-mm-thick section was fixed in Bouin's solution, and processed using routine histological techniques. The specimens were examined microscopically to develop a profile of gametogenesis, and the diameter of 100 oocytes was microscopically measured in sections from five animals to determine the degree of maturity every month. The stages of gonadal development were classified and scored on a 0 to 4 scale according to a scale of maturity (Ivell, 1979). Grade 0 represents the immature stage. At this grade, sexes are unidentifiable. At grade 1, sexes are just identifiable but very little gonad is developed. Grade 2 represents a stage of moderate gonadal development. At this grade, oocytes are beginning to develop. At grade 3, gonads are distended and ripe. At this stage, gametogenesis is complete but spawning has not yet occurred. Oocytes are liberated freely on dissection and free sperm are manifested as streaks. Grade 4 is the spawning stage of animals and in half-spent to completely spent condition. 2.3. Biochemical analysis For the biochemical characterization of the body components, the quantity of protein, glycogen, lipid,

754


3. Results 3.1. Environmental parameters

Fig. 1. Monthly average values of water temperature, salinity and chlorophyll a in Rushan Bay.

and nucleic acids was estimated. Total protein was determined by the Kjeldahl method. The dried, powdered samples were catalytically digested with sulphuric acid, and analyzed by an automatic Kjeldahl analysis instrument (Kjeltec™ 2300; Foss Tecator, Sweden). The amount of N was multiplied by 6.25 to estimate the amount of proteins (Stephen, 1980). For determining the lipid levels, extractions were made in chloroform–methanol (3:1) using a soxhlet apparatus (Folch et al., 1957). The glycogen content was determined with minor modifications to the anthrone, sulfuric acid method described by Horikoshi (1958). The powdered, freeze-dried samples were suspended in 60 volumes of 30% KOH, and saponified by heating to 100 °C for 30 min. After cooling, a portion of the saponified mixture was treated with the cold 0.2% anthrone-sulfuric acid solution for 10 min; absorbance of the resulting colored complex was measured at the wave length of 620 nm. After the samples were homogenized in 20 volumes of distilled water, 1 ml of each homogenate was used for determination of nucleic acid (DNA and RNA) contents according to the modification of the Schmidt–Thammhauser–Schnerder method by Nakano (1988). Nucleic acids were precipitated with ethanol and washed with a mixture of ethanol and ether. RNA was separated by alkaline hydrolysis, and DNA was hydrolyzed with perchloric acid. DNA and RNA contents were determined by measuring their absorbances at 260 nm.

Monthly water temperature and salinity values for the Rushan Bay are given in Fig. 1. A seasonal cycle in water temperature was observed, with the maximum value in summer (29.5 °C in August 2004), decreasing gradually until winter (1.8 °C in January 2005). Salinity remained relatively stable throughout the year with the exception of low salinity in the rainy summer season from June to August. The concentration of chlorophyll a exhibited a clear seasonal pattern characterized by two unequally sized peaks (Fig. 1). The small one was seen in April 2004 (17.7 μg/l), while the large one was seen in September 2004 (25.8 μg/l). During winter chlorophyll a concentration was low, and the mean of experimental period was 11.3 ± 8.2 (SD) μg/l. 3.2. Gametogenesis The gametogenic cycle of C. plicatula is characterized by a clear seasonal pattern (Fig. 2). The oocyte diameters increased from March (8.0 μm), reached a maximum value of 49.6 μm in July and then decreased after August (Fig. 2). Gonad development started in March and peaked during July to August when the oocyte diameters declined suddenly (Fig. 2). Ripe gonads were found from May to August. It was estimated from the histological observations that C. plicatula

2.4. Statistical analysis Student's t-tests were used to test for differences between mean values for males and females.

Fig. 2. Monthly variations in oocyte diameter (n = 100) and gonadal development of C. plicatula. Values are means ± SD.

Table 1 Montly variations in protein, glycogen and lipid contents (% dry weight) and RNA/DNA ratio of gonad, adductor muscle and mantle of C. plicatula Date

Sex Protein

Lipid

RNA/DNA ratio

Gonad

Adductor muscle Mantle

Gonad


Gonad


Gonad


39.9 ± 2.6 34.6 ± 2.1* 40.2 ± 2.1 35.6 ± 1.7* 44.6 ± 2.3 30.5 ± 3.2** 47.5 ± 2.3 25.0 ± 3.6** 49.1 ± 2.6 25.5 ± 2.4** 43.4 ± 2.4 25.8 ± 2.4** 35.0 ± 2.4 26.0 ± 3.6* 34.1 ± 2.2 31.0 ± 2.8* 33.3 ± 2.6 37.3 ± 3.3 39.6 ± 2.5 40.1 ± 1.9

47.3 ± 1.1 46.5 ± 1.6 49.4 ± 1.3 48.4 ± 2.1 50.2 ± 0.8 49.9 ± 2.2 40.4 ± 2.3 40.2 ± 2.4 38.4 ± 2.2 39.2 ± 2.1 37.3 ± 2.5 36.1 ± 2.6 44.1 ± 2.6 42.4 ± 2.9 46.8 ± 2.6 47.4 ± 2.7 50.2 ± 1.9 49.7 ± 1.7 51.3 ± 2.1 50.9 ± 0.9

34.3 ± 2.9 31.1 ± 2.5* 33.8 ± 2.9 31.0 ± 3.5* 23.0 ± 1.9 19.2 ± 2.4* 18.1 ± 2.7 14.9 ± 2.3* 13.8 ± 2.9 10.4 ± 2.1* 12.9 ± 2.4 10.2 ± 2.5* 21.2 ± 2.6 18.8 ± 2.8* 26.4 ± 2.8 24.6 ± 2.1 32.1 ± 2.3 33.7 ± 2.6 33.8 ± 2.5 34.6 ± 2.7

14.9 ± 1.6 14.1 ± 1.4 14.1 ± 1.5 13.1 ± 0.9 13.8 ± 2.1 13.0 ± 1.7 6.9 ± 1.3 5.9 ± 1.1 6.3 ± 1.4 5.4 ± 1.3 6.1 ± 1.4 5.5 ± 1.5 9.7 ± 1.6 9.1 ± 1.4 10.4 ± 0.9 10.1 ± 1.4 12.4 ± 1.9 14.7 ± 1.7 15.7 ± 1.0 15.9 ± 2.0

14.9 ± 2.3 12.7 ± 1.0* 15.1 ± 1.1 13.0 ± 1.0* 15.0 ± 1.6 14.1 ± 1.1* 14.7 ± 2.4 14.0 ± 1.2 18.2 ± 1.3 17.9 ± 4.2 19.9 ± 1.3 19.0 ± 0.9 20.8 ± 2.1 20.2 ± 1.9 19.0 ± 1.3 18.9 ± 1.6 18.8 ± 1.6 18.2 ± 0.7 16.2 ± 1.3 15.3 ± 1.2

4.0 ± 0.4 3.9 ± 0.5 4.1 ± 0.6 4.1 ± 0.5 4.4 ± 0.7 4.3 ± 0.6 4.6 ± 0.5 5.4 ± 0.6 5.6 ± 0.6 5.7 ± 0.7 5.1 ± 0.1 5.0 ± 0.4 4.9 ± 0.7 4.4 ± 0.6 4.3 ± 0.6 4.1 ± 0.4 4.1 ± 0.7 4.1 ± 0.8 3.9 ± 0.1 3.8 ± 0.6

2.2 ± 0.2 1.9 ± 0.3 2.8 ± 0.2 2.2 ± 0.2* 3.7 ± 0.2 2.2 ± 0.3** 5.0 ± 0.2 2.2 ± 0.3** 3.7 ± 0.4 2.2 ± 0.2** 3.5 ± 0.3 2.1 ± 0.2** 2.6 ± 0.3 2.2 ± 0.4* 2.5 ± 0.3 2.3 ± 0.2 1.8 ± 0.2 1.7 ± 0.2 1.6 ± 0.2 1.6 ± 0.2

1.3 ± 0.1 1.3 ± 0.1 1.6 ± 0.1 1.4 ± 0.2 2.0 ± 0.1 1.7 ± 0.2 1.6 ± 0.1 1.6 ± 0.1 2.0 ± 0.3 2.0 ± 0.2 1.9 ± 0.2 1.9 ± 0.2 1.9 ± 0.2 2.0 ± 0.2 1.7 ± 0.2 1.8 ± 0.2 1.8 ± 0.1 1.7 ± 0.1 1.3 ± 0.1 1.5 ± 0.1

49.4 ± 2.1 48.2 ± 1.5 51.1 ± 1.7 49.6 ± 1.4 52.4 ± 1.3 50.3 ± 1.4 55.4 ± 3.4 54.3 ± 2.0 57.2 ± 1.8 55.3 ± 1.6 57.4 ± 1.8 56.3 ± 1.7 48.6 ± 3.1 49.2 ± 1.5 46.4 ± 2.8 46.7 ± 2.4 48.3 ± 1.3 46.3 ± 2.5 49.2 ± 1.4 48.9 ± 2.0

20.1 ± 1.3 20.7 ± 1.4 21.8 ± 1.4 20.0 ± 1.5 22.4 ± 1.9 19.8 ± 0.6 15.0 ± 0.6 13.1 ± 1.4 13.1 ± 0.7 12.0 ± 0.7 11.6 ± 2.5 10.9 ± 1.6 16.3 ± 0.4 14.8 ± 1.3 18.9 ± 2.0 16.6 ± 1.4 22.5 ± 0.8 22.0 ± 1.1 23.3 ± 0.6 22.7 ± 1.3

9.8 ± 2.0 9.7 ± 1.4 10.2 ± 0.7 10.1 ± 1.1 10.4 ± 2.2 10.2 ± 1.9 10.7 ± 2.4 11.4 ± 3.5 11.4 ± 1.8 11.3 ± 2.7 12.4 ± 2.6 12.1 ± 2.9 11.5 ± 1.1 11.1 ± 2.1 11.4 ± 1.5 11.3 ± 1.6 10.9 ± 1.3 10.8 ± 0.8 10.7 ± 0.9 10.3 ± 1.8

1.6 ± 0.2 1.7 ± 0.2 1.8 ± 0.2 1.7 ± 0.2 1.4 ± 0.2 1.7 ± 0.2 1.4 ± 0.1 1.6 ± 0.2 1.6 ± 0.2 1.8 ± 0.2 1.8 ± 0.2 1.8 ± 0.1 1.5 ± 0.2 1.7 ± 0.2 1.6 ± 0.2 1.7 ± 0.2 1.6 ± 0.3 1.6 ± 0.2 1.8 ± 0.1 1.6 ± 0.1


♀ ♂ 4/19/2004 ♀ ♂ 5/20/2004 ♀ ♂ 6/22/2004 ♀ ♂ 7/20/2004 ♀ ♂ 8/23/2004 ♀ ♂ 9/23/2004 ♀ ♂ 10/23/2004 ♀ ♂ 11/24/2004 ND 12/29/2004 ND 1/25/2005 ND 2/23/2005 ND 3/23/2004

Glycogen

ND, sex not determined. Values are means ± SD (n = 5). *Indicates significant differences between males and females(*P b 0.05, **P b 0.01).

755

756


partially spawned between July and September. After spawning, there was a sexual resting period from November to February. 3.3. Biochemical composition The protein content in the ovaries increased gradually from 40.2% in April to a maximum value of 49.1% in July and decreased after August. In contrast, the protein content in the testes presented opposite up and down trends, and was significantly lower (P b 0.05) than that for the ovaries (Table 1). The protein content in the adductor muscles decreased gradually from May and reached its lowest level in August, then increased to a constant level from November to February. The protein content in the mantles showed a slight increase during sexual maturation and then declined after August. The protein content in the adductor muscles and mantles did not show significant differences between males and females. The glycogen content in the ovaries decreased gradually from March (34.3%) to August, when the minimum value (12.9%) was observed, and then increased from September to February (Table 1). In the testes, a similar pattern was observed, although significant differences were present (P b 0.05) between ovaries and testes in March–September. The glycogen content in the adductor muscles of each sex decreased from March until minima values (6.1% in females and 5.5% in males) were reached in August, showing the same tendency with those of the gonad. The annual means showed no significant difference between sexes. The glycogen content in the mantles of each sex showed a similar pattern to those in the gonad and adductor muscle, varying from 10.9% to 23.3%, higher than those of the adductor muscle. No significant difference was observed between the male and female mantles. The lipid content in the ovaries and testes showed a seasonal pattern. The lower values were observed from March to June (Table 1). An obvious increase was then observed from June, the highest values were attained in September, followed by an apparent decrease from October. Significant differences were observed between the ovaries and testes in March–May. The lipid content in the adductor muscle fluctuated between 3.9% and 5.7%, and did not change markedly throughout the year. The lipid content in the mantles varying form 9.7% to 12.4% was lower than that for the gonad but higher than that for adductor muscle, and showed a slight increase during sexual maturation. The lipid content in the adductor muscles and mantles had no significant difference between males and females.

The RNA/DNA ratio in the ovaries increased rapidly from March reaching a maximum level in June, similar to the changes in the protein content (Table 1). The RNA/DNA ratio in the testes remained significantly lower than that of the ovaries in April–September and showed no change during sexual maturation. The variations in the RNA/DNA ratio in the adductor muscles and mantles were similar to those of the testes, and showed no significant difference between males and females. 4. Discussion Although seasonal variations in the biochemical composition in relation to reproductive cycles and environmental factors have been well documented for some commercially important species of marine bivalves, most of the aspects concerning the storage capacity and mobilization of nutrients to satisfy metabolic needs related to gametogenesis seem to be species-specific (Thompson, 1977; Stephen, 1980; Urrutia et al., 2001; Saucedo et al., 2002). The present work demonstrated for the first time reproductive and biochemical cycles in relation to environment conditions for a species of C. plicatula in Rushan Bay. Based on histological data, the current study showed that the gametogenesis of C. plicatula in Rushan Bay was characterized by two distinctive phases: a resting phase in November–February and gametogenesis, including maturity and spawning in spring–summer. A new cycle of gametogenesis began at the low water temperature (6.5 °C) in March and spawning took place at the moment when water temperature and chlorophyll a levels were highest, and salinity lowest throughout the year. The variations in the temperature were consistent with the variations in the oocyte diameters, indicating that temperature may play an important role in inducing the gonadal development and spawning of C. plicatula. Similar results have been observed for other oyster species Ostrea edulis and C. gigas (Ruiz et al., 1992; Li et al., 2000). Nevertheless, food availability also may have an effect on initiation of spawning. Starr et al. (1990) demonstrated that phytoplankton levels during blooms should be sufficient to induce spawning in sea urchin and mussels. It is difficult to explicate the summer spawning event by an environmental factor that may trigger spawning. In Rushan Bay, the blooms of phytoplankton occurred in summer when C. plicatula spawned. Cui et al. (1999) reported that the addition of excessive dissolved inorganic nutrients via streams enhanced phytoplankton biomass greatly in the rainy summer period in Rushan Bay. Therefore, spawning in


summer enables larvae to be produced at the most opportune moment with regards to food availability (Navarro et al., 1989; Park et al., 2001). The present study demonstrated the seasonal changes in biochemical composition of gonad, adductor muscle and mantle in the male and female oyster C. plicatula. The analysis of body parts is often more instructive than analysis of the whole animal when studying biochemical composition in relation to reproductive cycles. In fact, measurement of the protein content of the different organs showed that they did not experience the same variation. The protein content in the ovaries showed a synchronous increase with the oocyte diameter during maturation, indicating that ovarian protein would be accumulated as a vitellin. Conversely, the protein content in the testes began to decrease since sperms were formed in May, suggesting that the protein provided a nutrient source and material for spermatogenesis in the oyster as reported in C. gigas and Pseudocentrotus depressus (Li et al., 2000; Unuma et al., 1998). This marked decrease during maturation was also observed in the male and female adductor muscle in May–August, indicating that adductor muscle protein may be used for supporting the process of gametogenesis. The important role of adductor muscle protein in supplying energy demands during reproduction has been reported for other marine bivalves, including Nodipecten subnodosus (Racotta et al., 2003), A. ventricosus (Racotta et al., 1998), C. opercularis (Taylor and Venn, 1979), Tagelus dombell (Urrutia et al., 2001), Pinctada mazatlanica (Saucedo et al., 2002), and A. irradians irradians (Epp et al., 1988). In the male and female mantle tissues, protein tended to increase slightly between March and August, despite the development of gonads, thus suggesting this organ did not transfer protein to the gonad during ripening. However, the mantle protein obviously decreased from September throughout February, suggesting that it may be important for supporting energetic costs during winter when the available food was scarce as indicated by low chlorophyll a levels. The glycogen content in the gonad, adductor muscle and mantle of males and females considerably decreased with increase in oocyte diameter during sexual development, clearly demonstrating that the gametogenesis of C. plicatula in spring–summer depended largely on the glycogen stored during the previous autumn and winter and thus behaved as a conservative species (Bayne, 1976). These results are in agreement with previous data concerning other bivalves like A. irradians concentricus (Barber and Blake, 1981), Mytilus edulis (Zandee et al., 1980), and A. purpuratus (Martinez, 1991). However, in the oyster O. edulis (Ruiz et al., 1992), the storage

757

reserves and the gametogenesis take place concurrently and reproductive activity depends minimally on previously stored reserves, showing an opportunistic behavior (Bayne, 1976). Both strategies can be adopted by geographically different populations of the same species to support gametogenesis. In the scallop N. subnodosus, while the glycogen stored is used in the gametogenesis of a population at the locality of Laguna Ojo de Liebre (Mexico) (Arellano-Martinez et al., 2004), the accumulation of reserve materials and the gametogenesis commenced simultaneously at a southern locality of Bahía Magdalena (Racotta et al., 2003). Luna-Gonzalez et al. (2000) also found that A. ventricosus uses the available food in the environment more than muscle reserves for the gonadal maturation when the food is abundant, but they use the muscle reserves when the food abundance is poor. Several authors have discussed the relative contribution of energy reserves vs. food intake to satisfy the metabolic demands of growth and gonadal production in marine bivalves (Bayne, 1976; Epp et al., 1988; Racotta et al., 1998). However, because these processes are highly dependent on several exogenous and endogenous factors, no pattern has yet been established. In the present study, the glycogen content in the adductor muscle and mantle showed no significant difference between males and females, however, the glycogen content in the ovaries was significantly higher than in the testes in March–October in periods of high food availability, suggesting that the females could take better advantage of food availability than males. The lipid content in the ovaries and testes faithfully reflected the course of sexual maturation in the oyster, showing the high levels in July–September when the gametes had grown sufficiently and were ready to be spawned. This result is consistent with the previous reports for C. tehuelcha (Pollero et al., 1978), Scapharca broughtonii (Park et al., 2001), and C. gigas (Berthelin et al., 2000), suggesting that lipid was converted from glycogen reserves and biosynthesized during the formation of gametes (Gabbott, 1975, 1983). Lipid level may be a good index of gonad maturity (Pazos et al., 1997), because lipid has been considered the basic energetic reserves for sustaining embryonic and larval development of most species of marine bivalves (Holland, 1978; Fraser, 1989). In this study, different lipid levels between the ovaries and testes were only observed in March–May during gonad development, with ovaries having higher levels than testes. In the adductor muscle and mantle the lipid content did not show seasonal variations or differences between sexes. Since DNA content per nucleus in somatic cells is the same for a given species and RNA participates in the

758


protein synthesis, the RNA/DNA ratio has been used as an index of the synthetic activity of protein in many species of animals (Nakata et al., 1994; Malloy and Targett, 1994). In the present study, the RNA/DNA ratio in the ovaries exhibited a seasonal variation which was synchronized with oocyte growth and protein content. The change in the RNA/DNA ratio in the ovary has been reported to correspond well with that in the vitellin content in the Pacific oyster during maturation (Li et al., 1998). Therefore, the results indicate that the RNA/ DNA ratios are valuable indicators of sexual maturation in the ovary and the increasing RNA/DNA ratio in the ovary appears to show the rising synthetic activity of vitellin as one of proteins produced within the ovary. However, the RNA/DNA ratio in the testes, adductor muscle and mantle exhibited no change throughout the year. In conclusion, C. plicatula from Rushan Bay, an eastern coastal bay of China, was characterized of a spawning period in summer due to the monocyclic gametogenesis. The annual variation in the biochemical compositions of the tissues was closely coupled to temperature cycles that regulate metabolism and to the processes of gamete synthesis and release. Gametogenesis took place in spring–summer at the expense of reserves (mainly glycogen) accumulated previously during winter. An inverse relationship between glycogen contents of different organs and oocyte diameter suggests that carbohydrates played an important role as the gametogenesis energy in the species. The data generated in this study provide useful information on reproductive strategy of C. plicatula from Rushan Bay. This information is valuable because of biological and commercial interests. Acknowledgments The authors are indebted to Mr. Jianzhong Song from Rushan Fishery Technique Promoting Station for his assistance with the collection of the samples. The study was supported by grants from Chinese Ministry of Education (NCET-04-0640), and National Natural Science Foundation of China (No. 30571442). References Arellano-Martinez, M., Racotta, I.S., Ceballs-vazquez, B.P., ElorduyGaray, J.F., 2004. Biochemical composition, reproductive activity and food availability of the lions paw scallop Nodipecten subnodosus in the Laguna ojo de liebre baja California sur, Mexico. J. Shellfish Res. 23, 15–23. Bao, J., Li, M., 1995. Raft culture techniques of shell strings of the Zhe oyster. J. Aquac. 1, 8–9 (in Chinese).

Barber, B.J., Blake, N.J., 1981. Energy storage and utilization in relation to gametogenesis in Argopecten irradians concentricus (Say). J. Exp. Mar. Biol. Ecol. 52, 121–134. Bayne, B.L., 1976. Aspects of reproduction in bivalve molluscs. In: Wiley, M.L. (Ed.), Estuarine Processes. Academic Press, New York, pp. 432–448. Beninger, P.G., Lucas, A., 1984. Seasonal variations in condition, reproductive activity, and gross biochemical composition of two species of adult clam reared in a common habitat: Tapes decussatus L. (Jeffreys) and Tapes philippinarum (Adams and Reeve). J. Exp. Mar. Biol. Ecol. 79, 19–37. Berthelin, C., Kellner, K., Mathieu, M., 2000. Storage metabolism in the Pacific oyster (Crassostrea gigas) in relation to summer mortalities and reproductive cycle (west coast of France). Comp. Biochem. Physiol., Part B Biochem., Mol. Biol. 125, 359–369. Cui, Y., Ma, S., Chen, B., Chen, J., Xin, F., Zhou, S., 1999. Study on the status of biological, physical and chemical environment in Rushan bay. J. Fish. Sci. China 6, 77–80 (in Chinese). Department of Fisheries (DOF), 2003. China Fisheries Statistic Yearbook 2002. China Agriculture Press, Beijing (in Chinese). Epp, J., Bricelj, V.M., Malouf, R.E., 1988. Seasonal partitioning and utilization of energy reserves in two age classes of the bay scallop Argopecten irradians irradians (L.). J. Exp. Mar. Biol. Ecol. 121, 113–136. Folch, J., Lees, M., Stanley-Sloane, G.H., 1957. A simple method for the isolation purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–507. Fraser, A.J., 1989. Triacylglycerol content as a condition index for fish, bivalve, and. crustacean larvae. Can. J. Fish. Aquat. Sci. 46, 1868–1873. Gabbott, P.A., 1975. Storage cycles in marine bivalve molluscs: a hypothesis concerning the relationship between glycogen metabolism and gametogenesis. In: Barnes, H. (Ed.), Proceedings of Ninth European Marine Biology Symposium. Aberdeen University Press, Scotland, pp. 191–211. Gabbott, P.A., 1983. Developmental and seasonal metabolic activities in marine molluscs. In: Hochachka, P.W. (Ed.), The Mollusca, vol. 2. Academic Press, New York, pp. 165–217. Guo, X., Ford, S.E., Zhang, F., 1999. Molluscan aquaculture in China. J. Shellfish Res. 18, 19–31. Holland, D.L., 1978. Lipid reserves and energy metabolism in the larvae of benthic marine invertebrates. In: Malin, D.C., Sargent, J.R. (Eds.), Biochemical and Biophysical Perspectives in Marine Biology. Academic Press, London, pp. 85–123. Horikoshi, H., 1958. Glycogen. Chem. Field 34, 36–39 (in Japanese). Ivell, R., 1979. The biology and ecology of a brackish lagoon bivalve, Cerastoderma glaucum Bruguiere, in an English lagoon, the Widewater, Sussex. J. Molluscan Stud. 45, 383–400. Li, K., 1996. Techniques for seed collection and culture of the Zhe oyster. J. Fish. Sci. 15, 29–30 (in Chinese). Li, Q., Osada, M., Suzuki, T., Mori, K., 1998. Changes in vitellin during oogenesis and effect of estradiol-17β on vitellogenesis in the Pacific oyster Crassostrea gigas. Invertebr. Reprod. Dev. 33, 87–93. Li, Q., Osada, M., Mori, K., 2000. Seasonal biochemical variations in Pacific oyster gonadal tissue during sexual maturation. Fish. Sci. 66, 502–508. Liu, S., 2005. Seed collection techniques of the Zhe oyster in natural sea area. China Fish. 51, 9 (in Chinese). Luna-Gonzalez, A., Caceres-Martinez, C., Zuniga-Pacheco, C., Lopez-Lopez, S., Ceballos-Vazquez, B.P., 2000. Reproductive cycle of Argopecten ventricosus (Sowerby II. 1842) (Bivalvia:

Q. Li et al. / Aquaculture 261 (2006) 752–759 Pectinidae) in the Rada of Pueblo de Pichilingue, B.C.S., Mexico and its relation to temperature, salinity, and quality of food. J. Shellfish Res. 19, 107–112. Malloy, K.D., Targett, T.E., 1994. The use of RNA:DNA ratios to predict growth limitation of juvenile summer flounder (Paralichthys dentatus) from Delaware and North Carolina estuaries. Mar. Biol. 118, 367–375. Martinez, G., 1991. Seasonal variation in biochemical composition of three size classes of the Chilean scallop Argopecten purpuratus Lamarck, 1819. Veliger 34, 335–343. Mathieu, M., Lubet, P., 1993. Storage tissue metabolism and reproduction in marine bivalves – a brief review. Invertebr. Reprod. Dev. 23, 123–129. Nakano, H., 1988. Techniques for studying on the early life history of fishes. Aquabiology 10, 23–26 (in Japanese). Nakata, K., Nakano, H., Kikuchi, H., 1994. Relationship between egg productivity and RND/DNA ratio in Paracalanus sp. in the frontal waters of the Kuroshio. Mar. Biol. 119, 591–596. Navarro, E., Iglesias, J.I.P., Larranaga, A., 1989. Interannual variation in the reproductive cycle and biochemical composition of the cockle Cerastoderma edule from Mundaca Estuary (Biscay, North Spain). Mar. Biol. 101, 503–511. Park, M.S., Kang, C.K., Lee, P.Y., 2001. Reproductive cycle and biochemical composition fo the ark shell Scapharca broughtonii (Schrenck) in a southern coastal bay of Korea. J. Shellfish Res. 20, 177–184. Parsons, T.R., Maita, Y., Lalli, C.M., 1984. A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press, New York. Pazos, A.J., Roman, G., Perez Acosta, C., Abad, M., Sanchez, J.L., 1997. Seasonal changes in condition and biochemical composition of the scallop Pecten maximus L. from suspended culture in the Ria de Arousa (Galicia, N.W. Spain) in relation to environmental conditions. J. Exp. Mar. Biol. Ecol. 211, 169–193. Pollero, R.J., Ré, M.E., Brenner, R.R., 1978. Seasonal changes of the lipids of the mollusc Chlamys tehuelcha. Comp. Biochem. Physiol. 64A, 257–263. Racotta, I.S., Ramirez, J.L., Avila, S., Ibarra, A.M., 1998. Biochemical composition of gonad and muscle in the catarina scallop, Argopecten ventricosus, after reproductive conditioning under two feeding systems. Aquaculture 163, 111–122. Racotta, I.S., Ramirez, J.L., Ibarra, A.M., Rodriguez-Jaramillo, M.C., Carreño, D., Palacios, E., 2003. Growth and gametogenesis in the lion-paw scallop Nodipecten (Lyropecten) subnodosus. Aquaculture 217, 335–349. Ruiz, C., Martínez, D., Mosquera, G., Abad, M., Sánchez, J., 1992. Seasonal variation in condition, reproductive activity and bio-

759

chemical composition of the flat oyster, Ostrea edulis, from San Cibran (Galicia, Spain). Mar. Biol. 112, 67–74. Saucedo, P., Racotta, I., Villarreal, H., Monteforte, M., 2002. Seasonal changes in the histological and biochemical profile of the gonad, digestive gland and muscle of the calafia mother-of-pearl oyster, Pinctada mazatlanica (Hanley, 1856) associated with gametogenesis. J. Shellfish Res. 21, 127–135. Starr, M., Himmelman, J.H., Therriault, J.C., 1990. Direct coupling of marine. invertebrate spawning with phytoplankton blooms. Science 247, 1071–1074. Stephen, D., 1980. The reproductive biology of the Indian oyster Crassotrea madrasensis (Preston). II. Gametogenic cycle and biochemical levels. Aquaculture 21, 147–153. Taylor, A.C., Venn, T.J., 1979. Seasonal variation in weight and biochemical composition of the tissues of the queen scallop Chlamys opercularis from the Clyde Sea area. J. Mar. Biol. Assoc. UK 59, 605–621. Thompson, R.J., 1977. Blood chemistry, biochemical composition, and the annual reproductive cycle in the giant scallop, Placopecten magellanicus, from southeast Newfoundland. J. Fish. Res. Board Can. 34, 2104–2116. Unuma, T., Suzuki, T., Kurokawa, T., Yamamoto, T., Akiyama, T., 1998. A protein identical to the yolk protein is stored in the testis in male red sea urchin, Pseudocentrotus depressus. Biol. Bull. 194, 92–97. Urrutia, G.X., Navarro, J.M., Clasing, E., Stead, R.A., 2001. The effects of environment factors on the biochemical composition of the bivalve Tagelus dombell (Lamarck.1818) (Tellinacea: Solecurtidae) from the intertidal flat of Coihuin, Puerto Montt, Chile. J. Shellfish Res. 20, 1077–1087. Wang, R., Li, Q., Yu, R., Wang, Z., Tian, C., Zheng, X., 2004. Techniques of Oyster Culture. Jindun Publishing Co., Beijing (in Chinese). Yang, R., Yu, Z., Chen, Z., Kong, X., Dai, J., 2000. Allozyme variation with Crassostrea plicatula and Crassostrea gigas from Shandong coastal waters. J. Fish. China 24, 130–133 (in Chinese). Yuan, X., Qin, X., Zhu, G., 1998. Techniques of suspended longline culture of the Zhe oyster. Fujian Agric. 4, 16 (in Chinese). Yuan, C., Liu, Z., Zhen, J., 2003. Feasibility of oyster (Crassostrea plicatula Gemlin) cultivation in Zhimao bay. J. Fish. Sci. 22, 34–36 (in Chinese). Zandee, D.I., Kluytmans, J.H., Zurburg, W., Pieters, H., 1980. Seasonal variations in biochemical composition of Mytilus edulis with reference to energy metabolism and gametogenesis. Neth. J. Sea Res. 14, 1–29. Zhang, Z., 1996. Prediction technique for spats collection of rocky oyster. Mar. Sci. 4, 70–71 (in Chinese).