J. Ocean Univ. China (Oceanic and Coastal Sea Research) DOI 10.1007/s11802-013-1979-1 ISSN 1672-5182, 2013 12 (3): 427-433 http://www.ouc.edu.cn/xbywb/ E-mail:
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Reproductive Cycle and Seasonal Variations in Lipid Content and Fatty Acid Composition in Gonad of the Cockle Fulvia mutica in Relation to Temperature and Food LIU Wenguang1), LI Qi2), *, and KONG Lingfeng2) 1) Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, P. R. China 2) Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, P. R. China (Received March 17, 2012; revised May 15, 2012; accepted November 27, 2012) © Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2013 Abstract From March 2004 to February 2005, seasonal variations in lipid content and fatty acid composition of gonad of the cockle Fulvia mutica (Reeve) were studied on the eastern coast of China in relation to the reproductive cycle and environment conditions (e.g., temperature and food availability). Histological analysis as well as lipid and fatty acid analyses were performed on neutral and polar lipids of the gonad. Results showed that gametogenesis occurred in winter and spring at the expense of lipids previously accumulated in summer and autumn, whereas spawning occurred in summer (20.4–24.6℃). The seasonal variation in lipid content was similar to that of the mean oocyte diameter. In both neutral and polar lipids, the 20:5n-3 and 22:6n-3 levels were relatively higher than saturated fatty acids, and polyunsaturated fatty acids were abundant, with series n-3 as the predominant component. Seasonal variations in the 20:5n-3 and 22:6n-3 levels and the principal n-3 fatty acids were clearly related to the reproductive cycle. The ∑(n-3) and ∑(n-6) values were relatively high during January–May, and the associated unsaturation index was significantly higher than that in other months. The results suggest that fatty acids play an important role in the gametogenesis of F. mutica. Key words Fulvia mutica; lipids; fatty acids; reproductive cycle; food; temperature
1 Introduction The cockle Fulvia mutica (Reeve) in the family Cardiidae is widely distributed in coastal waters of the northern Yellow Sea in China, southern Mutsu Bay to Kyushu in Japan, and the Korean Peninsula. It inhabits from intertidal to muddy-sand bottoms at a depth of 50 m, and is a functional hermaphrodite bivalve species with rapid growth rates (Xu et al., 1995; Liu et al., 2008). F. mutica is a food source of commercial importance, but current exploitation of this species is mainly based on natural populations (Li et al., 1994; 1999). For better management of F. mutica populations and successful development of relevant artificial breeding techniques, it is essential to understand its reproductive physiology. The reproductive activity of marine bivalves is affected by complex interactions between endogenous factors (e.g., energy storage-utilization cycle) and exogenous factors (e.g., food availability and water temperature) (Barber and Blake, 1981; Bayne, 1976). Gametogenesis requires a continuous supply of nutrients for the biosynthesis of reproductive materials (Brokordt and Guderley, 2004). * Correspondending author. Tel: 0086-532-82031622 E-mail:
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However, there are interspecific and intraspecific differences in the energy sources of gametogenesis (Bayne, 1976). In some species of bivalves, gametogenesis takes place at the expense of the recently ingested food, whereas in other species, gametogenesis occurs using the previously stored nutrient reserves (Taylor and Venn, 1979; Barber and Blake, 1981). Reproduction, on the other hand, is influenced by the environmental conditions such as temperature and food availability (Gabbott, 1983). In the reproductive processes of marine bivalves, lipids are important components as a major source of metabolic energy and essential materials for the formation of cell and tissue membranes (Holland, 1978). When food is abundant, the lipids are stored in the gonad prior to gametogenesis. They are then utilized for production of gametes when the metabolic demand is high (Mann, 1979; Li et al., 2000). In addition, the lipids have been shown to provide energy for growth in winter when carbohydrate sources are depleted (Li et al., 2006). The seasonal variations in lipid content and fatty acid composition of adult bivalves are thought to be closely linked to the reproductive cycle and affected by the quality and quantity of natural diet and water temperature (Beninger and Lucas 1984; Galap et al., 1997). The importance of fatty acids for maturation of bivalves has also been widely reported (Napolitano and Ackman, 1993; Soudant et al., 1996;
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Soudant et al., 1997; Soudant et al., 1999). Polyunsaturated fatty acids (PUFA) are considered to be the structural components during embryogenesis and the precursors of physiologically active molecules such as prostaglandins and other eicosanoids (Soudant et al., 1996; Delaunay et al., 1993; Langdon and Waldock, 1981). In case bivalves have a limited capacity to synthesize and modify fatty acids, the fatty acid composition of their lipids is markedly influenced by the food consumed. Therefore, the fatty acid composition may reflect useful information regarding trophic sources (Allan et al., 2010; Dalsgaard et al., 2003). Some fatty acids can be used as fatty acid trophic markers (FATMs) to trace the food sources of consumers (Kharlamenko et al., 2001; Pernet et al., 2012). Detailed knowledge of variations in the fatty acid composition of F. mutica in relation to gametogenesis and environment conditions is important. However, data is lacking regarding annual fatty acid variation in F. mutica during reproductive cycle in relation to environment variables. The aim of this study is to provide evidence for seasonal variations in the lipid content and fatty acid composition in gonad of F. mutica, with particular interest in their correlation with the reproductive cycle and environment parameters. Results can be used for F. mutica population management and mariculture.
a profile of gametogenesis. The diameter of 100 oocytes from five animals was measured monthly to determine the degree of maturity. There was no dioecism in the animals examined. According to the scale of maturity (Gallucci and Gallucci, 1982), five gametogenic stages were classified, including 1) Undifferentiated stage: this stage was characterized by a total absence of gametes; the connective tissue occupied almost all of the space (Fig.1A). 2) Developing stage: in the female, there were rounded oocytes along with oocytes attached to the follicle wall; some detached oocytes occurred. In the male, varying quantities of spermatogenic cells were present (Fig.1B). 3) Ripe stage: in the female, most oocytes were free-living within the follicles, with some oocytes attached to the follicle wall. In the male, follicles were filled by spermatozoa arranged in characteristic bands (Fig.1C). 4) Partially spawned: in the female, large spaces inside the follicles and between free oocytes were present. Some follicles were completely devoid of oocytes. In the male, a substantial decrease in the quantity of spermatozoa was observed. Large spaces inside the follicles occurred. In some follicles, only a few residual spermatozoa were present (Fig.1D). 5) Spent: at this stage, some unspawned oocytes and spermatozoa were observed within follicles (Fig.1E).
2 Materials and Methods
2.3 Lipid and Fatty Acid Analyses Five specimens from each gonad sample were used for lipid and fatty acid analyses. Total lipids were extracted from the gonad with chloroform/methanol 2:1 containing 0.1% butylated hydroxytoluene (BHT) as antioxidant following the Folch’s method (Folch et al., 1957). The lipid extracts were fractionated into neutral lipids (including triglycerides, free fatty acids, and sterols) and polar lipids (mainly including phospholipids with a small amount of glycolipids) using column chromatography on a silica gel hydrated with 6% water (Pernet et al., 2006). Briefly, the 100-mg columns were preconditioned with 1 mL of methanol and 1 mL of chloroform. The lipid aliquots (200 μL) corresponding to about 1 mg of lipid were loaded to the solid-phase extraction column. Samples were gently drawn into the solid phase under light vacuum, and the columns were washed with 1 mL of chloroform-methanol (98:2) to elute neutral lipids followed by 5 mL of methanol to elute polar lipids. The eluted fractions were collected in 7 mL tubes positioned in a vacuum manifold apparatus. The vacuum was adjusted to generate a flow rate of about 1 mL min−1. The fractions of polar lipids and neutral lipids were saponified with 1 mol L−1 KOH/methanol solution and esterified with 2 mol L−1 HCl/methanol solutions. Methyl esters were extracted with n-hexane. The fatty acid profile was determined by gas chromatography (HP 5890) using a Carbowax 20 m column (25 mm × 0.35 mm). The oven temperature was kept at 150℃ for 1 min, and then increased to 200℃ for 15℃ per min, followed by an increase to 250℃ for 2℃ per min. Injector and detector
2.1 Sample Preparation From March 2004 to February 2005, 30 cockles (shell height, 5.1cm ± 0.4 cm; shell length, 4.6 cm ± 0.3 cm; wet flesh weight, 13.4 g ± 2.2 g) were collected monthly on the eastern coast of China (Weihai, Shandong) (36˚41΄N– 37˚36΄N and 121˚11΄E–121˚42΄E). The cockles were immediately transported to the laboratory for measurements of the fresh weight and linear dimension (length, height). Then, the cockles were dissected and the gonad was collected and stored at −80℃ before use. During sampling, sea water temperature was measured in situ using centigrade thermometer, and the chlorophyll a content (0–1 m depth) was determined according to Parsons et al. (1984). Phytoplankton were sampled with a standard Shallow III Microplankton Net (diameter 37 cm, mesh fiber JF62, mesh size 0.077 mm) that was hauled vertically, strictly following the CSBTS standard method (CSBTS, 1991). The phytoplankton samples were fixed with Lugol’s solution, with cell number (counting units) counted under a light microscope. 2.2 Histology Fifteen specimens from each gonad sample were selected for histological examination. A 5-mm-thick cube of the gonad was fixed in Bouin’s solution, dehydrated using graded series of ethanol, cleared in xylol and embedded in paraffin. The 6-μm thick sections were cut on a rotary microtome, stained with haematoxylin and eosin (Humason, 1979), and then examined by microscopy to develop
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temperatures were 270℃. Helium was used as the carrier gas. The identification of fatty acids was performed by comparing retention times with a standard mixture of fatty acids. The quantification was based on the relative peak areas. Results are expressed as a percentage of the total fatty acid area (Xue et al., 1995).
2.4 Statistical Analysis The concentration of 16:1 represents the sum of two fatty acids: 16:1n-7 and 16:1n-9. All data are reported as mean ± standard error (n = 5). The relationships between the chlorophyll a concentration and the phytoplankton biomass and cell number were examined using a regression analysis. Pearson correlation was used to explore relationships between lipid content and the two environmental factors (sea water temperature and chlorophyll a concentration). One-way ANOVA followed by Tukey’s multiple comparison tests was used to assess the monthly differences in oocyte diameter, lipid content and fatty acids. Fatty acids from the neutral and polar fractions were analyzed separately.
3 Results 3.1 Environment Parameters The seasonal variation in phytoplankton biomass in Rushan Bay was typical double-peak type (Fig.1). The highest peaks appeared in August and September, and the second highest peaks appeared in May and April. The cell number of phytoplankton ranged from 248 × 104 to 3024 × 104 cells m−3, with an average of 1092 × 104 cells m−3. The phytoplankton biomass ranged from 2.0–40.7 mg m−3 annually, with an average of 13.3 mg m−3.
a exhibited a clear seasonal pattern characterized by two peaks, i.e., the lower one in April 2004 (17.7 μg L−1) and the higher one in September 2004 (25.8 μg L−1). The chlorophyll a concentration was low in winter, and averaged 11.3 ± 8.2 μg L−1 during the entire experimental period. Regression analysis showed that chlorophyll a concentration was significantly correlated with phytoplankton biomass and cell number (P
