Seasonal variations in biochemical composition

Marine Biology Research

ISSN: 1745-1000 (Print) 1745-1019 (Online) Journal homepage: http://www.tandfonline.com/loi/smar20

Seasonal variations in biochemical composition during the reproductive cycle of the veined rapa whelk Rapana venosa (Valenciennes, 1846) from the northern coast of China Jinhong Bi, Qi Li, Hong Yu, Zhixin Zhang, Yan Lian, Renjie Wang & Tingyu Wang To cite this article: Jinhong Bi, Qi Li, Hong Yu, Zhixin Zhang, Yan Lian, Renjie Wang & Tingyu Wang (2016) Seasonal variations in biochemical composition during the reproductive cycle of the veined rapa whelk Rapana venosa (Valenciennes, 1846) from the northern coast of China, Marine Biology Research, 12:2, 177-185, DOI: 10.1080/17451000.2015.1125002 To link to this article: http://dx.doi.org/10.1080/17451000.2015.1125002

Published online: 01 Mar 2016.

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Date: 01 March 2016, At: 21:52

MARINE BIOLOGY RESEARCH, 2016 VOL. 12, NO. 2, 177–185 http://dx.doi.org/10.1080/17451000.2015.1125002

ORIGINAL ARTICLE

Seasonal variations in biochemical composition during the reproductive cycle of the veined rapa whelk Rapana venosa (Valenciennes, 1846) from the northern coast of China Jinhong Bia,b, Qi Lia, Hong Yua, Zhixin Zhangb, Yan Lianc, Renjie Wangb and Tingyu Wangb Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, China; bRongcheng Fishery Technical Extension Station, Rongcheng, China; cRongcheng Marine Environmental Monitoring Center, Rongcheng, China

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a

ABSTRACT

ARTICLE HISTORY

Seasonal variations in the biochemical composition of Rapana venosa in relation to reproductive cycle and environment on the northern coast of China were investigated from March 2012 to February 2013. The results indicated that R. venosa has an annual reproductive cycle with synchronized gonad development in both females and males. Gametogenesis was initiated in September and gametes developed slowly during the winter, followed by rapid gonad development during spring and summer. Most individuals from this study were sexually mature between May and June, and gamete release occurred mainly between May and August. The peak of spawning was found in July and the recovery of the gonad was observed between August and November. The key biochemical components including glycogen, protein and lipid were analysed in four tissues, specifically the gonad, digestive gland, mantle and foot. The declining glycogen content in the gonad, digestive gland and mantle during maturation suggested that glycogen was consumed during the development of the gonad. Lipids and protein can be stored in the digestive gland and used during the winter in a period of food shortage. The protein and lipid contents in the ovaries increased during gonad development, which suggested that the protein and lipid had been accumulated as vitellin in oocytes.

Received 23 February 2015 Accepted 21 November 2015 Published online 1 March 2016

Introduction The veined rapa whelk Rapana venosa (Valenciennes, 1846), which belongs to the family Muricidae, is a large predatory marine gastropod native to the coast of China, Korea and Japan (Chung et al. 2002; Xue et al. 2014a). In the mid-1940s, it invaded the Black Sea and eventually became one of the dominant species in the benthic ecosystem (Drapkin 1963; Kos’yan 2013). Since then, R. venosa has been found from the Chesapeake Bay in USA (Harding & Mann 1999) to the Maldonado Bay in Uruguay (Carranza et al. 2010), and has been recognized as a typical invasive species around the world (Chandler et al. 2008). In China, R. venosa is an economically important mollusc species. Its production has decreased dramatically in the last 20 years due to over-exploitation and the deterioration of environmental conditions (Yang et al. 2008). The large-scale artificial production of whelk seeding has not been accomplished. Understanding the reproductive cycle of this species is crucial for establishing aquaculture techniques of seeding production.

CONTACT Qi Li © 2016 Taylor & Francis

[email protected]

RESPONSIBLE EDITOR

Eric Thompson KEYWORDS

Rapana venosa; gametogenesis; biochemical composition; environment

The development and maturation of gametes in molluscs is affected by exogenous (e.g. temperature, salinity, light and food) and endogenous (e.g. neuronal and hormonal regulators) factors (Chung et al. 2002; Louro et al. 2006; Magnesen & Christophersen 2008; Park et al. 2011). The reproductive changes in molluscs are associated with translocation of the biochemical constituents between somatic tissue and reproductive organs (Giese 1959, 1969). In general, when food is abundant, energy is stored in the form of glycogen, lipid and protein, which can be used for maintenance, growth and reproduction (Dridi et al. 2007). However, the storage location and the usage timing of energy varies among species and even among populations from the same species (Giese 1969; Bayne 1976). For instance, Pisaster ochraceus (Brandt, 1835) stores food reserves in the pyloric caeca and transfers it to the gonad during the breeding period (Farmanfarmaian et al. 1958), while Haliotis cracherodii Leach, 1814 uses the foot as a nutrient storage pool for one of the periods of gonad development (Webber 1970).

Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, China


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Studies on reproduction-related biochemical component changes have been reported in several gastropod species, including H. cracherodii (Webber 1970), Nucella lamellosa (Gmelin, 1791) (Lambert & Dehnel 1974), Haliotis varia (Linnaeus, 1758) (Najmudeen 2007) and Turbo bruneus (Röding, 1798) (Ramesh & Ravichandran 2008). Extensive work has been conducted on the veined rapa whelk for analysis of morphological characteristics (Kos’yan 2013), biological invasions (Carranza et al. 2010; Lezama et al. 2013), reproductive biology (Wei et al. 1996; Yang et al. 2007; Pan et al. 2013) and genetic studies (Chandler et al. 2008; Yang et al. 2008; Xue et al. 2014b). However, information on the reproductive cycle of R. venosa remains largely unknown, and alterations of reproduction-related biochemical components in an annual cycle have not been analysed. In this study, we investigated variations in reproduction-related biochemical components in one full reproductive cycle of R. venosa from Jinghai Bay of northern China. The concentrations of glycogen, lipid and protein from the gonad, digestive gland, mantle and foot tissue of the sample were determined in relation to the recorded environmental parameters (temperature, salinity and chlorophyll a) on a monthly basis. The observations in this study should be useful to develop techniques to facilitate seeding production for aquaculture in R. venosa.

Materials and methods Sample collection The sampling area (122°1’–122°8’;E, 36°52’–36°43’N) was located in Jinghai Bay, Rongcheng, Shandong Province of north China (Figure 1). In the middle of each month, 90–100 wild veined rapa whelks (85.1–119.5 mm in shell height and 97.5–230.5 g in wet weight) were collected at a water depth of 10–15 m using a trawl net from March 2012 to February 2013. The temperature and salinity of seawater were measured at the sampling site using a handheld multi-parameter

Figure 1. Location of collection site for Rapana venosa in Jinghai Bay of China.

water quality monitor (Horiba U-50 Series; Horiba Ltd, Kyoto, Japan) every month. The concentration of chlorophyll a at a depth of 10 m was determined according to Dai & Lu (1997). Four tissues, including gonad, digestive gland (hepatopancreas), mantle and foot, were dissected and flash-frozen at –80°C for further analysis.

Sex phenotype The sex of each specimen was distinguishable by the presence of a penis in males. The gonad lies on one side of the digestive gland in the upper coils of the visceral mass. The ovary is usually bright orange–red, whereas the testis is faint yellow. Each individual was also determined for sex using light microscopy (see below).

Histology Histological analysis was conducted to further determine the sex and gametogenic stage for each sample. A 5 mm thick section of gonad was removed from the whelk and fixed in Bouin’s solution for 24 h, then dehydrated with serial dilutions of alcohol and embedded in paraffin wax. Sections of 6 μm thickness were cut with a microtome and stained with haematoxylin and eosin. The sections were examined using an Olympus CX21 microscope at 40× magnification and each specimen was assigned to a stage that represented the gonad condition. The gametogenic stage was categorized into five stages according to Chung et al. (2002): early active stage (stage I), late active stage (stage II), ripe stage (stage III), partially spawned stage (stage IV) and recovery stage (stage V). To obtain quantitative data, the diameters of oocytes with visible nuclei from 10 randomly selected females (100 oocytes per specimen) were measured each month (Joaquim et al. 2008).

Biochemical analyses Glycogen, protein and lipid content were analysed in the four tissues of gonad, digestive gland, mantle and foot. To minimize inter-animal variability, 30–45 specimens of each sex were randomly divided into three groups with 10–15 specimens pooled in each replicate. Glycogen content was analysed with a minor modification of the anthrone–sulphuric acid method described by Horikoshi (1958). The 0.05 g of powdered, freeze-dried samples were suspended in 60 volumes of KOH (0.3 g/ml) and saponified by heating to 100°C for 30 min. After cooling, 0.5 ml of homogeneous solution was treated with 5 ml of cold 0.2% anthrone–sulphuric

MARINE BIOLOGY RESEARCH


acid solution for 10 min, and the absorbance of the resulting coloured complex was measured at a wavelength of 620 nm. Soluble protein levels in tissues were determined according to the method of Bradford (1976), using bovine serum albumin as the reference protein. Fresh tissue (100 mg) was homogenized in 2 ml saline solution. After being centrifuged and diluted, the crude extract sample was mixed with the dye reagent Brilliant Blue G and the absorbance value was measured at 595 nm. The lipid content was analysed using the gravimetric method. Lipid extraction was made in diethyl ether using an automatic Buchi extraction system (B-811; Buchi Co., Sweden). The dry tissue powders were reweighed after extraction and the lipid content was determined as the lipid loss.

Statistical analysis All statistical analyses were performed using SPSS software (version 19.0). The chi-square test was used to compare the population sex ratio with the parity (1 : 1). One-way ANOVA was used to assess the monthly differences in oocyte diameter followed by Duncan’s test (α = 0.05). Two-factor ANOVAs were employed to compare the biochemical parameter on each separate tissue using sex and month as the factors, followed by post-hoc comparisons using Duncan’s test (α = 0.05). When the values of each biochemical parameter were significantly different between sexes, the values of each biochemical composition of each separate tissue

Figure 2. Seasonal variations in seawater temperature, salinity and chlorophyll a concentration in Jinghai Bay of China.

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were compared in each sex for different months using one-way ANOVA followed by post-hoc comparisons using Duncan’s test (α = 0.05). Prior to analysis, raw data were tested for normality of distribution and homogeneity of variance.

Results Environmental parameters The monthly variations of the environmental parameters (seawater temperature, salinity and concentration of chlorophyll a) from Jinghai Bay during the studying period (March 2012–February 2013) are illustrated in Figure 2. The water temperature at the sampling site showed a typically seasonal pattern of the temperate zone, with a unimodal peak (25.8°C) in August and minimum value (0.9°C) in January. The concentrations of chlorophyll a exhibited a similar pattern to the water temperature, with an observed peak (10.6 μg l–1) in September and a minimum value (0.46 μg l–1) in January. The chlorophyll a concentrations were significantly related to the seawater temperature (Pearson correlation, r = 0.673, P < 0.01). Salinity was relatively stable throughout the year, varying from 30.2 to 32.0 PSU.

Gametogenic activity Based on histological observations of the gonads, both sexes displayed variable proportions of different gonad

Figure 3. Seasonal distribution of Rapana venosa at different stages of gonad development. Stage I: early active stage, stage II: late active stage, stage III: ripe stage, stage IV: partially spawned stage and stage V: recovery stage.


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stages throughout the year (Figure 3). The veined rapa whelks had an annual reproductive cycle with a synchronized gonad development of both females and males. Most individuals were sexually mature between May and June. Gamete release occurred mainly between May and August with the spawning peak in July (female: 71.4%, male: 68.2%), followed by a period of gonad recovery between August and December. Gametogenesis initiated in September and the early active stage (stage I) was detected between September 2012 and February 2013. The following period is the late active stage (stage II) and was recorded from March to May 2012 and January to February 2013. The oocyte diameter showed significant differences among months (P < 0.05), which increased from 134.9 μm in March to a maximum value of 258.7 μm in June, and decreased after August (Table I). The minimum value was recorded as 17.1 μm in September. From September 2012 to February 2013, the oocyte diameter increased to 100.3 μm.

Sex ratio Based on all 1093 specimens, 552 were females and 541 were males (Table II), resulting in a balanced sex ratio (female:male = 1.02:1, P > 0.05). The monthly sex ratio was balanced in March–June, September, November and December 2012, and January and February 2013 (P > 0.05), while July 2012 was dominated by males (P < 0.05) and August and October 2012 were dominated by females (P < 0.05).

Biochemical composition The glycogen content in the gonads, digestive glands and mantles of both sexes showed marked variations throughout the reproductive cycle, but did not show significant oscillations in the foot (Table III). In the ovaries and testes, glycogen content dropped sharply from March (female: 35.8%, male: 34.1%) to July (female: 13.0%, male: 10.5%), then rose up to the peak in October (female: 41.7%, male: 40.1%). After that, glycogen content declined dramatically. The general trend of the glycogen contents in digestive glands and mantles was similar in the gonads. The glycogen content in the foot ranged from 32.6% to 41.3% in females and 32.0% to 44.5% in males. Two-factor ANOVA analysis indicated that there was no significant difference between sexes in the glycogen content of the gonads, digestive glands and mantles (P > 0.05), although the glycogen content changed significantly over the year (P < 0.05).

The lipid content in the ovaries increased from March to a maximum value in June (15.3%) and decreased gradually to a minimum of 7.6% in September, followed by another increase to 13.5% in February 2013 (Table III). The lowest value of lipid content in the testis was in June (6.0%) and the highest value was in October (13.5%). In the digestive gland, there was an increase in lipid content from March and the highest value was obtained in August (female: 15.7%, male: 14.6%), followed by the decrease from September to February of the next year. In the foot, lipid level was very low and showed limited seasonal variation, with values ranging from 1.4% to 1.9% in females and from 1.4% to 2.3% in males. In the mantles, the lipid content ranged from 1.5% to 3.7% in females and from 1.3% to 4.0% in males with minor fluctuations. Two-factor ANOVA analysis indicated that lipid content did not change in the foot and mantles (P > 0.05). However, significant sexual and temporal variations were found in the gonads (P < 0.05) and a significant temporal variation was found in the digestive gland (P < 0.05). The protein content in the ovaries exhibited a clear seasonal variation, which increased from March to a maximum value of 64.5 mg g–1 in June and declined to a minimum of 22.6 mg g–1 in September, followed by another increase to 42.8 mg g–1 in February (Table III). The protein content in the testes rose from March to the maximum value of 54.4 mg g–1 in September and declined thereafter. The protein content in the digestive gland of females appeared to drop down to 35.5 mg g–1 in June and successively increased to 48.9 mg g–1 in October, and then decreased during winter. A similar pattern was found in the digestive gland of the males. There were no seasonal variations in the protein content of foot and mantles. Twofactor ANOVA analysis indicated that the protein content did not change in the foot and mantles (P > 0.05). However, significant sexual and temporal variations were found in the gonads (P < 0.05) and a significant temporal variation was found in the digestive gland (P < 0.05).

Discussion Many studies have reported the effect of environmental variables on the reproductive processes of molluscs (Rodriguez-Rua et al. 2003; Cantillanez et al. 2005; Uddin et al. 2007). Fluctuations in water temperature have a substantial and direct effect on gametogenesis (Gribben et al. 2004). In our study, gametogenesis was initiated in September, when the water temperature reached 22.4°C. Gametes grew slowly throughout the


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Table I. Seasonal variations in oocyte diameter of Rapana venosa. Month-Year

Stage I

Stage II

Stage III

Stage IV

Average value


Mar-2012 134.9 ± 6.7 134.9 ± 6.7bc Apr-2012 144.4 ± 16.6 241.8 ± 13.9 208.1 ± 15.8ab May-2012 138.2 ± 11.6 245.1 ± 11.8 231.8 ± 15.1 211.7 ± 14.2ab Jun-2012 262.3 ± 13.9 240.1 ± 16.5 258.7 ± 15.8a Jul-2012 251.3 ± 18.5 213.5 ± 14.6 249.9 ± 16.6a Aug-2012 234.6 ± 12.7 114.6 ± 9.5 134.6 ± 11.6bc Sep-2012 17.1 ± 2.5 17.1 ± 2.5d Oct-2012 37.6 ± 1.9 37.6 ± 1.9d Nov-2012 48.5 ± 2.4 48.5 ± 2.4cd Dec-2012 80.2 ± 4.0 80.2 ± 4.0cd Jan-2013 60.9 ± 10.8 121.7 ± 12.5 96.1 ± 11.2cd Feb-2013 82.6 ± 9.2 118.1 ± 14.5 100.3 ± 12.9cd Means not sharing the same superscript are significantly different (P < 0.05). Values are means ± SD.

winter as the water temperature gradually decreased, following by a rapid development of the gonad during spring and summer. Most individuals were sexually mature between May and June, and gamete release occurred mainly between May and August, with a water temperature of 18.0–25.8°C. The peak of spawning was found in July, with a water temperature of 24.5°C. Our findings are consistent with observations described by Chung et al. (2002) in Korea, where gametogenesis initiated in September and spawning occurred between May and early August, with seawater temperature rising to 18–26°C. The oocyte diameter measured in this study showed clearly seasonal variations, which increased at the beginning of gametogenesis, reached the highest value when the gonad matured and declined after gametes were released, indicating that oocyte size can be a good indicator of maturation in Rapana venosa. Quantitative analysis of oocyte diameter has been used as a good quantitative descriptor of the reproductive development and spawning in Crassostrea plicatula (Thunberg, 1793) (Li et al. 2006), Sinonovacula constricta (Lamarck, 1818) (Yan et al. 2010) and Mactra chinensis (Philippi, 1846) (Li et al. 2011). A balanced sex ratio was observed in the population of R. venosa from Jinghai Bay throughout the whole year in this study, which was consistent with the trend observed from the west coast of Korea (Chung et al. 2002) and Chesapeake Bay (Mann et al. 2006). Low proportions of females were detected in July, coinciding with the spawning peak and high proportions of females were detected at the end of the spawning season (in August) and the early active stage (in October). The monthly variation in the sex ratio could be caused by a combination of different factors, such as the reproductive behaviour, migrations and sample collection (Saglam et al. 2009; Lourenco et al. 2012). The carbohydrates in the molluscs are mainly composed of glycogen (Taylor & Venn 1979) and several

studies have indicated that glycogen plays an important role in the physiology of molluscs, particularly during the reproductive period (Lambert & Dehnel 1974; Liu et al. 2008; Park et al. 2011; Ke & Li 2013). In the present study, the glycogen content in all the sampled tissues except the foot decreased significantly during sexual maturation. This suggests that the gonads, digestive glands and mantles were the main storage sites of glycogen, and glycogen may be utilized in a considerable quantity for the development of gametes. Notably, the foot was not used as a glycogen storage site, and could not supply energy for gametogenesis, which is different from the observation in H. cracherodii, where the foot glycogen acts as a nutrient storage site for metabolic demands of gonad growth, maintenance and growth (Webber 1970). In Turbo bruneus, the carbohydrate content decreased in the gonad but increased in the digestive gland, which suggested that carbohydrate from the digestive gland may be used up when the gonad became mature (Ramesh and Ravichandran, 2008). In other bivalves, such as Fulvia mutica (Reeve, 1844) (Liu et al. 2008) and S. constricta (Yan et al. 2010), it was reported that glycogen content in all tissues decreased during gametogenesis. Lipids are also an important energy source for sustaining embryonic and early larval development in marine invertebrates (Giese 1966; Holland 1978; Gallager & Mann 1986). As for the veined rapa whelks in this study, the lipid content in the digestive gland of both sexes increased continuously from March to August, when the feeding quantity of whelks increased with the abundant food supply (such as bivalves). It is well known that the nutritional components in food are processed in the digestive gland, and accordingly, large amounts of lipids are accumulated. The lipid content in the digestive gland decreased from August 2012 to February 2013, while in the ovaries it increased from September 2012 to February 2013,

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Table II. Monthly variations in sex ratio of Rapana venosa between March 2012 and February 2013. Month-year Mar-2012 Apr-2012 May-2012 Jun-2012 Jul-2012 Aug-2012 Sep-2012 Oct-2012 Nov-2012 Dec-2012 Jan-2013 Feb-2013 Average

No. of specimens

No. of females

Percentage female (%)

No. of males

Percentage male (%)

Sex ratio female:male

84 90 90 85 93 80 100 99 96 89 96 91 91

48 50 42 40 33 50 45 60 42 39 57 46 46

57.1 55.6 46.7 47.1 35.5 62.5 45.0 60.6 43.8 43.8 59.4 50.5 50.5

36 40 48 45 60 30 55 39 54 50 39 45 45

42.9 44.4 53.3 52.9 64.5 37.5 55.0 39.4 56.2 56.2 40.6 49.5 49.5

1.33 1.25 0.88 0.89 0.55* 1.67* 0.82 1.54* 0.78 0.78 1.46 1.02 1.02

*Indicates significant differences between females and males (P < 0.05).

Table III. Monthly variations in glycogen contents (% dry weight), lipid contents (% dry weight) and protein contents (mg g–1 wet weight) of gonad, digestive gland, mantle and foot of Rapana venosa.


Glycogen Monthyear

Sex

Mar-2012 ♀

Apr-2012

May-2012

Jun-2012

Jul-2012

Aug-2012

Sep-2012

Oct-2012

Nov-2012

Dec-2012

Jan-2013

Feb-2013

Gonad

Digestive gland

Lipid Mantle

Foot

45.2 ± 4.2ab 44.6 ± 32.6 ± 35.8 ± 1.9a 0.1 2.8b ♂ 34.1 ± 7.0 43.3 ± 2.3 45.3 ± 3.6 38.4 ± 13.0 39.3 ± 2.8b 35.5 ± 37.5 ± ♀ 27.2 ± cd bc 3.6 11.4 0.5 ♂ 22.9 ± 5.6 41.1 ± 0.9 40.4 ± 4.7 32.0 ± 0.5 33.8 ± 41.3 ± ♀ 19.4 ± 4.6c 36.6 ± 5.4c cd 3.1 1.2 ♂ 18.9 ± 4.9 22.1 ± 3.1 35.5 ± 3.4 44.5 ± 9.7 17.6 ± 1.8d 14.8 ± 36.9 ± ♀ 18.4 ± ef fg 1.6 3.7 5.8 ♂ 15.3 ± 1.5 21.4 ± 1.5 15.3 ± 9.7 40.9 ± 9.2 11.9 ± 38.7 ± ♀ 13.0 ± 2.1f 7.6 ± 1.2e g 7.6 5.1 ♂ 10.5 ± 4.5 18.1 ± 3.8 14.2 ± 3.9 40.5 ± 3.4 17.3 ± 0.5d 15.8 ± 41.0 ± ♀ 16.7 ± ef f 2.0 0.4 2.9 ♂ 14.2 ± 5.7 29.0 ± 2.9 21.8 ± 4.9 40.0 ± 2.7 38.5 ± 0.8c 26.8 ± 40.7 ± ♀ 33.9 ± b de 2.3 0.5 5.6 ♂ 34.9 ± 1.8 30.0 ± 9.8 34.2 ± 8.5 40.8 ± 0.6 46.4 ± 40.5 ± ♀ 41.7 ± 4.7a 48.6 ± 6.5a a 8.1 0.5 ♂ 40.1 ± 4.4 45.4 ± 3.5 42.7 ± 1.5 42.4 ± 0.5 43.8 ± 4.7b 35.5 ± 40.0 ± ♀ 38.6 ± ab ab 2.1 1.3 10.8 ♂ 32.7 ± 3.9 36.5 ± 3.6 45.2 ± 3.0 38.4 ± 6.2 ♀ 24.9 ± 1.5c 37.8 ± 2.6c 33.1 ± 3de 34.6 ± 4.3 ♂ 28.5 ± 6.6 30.9 ± 0.6 27.0 ± 2.6 35.2 ± 5.5 29.6 ± 5.8c 31.5 ± 39.5 ± ♀ 14.5 ± de e 3.2 0.2 1.3 ♂ 25.1 ± 1.2 30.4 ± 4.8 25.5 ± 1.2 36.0 ± 3.3 14.5 ± 40.2 ± ♀ 11.4 ± 4.7f 25.3 ± 1.6d fg 1.2 0.7 ♂ 12.8 ± 2.4 19.6 ± 5.9 16.0 ± 0.9 40.2 ± 0.9

Digestive gland

Gonad 11.1 ± 3.9ABCD 9.4 ± 1.3bcd

3.6 ± 0.6h

12.0 ± 4.6ABC 8.2 ± 0.7cdef 14.8 ± 0.7A

gh

7.5 ± 1.2efg 15.3 ± 1.9

A

6.0 ± 0.4g 8.9 ± 0.5

CD

6.4 ± 0.7fg

6.2 ± 2.1 4.9 ± 1.5

7.4 ± 1.2 def

8.2 ± 0.7

7.6 ± 1.7 9.1 ± 0.8

cde

8.0 ± 0.4 10.1 ± 0.6

bcd

8.7 ± 0.3

CD

15.7 ± 0.1

8.6 ± 1.2bcde 7.6 ± 0.8D

14.6 ± 2.1 13.0 ± 2.5

10.4 ± 0.8b

13.0 ± 5.1

CD

11.9 ± 0.4

b

13.5 ± 1.3a

10.9 ± 1.3

CD

bc

8.4 ± 1.3

8.3 ± 0.9

9.0 ± 1.6

10.4 ± 0.6b 10.2 ± 1.5BCD 9.9 ± 1.2bc 11.5 ± 1.9ABCD 7.7 ± 0.5defg 13.5 ± 2.9AB 6.7 ± 1.3fg

9.8 ± 0.8

a

a

10.6 ± 1.7 9.5 ± 0.5

bcd

10.5 ± 0.9 8.7 ± 2.3

cd

8.9 ± 0.1 7.1 ± 1.2

fgh

6.1 ± 0.1

Protein Mantle

Foot

1.5 ± 0.4 1.5 ± 0.4 1.6 ± 0.2 2.0 ± 0.8 1.8 ± 0.1 2.2 ± 0.4 2.1 ± 0.2 2.2 ± 0.3 1.8 ± 0.4 1.6 ± 0.6 1.6 ± 0.1 2.7 ± 0.9 2.0 ± 1.0 3.8 ± 0.8 2.2 ± 1.4 1.6 ± 1.1 3.7 ± 1.1 4.0 ± 0.1 1.7 ± 0.7 2.1 ± 0.2 2.7 ± 0.4 2.7 ± 0.6 1.5 ± 0.1 1.3 ± 0.5

1.6 ± 29.3 ± 4.8EFG 0.4 1.7 ± 32.5 ± 0.7de 0.1 1.5 ± 35.3 ± 0.4 2.3CDEF 2.3 ± 35.0 ± 3.2de 1.8 1.4 ± 47.5 ± 1.4B 0.5 1.9 ± 38.9 ± 8.2cde 0.6 1.8 ± 64.5 ± 11.9A 0.8 2.3 ± 43.7 ± 0.8 2.8abcd 1.8 ± 41.9 ± 0.7 4.2BCD 1.7 ± 50.5 ± 9.4ab 0.5 1.9 ± 30.7 ± 0.9 10.5DEFG 1.4 ± 53.1 ± 2.5ab 0.1 1.6 ± 22.6 ± 2.0G 0.5 1.4 ± 54.4 ± 5.1a 0.1 1.8 ± 27.8 ± 2.2FG 0.6 2.2 ± 47.0 ± 1.1abc 0.1 1.5 ± 37.3 ± 0.3 9.3BCDEF 2.1 ± 42.6 ± 0.3 15.6bcd 1.4 ± 39.7 ± 0.4 6.9BCDE 1.5 ± 35.1 ± 2.0de 0.4 1.5 ± 41.4 ± 0.3 3.4BCD 1.4 ± 32.7 ± 1.8de 0.4 1.8 ± 42.8 ± 2.7BC 0.3 1.8 ± 30.4 ± 0.1c 0.1

Gonad

Digestive gland 48.4 ± 4.5a 49.3 ± 5.9 39.8 ± 6.3ab 46.1 ± 4.1 39.4 ± 3.2abc 44.1 ± 0.7 35.5 ± 1.0de 31.2 ± 1.8 38.7 ± 13.0bcd 35.0 ± 11.3 45.5 ± 4.1ab 40.0 ± 1.2 45.9 ± 0.6ab 40.4 ± 9.0 48.9 ± 5.1a 45.7 ± 2.7 37.3 ± 2.1bcd 40.1 ± 6.9 35.4 ± 2.8cde 34.3 ± 6.4 35.0 ± 3.4de 32.6 ± 2.2 30.4 ± 4.5e 27.1 ± 5.7

Mantle

Foot

37.1 ± 3.0 35.3 ± 1.7 35.1 ± 1.3 37.7 ± 5.2 41.9 ± 11.8 35.8 ± 6.8 36.3 ± 5.8 32.3 ± 4.3 32.8 ± 6.5 38.1 ± 10.0 36.1 ± 1.9 31.0 ± 7.1 33.4 ± 9.7 30.2 ± 5.3 39.0 ± 8.2 35.6 ± 4.8 41.5 ± 9.2 41.3 ± 3.9 33.7 ± 3.8 42.5 ± 2.1 35.5 ± 4.2 33.5 ± 3.4 35.1 ± 9.0 34.6 ± 4.8

52.0 ± 10.2 53.9 ± 5.3 45.9 ± 7.3 48.4 ± 1.8 50.9 ± 5.6 48.0 ± 7.7 57.8 ± 2.0 51.8 ± 4.7 55.1 ± 4.8 55.2 ± 12.7 50.2 ± 9.3 49.6 ± 9.0 45.1 ± 3.3 46.3 ± 1.0 46.7 ± 3.7 44.8 ± 0.9 53.4 ± 9.5 52.7 ± 12.3 51.7 ± 6.2 43.8 ± 2.5 51.5 ± 4.9 46.8 ± 9.0 49.5 ± 10.4 46.7 ± 7.2

Different lower-case superscript letters in the same row indicate a statistical difference (P < 0.05). Different capital superscript letters in the same column indicate a statistical difference (P < 0.05).



suggesting that the stored lipids in the digestive gland could be transferred to the ovaries. A similar phenomenon was reported by Sastry & Blake (1971), who recorded that the digestive gland in molluscs could not only store the nutrients but also transfer them to other tissues. The drop in the abundance of phytoplankton in the water can also lead to a diet with a lower amount of lipids. The lipid content in the ovaries increased gradually during gonad development, which suggested that the lipids formed the energy source for the egg yolk (Giese 1966). Similar trends were reported in gastropods such as Hexaplex trunculus (Linnaeus, 1758) (Gharsallah et al. 2010) and in bivalves such as Anadara broughtonii (Schrenck, 1867) (Park et al. 2001). Furthermore, we observed an inverse relationship between the lipid content and the glycogen content in the ovaries, indicating that the lipids were converted from glycogen reserves (Napolitano et al. 1992). The phenomenon of the glycogen being converted to lipid during gametogenesis was also reported in Haliotis varia (Najmudeen 2007) and Crassostrea gigas (Li et al. 2000). The lipid content in the testes decreased during gonad development, suggesting that the lipid was used as an energy source during spermatogenesis (Yan et al. 2010). The lipid content in the foot and mantles showed minor fluctuations, which supported the fact that the foot and mantles were rarely to be considered as lipid storage organs (Giese 1966). In this study, the increase of the protein content together with an increase in lipids in ovaries suggested a synchronous accumulation of protein and lipids as vitellin (Li et al. 2000). The protein content in the female digestive glands decreased during gonad development, suggesting that protein in the digestive glands could be utilized and transferred to the ovaries. It has previously been reported that the gonad protein accumulated in yolk granules was ferritin (Najmudeen 2007) which was derived from the digestive glands (Heneine et al. 1969). Proteins were accumulated in the testes between March and September and provided energy during winter in a period of food shortage. The protein content in the digestive gland of the males decreased during gonad development, demonstrating that they could serve as an energy source for spermatogenesis. The protein content of the mantles did not change considerably, indicating that there was no transfer of proteins between mantles and gonads. Among all the tissues analysed, the highest value of protein was observed in the foot and showed no clear seasonal variation, as noted in T. bruneus (Ramesh & Ravichandran 2008).

183

In conclusion, the present study reports for the first time the reproductive cycle and energy storage utilization in relation to environmental factors of R. venosa from the northern coast of China. Gametogenesis initiated in September with a water temperature of 22.4°C, most individuals were sexually mature between May and June, and spawning was observed between May and August. Three tissues (gonad, digestive gland and mantle) were sites for glycogen storage, which supplied energy for gametogenesis. The protein and lipid in the ovaries can be accumulated as vitellin in oocytes. The lipid in the testes can supply energy during spermatogenesis. The useful information obtained in this study is valuable not only for sustainable exploitation of wild populations in this area, but also for optimizing the hatchery-based seed production for aquaculture.

Disclosure statement No potential conflict of interest was reported by the authors.

Funding This study was supported by grants from the National Marine Public Welfare Research Program (201305005), the Scientific and Technical Supporting Program (2011BAD13B01), and the National Natural Science Foundation of China (31201998).

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