Seasonal changes of the fatty acid composition in the

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Apr 22, 2014 - and vitelline gland of the gastropod Onchidium struma. XUE-PING YING1†, HANS-UWE .... ducts are further desaturated to arachidonic (ARA).
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Seasonal changes of the fatty acid composition in the hepatopancreas and vitelline gland of the gastropod Onchidium struma a

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Xue-Ping Ying , Hans-Uwe Dahms , Xiao-Ying Ni , Cong-Cong Hou , Yu-Jie Zhang & WanXi Yang a

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School of Life and Environmental Science, Wenzhou University, Wenzhou, China

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Environmental Biology Laboratory, Green Life Science Department, College of Natural Science, Sangmyung University, Seoul, South Korea c

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The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, China Published online: 22 Apr 2014.

To cite this article: Xue-Ping Ying, Hans-Uwe Dahms, Xiao-Ying Ni, Cong-Cong Hou, Yu-Jie Zhang & Wan-Xi Yang (2014) Seasonal changes of the fatty acid composition in the hepatopancreas and vitelline gland of the gastropod Onchidium struma , Marine Biology Research, 10:8, 781-790, DOI: 10.1080/17451000.2013.853125 To link to this article: http://dx.doi.org/10.1080/17451000.2013.853125

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Marine Biology Research, 2014 Vol. 10, No. 8, 781–790, http://dx.doi.org/10.1080/17451000.2013.853125

ORIGINAL ARTICLE

Seasonal changes of the fatty acid composition in the hepatopancreas and vitelline gland of the gastropod Onchidium struma XUE-PING YING1†, HANS-UWE DAHMS2†,‡, XIAO-YING NI1, CONG-CONG HOU3, YU-JIE ZHANG1 & WAN-XI YANG3* School of Life and Environmental Science, Wenzhou University, Wenzhou, China, 2Environmental Biology Laboratory, Green Life Science Department, College of Natural Science, Sangmyung University, Seoul, South Korea, and 3The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, China

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Abstract We analysed the fatty acid composition of the hepatopancreas and vitelline gland of Onchidium struma by capillary gas chromatography. Our results showed that there are 22 fatty acids (FAs) in the hepatopancreas and 23 in the vitelline gland, all of which were even carbon fatty acids and long chain fatty acids. C16:0, C16:1n-7, C18:0, C18:1n-9, C20:1n-9, C20:4n6 and C20:5n-3 were the principal FAs. In the hepatopancreas, 4 saturated fatty acids (SFAs; 29.53–35.44%), 4 monounsaturated fatty acids (MUFAs; 30.73–34.34%) and 14 polyunsaturated fatty acids (PUFAs; 29.33–36.72%) were found. In spring, summer, autumn and winter, FA numbers in the hepatopancreas were 22, 19, 18 and 20, respectively. C22:2NIMD and C22:4n-6 were only detected in spring. The content of C18:1n-9, C18:2n-6, C20:5n-3 and ∑n3 was remarkably different with season. In the vitelline gland, 4 SFAs (26.33–38.03%), 4 MUFAs (18.97–32.67%) and 15 PUFAs (31.92–52.51%) were found. In spring, summer, autumn and winter, FA numbers in the vitelline gland were 23, 17, 18 and 18, respectively. C20:0, C22:2NIMD, C22:3n-3 and C22:4n-6 were only detected in spring. The content of C18:1n9, C18:2n-6, C18:3n-3, C20:4n-3, C20:4n-6, C20:5n-3 and ∑n3 was remarkably different for different seasons. From spring to summer, C20:4n-6, C20:5n-3 and C22:6n-3(DHA) in the vitelline gland decreased significantly, while C18:2n-6 and C18:3n-3 increased. These results showed that the seasonal variation of FAs is clearly related to the reproductive cycle, and suggested that FA in the hepatopancreas may be transferred to the vitelline gland of O. struma during maturation. Key words: Fatty acid, hepatopancreas, Onchidium struma, seasonal change, vitelline gland

Introduction The snail Onchidium struma belongs to the Onchidiidae, Stylommatophora, Pulmonata, Gastropoda, Mollusca (Shen et al. 2004). In China, it is also known as ‘tu hai sen’, ‘tu ji’ or ‘sea toad’. O. struma is rich in protein, amino acids and abundant mineral elements and has high levels of flavour and could be a prospective specialty seafood or upscale tonic for elderly people and teenagers (Huang & Wang 2008). Many investigators have studied various basic biological aspects of O. struma, including its reproductive behaviour (Wang et al. 2005), reproductive

system and gonad development (Wang et al. 2006; Chen et al. 2010), breeding (Huang et al. 2004), nutrient components (Wu et al. 2007, 2008; Huang & Wang 2008), ecological habits (Huang et al. 2004), and sperm ultrastructure (Ying et al. 2008). Other aspects of studies on Onchidium include its neurophysiology, hormone regulation (Katagiri 1984) and reproductive biology (Healy 1986; Smith & Kenny 1987). Nevertheless, few studies have determined the effect of transitory variation of seasons on the fatty acid profile of the species. The fatty acid profile is certainly influenced by temperature (Mateos

*Correspondence: Wan-Xi Yang, The Sperm Laboratory, College of Life Sciences, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou 310058, China. E-mail: [email protected] †Xue-Ping Ying and Hans-Uwe Dahms contributed equally to this work. ‡Current address: Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan. Published in collaboration with the Institute of Marine Research, Norway (Accepted 26 September 2013; Published online 22 April 2014; Printed 29 April 2014) © 2014 Taylor & Francis

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et al. 2010) and seasonal variations in lipid and fatty acid composition have been reported for several molluscs (Abad et al. 1995; Pazos et al. 2003; Dridi et al. 2007; Mateos et al. 2010; Ezgeta-Balić et al. 2012). Therefore, it is known that the biochemical contents of organisms undergo seasonal changes (Ozyurt et al. 2006). A strong relationship between the seasonal variation of fatty acids and sexual development has been reported for several mollusc species (Pazos et al. 1997; Palacios et al. 2005; Hurtado et al. 2012; Lazzara et al. 2012). However, there have been no studies that analysed the seasonal variations of fatty acids in relation to gametogenesis in Onchidium. Lipids are second only to protein as an important nutrient, and play an important role during biochemical metabolism and reproductive processes in aquatic animals (Castell et al. 2004; Grubert et al. 2004; Palacios et al. 2005, 2007). Moreover, the hepatopancreas, which is regarded as a major organ for lipid storage and processing, influences reproduction, egg growth and embryonic development (Cheng et al. 2001; Ying et al. 2004). The transfer of lipids from digestive gland to gonads during maturation has been observed in some molluscs (Pazos et al. 1997; Palacios et al. 2005, 2007; Ozyurt et al. 2006; Hurtado et al. 2012). However, molluscs have a limited capacity for the elongation and desaturation of long-chain polyunsaturated fatty acids (PUFA; Palacios et al. 2005, 2007; Mateos et al. 2010). Thus, PUFAs for gonad development are probably obtained directly from the diet or indirectly following previous accumulation in the digestive gland or other tissues (Grubert et al. 2004; Palacios et al. 2005). In marine animals, 18-carbon PUFA can be converted to LC-PUFA; likewise, 18:2n-6 is converted to 18:3n-6 and 18:3n-3 is converted to 18:4n-3. Then 18:3n-6 is converted to 20:3n-6 and 18:4n-3 converted to 20:4n-3. These elongated products are further desaturated to arachidonic (ARA) and eicosapentaenoic fatty acids (EPA), respectively. EPA is also substrate for further elongation and desaturation to produce docosahexaenoic acid (DHA; González-Durán et al. 2008; Carboni et al. 2013). The fatty acid pattern of the vitelline gland of O. struma is considered as critical for normal gonad development (Wu et al. 2007). Moreover, some essential fatty acids (EFA) have also been shown to be of special significance for

gonad maturation and brood quality and describe more precisely the main results found by Izquierdo et al. (2001) and Miliou et al. (2006). Investigation has already found that PUFAs such as ARA (20:4n6), EPA (20:5n-3) and DHA (22:6n-3) have effects on the physiological and biochemical processes of growth, development and reproduction (Khardin et al. 2003; Castell et al. 2004; Grubert et al. 2004). To date, studies have mainly focused on the fatty acid composition in muscle or other tissues in the Mollusca (Palacios et al. 2005; Miliou et al. 2006; Wu et al. 2007, 2008; Li et al. 2009; Koizumi & Hiratsuka 2009); therefore, systematic studies of the seasonal changes in fatty acid composition are lacking. In this article, we present: (1) fatty acid composition and seasonal changes of fatty acids in the hepatopancreas of O. struma, and (2) the fatty acid composition and seasonal changes of fatty acids in the vitellin gland of O. struma. The aims are to obtain a better understanding of reproductive performance at the biochemical level and to evaluate their nutritional value in order to advance our knowledge of shellfish nutrition.

Materials and methods Animals Individuals of Onchidium struma were collected from the Longguan Breeding Farm, Wenzhou, Zhejiang Province, China. The development cycle of the gonad of O. struma can be divided into five stages: resting stage, proliferating stage, growing stage, maturing stage and spawning stage (Ying et al. 2008; Chen et al. 2010), with the gonad development peak from early July to early August. The materials were gathered each month of the year from 2008 to 2009 to obtain different physiological maturation stages. The length and width of O. struma individuals were measured with vernier calipers; the wet weight was measured with an electronic balance (Table I). In each season, 180 similar-sized O. struma individuals were sampled and the 180 individuals were divided into 6 groups of 30 individuals. In the laboratory, the animals were dissected and the fresh hepatopancreas and vitelline gland were removed immediately. The fresh hepatopancreas and vitelline gland of each group (30 individuals) were

Table I. Weight, length and width of Onchidium struma (n = 60).

Weight (g) Length (mm) Width (mm)

Spring (April 2008)

Summer (July 2008)

Autumn (October 2008)

Winter (January 2009)

4.98 ± 0.71 (4.84–5.13) 38.33 ± 0.44 (37.43–39.24) 24.62 ± 0.40 (23.79–25.45)

14.09 ± 0.33 (13.42–14.76) 57.95 ± 1.18 (55.54–60.36) 40.31 ± 0.77 (38.73–41.88)

11.92 ± 0.24 (11.42–12.42) 58.01 ± 0.90 (56.17–59.86) 40.33 ± 0.72 (38.85–41.80)

7.63 ± 0.17 (7.28–7.97) 58.86 ± 0.61 (57.62–60.10) 41.97 ± 0.57 (40.80–43.14)

Fatty acids in Onchidium struma

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dried in an oven at 65°C for 48 h to constant weight. Each group of dried tissue was homogenated and the two organs were bagged separately. Tissues were kept in dry conditions until ready for use. Fatty acid analysis Fatty acid analysis was carried out by capillary gas chromatography, using a Shimadzu GC-9A equipped with an autosampler, which was also linked to a spectral data recording microcomputer (C-R2AX). Analysis was conducted under the following conditions: the carrier gas was nitrogen, the revolving rate was 20 ml min−1, the hydrogen was at 0.8 kg cm−2, and the air was at 0.8 kg cm−2. The analysis column was a Stabilwax capillary column (PEG-20 M, 25 m in length, 0.25 mm in diameter), and the detecting as well as sample tapping temperature was 260°C. The oven was programmed to rise from an initial temperature to 230 °C and the detection mode was a hydrogen flame ionization detector (FID). We used the potassium hydroxide–methanol high-temperature esterification method for extraction. The benzene: petroleum ether ratio was 1:1 to dissolve the lipids, and a potassium hydroxide–methanol solution was used to esterify (Metcalfe et al. 1966). Each fatty acid was determined according to the characteristics of standard fatty acid chromatograms using the same chromatogram conditions. Mixed standard fatty acids were purchased from Sigma Chemical Company (St. Louis, MO, USA). Statistical analysis The data were analysed using the SPSS Statistical Package (Version 12.0, Chicago, IL, USA for Windows). Differences among groups in different seasons were determined by one-way repeated measures analysis of variance (ANOVA) and P < 0.05 was taken to be statistically significant. Least significant difference (LSD) was used to conduct multiple comparisons among different seasons. All results were expressed as mean ± SE.

Results Seasonal profiles of hepatopancreas fatty acid composition in Onchidium struma There were 22 different fatty acids in the hepatopancreas of Onchidium struma, of which 4 were SFA (29.53–35.44%), 4 were MUFA (30.73–34.34%) and 14 were PUFA (29.33–36.72%). The major fatty acids in all seasons included C16:0 (17.53– 25.38%) in SFA and C16:1n-7 (11.06–24.80%) and C18: 1n-9 (4.98–7.40%) in the MUFA group. EPA (C20: 5n-3) was the predominant PUFA,

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accounting for 8.00–14.99%, but DHA (C22:6n-3) was maintained at a relatively low level (1.24– 2.57%), and only detected in spring and winter. The PUFA was richer in the n-3 series than that in the n-6 series (Table II). The percentage of most fatty acids in the hepatopancreas of O. struma showed significant differences in different seasons, especially in PUFA. C18:2n-6, C20:5n-3 and ∑n-3 showed significant differences with seasons. There was a continuous increase in C18:2n-6 fatty acid, which was followed by an inconsistent decrease in winter. From spring to summer the content of C18:3n-3 increased and then decreased in autumn. The fatty acids content of C20:4n-6, C20:5n-3, C22:6n-3 and ∑n-3 was at the highest levels in spring. The n3/n6 PUFA ratio showed no significant differences between spring, summer and winter. Two non-methyleneinterrupted-dienoic fatty acids, C20:2NIMD and C22:2NIMD, were detected in the hepatopancreas of O. struma. The content of C20:2NIMD increased from spring to summer, and then decreased in autumn. The C22:2NIMD was only detected in spring (Table II). The composition of fatty acids at different seasons showed certain differences. There were 22 kinds of fatty acids in the hepatopancreas of O. struma in spring, with the highest number of fatty acids of the four seasons. The C22:2NIMD and C22:4n-6 were only detected in spring. Fatty acids belonging to 19 kinds were found in the hepatopancreas in summer (excluding C22:2, C22:4 and DHA). There were 18 fatty acids in the hepatopancreas in autumn and at this time the number of fatty acids was the least, with C20:0, C22:2, C22:4 and DHA lacking. There were 20 kinds of fatty acids except C22:2 and C22:4 in winter (Table II). In the hepatopancreas of O. struma, the percentage content of SFA was remarkably different throughout the seasons, with the highest concentration being recorded in summer. The MUFA content was significantly different between spring, summer and autumn, while no difference was seen between autumn and winter, and the highest content was observed in summer. The PUFA content was significantly different between spring, summer and winter, whereas no difference was seen between spring and autumn. The variations of the total PUFA and n-3 PUFA showed the same trends, with the highest content being found in winter (Figure 1).

Seasonal profiles of vitelline gland fatty acid composition in Onchidium struma There were 23 kinds of fatty acids in the vitelline gland of Onchidium struma, of which 4 SFA

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Table II. Seasonal changes of fatty acid composition in the hepatopancreas of Onchidium struma.

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Fatty acids C14: 0 C14: 1 C14: 2 C14: 3 C16: 0 C16: 1n-7 C16: 2n-4 C16: 2n-9 C16: 3n-4 C18: 0 C18: 1n-7 C18: 1n-9 C18: 2n-6 C18: 3n-3 C18: 3n-6 C20: 0 C20: 1n-9 C20: 2n-6 C20: 2NIMD C20: 3n-3 C20: 3n-6 C20: 4n-3 C20: 4n-6 C20: 5n-3 C22: 2NIMD C22: 4n-6 C22: 5n-3 C22: 5n-6 C22: 6n-3 n-3 n-6 n-3/n-6

Spring (n = 6) 8.55 0.31 0.29 0.35 17.53 24.80 0.72 1.18 2.47 2.66 1.19 4.98 1.49 1.27 0.64 0.78 3.06 0.20 0.45 1.01 0.26 0.53 3.12 14.99 1.50 0.86 1.01 0.14 2.57 21.38 6.71 3.19

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Summer (n = 6)

Autumn (n = 6)

4.70 ± 0.01b (4.69–4.73) 3.89 ± 0.03c (3.85–3.95) 0.12a (8.41–8.79) d c 0.41 ± 0.01 (0.39–0.44) 0.68 ± 0.01a (0.65–0.70) 0.01 (0.30–0.32) 0.01d (0.27–0.30) 1.30 ± 0.01a (1.29–1.31) 0.79 ± 0.05b (0.71–0.87) d c 0.00 (0.35–0.36) 0.88 ± 0.03 (0.83–0.92) 1.92 ± 0.00a (1.91–1.92) 0.20c (17.17–17.85) 25.38 ± 0.04a (25.32–25.45) 21.52 ± 0.07b (21.39–21.62) 0.19a (24.43–25.01) 16.33 ± 0.04b (16.25–16.39) 11.06 ± 0.05d (10.98–11.14) 0.02a (0.69–0.75) 0.64 ± 0.02b (0.61–0.66) 0.63 ± 0.03b (0.59–0.69) 0.01c (1.16–1.20) 1.88 ± 0.04a (1.83–1.96) 1.44 ± 0.03b (1.41–1.49) 0.66 ± 0.03c (0.60–0.71) 2.00 ± 0.09b (1.83–2.12) 0.04a (2.42–2.54) 0.03d (2.62–2.73) 5.03 ± 0.01b (5.01–5.05) 7.12 ± 0.03a (7.16–7.15) 0.03d (1.13–1.25) 2.65 ± 0.01c (2.63–2.67) 2.80 ± 0.01b (2.79–2.81) 0.03d (4.93–5.02) 6.29 ± 0.01c (6.28–6.31) 6.63 ± 0.05b (6.57–6.73) 2.87 ± 0.00c (2.87–2.88) 4.18 ± 0.04a (4.11–4.23) 0.00d (1.48–1.49) 0.02c (1.23–1.31) 3.07 ± 0.03a (3.02–3.11) 2.02 ± 0.02b (1.98–2.05) 0.01b (0.61–0.66) 0.81 ± 0.03a (0.76–0.84) 0.49 ± 0.01c (0.47–0.51) 0.01a (0.75–0.80) 0.33 ± 0.02ab (0.30–036) ND 7.32 ± 0.03b (7.27–7.35) 9.71 ± 0.03a (9.65–9.74) 0.03d (3.00–3.11) 0.01bc (0.18–0.22) 0.09 ± 0.02c (0.07–0.12) 0.56 ± 0.02a (0.53–0.60) c b 0.02 (0.41–0.49) 1.21 ± 0.04 (1.15–1.30) 2.05 ± 0.04a (1.98–2.13) 0.02d (0.98–1.04) 4.09 ± 0.01c (4.07–4.11) 7.01 ± 0.03a (6.95–7.05) d c 0.98 ± 0.01 (0.96–1.00) 1.26 ± 0.03b (1.22–1.33) 0.02 (0.24–0.29) 0.01a (0.51–0.55) 0.11 ± 0.01b (0.09–0.13) 0.12 ± 0.02b (0.08–0.15) 0.05a (3.05–3.21) 0.36 ± 0.01c (0.35–0.38) 0.33 ± 0.02c (0.29–0.36) 8.83 ± 0.02c (8.79–8.87) 8.00 ± 0.19d (7.80–8.38) 0.16a (14.70–15.25) 0.03 (1.45–1.54) ND ND 0.01 (0.83–0.87) ND ND 0.01d (0.99–1.02) 1.21 ± 0.01c (1.19–1.23) 2.02 ± 0.03a (1.98–2.07) 0.33 ± 0.00b (0.33–0.34) 0.58 ± 0.03a (0.54–0.64) 0.01c (0.12–0.16) a 0.02 (2.54–2.62) ND ND 0.22b (20.98–21.73) 17.31 ± 0.05d (17.25–17.40) 19.17 ± 0.21c (18.94–19.58) 0.08b (6.59–6.82) 5.45 ± 0.05c (5.36–5.51) 7.40 ± 0.05a (7.30–7.47) 3.18 ± 0.03a (3.13–3.22) 2.59 ± 0.05b (2.54–2.68) 0.01a (3.18–3.20)

Winter (n = 6) 4.01 0.49 0.57 1.35 21.26 13.53 0.62 1.48 0.70 4.88 3.05 7.40 3.64 3.08 0.85 0.12 6.26 0.30 1.94 5.16 1.55 0.15 0.54 11.40

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.87 0.28 1.24 22.90 7.17 3.20

± ± ± ± ± ±

0.01c (3.99–4.03) 0.01b (0.48–0.50) 0.00c (0.56–0.57) 0.03b (1.29–1.38) 0.05b (21.16–21.32) 0.06c (13.42–13.59) 0.03b (0.58–0.67) 0.02b (1.45–1.52) 0.07c (0.57–0.79) 0.02c (4.85–4.91) 0.02a (3.02–3.08) 0.01a (7.37–7.42) 0.01b (3.63–3.65) 0.02a (3.05–3.12) 0.01a (0.83–0.87) 0.00c (0.12–0.12) 0.04c (6.21–6.34) 0.05b (0.20–0.36) 0.07a (1.81–2.05) 0.02b (5.12–5.20) 0.06a (1.44–1.62) 0.03b (0.11–0.20) 0.06b (0.46–0.66) 0.17b (11.22–11.74) ND ND 0.02b (1.85–1.90) 0.01b (0.26–0.30) 0.01b (1.23–1.25) 0.12a (22.75–23.13) 0.14a (6.92–7.39) 0.07a (3.09–3.34)

Notes: Values in the same row with different superscripts (a, b, c, and d) indicate significant differences (P < 0.05). NIMD, non-methyleneinterrupted-dienoic fatty acids; ND, not detected.

(26.33–38.03%), 4 MUFA (18.97–32.67%) and 15 PUFA (31.92–52.51%) were found. The content of C16:0 in SFA was the highest, accounting for 15.77– 26.87%. The content of C16:1n-7 (5.27–10.67%)

Figure 1. Seasonal changes of SFA, MUFA and HUFA in the hepatopancreas of Onchidium struma.

and C18:1n-9 (5.00–8.02%) was dominant in MUFA. The content of EPA was the highest in PUFA with 5.86–15.02%, while the content of DHA was only detected in spring (1.82%) and winter (0.98%). The PUFA was richer in the n-3 series than that in the n-6 series (Table III). In the vitelline gland of O. struma the content of most fatty acids showed significant differences in different seasons. In PUFA, C14:3, C18:2n-6, C18:3n-3, C20:3n-3, C20:4n-3, C20:4n-6, C20:5n-3, C22:5n-3, ∑n-3 and n3/n6 ratios showed significant differences with seasons. From spring to autumn, there was a continuous increase of C18:2n-6 fatty acids, which was followed by an inconsistent decrease in winter. A similar pattern also occurred with C18:3n-3, C18:3n-6, C20:3n-3 and C20:3n-6. The fatty acid content of C20:4n-3, C20:4n-6, C20:5n-3, ∑n-3 and ∑n-6 had their highest levels in spring. C20:2NIMD and C22:2NIMD was detected in the vitelline gland of O. struma. The content of C20:2NIMD increased from spring to summer, and the content in winter was not significantly

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Table III. Seasonal changes of fatty acid composition in the vitelline gland of Onchidium struma.

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Fatty acids C14: 0 C14: 1 C14: 2 C14: 3 C16: 0 C16: 1n-7 C16: 2n-4 C16: 2n-9 C16: 3n-4 C18: 0 C18: 1n-7 C18: 1n-9 C18: 2n-6 C18: 3n-3 C18: 3n-6 C20: 0 C20: 1n-9 C20: 2n-6 C20: 2NIMD C20: 3n-3 C20: 3n-6 C20: 4n-3 C20: 4n-6 C20: 5n-3 C22: 2NIMD C22: 3n-3 C22: 4n-6 C22: 5n-3 C22: 5n-6 C22: 6n-3 n-3 n-6 n-3/n-6

Spring (n = 6) 3.24 0.74 1.84 0.53 15.77 5.27 0.25 1.25 1.89 6.97 1.61 5.00 1.64 0.87 0.14 0.35 6.35 0.24 2.41 1.40 0.24 1.26 4.82 15.02 5.70 1.37 5.93 2.95 0.97 1.82 24.69 13.97 1.77

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Summer (n = 6)

Autumn (n = 6)

3.19 ± 0.03a (3.15–3.25) 0.05a (3.05–3.37) b ND 0.01 (0.72–0.76) 0.02a (1.76–1.92) 1.83 ± 0.01a (1.80–1.85) 0.03d (0.46–0.60) 1.31 ± 0.06c (1.20–1.40) 0.14c (15.27–16.14) 26.87 ± 0.23a (26.61–27.32) 9.51 ± 0.22b (9.06–9.77) 0.08c (4.97–5.47) 0.02b (0.22–0.28) 0.60 ± 0.03a (0.57–0.66) 0.02c (1.21–1.29) 2.18 ± 0.09a (2.08–2.35) 0.99 ± 0.07c (0.87–1.11) 0.02a (1.84–1.98) 0.06c (6.74–7.17) 7.97 ± 0.10b (7.86–8.17) 0.06c (1.53–1.72) 1.96 ± 0.01b (1.94–1.98) 0.13d (4.84–5.25) 8.02 ± 0.05a (7.92–8.10) 3.36 ± 0.04b (3.28–3.42) 0.06d (1.48–1.77) 0.03c (0.81–0.92) 1.05 ± 0.02b (1.02–1.08) 0.02b (0.12–0.17) 0.24 ± 0.01ab (0.22–0.25) 0.01 (0.31–0.37) ND 0.06d (6.09–6.48) 9.74 ± 0.06c (9.64–9.86) 0.01c (0.22–0.25) 1.03 ± 0.11a (0.88–1.25) c 0.05 (2.32–2.48) 4.04 ± 0.16a (3.82–4.35) 0.03d (1.36–1.45) 2.64 ± 0.03c (2.59–2.68) 0.88 ± 0.03b (0.83–0.91) 0.01c (0.21–0.26) 0.03a (1.21–1.32) 0.86 ± 0.02b (0.83–0.91) a 0.07 (4.75–4.95) 2.06 ± 0.02b (2.02–2.09) 5.86 ± 0.06d (5.75–5.97) 0.11a (14.67–15.41) 0.14 (5.27–6.18) ND 0.11 (0.99–1.74) ND 0.04 (5.80–6.07) ND 0.01a (2.93–2.97) 2.28 ± 0.02d (2.25–2.30) 0.05a (0.88–1.02) 0.72 ± 0.01b (0.69–0.74) 0.03 a (1.72–1.88) ND 0.50a (23.73–25.40) 12.69 ± 0.07d (12.62–12.82) 8.29 ± 0.06b (8.21–8.41) 0.25a (13.53–14.39) c 0.01 (1.75–1.78) 1.53 ± 0.02d (1.50–1.55)

2.71 0.94 1.61 2.20 16.12 9.73 0.66 1.59 1.96 9.75 2.13 7.07 3.58 1.21 0.31

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10.84 0.90 3.35 5.14 1.39 0.14 1.06 10.54

± ± ± ± ± ± ± ±

2.72 ± 0.89 ± 19.75 ± 8.13 ± 2.43 ±

0.07c (2.58–2.82) 0.01a (0.91–0.95) 0.02b (1.58–1.64) 0.05a (2.14–2.31) 0.15c (15.85–16.38) 0.01b (9.72–9.74) 0.03a (0.59–0.70) 0.04b (1.52–1.65) 0.08a (1.86–2.11) 0.13a (9.48–9.90) 0.05b (2.05–2.21) 0.08c (6.90–7.16) 0.04a (3.53–3.66) 0.04a (1.13–1.25) 0.02a (0.27–0.33) ND 0.24b (10.58–11.32) 0.04ab (0.83–0.96) 0.30b (3.02–3.95) 0.07a (5.02–5.25) 0.10a (1.23–1.56) 0.02c (0.12–0.17) 0.01c (1.03–1.08) 0.14b (10.26–10.71) ND ND ND 0.04c (2.63–2.77) 0.01a (0.87–0.91) ND 0.17b (19.42–19.98) 0.12b (7.88–8.28) 0.05b (2.35–2.52)

Winter (n = 6) 2.99 0.33 1.64 1.86 22.00 10.67 0.35 2.19 1.23 7.89 2.80 7.39 2.53 0.60 0.16

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

11.48 0.73 3.90 3.98 0.79

± ± ± ± ±

10.10 ±

2.82 0.97 0.98 17.51 5.18 3.38

± ± ± ± ± ±

0.02b (2.96–3.04) 0.01c (0.31–0.34) 0.01b (1.62–1.66) 0.06b (1.74–1.93) 0.10b (21.86–22.20) 0.44a (9.81–11.25) 0.03b (0.31–0.41) 0.09a (2.04–2.35) 0.13b (0.98–1.43) 0.08b (7.80–8.05) 0.12a (2.56–2.98) 0.01b (7.37–7.42) 0.03c (2.50–2.59) 0.16d (0.34–0.88) 0.05b (0.08–0.26) ND 0.08a (11.37–11.63) 0.04b (0.68–0.80) 0.03ab (3.85–3.95) 0.02b (3.95–4.01) 0.01b (0.77–0.81) ND ND 0.14c (9.86–10.36) ND ND ND 0.02b (2.80–2.85) 0.04a (0.92–1.04) 0.02b (0.95–1.03) 0.14c (17.25–17.75) 0.09c (5.02–5.32) 0.05a (3.29–3.44)

Notes: Values in the same row with different superscripts (a, b, c, and d) indicate significant differences (P < 0.05). NIMD, non-methyleneinterrupted-dienoic fatty acids; ND, not detected.

different between summer and autumn. Higher contents of C22:2NIMD (5.70%) were only detected in spring (Table II). The composition of fatty acids showed differences with seasons. The highest number of fatty acids (23) was present in the vitelline gland of O. struma in spring, with four fatty acids (C20:0, C22:2NIMD, C22:3n-3 and C22:4n-6) only detected in spring. Fatty acids belonging to 17 kinds were found in the vitelline gland in summer, while no C14:1, C20:0, C22:2 NIMD, C22:3n-3, C22:4n-6 or DHA was detected. There were 18 kinds of fatty acids in autumn, excluding C20:0, C22:2 NIMD, C22:3n-3, C22:4n-6 and DHA. There were also 17 kinds of fatty acids, with the exception of C20:0, C20:4n-3, C20:4n-6, C22:2 NIMD, C22:3n-3 and C22:4n-6, in winter (Table III). Furthermore, the content of SFA, MUFA and PUFA was also significantly different with seasons. The highest concentration of total SFA, MUFA and HUFA was found in summer, winter and spring, respectively. From spring to

summer (representing the breeding season), the content of SFA in the vitelline gland increased from 26.33 to 38.03%. The content of MUFA increased rapidly from 18.97 to 29.22%. However, the content of PUFA decreased from 52.51 to 31.92% (Figure 2).

Discussion There were 22 and 23 kinds of fatty acids in the hepatopancreas and vitelline gland of Onchidium struma, respectively, with the principal fatty acids being C16:0, C16:1n-7, C18:0, C18:1n-9, C20:1n9, C20:4n-6 and C20:5n-3 (Tables II, III). This is consistent with the fatty acid composition in the hepatopancreas and gonads of some other economically important invertebrates (Khardin et al. 2003; Castell et al. 2004; Ying et al. 2004; Ozyurt et al. 2006). While C22:6n-3 (DHA) was rather low, and was not detected in summer and autumn, this is different from other molluscs (Palacios et al. 2005;

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Figure 2. Seasonal changes of SFA, MUFA and HUFA in the vitelline gland of Onchidium struma.

Mateos et al. 2010; Lazzara et al. 2012). Interestingly, palmitic acid (16:0), the first fatty acid produced during lipogenesis and from which longer fatty acids are formed, was more abundant during spawning (summer), while palmitoleic acid (16:1n-7), the most abundant MUFA, was significantly higher in the hepatopancreas in spring and in the vitelline gland in winter. These results suggested that some kind of transformation between C16:0 and C16:1n-7 might have happened as the experiment progressed. A similar result was reported by other authors (Grubert et al. 2004; Ozyurt et al. 2006; Ying et al. 2006). During the rapid gonadal development (from spring to summer) of O. struma (Chen et al. 2010), the content of C16:1 in the hepatopancreas decreased rapidly from 24.80 to 16.33%, while the content of C16:1 in the vitelline gland increased from 5.27 to 9.51%. We suggest that at least some of the fatty acids (such as C16:1) in the hepatopancreas were transferred to the accessory sexual gland (vitelline gland) during the rapid maturation period. This was similar to findings in decapod crustaceans (Mourente et al. 1994; Lautier & Lagarrigue 1998; Ying et al. 2006) and molluscs (Wu et al. 2007; Mateos et al. 2010; Hurtado et al. 2012). The content of linoleic acid (18:2n-6) and linolenic acid (18:3n-3) is relatively lower in the hepatopancreas and vitelline gland of O. struma (Kanazawa et al. 1977). This finding is consistent with the results from Melicertus kerathurus (Forskål, 1775) and Marsupenaeus japonicus (Spence Bate, 1888) (Mourente 1996; Teshima & Kanazawa 1983). These authors mentioned that the lower content of C18:2 and C18:3 is a characteristic typical of marine animals. In O. struma the contents of C18:2 and C18:3 were inversely proportional to that of C20:4n-3, which is consistent with some marine animals (GonzálezDurán et al. 2008; Carboni et al. 2013). These authors thought that C18:2n-6 can convert to

C18:3n-6, C18:3n-6 to C20:3n-6 and C18:4n-3 to C20:4n-3. The more important effect in our study was an increase in the PUFA levels in the vitelline gland in spring, during gonad maturation, mainly of the series n-3, while the lowest levels were detected in summer after spawning. C20:5n-3 (EPA) showed a strong seasonal variation, reaching the highest levels in spring for the hepatopancreas (14.99%) and vitelline gland (15.02%) of O. struma. During the breeding season in summer, the content of EPA decreased rapidly to 8.83% in the hepatopancreas and 5.86% in the vitelline gland. DHA was only detected in spring and winter, and the content was lower, only 2.57% in the hepatopancreas and 1.82% in the vitelline gland in spring. Similarly, Lazzara et al. (2012) reported high levels of C20:5n-3 in the female gonads of Dreissena polymorpha (Pallas, 1771) during sexual maturity, while levels of C22:6n-3 showed no clear seasonal trend and significantly declined after the spawning period. Indeed, C22:6n-3 has been suggested to play a structural and functional role in the maintenance of cell membranes in bivalves, while C20:5n-3 is often related to energetic-type functions (Grubert et al. 2004; Ozyurt et al. 2006; Lazzara et al. 2012). Some authors thought that C20:5n-3 followed cycles related to oocyte maturation and spawning (Pazos et al. 1997) and to the accumulation of nutrients for ovarian development (Alava et al. 1993; Dridi et al. 2007). In spring, the high content of EPA in the hepatopancreas and vitelline gland of O. struma may provide essential nutrients for breeding. During summer, these nutrients may partly be transferred to the hermaphroditic gland for gonad development and the formation of germ cells. Before the breeding phase of O. struma, the hepatopancreas and vitelline gland accumulated a certain amount of EPA, whereas the content of EPA decreased rapidly during the breeding phase. After the breeding phase, the content of EPA recovered gradually. A similar pattern has also been confirmed by other authors in other molluscs (Palacios et al. 2005; Wu et al. 2007). It has to be noted that the content of DHA during different seasons was lower than that of EPA in the hepatopancreas and vitelline gland of O. struma. This is different from other marine molluscs and more similar to most freshwater and terrestrial molluscs (Liu 1991). We interpret this as the lowsalinity habitat of O. struma representing a more transitional habitat type than for marine and terrestrial molluscs (Huang et al. 2004). Furthermore, there was a quantity of DPA (C22:5) in the hepatopancreas and vitelline gland of O. struma. Grubert et al. (2004) found that there was some DPA (5–10%) in the muscle and testis of Haliotis

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Fatty acids in Onchidium struma rubra Leach, 1814 and Haliotis laevigata Donovan, 1808, and the content was much higher than that of DHA (0.4–2.8%). They guessed that DPA was converted by EPA or inverted by DHA. The role of DPA in the growth and gonad development of O. struma still needs further research. The contents of C20:4 in the hepatopancreas and vitelline gland of O. struma in spring were far higher than those in other seasons (Tables II, III). C20:4 is a precursor in the prostaglandin synthesis, and prostaglandin is an effective substance in the physiology of animals. It plays certain roles in the reproductive, digestive, and respiratory systems and also regulates membrane permeability and inhibits lipid decomposition (Koven et al. 2001; Chen et al. 2003). We assume that the high content of C20:4 in the hepatopancreas and vitelline gland in spring may inhibit lipid decomposition and contribute to gonad development. The gonad of O. struma in spring is still immature, and the accumulation of fatty acids is greater than their decomposition. Moreover, the high content of C20:4 in well-developed membrane systems may be propitious to the synthesis of prostaglandin, thereby contributing to the development of the gonad. During the gonad maturation phase, the content of C20:4 in the hepatopancreas and vitelline gland decreases. C20:4 may accelerate the lipid decomposition, which would provide more energy during this metabolically active stage. After gonad maturation, the content of C20:4 declines substantially. This is consistent with the study of the fatty acid decomposition in the hepatopancreas of Eriocheir sinensis H. Milne Edwards, 1853 by Ying et al. (2004). In spring, the content of C20:4 in the vitelline gland (6.08%) of O. struma was far higher than that in the hepatopancreas (3.65%). The C20:4 content decreased significantly in summer, and showed a significant difference to that in spring, but its content in the vitelline gland (2.92%) was still far higher than that in the hepatopancreas (0.47%; Tables II, III). This shows that the relationship between the vitelline gland and the gonad is very close. The high level of C20:4 in the vitelline gland in spring may provide the material needed for gonad development. In our study, we found two non-methylene-interrupted-dienoic fatty acids (NMID), C20:2 and C22:2, in the hepatopancreas and vitelline gland of Onchidium struma. The content of C20:2 (NMID) and C22:2 (NMID) in the vitelline gland is higher than that in the hepatopancreas, and the C22:2 (NMID) is only detected in spring. Several cases of 20 and 22 carbon NMID have been reported in a number of marine invertebrates such as molluscs (Grubert et al. 2004; Palacios et al. 2005; Dridi et al. 2007; Ezgeta-Balić et al. 2012; Lazzara et al. 2012)

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and echinoderms (Cook et al. 2000; Castell et al. 2004; González-Durán et al. 2008). The amount of NMID fatty acid in molluscs varies widely (from 0.7% to 20.7%) from species to species (Barnathan 2009). The function of NMID in molluscs is not clearly understood, but their predominance in the phospholipid fraction suggests that they may play a structural role maintaining membrane fluidity (Dridi et al. 2007). NMIDs and their precursors C16:1n-7 and C18:1n-9 (Joseph 1982), as well as n-3HUFA, were negatively correlated in the hepatopancreas and vitelline gland of O. struma. Ezgeta-Balić et al. (2012) thought that the fatty acid synthesis was stimulated by the presence of C16:1n-7 in the diet, and some authors suggested that it is possibly when there is a deficiency of PUFAs in the diet or biosynthesized by the molluscs, as NMID is the only PUFA synthesized by marine molluscs (Grubert et al. 2004; Palacios et al. 2005; Dridi et al. 2007; Lazzara et al. 2012). Bivalves showed some ability to elongate fatty acids, C16:1(n-7) to C18:1(n-7), C18:1(n-9) to C20:1(n-9), C20:5(n-3) to C22:5(n-3) and C20:4(n-6) to C22:4(n-6) or to desaturate C20:3 n-6 to C20:4 n-6 (Ezgeta-Balić et al. 2012). It is possible that NMID might be synthesized from C20:1n-9 by the action of the Δ9 and Δ5 (Joseph 1982; Zhukova & Svetashev 1986). A likely hypothesis for the NMID formation has been proposed, i.e. that the parallel formation during the synthesis of C18:2n-6 to C20:4n-6 (AA), depending on the synthesis of AA by the Δ6 and Δ5, might be mechanisms associated with the synthesis of NMID (Ackman & Hooper 1973; Takagi et al. 1979). Castell et al. (2004) have described the relationship between low levels of AA and the synthesis of NMID. High NMID and low AA contents have been seen in the test of sea urchins. In our results the content of AA was also inversely proportional to that of C20:2NMID. According to Castell et al. (2004), the results might suggest a certain degree of dependency on 20-carbon fatty acids for the synthesis of NMID. A biosynthetic route for the NMID in molluscs was proposed in which the 20:2n-6 acid, obtained by elongation of 18:2n-6, is converted to the 20:3Δ5,11,14 acid that gives the 22:3Δ7,13,16 acid by a subsequent chain elongation step (Saito 2004). Thus, while molluscs have active FA elongation and desaturation systems (Δ5-desaturase) permitting the de-novo synthesis of the NMID, they seem to be lacking Δ6-desaturase to synthesize common (n-6) and (n-3) long-chain PUFA (Barnathan 2009). There were significant differences in the content of SFA, MUFA and HUFA between spring and summer in the hepatopancreas of O. struma (Figure 1), whereas the content of SFA, MUFA and

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HUFA showed significant differences during all four seasons in the vitelline gland (Figure 2). Fatty acids in the vitelline gland showed more striking seasonal changes than those in the hepatopancreas, especially during the breeding phase (Figures 1, 2). With the gradual development of gonads, major changes take place in the content and composition of fatty acids in the hepatopancreas and vitelline gland. From spring to summer, when O. struma was gradually entering the breeding season, the fatty acids in the vitelline gland varied widely, and SFA increased rapidly from 26.33% to 38.03%. MUFA also instantly increased from 18.97% to 29.22%, while PUFA decreased from 52.51% to 31.92%. This is in confirmation with the results by Li et al. (1994) on the mud crab Scylla serrata (Forskål, 1775). The study on S. serrata by Cheng et al. (2001) found that the content of MUFA in ovaries increased substantially, while PUFA significantly decreased at the vitellogenesis stage. From spring to summer, the increase of SFA and MUFA in the vitelline gland of O. struma might be relative to the energy supply for cell development within the hermaphroditic gland. During the breeding phase, the content of UFAs in the hepatopancreas of O. struma decreased. Cheng et al. (1998) thought that in the process of ovarian development of Eriocheir sinensis, this crab might use the fatty acids in the hepatopancreas for the synthesis of UFA in yolk. For the full development of the ovary, this may require exogenous lipids that are rapidly transferred to the ovary. The long-chain PUFAs were used for the synthesis of phospholipids. The neutral lipids (mainly triglycerides) were supposed to be firstly converted into the phospholipids in use (Cheng et al. 2001; Ying et al. 2004). It appears that the fatty acids of O. struma play an important role in growth and development. Different fatty acids play different roles in the growth and reproduction of aquatic animals. PUFAs play a critical role in the breeding of O. struma and undergo substantial changes. During the gonadal development of O. struma, UFAs are the main substance transferred from the hepatopancreas. Several authors agreed that the ovary continued to accumulate high levels of UFAs, that provided the nutrients for the development of the eggs (Vogt et al. 1985; Chen et al. 2003). During the breeding stage of O. struma, the content of MUFAs in the hepatopancreas decreased, while it increased rapidly in the vitelline gland. This seems to be mainly due to MUFAs being transferred from the hepatopancreas to the ovary. In summary, the hepatopancreas and vitelline gland of O. struma are regarded as major organs for fatty acid storage. Their fatty acid composition turned out to be similar to those in other marine animals. From the rapid development of the ovary to

actual reproduction, great changes are taking place in the fatty acid composition and content in the hepatopancreas and vitelline gland of O. struma. Funding This study was supported in part by the following grants: Nature Science Foundation of Zhejiang Province, China (Grant number: No. LY13C040003) (to XPY), the National Natural Science Foundation of China (No. 41276151) (to WXY), the Bureau of Science-Technology of Zhejiang Province, China (Grant number: 2007C33066) (to XPY) and the Science-Technology Innovation Team of Wenzhou City (Grant number: 201210351012) (to XPY).

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