Differences in Sterol Composition of Gonads of the Lottiid Limpets

NOTE Differences in Sterol Composition of Gonads of the Lottiid Limpets Nipponacmea concinna and Nipponacmea fuscoviridis from Northeastern Japan Hideki Kawashima1＊ , Masao Ohnishi2 and Satoshi Ogawa3 1 Bioscience Laboratory, Miyako College, Iwate Prefectural University (1-5-1 Kanan, Miyako, Iwate 027-0039, JAPAN) 2 Department of Food Science, Obihiro University of Agriculture and Veterinary Medicine (2-11 Inada-cho, Obihiro, Hokkaido 080-8555, JAPAN) 3 Department of Chemistry and Bioengineering, Faculty of Engineering, Iwate University (4-3-5 Morioka, Iwate 020-8551, JAPAN) Abstract: This is the first report on the sterol composition in Nipponacmea concinna and Nipponacmea fuscoviridis, 2 dominant species of lottiid limpets. There were signiﬁ cant differences in sterol composition between male and female gonads of the limpets. Previous studies have shown that zymostenol and zymosterol are major lipid components of male gonads of the nacellid limpets Cellana grata and Cellana toreuma. In contrast, in this study, only trace amounts of zymosterol were detected in male gonads of N. fuscoviridis.

Key words: gastropod, limpet, Nipponacmea concinna, Nipponacmea fuscoviridis, sterol composition

1 INTRODUCTION gonads（the oocytes and sperm）of N. fuscoviridis13）are Limpets, which are among the most common mollusks of currently being used in a developmental study on fertiliza- the class Gastropoda living in most intertidal rock shores tion in invertebrate marine organisms, very little is known worldwide, are model invertebrate animals for ecological about the composition and distribution of sterols in these 2 and physiological research1, 2）as well as for environmental limpet species. In this study, we analyzed whether sexual assessment studies3－5）of potential pollution sources. differences in sterol composition exist in N. concinna and However, except for some species6－12）, comparative bio- N. fuscoviridis and compared the results with our previ- chemical studies of intrinsic characteristics of limpets are ous studies on the sterol composition of C. grata and C. limited. In previous studies on fatty acid and sterol compo- toreuma7）. The results may provide further key insights nents in limpets from northeastern Japan6－11）, we showed into mechanisms that regulate the reproductive cycle in marked sex-related differences in sterol composition gonads of marine mollusks. between male（testis）and female（ovary）gonads of 2 repre- sentative dominant limpet species, Cellana grata and Cellana toreuma from the family Nacellidae, which is one of the most primitive gastropod families. We identiﬁ ed cho- 2 EXPERIMENTAL PROCEDURES lesterol and desmosterol as major sterol components and 2.1 Sampling of limpets and extraction of lipids found high levels of the unusual cholesterol precursors Δ8- Sampling of the lottiid limpets N. concinna（mean shell sterols, namely, zymostenol and zymosterol, in male length, 28.2±3.4 mm, n＝35）and N. fuscoviridis（mean gonads7）. These findings prompted us to further evaluate shell length, 17.6±2.1 mm, n＝50）from Kirikiri, Iwate Pre- the sexual differences in the level of the most abundant fecture, northeastern Japan, was performed between sterols in other dominant limpet species widely distributed October and November 2009. After sampling, all limpets in Japan. Although Nipponacmea concinna and Nippon- were immediately dissected in the laboratory. The sex was acmea fuscoviridis are the most abundant and represen- determined by microscopic examination, and the gonads tative limpets of the Lottiidae family in Japan, and the were distinguished as male or female. The muscle and

＊Correspondence to: Hideki Kawashima, Bioscience Laboratory, Miyako College, Iwate Prefectural University, 1-5-1 Kanan, Miyako, Iwate 027-0039, JAPAN E-mail: [email protected] Accepted June 10, 2011 (received for review March 31, 2011) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs

501 H. Kawashima, M. Ohnishi and S. Ogawa

gonad from male and female of each limpet were dissected tissues of N. concinna and N. fuscoviridis are shown in and separated. Each group of muscle and gonad tissues Table 1. In this study, 10 types of sterols were identifi ed by （0.5－1.0 g）was suspended in 15 mL of a mixture of chlo- GC-MS analysis of their TMS derivatives（Table 2）. roform-methanol（2:1, v/v）and then homogenized for 30 s In muscle samples of N. concinna and N. fuscoviridis, at 13,000 rpm by using an IKA Ultra Turrax T25 Basic ho- cholesterol was the most abundant sterol, followed by des- mogenizer（IKA Japan KK, Nara, Japan）. Lipids were ex- mosterol. No signifi cant differences were observed between tracted by the methods described by Bligh and Dyer14）. The the sterol composition of male and female muscles of Nip- different lipid classes were analyzed by thin-layer chroma- ponacmea species, which is in agreement with our previ- tography on silica gel 60 G plates（Merck, Darmstadt, ous results on lipid composition in Cellana spp7）. In con- Germany）as described previously15）. trast, the cholesterol levels in the muscles of Nipponacmea species were higher than those of Cellana 2.2 Extraction of sterols spp. The difference in cholesterol level may be due to dif- Each sample of total lipids（1－2 mg）was saponified as ferences in sterol biosynthesis or in the limpets’ diet. described previosly7）. After extraction of sterols, samples No significant differences were observed in the sterol were converted into their TMS derivatives and analyzed as composition of muscle tissues in the 2 Nipponacmea spp. described below. In contrast, sexual differences in sterol composition in gonads between the 2 Nipponacmea spp., as well as 2.3 Preparation of TMS derivatives, and identification among those of the genus Cellana7）, were signifi cant. Cho- and determination of sterols lesterol and desmosterol, which belong to the Δ5-sterol Preparation of TMS derivatives and GC and GC-MS anal- group, are the dominant sterols in both the Cellana and yses were carried out as described previously7, 15）. The Nipponacmea genera. Zymostenol and zymosterol, which identifi cation of free sterols and their TMS derivatives was belong to the Δ8-sterol group, were present as major com- confirmed by comparing their mass spectral data with ponents of male gonad lipids of Cellana species7）. In con- those obtained for authentic and laboratory standards. The trast, in this study, only trace amounts of zymosterol were sterol contents in limpet tissues were determined by a Shi- found in male gonads of N. fuscoviridis, and neither zy- madzu GC-1700 chromatograph equipped with an FID and mostenol nor zymosterol were detected in those of N. con- an SAC-5（30 m×0.25 mm i.d., 0.25 μm film thickness; cinna. Supelco, Bellefonte, PA）. 5α-cholestane was used as an in- The existence of both cholesterol and desmosterol as ternal standard（Sigma-Aldrich, St. Louis, MO）. major sterol components has mostly been reported in muscle tissues of marine mollusks16）. In this study, at least 2.4 Statistical analyses 10 types of sterols, including 3 minor ones, were identifi ed The procedures were repeated 3 or 4 times for indepen- in limpet gonads. Desmosterol is generally abundant in dent sets of pooled male or female muscle or gonad many red algae17）living on rocky shores. The differences in samples. Data are expressed as mean±SD. Statistical com- sterol composition between different genera of limpets parisons between sterols of male and female limpets were may be linked either to the limpets’ ability to synthesize made using Student’s t-test. A value of P＜0.05 was con- sterols or to their diet12）; however, the various causes for sidered signifi cant. these differences have not been fully elucidated. Therefore, the identifi cation of fecal sterol components from limpets may provide further insights into the relationship between sterol composition and the limpets’ food supply. 3 RESULTS AND DISCUSSION As compared to our previous results7）, the sterol compo- Total lipid and sterol contents of the muscle and gonad sition in gonads of N. concinna and N. fuscoviridis was

Table 1 Total lipid and sterol contents of the lottiid limpets N. concinna and N. fuscoviridis from northeastern Japan. Muscle Gonads N. concinna N. fuscoviridis N. concinna N. fuscoviridis Male Female Male Female Male Female Male Female Total lipid (%) 0.9±0.2a 0.7±0.1 0.7±0.5 1.0±0.3 2.7±0.4 6.1±1.0 2.6±0.3 3.0±1.2 Sterol (%)b 14.7±1.1 15.2±2.5 14.2±1.8 17.5±1.1 10.6±2.0 4.7±0.3 9.4±0.6 5.3±0.7 a Data are mean±SD (n = 3). b Results are expressed as weight percent of total lipid.

502 J. Oleo Sci. 60, (10) 501-504 (2011) Differences in Sterol Composition of Limpets

Table 2 Sterol composition in muscle and gonads of the lottiid limpets N. concinna and N. fuscoviridis from northeastern Japan. Muscle Gonads Sterol N. concinna N. fuscoviridis N. concinna N. fuscoviridis Male Female Male Female Male Female Male Female Cholesterol 93.5±0.8 92.9±1.2 91.4±1.2 92.8±1.5 68.1±1.1＊ 90.5±2.0 68.7±1.5＊ 86.7±3.6 Cholestanol 1.4±0.1 1.2±0.2 0.6±0.4 ＜0.1 0.7±0.2 1.0±0.2 ＜0.1 ＜0.1 Zymostenol －－－－－－－－ Desmosterol 3.3±0.4 3.7±0.5 4.8±0.8 4.0±0.2 27.1±1.9＊ 4.9±1.1 25.1±3.1＊ 8.9±3.7 Brassicasterol/Crinosterol 0.7±0.5 1.1±0.1 0.8±0.2 0.8±0.5 1.7±0.5 1.2±0.5 1.2±0.7 1.1±0.3 Lathosterol －－－－－－＜0.1 ＜0.1 Zymosterol －－－－－－＜0.1 － 24-Methylenecholesterol 0.5±0.5 0.5±0.4 1.1±0.3 0.5±0.5 1.0±0.2 1.3±0.2 1.4±0.5 1.5±0.3 24-Methylcholesterol/ 0.2±0.2 0.4±0.5 1.2±0.1 1.3±0.2 0.4±0.4 0.8±0.4 1.6±0.3 1.4±0.9 Dihydrobrassicasterol Sitosterol/Clionasterol －－＜0.1 ＜0.1 ＜0.1 ＜0.1 0.3±0.3 ＜0.1 Isofucosterol －－＜0.1 ＜0.1 ＜0.1 ＜0.1 0.2±0.1 ＜0.1 Unidentiﬁ ed 0.4±0.3 0.2±0.2 0.1±0.1 0.6±0.5 1.0±0.2 0.3±0.1 1.5±1.4 0.4±0.5 Values are presented as mean±SD (n = 3 or 4).

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