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Home , Aplysia juliana, Common periwinkle, Docosatetraenoic acid

Journal of Oleo Science Copyright ©2019 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess19137 J. Oleo Sci. 68, (12) 1199-1213 (2019)

Lipids and Fatty Acids of Sea Hares kurodai and Aplysia juliana: High Levels of Icosapentaenoic and n-3 Docosapentaenoic Acids Hiroaki Saito1, 2＊ and Hisashi Ioka1† 1 SA Laboratory, 2-1-12, Koyanagi, Aomori 030-0915, JAPAN 2 Japan Inspection Institute of Fats and Oils, 1-8-2, Shinobashi, Koto-ku, Tokyo 135-0007, JAPAN † Present address, Shimane Prefectural Fisheries Technology Center, Hamada 697-0051, Shimane, JAPAN

Abstract: The lipid and compositions of two of gastropods, Aplysia kurodai and Aplysia juliana (collected from shallow sea water), were examined to assess their lipid profiles, health benefits, and the trophic relationships between herbivorous gastropods and their diets. The primary polyunsaturated fatty acids (PUFAs) found in the neutral of all gastropod organs consisted of four shorter chain n-3 PUFAs: linolenic acid (LN, 18:3n-3), icosatetraenoic acid (ITA, 20:4n-3), icosapentaenoic acid (EPA, 20:5n- 3), and (DPA, 22:5n-3). The PUFAs found in polar lipids were various n-3 and n-6 PUFAs: (ARA, 20:4n-6), adrenic acid (docosatetraenoic acid, DTA, 22:4n-6), icosapentaenoic acid (EPA, 20:5n-3), and docosapentaenoic acid (DPA, 22:5n-3) in addition to trace levels of (DHA, 22:6n-3). Various n-3 and n-6 PUFAs (18:2n-6, 20:2n-6, 18:3n-6, 20:3n-6, 18:3n-3, 18:4n-3, 20:3n-3, n-3 ITA, and 22:3n-6,9,15) comprised the biosynthetic profiles of A. kurodai and A. juliana. Both Aplysia species have traditionally been eaten as local foods in Japan, and the high levels of n-3 (EPA and n-3 DPA) and n-6 (ARA and DTA) PUFAs indicate that they are a healthful addition to a human’s diet.

Key words: arachidonic acid, docosapentaenoic acid, docosatetraenoic acid, herbivore, icosapentaenoic acid, icosatetraenoic acid, marine gastropod, marine lipid, marine-grazing food chain, polyunsaturated fatty acids

1 Introduction dae8）; , Veneridae9－12）; and , Mytilidae11, 13））. Many studies have identified the importance of n-3 long- In contrast, relatively little information is available re- chain polyunsaturated fatty acids（LC-PUFAs）, such as garding the fatty acid composition of other mollusk species; icosapentaenoic acid（EPA, 20:5n-3）and docosahexaenoic only a few reports on the lipid and the fatty acid composi- acid（DHA, 22:6n-3）, which provide health benefits1）. tions of have been pubished（nudibranch: Recent studies have also proposed the beneficial health sphingolipids of Aplysia kurodai14）; Chromodoris sp. and effects of docosapentaenoic acid（n-3 DPA, 22:5n-3）, which Phyllidia coelestis15）; Dendrodoris nigra16）; Aeolidiella is involved in platelet aggregation inhibition and endothelial Abbreviations: ARA, arachidonic acid; CAEP, ceramide ami- 2） cell migration . Searching for sources of these compounds noethyl phosphonate; DMA, dimethylacetals; DMOX, 4,4-di- is an area of active interest. Although DHA sources, such methyloxazoline; DHA, docosahexaenoic acid; DPA, docosa- as tuna and krill oils, are already known throughout the pentaenoic acid; DTA, docosatetraenoic acid (adrenic acid); world3）, a high-quality source of other n-3 LC-PUFAs（EPA EPA (IPA), icosapentaenoic acid; ITA, icosatetraenoic acid; and n-3 DPA）has not been discovered. It is well established GC-MS, gas chromatography-mass spectroscopy; LC, long- that many high-trophic-level marine accumulate chain; LN, linolenic acid; MUFA, monounsaturated fatty acids; NMI(D), non-methylene interrupted (dienoic acids); NMR, nu- LC-PUFAs3, 4）. Some commercially important bivalves Bi- （ clear magnetic resonance; PC, phosphatidylcholine; PCA, prin- valvia）, such as and mussels, have been investigat- cipal component analysis; PE, phosphatidylethanolamine; ed in detail to determine their fat and lipid profiles（oysters, PUFA, polyunsaturated fatty acids; TAG, triacylglycerols; TFA, Ostreidae5, 6）; oysters, Pteriidae7）; , Pectini- total fatty acids.

＊Correspondence to: Hiroaki Saito, SA Lipid Laboratory, 2-1-12, Koyanagi, Aomori 030-0915, JAPAN E-mail: [email protected] Accepted September 25, 2019 (received for review May 22, 2019) 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

1199 H. Saito and H. Ioka

stephanieae17）; limpet18）; vent , Provaniidae19）; muscular tissues, gonads, digestive glands, and other common periwinkle, Littorinidae20）; , Haliotidae21, 22）; viscera; in addition, the stomach contents were analyzed and turban shell, cornutus23））. （Table 1）. The muscular tissues included the foot, mantle, The herbivorous gastropod, the sea hare（genus Aplysia, tail, parapodia, rhinophore, and other muscular tissues. , , ）, grazes on small Aplysia spp. are hermaphrodites, therefore, the gonad macroalgae in the tidal and subtidal zones of tropical and were separated into ovaries and testis. Each sample was temperate regions. Near Japan, two Aplysia species, homogenized in a mixture of chloroform and methanol（2:1, Aplysia kurodai Baba（the Northwestern Pacific）and vol/vol）, and a portion of the homogenized sample was ex- Aplysia juliana Quoy & Gainard（cosmopolitan, circum- tracted according to the Folch procedure27）. The Folch ex- tropical in all warm seas）, inhabit the sea surrounding the traction was performed as follows: 10 g of sample tissue Japanese archipelago24, 25）. In some coastal areas of Japan, was homogenized in 60 mL of methanol, 55 mL of chloro- Aplysia species are traditional, local foods that are consid- form was added, and the mixture was homogenized again. ered healthy and medicinal. Although the ecological func- The mixture was filtered, and the residual precipitate was tions of these species have been well documented24, 25）, the washed three times with 5 mL of chloroform（filtrate 1: ap- fatty acid and lipid profiles have yet to be rigorously eluci- proximately 120 mL）. The residue was homogenized with dated（however, sphingolipids have been studied14, 26））; in 55 mL of chloroform and washed with 5 mL of chloroform particular, a detailed analysis of their PUFAs is lacking. To （filtrate 2: approximately 60 mL）. Then, 0.8％ of brine（35 clarify the health benefits and lipid physiologies of A. mL）was added to the combined filtrate（1 and 2）at a final kurodai and A. juliana, the present study presents an ratio of 8:4:3 chloroform/methanol/brine in a separatory analysis of the lipid and fatty acid compositions of these funnel. After allowing to stand for 3 h, the chloroform layer two species. was collected and dried over anhydrous sodium sulfate; after evaporation, the crude lipids were stored in an argon atmosphere. The crude lipids of the organs of A. kurodai and A. juliana were separated on silicic acid columns 2 Materials and Methods （Merck and Co. Ltd., Kieselgel 60, 70-230 mesh）, and a 2.1 Materials quantitative analysis of the lipid constituents was per- The samples of A. kurodai（9 individuals, 502.1±118.0 formed via gravimetric analysis of each collected fraction12）. g）and A. juliana（13 individuals, 336.4±33.1 g）were col- lected from the sea off the Japanese coast of Honshu Island 2.3 NMR spectroscopy and the determination of lipid （34°54’N; 132°04’E, in Hamada City, Sea of Japan）. classes Spectra were recorded on a GSX-270 NMR spectrometer 2.2 Lipid extraction and analysis of lipid classes （JEOL Co. Ltd., Tokyo, Japan）in a pulsed Fourier trans- All samples of A. kurodai and A. juliana were dissect- form mode at 270 MHz in a deuterochloroform solution ed, and the organs were divided into 4 groups: foot and using tetramethylsilane as the internal standard28）.

Table 1 The lipid contents and lipid classes of the two Aplysia species.

1200 J. Oleo Sci. 68, (12) 1199-1213 (2019) Lipids and Fatty Acids of Sea Hares Aplysia spp.

2.4 Preparation of methyl esters and gas-liquid chroma- with Shimadzu Model C-R3A（Shimadzu Seisakusho Co. tography analysis Ltd.）and HP ChemStation System（A. 06 revision, Yokoga- The individual components of the TAG, PE and PC frac- wa HP Co. Ltd.）electronic integrators. tions were converted into fatty acid methyl esters by direct transesterification with methanol and 1％ concentrated 2.5 Preparation of 4,4-dimethyloxazoline（DMOX）deriva- hydrochloric acid under reflux for 1.5 h12）. The methyl tives and their analysis by gas chromatography - esters were purified using silica gel column chromatogra- mass spectrometry（GC-MS） phy via elution with n-hexane/diethyl ether（10/1, v/v）. DMOX derivatives were prepared by adding excess The compositions of the fatty acid methyl esters were 2-amino-2-methylpropanol to a small amount of the fatty determined by gas-liquid chromatography. Analysis was acid methyl esters（for example, 30 mg of fatty acid methyl performed on a Shimadzu GC-8A（Shimadzu Seisakusho esters and 1 mL of 2-amino-2-methylpropanol）in a test Co. Ltd., Kyoto, Japan）and an HP-6890（Hewlett Packard tube in an argon atmosphere. The mixture was heated at Co., Yokogawa Electric Corporation, Tokyo, Japan）gas 180℃ for 18 h. The reaction mixture was cooled, poured chromatograph equipped with an Omegawax-250 fused onto saturated brine, and extracted with n-hexane. Tripli- silica capillary column（30 m×0.25 mm i.d.; 0.25 µm film, cate extractions with n-hexane were perfomed; the ex- Supelco Japan Co. Ltd., Tokyo, Japan）. The temperatures tracts were washed with saturated brine and dried over an- of the injector and the column were held at 230℃ and hydrous sodium sulfate. The solvent was removed under 215℃, respectively, and the split ratio was 1:76（FID detec- reduced pressure, and the samples were again dissolved in tor at 240℃）. Helium was used as the carrier gas at a con- n-hexane for analysis by GC-MS12, 28）. stant inlet rate of 0.7 mL/min（Tables 2–4）. The phosphati- Analysis of the DMOX derivatives and the methyl esters dylethanolamine（PE）contained low levels of various was performed on an HP G1800C GCD Series II GC-MS dimethylacetals（DMAs）, such as DMA 16:0, DMA 17:0, （Hewlett Packard Co., Yokogawa Electric Corporation） DMA 18:0, and DMA 19:0（Fig. 1）. The theoretical values of equipped with the same capillary column for determining the fatty acid compositions were obtained by subtracting the fatty acids with an HP WS（HP Kayak XA, G1701BA the DMAs from the total fatty acids（TFAs）of the PE11, 12, 28） version, PC workstations）. The temperatures of the injec- （Tables 2–4）. tor and the column were held at 230℃ and 215℃, respec- Quantification of individual components was performed tively. The split ratio was 1:75, and the ionization voltage

a, b, c, d Table 2 Fatty acid composition of digestive gland and stomach content lipids .

1201 J. Oleo Sci. 68, (12) 1199-1213 (2019) H. Saito and H. Ioka

a, b, c, d Table 3 Fatty acid composition of muscle and other visceral lipids .

a, b, c, d Table 4 Fatty acid composition of testis and ovary lipids .

1202 J. Oleo Sci. 68, (12) 1199-1213 (2019) Lipids and Fatty Acids of Sea Hares Aplysia spp.

Fig. 1 The chromatograms of the DMOX derivatives（above chromatogram）and methyl esters（below one）of the fatty acids in PE of A. kurodai testis lipids. Analysis of the DMOX derivatives was performed on a HP G1800C GCD Series II gas chromatograph mass spectrometer equipped with an Omegawax-250 fused silica capillary column with the HP WS （HP Kayak XA, G1701BA version, personal computer workstations）. The temperatures of the injector and the column were held at 230 and 215℃, respectively. The split ratio was 1:75, and the ionization voltage was 70 eV, respectively. Helium was used as the carrier gas at a constant inlet rate of 0.7 mL/min. Each MS spectrum was obtained by every 0.009 min.

1203 J. Oleo Sci. 68, (12) 1199-1213 (2019) H. Saito and H. Ioka

was 70 eV. Helium was used as the carrier gas at a constant 153, 140, 126, 113（base peak）; in addition, two peak pairs inlet rate of 0.7 mL/min12, 28）. M-274/M-262 and M-234/M-222 reflected two double bonds: Δ-14（n-6）and Δ-11（n-9）, respectively. The peak pair 2.6 Chromatogram of the DMOX derivatives and the （M-153/M-136）indicated a Δ-5（n-15）double bond28）. In structural elucidation of the fatty acid DMOX deriva- Figs. 1 and 3, the spectrum at 31.47 min is one of the rep- tives resentative spectra of 22:3n-6,9,15, and more than 70 The representative chromatogram of the DMOX deriva- spectra（M＋-387）were obtained by scanning the peak tives for the PE from the Aplysia samples is shown in Fig. （31.3–32.0 min）. MS peaks（31.518 min, Scan No. 3995）of 1 and Table 5. The MS spectra of two characteristic but a DMOX derivative of 22:3n-3,6,15（Δ7,13,16-22:3）were M＋ unusual fatty acids are shown in Figs. 2 and 3 along with -387, 372, 358, 344, 330, 316, 302, 290, 276, 262, 250, 236, the chromatogram of the DMOX derivatives of the sea hare 222, 208, 194, 180, 168, 154, 140, 126（base peak）, and 113; lipids. Each MS spectrum was obtained every 0.009 min three peak pairs（M-302/M-290, M-262/M-250, and M-180/ （Figs. 2 and 3）. For example, the spectrum at 18.085 min M-168）reflected three double bonds: Δ-16（n-6）, Δ-13 （Scan No. 2172 in Fig. 2）is one of the representative （n-9）, and Δ-7（n-15）, respectively. spectra of 20:3n-6,9,15（Δ5,11,14-20:3）because 20:3n- 6,9,15 was detected near 18.08 min as one peak in the 2.7 Statistical analyses chromatogram（Fig. 1, Table 5）; more than 50 spectra（M＋ More than two experimental replicates（2–5）were com- -359）were obtained by scanning the peak（17.9–18.4 min）. pleted for each lipid class. For all samples of fatty acids an- In Fig. 2（18.085 min, Scan No. 2172）, the MS peaks of a alyzed by GLC, 4 to 12 replicates were completed. The DMOX derivative of 20:3n-6,9,15 were M＋-359, 344, 330, mean DHA, total n-6 PUFA, total n-3 PUFA, and total 316, 302, 288, 274, 262, 248, 234, 222, 208, 194, 180, 166, PUFA levels in the TAG and phospholipid（PE and PC）

Table 5 Retention time and fatty acid composition of testis PE lipids.

1204 J. Oleo Sci. 68, (12) 1199-1213 (2019) Lipids and Fatty Acids of Sea Hares Aplysia spp.

Fig. 2 The MS peaks（18.085 min, Scan No. 2172）of the DMOX derivative of 20:3n-6,9,15（Δ5,11,14-20:3）are M＋-359, 344, 330, 316, 302, 288, 274, 262, 248, 234, 222, 208, 194, 180, 166, 153, 140, 126, 113（base peak）and two pairs of the peaks（M-274/M-262 and M-234/M-222）are respectively reflected by two double bonds: Δ-14（n-6）and Δ-11（n-9）. A pair of the peak（M-153/M-136）shows a Δ-5（n-15）double bond.

Fig. 3 The MS peaks（31.518 min, Scan No. 3995）of the DMOX derivative of 22:3n-6,9,15（Δ7,13,16-22:3）are M＋-387, 372, 358, 344, 330, 316, 302, 290, 276, 262, 250, 236, 222, 208, 194, 180, 168, 154, 140, 126（base peak）, and 113, and three pairs of the peaks（M-302/M-290, M-262/M-250, and M-180/M-168）are respectively reflected by three double bonds as Δ-16（n-6）, Δ-13（n-9）, and Δ-7（n-15）. samples were compared with those from other organ dation of species differences. Tukey’s multiple procedure samples. Significant mean differences were determined was used to compare the differences among mean values. using a one-way analysis of variance（ANOVA）for the eluci- Differences were regarded as significant when p＜0.05.

1205 J. Oleo Sci. 68, (12) 1199-1213 (2019) H. Saito and H. Ioka

Fig. 5 Loading plot, describing the relationships among two Aplysia stomach contents PUFAs derived from Fig. 4 Loading plot, describing the relationships among a principal component analysis based on two Aplysia digestive gland PUFAs derived from a proportions of PUFA（n＝20）in the two Aplysia principal component analysis based on proportions species from 6 lipid classes（1StTAG, 2StTAG, （percentage of total fatty acids）of PUFAs（n＝20） 1StPE, 2StPE, 1StPC, and 2StPC）. The 1st and in the two Aplysia species from 6 lipid classes 2nd principal components described 74.3％ of total （1DTAG, 2DTAG, 1DPE, 2DPE, 1DPC, and 2DPC）. variation of the 6 lipid classes of FAs from stomach The 1st and 2nd principal components described content samples. We observed various PUFAs（total 80.4％ of the total variation of the 6 lipid classes of polyenoic, total n-6 polyenoic, 20:4n-6, 20:5n-3, FAs of digestive gland samples. We observed 22:5n-3, and 22:4n-6）in the positive quadrant of various PUFAs（total polyenoic, total n-6 polyenoic, the loading plot. 20:4n-6, 22:5n-3, 22:4n-6, and 18:3n-6）in the positive quadrant（positive loadings for both PC1 3 Results and PC2）of the loading plot. 3.1 Lipid content and lipid classes of A. kurodai and A. juliana Relationships between the fatty acid compositions of The lipid contents and lipid classes of A. kurodai and A. both Aplysia species were evaluated using the loading juliana samples are shown in Table 1. The lipid contents plots of principal component analysis（PCA）, based on the of the muscles ranged from 0.3％ to 0.6％ of wet weight in correlation matrix of each fatty acid. The first two principal all samples, while those of the viscera ranged from 0.4％ to components were plotted, both for loading and score plots, 3.9％. because they represented the majority of total variation TAGs and sterols were both primary components of the （Figs. 4–9）. For example, PCA was performed on six lipid neutral muscle lipids of all specimens; low levels of other classes（1MTAG, 2MTAG, 1MPE, 2MPE, 1MPC, and 2MPC） neutral lipids, such as wax esters, steryl esters, diacylglyc- from the muscles of both Aplysia species（Table 3）; specifi- eryl ethers, and diacylglycerols（Table 1）were detected as cally, PUFAs（total PUFA, total n-6 PUFA, total n-3 PUFA, well. In particular, TAG was the only major component of 18:2n-6, 18:3n-6, 20:2n-6, 20:3n-6, 20:4n-6, 22:4n-6, the neutral lipids of the digestive glands（A. kurodai: 63.8 18:3n-3, 18:4n-3, 20:3n-3, 20:4n-3, 20:5n-3, 22:4n-3, ±1.4％ and A. juliana: 45.9±2.4％）and ovaries（A. 22:5n-3, and 22:6n-3）were used along with 3 NMIDs for a kurodai: 62.3±3.7％ and A. juliana: 46.1±2.3％）; the total of 20 variables（Fig. 6）. Statistical calculations were lipid contents of the digestive glands（2.1–2.7％）and performed using statistical software（Ekuseru-Toukei 2010, ovaries（3.1–3.9％）were higher than those of other organs. Social Survey Research Information Co., Ltd., Tokyo, The polar lipids of A. kurodai and A. juliana（except for Japan）. digestive gland lipids）contained moderate levels of phos- pholipids（PE: 13.2–14.1％ and PC: 13.5–18.0％ of the total lipids）and noticeable levels of ceramide aminoethyl phos-

1206 J. Oleo Sci. 68, (12) 1199-1213 (2019) Lipids and Fatty Acids of Sea Hares Aplysia spp.

Fig. 7 Loading plot, describing the relationships among Fig. 6 Loading plot, describing the relationships among two Aplysia visceral PUFAs derived from a two Aplysia muscle PUFAs derived from a principal component analysis based on proportions principal component analysis based on proportions of PUFAs（n＝20）in the two Aplysia species from of PUFAs（n＝20）in the two Aplysia species from 6 lipid classes（1OGTAG, 2OGTAG, 1OGPE, 6 lipid classes（1MTAG, 2MTAG, 1MPE, 2MPE, 2OGPE, 1OGPC, and 2OGPC）. The 1st and 2nd 1MPC, and 2MPC）. The 1st and 2nd principal principal components described 84.9％ of total components described 88.4％ of total variation of variation of the 6 lipid classes of FAs from visceral the 6 lipid classes of FAs from muscle samples. We samples. We observed various PUFAs（total observed various PUFAs（total polyenoic, total n-6 polyenoic, total n-6 polyenoic, 20:4n-6, 20:5n-3, polyenoic, 20:4n-6, 20:5n-3, 22:5n-3, and 22:4n-6） and 22:4n-6）in the positive side quadrant of the in the positive quadrant of the loading plot. loading plot.

phonate. were found in the stomach contents of the samples: 14:0, 16:0, 18:0, 16:1n-7, 18:1n-7, 20:1n-13, 18:2n-6, ARA, 3.2 Fatty acid composition and its similarity to depot TAG 18:3n-3, 18:4n-3, n-3 ITA, and EPA（Table 2）. In the lipids in both the mantle and viscera of A. kurodai and neutral lipids of all organs, very low levels of DHA were ob- A. juliana served, which is unusual for marine animals. The TAG fatty acids（more than 0.3％ of TFA）of all organ lipids are shown in Tables 2–4. Although the TAG fatty 3.3 Fatty acid composition of phospholipids from A. kuro- acid compositions varied slightly between the muscles and dai and A. juliana viscera, the types of major fatty acids were almost identical The primary fatty acid compositions of all phospholipids in all organs. Furthermore, there were small differences in from A. kurodai and A. juliana are presented in Tables the fatty acid compositions of the two species（Sample No. 2–4. The PE fatty acid composition included low levels of 1–2）. Twelve dominant TAG fatty acids（more than approx- DMAs, such as DMA 18:0 and DMA 19:0, and this finding imately 3％ of TFA）of digestive glands, muscles, gonads, suggests that plasmalogen-type PEs are biosynthesized in and other organs were found in the samples: saturated A. kurodai and A. juliana tissue（Fig. 2）. fatty acids 14:0, 16:0 and 18:0; monounsaturated fatty acids Eleven major PE fatty acids were found: 14:0（digestive （MUFAs）16:1n-7, 18:1n-7, and 20:1n-13（muscles and glands）, 16:0, 18:0, 20:1n-13, 22:2n-9,15（Δ7,Δ13-22:2）, other organ lipids）; n-6 PUFAs 18:2n-6（ from 22:2n-7,15（Δ7,Δ15-22:2; mantle lipid）, ARA, 22:4n-6 the digestive glands and gonads）and ARA; and n-3 PUFAs （adrenic acid, docosatetraenoic acid; DTA）, 18:3n-3（diges- 18:3n-3（α-linolenic acid, LN）, 18:4n-3（digestive glands, tive gland and other visceral lipids）, EPA, and n-3 DPA mantle, and other visceral lipids）, n-3 ITA, and EPA（Tables along with trace levels of DHA. This was in contrast to the 2–4）. In particular, high levels of LN（6.8–10.6％）and EPA noticeable levels of shorter chain PUFAs such as 18:3n-3 （4.1–9.1％）with noticeable levels of ITA were observed in and n-3 ITA found in the TAG and the comparatively high the neutral lipids. Twelve similar dominant TAG fatty acids levels of LC-PUFAs such as DPA（6.6–8.3％ for muscle PE,

1207 J. Oleo Sci. 68, (12) 1199-1213 (2019) H. Saito and H. Ioka

Fig. 8 Loading plot, describing the relationships among Fig. 9 Loading plot, describing the relationships among two Aplysia testis PUFAs derived from a principal two Aplysia ovary PUFAs derived from a principal component analysis based on proportions of PUFAs component analysis based on proportions of PUFAs （n＝20）in the two Aplysia species from 6 lipid （n＝20）in the two Aplysia species from 6 lipid classes（1TTAG, 2TTAG, 1TPE, 2TPE, 1TPC, and classes（1OvTAG, 2OvTAG, 1OvPE, 2OvPE, 1OvPC, 2TPC）. The 1st and 2nd principal components and 2OvPC）. The 1st and 2nd principal described 74.7％ of total variation of the 6 lipid components described 76.5％ of total variation of classes of FAs from testis samples. We observed the 6 lipid classes of FAs from ovary samples. We various PUFA（total polyenoic, total n-6 polyenoic, observed various PUFA（Total n-3 polyenoic, 20:2n- 20:4n-6, 20:5n-3, and 22:2n-9,15）in the positive 6, 20:3n-6, 22:6n-3, 18:2n-6, and 18:3n-3）in the side quadrant of the loading plot. positive side quadrant of the loading plot.

4.6–7.5％ for other visceral PE, and 4.7–6.9％ for gonad variation of the 6 lipid classes of FAs from muscle samples. PE）found in the phospholipids. Similar to the fatty acid We observed various PUFAs（total polyenoic, total n-6 composition of the tissue PE, ten major fatty acids were polyenoic, 20:4n-6, 20:5n-3, 22:5n-3, and 22:4n-6）in the also found in the PE of the stomach contents（Table 2）: positive quadrant（positive loadings for both PC1 and PC2） 14:0, 16:0, 18:0, 18:1n-7, 18:1n-9, 22:2n-9,15（Δ7,Δ13- of the loading plot. A similar trend was found in other 22:2）, ARA, n-6 DTA, 18:3n-3, EPA, and n-3 DPA. viscera（total n-6 polyenoic, 20:4n-6, 20:5n-3, and 22:4n- Significant levels of NMI（D）were found in the PE, 6）, digestive glands（total polyenoic, total n-6 polyenoic, however, in contrast, fourteen fatty acids comprised the 20:4n-6, 22:4n-6, 18:3n-6, 20:2n-6, 22:4n-6, and 22:4n-3）, major components of the tissue PC: 16:0, 18:0, 18:1n-7, ovaries（total n-3 polyenoic, 22:6n-3, 20:3n-6, 20:2n-6, 18:1n-9, 20:1n-13, 18:2n-6, 20:2n-6, ARA, n-6 DTA, 18:3n- 18:3n-3, and 18:2n-6）, testis（total polyenoic, total n-6 3, 20:3n-3（mantle lipid）, n-3 ITA, EPA, and n-3 DPA in ad- polyenoic, total n-3 PUFA, 20:4n-6, 20:5n-3, and 22:2n- dition to an unusually low level of DHA. Similarly, twelve 9,15）, and the stomach contents（total polyenoic, total n-6 major fatty acids were found in the PC of the stomach con- polyenoic, 20:4n-6, 20:5n-3, 22:5n-3, 22:4n-6, 20:3n-3, tents: 16:0, 18:0, 18:1n-7, 18:1n-9, 20:1n-13, 18:2n-6, 20:2n-6, 22:4n-3, and 18:3n-6）. The two variables“ total 20:2n-6, ARA, n-6 DTA, 18:3n-3, EPA, and n-3 DPA in ad- n-6 polyenoic” and“ 20:4n-6” were highly correlated in dition to very low levels of DHA. almost all organs.

3.4 PCA of A. kurodai and A. Juliana fatty acid composi- tions PCA was used to compare the PUFA profiles of the two 4 Discussion Aplysia species. The PCA results showed that 74.3–88.4％ 4.1 Lipid content and lipid classes of A. kurodai and of the total variation was explained by the first two princi- A. juliana pal components（Figs. 4–9）. For example, in Fig. 6, the 1st In general, A. kurodai and A. juliana accumulated their and 2nd principal components described 88.4％ of the total lipids in the ovaries and digestive glands（Table 1）, similar

1208 J. Oleo Sci. 68, (12) 1199-1213 (2019) Lipids and Fatty Acids of Sea Hares Aplysia spp.

to other Gastropoda（digestive glands for blacklip and due to the similarity of their diets. Both Aplysia species greenlip abalones22）and digestive glands of T. cornutus23））. probably accumulated intact fatty acids in their digestive The lipid contents of the viscera of both Aplysia species glands upon the absorption of the dietary lipids. Therefore,

were markedly higher than those of the dietary , C2 elongation of the depot lipids was likely the only enzy- whose lipids are generally very low（approximately 0.1– matic biosynthesis performed by A. kurodai and A. 0.6％ of dry weight29）; approximately 0.3–0.7％30）; approxi- juliana. Interestingly, two bioactive compounds from tidal mately 0.3–0.7％ of dry weight31）; approximately 0.1％32））. macroalga, n-3 ITA and EPA, were found to be concentrat- In fact, the lipid contents of the gastropod digestive glands ed in the neutral lipids of both species; in addition, the n-3 were 21-27 times more concentrated than those of the shorter chain PUFAs（18:4n-3 and n-3 ITA）have anti-in- dietary algae. In the visceral lipids（Table 1）, TAGs, at flammatory and anti-arthritic effects38）. moderate levels, were identified as the major component. Except for the high levels of n-3 ITA in both Aplysia For example, the digestive gland and ovary TAGs of A. TAGs, both species retained similar types of PUFAs from kurodai and A. juliana were approximately 50％ of the the dietary algal lipids. Short-chain fatty acids are mostly TL. The high levels of neutral depot TAGs in A. kurodai limited to green macroalgae, and 16:0, 18:2n-6, 18:3n-3, were probably influenced the high lipid content; depot and 18:3n-4 are often their primary lipid components lipids typically change according to lipid accumulation. In without DHA31, 32）. In addition to green macroalgae, other contrast, the lipids of A. kurodai and A. juliana muscles macroalgae, which comprise the majority of the Aplysia （foot, mantle, tail, parapodia, rhinophore, and other mus- diet, generally contain only trace levels of DHA or lack cular tissues）were similar to those of other common gas- DHA altogether23, 29－32）. The muscle tissues（mantle, foot, tropod species（ Haliotis fulgens21）, T. cornutus23）, and other muscular parts）of both species contained the whose neutral lipid levels are also low）; their muscle lipids same PUFAs（Table 2）, although the levels of these fatty were consistently composed of phospholipids at moderate acids differed slightly. These findings suggest that the levels（41.5–46.6％）, which were the major components dietary algae lipids directly influenced the compositions of （Table 1）. The major lipid components of feet（muscle）of the muscle TAGs and the digestive gland lipids found in 19, 21, 23） common shallow-water gastropods are phospholipids . both species; it seems that with the exception of simple C2 PE and PC were the primary polar lipid classes of A. elongation, both gastropods merely accumulated intact kurodai and A. juliana. The PE fatty acids contained low lipids. levels of DMA, similar to other mollusk species7, 10, 11, 23）. In general, high levels of n-3 LC-PUFAs, such as DHA This is a general characteristic of mollusks related to A. and EPA, are found in many marine animals, such as , kurodai and A. juliana, and they are also known to crustaceans, and bivalves: even their depot TAGs contain 33） 3, 4） contain low levels of plasmalogen type PE . In addition to high DHA levels . High levels of various n-6 and n-3 C18 low levels of other minor phospholipids, ceramide amino- PUFAs（18:2n-6, 18:3n-3, and 18:4n-3）in addition to n-3 ethyl phosphonate（CAEP）, which is used in skin care ITA and EPA were found to be the dominant fatty acids in products, was the major component of the sphingolipid both the A. kurodai and A. juliana muscle and visceral fraction6, 7, 10, 13, 14, 19, 23, 34）. The presence of CAEP indicates TAGs（Tables 2–4）; this was in contrast to other common that the lipids of A. kurodai and A. juliana are similar to marine animals, which contain low levels of these short- typical mollusk lipids. chain n-6 and n-3 PUFAs3, 4, 33）. Only four n-3 PUFAs （18:3n-3, 18:4n-3, n-3 ITA, and EPA）of A. kurodai and A. 4.2 Unusual TAG fatty acid composition in the muscle juliana TAGs were found at high levels; very low levels of and viscera of A. kurodai and A. juliana: in uence of DHA（0.1–0.5％）were found in all organ lipids, which is un- dietary algal lipids common in marine animals. In particular, high levels of n-3 Partially digested green and brown algae, such as Ulva ITA were characteristically observed in all organ TAGs of A.

spp., were clearly identified in the stomach contents of juliana. This phenomenon indicates that the process of C2 both Aplysia species, similar to other reports23, 24）. These elongation of 18:4n-3 is active in the mollusk tissue;

macroalgae contain high levels of short-chain（C16–C18）fatty 18:4n-3 is a common macroalgal lipid while n-3 ITA is a acids（16:0, 16:1n-7, 16:3n-3, 18:1n-7, 18:2n-6, ARA, rare algal lipid6, 31, 32）. 18:3n-3, 18:4n-3, and EPA）with lower levels of other short- Furthermore, high levels of 20:1n-13 were found in both chain n-3 PUFAs（20:2n-6, 20:3n-6, and n-3 ITA in brown A. kurodai and A. juliana（Tables 2–4）. The levels of algae23, 31, 35, 36）; 18:3n-6, 20:2n-6, and n-3 ITA in green 20:1n-13 in the TAG stomach contents and lipids from all algae31, 35, 37）; 18:3n-6 and n-3 ITA in brown and green their tissues of both species were similar to each other. algae32））. High levels of similar shorter-chain PUFAs（18:2n- 20:1n-13 was likely derived from 18:0 by Δ5-desaturation

6, 18:3n-3, and 18:4n-3）were found in both species（Table and C2 elongation. LC-MUFAs may also be biosynthesized

2）. The same dominant fatty acids were observed in both in the Aplysia tissues by Δ5-desaturation and C2 elonga- Aplysia TAGs of the digestive glands and stomach contents tion6, 23）. In addition, the presence of two unusual non-

1209 J. Oleo Sci. 68, (12) 1199-1213 (2019) H. Saito and H. Ioka

6） methylene-interrupted PUFAs（20:3n-6,9,15 and 22:3n- species（C2 elongation and Δ5-desaturation ）. The n-6

6,9,15）suggested active C2 elongation and Δ5-desaturation PUFAs could have been biosynthesized in Aplysia tissues from 18:2n-6（Figs. 1–3）. In particular, various C22 LC-PU- by C2 elongation and Δ5-desaturation, similar to the biosyn-

FAs in both Aplysia species suggest biosynthetic C2 elon- thesis of LC-MUFAs, such as 20:1n-13 in the depot TAGs. gation because the dietary macroalgae generally lack C22 All A. kurodai and A. juliana polar lipids contained LC-PUFAs23, 29－32, 35－37）. high levels of total PUFAs（PE: 46.1–55.2％ and PC: 41.2– 55.3％）, which were probably concentrated in their tissues. 4.3 High levels of n-6 PUFAs（ARA and n-6 DTA）in the However, the levels of total n-3 PUFAs（PE; 20.0–29.1％, phospholipids: similarity to other herbivorous marine PC; 24.8–30.8％）were similar to those of the TAGs（19.4– animals and useful lipid biomarkers for herbivorous 33.1％）. In contrast, the levels of total n-6 PUFAs（PE: marine animals 19.9–31.4％, PC: 15.4–28.5％）found in all muscle and The primary fatty acids found in the viscera and muscle viscera phospholipids of A. kurodai and A. juliana were phospholipids of A. kurodai and A. juliana are presented higher than those in TAGs（10.0–13.9％）. This finding in Tables 2–4. ARA and n-6 DTA were typically found to implies that n-6 PUFAs were accumulated or biothynthe- compose greater than 5％ of A. kurodai and A. juliana sized in the Aplysia tissues. For example, the high levels tissue phospholipids, however, these n-6 LC-PUFAs are of n-6 LC-PUFAs（C20–C22: ARA and n-6 DTA）in their phos- generally only minor components in other marine animals. pholipids（11.6–23.4％ in muscles, 10.6–28.1％ in gonads, The high levels of n-6 LC-PUFAs found in the phospholip- and 13.8–21.5％ in other viscera, shown in Tables 3 and 4） ids were unusual for marine animals; the PUFA profiles of differed from those found in their TAGs, which primarily all polar lipids from A. kurodai and A. juliana were mark- contained 18:2n-6 and ARA in addition to low n-6 DTA edly different from those in other common marine animals, levels（0.9–2.5％）. This also indicates that C2 elongation such as marine fishes3, 4）, which have high levels of only n-3 activity（ARA to n-6 DTA）in their tissues. PUFAs and very low levels of n-6 PUFAs. In contrast, herbivorous marine animals, whose diets are 4.5 Application of PCA for assessing correlations be- mostly composed of macroalgae, often contain high levels tween the fatty acid compositions of A. kurodai and of n-6 PUFAs（abalones, Haliotidae18, 21, 39）; T. cornutus23））. A. juliana For example, high proportions of ARA in the foot lipids of The PCA loading plot of various fatty acids indicated a common shallow-water gastropods are often found（ARA relationship and an importance of fatty acids in both levels; 10.6–38.8％ for Patella peroni and Cellana species. A positive correlation of various n-3 and n-6 tramoserica and 12.6–30.9％ for Ponerplax PUFAs between both species was determined from PCA. costata18）; 10.7–14.3％ for H. laevigata and H. rubra22, 39）; Using the PCA of the primary PUFAs in both Aplysia 9.8–14.2％ for foot of H. fulgens21）; 11.2–34.3％ for T. cor- phospholipids, we estimated the similarity of the PUFA nutus23））. The levels of n-6 LC-PUFAs in A. kurodai and A. compositions of the Aplysia species（Figs. 4–9）. The PCA juliana were similar to those found in the foot muscles of suggested that n-3 and n-6 PUFAs are the most important herbivorous gastropods. Various n-6 PUFAs（20:2n-6, compounds in both Aplysia species, which have the same 20:3n-6, ARA, and n-6 DTA）were the characteristic fatty PUFAs as major compounds. This similarity also suggests a acids of all organ PEs and PCs of both Aplysia species. similar diet and mechanism of LC fatty acid biosynthesis in The high levels of both ARA and n-6 DTA found in their the two sea hare. phospholipids could be used as lipid biomarkers for herbiv- orous marine animals. In particular, high levels of n-6 DTA 4.6 DHA is likely nonessential to A. kurodai and A. juliana in the phospholipids is an unusual and interesting charac- Regarding the n-3 PUFAs found in A. kurodai and A. teristic of Aplysia spp. This characteristic indicates com- juliana tissue（muscle, ovary, testis, and other organs plete herbivorous feeding behavior that is directly influ- shown in Tables 3 and 4）, higher levels of LC-PUFAs（EPA enced by macroalgae lipids. and n-3 DPA）were found in the phospholipids compared to the TAGs, which primarily contained shorter chain PUFAs 4.4 Biosynthetic elongation and desaturation in the such as 18:3n-3 and 18:4n-3. This finding also suggests Δ-5 6, 15, 16, 19） tissues of A. kurodai and A. juliana desaturation（n-3 ITA to EPA ）and C2 elongation High levels of n-3 DPA and low levels of 18:3n-3 and （18:4n-3 to n-3 ITA and EPA to n-3 DPA）in the Aplysia 18:4n-3 were found in A. kurodai and A. juliana phos- tissues, similar to that of n-6 LC-PUFAs. Four PUFAs（n-6 pholipids. ARA and EPA were likely biosynthesized to n-6 PUFA; ARA and n-6 DTA, n-3 PUFA; EPA and n-3 DPA）

DTA and n-3 DPA, respectively, by C2 elongation. Longer were found to be the primary components of both PE and chain n-3 PUFAs, such as EPA, might have been derived PC of A. kurodai and A. juliana. The trace levels of DHA from shorter chain n-3 PUFAs, such as C18 PUFAs, as previ- in all organ tissues of A. kurodai and A. juliana also indi- ously described; this would be similar to other mollusk cate that their nutritional source was the macroalgae lack

1210 J. Oleo Sci. 68, (12) 1199-1213 (2019) Lipids and Fatty Acids of Sea Hares Aplysia spp.

DHA. Many studies have reported trace levels of DHA in more specifically, n-3 ITA, EPA, and n-3 DPA are charac- other herbivorous gastropods（DHA: 0.21％ for periwinkle teristic fatty acids of both the depot TAGs and tissue PEs littorea20）; non-detectable and 0.4％ for P. and PCs of A. kurodai and A. juliana. A. kurodai and A. peroni and Cellana tramoserica, respectively, and 0.2– juliana are locally processed and safely consumed as me- 0.9％ for P. costata18）; 0.2–0.3％ for wild H. laevigata and dicinal . In addition, their bodies are markedly H. rubra39, 40）; non-detectable for H. fulgens21）; 0.2–0.3％ larger than those of nudibranch mollusks15－17, 38）. There- for T. cornutus23））. The low levels of DHA accompanied by fore, they could be a valuable source of n-3 ITA, EPA, and high levels of EPA and n-3 DPA in A. kurodai and A. n-3 DPA, which could be used in pharmaceuticals or health juliana phospholipids suggest the possibility of an inert supplements. enzyme for DHA synthesis, which is similar to marine fish; they are unable to biosynthesize DHA from DPA41）. These findings suggest that DHA is not essential for these herbivorous mollusks, which is similar to findings in Acknowledgments some gastropods and bivalves10, 13, 19, 20, 22）. This is in contrast The authors thank Profs. Mutsumi Sugita and Saki to marine and some bivalves, to whom n-3 PUFAs Itonori, Shiga University, for kindly donating the authentic are essential40, 42）. It is possible that A. kurodai and A. ceramide aminoethyl phosphonate which originated from juliana accumulate n-6 PUFAs as a substitute for n-3 the freshwater Corbicula sandai and for his valuable PUFAs. All marine animals show a tendency to accumulate discussion of the bivalve lipids. The author also thanks Ms. PUFAs in their tissues; however, the differences in the Sumiko Terada, Ms. Mikiko Tanaka, and Mr. Akihito Ta- degree of accumulation of n-6 and n-3 PUFAs among kashima for their skilled technical assistance. This work mollusk species may be influended by their position in the was supported in part by the research projects from food chain18, 21, 39）. It has been reported4）that many pelagic Ishikawa Prefectural University, JSPS KAKENHI fishes primarily accumulate n-3 PUFAs such as DHA JP15K07583, and the Fisheries Research Agency of Japan. through grazing food chain from phytoplankton in addition H. S. performed all aspects of the study including designing to the elongation of shorter PUFAs. In general, DHA is the the experiments, analyzing the data, and writing the manu- dominant PUFA found in both the PE and PC of almost all script. H. I. assisted in collecting and measuring the higher trophic marine animals, who maintain consistently samples. high DHA levels by the continuous exploitation of their food and subsequent DHA accumulation in vivo4）. In addi- tion, all marine fishes require DHA because it is the most important that they are unable to syn- Rrferences thesize, which differs from the ability of freshwater 1） Saravanan, P.; Davidson, N.C.; Schmidt, E.B.; Calder, fishes40）. P.C. Cardiovascular effects of marine omega-3 fatty It is possible that while DHA may be nonessential, A. acids. Lancet 376, 540-550（2010）. kurodai and A. juliana use it to maintain the fluidity of 2） Kaur, G.; Cameron-Smith, D.; Garg, M.: Sinclair, A.J. cell membranes. In addition, the total PUFA levels in the Docosapentenoic acid（22:5n-3）: a review of its biolog- PEs（46.1-52.7 and 53.1–55.2％ for muscle and gonadal ical effects. Prog. Lipid Res. 50, 28-34（2011）. lipids）and the PCs（51.5–53.3 and 47.0–55.3％ for muscle 3） Arts, M.T.; Ackman, R.G.; Holub, B.J.“ Essential fatty and gonadal lipids）of A. kurodai and A. juliana were acids” in aquatic ecosystems: a crucial link between consistently high（Tables 3 and 4）, and the accumulation diet and human health and evolution. Can. J. Fish. of n-6 PUFAs in phospholipids was observed as mentioned Aquart. Sci. 58, 122-137（2001）. above. Similar to the ARA essentiality for larvae of marine 4） Saito, H.; Seike, Y.; Ioka, H.; Osako, K.; Tanaka, M.; Ta- fishes43）, these findings suggest a physiological role of n-6 kashima, A.: Keriko, J.M.; Kose, S.; Souza, J.C.R. High PUFAs in compensation for a deficiency of n-3 PUFAs. This docosahexaenoic acid levels in both neutral and polar finding was also supported by very high levels of ARA lipids of a highly migratory fish: Thunnus tonggol （11.1–14.5％ in the testes and 13.4–14.1％ in the ovaries） Bleeker. Lipids 40, 941-953（2005）. in the gonadal PEs of both Aplysia spp.（Table 4）. 5） Thompson, P.A.; Harrison, P.J. Effects of monospecific algal diets of varying biochemical composition on the 4.7 Useful sources of various n-3 PUFAs, such as n-3 growth and survival of Pacific （ gi- ITA, EPA and n-3 DPA gas）larvae. Mar. Biol. 113, 645-654（1992）. Although DHA sources, such as tuna and krill oils, are 6） Saito, H.; Marty, Y. High levels of icosapentaenoic acid well known throughout the world3）, there is no established in the lipids of oyster Crassostrea gigas ranging over source of n-3 ITA, EPA, or n-3 DPA2, 38）. The lipids of the both Japan and France. J. Oleo Sci. 59, 281-292 two Aplysia spp. contain high levels of various n-3 PUFAs; （2010）.

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