Lipids and fatty acids of two pelagic cottoid fishes (Comephorus spp.) endemic to Lake Baikal

Comparative Biochemistry and Physiology Part B 126 (2000) 477 – 485 www.elsevier.com/locate/cbpb

Lipids and fatty acids of two pelagic cottoid fishes (Comephorus spp.) endemic to Lake Baikal T.A. Kozlova a,*, S.V. Khotimchenko b a

Limnological Institute, SB RAS, P.O. Box 4199, Irkutsk 664033, Russia b Institute of Marine Biology, FEB RAS, Vladi6ostok 690041, Russia

Received 9 March 1999; received in revised form 11 February 2000; accepted 20 March 2000

Abstract Matured females of two Lake Baikal endemic fish species, Comephorus baicalensis and Comephorus dybowski, have been investigated for lipid of the whole body and specific tissues (liver, muscles, ovaries), phospholipid classes and fatty acids of neutral and polar lipids. Total lipid in the body (38.9% fresh weight), liver (23.5%) and muscles (14.5%) of C. baicalensis were greater than those of C. dybowski (4.7, 8.7 and 2.6%, respectively); only their ovaries were similar (5.3 and 5.6% lipid, respectively). In both species, phosphatidylcholine and phosphatidylethanolamine were the major phospholipids, ranging from 60.7 to 75.1% of total phospholipid and 14.5 – 25.7%, respectively. In most cases, monounsaturated fatty acids (MUFA) were the major fatty acid group in C. baicalensis, whereas polyunsaturated fatty acids (PUFA) were the major group in C. dybowski. The MUFA 18:1(n-9) prevailed over other fatty acids in C. baicalensis and varied from 19% in polar lipids of muscles to 56.1% in neutral lipids of muscles. In polar lipid of C. dybowski, the PUFA 22:6(n-3) prevailed over other fatty acids in muscles and ovaries, while 16:0 dominated polar liver lipids and neutral lipids of all tissues. Other major fatty acids included 16:1(n-7), 18:1(n-7), and 20:5(n-3). Values of the (n-3)/(n-6) fatty acid ratio for neutral lipids of C. baicalensis (0.5 – 0.9) are well below the range of values characteristic either for marine or freshwater fish, while these values for polar lipids (1.6 – 1.8) are in the range typical of freshwater fish. Neutral lipid fatty acid ratios in C. dybowski (2.5 – 3.1) allow it to be assigned to freshwater fish, but polar lipids (2.8–3.7) leave it intermediary between freshwater and marine fish. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Lake Baikal; Cottoid fish; Lipid content; Fatty acid; Phospholipid

1. Introduction Lake Baikal is unique among the world’s freshwater lakes because of its ancient origin (27 – 28 Ma) (Mats, 1993) and many endemic species (of the total 2565 species, 64% are endemic; Timoshkin, 1997). Of the three families of cottoid fishes known in Lake Baikal, the family Comephoridae is entirely endemic and consists of two species * Corresponding author. E-mail address: [email protected] (T.A. Kozlova).

only: the big golomyanka, Comephorus baicalensis (Pallas), and the small golomyanka, Comephorus dybowski Korotneff (Sideleva, 1982; Reshetnikov et al., 1997). They are typical pelagic species, inhabiting the whole water column down to the lake’s maximum depth, 1636 m (Shimaraev et al., 1994). They are the only viviparous species within the Cottoidei (Taliev, 1955). Like all other cottoid fishes in Lake Baikal, C. baicalensis and C. dybowski lack a swimbladder. They are assigned to secondary pelagic forms (termed by A.P. Andriyashev), which means

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forms that originated from benthic fish but later adapted to life in the water column via morphophysiological adaptations (Andriyashev, 1986; Sideleva and Kozlova, 1989). Lipids are known to play a major role as regulators of body density while organisms adapt themselves to specific habitat conditions (Eastman and DeVries, 1982; Clarke et al., 1984; Kozlova, 1997, 1998). C. baicalensis stores large amounts of lipids, up to 40% fresh weight (Taliev, 1955). Local people at Lake Baikal used to melt the fat from C. baicalensis for medicinal purposes. Nevertheless, data on the chemical composition of the golomyankas were only available in the literature as technochemical characteristics (Taliev, 1955; Koryakov, 1964; Starikov, 1977). It was not until the work of Morris (1984) that fatty acid composition in adult C. baicalensis and juvenile C. dybowski were published, and lipid metabolism in the Lake Baikal hydrobionts examined (mainly crustaceans) revealed dominance of the (n-3) series fatty acids. Later, Kozlova and Khotimchenko (1993) discovered that the (n-3) series fatty acid content in two benthic-pelagic cottoid fish endemic to Lake Baikal, Cottocomephorus grewingki and Cottocomephorus inermis, was unusually high for freshwater fish. Fundamental research on evolution and adaptations of cottoid fish in Lake Baikal requires a more detailed ecological and biochemical investigation of C. baicalensis and C. dybowski. Both species play a major role in the lake’s trophic web. This paper presents data on whole body lipid content; lipid content, phospholipid composition, and fatty acid composition of polar and neutral lipids in liver, muscles, and gonads of C. baicalensis and C. dybowski. The results are discussed in relation to differences in the development of these fish species’ specialisation to life in pelagic waters, and to specific features of fatty acid composition in the Lake Baikal endemic fauna.

2. Materials and methods

2.1. Animals Female C. baicalensis were caught by trawling in Frolikha Bay (Northern Lake Baikal) at 250 – 350 m depths, female C. dybowski in Senogda Bay (Northern Lake Baikal) at 150 – 200 m depths in October 1997. All were sexually mature. Mean

body lengths: C. baicalensis, 215 9 1.5 mm; C. dybowski, 1409 1.0 mm. Mean body weights: C. baicalensis, 44.59 1.9 g; C. dybowski, 9.19 0.9 g. Samples were stored at − 80°C for a short period prior to analysis. Ten females of each species were analysed for lipid body content. Each specimen was homogenised individually. Tissues and organs (liver, muscles, ovaries) were taken from ten specimens of each species to determine their lipid content. Muscle samples of both species consist mainly of white muscle. Comephorus have no red muscle band along the lateral line, so characteristic for many other fish species. Lipid extracts from each tissue were then pooled into three samples (three or four fishes) to determine phospholipid class composition and fatty acid composition in the liver, muscles and ovaries.

2.2. Lipid extraction and analysis Total lipids were extracted using the method of Folch et al. (1957). The extracts were then mixed and divided into layers by adding water. The lower layer was dried under vacuum in a rotary evaporator for gravimetrical weight measurement and redissolved in chloroform. A portion of lipid extracts from each of liver, muscles, and gonads was used to determine phospholipid class composition by a thin-layer microchromatography method (Svetashev and Vaskovsky, 1972). The remainder was used to investigate fatty acid composition. Total lipids were separated into polar and neutral fractions by silica-gel column chromatography. Neutral lipids were eluted with 5 column volumes of chloroform, and polar lipids were then eluted with 10 column volumes of methanol. Neutral and polar lipids were dried under vacuum (Rouser et al., 1967). Fatty acid methyl esters (FAMEs) were prepared by transmethylation of lipid samples by adding 1% Na in methanol, followed by heating for 15 min at 55°C and then adding 5% HCl in methanol, followed by heating for 15 min at 55°C (Carreau and Dubacq, 1978). FAMEs were purified by thin-layer chromatography using benzene as solvent. A Shimadzu GC-9A gas chromatograph with flame ionisation detection and a Chromatopac C-R3A data station (Shimadzu, Japan) were used for FAME analysis. The FAME were analysed on an open-tube column of flexible fused silica (30 m ×0.25 mm i.d.) with a bonded coating (SUPELCOWAX-10; Supelco Inc., Bellefonte,

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PA). Separation temperature was 210°C. Identification was performed by applying standards and equivalent chain lengths values (Jamieson, 1975; Flanzy et al., 1976).

3. Results and discussion

3.1. Lipid content and phospholipid class composition Total lipid contents of the whole body and tissues of C. baicalensis and C. dybowski are shown in Table 1. C. baicalensis differs greatly from C. dybowski in lipid content of the body (38.9 and 4.7%, respectively), liver (23.5 and 8.7%, respectively), and muscle (14.5 and 2.6%, respectively), and only the gonads exhibit similar lipid content (5.3 and 5.6%, respectively). Our results for body lipid contents of C. baicalensis and C. dybowski are consistent with those reported for these fish species. Taliev (1955) noted great differences in meat fattiness between C. baicalensis and C. dybowski: the lipid content was 33.7% of fresh weight in C. baicalensis, while it was 1.5% in C. dybowski. Starikov (1977) also noted that fat content in C. baicalensis (44.3%) was much higher than in C. dybowski (8.8%). Some inconsistencies in the figures presented appear to have been caused by different methods used for lipid extraction and different sampling seasons. Comparison of body lipid content in C. baicalensis and C. dybowski with scarce literature data on the lipid content of other Lake Baikal Table 1 Lipid content of tissues from two species of cottoid fish from Lake Baikala Tissues

Total lipid (% fresh weight)

Comephorus baicalensis Whole body Liver Muscle Ovaries

38.9 9 1.2 23.5 9 2.3 14.5 9 0.5 5.39 0.3

Comephorus dybowski Whole body Liver Muscle Ovaries a

4.7 9 0.5 8.7 90.9 2.6 9 0.1 5.6 90.3

Values are mean 9 S.D.; n=10.

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cottoid fish reveals a much higher level of lipid in C. baicalensis, whereas in C. dybowski they are approximately at the level observed in the other fish. For example: average lipid in benthopelagic C. grewingkii and C. inermis is 9% in the sexual maturation period (Kozlova, 1997); in benthic Limnocottus megalops, it is 2.4%; in near-shore Cottus kessleri, 3% (Sideleva and Kozlova, 1989); in near-shore Paracottus kneri, 2.6%; and Batrachocottus baicalensis, 4% (Taliev, 1955). Although C. baicalensis and C. dybowski are two closely related congeners and have many features in common, they differ in their biology and ecology. Even though both species can be found at maximum depths, a number of workers (Koryakov, 1964; Starikov, 1977) considered C. baicalensis to be more abyssal as their data showed this species to keep much of the year in deeper waters than C. dybowski. C. baicalensis is a predator, its diet being dominated by Comephorus juveniles (76.1% in December), and the pelagic amphipod Macrohectopus branickii (23.6%) (Volerman and Kontorin, 1983). C. dybowski feeds largely on M. branickii (98.2% in December), while the copepod Epischura baicalensis makes up a smaller part of its diet (1.8%) (Volerman and Kontorin, 1983). Sideleva and Kozlova (1989), Sideleva et al. (1992) have discussed the specialisation of these fish to life in the water column, which developed differently in C. baicalensis and C. dybowski. Both species are sparsely dispersed throughout the water column, and spend most of their time in a slanted position, head down, as if soaring in the water column, and they do not school. Buoyancy of fish lacking a swimbladder can be related to lipid content (DeVries and Eastman, 1978; Eastman and DeVries, 1981, 1982; Neighbors and Nafpaktitis, 1982; Clarke et al., 1984). C. dybowski has a medium lipid content (5–8%) and weak negative buoyancy, and attains the capacity to live in pelagic waters thanks to significantly developed carrying planes, pectoral, dorsal and anal fins, which total over 200% of its body surface area (Sideleva and Kozlova, 1989; Sideleva et al., 1992). By its morphological features, C. baicalensis is not as well adapted to life in pelagic waters as C. dybowski, but it approaches neutral buoyancy owing to low body density (1.010) (Taliev, 1955) and considerable lipid accumulated in the body (40%). Fat inclusions

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Table 2 Phospholipid class composition of tissues from two species of cottoid fish from Lake Baikala Tissues

Phospholipid classes (% total phospholipid) PC

LPC

PE

PI

PS

SPM

Comephorus baicalensis Liver 62.8 Muscle 60.7 Ovaries 68.1

3.6 3.6 4.8

25.7 21.4 14.5

Trace 5.0 2.5

Trace 3.6 2.5

7.9 5.7 7.6

Comephorus dybowski Liver 65.3 Muscle 69.2 Ovaries 75.1

3.3 3.2 2.1

24.0 21.3 18.0

2.7 3.2 2.1

Trace 1.5 Trace

4.6 1.6 2.7

a Values are for pooled samples (ten fish). PC, Phosphatidylcholine; LPC, lysoPC; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SPM, sphingomyeline.

(deposits) are located under the skin, between muscle fibres, and in the body cavity. Anoshko (2000) has investigated changes of morphometric features that occur in C. baicalensis and C. dybowski during their growth, and shown that after the formation of adult features (100 – 120 mm fish length), maximum body height sharply increases in C. baicalensis, while absolute growth of fins (pectoral excluded) practically stops. These data correlate well with our joint research (Ju et al., 1997), which showed that it is at this period (age 3 to 4 – 5 years) that lipid content increases considerably (from 1.63% of wet weight in juveniles (3 years) to 28.3% in mature adults (5 years)) due to sharply increased content of triacylglycerols (from 18.0 to 86.6% of total lipids, respectively). This suggests that differences in lipid content between C. baicalensis and C. dybowski may reflect differences in the ways in which they adapted to pelagic life in Lake Baikal. The principal phospholipids are phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (Table 2). The content of PC in the tissues of both species varied from 60.7 to 75.1%, and that of PE from 14.5 to 25.7%. Phosphatidylinositol, phosphatidylserine, sphingomyelin and lysoPC contents were much lower (under 8%). Data obtained for phospholipid class ratios in C. baicalensis and C. dybowski are consistent with other data available for freshwater fish species (Henderson and Tocher, 1987). We have found no diphosphatidylglycerol in C. baicalensis or C. dybowski, in contrast to the Lake Baikal benthopelagic fishes C. grewingkii and C. inermis (Kozlova, 1998).

3.2. Fatty acid composition Differences in lipid between C. baicalensis and C. dybowski may affect fatty acid ratios. Total lipid fatty acid composition depends on the lipid class. Lipid-rich tissues are typically known to contain triacylglycerols as principal lipids, while tissues low in lipid may be dominated by phospholipids. Triacylglycerols account for \ 90% in C. baicalensis (Morris, 1984; Ju et al., 1997), while phospholipids make up 60% in C. dybowski (Ju et al., 1997). Therefore, for comparison between the two species, we investigated fatty acid composition in neutral and polar lipid. Table 3 shows fatty acid composition of neutral lipid in the liver, muscle, and ovaries of C. baicalensis and C. dybowski. We have identified 64–69 fatty acids, but included in the table only those exceeding 1.0%. In C. baicalensis, fatty acid group contents in neutral lipids of all tissues examined decreased in the following order: monounsaturated fatty acids (MUFA)\ saturated\ polunsaturated fatty acids (PUFA), which is characteristic for neutral lipids of most freshwater fish species (Ota, 1976; Wills and Hopkirk, 1976; Gunstone et al., 1978). This is also in concordance with the common opinion that fatty fish species accumulate depot lipids composed mainly of saturated and monoene fatty acids. C. dybowski, which have much less fat than C. baicalensis, display a converse pattern: in their muscles and ovaries, PUFA prevail, and MUFA are at the lowest level. In C. baicalensis, 18:1(n-9) is the dominant MUFA, accounting for 35.3% (of total fatty acids) in ovaries to 56.1% in muscles. It is also the dominant fatty acid among all fatty

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showed that the (n-3) PUFA level in the benthicpelagic species C. grewingki and C. inermis are unusually high for freshwater fish (51% of total fatty acids in polar lipids). According to the (n-3)/ (n-6) ratio in polar lipids, they occupy an intermediate place between freshwater and marine fishes, and, in regard to neutral lipids, they refer to typically freshwater species. There is still no consensus on whether the Lake Baikal fauna had marine or freshwater origin, so characters which discriminate between marine and freshwater organisms have a decisive value in this respect. In C. baicalensis examined here, the (n-3) fatty acid content exceeds that of the (n-6) fatty acid only in ovaries; in liver, the (n-3) content is equal to, and in muscles, it is twice as low as, the (n-6) content. The (n-3)/(n-6) ratio is 0.9 and 0.5 for liver and muscles, respectively, which is lower than the lowest published limit for freshwater fish. It is only in ovaries that the value of this index (1.7) is within the limits of the above values. In C. dybowski, the (n-3) fatty acid content was always

acids. Its very high level predetermines a high level of total MUFA reaching 68% in muscle. Next by content is 16:0, responsible for 11 – 14% (of total fatty acids) in the tissues examined. The 16:1(n-7) is in nearly the same proportion, accounting for 7–11%. This fatty acid distribution is characteristic for neutral lipids of freshwater fish (Ackman and Takeuchi, 1986). In C. dybowski, 18:1(n-9) dominates among MUFA from 10.2% (of total fatty acid) in ovaries to 18.1% in liver. Palmitate dominates among all fatty acids, from 16% in muscle and gonads to 23% in liver. Then, 16:1(n-7) is third by content; it accounts for from 8.9% in ovaries to 10.7% in liver. Among PUFA in neutral lipids of freshwater fishes, the (n-3) fatty acids generally prevail over the (n-6) fatty acid. The (n-3)/(n-6) ratio serves to discriminate between the ‘freshwater’ and ‘marine’ types of lipids: in freshwater fish, 1.08 – 3.3; in marine fish, 8.3–11.4 (Henderson and Tocher, 1987). Earlier, Kozlova and Khotimchenko (1993)

Table 3 Fatty acid composition (% total fatty acids) of neutral lipids from two species of cottoid fish from Lake Baikala Fatty acids

14:0 i-15:0 16:0 18:0 16:1(n-7) 18:1(n-9) 18:1(n-7) 16:2(n-6) 16:2(n-4) 18:2(n-6) 18:3(n-3) 18:4(n-3) 20:4(n-6) 20:5(n-3) 22:5(n-3) 22:6(n-3) Othersb Saturated Monounsaturated Polyunsaturated (n-3) (n-6) (n-3)/(n-6) a

Comephorus baicalensis

Comephorus dybowski

Liver

Muscles

Ovaries

Liver

Muscles

Ovaries

2.3 0.5 11.8 2.2 7.7 45.6 10.5 0.8 1.1 3.8 1.6 0.8 0.6 0.8 0.5 1.3 8.1 16.8 63.8 11.3 5.0 5.2 0.9

2.9 0.2 14.3 2.9 11.1 56.1 1.0 1.1 0.9 2.0 0.7 0.3 Trace 0.1 Trace 0.3 6.1 20.3 68.2 5.4 1.4 3.1 0.5

3.6 0.6 14.2 2.4 11.1 35.3 8.3 0.8 1.2 4.1 1.9 1.1 1.2 2.7 0.3 4.6 6.6 20.8 54.7 17.9 10.6 6.1 1.7

5.1 0.9 23.0 3.2 10.7 18.1 5.1 1.0 1.3 3.3 2.4 2.2 1.8 4.2 1.5 5.4 10.8 32.2 33.9 23.1 15.7 6.1 2.5

9.0 1.3 16.5 2.7 10.1 12.1 3.1 1.1 2.0 4.5 4.5 5.2 1.9 6.5 0.9 6.6 12.0 29.5 25.3 33.2 23.7 7.5 3.1

6.5 1.1 16.0 1.3 8.9 10.2 2.8 1.0 1.6 3.9 3.8 3.9 2.3 9.8 1.4 14.0 11.5 24.9 21.9 41.7 32.9 7.2 4.5

Values are for pooled samples (ten fish). Others: 12:0, 15:0, ai-15:0, i-16:0, ai-16:0, i-17:0, i-18:0, ai-18:0, 20:0, 14:1, 16:1(n-5), 17:1, 18:1(n-5), 19:1, 20:1(n-7), 20:1(n-9), 20:1(n-11), 22:1, 18:2(n-4), 18:3(n-1), 18:3(n-6), 20:2(n-6), 20:3(n-3), 20:4(n-3), 22:4(n-6), 22:4(n-3), 21:5(n-3). b

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Table 4 Fatty acid composition (% total fatty acids) of polar lipids from two species of cottoid fish from Lake Baikala Fatty acids

14:0 16:0 18:0 16:1(n-7) 18:1(n-9) 18:1(n-7) 20:1(n-9) 16:2(n-6) 18:2(n-6) 18:3(n-3) 18:4(n-3) 20:2(n-6) 20:4(n-6) 20:5(n-3) 22:4(n-3) 22:5(n-3) 22:6(n-3) Othersb Saturated Monounsaturated Polyunsaturated (n-3) (n-6) (n-3)/(n-6)

Comephorus baicalensis

Comephorus dybowski

Liver

Muscles

Ovaries

Liver

Muscles

Ovaries

0.9 9.0 1.6 6.8 42.0 12.6 0.6 0.7 3.6 2.3 0.9 0.4 1.9 2.6 0.3 1.0 4.1 8.7 11.5 62.0 17.8 11.2 6.6 1.6

1.9 15.2 6.2 6.5 19.0 7.0 0.9 0.4 2.8 1.3 0.5 0.5 9.6 8.0 1.3 0.9 13.2 4.8 23.3 33.4 38.5 25.2 13.3 1.8

2.2 15.8 5.3 6.1 19.2 8.2 1.2 0.7 3.3 1.1 0.5 0.6 7.9 8.7 1.2 1.0 8.1 8.9 23.3 34.7 33.1 20.6 12.5 1.6

2.2 17.4 4.5 10.7 15.8 6.0 0.4 1.2 2.7 2.0 1.7 0.7 4.2 6.6 1.7 2.0 11.5 8.7 24.1 32.9 34.3 25.5 8.8 2.8

2.0 14.9 6.7 3.8 9.7 4.2 0.6 0.5 3.2 1.9 0.9 1.1 8.5 11.4 2.0 1.5 20.8 6.3 23.6 18.3 51.8 38.5 13.3 2.8

2.9 16.1 4.1 5.0 8.9 4.9 0.7 0.9 2.4 1.9 1.6 0.9 5.9 12.7 2.3 1.9 17.5 9.4 23.1 19.5 48.0 37.9 10.1 3.7

a

Values are for pooled samples (10 fish). Others: 12:0, i-15:0, ai-15:0, 15:0, i-16:0, ai-16:0, i-17:0, 17:0, i-18:0, ai-18:0, 20:0, 22:0, 14:1, 16:1(n-5), 17:1, 18:1(n-5), 19:1, 20:1(n-7), 22:1, 24:1; 16:2(n-4),18:2(n-4), 18:3(n-1), 18:3(n-6), 20:3(n-3), 20:4(n-3), 21:5(n-3), 22:4(n-6). b

three to four times higher than (n-6). The (n-3)/(n6) ratio is 2.5 and 3.1 for liver and muscles, respectively, which is within the limits for freshwater fish, but the value for ovaries slightly exceeds the upper limit. Phospholipids serve as structural components in membranes and are never stored in large quantities. One of the important biological functions of phospholipids is compensation for, or balance of, changing or unusual ambient conditions. In representatives of various trophic levels, adaptation to colder living conditions and greater habitat depths is also accompanied by greater fatty acid unsaturation in phospholipids and other lipids of various tissues (Phleger et al., 1976; Sargent, 1976; Kreps, 1981; Sidorov, 1983). Both C. baicalensis and C. dybowski live at low temperatures: the upper thermal limit is 5°C for C. baicalensis and 6 – 8°C for C. dybowski (Starikov, 1977). Both species are characterised by pronounced vertical migrations.

Fatty acid composition of polar lipids in the liver, muscles and ovaries of C. baicalensis and C. dybowski are listed in Table 4. We identified 50– 70 fatty acids, but in the table, we present data only for those accounting for greater than 1.0%. Fatty acid group content decreases in polar lipids of muscles and gonads of C. baicalensis in the order PUFA \MUFA\ saturated; in liver, the order is MUFA\ PUFA\ saturated, which is determined by a very high content in the liver of the monounsaturated 18:1(n-9), making up 42%; whereas in muscles and gonads, it accounts for only half this value. In C. dybowski, PUFA dominate in all tissues, comprising 34.3% in liver to 51.8% in muscle. Saturated fatty acids are the second group by content in muscles and ovaries, followed by MUFA, but in the liver, MUFA predominate over saturates. In the polar lipids of C. baicalensis, 18:1(n-9) is the dominant fatty acid. Next follows 16:0, 9% in the liver to 15% in the muscles and gonads, 18:1(n-7) comprising 7–12%, and 22:6(n-3) com-

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prising 4–13%. In C. dybowski, 16:0 is the dominant fatty acid in the liver (17.4% of all fatty acids), but 22:6(n-3) is dominant in the muscles and ovaries (20.8 and 17.5%, respectively). Polar lipids in most freshwater fish contain higher PUFA levels and lower MUFA levels, compared with neutral lipids, but approximately similar levels of saturated fatty acids (Ota and Takagi, 1977; Gunstone et al., 1978; Ando et al., 1985; Ackman and Takeuchi, 1986). In C. baicalensis, the contents of saturated fatty acids were in approximately equal proportions between polar and neutral lipids of all tissues examined, while PUFA content was much higher in polar lipids of muscles and ovaries, and MUFA content lower than in neutral lipids. Liver contained approximately equal proportions of PUFA and MUFA in both polar and neutral lipids. In C. dybowski, PUFA content was much higher in polar lipids of all tissues examined than in neutral lipids, whereas the contents of saturates and MUFA were in approximately equal proportions. Generally, PUFA have longer chains in polar than in neutral lipids, and the C20+ C22 PUFA to C18 PUFA ratio is 4.3 – 9.8 greater in polar than in neutral lipids (Ota and Takagi, 1977; Gunstone et al., 1978; Ando et al., 1985; Ackman and Takeuchi, 1986). In the C. baicalensis and C. dybowski we studied, the C20 +C22 PUFA to C18 PUFA ratio was two to six times greater in polar than in neutral lipids of all tissues examined, except muscles in C. baicalensis, for which this ratio was 21 times greater. Of C18 PUFA, 18:2(n-6) was higher than 18:3(n-3) in all tissues of both fish species. The PUFA, icosapentaenoic acid 20:5(n-3) prevails over 20:4(n-6) in both fish species, except for C. baicalensis muscles, where it is somewhat lower. Other C20 PUFA, such as 20:2(n-6), 20:3(n-3), and 20:4(n-3), account for no more than 1.1% each in both species. The (n-3)/(n-6) PUFA ratio is 1.6 – 2.0 for polar lipids of freshwater fish and 7.8 – 18.5 for marine fish (Henderson and Tocher, 1987). The (n-3)/(n6) PUFA ratio for polar lipids in C. baicalensis tissues is 1.6–1.8, being within the range typical of freshwater fish. In C. dybowski, this ratio equals 2.8–3.7, i.e. higher than values for freshwater fish, but lower than those for marine fish. Liver is the principal organ for biosynthesis in animals; in some fish, it serves as a lipid depot. Liver differs significantly from muscles and

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ovaries of both species in fatty acid content. These differences are especially pronounced in polar lipids of C. baicalensis liver where 18:1(n-9) is two times greater, while palmitate content is nearly two times lower than in muscle and ovaries. Liver contains considerably less 20:4(n-6), 20:5(n-3), and 22:6(n-3) compared with muscles and ovaries. C. dybowski exhibits the same pattern, except for 16:0, which is roughly equal in all tissues. Morris (1984) studied the lipid and fatty acids of adult C. baicalensis and juvenile C. dybowski. Among phospholipids, three fatty acids dominate in C. baicalensis: 16:0 (21–23%), 18:1 (19–20%), and 22:6 (n-3) (14–22%). In C. dybowski, 22:6 (n-3) was the dominant fatty acid (34%). Among triacylglycerols, 18:1 (29–46%) dominated in C. baicalensis, while 16:0 (20%) dominated in C. dybowski. We have found a higher level of 18:1(n-9) (from 42% in polar lipids of the liver to 56% in neutral lipids of muscles) in C. baicalensis than Morris (1984), which determined different fatty acid group ratios for this species. In C. dybowski, fatty acid group ratios were generally similar to those reported by Morris (1984). The discrepancies encountered are most likely attributable to different age and maturity stage of fish examined by both us and Morris (1984). We compared fatty acid composition in C. baicalensis and C. dybowski to results of earlier investigations of the benthic-pelagic C. grewingkii and C. inermis (Kozlova and Khotimchenko, 1993). It was shown (Kozlova and Khotimchenko, 1993) that despite considerable differences in their diets, fatty acid contents in the organs and tissues (liver, muscle, gonads) of both the latter species are very similar. C. baicalensis is close to C. inermis in ecology and dietary spectrum (their diets are based on M. branickii and young cottoid fish), but they differ greatly in dominant fatty acid contents. C. dybowski is much closer to C. grewingkii and C. inermis in fatty acid composition, except for minor differences. Polar lipids of C. grewingkii and C. inermis are dominated by 22:6(n-3) (up to 38.9% in red muscle); 18:1 prevails in neutral lipids (up to 21.9% in the liver of C. inermis). In C. dybowski, 16:0 prevails in polar lipids of the liver and neutral lipids of all tissues examined. C. dybowski have a lipid metabolism strategy oriented towards dominance of the (n-3) PUFA, similarly to C. grewingkii and C. inermis. C.

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baicalensis do not follow this pattern: the (n-6) fatty acids prevailed in neutral lipids in many cases. We conclude that C. baicalensis differs considerably from C. dybowski not only in total lipid content of whole body and tissues, but also in the content of dominant fatty acids and in the fatty acids group ratios, which is mainly determined by a very high content of 18:1(n-9) in polar, and especially neutral, lipids. We attribute these differences to the different ways of the specialisations of these species to life in pelagic waters.

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