ISSN 0022-0930, Journal of Evolutionary Biochemistry and Physiology, 2018, Vol. 54, No. 2, pp. 109—118. © Pleiades Publishing, Ltd., 2018. Original Russian Text © E.Ya. Kostetsky, P.A. Velanskii, N. M. Sanina, 2018, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2018, Vol. 54, No. 2, pp. 96—104.

COMPARATIVE AND ONTOGENIC BIOCHEMISTRY

Phospholipid and Fatty Acid Composition of Phosphatidylcholine and Phosphatidylethanolamine in the Black Plaice Pleuronectes obscura during Thermoadaptation E. Ya. Kostetskya*, P. A. Velanskiia, and N. M. Saninaa a Far Eastern Federal University, Vladivostok, Russia

*e-mail: [email protected] Received May 30, 2017

Abstract—The phospholipid (PL) and fatty acid (FA) composition of major membrane lipid constituents, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), as well as the cholesterol/phospholipid (CL/PL) ratio were assayed in the muscles, gills and liver of the black plaice Pleuronectes (Liopsetta) obscura at different ambient temperatures (18, 9 and 0°C). PL and CL were shown to be actively involved in adaptation of the fish to changes in the seawater temperature. As temperature declines, the monounsaturated FA (MUFA) level increases while the polyunsaturated FA (PUFA) fraction in gills and liver PC and PE, on the contrary, decreases, resulting in diminished functional activity of the fish. However, in muscles this correlation is lacking. The PC and PE composition was shown to be organ- and ambient temperature-dependent. Major PC forms are saturated FA (SFA)/PUFA and MUFA/PUFA composed of a relatively small number of major molecular species. A temperature drop results in an increased SFA/PUFA level and decreased MUFA/PUFA and PUFA/PUFA levels in muscles and gills, and this may promote a drop in the viscosity of the outer lipid monolayer of membranes and in their functional activity. In contrast to PC, the PE composition in all organs tested is characterized by a decrease in the SFA/ PUFA level and an increase in MUFA/PUFA and PUFA/PUFA levels. Such changes promote the retention of functional activity of the inner lipid monolayer of membranes and are not synchronized with rearrangements in their outer monolayer. Due to intermolecular transfer of acyl radicals at a constancy of their composition, functional rearrangement of the lipid matrix appears to be achieved through changes in the membrane viscosity. Our data support the idea that different adaptation strategies in fish are driven by certain sets of PL molecular species. DOI: 10.1134/S0022093018020035 Key words: phospholipids, fatty acids, phosphatidylcholine and phosphatidylethanolamine molecular forms and species, marine fish, thermoadaptation.

INTRODUCTION Temperature is one of the major environmental factors that affect membranes in poikilothermic organisms including fish. To function properly, these animals have to maintain the functionally

active liquid-crystalline state of cell membranes as achieved due to compensatory rearrangements in the composition of acyl radicals [1] and molecular species of phospholipids (PL) [2], in the ratio of diacyl and ether PL species [3], size, hydrophobicity and charge of polar groups in membrane PL

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[4, 5], in the cholesterol (CL) composition [6]. Global changes in temperature regime of the fish habitat as well as aquaculture and fishery problems require deeper understanding of fish behavior during thermoadaptation. In the recent years, there has appeared a large body of studies on the role of the lipid matrix in poikilotherms (including fish) during their thermoadaptation [7–14]. Thus, as exemplified by 9 cold-water fish species from 6 families, it has been shown that in muscles, gills and liver the repertoire of major membrane PL as well as CL and PL fatty acids (FA) varies considerably in the tested organisms [9], suggesting different ways of adaptation to low temperatures in fish. A study of thermoadaptation in the Far Eastern redfin Tribolodon brandti to various temperatures under natural and experimental conditions has demonstrated that lowering of temperature is accompanied by an increase in the polyunsaturated FA (PUFA) level and unsaturation index (USI) as well as a decrease in the saturated FA (SFA) level in phosphatidylcholine (PC) and especially in phosphatidylethanolamine (PE) [10]. A study of phase transitions in major PL of 6 fish species from 4 families, caught in the Peter the Great Bay (Sea of Japan) and living at low temperatures (0–4°C), has shown that each fish species has its own adaptation strategy realized through a certain set of molecular species of FA contained in PC and PE [11]. The aim of this study was to analyze the molecular mechanisms of adaptation in one of the most common commercial fish species in the Peter the Great Bay (Sea of Japan), the black plaice Pleuronectes obscura (Pleuronectidae), to changes in thermal regime of their natural habitat. In the focus of the study there were the PL composition and the CL/PL ratio as well as the composition of FA, molecular species and forms of major polar lipids (PC and PE) in cell membranes of muscles, gills and liver. MATERIAL AND METHODS The object of this study was the black plaice Pleuronectes (Liopsetta) obscura Herzenstein, 1891 (Pleuronectiformes, Pleuronectidae). The fish was caught: in March—at a depth of 3 m and 0°C in the East Bay, in June—at a depth of 4 m and 9°C in the

Kievka Bay, and in August—at a depth of 4 m and 18°C in the Kievka Bay (all in the Sea of Japan). Muscle, liver and gill tissues were sampled from 3–5 fish, with individual tissues pooled separately. Samples were minced immediately upon capture and fixed in a chloroform–methanol mixture (2:1, v/v). Lipid extract was obtained using the Folch’s method [12]. PL and CL were assayed as described elsewhere [9]. FA were analyzed as methyl ethers on a gas chromatograph Agilent 6890GC equipped with a flame ionization detector as instructed previously [11]. FA were identified using the carbon number method. FA composition was computed for three chromatograms. Analytical separation of PC and PE molecular species was carried out by high-performance liquid chromatography on a chromatograph Shimadzu LC20 with a mass detector LCMS-2010EV [12]. Levels of PL molecular species were determined on chromatograms by squares of [M+H]+ quasi-molecular ions peaks corresponding to each individual molecular species. Each sample was analyzed three times. Statistical treatment was carried out using a Microsoft Excel program. Results were presented as – – x ± s, где x—arithmetic mean and s—standard deviation. RESULTS AND DISCUSSION Phospholipid composition and cholesterol/phospholipid ratio in organs of the black plaice during thermoadaptation. The black plaice is a littoral marine species that dwells all the year round at small depths and perform no seasonal migrations. The fish tolerates wide thermal and salinity gradients and does not avoid desalinated water. In summer, the black plaice holds to depths from 3 to 15 m with a ground-water temperature of 10–18°C and a salinity around 32 ‰. In fall, the fish moves away toward deeper water (40–60 m), where it overwinters at temperatures from –1.7 to +1.7°С. Most common ingredients of its diet are polychaetes, bivalves, crustaceans and other benthic animals. Depending on the temperature of the natural habitat, the black plaice’s lipid spectrum displays certain specific patterns characteristic of all its organs (Table 1). As temperature declines from 18°C to 0°C, the muscle PC level falls 1.3 times,

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Table 1. Composition of phospholipids (PL) and cholesterol (CL)/PL ratio in muscles, liver and gills of the black plaice under different environmental temperatures* PL and CL CL/PL

Muscles

Liver

Gills

18°С

9°С

0°С

18°С

9°С

0°С

18°С

9°С

0°С

0.17 ± 0.03

0.12 ± 0.02

0.13 ± 0.02

0.34 ± 0.02

0.62 ± 0.09

0.24 ± 0.04

0.38 ± 0.04

0.48 ± 0.02

0.71 ± 0.03

PC

70.7 ± 0.9 65.5 ± 0.6 55.9 ± 1.1 59.3 ± 0.6 62.3 ± 2.4 60.8 ± 0.9 45.5 ± 0.2 47.7 ± 2.8 43.9 ± 1.4

PE

17.7 ± 1.1 20.5 ± 0.7 28.2 ± 0.6 20.3 ± 0.7 24.4 ± 1.2 26.3 ± 1.0 26.5 ± 0.2 27.9 ± 0.4 27.4 ± 0.8

SM

3.3 ± 0.1

PS

3.0 ± 0.2

3.2 ± 0.2

7.4 ± 0.2

5.6 ± 0.2

5.2 ± 0.1 15.4 ± 0.2 11.9 ± 0.1 15.6 ± 0.1

2.1 ± 0.3 2.5 ± 0.3

3.0 ± 0.4 4.9 ± 0.3

1.0 ± 0.1

2.1 ± 0.1

6.5 ± 0.0

5.8 ± 0.1

4.8 ± 0.1

PI

5.0 ± 0.5

7.0 ± 0.2

7.2 ± 0.5 6.0 ± 0.6

4.0 ± 0.2

4.3 ± 0.0

4.3 ± 0.2

4.8 ± 0.2

4.1 ± 0.3

DPG

0.6 ± 0.2

0.9 ± 0.1

1.8 ± 0.2

1.8 ± 0.2

2.2 ± 0.2

0.8 ± 0.1

1.2 ± 0.1

0.7 ± 0.1

0.8 ± 0.0

LPC

0.1 ± 0.0

0.2 ± 0.0

0.3 ± 0.0

0.3 ± 0.0

0.2 ± 0.0

0.1 ± 0.0

0.4 ± 0.0

0.3 ± 0.0

0.7 ± 0.0

PA

0.4 ± 0.1

0.4 ± 0.1

0.4 ± 0.1

0.1 ± 0.0 0.3 ± 0.1

0.4 ± 0.1

0.2 ± 0.0

1.0 ± 0.0

2.7 ± 0.1

PE/PC

0.25 ± 0.04

0.31 ± 0.03

0.50 ± 0.04

0.43 ± 0.04

0.58 ± 0.02

0.58 ± 0.06

0.62 ± 0.05

0.34 ± 0.03

0.39 ± 0.07

– ± s, n = 3–7). * Individual PL levels are expressed in mol % of total PL; CL/PL and PE/PC ratios—in mol/mol, (x

whereas in the liver and gills it remains practically intact. Muscle and liver PE levels increase 1.6 and 1.3 times, respectively, but in the gills they do not change. The PE/PC ratio increases steadily in muscles and the liver but quite insignificantly in the gills. Lowering the environmental temperature decreases the CL/PL ratio in muscles and the liver (1.3–1.4 times) and increases it in the gills (1.9 times). The sphingomyelin (SM) level does not change at lowered temperatures in the gills and muscles but falls 1.4 times in the liver. Interestingly, in the liver as well as in muscles the PC level is intact, however, the SM level falls, i.e. the total choline-containing PL content decreases (Table 1). All the above-mentioned changes in the membrane lipid composition induced by lowering the ambient temperature indicate the existence of some mechanisms aimed at maintaining a functionally active liquid-crystalline status of the membrane lipid matrix due to changes in CL/ PL and PE/PC ratios. If adaptation processes in muscles are accompanied by changes in the PE/ PC ratio, in the gills they involve the CL/PL ratio while in the liver—changes in the SM content along with the CL/PL ratio. These are well-known factors that regulate the physical condition of cell membranes [4–6].

Phosphatidylserine (PS), phosphatidylinositol (PI), diphosphatidylglycerol (DPG) and phosphatidic acid (PA) total in each organ from 7 to 12% depending on the environmental temperature. As the latter goes down, PS, PI and DPG levels rise in muscles and fall in the liver and gills, while the PA level remains intact in muscles but rises in the liver and gills. The DPG level in muscles increases 3 times and decreases in the liver and gills 2.2 and 1.5 times, respectively. Cold adaptation in fish is known to be accompanied by a compensatory adaptation of aerobic processes, leading to a mitochondrial multiplication and an increased activity of mitochondrial enzymes and DPG level [15– 17]. Increased DPG levels in black plaice muscles under the lowered temperature indicates a possible compensatory adaptation of cell respiration. A drop in the DPG concentration in the liver and gills of the black plaice can be accounted for by a declined physiological activity in these organs. A comparison of the results obtained for the black plaice with the analogous data on the Far Eastern redfin [10] implies somewhat dissimilar ways of adaptation to lowered environmental temperature in organs of these fish. Thus, in the redfin, SM, DPG and PS levels decrease in muscles, PC levels—in the liver, while PC, PI and PA levels—in the gills, but in the latter case, in contrast

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Table 2. Fatty acid (FA) composition of phosphatidylcholine in muscles, liver and gills of the black plaice under different environmental temperatures* Muscles

FA

18°С

16:0

9°С

Liver

0°С

18°С

9°С

Gills

0°С

18°С

9°С

0°С

28.8 ± 0.6 27.7 ± 2.1 28.7 ± 2.0 21.9 ± 0.7 24.1 ± 1.4 30.1 ± 1.6 18.1 ± 1.1 16.8 ± 1.4 21.0 ± 0.8

18:0

3.2 ± 0.1

2.5 ± 0.1

4.2 ± 0.4

2.7 ± 0.1

3.1 ± 0.2

7.0 ± 0.3

16:1ω9

0.9 ± 0.0

0.5 ± 0.0

1.7 ± 0.1

1.4 ± 0.1

0.5 ± 0.0

3.0 ± 0.2 1.9 ± 0.1

1.6 ± 0.1

3.7 ± 0.4

16:1ω7

2.2 ± 0.0

2.4 ± 0.3

1.4 ± 0.1

1.6 ± 0.1

2.6 ± 0.1

2.3 ± 0.2 2.2 ± 0.1

2.7 ± 0.2

4.1 ± 0.5

18:1ω9

5.0 ± 0.4

3.0 ± 0.3

3.6 ± 0.1

4.5 ± 0.4

4.5 ± 0.4 3.7 ± 0.3

9.8 ± 1.0

8.3 ± 0.9

8.0 ± 0.9

18:1ω7

3.7 ± 0.4

2.6 ± 0.2

2.0 ± 0.1

2.4 ± 0.1

1.7 ± 0.1 2.1 ± 0.0

3.5 ± 0.3

3.6 ± 0.1

2.6 ± 0.3

20:1ω7

0.3 ± 0.0

0.6 ± 0.0

0.1 ± 0.0

0.2 ± 0.0

0.7 ± 0.1 0.4 ± 0.0

0.7 ± 0.1

1.1 ± 0.1

0.5 ± 0.1

18:2ω6

1.3 ± 0.1

0.8 ± 0.1

0.3 ± 0.0

1.0 ± 0.1

0.9 ± 0.0 0.8 ± 0.1

1.1 ± 0.0

0.7 ± 0.0

0.1 ± 0.0

20:4ω6

4.4 ± 0.3

5.3 ± 0.3

5.7 ± 0.2

7.4 ± 0.5

4.9 ± 0.5

3.7 ± 0.1 12.4 ± 1.0 10.7 ± 0.3

6.1 ± 0.4

20:5ω3

7.4 ± 0.3

6.6 ± 0.4 11.6 ± 0.6

24.7 ± 1.5 30.0 ± 2.0 24.3 ± 0.6 23.8 ± 0.8 33.1 ± 2.1 15.0 ± 1.4 17.8 ± 0.8 23.0 ± 1.9 10.6 ± 0.6

22:5ω3

4.9 ± 0.5

4.0 ± 0.2

3.2 ± 0.4

3.8 ± 0.2

3.3 ± 0.2

2.6 ± 0.2

2.9 ± 0.1

3.9 ± 0.1 2.6 ± 0.3

22:6ω3

8.7 ± 0.6 10.6 ± 0.9 16.5 ± 0.8 16.1 ± 0.8 10.1 ± 0.9 14.7 ± 0.8

8.2 ± 0.6

7.8 ± 0.2 12.5 ± 1.1

SFA

35.4 ± 0.3 34.4 ± 2.3 35.9 ± 0.8 30.1 ± 0.8 30.6 ± 1.4 45.4 ± 1.9 31.6 ± 1.2 27.7 ± 2.2 42.2 ± 1.7

MUFA

15.8 ± 0.6 12.2 ± 0.9 10.5 ± 0.3 12.2 ± 0.9 12.4 ± 0.3 13.8 ± 0.7 21.4 ± 0.8 20.8 ± 1.7 21.7 ± 1.5

PUFA

48.8 ± 1.2 53.4 ± 1.6 53.6 ± 1.8 57.6 ± 1.3 57.0 ± 3.0 40.8 ± 1.3 47.0 ± 0.8 51.5 ± 3.1 36.0 ± 0.7

ω6 PUFA

7.6 ± 0.6

7.9 ± 0.4

7.6 ± 0.4 10.3 ± 1.1

7.8 ± 0.3

6.0 ± 0.2 15.8 ± 1.2 13.9 ± 0.6

7.9 ± 0.8

ω3 PUFA 40.5 ± 1.9 46.1 ± 2.7 44.2 ± 1.8 46.0 ± 1.0 47.8 ± 1.7 32.6 ± 1.8 30.3 ± 1.0 35.9 ± 1.6 26.3 ± 2.2 ω3/ω6

5.3

5.8

5.8

4.5

6.1

5.4

1.9

2.6

3.3

USI

251

293

296

298

297

238

237

254

208

Here and in Table 3: * FA levels expressed in % of total FA as – x ± s, n = 3–7. FA with levels in all samples below 1% are not shown but considered while computing cumulative parameters; SFA, MUFA and PUFA—saturated, monounsaturated and polyunsaturated FA, respectively.

to the black plaice, there occurs an increase in PE and SM levels. This indicates the existence of the specific ways of adaptation to natural temperature changes in different fish species as well as the active involvement of polar membrane lipids in these processes. Fatty acid composition of phosphatidylcholine and phosphatidylethanolamine in organs of the black plaice during thermoadaptation. In muscle, liver and gill PC, dominant FA are 16:0 and 20:5ω3. Much fewer are 22:6ω3, 20:4ω6, 18:1ω9, 18:0 and 18:1ω7 (arranged in descending order of their concentrations) (Table 2). In the black plaice, lowering the environmental temperature from 18 to 0°С is accompanied by the following changes in parameters of FA in PC: total SFA in the liver and gills increase approximately

by 15 and 10%, respectively; that of MUFA remains intact while that of PUFA decreases by 17 and 11%, respectively. Simultaneously, the ω3 PUFA level falls in these organs, especially in the liver (by about 14%), and USI falls from 298 to 238 in the liver and from 237 to 208 in the gills. In organs of the black plaice, among FA of PE, two PUFA are dominant—22:6ω3 and 20:5ω3, reaching totally 40% (Table 3). Then follow (in descending order) 16:0, 18:0, 18:1ω9, 20:4ω6, 18:1ω7, 22:5 ω3. The FA composition of PC and PE is little different from that in organs of the Far Eastern redfin [10]; nevertheless, the available quantitative differences entail appreciable changes in total parameters of the lipid composition, especially their molecular species and molecular forms, as will be reported below.

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Table 3. Fatty acid (FA) composition of phosphatidylethanolamine in muscles, liver and gills of the black plaice under different environmental temperatures FA

Muscles 18°С

9°С

Liver 0°С

18°С

9°С

Gills 0°С

18°С

9°С

0°С

16:0

8.7 ± 0.4 10.2 ± 0.2 9.5 ± 0.6 10.6 ± 0.5 13.0 ± 0.7 20.1 ± 0.8 6.8 ± 0.4

18:0

6.2 ± 0.5

5.7 ± 0.3 5.0 ± 0.5 4.5 ± 0.1

7.6 ± 0.4 6.8 ± 0.3

16:1ω9

0.4 ± 0.1

0.3 ± 0.0 0.6 ± 0.1 0.4 ± 0.1

0.3 ± 0.0 1.7 ± 0.1 0.6 ± 0.0

0.8 ± 0.0 0.9 ± 0.1

16:1ω7

1.1 ± 0.2

2.0 ± 0.1 1.5 ± 0.1 1.3 ± 0.1

5.0 ± 0.2 1.7 ± 0.2

1.7 ± 0.1

1.8 ± 0.1

18:1ω9

5.4 ± 0.3

5.6 ± 0.3

8.4 ± 0.7 7.3 ± 0.7

8.5 ± 1.2 5.8 ± 0.2

6.6 ± 0.5

6.7 ± 0.7 11.4 ± 1.0

18:1ω7

6.0 ± 0.6

7.2 ± 0.9

7.3 ± 0.8 5.2 ± 0.9

5.7 ± 0.3 6.2 ± 0.3

4.0 ± 0.4

5.9 ± 0.2

20:1ω9

1.3 ± 0.2

1.5 ± 0.1

1.2 ± 0.1 2.0 ± 0.3

3.4 ± 0.1 1.3 ± 0.1

2.5 ± 0.2

4.7 ± 0.6 2.8 ± 0.1

20:1ω7

1.0 ± 0.3

2.1 ± 0.1

1.0 ± 0.0 1.1 ± 0.1

3.4 ± 0.2 1.4 ± 0.1

0.9 ± 0.0

1.9 ± 0.2 1.0 ± 0.1

18:2ω6

1.2 ± 0.2

1.1 ± 0.1

1.1 ± 0.0 1.1 ± 0.1

0.8 ± 0.0 1.4 ± 0.1

1.5 ± 0.2

0.9 ± 0.1 1.5 ± 0.1

20:4ω6

4.4 ± 0.2

5.3 ± 0.3

5.5 ± 0.1 7.8 ± 0.1

3.9 ± 0.3

20:5ω3 22:5ω3

5.2 ± 0.2 14.2 ± 0.3

9.8 ± 0.3 10.1 ± 0.8

9.3 ± 0.4 2.5 ± 0.1 6.5 ± 0.5

3.0 ± 0.3 14.0 ± 0.3 13.8 ± 0.3 6.9 ± 0.2

18.8 ± 0.4 19.1 ± 0.4 16.8 ± 0.4 15.9 ± 0.3 14.7 ± 0.4 10.7 ± 0.3 15.0 ± 0.8 15.3 ± 0.8 12.6 ± 0.3 8.6 ± 0.2

6.3 ± 0.4

5.0 ± 0.3

4.5 ± 0.2

4.2 ± 0.3

2.7 ± 0.1

6.7 ± 0.4

6.6 ± 0.3

2.9 ± 0.2

22:6ω3

26.9 ± 0.5 26.4 ± 0.9 29.1 ± 1.0 29.7 ± 0.4 18.2 ± 0.3 21.8 ± 1.0 20.6 ± 0.4 18.4 ± 0.4 18.3 ± 0.6

SFA

18.2 ± 1.0 18.8 ± 1.1 18.0 ± 0.9 20.4 ± 0.7 26.4 ± 1.2 36.4 ± 1.1 22.3 ± 1.1 19.6 ± 0.6 29.0 ± 0.8

MUFA

21.0 ± 1.6 21.8 ± 1.0 22.2 ± 1.3 18.5 ± 1.1 31.0 ± 1.2 21.9 ± 0.8 17.9 ± 2.0 24.2 ± 1.4 27.2 ± 1.8

PUFA

60.8 ± 1.6 59.4 ± 2.2 59.8 ± 1.8 61.1 ± 1.5 42.6 ± 4.2 41.6 ± 2.0 59.8 ± 2.0 56.2 ± 1.8 43.8 ± 1.4

ω6 PUFA

8.9 ± 0.3

9.3 ± 0.7

7.6 ± 0.8 10.8 ± 0.3

7.2 ± 0.6

5.6 ± 0.1 18.4 ± 1.8 17.3 ± 1.8

9.7 ± 0.2

ω3 PUFA 53.1 ± 2.1 48.1 ± 3.9 51.4 ± 2.0 48.5 ± 2.6 27.4 ± 3.2 33.6 ± 0.7 39.4 ± 1.7 36.7 ± 2.5 31.1 ± 3.3 ω3/ω6

6.0

5.2

5.1

4.5

3.8

6.0

2.1

2.1

3.2

USI

358

329

326

335

247

250

314

298

255

The content of dominant FA (22:6ω3 and 20:5ω3) is particularly high in muscle PE (up to 29 and 19%, respectively). With a decrease in the environmental temperature, there are similar tendencies in the dynamics of FA composition both of PE and PC in the liver and gills. Thus, in the liver and gills, total SFA rise by 16 and 7% while that of PUFA falls by 20 and 16%, respectively; however, total MUFA, in contrast to that in PC, rise approximately by 3 and 9%, respectively. Simultaneously, the ω3 PUFA level falls in these organs, especially in the liver, by about 15%, and the USI decreases from 335 to 250 in the liver and from 314 to 255 in the gills. Similarity of these rearrangements in the FA composition of PC and PE, located mainly in different membrane monolayers, may lead to symbate alterations in their physical condition and to a reduced functional activity of

the fish in the winter period, as supported by their restricted mobility which is close to anabiosis. At the same time, total SFA in PC remain unchanged while that of MUFA falls. Total PUFA rise insignificantly (by about 5%), and this rise also applies to total ω3 PUFA (by about 4%) and USI (from 251 to 296). The FA composition of muscle PE shows no statistically significant changes. The lack of symbate membrane-associated physicochemical processes in the outer and inner membrane monolayer in black plaice muscle cells may account for a decline in physiological activity during the winter season. Previously, we carried out a study of the Far Eastern redfin Tribolodon brandti in the similar manner [10]. There were revealed synchronous changes in SFA, MUFA and PUFA composition

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of PC and PE, and this is well consistent with a high physiological activity of the redfin in winter. Similar changes in the FA composition of PC and PE can also be observed in other fish species [18, 19]. Composition of molecular forms of phosphatidylcholine and phosphatidylethanolamine in organs of the black plaice during thermoadaptation. It was established that a major mechanism for maintaining membrane viscosity under lowered temperature is a buildup of molecular forms of PL containing MUFA at the sn-1 and PUFA at the sn-2 positions. This phenomenon is typical both for fish and marine invertebrates [12, 20–22]. However, in different fish species this mechanism can be implemented via different ways. Five basic FA of PC (16:0, 20:5ω3, 22:6ω3, 20:4ω6, 18:1ω9) in muscles, liver and gills of the black plaice make up the following major molecular species: 16:0/20:5, 16:0/22:6, 16:0/20:4, 16:0/18:1, 16:0/16:1, 18:1/20:5, 20:5/20:5 (in descending order of their concentrations) (Table 4). A dominant molecular form of diacyl-PC is SFA/PUFA (Table 4). Then, in descending order of their concentrations, follow MUFA/PUFA, SFA/MUFA, PUFA/PUFA, and SFA/SFA. In the composition of molecular forms, SFA/PUFA and MUFA/PUFA are presented mainly by PUFA 20:5, 22:6 and 20:4. The role of PC in adaptation to varied environmental temperatures can be assessed by changes in levels of individual molecular species or forms of this PL. Under lowering the temperature from 18 to 0°C, the fraction of SFA/PUFA rises in muscles and gills by 18 and 7.4%, respectively, and accordingly falls by 4.5% in the liver, while changing predominantly due to SFA/20:5, SFA/22:6 and SFA/20:4. The MUFA/PUFA level falls in muscles by 5% and rises in the liver and gills by 6–7%, while changing mainly due to MUFA/20:5 and MUFA/22:6. The PUFA/PUFA level in muscles and gills falls by 10 and 2.4%, respectively, but rises in the liver by 3%. The small concentration of MUFA/MUFA and SFA/SFA changes insignificantly and ambiguously in transition from one season to the other. In PC, alkylacyl forms dominate over alkenylacyl ones, with the latter reaching their maximum level in PC of the gills (20.7– 23.6%). Their level in PC of muscles and liver falls

by 2.5–3.7%, while rising in the gills by 2.9% under lowering the seawater temperature. It is necessary to point out that replacement of a common ether bond for alkyl or alkenyl bonds in PL molecules does not affect their phase condition [23]. In the formation of molecular species and their forms in PE of black plaice muscles, liver and gills, such FA as 22:6ω3, 20:5ω3, 16:0, 18:0, 18:1ω9, 20:4ω6, 18:1ω7, and 22:5 ω3 are involved most actively (Table 5). The set of PE molecular species and forms differs materially from those of PC, although the set of basic FA is nearly the same (Tables 2, 3). Major PE molecular species can be arranged in the following succession: 18:1/22:6, 18:1/20:5, 16:0/22:6 (from 11 to 7%), the rest are 16:0/20:5, 18:0/20:5, 18:0/22:6, 20:5/22:6, 20:1/22:6, 18:0/20:4, 18:1/22:5 (from 2 to 5%), in contrast to PC where only 3–4 molecular species are pronounced distinctly. This, however, does not prevent the formation of the three large PE molecular forms: MUFA/PUFA (29.8–68.8%), SFA/ PUFA (22.7–47.8%) and PUFA/PUFA (2.9– 23.2%), diacyl-PE (54.2–96.3%) and alkenyl-PE (1.7–42.3%, depending on the organ and environmental temperature). With lowering the temperature from 18 to 0°C, the level of MUFA/PUFA molecular forms appreciably rises in PE of all organs mainly due to the 18:1/20:5 and 18:1/22:6 molecular species. The MUFA/PUFA level in PE increases in the gills from 44.0 to 67.8%, in the liver—from 41.8 to 55.5%, while in muscles this parameter changes to the least extent (from 29.8 to 38.3%). An opposite tendency characterizes the SFA/PUFA level in PE of all organs, as occurs mainly due to a fall in levels of форм SFA/22:6, SFA/22:5 and SFA/20:5 molecular forms. The SFA/PUFA level in PE decreases in the gills from 46.9 to 22.7%, in the liver—from 47.8 to 34.3%, and in muscles— from 47.4 to 34.9%. Consequently, MUFA/ PUFA and SFA/PUFA molecular forms in PE are mutually antagonistic. Such a tendency in changing key molecular forms of PL in the black plaice indicates their importance for thermal adaptation. With lowering the environmental temperature, also observed is an insignificant rise in the PUFA/ PUFA level in PE of muscles and gills (by 5 and 1%, respectively). The contribution of the mi-

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Table 4. Composition of phosphatidylcholine molecular species in muscles, liver and gills of the black plaice under different environmental temperatures* Muscles Liver Gills Mol. species 18°С 9°С 0°С 18°С 9°С 0°С 18°С 9°С 0°С 16:0/16:0 0.3 0.1 0.2 0.2 0.1 0.1 1.4 0.6 0.2 16:0/16:1 2.5 1.7 2.4 1.4 4.8 2.2 4.5 4.0 4.6 16:0/17:1 0.2 0.1 0.3 0.5 0.3 0.4 0.7 0.3 1.0 16:0/18:1 4.6 2.4 3.6 6.9 6.1 2.5 10.8 10.5 7.6 16:0/18:2 1.7 0.9 0.3 1.2 1.2 0.2 1.2 0.8 0.2 16:0/20:4 5.2 6.2 7.4 4.4 4.2 4.3 3.9 4.6 4.3 18:0/20:4 0.5 0.2 0.1 0.2 0.1 0.2 1.3 2.4 1.3 16:0/20:5 17.5 29.3 33.3 20.2 27.7 20.0 8.2 11.1 11.9 18:0/20:5 2.4 2.1 1.3 1.1 2.1 1.1 2.6 4.1 2.2 16:0/22:5 6.6 5.1 5.1 3.4 2.6 3.9 2.2 2.4 2.9 14:0/22:6 0.9 0.8 1.6 2.3 0.4 2.4 0.6 0.2 1.1 16:0/22:6 8.9 10.6 16.9 14.6 5.2 14.7 3.3 2.7 7.6 18:0/22:6 0.4 0.4 0.2 0.3 0.1 0.4 0.5 0.6 0.7 17:0i/22:6 0.2 0.2 0.1 1.0 0.0 0.4 0.1 0.0 0.1 16:1/18:1 0.5 0.4 0.2 0.3 0.3 0.4 1.0 0.9 1.4 18:1/18:1 0.3 0.1 0.1 0.2 0.1 0.2 0.9 0.8 0.7 18:1/20:4 0.5 0.1 0.1 0.3 0.2 0.4 0.7 0.7 0.4 16:1/20:5 2.2 2.7 1.4 1.4 1.3 3.0 1.1 1.2 1.4 18:1/20:5 6.1 4.8 3.3 2.7 2.0 4.0 3.2 3.8 3.6 18:1/22:5 0.7 0.4 0.3 0.1 0.1 0.4 0.4 0.4 0.5 16:1/22:6 0.7 0.7 1.0 1.3 0.2 2.4 0.4 0.2 1.1 17:1/22:6 0.2 0.2 0.7 1.0 0.0 0.6 0.1 0.0 0.5 18:1/22:6 1.6 1.5 2.0 1.1 0.2 2.6 1.1 0.8 2.2 20:5/20:5 4.1 4.6 0.9 0.7 1.4 0.4 1.3 1.8 0.6 20:5/22:6 2.6 2.0 1.0 0.9 0.5 3.8 1.2 1.1 1.0 16:0a/16:1 0.2 0.1 0.3 0.0 0.1 0.1 0.8 0.5 0.6 16:0a/20:4 0.4 0.2 0.1 0.5 1.6 0.2 2.1 2.4 1.8 16:0a/20:5 1.6 1.1 0.7 2.4 10.9 1.0 4.3 5.9 3.6 16:0a/22:6 0.9 0.5 0.7 2.3 1.5 0.7 2.4 2.4 4.0 18:1a/20:5 0.4 0.1 0.2 0.6 1.6 0.8 2.4 2.7 3.5 18:1a/22:6 0.1 0.0 0.2 0.5 0.3 1.2 0.8 0.8 3.1 Diacyl-PC 89.0 91.7 92.2 80.1 69.0 87.5 70.3 69.0 68.9 Alkenyl-PC 0.4 0.7 0.5 0.3 0.1 0.1 0.6 0.4 0.5 Alkyl-PC 5.3 2.6 2.8 8.6 21.1 4.9 20.7 22.5 23.6 Molecular species and forms of diacyl-PC SFA/SFA 0.9 0.3 0.4 0.5 0.5 0.2 1.7 2.2 0.7 SFA/MUFA 8.5 4.8 6.9 9.2 11.7 5.3 20.3 17.5 14.9 SFA/PUFA 55.2 64.6 73.8 62.1 65.5 57.6 41.3 46.4 48.7 SFA/20:4 6.9 7.4 8.2 5.8 6.6 5.2 8.9 10.4 8.1 SFA/20:5 25.3 36.0 37.7 28.1 45.3 26.5 18.5 24.2 20.3 SFA/22:5 7.0 5.3 5.2 3.7 3.5 4.1 3.0 3.4 3.9 SFA/22:6 11.9 13.1 20.3 21.6 8.0 19.7 7.9 6.6 14.8 MUFA/MUFA 1.0 0.6 0.4 0.5 0.4 0.7 2.3 2.0 2.4 MUFA/PUFA 16.0 13.1 10.8 12.8 7.8 19.7 16.5 16.7 22.4 MUFA/20:4 1.5 0.9 0.5 1.4 0.6 1.5 3.0 3.3 2.9 MUFA/20:5 10.1 8.8 5.7 6.4 5.9 9.8 9.1 10.0 10.2 MUFA/22:5 1.0 0.6 0.6 0.4 0.2 1.1 0.8 0.8 1.2 MUFA/22:6 2.8 2.5 3.9 4.1 0.9 6.9 2.9 2.3 7.7 PUFA/PUFA 13.2 11.6 3.2 3.6 2.9 8.8 6.0 6.5 3.6 *Values expressed in % of total molecular species as x–. Standard deviation S < 0.1, n = 3; а—alkylacyl forms. Molecular species with levels not exceeding 3% are not shown but considered while computing cumulative parameters.

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Table 5. Composition of phosphatidylethanolamine molecular species in muscles, liver and gills of the black plaice under different environmental temperatures* Muscles Liver Gills Mol. species 18°С 9°С 0°С 18°С 9°С 0°С 18°С 9°С 0°С 16:0/18:1 0.2 0.1 0.1 0.0 0.0 0.1 0.2 0.0 0.0 16:0/20:4 1.2 1.5 1.7 2.3 1.7 1.7 1.6 0.3 1.5 18:0/20:4 1.2 1.0 0.7 1.6 1.1 0.7 6.6 6.7 3.6 16:0/20:5 5.1 8.3 7.7 4.8 14.1 6.3 1.7 0.4 2.3 17:0i/20:5 0.3 0.3 0.2 1.3 0.7 0.7 0.3 0.2 0.4 18:0/20:5 4.0 3.9 2.3 2.7 7.3 1.9 5.1 6.6 3.0 16:0/22:5 3.8 3.1 2.6 4.9 3.3 2.3 1.3 1.1 1.8 18:0/22:5 2.8 1.3 0.6 0.7 0.6 0.2 1.7 2.0 0.4 16:0/22:6 8.3 6.4 7.0 16.9 5.8 14.3 2.7 1.7 2.3 18:0/22:6 5.0 3.8 4.2 3.8 1.2 1.8 3.9 3.9 2.6 18:1/20:4 1.1 1.1 1.4 1.8 1.8 3.6 5.5 6.5 7.4 20:1/20:4 0.3 0.5 0.3 0.8 0.9 0.8 2.8 4.4 3.9 16:1/20:5 0.3 1.2 0.9 0.5 5.2 1.8 0.3 0.3 2.4 18:1/20:5 6.5 9.3 8.9 8.2 22.0 15.2 4.8 7.2 13.1 20:1/20:5 1.1 1.6 1.3 1.5 5.9 3.4 1.6 4.3 5.3 18:1/22:5 3.6 2.4 2.3 1.9 2.4 1.6 1.1 1.8 1.4 16:1/22:6 0.4 0.9 1.5 1.3 1.7 2.3 0.4 0.4 1.7 18:1/22:6 11.2 10.8 16.4 18.6 7.6 19.5 5.0 6.6 10.6 20:1/22:6 2.3 2.8 2.3 3.8 1.8 4.0 0.9 2.4 2.5 20:4/22:6 0.5 1.0 1.5 0.3 0.1 0.4 0.1 0.5 0.4 20:5/20:5 1.2 4.0 2.2 0.4 1.2 0.4 0.2 0.7 0.2 20:5/22:5 1.3 1.5 1.3 0.2 0.3 0.2 0.1 0.4 0.2 20:5/22:6 5.8 8.3 6.1 1.7 0.7 1.8 0.3 1.4 0.7 22:5/22:6 1.7 1.8 2.9 0.4 0.1 0.6 0.1 0.3 0.3 22:6/22:6 1.6 1.5 5.3 0.4 0.1 1.5 0.1 0.1 0.5 16:0p/20:4 0.5 0.4 0.3 0.2 0.0 0.0 0.9 0.3 0.1 16:0p/20:5 2.2 1.2 1.0 0.3 0.2 0.1 2.0 0.7 0.3 18:0p/20:5 0.3 0.2 0.1 0.1 0.1 0.0 3.1 1.2 0.5 16:0p/22:5 2.0 1.0 0.4 0.4 0.2 0.3 1.8 2.4 0.4 18:0p/22:5 0.1 0.0 0.0 0.0 0.1 0.0 1.6 1.2 0.3 16:0p/22:6 5.6 3.8 3.5 0.3 0.3 0.2 3.2 1.1 0.6 18:0p/22:6 0.2 0.2 0.1 0.1 0.0 0.0 3.1 1.4 1.1 18:1p/20:4 0.2 0.1 0.3 0.3 0.4 0.1 5.3 3.7 2.3 18:1p/20:5 0.5 0.3 0.4 0.4 0.2 0.3 7.0 7.0 4.2 18:1p/22:6 0.8 0.4 0.8 0.4 0.2 0.4 5.6 7.3 8.0 Diacyl-PE 80.4 88.3 88.8 94.5 95.6 96.3 54.2 66.3 75.0 Alkenyl-PE 16.4 9.4 9.1 2.9 1.8 1.7 42.3 29.8 20.1 Alkyl-PE 0.2 0.1 0.1 0.3 0.9 0.1 0.6 0.9 0.3 Molecular species and forms of diacyl-PE SFA/MUFA 1.0 0.4 0.5 0.1 0.0 0.2 1.4 0.0 0.6 SFA/PUFA 47.4 39.2 34.9 47.8 39.9 34.3 46.9 34.4 22.7 SFA/20:4 3.4 3.1 2.9 4.7 3.0 2.7 11.9 8.3 5.9 SFA/20:5 12.9 14.6 11.9 9.8 23.6 9.5 13.7 10.1 6.8 SFA/22:5 9.1 5.6 3.7 6.1 4.3 2.8 6.6 7.1 3.0 SFA/22:6 21.1 15.4 16.2 26.5 8.3 19.1 14.1 8.6 6.9 MUFA/MUFA 1.1 0.6 1.0 0.1 0.2 0.2 0.8 0.0 0.4 MUFA/PUFA 29.8 32.6 38.3 41.8 52.6 55.5 44.0 55.7 67.8 MUFA/20:4 1.7 1.9 2.3 3.2 3.8 5.0 14.2 14.8 15.5 MUFA/20:5 8.5 12.5 11.9 10.8 33.7 21.4 14.1 19.1 25.7 MUFA/22:5 4.3 3.0 2.5 2.6 3.5 1.8 3.2 4.6 2.9 MUFA/22:6 14.8 15.0 21.5 24.7 11.4 27.1 12.0 17.0 23.3 PUFA/PUFA 17.7 25.0 23.2 8.0 5.5 7.8 2.9 6.8 3.9 * p—alkenylacyl (plasmalogen) forms. Other designations as in Table 4.

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nor molecular forms (MUFA/MUFA and SFA/ MUFA) to thermoadaptation seems to be insignificant. SFA/SFA molecular forms in PE of the black plaice organs were not detected. A major ether form of PE is alkenylacyl-PE, while the level of the other form (alkylacyl-PE) does not reach 1%. With lowering the temperature, alkenylacyl-PE falls in muscles and gills about 2 times. In liver PE, there also occurs a fall, despite an initially low level of the alkenyl form in this organ (2.9%). Our investigation has shown that in the composition of PC molecular forms in the black plaice organs the dominant forms are SFA/PUFA and MUFA/PUFA composed of a relatively small number of major molecular species. Lowering the environmental temperature from 18 to 0°C leads to an increase in the level of SFA/PUFA molecular forms in muscles and gills, a small increase in the MUFA/PUFA level in the gills, and a decrease in MUFA/PUFA and PUFA/PUFA levels in muscles and gills, apparently promoting a fall in viscosity of the outer membrane monolayer and hence decreasing functional activity of the latter. Simultaneously, in PE of all black plaice organs there is a decrease in the SFA/PUFA level (as opposed to PC) but an increase in MUFA/PUFA and PUFA/PUFA levels. Similar changes are directed toward maintaining functional activity of the inner membrane monolayer being unsynchronized with rearrangements in the outer monolayer. Presumably, a sharp drop in the level of alkenylacyl PE forms and their replacement for diacyl forms in muscles and gills play a certain regulatory role in functional activity of the outer and inner membrane parts. Despite a close composition of major FA in PC and PE, that of their molecular forms differs depending on the organ and environmental temperature. Rearrangement of molecular forms of major PL in the black plaice due to a molecular transfer of acyl radicals leads to a change in viscosity of the membrane lipid matrix, however, does not affect the total FA composition of individual PL. Our results obtained herein support the previous conclusions that each fish species follows its own adaptation strategy realized by means of rearrangements in a certain set of FA molecular species in the PL composition [11].

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Supported by the Russian Scientific Foundation (project no. 14-50-00034). Part of studies concerned with phospholipid composition analysis was supported by the Ministry of Education and Science of the Russian Federation within the frames of the State assignment no. 6.5736.2017/6.7. REFERENCES 1.

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