DOI 10.1007/s10600-015-1486-2 Chemistry of Natural Compounds, Vol. 51, No. 6, November, 2015
FATTY-ACID COMPOSITION OF THE DEEP-WATER BAIKAL AMPHIPOD Polyacanthisca calceolata
S. V. Bazarsadueva,1* L. D. Radnaeva,1,2 T. Ya. Sitnikova,3 and V. V. Taraskin2
The fatty-acid composition of the deep-water Baikal amphipod Polyacanthisca calceolata Baz., 1937, that was collected in the vicinity of hydrocarbon-gas releases (near the submerged mud volcano St. Petersburg) at depths of 1300–1400 m during the international expedition Miry in Baikal was studied for the first time. Gas-chromatography–mass-spectrometry (GC–MS) detected in the amphipod tissues 46 fatty acids, the principal ones of which were 16:0, 14:0, 18:0, 18:1n9, 16:1n7, 18:3n3, and 20:4n6. The high 18:1n9/ 18:1n7 ratio and low 16:1n7/16:0 and 20:5n3/22:6n3 ratios confirmed that the deep-water amphipod P. calceolata was a necrophage. Keywords: Polyacanthisca calceolata, fatty-acid composition, Lake Baikal, deep-water amphipods. Amphipods (Crustacea, Amphipoda) represent one of the most numerous groups of hydrobionts (>272 species and 76 subspecies) that is typically endemic almost totally to Lake Baikal. Polyacanthisca calceolata Baz. is a variety of Baikal amphipods, small populations of which are found in only three regions of the lake at depths from 1195 m to the bottom [1, 2]. Schools of P. calceolata were discovered in the vicinity of natural methane releases from the submerged mud volcano St. Petersburg (middle Baikal basin) during submersion of Mir deep-water probes (DWP) in 2009. The karyotype of only these crustaceans has currently been studied. It was shown that the diploid set in them contains 52 chromosomes (2n = 52) [3], like in the majority of previously studied Baikal amphipod species [4]. The lipid composition of this species has escaped report. Amphipods are known to be an important component in mass- and energy-exchange processes because they are an important food source for many hydrobionts. However, their lipid biochemistry and fatty-acid (FA) composition have been studied in only 2% of Baikal amphipod species [5–7]. As a rule, different habitats are responsible for variations in the compositions and functions of the structures isolating the organism from the environment, i.e., biomembranes, the principal components of which are proteins, carbohydrates, and lipids, the principal energy source for hydrobionts. Therefore, the functioning of any ecosystem is largely related to foodchain lipid biosynthesis and transport. Much research proved that lipids are involved in the adaptation of hydrobionts to variously changing environmental conditions [8]. The goal of the present work was to study the FA composition of the deep-water Baikal amphipod P. calceolata. Analysis of the FA composition of these amphipods identified 46 fatty acids with carbon chains from C14 to C22. Table 1 lists acids for which the relative amounts were >0.1%. The saturated FA content in the amphipods varied in the range 23.6–29.9 rel%. The principal saturated FA in P. calceolata were palmitic 16:0 (10.8 rel%), myristic 14:0 (~8.7), and stearic 18:0 (~4.0). Similar ratios of these dominant saturated acids were also found in the deep-water Baikal amphipod Acanthogammarus (Brachyuropus) grewingkii [6].
1) Baikal Institute of Nature Management, Siberian Branch, Russian Academy of Sciences, 670047, Ulan-Ude, Ul. Sakhcyanovoi, 6, e-mail:
[email protected]; 2) Buryatia State University, 670000, Ulan-Ude, Ul. Smolina, 24a; 3) Limnological Institute, Siberian Branch, Russian Academy of Sciences, 664033, Irkutsk, Ul. Ulan-Batorskaya, 3. Translated from Khimiya Prirodnykh Soedinenii, No. 6, November–December, 2015, pp. 899–901. Original article submitted April 29, 2015. 1042
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©2015
Springer Science+Business Media New York
TABLE 1. Principal Fatty Acids of the Amphipod Polyacanthisca calceolata, rel% Acid
Content
Acid
Content
Acid
Content
14:0 i15 ai15 15:0 16:0 i17 17:0 18:0 14:1n5 16:1n9
8.69 r 0.91 0.13 r 0.04 0.70 r 0.61 0.60 r 0.11 10.84 r 1.46 0.10 r 0.02 0.43 r 0.10 4.04 r 0.82 0.30 r 0.06 0.31 r 0.09
16:1n7 18:1n11 18:1n9 18:1n7 18:1n5 16:2n6 16:3n4 18:2n6 18:2n4 18:3n6
14.56 r 1.50 4.73 r 0.21 22.24 r 4.37 4.32 r 0.49 0.62 r 0.03 0.40 r 0.06 0.67 r 0.12 0.38 r 0.07 0.49 r 0.08 0.30 r 0.06
18:3n3 18:4n3 20:2n6 20:4n6 20:3n3 20:5n3 22:6n3 6sat. 6 monounsat. 6polyunsat.
11.31 r 0.68 1.55 r 0.17 0.48 r 0.11 8.89 r 0.96 0.18 r 0.01 0.21 r 0.03 0.85 r 0.16 26.7 r 3.3 47.3 r 4.6 25.8 r 2.5
Unsaturated FA were the principal ones (~73% of total FA). Monounsaturated FA made up ~47% of them and were dominated by monounsaturated oleic acid 18:1n9 (~22.2 rel%). Another principal acid was palmitoleic 16:1n7 (14.6 rel%). Monounsaturated 16:1 acids were present as three isomers (n9, n7, and n5); 18:1, four (n11, n9, n7, and n5); and 20:1, three (n11, n9, and n7). The total amount of monounsaturated 18:1 isomers in P. calceolata FA was ~32 rel%. High levels of 18:1n9 oleic acid (30 rel%) and a total amount of 18:1 isomers of ~37 rel% were also found in the deep-water Baikal amphipod Ommatogammarus albinus from Frolikha Bay [7]. Total 18:1 isomers were also the dominant component with contents from 30.2 to 46.6 rel% in FA from three species of deep-water Baikal gammarid species (Ceratogammarus dybowski, Abyssogammarus sp., and Polycotilus sp.) [5]. Therefore, isomers of 18:1 acids dominated in deep-water Baikal amphipod species. However, total 18:1 isomers also had high contents in FA from the deep-water Baikal amphipod A. grewingkii although the dominant isomer was 18:1n7 acid [6]. Polyunsaturated FA (PUFA) play an important role in sustaining membrane functions. Essential acids such as linolenic 18:3n3 (11.3 rel%) and arachidonic 20:4n6 (8.9 rel%) dominated the PUFA. Lipids and FA from the Baikal amphipods C. dybowski, Abyssogammarus sp., and Polycotilus sp. collected at depths of 1100–1300 m were reported by Morris [5]. The FA were dominated by isomeric 18:1 (22.4–45.2%), eicosapentaenoic 20:5n3 (12.4–17.8%), and docosahexaenoic 22:6n3 (5.8–20.9%) acids. Long-chain n3 PUFA (mainly 20:5n3 and 22:6n3) are usually related to marine hydrobionts and, as a rule, are uncharacteristic of fresh-water faunae. Fresh-water hydrobionts cannot synthesize long-chain PUFA and acquire them from food [8]. Dembitsky et al. observed that the contents of eicosapentaenoic and docosahexaenoic acids in deep-water Baikal amphipod A. grewingkii were 0.07 and 0.11 rel%, respectively [6]. Low levels of these acids (0.21 and 0.85 rel%, respectively) were also detected by us in P. calceolata. The high contents of unsaturated FA and the low contents of saturated FA were apparently related to the low temperature and the need to maintain the liquid-crystalline condition of membrane structures at a certain level. Thus, high PUFA contents were found in specimens of the amphipod Caprellidea from the Straits of Gibraltar that were collected at lower temperatures [9]. Salinity in the environment is known to be a leading abiotic factor that affects hydrobionts. Research on the effect of salinity on amphipod FA composition showed that the fractions of saturated and polyunsaturated acids decreased whereas that of monoenoic acids, especially 16:1 and 18:1, increased progressively as the salinity decreased [10]. Higher levels of monounsaturated and relatively low levels of saturated and polyunsaturated FA were found by us in P. calceolata. This was also probably related to the low salinity of Lake Baikal. The trophic structure in aquifers also plays a large role in hydrobiont evolution. In particular, different food sources are responsible for the rather high taxonomic specificity of deep-water faunae. This factor manifests in Lake Baikal as variations in the amounts and types of available food sources. Miniature species (hypomorphs) develop because of food deficits in organic-depleted sediments. Most Baikal amphipods are omnivores regardless of the way of life and form. Their food sources include detritus, benthic and planktonic diatoms, filamentary and gold algae, oligochaete bristles, remains of lower and higher crustaceans, rotifers, and ciliates. Spicules from various sponges and their pieces are often included. However, the spicules were evidently absorbed from sediments and not the living sponge because there were no fused spicules [11]. A very specialized diet has developed in several amphipods. In particular, benthopelagic “vultures” such as Ommatogammarus and Polyacanthisca species feed at great depths where the depleted food sources make this feeding method 1043
one of the most advantageous. Feeding on detritus at great depths is advantageous because of the deluge of corpses from above in combination with seriously decreased amounts or even the complete disappearance of other food sources (phytobenthos). Thus, it can be rather certainly asserted that trophic differentiation is observed only for a small part of Baikal amphipods [11]. Biomarker-acid ratios can inform about the food trophic level, composition, and type. High contents of specific lipids, e.g., 16:1n7, 18:1n7, and 20:5n3 acids, which are used as trophic biomarkers, are considered to be indicative of diatom algae food [12, 13]. Conversely, flagellates contain large amounts of docosahexaenoic acid 22:6n3 [14] whereas arachidonic acid 20:4n6 of microalgal origin is a constant component of the amphipods [12]. Oleic acid 18:1n9 is considered to be indicative of carnivores [13]. Animals can be classified as necrophages by using this parameter [7, 13]. Thus, the biomarkeracid ratio in deep-water Baikal O. albinus confirmed that it is a necrophage [7]. The amphipod P. calceolata is known to be a benthopelagic vulture [1]. Significant amounts of marker acids from algal feed in addition to the carnivorous food marker 18:1n9 were found in its FA composition. However, the ratios of biomarkers 20:5n3/22:6n3 and 16:1n7/16:0, which are indicative of herbivorous feeding, were low at 1.3 and 0.2, respectively. The ratio 18:1n9/18:1n7 was 5.1. This confirmed that P. calceolata was a necrophage [1]. Thus, we described for the first time the FA composition of the endemic Baikal amphipod P. calceolata and compared it with those of other deep-water Baikal amphipod species. High levels of unsaturated acids, in particular oleic 18:1n9, were found. The biomarker-acid ratio showed that P. calceolata was a necrophage.
EXPERIMENTAL The Baikal amphipod P. calceolata (n = 20) was collected in the emergence zone of hydrocarbon gases in the vicinity of the submerged mud volcano St. Petersburg at a depth of 1400 m using a Mir DWP in July 2009. A weighed portion of amphipod tissue (0.5–1.0 mg) was treated with HCl solution (1 mL, 2 M) in MeOH in order to produce the FA methyl esters (FAME). The reaction was carried out in thin-walled tubes with Teflon caps for 2 h at 90°C in a muffle furnace. The resulting solution was evaporated in a stream of Ar to half the volume and treated with distilled H2O (0.5 mL) and hexane (1 mL). The upper hexane layer was separated. The extraction was repeated twice [15]. The composition of the FAME was studied using GC–MS on an Agilent 6890 GC with an MSD 5973N quadrupole MS as the detector; a DB-Wax column (0.25 Pm ID); He carrier gas (constant flow rate 1.5 mL/min); column temperature 90°C (4 min isothermal), 90–165°C (30°C/min), and 165–225°C (3°C/min and 10.5 min isothermal); and vaporizer temperature 250°C. The sample volume was 1 PL with 40:1 flow division. The percent composition of the mixture was calculated from the areas of GC peaks. Qualitative analysis was based on comparisons of retention times and mass spectra with those of the pure compounds using the NIST02.L library and GLC-68D standard mixtures (Nu-Chek-Prep; Elysian, Minnesota, USA).
ACKNOWLEDGMENT Specimens were collected by the Mir DWP with organizational and financial support of the Foundation for Preservation of Lake Baikal and the Metropol group of companies. The scientific part of the work was sponsored by RFBR Grants Nos. 14-05-00516 and 14-44-04126 and the Basic Research Program of the RAS (Project V.46.5.2). The authors thank Cand. Biol. Sci. I. V. Mekhanikova, Limnological Institute, SB, RAS for determining the amphipod species.
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