Comp. Biochem. PhysioL, 1975, VoL 50B, pp. 479 to 484. Pergamon Press. Printed in Great Britain LIPID COMPOSITION OF TWO SPECIES OF ANTARCTIC KRILL: EUPHA USIA SUPERBA AND E. CR YSTALLOROPHIAS NESTOR R. BoTrn~o Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77843, U.S.A. (Received 21 February 1974) Al~raetml. The lipids of two Antarctic euphausiids were characterized. 2. In Euphausia superba complex lipids were the major lipid class followed by triglycerides. 3. In E. crystallorophias the complex lipids were also the major lipid class, but the second major constituent was waxes. 4. The complex llpids of both euphausiids consisted mostly of phosphatidylcholine with smaller mounts of phosphatidylethanolamine and lysophosphatidylcholine. The phespholipids of E. crystallorophias were less unsaturated than those of E. superba. 5. The waxes of E. crystallorophias were mostly esters of oleic (84 %) and palmitoleic (10~o) acids with n-tetradecanol (69 Yo) and n-hexadecanol (28 ~o). INTRODUCTION THE WORD "kriU" in Norwegian means "whale food", and it is with this meaning that the term is generally used, involving the various crustaceans (mostly euphausiids, but also amphipods and decapods) which whales eat (Nemoto, 1970). However, when one refers to Antarctic krill, one generally means Euphausia superba, which is the most abundant and far better known species of krill in the Antarctic Oceans. E. superba is usually found in open, turbulent waters at the confluences of oceanic streams (Ivanov, 1970). But E. superba is rarely found in the colder waters in close proximity to the ice where the smaller E. crystalorophias seems to predominate (Knox, 1970). Both E. superba and E. crystallorophias are considered phyto- plankton feeders (Mauchline & Fisher, 1969 for E. superba; Andriashev, 1968 and Knox, 1970 for E. crystallorophias). Both euphausiids are consumed by whales, seals, fish, penguins and petrels (Andriashev, 1968; Knox, 1970) thus occupying a central position in various food chains in open waters and in waters close to or under the ice. During cruise 51 of the USNS Eltanin to the Ross Sea, January-February 1972, I was able to collect specimens of E. superba in stations 8, 9 and 11 (Fig. 1) and specimens of E. crystallorophias in practically all of the stations located along the Ross Ice Shelf (stations 11-18). Since the lipids of E. superba have been studied only superficially and those of E. crystallorophias have not been studied at all, I decided to scrutinize them in more detail. The results are the subject of the present report. A preliminary presentation of these results has been made (Bottino, 1973). Fig. I MATERIALS AND METHODS Euphausiids were collected with a 1 m mid-water trawl at depths from 0 to 300 m. Once on board the ship the samples were rapidly sorted by hand and extracted with the chloroform-menthanol (2:1, v/v) mixture of 479
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`480 NESTOR R. BoTr~o Folch et al. (1957). Portions of 5-10mg lipids were transesterified with methanol in the presence of boron trifiuoride (American Oil Chemists' Society, 1970). The fatty acid methyl esters thus formed were separated from wax alcohols by thin-layer chromatography (TLC) on silica gel without binder (Adsorbosil-5, Applied Science Co., State College, Pa,) using a mixture of benzene-ethyl acetate (95 : 5, v/v) as developing solvent. Fatty acid compositions were determined by gas-liquid chromatography on a 6 ft x ~ in. column of siliconized polyethyleneglycol succinate (DGSS-X, Applied Science) 10yo (w/w) on Gas Chrom P 100-120 mesh (Johns- Manville, Denver, Colo.) at 170°C. A dual flame Model GC-5 Beckman gas chromatograph (Beckman Co., Fallerton, Calif.) was used connected to an Infotronic digital integrator (Columbia Scientific Industries, Austin, Texas). Final results were calculated on a Hewlett- Packard Desk Programmable Calculator Model 7810A. Results were expressed as weight per cent. Fatty acids were identified by c,o-chromatography with known standards and by plotting relative retention times vs. chain length before and after hydrogenation. For quantitative fractionation of lipid classes 20-40 mg of lipids were separated by the method of Freeman & West (1966) modified as follows: (a) Silica gel without binder (Adsorbosil-5, Applied Science) was used instead of silica gel-G. (b) Acetic acid was eliminated from the solvent mixture No. 1 to simplify the drying between Table 1. Fatty acids of Antarctic krill* E. superba E. crystailorophias Fatty acid Station 8 Station 11 Station 13 Station 16 weight ~o 12:0 0.3 0.2 0.2 tr 14:0 14.9 14.3 2.3 2.4 15:0 brt 0.3 0-2 0.1 15:0 0.5 0.2 0.2 0.1 16:0 21.2 24.7 13.8 14.8 18:0 0-7 1-4 1.2 1-3 22:0 0.1 0.1 14:1 (n- ?) 0-3 0-2 15:1 (n- ?) tr 0"1 16:1 (n-7) 9"0 8"9 8"4 10"8 17:1 (n-8) 0-7 0.3 0.4 0"4 18:1 (n-9) 18.2 21"7 47"5 45"2 20:1 (n-9) 0"6 0"9 0"2 0"5 18:2 (n- 6) 0"3 0"1 18:2 (n-3) 2'6 2"0 3"3 2"7 20:2 (n- 3) 0.1 18:3 (n-6) 0.3 0.2 0"2 0"3 18:3 (n--3) 1.1 1"0 0-9 0-9 20:3 (n- 3) 0"6 0"5 0"5 18:4 (n-- 3) 2-2 3"3 1.5 0"9 20:4 (n--6) 0"5 0"4 0"7 20:4 (n- 3) 0.5 0"2 0"1 22:4 (n- 6) 0.2 0"3 20:5 (n-3) 16-0 11"4 11"8 13"4 22:5 (n- 3) 0"3 0"1 0"1 22:6 (n-- 3) 8"6 7"3 7"3 5-5 * Data from Bottino (1974). "[" br, Branched-chain fatty acids. developments. (c) Quantitation of the spots was done by gravimetry and not by colorimetry of dichromate reduction. The gravimetric estimation required the use of the following procedure: Once separated by TLC and located with dichlorofluorescein spray, the spots were scraped off the plates, extracted six times with 4 ml (each) of a mixture of chloroform-methanol-acetic acid-water (50 : 39 : 1 : 10, v/v) (Arvidson, 1968). The extracts were filtered through fine pore sintered glass funnels into test-tubes, then 8 ml of 4 M ammonium hydroxide were added and the mixture shaken and centrifuged. The resulting upper phase, which contained the DCF, was discarded and to the lower phase was added 8 ml of 50yo (v/v) methanol in water. After the addition of anhydrous sodium sulfate, the liquid was filtered through a fine pore sintered glass funnel containing a layer of anhydrous sodium sulfate. The filtrate was then evapor- ated under vacuum and the residue was weighed. Using known amounts of total lipids in each determination, it was possible to estimate the recovery. Any result showing less than 80 per cent recovery was discarded and the fractionation repeated. The fatty acids of the lipid classes were converted into their methyl esters and studied by GLC as indicated above. In the case of the wax fraction the methylation procedure using boron trifluoride catalyst was found inetf~°ient and was replaced by a 1-hr reflux with a 2~o solution of sulfuric acid in methanol. The alcohol components of the waxes were studied by GLC without any previous treatment. The major component of the alcohol mixture was also isolated by preparative GLC and examined with an LKB-9000 mass spectrometer. The spectrum was iden- tical to that of authentic 1-n-tetradecanol. Other alcohols were identified by their GLC behavior in comparison with known standards and by plotting relative retention times vs. chain length. RESULTS AND DISCUSSION Table 1 shows the fatty acid compositions of the unfractionated lipids of E. superba and E. crystal- lorophias. The fatty acid patterns of the samples of E. superba were quite similar to each other and to that previously reported for another sample of the same species. In fact, differences among samples were only slightly higher than those found between duplicate analyses of the same sample (Bottino, 1974). Similarly, the fatty acid patterns of six samples of E. crystallorophias differed very little from each other (Bottino, 1974). Table 1 shows one example of this. However, there are noticeable quantitative differences between the fatty acid compositions of the two species. For example, the level of oleic acid is about twice as high in E. crystallorophias as in E. superba, while the reverse is true for the content of the saturated acids. Previous studies from this laboratory (Bottino, unpublished) have demonstrated that complex lipids constitute 50 per cent or more of the lipids of most Antarctic plants and animals other than mammals. The data in Table 2 show that complex lipids are also the major lipid class in the two euphausiids of the present study, comprising between 42 and 58 per cent of the total. In both organisms,
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`Lipids of Antarctic krill Table 2. The lipids of Antarctic krill E. superba E. crystallorophias Station 8 (1)* Station 11 (2) Station 13 (4) Station 16 (2) weight % Waxes -- -- 44 + 10:[: 20 +- 1 Steroid esters -- -- 2 + 3 27 + 9 Triglycerides 8 36 + 6 -- -- Diglycerides 17 4 + 5 -- 4 _+ 1 Complex lipids 54 58 _+ 14 53 +_ 8 42_+ 8 PCt 48 46 PEt 8 6 Lyso PC 1 1 PGt 1 Unknown§ 21 2+_22 1 +_2 7+ 1 * Number of determinations in parentheses. t PC, Phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglyccrol. ~/Weight per cent plus or minus the standard deviation. § R t between those of triglycerides and diglycerides. The recovered amount of this fraction was too small for further characterization. Table 3. Composition of the waxes of E, erystallorophias Fatty acids Station 13 Station 16 Waxes Waxes Steroid esters Wax alcohols Station 13 Station 16 8:0 0.3 10:0 0.1 0.1 12:0 tr 13:0 tr 14:0 0.5 15:0 br 0.5 15:0 0.1 16:0 0.8 0.6 18:0 0.3 0.2 20:0 22:0 11:1 (n- ?) 12:1 (n- 7) 13:1 (n- 2) 0.6 14:1 (n- 2) 15:1 (n-- ?) 0.1 1.3 16:1 (n-7) 8.8 11.2 17:1 (n--8) 0-3 0-3 18:1 (n--9) 84.1 83.1 20:1 (n-9) 0.6 0-4 18:2 (n- 3) 3.1 2.2 18:3 (n--3) 0.4 20:3 (n-3) 18:4 (n- 3) 20:5 (n- 3) 22:6 (n- 3) Unknown weight % 1.4 0.6 0.4 0.1 8.3 0.3 14"0 2"4 0.1 0.3 0.8 0.3 11.0 14.0 1.I 22.1 1.9 0.9 0.3 1.5 16.5 1.4 0.3 0"1 tr tr 72.5 69.2 25 "3 2"2* tr tr 28.0 2.2* 0.5 * 18:0+ 17:1. 481
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`482 NESTOR R. BOTrlNO Table 4. Fatty acids of the complex lipids of Antarctic krill E. superba---Station 11 E. crystaliorophias--Station 13 CL* PC PE Lyso PC CL PC PE Lyso PC 9:0 tr 10:0 11:0 12:0 13:0 tr 14:0 3.0 15:0 0.7 16:0 br 16:0 25.1 17:0 18:0 1.2 20:0 22:0 0.3 11:1 (n- .9) 12:1 (n- .9) 0.1 13:1 (n- 9.) 14:1 (n- ?) tr 15:1 (n- 9.) 16:1 (n-7) 2.9 17:1 (n--8) 0.1 18:1 (n-9) 14.2 20:1 (n-9) 0.8 22:1 (n--9) 18:2 (n- 3) 2.3 21:2 (n- 6) 18:3 (n-6) 0-2 18:3 (n-3) 1"6 20:3 (n-6) 0"1 20:3 (n- 3) 1"0 21:3 (n-3) tr 22:3 (n- 3) 0.5 18:4 (n- 3) 3"8 20:4 (n- 6) 1.5 20:4 (n- 3) 20:5 (n- 3) 25.3 22:5 (n- 3) 0.1 22:6 (n- 3) 15-2 Saturated 30-3 Monounsaturated 18.1 Polyunsaturated 51.6 Weight 0.1 0.2 0.1 tr 0.1 0.6 0-3 0.5 1.8 tr 1"4 2.7 0.1 0.2 0.1 0.6 tr 0.2 1.3 1"4 1.7 0.1 0.2 4-2 tr tr 1"6 6.2 3.4 2.6 8-3 2.9 4.2 4.6 I1.1 1.1 1.5 5.9 0.9 1.0 1.2 5.2 1.6 25.9 18.8 32.6 29.1 29.8 26-2 37.8 1.4 tr 1.1 1.7 4.1 1.0 1.0 2.9 1.9 0.2 0.4 1.8 0.2 0.1 tr 0.1 2.0 0.1 0.1 2.3 3.9 0.1 2.2 tr 0.1 2.8 0.2 2.5 tr 1.6 tr 0.1 2.8 1-3 4.2 5"3 6.9 2"0 2.4 0.1 tr 0-2 0.2 13.2 21.8 12.3 21.2 21.0 31"0 13-0 0.9 0.3 0.2 0.1 tr 0.5 2.4 1.4 1.1 2.1 2.5 1-3 0.7 0.1 0.4 0.1 1.8 0-8 0.2 1-2 1-5 0.3 0.4 0.1 0.1 0.7 1.2 tr 1"4 0.3 0-3 3.8 4.5 1.6 1.1 8.4 0-5 0.1 3.6 4-7 0.6 1.2 1.7 2-3 tr 1.5 0.2 1.9 0.1 0.3 0.2 0.4 0.2 0.2 0.2 22.5 20-3 6.5 19.3 14.8 15.1 2.4 11.8 20"8 2.5 8.6 5.2 10.3 tr 32.3 26.1 61.3 34.0 36.5 39.5 71.6 17.1 23"7 23.4 26"8 28.2 33"2 ~'5 50.6 50"2 15-3 39"2 35"3 27.3 3"9 * CL, Complex lipids. PC, phosphatidylcholine; PE, phosphatidylethanolamine. the major complex lipid was phosphatidylcholine (PC), 49 per cent in E. superba and 46 per cent in E. crystallorophias. Phosphatidylethanolamine (PE) and lysophosphatidylcholine were also found in both euphausiids but in much lower concentrations. Unexpected large amounts of waxes were found in E. crystallorophias while none was found in E. superba. In the specimens of E. crystaUorophias from station 16, slightly more than half of the waxes were steroid esters.* The waxes of each sample of E. crystaUorophias were isolated by TLC and the fatty acids and alcohols were examined indepen- dently by GLC. Both samples had very similar compositions, as shown by the data in Table 3. Two * For the sake of simplicity in the following discussion I shall call waxes the esters of long-chain aliphatic alcohols only.
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`Lipids of Antarctic krill 483 monoenoic acids, oleic and palmitoleic, con- stituted about 94 per cent of the fatty acids of these waxes. Of the four alcohol components, about 70 per cent was tetradecanol and 28 per cent was hexa- decanol. Thus the waxes of E. crystallorophias are less unsaturated than the copepod waxes examined by Lee et al. (1971a, b) and by Benson et al. (1972). The waxes of the present study resemble many other waxes of marine animals in their high content of oleic acid (Nevenzel, 1970) but differ from the rest in having tetradecanol as their major alcohol com- ponent. In most other marine waxes so far in- vestigated, hexadecanol is the predominant alcohol. The fatty acids of the steroid esters of E. crystal- lorophias (Table 3) contain polyunsaturated fatty acids while those of the aliphatic waxes do not. Table 4 shows the fatty acids of the major phos- pholipids of E. superba and E. crystallorophias. About half of the fatty acids in E. superba PC and PE were polyunsaturated, mostly 20:5 and 22:6. Only about one-third of the fatty acids of the PC and PE of E. crystaUorophias were polyunsaturated. Again, 20:5 and 22:6 were the predominant poly- unsaturated acids. With few exceptions, the experimental evidence indicates that the lipids of aquatic animals living at lower temperatures tend to be more polyunsaturated than those of aquatic animals living at higher temperatures (Johnson & Roots, 1964; Knipprath & Mead, 1966; Kemp & Smith, 1970). In the case of the lipids of E. superba and E. crystallorophias, a peculiar situation exists. Although it is true that the total lipids of E. crystallorophias, which lives in colder waters, are more unsaturated than those of E. superba, it is due to a higher level of mono- unsaturated acids. In fact, the phospholipids of E. crystallorophias are less unsaturated than those of E. superba (Table 4). It is possible that the monoenoic waxes of E. crystallorophias may have a role in the adaptation of these animals to their extremely cold environ- ment. Benson et al. (1972) have found that cold water copepods accumulate waxes. According to their views, the energy stored as waxes may be liberated slowly during periods in which food is scarce or not available. These terms certainly apply to E. crystallorophias since this euphansid not only lives in the cold waters near the ice but also under the ice, almost all year around. SUMMARY The lipids of specimens of E. superba and E. crystallorophias caught in various locations of the Ross Sea, Antarctica, were studied. The fatty acid composition of E. superba differed quantitatively from that of E. crystallorophias, the former con- taining more saturated acids, the latter containing more monoenoic acids. In both euphausiids the major lipid class was complex lipids (about 20 per cent of the total) consisting mostly of phosphatidylcholine with smaller amounts of phosphatidylethanolamine and lysophosphatidylcholine. The two species differ, however, in their neutral lipids, those of E. superba being triglycerides and diglycerides, those of E. crystallorophias being waxes, including steroid esters. The alcohols of the waxes were identified by mass spectrometry and gas-liquid chromatography, before and after hydrogenation, and found to be 6970 n-tetradecanol and 2870 n-hexadecanol. Wax fatty acids were about 8470 18:1 (n-9) and 1070 16:1 (n-7). The high levels of waxes in E. crystal- lorophias may be related to their year-round very cold environment. Acknowledgements--I wish to thank Dr. Karl Dahm for the mass spectrographic analyses and Mrs. Claudia Wiltrout for her technical assistance. These studies were supported by a grant from the National Science Foundation (GV-30413). REFERENCES AS~SmCAN On~ ~'s' SOCIETY (1970) Official and Tentative Methods, 3rd edn. Method Ce-2-66. Am. Oil Chemists' Soc., Chicago. Abn31UASH~ A. P. (1968) The problem of life community associated with the Antarctic fast ice. In Symposium on Antarctic Oceanography, pp. 147-154, Scott Polar Research Institute, Cambridge. ARVIVSON G. A. E. (1968) Structural and metabolic heterogeneity of rat liver glycerophosphates. Fur. J. Biochem. 4, 478--486. BaNSON A. A., L~ R. F. & N~m, lz~ J. C. (1972) Wax esters: major marine metabolic energy forces. In Current Trends in the Biochemistry of Lipids (Edited by GANOULY J. & S~LL~ R. M. S.), pp. 175-187. Academic Press, New York. BoTr~o N. R. (1973) The lipids of Antarctic krill. Presence of waxes in Euphausia crystallorophias but not in Euphausia superba lipids. Fedn Proe. Fedn Am. Socs. exp. Biol. 32, 562. BOTT~O N. R. (1974) The fatty acids of Antarctic phytoplankton and euphansiids. Fatty acid exchange among trophic levels of the Ross Sea. Mar. BioL On Press.) FOLCH J., LEES M. & SLO~NE-STASLL~ G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. Fp.~.~ C. P. & W~s'r D. (1966) Complete separation of lipid classes on a single thin-layer plate. J. LipidRes. 7, 324-327. Iv,~ov B. G. (1970) On the biology of the Antarctic krill Euphausia superba. Mar. Biol. 7, 340-351. JOHSSrON P. V. & ROOTS B. I. (1964) Brain lipid fatty acids and temperature acclimation. Comp. Bioehem. Physiol. 11, 303-309. P. & SMrrH M. W. (1970) Effect of temperature acclimatization on the fatty acid composition of goldfish intestinal lipids. Biochem. J. 117, 9--15.
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`484 NESTOR R. BOTTINO Ka, arPnA~-i W. G. & MEAD J. F. (1966) Influence of temperature on the fatty acid pattern of mosquitofish (Gambus/a a.~n/~) and guppies (Lebistes reticulatus). Lipids 1, 113-117. KNox G. A. (1970) Antarctic marine ecosystems. In Antarctic Ecology (Edited by HOLDGAT~ M. W.), VO1. 1, pp. 69-96. Academic Press, New York. LEE R. F., NS3nSNZ~ J. C. & P~6tazn G. A. (1971a) Importance of wax esters and other lipids in the marine food chain: phytoplankton and copepods. Mar. Biol. 9, 99-108. Lw, R. F., I-Imo'rA J. & BAR~S'r'r A. M. (1971b) Distribu- tion and importanc¢ of wax esters in marine copepods and other zooplankton. Deep Sea Res. 18, 1147-1165. MAUCHLINE J. & Ftsi~a L. R. (1969) The biology of euphausiids. In Advances in Marine Biology, Vol. 7. p. 205. Academic Press, New York. N~OTO T. (1970) Feeding pattern of balee~ whales in the ocean. In Marine Food Cha/ns (Edited by ST~LE J. F.), pp. 241-252. Oliver & Boyd, Edinburgh. N~rmqz~ J. C. (1970) Occ~ce~ce, function and bio- synthesis of wax esters in marine organisms. Lipids 5, 308-319. Key Word Index--Waxes; Antarctic crustacea; Antarctic zooplankton; food chains; marine phos- pholipids; fatty acids; tr/glycerides; steroid esters.
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