Marine Biology 27, 197-204 (1974) (cid:14)9 by Springer-Verlag 1974 The Fatty Acids of Antarctic Phytoplankton and Euphausiids. Fatty Acid Exchange among Trophic Levels of the Ross Sea N.R. Bottino Department of Biochemistry and Biophysics, Texas A&M University; College Station, Texas, USA Abstract The fatty acids of 3 samples of Euphausia superba, 7 samples of E. crystallorophias, and 12 samples of phytoplankton collected in the Ross Sea, Ant- arctica, during Eltanin Cruise 51 were examined. The fatty acid profiles of the samples of E. su- perba resembled each other closely. The fatty acid profiles of the E. crystallorophias samples were also similar to each other but different quanti- tatively from those of E. superba. Phytoplankton fatty acid patterns varied with the geographical location and species composition of the samples. The fatty acids of euphausiids were compared to those of the phytoplankton from the corresponding locations. Rather similar fatty acid patterns in phytoplankton and E. superba corroborate the herbivorous nature of this euphausiid. On the other hand, phytoplankton and E. crystallorophias showed quite different fatty acid patterns. The differ- ences were mostly due to the presence of waxes among the lipids of E. crystallorophias. It is not clear whether these waxes are of dietary origin or are synthesized endogenously. Introduction The study of krill has become intensive in recent times, perhaps as a result of its potential im- portance as food. A variety of organisms is usual- ly included under that generic name, but in the Southern Oceans the name Euphausia superba has been considered almost a synonym for krill. How- ever, due to a quite defined geographical distri- bution, there are certain areas in which E. su- perba is replaced by other members of the same genus. For example, E. superba is very seldom found in shallow waters close to ice, such as the Ross Ice Shelf, but the smaller E. crystalloro- phias predominates in such areas (Marr, 1962; Mauchline and Fisher, 1969). The chemical composition of Euphausia superba is reasonably well known. Its fatty acids, in par- ticular, have been the subject of several studies in the past few years (Nonaka and Koizumi, 1964; Tsuyuki et al., 1964a, b; Hansen, 1969; Pierce et al.~ 1969; Hansen and Meiklen, 1970; Sidhu et al., 1970; Van der Veen et al., 1971). On the other hand, very little is known about the lipids of E. crystallorophias, except that they increase in the late austral summer and decrease gradually during the winter (Littlepage, 1964). The present report describes studies on the fatty acids of E. superba and E. crystallorophias from various locations in the Ross Sea. The fatty acid patterns of the two euphausiids are compared to each other as well as to the fatty acids of phytoplankton from corre- sponding locations. An attempt is made to deter- mine the flow of fatty acids through trophic levels. Materials and Methods Phytoplankton samples were collected with a 35 net, by vertical hauls to a depth of 200 m. After microscopic examination, samples containing less than 80% phytoplankton were discarded. Microzoo- plankton constituted the major contaminant. Euphausiids were collected with a ! m mid-water trawl, at depths varying between 0 and 300 m. Shortly after being sorted by hand the samples were extracted for lipids with a chloroform:meth- anol (2:1, v/v) mixture (Folch et al., 1957). Quantities of 5 to I0 mg total lipids were con- verted into fatty acid methyl esters by saponifi- cation, followed by reflux with methanol in the presence of boron trifluoride (American Oil Chem- ists' Society, 1970). The fatty acid methyl esters were studied by gas-liquld chromatography on a 6' I/8" column of siliconized polyethylene glycol succinate (DGSS-X, Applied Science Co., State Col- lege, Pennsylvania) 10% w/w on Chromosorb (Johns- Manville, Denver, Colorado) at 170~ A dual-flame model GC-5 Beckman gas chromatograph (Fullerton, California) was used connected to an Infotronics (Columbia Scientific Industries, Austin, Texas) digital integrator. Results are expressed as weight percent. Fatty acid methyl esters were identified by co-chromatography with known standards and by plotting relative retention times versus chain length before, and in most cases, after hydro- genation. Results and Discussion Fatty Acids of Euphausia superba Samples of Euphausia superba were collected from Stations 8, 9 and II of Eltanin Cruise 51 (Fig. l).
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`198 N.R. Bottino: Fatty Acids of Antarctic Phytoplankton and Euphausiids Fig. I. Ross Sea, Antarctica: track of Eltanin Cruise 51, showing stations These stations are located in an area in which two currents of water mix (Marr, 962), thus providing the turbulent environment that E. superba seems to prefer for a habitat (Ivanov, 1970). There is a remarkable similarity in the fatty acid compositions of the samples collected from the three stations (Table I). In addition, these pat- terns resemble quite closely those reported by Hansen and Meiklen (1970) and by Sidhu et al. (1970). The present results, however, differ some- what from those of Nonaka and Koizumi (1964) and of Van der Veen et al. (1971). Of all samples of Euphausia superba studied so far, only those of the present study were extracted immediately after capture. The others were frozen, transported and then extracted. This might explain some of the dif- ferences. In two groups of krill (Stations 8 and 11), the hepatopancreas and stomach were excised and their fatty acids were studied separately from those of the whole animal. In one case (Station 11), the fatty acids of the remaining carcass were also studied. Since the stomachs were empty in all cases, the values in Table I under the heading hepatopancreas and stomach correspond essentially to hepatopancreas lipids only. The remarkable simi- larity among organ, remaining carcass, and whole Table I. Euphausia superba. Fatty acids (as weight per cent of total acids) Fatty acid a Station 8 Station 9 Station 11 Whole krill HP+S b Whole krill Whole krill HP+S Remaining carcass 14:0 14.9 10.7 12.9 14.3 12.9 13.5 16:0 21.2 21.2 20.9 24.7 22.3 23.4 18:0 0.7 1.2 0.9 1.4 1.3 1.4 16:1 (n-7) 9.0 6.7 10.7 8.9 8.2 8.0 18:1 (n-9) 18.2 17.1 22.8 21.7 21.8 21.5 20:1 (n-9) 0.6 0.9 1.1 0.9 1.2 1.1 18:2(n-3) 2.6 2.5 2.7 2.0 2. 1 1.9 18:3(n-3) 1. 1 1.2 1.4 1.0 1.0 1. 1 18:4(n-3) 2.2 1.9 2.6 3.3 3.6 3.8 20:5 (n-3) 16.0 22.2 11.8 11.4 13.9 11.6 22:6 (n-3) 8.6 9.4 8.3 7.3 8.1 9.4 Minor fatty acids c 4.9 5.0 3.9 3.1 3.6 3.3 aThe number preceding the colon gives the number of carbon atoms in the chain, the number following the colon the number of double bonds; (n-x): number of carbons in the chain minus number of carbons between the methyl end and the nearest double bond. bHepatopancreas plus stomach. COnly those fatty acids present at a level of I% or more are included.
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`N.R. Bottino: Fatty Acids of Antarctic Phytoplankton and Euphausiids 199 body composition suggest that there is very little differentiation of organ lipids in Euphausia su- perba. Fatty Acids of Euphausia crystallorophias In contrast to Euphausia superba, which prefers turbulent waters, E. erystallorophias is usually found in shallow waters in the proximity of the Continental Shelf (Marr, 962; Mauchline and Fisher, 1969). We know from Littlepage (1964) that E. crystallorophiaa' lipids decrease in amount at the end of the austral winter and rise in late summer. My own studies (Bottino, in press) show that 20 to 40% of the lipids of E. crystalloro- phias are waxes, the rest being mostly complex lipids and small amounts of neutral lipids. The Euphaus~a crystallorophias collected from Stations 11, and 13 through 17 during Eltanin Cruise 5! show closely similar fatty acid patterns (Table 2). Comparison of the fatty acids of the two euphausiids show (Table 3) that E. crystalloro- phias contains about twice as much oleic acid (average 44% of total fatty acids) as E. superba (average 21%). Most of this oleic acid comes from the waxes, since about 83% of the wax fatty acids is oleic acid (Bottino, in press). The levels of highly unsaturated fatty acids (HUFA), mostly 20:5(n-3) and 22:6(n-3) are quite similar in both euphausiids. In conclusion, comparison of the fatty acids of both euphausiids shows that Euphausia crystalloro- phias lipids are more unsaturated than E. superba lipids on account of the larger amount of oleic acid in the former. This different degree of lipid unsaturation might be related to the different en- vironment in which the two euphausiids live. Where- as E. erystallorophias dwells near the ice all year around, E. superba is probably in contact with the ice only during the winter months (Mack- intosh, 1970). Phytoplankton Fatty Acids Phytoplankton samples from Stations 8, 9, I, 13- 15, and 18 were studied. According to microscopic and macroscopic observations , the nature of the phytoplankton population changes with the stations, and this is reflected in quantitative variations in the fatty acid patterns (Table 3). Qualitative- ly, however, the fatty acid patterns showed some common characteristics: () The presence in most samples of significant amounts (up to 5%) of C 8 to C13 fatty acids with both even and odd carbon chains (Table 3); most of these acids are not de- tected or are present at much lower levels in euphausiid lipids (Tables and 2). (2) All phyto- plankton samples contained HUFA, mainly 20:5(n-3) and 22:6(n-3) in levels ranging from less than 1% to about 23% (Table 3). This suggests that the lDr. S. EI-Sayed provided qualitative and semi- quantitative microscopic data on the composition of the phytoplankton samples. Table 2. Euphausia crystallorophias. Fatty acids (as weight per cent of total acids) Fatty acid Station 11 s~ation 13 Station 14 Station 15 Station 16 Station 17 Adults Juvenile 4:0 16:O 18:0 16:l(n-7) 8:1(n-9) 18:2(n-3) 18:3(n-3) 8:4 (n-3) 20:4 (n-6) 20:5(n-3) 22:6 (n-3) Minor fatty acids a 2.3 2.2 2.4 2.6 2.6 2.2 2.6 15.3 12. I 3.3 17.2 5.5 13. 16.0 0.3 0.4 0.6 0.3 0.5 0.5 0.8 8.6 6.5 7.9 7.9 8.4 9.3 6.5 39.6 49.8 48.8 47,8 49.0 47.6 40.7 1.7 2.4 2.2 2.1 2.3 2.3 1.6 1.0 1.0 1.0 l.O 0.8 0.7 0.8 0.7 1.8 .5 1.2 1.3 0.8 .0 0.9 - - 0.7 0.4 - 1.0 18.2 14. I 12.5 12.3 12.8 14.9 16.5 9.9 7.2 7.7 6.0 5.3 5.6 1.1 1.5 2.5 2.1 0.9 . 3.0 1.4 aExcept for stearic acid (18:0) only those fatty acids present at a level of % or more are included. -: not detected. See footnotes to Table for further explanation.
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`202 N.R. Bottino: Fatty Acids of Antarctic Phytoplankton and Euphausiids HUFA of the marine food-chain originate in phyto- plankton. Holz (1969) has reached a similar con- clusion from studies on laboratory-grown Protozoa. Comparison of Trophic Levels Whereas most euphausiids are considered omnivores, there is good evidence that Euphausia superba feeds on phytoplankton (Mauchline and Fisher, 1969). Whether the preferred food is Fragilaria as stated by many (Raymont, 1963; Nemoto, 1968), or whether E. superba has a wider range of selec- tivity (Pavlov, 1970), is still in doubt. Although generally considered a phytoplankton feeder (An- driashev, 1968; Knox, 1970), the evidence on the food preference of E. crystallorophias seems to be only of indirect nature, e.g. the finding by Littlepage (1964) of parallel changes in seasonal phytoplankton abundance and the lipid content of E. crystallorophias, and the study by Mauchline (1967) showing striking similarities between the feeding appendages of E. crystallorophias and E. superba. It was of interest, therefore, to compare the fatty acid patterns of Euphausia superba and E. crystallorophias collected during Eltanin Cruise 51 with the fatty acids of phytoplankton from the corresponding stations. It was hoped that the feeding habits would be reflected in similarities in the fatty acid patterns of the related trophic levels. This obviously assumes that euphausiids add very little endogenously-made fatty acids to those they acquire from the diet. Since the lipid content of phytoplankton is relatively high, be- tween 2 and 20% of their dry weight (Parsons et al~, 1961; Andriashev, 968), their ingestion by zooplankton should markedly diminish the lipo- genetic activity of the zooplankton. Recent ex- periments by Jeffries (972) show that the dietary origins of the body lipids of omnivorous fish can be determined with a simple budget of fatty acid flow from diet to body lipids. The feasibility of these determinations is supported by the findings of Owen et al. (1972) who, confirming previous work by others, have shown that the fatty acid composition of fish lipids reflects the compo- sition of the dietary lipids. Additional support is given by experiments in my laboratory (Warman and Bottino, 1974) which indicate that, in salt- water fish-liver the degree of lipogenesis is equally low whether the animals are starved, fed fat, or fed a fat-free diet. Most probably, the same is true for other marine organisms including euphausiids. It is possible that certain fish-lipid classes such as triglycerides may reflect changes in diet- ary lipids better than other classes of a more structural nature. It is shown below that this is not true in the present case. Finally, the similar fatty acid composition of euphausiid whole body and hepatopancreas (shown above) rules out the possibility of a preferential incorporation of dietary fatty acids into certain tissues. Owen et al. (1972) reached the same conclusion while study- ing the ingestion of polyunsaturated fatty acids by fish. Table 3 includes in the columns to the right av- erages of the Euphausia superba and E. crystalloro- phias fatty acids to facilitate comparisons. Com- parisons can be made quantitative by determining the "distance" between fatty acid profiles with the formula (McIntire et al., 1969; De Mort et al., 1972) : Djh = I where Djh is the degree of difference between the jth and hth species and Pij is the percentage of the total fatty acid content represented by the ith fatty acid in the jth species. For example, the distance between the results of two gas- chromatographic analyses of the same sample of mar- ine fatty acids is about 2.2 + 1.8 (average -+ l standard deviation). The average distance between the 3 samples of E. superba fatty acids (Table 1) was 6.3 _+ 1.2. The average distance among the 6 samples of E. crystallorophias fatty acids (Table 2) was 7.7 + 3.5. The distance between the average of E. superba and the average of E. crystalloro- phias (Table 3) was 29.0. In comparing the euphausiids with the phyto- plankton samples of the corresponding stations, the distances between the average of Euphausia superba and the phytoplankton profiles (Stations 8, 9a and 9b) ranged between O and 15, whizh are reasonably low values. The distances between the average of E. crystallorophias and phytoplankton (Stations 11, 3, 4, 15 and 182 ) were in the 30 to 40 range. Thus, the fatty acids of E. superba compare well both in nature and concentration with those of phytoplankton of the corresponding lo- cations. However, there are marked quantitative differences between the fatty acid profiles of E. crystallorophias and those of phytoplankton. It was mentioned earlier that the lipids of E. crystallorophias differ from those of E. superba in that they contain 20 to 40% waxes very rich in oleic acid (Bottino, in press). Thus, the dif- ference in fatty acid profiles between the two euphausiids and between E. crystallorophias and phytoplankton may be due to the presence of these waxes. To assess this possibility, the average fatty acid profile of E. crystallorophias was re- calculated eliminating the oleic acid contributed by the waxes, and distances were determined be- tween the new set of values and those of E. super- ba and phytoplankton. The distance between the corrected average of E. crystallorophias and the average of E. supembc was 14.1. The distances be- tween the corrected values of E. crystallorophias 2Despite the fact that no samples of Euphausia crystallorophias from Station 18 were analyzed, the phytoplankton data from that station are in- cluded in view 6f the short distance between Stations 18 and 17.
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`N.R. Bottino: Fatty Acids of Antarctic Phytoplankton and Euphausiids 203 and phytoplankton ranged between 22 and 32 for Stations 11 to 15 and between lO and 15 for Station 18. These results indicate that the pres- ence of waxes in E. crystallorophias is what makes the fatty acid profile of this euphausid different. It is possible that certain lipid classes such as triglycerides may be more susceptible than, for example, complex lipids to changes in dietary lipids. In fact, it has been accepted for many years that depot lipids are more sensitive than structural lipids to changes in dietary fats. To test this possibility, distances were determined between the major lipid classes of both euphausiids and the total lipids of the phytoplankton obtained in corresponding stations. The results showed that the waxes of Euphausia crystallorophias were the most distant from phytoplankton (aversge D = 76). The complex lipids of both euphausiids and the steroid esters of E. crystallorophias showed an average distance of about 30 with respect to phyto- plankton. The triglycerides of E. superba were the closer to phytoplankton with an average D = 17, a value which is in the same range as that of E. su- perba total lipids versus phytoplankton. These re- sults show that, in the present case, measuring distances between diet lipids and individual lipid classes is not better than comparing the total lipid fatty acids of the diet with those of total body lipids. In addition, the data seem to in- dicate that neither the complex lipids nor the steroid esters, and even less the waxes of E. crystallorophias reflect very well the composition of the dietary fatty acids. The similarity between the corrected values for Euphausia crystallorophias and some of the phyto- plankton values suggest that E. crystallorophias may be, in fact, herbivorous. Since most of the phytoplankton samples contained relatively small amounts of waxes, it is possible that E. crystal- lorophias may be able to incorporate and concen- trate dietary waxes. Alternatively, waxes could have been synthesized, partially or totally by the euphausiid. Holtz et al. (1973) and Sargent et al. (1974) have recently characterized wax synthesiz- ing systems in copepods. Acknowledgements. The author wishes to acknowl- edge the identification of the species by Drs. M. A. McWhinnie and S.Z. Ei-Sayed and the skilled technical assistance of Mrs. C. Wiltrout. This re- search was supported by a grant from the National Science Foundation (GV-30413). Literature Cited American Oil Chemists' Society: Official and ten- tative methods, 3rd. ed., with additions and re- visions, 2 pp. Chicago: Am. Oil Chemists' Soc. (Method Ce-2-66) 1970 Andriashev, A.P.: The problem of the life com- munity associated with the Antarctic fast ice. In: S.C.A.R. Symposium on Antarctic Oceanog- raphy, Santiago - Chile, 13-6 September 1966, pp 147-154. Cambridge: Scott Polar Research In- stitute 1968 Bottino, N.R.: Lipid composition of two species of Antarctic krill. Euphausia superba and E. cry- stallorphias. Comp. Biochem. Physiol. (In press) De Mort, C.L., R. Lowry, I. Tinsley and H.K. Phinney: The biochemical analysis of some estu- arine phytoplankton. I. Fatty acid composition. J. Phycol. 8, 211-216 (1972) Folch, J., M. Lees and G.H. Sloane-Stanley: A simple method for the isolation and purifi- cation of total lipids from animal tissues. J. biol. Chem. 226, 497-509 (1957) Hansen, R.P.: The occurrence of phytanic acid in Antarctic krill (Euphausia superba). Aust. J. Sci. 32, 160-161 (1969) - and S.M. Meiklen: Isopropenoid fatty acids in Antarctic krill (Euphausia superba). J. Sci. Fd Agric. 21, 203-206 (1970) Holz, G.G., Jr.: Polyunsaturated fatty acids of marine Protozoa. In: Progress in Protozoology, Int. Congr. Protozool. (III, Leningrad) pp 126- 27. Nauka 1969 Holtz, R.B., E.D. Marquez and A.A. Benson: Wax ester biosynthesis by isolated membrane frac- tions from calanoid copepods. Comp. Biochem. Physiol. 45B, 585-591 (1973) Ivanov, B.G.: On the biology of the Antarctic krill Euphausia superba. Mar. Biol. 7, 340-351 (1970) Jeffries, H.P.: Fatty acid ecology of a tidal marsh. Limnol. Oceanogr. 17, 433-440 (972) Knox, G.A.: Antarctic marine ecosystems. In: Ant- arctic ecology, Vol. 1. pp 69-96. Ed. by M.W. Holdgate. New York: Academic Press 1970 Littlepage, J.L.: Seasonal variation in lipid con- tent of two Antarctic marine crustacea. In: Biologie antarctique, pp 463-470. Ed. by R. Carrick, M.W. Holdgate and J. Prevoost. Paris: Hermann 1964 Mackintosh, N.A.: Whales and krill in the twentieth century. In: Antarctic ecology, Vol. I. pp 195- 212. Ed. by M.W. Holdgate. New York: Academic Press 970 Marr, J.W.S.: The natural history and geography of the Antarctic krill (Euphausia superba Dana). 'Discovery' Rep. 32, 33-464 (1962) Mauchline, J.: Feeding appendages of the Euphausia- cea (Crustacea). J. Zool. Lond. 153, 1-43 (967) - and L.R. Fisher: The biology of euphausiids. In: Advances in marine biology, Vol. 7. 454 pp. New York: Academic Press 969 McIntire, C.D., I.J. Tinsley and R.R. Lowry: Fatty acids in lotic periphyton: another measure of cormmunity structure. J. Phycol. 5, 26-32 (969) Nemoto, T.: Feeding of baleen whales and krill, and the value of krill as a marine resource in the Antarctic. In: S.C.A.R. Symposium on Ant- arctic Oceanography, Santiago - Chile, 3-16 September 1966, pp 240-253. Cambridge: Scott Polar Research Institute 1968 Nonaka, J. and C. Koizumi: Component fatty acids and alcohols of Euphausia superba lipid by gas- liquid chromatography. Bull. Jap. Soc. scient. Fish. 30j 630-634 (1964)
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`204 N.R. Bottino: Fatty Acids of Antarctic Phytoplankton and Euphausiids Owen, J.M., J.W. Adron, J.R. Sargent and C.B. Cowey: Studies on the nutrition of marine flat- fish. The effect of dietary fatty acids on the tissue fatty-acids of the plaice Pleuronectes platessa. Mar. Biol. 13, 160-166 (1972) Parsons, T.N., K. Stephens and J.D.H. Strickland: On the chemical composition of eleven species of marine phytoplankton. J. Fish. Res. Bd Can. 18, 1001-1016 (1961) Pavlov, V.Y.: On the physiology of feeding in Eu- phausia superba. Russ.. Dokl. Akad. Nauk SSSR. 196, 1477-1480 (1970). Engl. transl. Dokl. (Proc.) Acad. Sci. U.S.S.R. 196, 147-150 (1971) Pierce, R.W., J. Vander Veen and H.S. Olcott: Proximate and lipid analyses of krill (Euphausia species) and red crab (Pleurocodes planipes). J. agric. Fd Chem. 17, 367-369 (1969) Raymont, J.E.G.: Plankton and productivity in the oceans, 660 pp. Oxford: Pergamon Press 1963 Sargent, J.R., R.R. Gatten and R. McIntosh: Bio- synthesis of wax esters in cell-free prep- arations of Euchaeta norvegica. Comp. Biochem. Physiol. 47B, 217-227 (1974) Sidhu, G.S., W.A. Montgomery, G.L. Holloway, A.R. Johnson and D.M. Walker: Biochemical composition and nutritive value of krill (Euphausia superba Dana). J. Sci. Fd Agric. 21, 293-296 (1970) Tsuyuki, H., V. Naruse, A. Mochizuki and S. Ito: Studies on the lipids of Euphausiacea, Euphausia swperba. I. Acetone-soluble lipid. J. Japan Oil Chem. Soc. 13, 203-206 (1964a) .... Studies on the lipids of Euphausiaceae, Eu- phausia superba. II. Properties of phospho- lipids. J. Japan Oil Chem. Soc. 13, 477-480 (1964b) Van der Veen, J., B. Medwadowski and H.S. Olcott: The lipids of krill (Euphausia species) and red crab (Pleuroncodes planipes). Lipids 6, 481-485 (1971) Warman, A.W. and N.R. Bottino: Acetyl-CoA carboxy- lase and fatty acid synthetase from fish liver: properties and response to dietary fats. Proc. Am. Oil Chem. Soc., Philadelphia, September 1974 Dr. N.R. Bottino Department of Biochemistry and Biophysics Texas A & M University College Station, Texas 77843 USA Date of final manuscript acceptance: June 28, 1974. Communicated by J. Bunt, Miami
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