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Species-specific differences in intact wax esters of Calanus hyperboreus and C. finmarchicus from Fram Strait — Greenland Sea
Marine Chemistry, 39 (1992) 269-281
269
Elsevier Science Publishers B.V., A m s t e r d a m
Species-specific differences in intact wax esters of Calanus hyperboreus and C. finmarchicus from Fram S t r a i t - Greenland Sea Martin Graeve and Gerhard Kattner Alfred-Wegener-lnstitut J~r Polar- und Meeresforschung, Sektion Chemie, Am Handelshafen 12, 2850 Bremerhaven, Germany (Received 6 September 1991; accepted 27 March 1992)
ABSTRACT Graeve, M. and Kattner, G., 1992. Species-specific differences in intact wax esters of Calanus hyperboreus and C. finmarchicus from Fram Strait - - Greenland Sea. Mar. Chem., 39:269-281. Intact wax esters were studied in the herbivorous calanoid copepods Calanus hyperboreus and C. finmarchicus from the close pack ice region and the marginal ice zone (MIZ) of the Fram Strait in the northern Greenland Sea. Wax esters ranging from 30 to 44 carbon atoms with up to seven double bonds were identified. In the MIZ both species had significantly higher proportions of polyunsaturated wax esters than those in the pack ice zone, as a result of the different nutritional regimes. Despite this spatial variability, pronounced species-specific differences were found. Calanus hyperboreus lipids contained more diunsaturated long-chain wax esters of 38 : 2, 40 : 2, 42 : 2 and 44 : 2 structure, whereas C.finmarchicus lipids were dominated more by the monounsaturated short-chain wax esters, 34 : 1 and 36 : 1. The identification and molecular structure o f the wax esters were confirmed by analysing fatty acid and alcohol moieties separately. Only small differences were detected between the wax ester compositions of females and copepodid stages IV and V. There was a tendency towards long-chain wax esters in the older stages.
INTRODUCTION
Calanus hyperboreus and C.finmarchicus are dominant zooplankton species in the Greenland Sea, where they play an important role in the food web. Herbivorous copepods from high latitudes synthesize large lipid stores to compensate for the short period of food availability (Sargent et al., 1981; Kattner et al., 1989). Both species produce large amounts of wax esters as energy reserves (reviewed by Sargent et al., 1976; Sargent and Henderson, 1986). The alcohol moiety of the wax esters in both copepods is rather Correspondence to: G. Kattner, Alfred-Wegener-Institut fiir Polar- und Meeresforschung, Sektion Chemie, A m Handelshafen 12, 2850 Bremerhaven, Germany.
0304-4203/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
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M. GRAEVE AND G. KATTNER
uniform, with principal alcohols 20:1 and 22: 1. In contrast, the fatty acid moiety is more variable, and may to some extent reflect the input of dietary fatty acids (Kattner et al., 1989). Few studies have been carried out to analyse intact wax esters in the marine environments (Lee et al., 1971, 1974; Takagi et al., 1976; Boon and De Leeuw, 1979; Wakeham and Frew, 1982; Lawrence et al., 1982; Itabashi and Takagi, 1984). To date, the chemical structure of the copepod wax esters has usually been determined after transmethylation into fatty acid and alcohol derivatives because the high temperatures necessary for gas chromatographic analysis of intact wax esters may destroy parts of the polyunsaturated components. However, the method described by Kattner et al. (1990) allows a good separation of intact polyunsaturated wax esters. Most of the gas chromatographic peaks could be determined as one major component. Thus, it is now possible to investigate the variability of copepod wax esters with respect to abiotic parameters such as geography and hydrography, and to biotic parameters, such as feeding history and taxonomic affinity. The composition of intact wax esters as well as their fatty acid and alcohol moieties from two copepod species will be compared, considering the incorporation of dietary fatty acids into wax esters, de novo synthesis of wax esters and change in their composition during food deprivation. MATERIALS
AND
METHODS
Calanus hyperboreus and C. finmarchicus were collected in the Fram Strait area of the Greenland Sea during the M I Z E X cruise of the R/V "Polarstern" and R/V "Valdivia" in 1984. Sampling sites are shown in Fig. 1. The copepods were caught by vertical Bongo net hauls (300 and 500/~m mesh size) in the upper 100 m. Animals were immediately sorted into species and developmental stages, transferred into glass tubes containing chloroform-methanol (2 : 1) and stored at - 2 0 ° C until wax ester analysis. Lipid extracts were obtained by homogenizing the copepods in the storage solution with a Potter homogenizer (Braun, Melsungen, Germany). Intact wax esters were separated by thin-layer chromatography on silica gel 60 (Merck, Darmstadt, Germany) using hexane-diethylether-acetic acid (90:10 : 1). After development the lipid bands were visualized with a solution of 0.1% 2',7'-dichlorofluorescein in ethanol. Under UV light the wax ester bands were scraped off and eluted with chloroform. The chloroform solution was separated from the silica gel by slight centrifugation. An aliquot of the chloroform solution was evaporated and dissolved in n-hexane for analysis by gas-liquid chromatography (GLC), using a Carlo Erba 5370 Mega Series gas chromatograph (Carlo Erba Instruments, Milan, Italy) equipped with a flame ionization detector (FID) on a 25 m x 0.25 mm i.d. wall coated open tubular (WCOT) bonded fused silica column coated with
WAX ESTERSOF CALANUS SPECIES
271
Fig. 1. Location of stations in Fram Strait between Greenland and Spitsbergen where copepods were collected, o, C. hyperboreus; O, C.finrnarchicus. Continuous line: marginal ice zone, indicated by 0°C isotherm at 5 m depth.
a 0.10#m film of a 50% methyl-50% phenyl polysiloxane (Triglyceride Analysis Phase (TAP), Chrompack, Frankfurt, Germany). The carrier gas was hydrogen (100 kPa). Separations were made using on-column injection at 280°C with auxiliary cooling (30 s) and temperature programming (2 min at 280°C, l ° C m i n -t to 320°C). The F I D was set at 380°C. (For further details, see Kattner et al. (1990).) Wax esters were transesterified to give fatty acid methyl esters and alcohols, using 3% sulphuric acid in methanol. After extraction with n-hexane the mixture of fatty acid methyl esters and alcohols were analysed in a single run on a 30 m x 0.24 m m i.d. W C O T fused silica column coated with a film of OV 351 (J&W Scientific, San Jose, CA, USA), using temperature programming according to the method of Kattner and Fricke (1986). RESULTS
The wax esters from C. hyperboreus and C. finmarchicus showed speciesspecific differences as well as a pronounced spatial variability between stations. Gas chromatograms o f intact wax esters from lipid extracts of C.
272
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Fig. 2. Gas chromatograms of intact wax esters of C. hyperboreus (a) and C.finmarchicus (b) using the TAP phase.
hyperboreus and C. finmarchicus are presented in Fig. 2. The number of carbon atoms of these wax esters ranged from C30 to C44, including components with up to seven double bonds. Saturated wax esters were not detected. The amounts of wax esters were generally high; in C. hyperboreus the wax esters were more than 90% o f the total lipids. The proportion of wax esters increased from copepodid stage IV to females, except for C.finmarchieus females, which contained fewer wax esters than the copepodid stage V.
WAX ESTERS OF C A L A N U S SPECIES
273
Calanus hyperboreus The Arctic species C. hyperboreus dominated the zooplankton of the East Greenland Current. It also occurred in considerable numbers at all stations near the ice edge. The wax ester composition of the females together with their alcohol-fatty acid combinations are presented in Table 1, and in Table 2 the corresponding fatty acid and alcohol patterns are given. The major wax esters from the close pack ice region extending from the Northeast Water Polynya to the marginal ice zone (MIZ) were diunsaturated compounds, mainly 38 : 2 (20.2%) and 42:2 (34.7%) (number of carbon a t o m s : n u m b e r of double bonds). These wax esters resulted from combinations of 16 : 1, 20 : 1 and 22 : 1 fatty acids with 20:1 and 22:1 alcohols. The latter are by far the dominant alcohols (92.8%) o f C. hyperboreus; 14:0, 16:0 and a small proportion of 16:1 alcohol also contribute to the total alcohols. In the pack ice region the level of polyunsaturated compounds was remarkably low (8.9%). The proportion of the m o n o u n s a t u r a t e d wax esters was 19.7% o f total wax esters, with 36 : 1 as the main c o m p o u n d (9.0%), biosynthesized by a combination of the 16:0 and 14:0 fatty acids with the dominant 20:1 and 22:1 alcohols, respectively. The major wax esters of C. hyperboreus from the M I Z (Table 1) were the long-chain wax esters 40 : 2, 42 : 2 and 44 : 2 (57.7% of the total wax esters). The proportion of m o n o u n s a t u r a t e d wax esters (10.2%) was lower than in the pack ice region, whereas the polyunsaturated wax esters increased considerably. They reached 24.8%, with the 40 : 5 wax ester as the main component (10.2%), resulting from the combination of the 18 : 4 fatty acid and the 22 : 1 alcohol.
Calanus finmarchicus The boreal species C. finmarchicus dominated the samples in the Atlantic Water o f the West Spitsbergen Current but was also found in lower numbers at all stations under the pack ice and on the East Greenland shelf. The major wax esters of the females of C. finmarchicus from the pack ice region were 34 : 1 and 36 : 1 (42.5%, Table 1), as a result o f high amounts of 14 : 0 fatty acid in combination with a 20:1 or 22:1 alcohol (Table 2). The 14:0 fatty acid reached 28% of total fatty acids, whereas the 14 : 0 alcohol occurred only in trace amounts. The high proportion of the 14:0 acid, which has been found in C. finmarchicus, is the main difference between the two Calanus species. Calanus hyperboreus contained on average a lower percentage of wax esters with 14 : 0 as acid moiety at both stations. The diunsaturated wax esters of C. finmarchicus comprised only half o f those found in C. hyperboreus. The quantity of polyunsaturated compounds in C.finmarchicus was conspicuously low (5.5%).
274
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TABLE 2
Composition of fatty acids and alcohols (wt.%) for Calanus hyperboreus females and C. finmarchicus females Fatty acid
C. hyperboreus Pack ice
14 : 0 16:0 16:1 ( n - 7 ) 16: I ( n - 5 ) 16:2(n-6) 16:3(n-3) 1 6 : 4 ( n - 3) 18:0 18:1 ( n - 9 ) 18:1 ( n - 7 )
18 : 2 ( n - 6) 18:3(n-3) 18 : 4 ( n - 3) 20: I ( n - 9 ) 20:1 (n-7) 20 : 4 ( n - 6) 20 : 5 ( n - 3) 22 : 1 ( n - 11) 22:1 (n-9) 22: 5 ( n - 3 ) 22:6(n-3)
5.4 3.0 21.8 0.3 0.1 0.9 3.8 1.4 1.4 1.5 1.4 22.7 2.2 0.8 4.3 20.0 6.4 0.6 1.9
C. finmarchicus MIZ
Pack ice
M1Z
3.9 2.2 5.2 0.2
27.9 8.1 9.3 0.8
0.5
-
1.2
18.3 7.2 7.3 0.9 0.8 0.9 0.5 3.8 0.5 1.4 1.1 14.4 12.7 1.0 1.2 8.0 12.1 3.2 1.5 1.8
0.3 1.7 0.5
2.2 7.3 2.8
43.1 54.4
41.7 46. I
-
2.8 0.5 2.5 1.5 22.5 18.9 1.2 5.2 18.5 6.0 0.8 6.6
0.3 5.3 0.3 1.2 0.8 1.8 17.0 0.5
24.0 1.5
Alcohol 14:0 16:0 16:1 ( n - 7 ) 18:0 20 : 1 ( n - 9 ) 22:1 (n-11)
1.8 4.3 1.1 26.9 65.9
2.1 3.0 32.0 62.9
The major wax esters of C.finmarchicus from the MIZ were 34 : 1 and 36 : 1 (40%), as in the pack ice region (Table 1). The diunsaturated wax ester levels (31.2%) were the lowest o f all samples. At all stations near the ice edge the amount of polyunsaturated fatty acid moieties o f the wax esters increased to 23.1%. Here the 18:4 acid was one of the major fatty acid moieties in C. finmarchicus and in C. hyperboreus (Table 2).
The wax ester composition of the copepodid stages The wax ester composition o f C. hyperboreus was analysed separately from females and copepodid stages V and IV (Fig. 3). The specimens were all caught in the MIZ at the same station. All stages were dominated by high
276
M. G R A E V E A N D G. K A T T N E R C hyperboreus females
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Fig. 3. Wax ester compositions of females and copepodid stages V and IV of C. hyperboreus (a) and C. finrnarchicus (b) from the MIZ.
percentages o f polyunsaturated wax esters, especially 40 : 5 (up to 30%). The level of polyunsaturated wax esters decreased from female to copepodid stage IV, whereas the level of short-chain monounsaturated wax esters and shortchain polyunsaturates increased. The quantities of diunsaturated long-chain wax esters were similar in all stages. In females and copepodid stages V and IV of C.finmarchicuscollected from the open water in the vicinity o f the ice edge the wax esters showed the typical species-dependent composition, with high percentages of 34 : 1 and 36 : 1 wax esters (Fig. 3). The distribution of long-chain diunsaturated wax esters and those containing polyunsaturated moieties was similar within the stages, with
WAX ESTERS OF C A L A N U S SPECIES
277
slightly higher values of the long-chain ones in the older stages. Only copepodid stage V of C. finmarchicus showed a slight increase of polyunsaturated compounds. DISCUSSION
It is well established that calanoid copepods from high latitudes are able to synthesize large lipid stores (Lee, 1974; Sargent et al., 1981; Tande and Henderson, 1988; Kattner et al., 1989). Because of the herbivorous feeding mode of C. hyperboreus and C. finmarchicus, their life and the accumulation of lipid reserves are closely linked to the phytoplankton production. They synthesize a large pool of energy-rich wax esters to survive the long period of low food availability in polar regions and to preserve energy for reproduction. Wax esters have to be biosynthesized de novo from fatty acids and long-chain alcohols, because they are absent in the phytoplankton (e.g. Ackman et al., 1968). The product is a complex mixture of wax esters with different chain lengths and different degrees of unsaturation. Analysis by G L C of wax esters using the method described by Kattner et al. (1990) allows the differentiation of wax esters according to the number of carbon atoms and the degree of unsaturation. However, separation of wax esters with the same carbon number and the same degree of unsaturation but different alkyl and acyl moieties has some limitations. With the additional analysis of fatty acids and alcohols, most of the gas chromatographic peaks could each be identified as one major single wax ester, as illustrated in Table 1. The wax ester compositions of C. hyperboreus and C. finmarchicus were dominated by different major wax esters for each species. Thus, C. hyperboreus contained generally higher proportions of long-chain wax esters than C.finmarchicus. Wax ester combinations of the 14 : 0 fatty acid and the 14 : 0 alcohol, which have been identified after transesterification, were generally not detected. Despite this, species-specific compositions were strongly influenced by spatial variabilities, i.e. the different phytoplankton communities on which the species were feeding. Calanusfinmarchicus, normally a boreal Atlantic species, was expatriated under the pack ice as a result of cross-frontal mixing, eddy advection or transport with the Return Atlantic Current (Hirche, 1989; Smith, 1990). Under the ice, primary production is very low (Spies, 1987; Smith et al., 1987), and is dominated by flagellates (Baumann, 1990). In this region both species of copepods showed conspicuously low levels of polyunsaturated wax esters, compensated by higher amounts of diunsaturates. In fact, C. hyperboreus contained more than half of its wax esters as diunsaturates. It seems that polyunsaturated wax esters were preferentially depleted during starvation or converted into long-chain wax esters or phospholipids. This species had also
278
M. GRAEVE AND G. KATTNER
a high amount of wax esters containing 16:1 fatty acid moieties, which indicates an uptake of diatom fatty acids before starvation. The 16:1 and 20 : 5 acids are known to be the dominant fatty acids of diatoms (e.g. Kates and Volcani, 1966; Ackman et al., 1968; Kattner et al., 1989). At the MIZ, phytoplankton and zooplankton production is often enhanced (Smith et al., 1987). The phytoplankton in this area was mainly composed of a summer population of dinoflagellates and large amounts of Phaeocystis pouchetii in a subsurface layer (Smith et al., 1987; Baumann, 1990). In contrast to the pack ice region, the wax ester composition of both Calanus species at the MIZ showed the dominant influence of the 18:4 ( n - 3) fatty acid moiety, which is the major fatty acid of Phaeocystis sp. (Sargent et al., 1985) and of dinoflagellates (Harrington et al., 1970). The MIZ C. hyperboreus biosynthesized de novo high levels of energy-rich long-chain wax esters such as 40:2, 42:2 and 44:2. High concentration of food may provide the energy for the biosynthesis of long-chain wax esters. On the other hand, the incorporation of unchanged dietary fatty acids into wax esters or their use as precursors for longer-chain moieties are less energyconsuming processes. If dietary fatty acids really serve as precursors for long-chain fatty acids and finally alcohols, only saturated acids are used. An indication of this conversion may be the small amounts of saturated 14 : 0 and 16:0 acids in wax esters of C. hyperboreus. Other fatty acids do not seem to be usable, because polyunsaturated alcohols have not been found in marine wax esters. Also, the large amount of 16:1 ( n - 7 ) acid moiety in wax esters of C. hyperboreus from the pack ice region was not preferentially used in the biosynthesized fatty acids and alcohols. A small part of the 16:1 ( n - 7 ) acid may have been converted into 20:1 ( n - 7 ) , which is generally less than 10% of the 20:1 fatty acid isomers. This chain elongation within the same fatty acid family is a possible biosynthetic pathway; the further elongation to the 22 : 1 ( n - 7) acid was not detected. A small portion of the 16 : 1 acid may also be converted into the corresponding alcohol. In contrast, C. finmarchicus contained high levels of saturated fatty acids and generally very low amounts of the 20 : 1 ( n - 7) acid (Kattner et al., 1989). Calanus hyperboreus seems to store more energy-rich lipids than C.finmarchicus, which may be explained by their different life strategies. The ratio between the wax esters of the dietary fatty acids 16:1 and 18:4 esterified with the 20:1 and 22:1 alcohols corresponded to the ratio of the 20:1 and 22:1 alcohols, which were analysed separately. Hence, it appears that the wax esters were synthesized according to the abundance of the alcohol moieties available. We found no preference for any particular combination. In C. hyperboreus the amount of long-chain wax esters increased from copepodid stage IV to females, whereas C. finmarchicus synthesized the energy-richest wax esters mainly in the copepodid stage V. The Arctic species C. hyperboreus is at least biennial (Dawson, 1978) and its spawning is not
WAX ESTERS OF C A L A N U S SPECIES
279
dependent on food (Conover, 1967), whereas the boreal species C. finmarchicus has only one generation per year (Lie, 1965). Reproduction in C. finmarchicus is coupled with the onset of phytoplankton blooms (Marshall and Orr, 1955) and thus is dependent on the food availability. The developmental time of copepods seems to play an important role in the production or the accumulation of long-chain wax esters. Only the older copepodid stages are able to accumulate large amounts of these lipids with a high calorific value. This would explain the species-dependent differences in the wax esters, which were obvious in spite of the spatial variability. CONCLUSION
The determination of intact wax esters allows an extended study of the lipid biochemistry of copepods especially from high latitudes, where herbivorous calanoid copepods are able to synthesize large wax ester stores. The wax ester compositions of C. hyperboreus and C. finmarchicus are clearly different in each species. Habitat and food availability considerably influenced the fatty acid and alcohol moieties of the wax esters. The copepods' wax ester compositions often reflect the fatty acid composition of their food. The incoporation of dietary fatty acids into wax esters and the alterations during, for instance, change of food are not yet clear and require further research. Obviously, wax esters containing dietary fatty acids decrease during starvation, which means that they are utilized preferentially or converted into other components. The different life cycles are also clearly reflected in the wax ester compositions. C. hyperboreus is better adapted to the unfavourable life in Arctic regions because it can produce large amounts of long-chain wax esters. These esters have the highest calorific value of those investigated in copepods to date. ACKNOWLEDGEMENTS
The authors would like to thank W. Hagen for helpful discussions. This study is Contribution 474 of the Alfred Wegener Institute for Polar and Marine Research. REFERENCES Ackman, R.G., Tocher, C.S. and McLachlan, J., 1968. Marine phytoplankter fatty acids. J. Fish. Res. Board Can., 25: 1603-1620. Baumann, M.E.M., 1990. Untersuchung zur Primfirproduktion und Verteilung des Phytoplanktons der Gr6nlandsee mit Kulturexperimenten zum EinfluB des Lichtes und der Temperatur auf Wachstum und Photosyntheseleistung arktischer Diatomeen. Dissertation, Rheinisch-Westf'filische Technische Hochschule, Aachen, 129 pp. Boon, J.J. and de Leeuw, J.W., 1979. The analysis of wax esters, very long mid-chain ketones and sterol ethers isolated from Walvis Bay diatomaceous ooze. Mar. Chem., 7: 117-132.
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Conover, R.J., 1967. Reproductive cycle, early development, and fecundity in laboratory populations of the copepod Calanus hyperboreus. Crustaceana, 13: 61-72. Dawson, J.K., 1978. Vertical distribution of Calanus hyperboreus in the central Arctic Ocean. Limnol. Oceanogr., 23: 950-957. Harrington, G.W., Beach, D.H., Dunham, J.E. and Holz, Jr., G.G., 1970. The polyunsaturated fatty acids of marine dinoflagellates. J. Protozool., 17: 213-219. Hirche, H.J., 1989. Spatial distribution of digestive enzyme activities of Calanusfinmarchicus and C. hyperboreus in Fram Strait/Greenland Sea. J. Plankton Res., l l: 431-443. Itabashi, Y. and Takagi, T., 1984. Gas chromatographic resolution on polar open-tubular columns of saturated and unsaturated wax ester isomers differing in combinations of acyl and alcoholic groups. J. Chromatogr., 299: 351-363. Kates, K. and Volcani, B.E., 1966. Lipid components of diatoms. Biochim. Biophys. Acta, 116: 264-278. Kattner, G. and Fricke, H.S.G., 1986. Simple gas liquid chromatographic method for the simultaneous determination of fatty acids and alcohols in wax esters of marine organisms. J. Chromatogr., 361: 263-268. Kattner, G., Hirche, H.J. and Krause, M., 1989. Spatial variability in lipid composition of calanoid copepods from Fram Strait, the Arctic. Mar. Biol., 102: 473-480. Kattner, G., Graeve, M. and Ernst, W., 1990. Gas-liquid chromatographic method for the determination of marine wax esters according to the degree of unsaturation. J. Chromatogr., 513: 327-332. Lawrence, J.F., Iyengar, J.R., Page, B.D. and Conacher, H.B.S., 1982. Characterization of commercial waxes by high-temperature gas chromatography. J. Chromatogr., 236: 403-419. Lee, R.F., 1974. Lipid composition of the copepod Calanus hyperboreus from the Arctic Ocean. Changes with depth and season. Mar. Biol., 26: 313-318. Lee, R.F., Nevenzel, J.C. and Paffenh6fer, G.A., 1971. Importance of wax esters and other lipids in the marine food chain: phytoplankton and copepods. Mar. Biol., 9: 99-108. Lee, R.F., Nevenzel, J.C. and Lewis, A.G., 1974. Lipid changes during life cycle of marine copepod, Eucheta japonica Marukawa. Lipids, 9: 891-898. Lie, U., 1965. Quantities of zooplankton and propagation of Calanusfinmarchicus at permanent stations on the Norwegian coast and at Spitsbergen, 1958-1962. Rep. Norw. Fish. Mar. Invest., 13: 5-19. Marshall, S.M. and Orr, A.P., 1955. The Biology of a Marine Copepod Calanus finmarchicus Gunnerus. Oliver and Boyd, Edinburgh, 188 pp. Sargent, J.R. and Henderson, R.J., 1986. Lipids. In: E.D.S. Corner and S.C.M. O'Hara (Editors), The Biochemical Chemistry of Marine Copepods. Clarendon, Oxford, pp. 59-108. Sargent, J.R., Lee, R.F. and Nevenzel, J.C., 1976. Marine waxes. In: P.E. Kollattukudy (Editor), Chemistry and Biochemistry of Natural Waxes. Elsevier, Amsterdam, pp. 50-91. Sargent, J.R., Gatten, R.R. and Henderson, R.J., 1981. Lipid biochemistry of zooplankton from high latitudes. Oceanis, 7: 623-632. Sargent, J.R., Eilertsen, H.C., Falk-Petersen, S. and Taasen, J.P., 1985. Carbon assimilation and lipid production in phytoplankton in northern Norwegian Ijords. Mar. Biol., 85:109-116. Smith, S.L., 1990. Egg production and feeding by copepods prior to the spring bloom of phytoplankton in the Fram Strait area of the Greenland Sea. Mar. Biol., 106: 59-69. Smith, W.O., Baumann, M.E., Wilson, D.L. and Aletsee, L., 1987. Phytoplankton biomass and productivity in the marginal ice zone of the Fram Strait during summer, 1984. J. Geophys. Res., 92: 6777-6786. Spies, A., 1987. Phytoplankton in the marginal ice zone of the Greenland Sea during summer, 1984. Polar Biol., 7: 195-205. Takagi, T., Itabashi, Y., Ota, T. and Hayashi, K., 1976. Gas chromatographic separation of wax esters based on the degree of unsaturation. Lipids, 11: 354-356.
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Tande, K.S. and Henderson, R.J., 1988. Lipid composition of copepodite stages and adult females of Calanus glacialis in Arctic waters of the Barents Sea. Polar Biol., 8: 333-339. Wakeham, S.G. and Frew, N.M., 1982. Glass capillary gas chromatography-mass spectrometry of wax esters, steryl esters and triacylglycerols. Lipids, 17: 831-843.
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Species-specific differences in intact wax esters of Calanus hyperboreus and C. finmarchicus from Fram S t r a i t - Greenland Sea Martin Graeve and Gerhard Kattner Alfred-Wegener-lnstitut J~r Polar- und Meeresforschung, Sektion Chemie, Am Handelshafen 12, 2850 Bremerhaven, Germany (Received 6 September 1991; accepted 27 March 1992)
ABSTRACT Graeve, M. and Kattner, G., 1992. Species-specific differences in intact wax esters of Calanus hyperboreus and C. finmarchicus from Fram Strait - - Greenland Sea. Mar. Chem., 39:269-281. Intact wax esters were studied in the herbivorous calanoid copepods Calanus hyperboreus and C. finmarchicus from the close pack ice region and the marginal ice zone (MIZ) of the Fram Strait in the northern Greenland Sea. Wax esters ranging from 30 to 44 carbon atoms with up to seven double bonds were identified. In the MIZ both species had significantly higher proportions of polyunsaturated wax esters than those in the pack ice zone, as a result of the different nutritional regimes. Despite this spatial variability, pronounced species-specific differences were found. Calanus hyperboreus lipids contained more diunsaturated long-chain wax esters of 38 : 2, 40 : 2, 42 : 2 and 44 : 2 structure, whereas C.finmarchicus lipids were dominated more by the monounsaturated short-chain wax esters, 34 : 1 and 36 : 1. The identification and molecular structure o f the wax esters were confirmed by analysing fatty acid and alcohol moieties separately. Only small differences were detected between the wax ester compositions of females and copepodid stages IV and V. There was a tendency towards long-chain wax esters in the older stages.
INTRODUCTION
Calanus hyperboreus and C.finmarchicus are dominant zooplankton species in the Greenland Sea, where they play an important role in the food web. Herbivorous copepods from high latitudes synthesize large lipid stores to compensate for the short period of food availability (Sargent et al., 1981; Kattner et al., 1989). Both species produce large amounts of wax esters as energy reserves (reviewed by Sargent et al., 1976; Sargent and Henderson, 1986). The alcohol moiety of the wax esters in both copepods is rather Correspondence to: G. Kattner, Alfred-Wegener-Institut fiir Polar- und Meeresforschung, Sektion Chemie, A m Handelshafen 12, 2850 Bremerhaven, Germany.
0304-4203/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
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M. GRAEVE AND G. KATTNER
uniform, with principal alcohols 20:1 and 22: 1. In contrast, the fatty acid moiety is more variable, and may to some extent reflect the input of dietary fatty acids (Kattner et al., 1989). Few studies have been carried out to analyse intact wax esters in the marine environments (Lee et al., 1971, 1974; Takagi et al., 1976; Boon and De Leeuw, 1979; Wakeham and Frew, 1982; Lawrence et al., 1982; Itabashi and Takagi, 1984). To date, the chemical structure of the copepod wax esters has usually been determined after transmethylation into fatty acid and alcohol derivatives because the high temperatures necessary for gas chromatographic analysis of intact wax esters may destroy parts of the polyunsaturated components. However, the method described by Kattner et al. (1990) allows a good separation of intact polyunsaturated wax esters. Most of the gas chromatographic peaks could be determined as one major component. Thus, it is now possible to investigate the variability of copepod wax esters with respect to abiotic parameters such as geography and hydrography, and to biotic parameters, such as feeding history and taxonomic affinity. The composition of intact wax esters as well as their fatty acid and alcohol moieties from two copepod species will be compared, considering the incorporation of dietary fatty acids into wax esters, de novo synthesis of wax esters and change in their composition during food deprivation. MATERIALS
AND
METHODS
Calanus hyperboreus and C. finmarchicus were collected in the Fram Strait area of the Greenland Sea during the M I Z E X cruise of the R/V "Polarstern" and R/V "Valdivia" in 1984. Sampling sites are shown in Fig. 1. The copepods were caught by vertical Bongo net hauls (300 and 500/~m mesh size) in the upper 100 m. Animals were immediately sorted into species and developmental stages, transferred into glass tubes containing chloroform-methanol (2 : 1) and stored at - 2 0 ° C until wax ester analysis. Lipid extracts were obtained by homogenizing the copepods in the storage solution with a Potter homogenizer (Braun, Melsungen, Germany). Intact wax esters were separated by thin-layer chromatography on silica gel 60 (Merck, Darmstadt, Germany) using hexane-diethylether-acetic acid (90:10 : 1). After development the lipid bands were visualized with a solution of 0.1% 2',7'-dichlorofluorescein in ethanol. Under UV light the wax ester bands were scraped off and eluted with chloroform. The chloroform solution was separated from the silica gel by slight centrifugation. An aliquot of the chloroform solution was evaporated and dissolved in n-hexane for analysis by gas-liquid chromatography (GLC), using a Carlo Erba 5370 Mega Series gas chromatograph (Carlo Erba Instruments, Milan, Italy) equipped with a flame ionization detector (FID) on a 25 m x 0.25 mm i.d. wall coated open tubular (WCOT) bonded fused silica column coated with
WAX ESTERSOF CALANUS SPECIES
271
Fig. 1. Location of stations in Fram Strait between Greenland and Spitsbergen where copepods were collected, o, C. hyperboreus; O, C.finrnarchicus. Continuous line: marginal ice zone, indicated by 0°C isotherm at 5 m depth.
a 0.10#m film of a 50% methyl-50% phenyl polysiloxane (Triglyceride Analysis Phase (TAP), Chrompack, Frankfurt, Germany). The carrier gas was hydrogen (100 kPa). Separations were made using on-column injection at 280°C with auxiliary cooling (30 s) and temperature programming (2 min at 280°C, l ° C m i n -t to 320°C). The F I D was set at 380°C. (For further details, see Kattner et al. (1990).) Wax esters were transesterified to give fatty acid methyl esters and alcohols, using 3% sulphuric acid in methanol. After extraction with n-hexane the mixture of fatty acid methyl esters and alcohols were analysed in a single run on a 30 m x 0.24 m m i.d. W C O T fused silica column coated with a film of OV 351 (J&W Scientific, San Jose, CA, USA), using temperature programming according to the method of Kattner and Fricke (1986). RESULTS
The wax esters from C. hyperboreus and C. finmarchicus showed speciesspecific differences as well as a pronounced spatial variability between stations. Gas chromatograms o f intact wax esters from lipid extracts of C.
272
M, G R A E V E A N D G. K A T T N E R
~1
42:2
44:2
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I
.
_-.2..
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] 34"4
~IL:S 36;5
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112
40:2
44"2
38:4
36:6
114
16
42:6 42;)'
40:8
16
20
22
24
"
26
rlli n
Fig. 2. Gas chromatograms of intact wax esters of C. hyperboreus (a) and C.finmarchicus (b) using the TAP phase.
hyperboreus and C. finmarchicus are presented in Fig. 2. The number of carbon atoms of these wax esters ranged from C30 to C44, including components with up to seven double bonds. Saturated wax esters were not detected. The amounts of wax esters were generally high; in C. hyperboreus the wax esters were more than 90% o f the total lipids. The proportion of wax esters increased from copepodid stage IV to females, except for C.finmarchieus females, which contained fewer wax esters than the copepodid stage V.
WAX ESTERS OF C A L A N U S SPECIES
273
Calanus hyperboreus The Arctic species C. hyperboreus dominated the zooplankton of the East Greenland Current. It also occurred in considerable numbers at all stations near the ice edge. The wax ester composition of the females together with their alcohol-fatty acid combinations are presented in Table 1, and in Table 2 the corresponding fatty acid and alcohol patterns are given. The major wax esters from the close pack ice region extending from the Northeast Water Polynya to the marginal ice zone (MIZ) were diunsaturated compounds, mainly 38 : 2 (20.2%) and 42:2 (34.7%) (number of carbon a t o m s : n u m b e r of double bonds). These wax esters resulted from combinations of 16 : 1, 20 : 1 and 22 : 1 fatty acids with 20:1 and 22:1 alcohols. The latter are by far the dominant alcohols (92.8%) o f C. hyperboreus; 14:0, 16:0 and a small proportion of 16:1 alcohol also contribute to the total alcohols. In the pack ice region the level of polyunsaturated compounds was remarkably low (8.9%). The proportion of the m o n o u n s a t u r a t e d wax esters was 19.7% o f total wax esters, with 36 : 1 as the main c o m p o u n d (9.0%), biosynthesized by a combination of the 16:0 and 14:0 fatty acids with the dominant 20:1 and 22:1 alcohols, respectively. The major wax esters of C. hyperboreus from the M I Z (Table 1) were the long-chain wax esters 40 : 2, 42 : 2 and 44 : 2 (57.7% of the total wax esters). The proportion of m o n o u n s a t u r a t e d wax esters (10.2%) was lower than in the pack ice region, whereas the polyunsaturated wax esters increased considerably. They reached 24.8%, with the 40 : 5 wax ester as the main component (10.2%), resulting from the combination of the 18 : 4 fatty acid and the 22 : 1 alcohol.
Calanus finmarchicus The boreal species C. finmarchicus dominated the samples in the Atlantic Water o f the West Spitsbergen Current but was also found in lower numbers at all stations under the pack ice and on the East Greenland shelf. The major wax esters of the females of C. finmarchicus from the pack ice region were 34 : 1 and 36 : 1 (42.5%, Table 1), as a result o f high amounts of 14 : 0 fatty acid in combination with a 20:1 or 22:1 alcohol (Table 2). The 14:0 fatty acid reached 28% of total fatty acids, whereas the 14 : 0 alcohol occurred only in trace amounts. The high proportion of the 14:0 acid, which has been found in C. finmarchicus, is the main difference between the two Calanus species. Calanus hyperboreus contained on average a lower percentage of wax esters with 14 : 0 as acid moiety at both stations. The diunsaturated wax esters of C. finmarchicus comprised only half o f those found in C. hyperboreus. The quantity of polyunsaturated compounds in C.finmarchicus was conspicuously low (5.5%).
274
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TABLE 2
Composition of fatty acids and alcohols (wt.%) for Calanus hyperboreus females and C. finmarchicus females Fatty acid
C. hyperboreus Pack ice
14 : 0 16:0 16:1 ( n - 7 ) 16: I ( n - 5 ) 16:2(n-6) 16:3(n-3) 1 6 : 4 ( n - 3) 18:0 18:1 ( n - 9 ) 18:1 ( n - 7 )
18 : 2 ( n - 6) 18:3(n-3) 18 : 4 ( n - 3) 20: I ( n - 9 ) 20:1 (n-7) 20 : 4 ( n - 6) 20 : 5 ( n - 3) 22 : 1 ( n - 11) 22:1 (n-9) 22: 5 ( n - 3 ) 22:6(n-3)
5.4 3.0 21.8 0.3 0.1 0.9 3.8 1.4 1.4 1.5 1.4 22.7 2.2 0.8 4.3 20.0 6.4 0.6 1.9
C. finmarchicus MIZ
Pack ice
M1Z
3.9 2.2 5.2 0.2
27.9 8.1 9.3 0.8
0.5
-
1.2
18.3 7.2 7.3 0.9 0.8 0.9 0.5 3.8 0.5 1.4 1.1 14.4 12.7 1.0 1.2 8.0 12.1 3.2 1.5 1.8
0.3 1.7 0.5
2.2 7.3 2.8
43.1 54.4
41.7 46. I
-
2.8 0.5 2.5 1.5 22.5 18.9 1.2 5.2 18.5 6.0 0.8 6.6
0.3 5.3 0.3 1.2 0.8 1.8 17.0 0.5
24.0 1.5
Alcohol 14:0 16:0 16:1 ( n - 7 ) 18:0 20 : 1 ( n - 9 ) 22:1 (n-11)
1.8 4.3 1.1 26.9 65.9
2.1 3.0 32.0 62.9
The major wax esters of C.finmarchicus from the MIZ were 34 : 1 and 36 : 1 (40%), as in the pack ice region (Table 1). The diunsaturated wax ester levels (31.2%) were the lowest o f all samples. At all stations near the ice edge the amount of polyunsaturated fatty acid moieties o f the wax esters increased to 23.1%. Here the 18:4 acid was one of the major fatty acid moieties in C. finmarchicus and in C. hyperboreus (Table 2).
The wax ester composition of the copepodid stages The wax ester composition o f C. hyperboreus was analysed separately from females and copepodid stages V and IV (Fig. 3). The specimens were all caught in the MIZ at the same station. All stages were dominated by high
276
M. G R A E V E A N D G. K A T T N E R C hyperboreus females
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30
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32
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36
38
40
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32
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C. f l n m a r c h l c u s
40
42
44
30
30
20
20
20
I
I
10
36
38
40
42
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30
32
34
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3B
40
36
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40
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0 34
34
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ClV
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32
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C.flnmarchlcus
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30
30
I
IO
42
CHAIN LEN6TH IN C-ATOMS
44
30
~2
34
IS
I
]8
• • r'l
40 42
44
monounsaturatea alunsaturatea
polyunsat uratea
Fig. 3. Wax ester compositions of females and copepodid stages V and IV of C. hyperboreus (a) and C. finrnarchicus (b) from the MIZ.
percentages o f polyunsaturated wax esters, especially 40 : 5 (up to 30%). The level of polyunsaturated wax esters decreased from female to copepodid stage IV, whereas the level of short-chain monounsaturated wax esters and shortchain polyunsaturates increased. The quantities of diunsaturated long-chain wax esters were similar in all stages. In females and copepodid stages V and IV of C.finmarchicuscollected from the open water in the vicinity o f the ice edge the wax esters showed the typical species-dependent composition, with high percentages of 34 : 1 and 36 : 1 wax esters (Fig. 3). The distribution of long-chain diunsaturated wax esters and those containing polyunsaturated moieties was similar within the stages, with
WAX ESTERS OF C A L A N U S SPECIES
277
slightly higher values of the long-chain ones in the older stages. Only copepodid stage V of C. finmarchicus showed a slight increase of polyunsaturated compounds. DISCUSSION
It is well established that calanoid copepods from high latitudes are able to synthesize large lipid stores (Lee, 1974; Sargent et al., 1981; Tande and Henderson, 1988; Kattner et al., 1989). Because of the herbivorous feeding mode of C. hyperboreus and C. finmarchicus, their life and the accumulation of lipid reserves are closely linked to the phytoplankton production. They synthesize a large pool of energy-rich wax esters to survive the long period of low food availability in polar regions and to preserve energy for reproduction. Wax esters have to be biosynthesized de novo from fatty acids and long-chain alcohols, because they are absent in the phytoplankton (e.g. Ackman et al., 1968). The product is a complex mixture of wax esters with different chain lengths and different degrees of unsaturation. Analysis by G L C of wax esters using the method described by Kattner et al. (1990) allows the differentiation of wax esters according to the number of carbon atoms and the degree of unsaturation. However, separation of wax esters with the same carbon number and the same degree of unsaturation but different alkyl and acyl moieties has some limitations. With the additional analysis of fatty acids and alcohols, most of the gas chromatographic peaks could each be identified as one major single wax ester, as illustrated in Table 1. The wax ester compositions of C. hyperboreus and C. finmarchicus were dominated by different major wax esters for each species. Thus, C. hyperboreus contained generally higher proportions of long-chain wax esters than C.finmarchicus. Wax ester combinations of the 14 : 0 fatty acid and the 14 : 0 alcohol, which have been identified after transesterification, were generally not detected. Despite this, species-specific compositions were strongly influenced by spatial variabilities, i.e. the different phytoplankton communities on which the species were feeding. Calanusfinmarchicus, normally a boreal Atlantic species, was expatriated under the pack ice as a result of cross-frontal mixing, eddy advection or transport with the Return Atlantic Current (Hirche, 1989; Smith, 1990). Under the ice, primary production is very low (Spies, 1987; Smith et al., 1987), and is dominated by flagellates (Baumann, 1990). In this region both species of copepods showed conspicuously low levels of polyunsaturated wax esters, compensated by higher amounts of diunsaturates. In fact, C. hyperboreus contained more than half of its wax esters as diunsaturates. It seems that polyunsaturated wax esters were preferentially depleted during starvation or converted into long-chain wax esters or phospholipids. This species had also
278
M. GRAEVE AND G. KATTNER
a high amount of wax esters containing 16:1 fatty acid moieties, which indicates an uptake of diatom fatty acids before starvation. The 16:1 and 20 : 5 acids are known to be the dominant fatty acids of diatoms (e.g. Kates and Volcani, 1966; Ackman et al., 1968; Kattner et al., 1989). At the MIZ, phytoplankton and zooplankton production is often enhanced (Smith et al., 1987). The phytoplankton in this area was mainly composed of a summer population of dinoflagellates and large amounts of Phaeocystis pouchetii in a subsurface layer (Smith et al., 1987; Baumann, 1990). In contrast to the pack ice region, the wax ester composition of both Calanus species at the MIZ showed the dominant influence of the 18:4 ( n - 3) fatty acid moiety, which is the major fatty acid of Phaeocystis sp. (Sargent et al., 1985) and of dinoflagellates (Harrington et al., 1970). The MIZ C. hyperboreus biosynthesized de novo high levels of energy-rich long-chain wax esters such as 40:2, 42:2 and 44:2. High concentration of food may provide the energy for the biosynthesis of long-chain wax esters. On the other hand, the incorporation of unchanged dietary fatty acids into wax esters or their use as precursors for longer-chain moieties are less energyconsuming processes. If dietary fatty acids really serve as precursors for long-chain fatty acids and finally alcohols, only saturated acids are used. An indication of this conversion may be the small amounts of saturated 14 : 0 and 16:0 acids in wax esters of C. hyperboreus. Other fatty acids do not seem to be usable, because polyunsaturated alcohols have not been found in marine wax esters. Also, the large amount of 16:1 ( n - 7 ) acid moiety in wax esters of C. hyperboreus from the pack ice region was not preferentially used in the biosynthesized fatty acids and alcohols. A small part of the 16:1 ( n - 7 ) acid may have been converted into 20:1 ( n - 7 ) , which is generally less than 10% of the 20:1 fatty acid isomers. This chain elongation within the same fatty acid family is a possible biosynthetic pathway; the further elongation to the 22 : 1 ( n - 7) acid was not detected. A small portion of the 16 : 1 acid may also be converted into the corresponding alcohol. In contrast, C. finmarchicus contained high levels of saturated fatty acids and generally very low amounts of the 20 : 1 ( n - 7) acid (Kattner et al., 1989). Calanus hyperboreus seems to store more energy-rich lipids than C.finmarchicus, which may be explained by their different life strategies. The ratio between the wax esters of the dietary fatty acids 16:1 and 18:4 esterified with the 20:1 and 22:1 alcohols corresponded to the ratio of the 20:1 and 22:1 alcohols, which were analysed separately. Hence, it appears that the wax esters were synthesized according to the abundance of the alcohol moieties available. We found no preference for any particular combination. In C. hyperboreus the amount of long-chain wax esters increased from copepodid stage IV to females, whereas C. finmarchicus synthesized the energy-richest wax esters mainly in the copepodid stage V. The Arctic species C. hyperboreus is at least biennial (Dawson, 1978) and its spawning is not
WAX ESTERS OF C A L A N U S SPECIES
279
dependent on food (Conover, 1967), whereas the boreal species C. finmarchicus has only one generation per year (Lie, 1965). Reproduction in C. finmarchicus is coupled with the onset of phytoplankton blooms (Marshall and Orr, 1955) and thus is dependent on the food availability. The developmental time of copepods seems to play an important role in the production or the accumulation of long-chain wax esters. Only the older copepodid stages are able to accumulate large amounts of these lipids with a high calorific value. This would explain the species-dependent differences in the wax esters, which were obvious in spite of the spatial variability. CONCLUSION
The determination of intact wax esters allows an extended study of the lipid biochemistry of copepods especially from high latitudes, where herbivorous calanoid copepods are able to synthesize large wax ester stores. The wax ester compositions of C. hyperboreus and C. finmarchicus are clearly different in each species. Habitat and food availability considerably influenced the fatty acid and alcohol moieties of the wax esters. The copepods' wax ester compositions often reflect the fatty acid composition of their food. The incoporation of dietary fatty acids into wax esters and the alterations during, for instance, change of food are not yet clear and require further research. Obviously, wax esters containing dietary fatty acids decrease during starvation, which means that they are utilized preferentially or converted into other components. The different life cycles are also clearly reflected in the wax ester compositions. C. hyperboreus is better adapted to the unfavourable life in Arctic regions because it can produce large amounts of long-chain wax esters. These esters have the highest calorific value of those investigated in copepods to date. ACKNOWLEDGEMENTS
The authors would like to thank W. Hagen for helpful discussions. This study is Contribution 474 of the Alfred Wegener Institute for Polar and Marine Research. REFERENCES Ackman, R.G., Tocher, C.S. and McLachlan, J., 1968. Marine phytoplankter fatty acids. J. Fish. Res. Board Can., 25: 1603-1620. Baumann, M.E.M., 1990. Untersuchung zur Primfirproduktion und Verteilung des Phytoplanktons der Gr6nlandsee mit Kulturexperimenten zum EinfluB des Lichtes und der Temperatur auf Wachstum und Photosyntheseleistung arktischer Diatomeen. Dissertation, Rheinisch-Westf'filische Technische Hochschule, Aachen, 129 pp. Boon, J.J. and de Leeuw, J.W., 1979. The analysis of wax esters, very long mid-chain ketones and sterol ethers isolated from Walvis Bay diatomaceous ooze. Mar. Chem., 7: 117-132.
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Tande, K.S. and Henderson, R.J., 1988. Lipid composition of copepodite stages and adult females of Calanus glacialis in Arctic waters of the Barents Sea. Polar Biol., 8: 333-339. Wakeham, S.G. and Frew, N.M., 1982. Glass capillary gas chromatography-mass spectrometry of wax esters, steryl esters and triacylglycerols. Lipids, 17: 831-843.