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`Composition and Physical State of Phospholipids in Calanoid Copepods from India and Norway T. Farkasa.*, T. Storebakkenb and N.B. Bho$1ec
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`aBiological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Hungary, blnstltute or Aquaculture Research, The
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`Agricultural Center of Norway, N-1432 As-NLH, No~way, and CNatloflal Institute Oceanography, Dana Paula, Goa-403OO4, India The fatty acid composition and physical state of isolated phosphollpids obtained from marine copepods collected on the Southwest coast of India (Calanus ssp.) and the West coast of Norway (Calanus finmarchicus) were in- vestigated to compare the adaptation of membrane lipids with seawater temperatures. Phospholipid vesicles o1~ talned from the tropic copepods proved more rigid than those from C. finmarchicus, as assessed by diphenylhex- atriene fluorescence polarization techniques. In each case, there were two breaks present on the fluorescence polar- ization vs lfr plots, suggesting that the onset and com- pletion of phase separation to occurred above 0 C. For the tropic eopepods, the onset of phase separation roughly corresponded to the ambient water temperature, while for C. finmarchicus some discrepancies were observed, depending on the time of the year. Phospholipids in copepods from both habitats contained more than 50% unsaturated fatty adds, the animals from Norway con- tainlng slightly higher amounts. The data indicate an adaptation of membranes to temperature. Lipids 23, 619-622 {1988). Most of our knowledge concerning adaptation of the com- position and physical state of membranes to temperature in higher eukariotic systems is derived from observations made on freshwater organisms (1,2). The majority of these investigations demonstrate an inverse relationship be- tween the unsaturation of the constituent phospholipids and temperature (1,3-6), and also a varying degree of homeoviscosus response of membrane physical state to the temperature (7,8). Fatty acids are regarded as the most important factors controlling the physical state of these structures. A major difference between freshwater and marine species is the high level of long~hain polyun- saturated fatty acids in the latter. This should render their membranes more fluid. Based on the data available {2,9,10), the question remains whether they are able to control fatty acid composition according to temperature, by homeoviscosus adaptation. The oil sardine, Sardinella longiceps, responded by an increase in the level of docosahexaenoic acid and a decrease of saturated fatty acids when the temperature decreased from 30-31 C to 25-26 C from summer to winter ill). Absence of seasonal variation of phospholipid fatty acids was noted with two marine clam species (12} and with shrimp {13) as well as with Porphyra yezoensis exposed to cold for a prolonged time {14). However, in these investigations there was no direct determination of the effect of temperature on the fluidity of membrane phospholipids. In this study, the fatty acid composition and the physical state of phos- pholipids obtained from marine calanoid copepods col- lected, respectively, from tropic and temperate seas was investigated. *To whom correspondence should be addressed. Abbreviations: BHT, butylated hydroxytoluene; DPH, 1,6-diphenyl 1,3,5-hexatriene; P0 fluorescence polarization. MATERIALS AND METHODS Animals. Ca]anus finmarchicus were collected at the West coast of Norway, at Austevoll {60~ 5~ and Stangvik Fjord {62~ 8~ on April 13, and November 26, 1984, respectively. The water temperature was near 10 C in both cases. Calanoid copepods Calanus ssp.) also were collected on the West coast of India. One sample originated from the coastal waters off Bombay, collected one km offshore of the Gateway of India [19~ 72~ on February 16, 1981. Another sam- ple was collected at Dona Paula, Goa (15~ 72~ on January 20, 1986. The surface temperatures were 26 C and 25 C, respectively. No species were identified from the samples collected in India. The copepods originating from the Bombay area were large specimens resembling the size of C. finmarchicus Stage V, while those collected at Dona Paula were considerably smaller. The hauls of C. finmarchicus were almost 100% copepods, while those made in India were 90-95% copepods with some con- taminating decapod larvae. Analysis oflipids. The animals were fixed in chloroform/ methanol {2:1, v/v) cont~iulng 0.01% butylated hydroxy- toluene {BHT) and frozen until transferred to the labora- tory. The Folch procedure {15} was used to extract the total lipids. Phospholipids were separated by silicic acid column chromatography using chloroform to remove the neutral lipids and methanol to obtain the phospholipid fraction. The polar head group composition of the latter was determined according to Rouser et al. {16}. Phosphatidylcholine and phosphatidylethanolamlne were separated for further analysis by preparative thin layer chromatography using chloroform/methanol]water {65:25:4, v/v/v) as solvent. Identification of the spots was by comparing the R~ values to those of known standards {Supelco, Bellefonte PAL The spots were detected by spraying the plates with 0.05% 8-anillno-l-naphtalene sulfonic acid in 50% methanol and viewed under UV light. Total or individual phospholipids were transmethylated in the presence of 5% HC1 in absolute methanol at 80 C in sealed vials for 2.5 hr. A Hitachi 263-80 type gas chromatograph connected to a Hitachi M263 data proces- sor was used to separate the fatty acid methyl esters. The polar phase was 10% Carbowax 20M on 100-120 mesh Supelcoport in 2 m long stainless steel columns (3 mm i.&). The oven temperature was programed to rise linearly from 180 to 215 C, at 1 C/min. Each run was made in triplicate, and the error was not more than 1% in the case of the major fatty acids such as 22:6{n-3). Fluorescence polarization. Phospholipid vesicles were prepared and labeled with 1,6-diphenyl 1,3,5-hexatriene {Sigma Chemical Co., St. Louis, MO) as described by Montaudon et al. (17). A Perkin Elmer Model 44A fluorescence spectrophotometer equipped with a polariza- tion accessory and fitted with a temperature regulation unit was used for the measurements. The excitation wavelength was 370 nm, and the fluorescence emission
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`COMMUNICATIONS was monitored at 420 nm. Fluorescence polarization {P) was calculated from the equation, P -- Iw - Ivh/Ivv + Ivh (cid:12)9 Z, in which Iw and Ivh are the fluorescence inten- sities measured with emission analyzer parallel or perpen- dicular, respectively, to the polarization of the detection system for vertically and horizontally polarized light. RESULTS AND DISCUSSION Figure 1 presents a Vant Hoffs representation of 1,6- diphenyl 1,3,5-hexatriene (DPH) fluorescence polariza- tion, P, of vesicles of phospholipids prepared from marine calanoid copepods collected either in the North Atlantic or in tropic seas. Lower P-values represent more fluid structures. From these data, it may be inferred that the most fluid membranes were present in C. finmarchicus collected in the spring and the most rigid ones in the copepods inhabiting the tropic seas. C. finrnarchicus sampled in early fall revealed values in between these two extremes. In addition, there are two distinct breaks in the curves on the P vs 1/T plots. The break at the higher temperature indicates onset, while that at the lower temperature indicates completion of phase separation of these phospholipids. The sea water temperature at the
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`33 3, 3s 36 37 x_ K T-- FIG. 1. Temperature dependency (liT) of DPH fluorescence polariza- tion (P) in phosphoHpids of marine calanoid copepods. Calanus spp. were collected at Dona Paula, Goa (--V--) and Bombay (--A--) at the Southwest coast of India, while C. Fmmarchicus were collected at the West coast of Norway in the fall (--D--) and in the spring (--O--), respectively. time of collecting C. finrnarchicus was about 10 C but varied between 2 and 20 C during the year. The tempera- ture in the tropic seas was about 25-26 C at the time of sample collecting and varied from 25 to 30-31 C during the year. The temperature at the onset of phase separa- tion of phospholipids from each sample was close to the temperature at which the organism lived, except in the C. finmarchicus collected in the fall. However, it should be remembered that the above values for phospholipid vesicles might be modulated if other membrane consti- tuents (proteins, sterols, etc.} also were present. The observation that the temperature at onset of the phase separation of the membrane lipids coincides with growth temperature has been made with other poikilo- therms (10,18-20}, but it is not documented as a general phenomenon {7,21}. Cossins and Prosser (18} reported that the onset of phase separation of phospholipids from synaptosomal membranes of arctic sculpin adapted to 0 C occurred at 5 C while that for goldfish acclimated at 5 C occurred at 10 C. It can be inferred from Figure 1 of Prosser and Cossin's paper that phospholipids of synap- tosomal membranes of goldfish adapted at 25 C show phase separation around the growth temperature {18}. In a current study of liver phospholipids of the carp, Cyprinus carpio L., we found that the onset of phase separation of phospholipids of summer-adapted fish oc- curred around 25 C, while that of winter-adapted fish oc- curred around 6 C (unpublished observations}. In earlier work on the freshwater copepod Cyclops vicinus, we also found that phase separation temperatures were similarly related to the actual growth temperature (10). Although the water temperature at the time of collecting C. fin- marchicus was the same in the fall and the spring (10 C), the higher polarization value and phase separation tem- perature of phospholipids of the fall sample may suggest that the fall specimens retained a "summer" state in their lipids. Whether marine species similar to C. vicinus can regulate the physical state of their phospholipids accord- ing to the temperature or whether they lack this prop- erty, like the freshwater crustacean Daphnia magna, re- quires further investigation. Some freshwater crustaceans and fish are exposed to fluctuation in their environmen- tal temperature. In cases of tropic seas, this is less pro- nounced. Judging from the temperature range at which the phase separation occurs, it may be inferred that C. finmarchicus can tolerate less changes in the water tem- perature than the copepods in the tropic seas. Spring-collected C. finmarchicus did not survive ex- posure to 20 C longer than two hr, and copepods collected at Dona Paula lost their swimming activity but did not die when exposed to 17 C for six hr {unpublished obser- vations}. Because the former can be regarded as a cold stenothermic and the latter as a warm stenothermic species, it is tempting to speculate that this is at least partially related to the phase behavior of their membranes. Table 1 shows that the above differences in the phase behavior are not easily explained by the fatty acid composition of the phospholipids. An inverse relationship between environmental temperature and fatty acid un- saturation also was observed in this case. This was due mainly to a higher level of docosahexaenoic acid in phos- pholipids of C. finmarchicus. Despite these differences,
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`TABLE 1 Fatty Acid Composition (mol %) in Phospholipids of Calanoid Copepods Species Calanus app. Origin India a India b Water temperature (~ 25 26 C. finmarchius Norway c Norway d 10 10 14:0 4.7 3.9 3.4 2.4 14:1 0.2 0.1 0.5 0.1 15:0 0.2 0.3 0.7 0.2 15:1 0.2 tr 0.1 tr 16:0 19.6 16.0 16.5 15.2 16.1 1.4 1.6 0.3 0.7 16:2 0.9 0.3 0.1 0.3 16:3 1.3 0.2 0.7 0.1 18:0 8.7 4.0 2.0 0.8 18:1 4.5 3.7 2.9 4.5 18:2 1.5 1.5 0.8 0.6 18:3 1.7 1.8 0.7 tr 18:4 0.3 2.5 1.9 0.2 20:1 tr 0.2 0.I 1.2 20:4 2.5 4.5 1.2 1.9 20:5 15.7 25.2 24.1 30.2 22:3 0.5 0.2 0.2 0.2 22:4 0.2 0.1 0.2 0.3 22:5 2.4 2.3 0.4 0.5 22:6 33.4 31.2 42.9 40.0 Sat/unsat 0.49 0.31 0.29 0.22 Total polyen(%) 55.0 66.1 71.0 73.8 aBombay. bDona Paula, Go& cSpring. dFall. the spring~ollected C finmarchicus and the copepods col- lected at Dona Paula showed similar saturated to un- saturated ratios but great differences in the phase b~ havior of their phospholipids. Moreover, the two samples of copepods from the tropic seas showed differences in phospholipid fatty acid compositions as well as in the saturated to unsaturated fatty acid ratios, although the P-values and the phase separation temperatures were almost identical {Fig. 1). Thus, it is highly probable that control occurs at a level beyond the overall distribution of fatty acids in phospholipids. Table 2 shows that the phospholipids of copepods from tropic seas were poorer in sphingomyelin and phosphatidic acid, and richer in phosphatidylethanolarnlne than those in C. finmarchicus. Phosphatidylcholines in spring-collected C finmarchicus contained more polyunsaturated acids (82% vs 62%) and had a lower saturated-to-vn~aturated fatty acid ratio {0.15 vs 0.38) than those of the tropic copepods (Table 3). Even though phospholipids were not separated according to molecular-species composition, one could expect that di- nn.qaturated phospholipids would be present whenever the level of total unsaturated fatty acids exceeds 50 tool %. As shown in Table 3, the phosphatidylcholines and phos- phatidylethanolamines were richer in diunsaturated phos- pholipids than were the phospholipids of copepods in the tropic seas (32% vs 12% and 17% vs 11%, respectively), and this could explain the observed differences in the P- values {Fig. 1). Because the phase separation temperature TABLE 2 Composition of PhosphoHpids (% wt) in CAIAnoid Copepods Species: Calanus app. Calanus finmarchius Origin: India a Norway b Phosphatidic acid 3.6 10.1 Phosphatidylserine 3.1 8.9 Phoaphatidylinositol 3.9 6.4 Lysophosphatidyl- ethanolsmine -- 5.2 Lysophosphatidyl- choline 6.8 I. 1 Sphingomyelin 3.9 7.6 Phosphatidylcholine 35.6 29.3 Phosphatidyl- ethanolamine 28.9 22.3 Cardiolipin 6.8 9.8 aDona Paula, Goa. bSpring. TABLE 3 Fatty Acid Composition (mol %) of Phosphatidylcholines and Phosphatidylethanolamines in Calenoid Copepods Phospholipid Phosphatidyl- choline Origin India a Norway b Phosphatidyl- ethanolamine India a Norway b 14:0 3.6 2.5 1.0 0.5 14:1 tr tr tr tr 15:0 0.4 0.1 tr tr 16:0 20.9 10.2 20.4 24.6 16:1 1.0 1.1 0.4 0.1 18:0 2.6 0.2 12.4 2.2 18:1 6.4 2.6 2.6 4.3 18:2 1.8 0.7 1.3 1.3 18:3 2.0 0.2 0.9 0.3 18:4 2.4 1.7 0.5 1.5 20:3 tr tr tr tr 20:4 4.9 5.0 6.0 3.3 20:5 21.9 42.8 14.3 9.8 22:4 0.7 tr 0.3 0.7 22:5 2.2 0.5 3.0 1.6 22:6 28.2 32.0 36.7 50.00 Sat/unsat 0.38 0.15 0.51 0.38 Total polyen(%) 62.0 82.0 61.0 67.0 aCalanus sap., Dona Paula, GoeL bc finmarchicus, spring. of 1-palmitoyl,2~iocosahexaenoyl phosphatidylcholine is about - 10 C (22) and that of diunsaturated molecules is even lower, phospholipids of marine copepods should ex- hibit lower phase-separation temperatures than those observed. Phospholipids of these copepods behave simi- larly to those of the bovine retinal rod outer segment membranes. Although the latter are as rich in polyenes as the phospholipids of copepods investigated here, they too contain fair amounts of supraenes (dipolyunsaturated phospholipids) (23,24) and exhibit phase-separation temperature between 15 and 5 C (25).
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`COMMUNICATIONS It has been proposed that this results from a precise balance between disaturated and diunsaturated phospho- lipid molecules (25). The differences demonstrated in physical parameters of phospholipid vesicles indicate an adaptation of membrane physical states to temperatures. ACKNOWLEDGMENTS We thank S. K. Chekraborty, Central Institute of Marine Fisheries, Bombay, India, for making possible the collecting of marine copepods. Than~s go also to E. Lehoczki (Jozsef A. University, Szeged, Hungary) fox help in fluorescence polarization measur~m,~ts. REFER1ENCES 1. Hazel, J., and Prosser, C.L. (1974) PhysioL Rev. 54, 620-667. 2. Dutta, H., Das, A., Das, A.B., and Farkas, T. 11985) Comp. Biocher~ PhysioL 81B, 341-347. 3. Hazel, J. (1979} Am. J. Physiol 236, 91-101. 4. Farkas, T., and Csengeri, I. (1976) Lipids 11, 401-407. 5. Wodtke, E. (1978) Biochir~ Biophys. Acta 629, 280-291. 6. Van dan Thillart, G., and Bruin, G. (1981) Biochir~ Biophys. Acta 640, 439-447. 7. Co~i~ A.R., Christiansen, J., and Pross~, C.L. (1978) B/och/r~ Biophys. Acta 511, 442-454. 8. Cossins, A.R., and Prosser, C.L. (1982) Biochim. Biophys. Acta 687, 303-309. 9. Farkas, T. (1979) Comp. Biochem. PhysioL 64B, 71-76. 10. Farkas, T., Nemecz, Gy., and Csengeri, I. (1984) Lipids 19, 436-442. 11. Gopakumar, K. (1973) J. Mar. BioL Assoc. (India)14, 2-5. 12. B~nlnoer, P.G., and StePhan, G. (1985) Comp. Biochent PhysioL 81B, 591-601. 13. Bottino, N.R., Gennity, J., Lilly, M.I.,, Simons, E., and Finns, G. (1980) Aquaculture 19, 139-148. 14. Kayama, M., Iijima, N., Kuwahara, M., Sado, T., Araki, S., and Sakural, T. (1985) Bull Jpra Soc ScL Fisk 51, 687. 15. Folch, J., Lees, M., and Sloans-Stanley, G.H. (1957) J. BioL Chem. 226~ 497-507. 16. Rouser, G., Fleischer, S., and Yamamoto, A. (1970) L/p/ds 5, 494-496. 17. Montaudon, D., Robert, J., and Canguilhe~ B. (1984) B/ochem. Biophys~ Res. Commu~ 119, 396-400. 18. Cossins, A.P~, and Prosser, C.L. (1978) Pro~ NatL AcacL ScL USA 75, 2040-2043. 19. Janoff, A.S., Gupte. S., and McGroaty, E.J. (1980) Biochim. Biophys. Acta 59~ 641-644. 20. Durairay, G.I., and Vijakumar, I. (1964)BiochitrL Biophys. Ac- ta 770, 7-14. 21. Cossins, A.R., and Prosser, C.L (1982) Biochim. Biophy~ Ac- ta 687, 303-309. 22. Coolbear, K.P., Redde, C.B., and Keough, K.M.W. (1983) Biochemistry 22, 1466-1473. 23. Milajanich, G.P., Sklar, L.A., White, D.L., and Dratz, E.A. (1979) Biochir~ Biophys. Acta 552, 294-306. 24. Aveldano, M.I., and Bazan, N.G. (1983) J. L/p/dRea 24 620-627. 25. Sklar, L.A., Miljsnich, G.P., Bruster~ S.L., and Drat~ E.A. (1979) B/ochem/stry 254, 9583-9591. [Received January 26, 1987; Revision accepted February 16, 1988]
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