Short communication
Temperature eects on lipid composition of the earthworms
Lumbricus rubellus
and
Eisenia nordenskioeldi
Sùren O. Petersen
a,*, Martin Holmstrup
ba
Danish Institute of Agricultural Sciences, Department of Crop Physiology and Soil Science, P.O. Box 50, DK-8830 Tjele, Denmark b
National Environmental Research Institute, Department of Terrestrial Ecology, Vejlsùvej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark
Accepted 16 February 2000
Abstract
Two earthworm species, the freeze-intolerant Lumbricus rubellus (Homeister) and the freeze-tolerant Eisenia nordenskioeldi
(Eisen), were compared with respect to the eects of temperature on lipid composition. The animals were incubated at either 08C or 208C for 28 days, and then analyzed for the composition of free and ester-linked long-chain fatty acids, as well as other lipids with similar properties. Inter-species dierences dominated the patterns of lipid composition, and the two species responded similarly to incubation temperature with respect to changes in lipid composition. In particular, polyunsaturated fatty acids were present in higher concentrations at 08C, while straight-chain saturated fatty acids were more common at 208C. The results did not implicate specialized lipid adjustments in the freeze-tolerance ofE. nordenskioeldi.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Earthworm; Freeze-tolerance; Fatty acid; Unsaturation; Cholesterol; Adaptation
1. Introduction
Like other poikilotherms, earthworms use one of two main strategies for coping with environmental temperatures below the freezing point of their body ¯uids (Holmstrup and Zachariassen, 1996). One strat-egy is based on freeze-avoidance by either migration or physiological adaptation, such as the synthesis of cryo-protectants to avoid formation of ice within the animal. The second strategy, termed freeze-tolerance, allows formation of extracellular ice, which leads to dehydration (and thus, a lower freezing point) of cells due to freeze concentration of unfrozen extracellular ¯uids.
Because of their intimate contact with the soil water phase, earthworms are prone to inoculative freezing, and they survive freezing soil temperatures mainly by
migration to unfrozen soil layers. Many species also produce cold-tolerant cocoons which may enable populations to persist in cold regions. However, pre-vious studies (Berman and Leirikh, 1985; Holmstrup and Petersen, 1997; Holmstrup et al., 1999) have docu-mented that a Siberian species, Eisenia nordenskioeldi
(Eisen), living in areas with permafrost, has developed freeze-tolerance as a mechanism for survival in this extreme habitat, where more than one summer season is required to complete a life cycle (Mazantzeva, 1985; Holmstrup and Petersen, 1997). A recent study has shown that E. nordenskioeldi synthesizes and accumu-lates glucose as an immediate response to ice for-mation in extracellular body ¯uids (Holmstrup et al., 1999). In the freeze-intolerant Lumbricus rubellus
(Homeister) glucose accumulation was also detected upon freezing, but at much lower concentrations. These observations suggest that glucose loading is essential for freeze-tolerance in earthworms, but other factors could also be involved.
Cell membranes contribute to the maintenance of
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* Corresponding author. Tel.: 1723; fax: +45-8999-1619.
concentration gradients between intracellular and extracellular spaces through selective transport mech-anisms. These mechanisms are in¯uenced by mem-brane constitution, which in turn is in¯uenced, among several factors, by temperature (Hazel, 1995). A litera-ture search revealed no information regarding inter-actions between temperature and lipid composition in earthworms; but other invertebrates, as well as higher organisms and microbes, have been shown to respond to temperature changes by adjusting the composition of membrane lipid fatty acids (Hazel, 1995). The ex-periment reported here was carried out to examine whether the unique freeze-tolerance of E. nordens-kioeldi compared to that of other species was also re¯ected in their ability to modify lipid composition when incubated at low temperature.
2. Materials and methods
Freeze-tolerant specimens of E. nordenskioeldi and freeze-intolerant specimens of L. rubellus were kept at 208C in the laboratory in soil cultures (Holmstrup et
al., 1999). E. nordenskioeldi was collected at the Tai-myr Peninsula, Siberia, in 1997 and a laboratory cul-ture was established. L. rubellus was collected in the vicinity of Silkeborg, Denmark. The animals used in this study had been kept for 2 months at 208C. This is above the optimal temperature (158C), but both species grow and reproduce at 208C. Groups of each species were transferred to 08C and incubated for 28 days, and then three specimens of both species were killed by quick freezing at ÿ808C, freeze-dried, and
®nely ground in a mortar. Lipid composition was determined on 30±100 mg subsamples from each indi-vidual; in three cases the number of detectable peaks was so low that the samples had to be omitted from further data analysis.
Pro®les of long-chain fatty acids and other lipids of similar molecular size were produced according to a standard procedure for preparation of whole-cell fatty acid methyl esters (Kennedy, 1994). Samples were saponi®ed (hydrolyzed) in 10 ml test tubes with te¯on-lined screw caps by adding 1 ml 4 N NaOH in 50% methanol and heating for 30 min in a 1008C water bath. After cooling, 2 ml 6 N HCl in methanol was added for methylation of dissolved fatty acids in an 808C water bath (10 min). Then 1 ml
hexane:methyl-tert-butyl ether (1:1, v/v) was added and lipid material extracted by shaking end-over-end for 10 min. The or-ganic phase was transferred to a new test tube and the extraction repeated. The combined organic phase was washed once with 0.25 N NaOH, and subsequently transferred to 2-ml vials for analysis on a Hewlett-Packard 5890 gas chromatograph equipped with a 25 m fused silica capillary column (Ultra 2;
Hewlett-Pack-ard, Birkerùd, Denmark) and FID detector. The initial temperature was 1708C, increasing at 58C per minute
until 2708C. Temperatures of injection port and
detec-tor were 1708C and 3008C, respectively, and He was
used as carrier. Automated integration and peak identi®cation was done by the software supplied by the Microbial Identi®cation System (M.I.S.; Newark, Delaware, USA) using a calibration mixture before and during the analytical sequence for con®rmation of retention times.
Fatty acids are designated asX:YoZ, whereXis the
number of carbon atoms, Y the number of double bonds, and Zindicates the position of the ®rst double bond from the methyl end of the molecule, if known. Branching is indicated by pre®xes i or a, and c stands for cis con®guration around double bonds. The mol percentage distribution of identi®ed compounds was calculated by dividing integrated areas by molecular weights. These data were used for a principal com-ponent analysis (PCA) of mol percentage distributions after log n1 transformation. The degree of unsa-turation was calculated as (Kates, 1986):
X
%monoenes2%dienes3%trienes =100:
3. Results and discussion
A total of 15 long-chain fatty acids and eight other identi®ed compounds were consistently present after saponi®cation and acid methylation, and no com-pound was associated exclusively with one species
Fig. 1. Mol percentages of the lipid compounds extracted from E. nordenskioeldi and L. rubellus (listed in Table 1) were log n1
transformed and used for a principal component analysis. A Ð Scores for PC1 and PC2, with the percentage variation explained by each component in parentheses. Key: R0:L. rubellusat 08C; R20:L.
rubellusat 208C; N0:E. nordenskioeldiat 08C; N20:E. nordenskioeldi
at 208C. B Ð Component loadings. The key to lipid compounds is
given in the left-hand column of Table 1.
S.O. Petersen, M. Holmstrup / Soil Biology & Biochemistry 32 (2000) 1787±1791
Mol percentage distribution of long-chain fatty acids and other lipids detected after saponi®cation and acid methylation ofE. nordenskioeldiandL. rubellus
Code E. nordenskioeldi L. rubellus
08C 208C 08C 208C
Mean Standard Deviation Mean Standard Deviation Mean Standard Deviation Mean Standard Deviation
Straight chain fatty acids
S1 16:0 0.83 0.14 1.07 0.00 1.62 0.12 2.09 0.22 S2 17:0 1.69 0.01 2.14 0.07 1.92 0.02 1.96 0.08 S3 18:0 5.41 0.40 5.89 0.32 5.68 0.90 6.35 0.66 Branched chain fatty acids
B1 i15:0 2.05 0.11 3.20 0.07 1.58 0.10 1.99 0.21 B2 i16:0 0.82 0.07 0.72 0.06 0.52 0.07 0.73 0.09 B3 i/a17:1 1.62 0.17 1.93 0.07 3.14 0.09 3.02 0.26 Monounsaturated fatty acids
M1 18:1o9c 2.58 0.22 3.31 0.17 4.89 0.56 4.60 0.40 M2 17:1o7c 7.71 0.27 7.66 0.21 5.28 0.14 7.07 0.87 M3 20:1o11/12c 7.92 0.85 8.88 0.08 7.91 0.19 10.73 0.34 Polyunsaturated fatty acids
P1 18:2o9c 4.59 0.35 3.45 0.22 2.32 0.32 2.49 0.19 P2 20:4o6c 10.54 2.83 9.83 0.10 14.67 0.17 11.44 1.36 P3 20:5o3c 11.54 0.61 9.68 0.98 12.57 0.08 10.49 1.29 P4 20:3o6c 1.90 0.03 2.19 0.31 1.85 0.47 1.38 0.23 P5 22:4o6c 1.90 0.52 1.66 0.37 1.18 0.13 1.21 0.10 P6 22:5o3c 1.51 0.17 0.97 0.11 1.21 0.04 0.96 0.16 Degree of unsaturation 1,48 0,11 1,33 0,08 1,61 0,03 1,39 0,02 Sterols
St1 Cholesterol 21.94 0.99 21.92 1.74 22.95 2.14 21.21 2.50 St2 Cholestanol 2.06 0.32 2.59 0.35 1.99 0.57 1.88 0.21 St3 Campesterol 2.15 0.09 2.31 0.05 2.54 0.19 3.26 0.48 Dimethyl acetals
D1 i17:0 5.35 0.10 4.97 0.29 2.06 0.39 2.36 0.20 D2 18:1o7c 1.80 0.22 1.59 0.23 1.20 0.05 1.23 0.46 Alcohols
A1 C17 1.71 0.13 1.94 0.18 1.19 0.17 1.44 0.16
A2 19:1 0.75 0.03 0.75 0.24 0.60 0.00 0.75 0.12
A3 C20 1.62 0.10 1.33 0.29 1.11 0.13 1.38 0.21
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(Table 1). The predominant compound was always cholesterol, followed by the polyunsaturated fatty acids arachidonic acid (20:4o6c) and eicosapentanoic
acid (20:5o3c).
Inter-species dierences in fatty acid composition were evident and samples fromL. rubellus and E. nor-denskioeldi species were separated along the PC 1 axis (Fig. 1A). However, an eect of incubation tempera-ture was also seen for both species along PC 2. The loadings (Fig. 1B), as well as the percentages given in Table 1, show that polyunsaturated fatty acids tended to be present at higher concentrations in animals incu-bated at 08C, while straight-chain saturated fatty acids
were more common at 208C. This is in agreement with
the theory of homeoviscous adaptation, according to which organisms will introduce double bonds at decreasing temperatures in order to counteract mem-brane crystallization (Hazel, 1995). Speci®cally, the proportion of 20:5o3c and 20:4o6c increased at 08C
compared to that at 208C. Albro et al. (1992) reported
that 20:5o3ccan be biosynthesized by L. terrestris, but
its metabolic precursor, 18:3o3c, was not detected at
all. It indicates that the changes observed may have been partly due to selective biodegradation rather than biosynthesis.
The statistical signi®cance of the temperature eects observed is not easily determined for a data set with many variables, as discussed by Petersen and Klug (1994). Instead the degree of unsaturation, a weighted expression for the number of double bonds, was used to test the dierences (Table 1). For L. rubellus the degree of unsaturation was signi®cantly lower at 20 than at 08C (P= 0.003), but not forE. nordenskioeldi
(P= 0.24), although the tendency was the same as for
L. rubellus.
The fatty acids determined may have been derived from several dierent lipid classes, among which only phospholipids, the main component of cell membranes, are involved in temperature adaptation. No infor-mation about lipid composition is available for the two species used in this study, but Kholodova et al. (1991) reported that phospholipids constituted ca. 50% of total lipids in E. fetida, while Lee et al. (1988) reported that phospholipids represented 23% of total lipids in L. terrestris. In contrast, Albro et al. (1992) found that lipids in L. terrestris contained ca. 60% phospholipids. Both of these species have been reported to have a high proportion of ether-containing phospholipids (Okamura et al., 1985; Sugiura et al., 1995) which are not released by the saponi®cation (Kates, 1986); this is likely to be the case also for L. rubellusandE. nordenskioeldi. This would suggest that phospholipids were only one of several sources of the fatty acids observed. On the other hand, among seven lipid classes isolated from L. terrestris by Albro et al. (1992), the fatty acid composition of their
phospholi-pid class was much more similar to the fatty acid com-positions observed in the present study than any of the other classes described by Albro et al. (1992).
The limited temperature eects on lipid composition may be partly due to the lipid biochemistry of earth-worms. Cholesterol is an important membrane con-stituent in earthworms (McLaughlin, 1971; Okamura et al., 1985) and involved in regulating membrane ¯uidity, but no signi®cant dierence was observed in response to temperature with either species (Table 1). Earthworms rely on an external supply of sterols via their diet (Albro et al., 1993), and the absence of changes in this component may, therefore, re¯ect the absence of cholesterol intake. Also, Albro et al. (1993) observed a slow turnover rate for the polar lipid class phosphatidyl ethanolamine which would mediate against temperature adjustments of membrane lipid composition.
In order to avoid physiological stresses other than the imposed temperature the animals used in this study were not aseptic. In theory, the earthworm gut micro-¯ora may have contributed to the results presented above. The earthworm gut contains a somewhat specialized micro¯ora typically dominated by Gram-negative bacteria and actinomycetes (Brown, 1995) which may even proliferate during the passage in some earthworm species (Kristufek et al., 1994). However, the absence of common bacterial fatty acids such as 16:1o7c, and hydroxy fatty acids from cell wall
lipopo-lysaccharides, as well as the actinomycete biomarker 10Me18:0 (Lechevalier, 1977), suggests that the fatty acids observed were predominantly derived from worm lipids.
In summary, the two species responded similarly to the temperature dierence, and there was no evidence for unique ways of temperature adaptation via changes in membrane lipid composition in the freeze-tolerant
E. nordenskioeldi. Hence, the results presented here support the hypothesis that glucose loading is the most important physiological adaptation promoting freeze-tolerance.
Acknowledgements
We would like to thank Ann C. Kennedy, USDA-ARS, Pullman, WA, for the opportunity to analyze these samples during a working stay of SOP. We also thank Dr. P.W. Albro and Dr. V. Kristufek for valu-able comments to the manuscript.
References
Albro, P.W., Schroeder, J.L., Corbett, J.T., 1992. Lipids of the earthwormLumbricus terrestris. Lipids 27, 136±143.
S.O. Petersen, M. Holmstrup / Soil Biology & Biochemistry 32 (2000) 1787±1791
Albro, P.W., Corbett, J.T., Schroeder, J.L., 1993. Endogenous lipids of the earthworm Lumbricus terrestris. Biochemistry and Cell Biology 71, 220±221.
Brown, G.G., 1995. How do earthworms aect micro¯oral and fau-nal community diversity? Plant and Soil 170, 209±231.
Berman, D.I., Leirikh, A.N., 1985. The ability of the earthworm
Eisenia nordenskioldi (Eisen) (Lumbricidae, Oligochaeta) to endure subfreezing temperatures. Proceedings of the Academy of Sciences USSR 285, 1258±1261.
Hazel, J.R., 1995. Thermal adaptation in biological membranes: Is homeoviscous adaptation the explanation? Annual Reviews of Physiology 57, 19±42.
Holmstrup, M., Petersen, B.F., 1997. Freeze-tolerance in the subarc-tic earthworm Eisenia nordenskioeldi (Eisen). Cryo-Letters 18, 153±156.
Holmstrup, M., Zachariassen, K.E., 1996. Physiology of cold hardi-ness in earthworms. Comparative Biochemistry and Physiology 115A, 91±101.
Holmstrup, M., Costanzo, J.P., Lee, R.E., 1999. Cryoprotective and osmotic responses to cold acclimation and freezing in freeze-toler-ant and freeze-intolerfreeze-toler-ant earthworms. Journal of Comparative Physiology B 169, 207±214.
Kates, M., 1986. Techniques in Lipidology, 2nd ed. Elsevier, Amsterdam.
Kennedy, A.C., 1994. Carbon utilization and fatty acid pro®les for characterization of bacteria. In: R.W. Weaver et al. (Eds.), Methods of Soil Analysis. Part 2: Microbiological and Biochemical Properties. Soil Science Society of America, Madison, WI, USA, pp. 543±556.
Kholodova, I.D., Mironova, V.N., Povkhan, M.F., Golodnii, N.M.,
Berdyshev, A.G., Bulevskii, N.V., Melnik, I.A., 1991. Lipid and fatty acid composition of Eisenia foetida. Ukarinskii Biokhimicheskii Zhurnal 63, 76±81 (in Russian, cited from Abstract in the database Medline).
Kristufek, V., Tajovsky, K., Pizl, V., 1994. Ultrastructural analysis of the intestinal content of earthwormLumbricus rubellusHom. (Annelida, Lumbricidae). Acta Microbiologica et Immunologica Hungarica 41, 283±290.
Lechevalier, M.P., 1977. Lipids in bacterial taxonomy: a taxono-mist's view. Critical Reviews in Microbiology 5, 109±210. Lee, H.K., Kim, H.S., Adachi, S., Ito, S., Okamoto, K., Kametaka,
M., 1988. Occurrence of unusual fatty acids in the earthworm
Lumbricus terrestris. Agricultural and Biological Chemistry 52, 2379±2380.
Mazantzeva, G.P., 1985. Soil fauna and soil fertility. In: Striganova, B.R. (Ed.), Proceedings of the 9th International Colloqium on Soil Zoology. Nauka, Moscow, pp. 139±143.
McLaughlin, J., 1971. Biochemical studies on Eisenia foetida
(Savigny, 1826), the brandling worm. Part I: Tissue lipids and sterols. Comparative Biochemistry and Physiology 38B, 147±163. Okamura, N., Stoskopf, M., Yamaguchi, H., Kishimoto, Y., 1985.
Lipid composition of the nervous system of earthworms (Lumbricus terrestris). Journal of Neurochemistry 45, 1875±1879. Petersen, S.O., Klug, M.J., 1994. Eects of sieving, storage, and
in-cubation temperature on the phospholipid fatty acid pro®le of a soil microbial community. Applied and Environmental Microbiology 60, 2421±2430.
