Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster

ARTICLE IN PRESS

Journal of Insect Physiology 51 (2005) 1173–1182 www.elsevier.com/locate/jinsphys

Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster Johannes Overgaarda,b, Jesper G. Sørensenb, Søren O. Petersenc, Volker Loeschckeb, Martin Holmstrupa,b, a Department of Terrestrial Ecology, National Environmental Research Institute, Vejlsøvej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark Aarhus Centre for Environmental Stress Research (ACES), Department of Genetics and Ecology, Institute of Biological Sciences, University of Aarhus, Building 540, DK-8000 Aarhus, Denmark c Department of Agroecology, Danish Institute of Agricultural Sciences, P.O. Box 50, DK-8830 Tjele, Denmark

b

Received 21 March 2005; received in revised form 13 June 2005; accepted 15 June 2005

Abstract Naturally occurring diurnal variations in temperature are sufficient to induce a rapid cold hardening (RCH) response in insects. RCH can increase cold tolerance by 1–2 1C and extend the temperature interval at which insects can remain active. While the benefits of RCH are well established, the underlying physiological mechanisms remain unresolved. In this study we investigated the role of RCH on expression of heat shock proteins (Hsp70) after a cold shock, and the effect of RCH on the composition of phospholipid fatty acids (PLFAs) in membranes of Drosophila melanogaster. These experiments were performed on both ‘‘control’’ flies and flies selected for cold resistance in order to additionally examine a possible target for selection for cold tolerance. RCH improved survival following cold shock at
4,
6 and
8 1C. No induction of Hsp70 was found following cold shock irrespective of the pre-treatment. In contrast, a 5 h RCH treatment was sufficient to induce small, but significant, changes in the composition of PLFAs. Here, the polyunsaturated linoleic acid, 18:2(n
6), increased while monounsaturated (18:1) and saturated (14:0) PLFAs decreased in abundance. These changes were observed in both selection groups and caused a significant increase in the overall degree of unsaturation. This response is consistent with the membrane response typically found during cold acclimation in ectothermic animals and it is likely adaptive to maintain membrane function during cold. Cold selection resulted in PLFA changes (decrease of 18:0 and 18:1 and increase of 14:0 and 16:1), which may improve the ability to harden during RCH. r 2005 Elsevier Ltd. All rights reserved. Keywords: Rapid cold hardening; Phospholipid composition; Polyunsaturated fatty acids; Hsp70; Cold selection

1. Introduction Short episodes of subzero temperatures are potentially lethal for many insect species including the fruitfly, Drosophila melanogaster. The mortality caused by cold shock is not due to internal ice formation and occurs at temperatures well above their supercooling point (the Corresponding author. Department of Terrestrial Ecology, National Environmental Research Institute, Vejlsøvej 25, PO Box 314, 8600 Silkeborg, Denmark. E-mail address: [email protected] (M. Holmstrup).

0022-1910/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2005.06.007

temperature at which spontaneous formation of ice occurs) (Lee and Denlinger, 1985; Knight et al., 1986). For example, a 2 h exposure to
5 1C induces 50% mortality in D. melanogaster even though the supercooling point of this species is around
20 1C (Czajka and Lee, 1990). Instead, mortality following cold shock is likely to be associated with loss of membrane function as cold shock causes dissipation of trans-membrane gradients of Na+ and K+ and a depolarisation of the membrane (Kelty et al., 1996; Kostal et al., 2004). Tolerance to cold shock is improved by prior exposure to sub-lethal low temperatures (acclimation/

ARTICLE IN PRESS 1174

J. Overgaard et al. / Journal of Insect Physiology 51 (2005) 1173–1182

hardening). D. melanogaster survived 1 h at either
7 or
8 1C when the rate of cooling to these temperatures was slowed to ecologically relevant rates (0.05–0.1 1C min
1) while flies cooled directly to the target temperature only survived 1 h at
6 1C (Czajka and Lee, 1990; Kelty and Lee, 1999, 2001). The protective effect gained by gradual cooling is termed rapid cold hardening (RCH), a type of cold hardening that has also been demonstrated in several other species of insects (Burks and Hagstrum, 1999; Chen et al., 1987; Larsen and Lee, 1994; Powell and Bale, 2004). It has been demonstrated that RCH lowers the critical thermal minimum in D. melanogaster (i.e. the lowest temperature at which the flies can remain active) (Kelty and Lee, 1999), and RCH preserves reproductive behaviour during modest cooling (Shreve et al., 2004). Thus, the physiological adjustments associated with rapid cold hardening are important for the ability of flies to maximize survival and activity in an environment with fluctuating temperatures. Although RCH responses have been demonstrated in many insect species, the physiological basis remains elusive (Kelty and Lee, 1999, 2001; Sinclair et al., 2003). The hardening is part of a fundamental cellular response rather than a centrally regulated process as the protection afforded by RCH is similar for in vitro tissue cultures and for samples obtained following in vivo hardening (Yi and Lee, 2004). Previous efforts have aimed at determining possible roles of both cryoprotectants and stress proteins such as heat shock proteins, particularly, Hsp70 (Chen et al., 1987; Kelty and Lee 1999, 2001; Sejerkilde et al., 2003). Cryoprotectants such as glycerol and trehalose play an important part in the long-term cold tolerance found in winter hardy insects (Zachariassen, 1985; Lee, 1991), but the role of cryoprotectants in RCH is ambiguous. Thus, Chen et al. (1987) found only slight increases in glycerol during RCH in the fleshfly, Sarcophaga crassipalpis, while Kelty and Lee (1999) did not find any increase in glycerol levels of hardened D. melanogaster. A similar ambiguity characterizes the role of heat shock proteins during cold hardening and cold stress where induction of the heat shock response markedly improved survival in larvae of D. melanogaster following a subsequent cold shock (Burton et al., 1988). However, cold-induction of Hsp70 is only seen when cold exposures last more than 8 h (Burton et al., 1988; Sejerkilde et al., 2003) and Hsp70 levels following cold are considerably lower than those found following heat hardening (Goto and Kimura, 1998). Moreover, expression of Hsp70 was absent in flies exposed to RCH by a protocol mimicking diurnal temperature variation (Kelty and Lee, 2001). While it seems unlikely that Hsps are expressed during the actual hardening period, the present study investigates the possibility that RCH alters the Hsp70 expression level following RCH and a subsequent cold shock.

Given that previous investigations have failed to produce a mechanistic explanation of RCH effects, other avenues must be explored. A possible physiological basis of RCH could be achieved by rapid changes in the cell membrane lipid composition (Watson and Morris, 1987; Cossins and Raynard, 1987; Hazel, 1995; Cossins et al., 2002). Changes in membrane composition during cold acclimation to maintain membrane viscosity and function is termed homeoviscous adaptation (Cossins and Raynard, 1987; Hazel, 1995; Cossins et al., 2002). Seasonal changes in cold tolerance are clearly associated with such alterations of membrane lipids (Bennett et al., 1997; Kostal and Simek, 1998; Ohtsu et al., 1998). Here we have investigated the influence of RCH on phospholipid fatty acid (PLFA) composition, cold tolerance and the expression levels of Hsp70 following a cold shock. To include the possible effects of long-term selection for cold tolerance, these experiments were performed on adult flies from two selection regimes (i) control (reared at constant 25 1C) and (ii) cold stress survival (selected for survival following 1–2 days exposure to 0 1C).

2. Materials and methods 2.1. Maintenance and origin of experimental flies D. melanogaster Meigen used in this experiment were derived from a mass population based on four preexisting laboratory stocks originating from different geographical regions. The mass population was created in September 2002 and was kept at Aarhus University in high numbers. The flies were held under standard laboratory conditions (25 1C and 12/12 h light–dark cycle) on a standard agar–sugar–yeast–oatmeal medium. The newly established mass population was held as one interbreeding population for four generations, before independent selection lines were established (Bubliy and Loeschcke, 2005). 2.2. Selection regimes In the present study, flies from two selection regimes were used (control and cold-selection). We used 3 independent replicate lines from each regime. The control lines were kept at standard laboratory conditions with a population size of at least 300 pairs per replicate line. The cold selected lines were only selected every second generation in order to allow the population to recover in size, and to avoid carry-over effects (Watson and Hoffmann, 1995). The selection regime for the cold-selected flies aimed at creating a mortality of approximately 50% while maintaining a minimum population size of at least 300 pairs per replicate line. Briefly, after emergence flies were put in food vials (30

ARTICLE IN PRESS J. Overgaard et al. / Journal of Insect Physiology 51 (2005) 1173–1182

pairs in each) until they were 2 days old. The flies were then acclimated at 11 1C for 5 days and subsequently exposed to 0 1C for 27 to 50 h (the intensity of the coldshock had to be increased as selection progressed). Both the control and the cold-selected lines passed more than twenty generations before onset of the experimental protocol. Thus, the cold-selected lines had experienced more than 10 events of selection for cold resistance. 2.3. Collection of flies Flies were maintained for one generation without selection before they were collected for experimentation. Females were collected under light CO2 anaesthesia soon after emergence (less than 1 day old). Batches of 20 female flies were then transferred to food vials for 2 days. For each parameter measured, three samples were taken from each of the three replicate lines within each of two selection regimes, in total 18 samples with 20 flies each. 2.4. Experimental protocol The experiments were run in two series (see Fig. 1). The first series was designed to evaluate the effects of RCH and/or cold selection on the composition of

Samples taken for PLFA measurements Estimation of survival

Temperature

PLFA measurements Untreated control 25°C Ra pid co ld ha rd en ing 0°C

Cold shock

Time Hsp70 measurements

Temperature

25°C

0°C

Untreated control Ra pid co ld ha rd en ing

Samples taken for survival and Hsp70 measurements

Cold shock Time

Fig. 1. Experimental protocol used to evaluate the effects of rapid cold hardening on phospholipids fatty acids composition (Top) and the effects of RCH on Hsp70 expression following cold shock (Bottom). Cold selected and control flies were either exposed to RCH or were left at 25 1C before they were exposed to a cold shock. Samples used for PLFA analysis were taken after the RCH treatment but before the cold shock. Samples for Hsp70 measurements were taken at several time points after the cold shock (see text for further details).

1175

membrane PLFAs and on survival 24 h following a 1 h cold shock at
4,
6 and
8 1C, respectively. The second series was designed to evaluate the effects of RCH and/or cold selection on the expression of Hsp70 following cold shock. In the latter experimental series we also measured survival rates at different time points following a 1 h cold shock at
6 1C (from 0 to 24 h after the cold shock). In both series the following four experimental groups were examined in order to evaluate the effects of cold selection and RCH: (A) untreated flies from the control regime; (B) flies from the control regime that were subjected to RCH; (C) untreated flies from the cold selection regime and (D) flies from the cold selection regime that were subjected to RCH.

2.4.1. Protocol for rapid cold hardening (RCH) The RCH protocol was similar to that described by Kelty and Lee (1999). Briefly, flies were transferred to 5 ml sealed plastic vials and were cold hardened by immersing the vials into a temperature bath set at the acclimation temperature of the flies (25 1C). The temperature was then lowered from 25 to 0 1C at a rate of 0.1 1C min
1 and, once at 0 1C, the flies were held at this temperature for 1 h. Hence, the duration of the entire cold hardening procedure was 5 h and 10 min. It should be noted that hardening processes are likely to take place in the lower temperature range of this procedure.

2.4.2. Phospholipid fatty acids and survival 24 h after cold shock Immediately after the RCH procedure, three vials from each replicate line of both control and cold selected flies were frozen at
80 1C for subsequent measurement of membrane lipids. Similarly, three vials from each line with untreated flies from both selection regimes were sampled and frozen. Survival following cold shock (1 h at
4,
6 or
8 1C, respectively) was examined for all four experimental groups. Immediately after cold hardening, flies from both control and cold-selected lines were transferred directly from 0 1C to water baths at
4,
6 or
8 1C. Non-hardened flies from both selection regimes were exposed to the same cold shock treatments, but these flies were transferred directly from their rearing temperature (25 1C) to
4,
6 or
8 1C. After the cold shock all vials were gradually returned to 25 1C over a 20 min period. The flies were then transferred to food vials, and survival was scored as the number of flies that were able to move after gentle tactile stimuli 24 h after the cold shock treatment (see Fig. 1).

ARTICLE IN PRESS 1176

J. Overgaard et al. / Journal of Insect Physiology 51 (2005) 1173–1182

2.4.3. Time-dependent survival and expression of Hsp70 protein following cold shock Survival and expression of Hsp70 were examined following a 1 h cold shock exposure at
6 1C in the same four experimental groups as those used for PLFA analysis. Survival could not be assessed immediately after cold shock as all flies were in cold torpor. Consequently, survival was only scored 1, 2, 4, 8, 16 and 24 h after cold shock. Surviving flies were separated from dead and frozen at
80 1C for subsequent measurement of Hsp70 at 0, 1, 2, 4, 8, 16 and 24 h after the
6 1C cold shock. At time 0 all flies were frozen immediately and dead flies could not be scored, however, as the survival assay revealed that mortality was not observed before 4–8 h after the treatment all flies can be assumed to be alive at time 0 (See Fig. 1). 2.5. Measurement of Hsp70 expression level For the measurement of inducible Hsp70, whole flies were homogenized and the Hsp70 level was determined by the ELISA technique utilizing the antibody 7 FB, which is specific for the inducible Hsp70 in D. melanogaster (Welte et al., 1993). The ELISA procedure is described in more detail in Sørensen et al. (1999). A heat-treated sample was included in each assay as a positive control for Hsp70 expression, and expression levels were related directly to this internal reference. The heat hardening response was induced by exposing flies from each selection regime to 3570.1 1C for 1 h in a preheated water bath. After induction the vials were transferred to 25 1C for 1 h and then stored at
80 1C until analysis. The ELISA assay worked satisfactorily as confirmed by a large response from the internal reference (the heat treated flies). However, as we did not find any significant Hsp70 expression following cold shock we only measured four replicates from the first four time points (0, 1, 2 and 4 h after cold shock) and one replicate for the final three time points (8, 16 and 24 h after cold shock) in each of the four experimental groups. 2.6. Composition of membrane phospholipid fatty acids The PLFA composition was determined for control flies and cold selected flies, both with and without rapid cold hardening. As 10 flies were sufficient for a PLFA measurement, we could perform 15–18 independent measurements of PLFA for each of the 4 experimental groups. Ester-linked PLFAs were extracted from macerated flies (n ¼ 10) using a modified Bligh–Dyer single-phase lipid extraction (Bligh and Dyer, 1959; White et al., 1979) with the extraction carried out in two steps as described by Kates (1986). Following phase separation, the chloroform layer was transferred to a test tube and evaporated to dryness under N2.

Phospholipids were then isolated from the crude lipid extract by solid-phase extraction (100 mg silicic acid; Varian, Harbor City, CA, USA). Lipids of low and intermediate polarity were eluted with 1.5 ml chloroform and 6 ml acetone, respectively. Polar lipids (mainly phospholipids) were then eluted with 1.5 ml methanol (Kates, 1986) and collected in a test tube, dried under N2, and trans-methylated (Dowling et al., 1986). Fatty acid methyl esters were washed into hexane (Petersen and Klug, 1994), dried and re-dissolved in 1 ml hexane for gas chromatographic analysis on a Hewlett-Packard 6890 GC system with auto-sampler. The column was a HP-5 (Hewlett-Packard; 50 m fused silica, ID, 0.32 mm) with helium as carrier. The oven was programmed to increase temperature from 60 to 300 1C (Frostegard et al., 1993). Identification of individual fatty acids was based on retention times and cross-reference with samples analysed by GC–MS (Frostegard et al., 1993). Fatty acids are designated as X:Y(n
Z), where X indicates the number of C atoms, Y the number of double bonds, and Z the position of the first double bond counting from the methyl end of the molecule. Areas of identified peaks were quantified using 19:0 as internal standard, corrected for molecular weight, and mol% distributions and quantitative yields calculated. 2.7. Statistics We examined the effects of rapid cold hardening and/ or selection for cold tolerance on survival 24 h after a 1 h cold shock exposure at either
4,
6 or
8 1C. Survival data were arcsin–square-root transformed to improve normality and homogeneity of variances, as cold tolerance was evaluated from the survival proportion. At each cold shock temperature separately, survival was analysed with ANOVA using a nested design with the effects of rapid cold hardening and selection regime as fixed factors and line nested within selection regimes. As no significant differences in survival were found between the independent lines within each selection regime, the lines were pooled and the test reduced to a two-way ANOVA to increase the power. Mol percentages of individual PLFAs were, similarly, tested using nested ANOVA with a similar design for each PLFA separately and for overall PLFA unsaturation, ratio of unsaturated and saturated PLFAs (UFA/SFA) and chain length. No line effects were found in any of these tests either and this analysis was therefore also reduced to a two-way ANOVA for effects of selection and RCH. The expression level of Hsp70 was tested relative to a control level of 0 using three-way ANOVA for cold selection, RCH and time after the cold shock. The timedependent survival following a
6 1C cold shock was examined using a one-way ANOVA. The latter test was designed to examine when survival fell below the control

ARTICLE IN PRESS J. Overgaard et al. / Journal of Insect Physiology 51 (2005) 1173–1182

3. Results 3.1. Effects of rapid cold hardening and cold selection on survival after cold shock Survival 24 h after a cold shock was reduced as cold shock temperature decreased. After exposure to
8 1C none of the unhardened flies survived, while only 12% of the hardened flies were alive. Survival was higher after both
6 and
4 1C cold shock treatments, and this was especially true for flies that were subjected to RCH (Fig. 2). Indeed, RCH had a large positive effect at all cold shock temperatures (Po0:001). In this experimental run, we found no significant effects of long term cold tolerance selection as survival and effects of RCH were almost identical in flies from both selection regimes. The large and significant effect of RCH was confirmed in the second experimental run (Fig. 3). Here 100

Control No Hardening Cold Selec. No Hardening Control Hardening Cold Selec.Hardening

* 80 Survivors (%)

60

40

*

0 -4°C

-6°C Cold shock temperature

80

Control Hardening Control No Hardening Cold Selc.Hardening Cold Selec. No Hardening

a a,c,d

60

a,b, c,d

40

*,† *

20

a,b, c,d

0 0

4

8 12 16 Hours after cold shock

20

24

Fig. 3. Delayed mortality during 24 h after a 1 h cold shock at
6 1C. Survival was estimated from the percentage of flies that responded after gentle tactile stimuli. N ¼ 9 vials of 20 flies each at all time points. Letters indicate a drop in survival below the 1 h survival level for (a) unhardened and cold selected flies, (b) unhardened control flies, (c) hardened control flies and (d) hardened and cold selected flies. An asterisk indicates a significant effect of rapid cold hardening on survival 24 h after cold shock and a cross indicates a significant effect of cold selection on survival 24 h after cold shock. All values are mean7SEM.

the effect was observed both as an improved survival 24 h after cold shock, but also as a delayed mortality in the hardened flies. In contrast to the first experimental run we did observe a significant effect of cold selection. This effect was, however, only present in hardened flies, where cold selected flies had a higher survival rate 24 h after a cold shock compared with control flies (Po0:008; Fig. 3). Fig. 3 demonstrates that death due to cold shock did not occur until several hours after the animals had been returned to 25 1C. Even in unhardened flies death did not occur until 2 to 4 h after the cold shock. Death of rapidly cold hardened flies began within 4 to 8 h after cold shock. 3.2. Effects of rapid cold hardening and cold selection on the expression level of Hsp70

*

20

100

Survivors (%)

level (the level observed 1 h after cold shock) and this test was performed for both selection regimes with and without a preceding RCH treatment. Furthermore a two-way ANOVA was used to evaluate the effects of RCH and cold selection on survival 24 h after cold shock. All statistics were performed with the program package SPSS (SPSS, Inc., Chicago, Illinois, USA), while F-values for the nested variables were calculated by hand according to Sokal and Rohlf (1995). For all tests post hoc Bonferroni tests were used to identify groups that differed. Effects were considered significant at the Po0:05 level. All data are presented as mean7SEM.

1177

-8°C

Fig. 2. Survival 24 h after a 1 h cold shock at
4,
6 or
8 1C. Survival was estimated from the percentage of flies that responded after gentle tactile stimuli. N ¼ 9 vials of 20 flies in all experimental situations. An asterisk indicates a significant effect of rapid cold hardening. All values are mean7SEM.

The temporal Hsp70 expression level following a 1 h cold shock at
6 1C is depicted in Fig. 4 for all fourtreatment groups. Expression levels of Hsp70 were always below 2% of the reference level (i.e. the Hsp70 expression levels following a 35 1C heat shock). Hsp70 levels were never significantly different from 0 irrespective of selection, RCH treatment or time after cold shock. 3.3. Effects of rapid cold hardening and cold selection on composition of PLFA The observed changes in membrane lipid fatty acid composition due to rapid cold hardening or cold

ARTICLE IN PRESS J. Overgaard et al. / Journal of Insect Physiology 51 (2005) 1173–1182

1178

selection were in the order of 1 mol% or below (Table 1). Even so, there were significant effects of both RCH and cold selection. A 5 h RCH treatment resulted in significant changes in PLFAs through reductions in 14:0, 18:1(n
9), and 18:1(n
7) PLFAs (P ¼ 0:042, 0.006, 0.004, respectively) while the abundance of 18:2(n
6) was increased (P ¼ 0:009). These changes were accompanied by an increase in the overall degree of unsaturation (P ¼ 0:024) while the ratio of unsaturated and saturated PLFAs (UFA/SFA) did not change significantly (P ¼ 0:13). The changes observed due to hardening were seen in both selection groups and were independent of the preceding selection regime.

Hsp70 expression level (% of Hsp70 level after heat stress)

10 Control No hardening Cold selec. No hardening Control Hardening Cold Selec.Hardening

8 6 4 2 0

0h

1h 2h Time after cold shock

4h

Fig. 4. Expression levels of Hsp70 at time 0, 1, 2 and 4 h after a 1 h cold shock at
6 1C. The level is expressed as percentage of the Hsp70 level after a 1 h heat shock at 35 1C in similar flies. N ¼ 4 samples at each time point. None of the expression levels are significantly above 0. All values are mean7SEM.

Cold selection resulted in significant decreases in 18:0 and 18:1(n
7) (Po0:001), while 14:0 and cis-16:1(n
7) increased (P ¼ 0:035 and 0.044, respectively). These changes however, did not result in a significant decrease of the average length of the PLFAs (P ¼ 0:18), nor had the degree of unsaturation changed (Table 1).

4. Discussion The ability to survive acute exposures to subzero temperatures is likely to be of little ecological relevance in D. melanogaster. The physiological adjustments initiated during RCH in D. melanogaster are, however, not only important for survival following cold shock, but physiological adjustments are also important for sustained activity at low temperature, including courting/mating activity (Kelty and Lee, 1999; Shreve et al., 2004). Thus, RCH is central for the ability to respond dynamically to diurnally fluctuating temperature. In spite of the ecological relevance of RCH, the mechanisms behind RCH have so far been unresolved (Sinclair et al., 2003). In this study we explored if changes in membrane composition and/or induction of stress proteins formed part of the RCH response. These parameters were chosen because membrane thermotropism and protein denaturation are possible causes of cold shock injury (Watson and Morris, 1987). Moreover, adjustment of the composition of membrane lipids is a recurring theme of studies concerning acclimatory changes in ectothermic organisms in response to low temperature.

Table 1 Composition of phospholipids fatty acids (PLFAs) in flies from control lines and flies that were selected for cold tolerance Control line No hardening (N ¼ 16) 14:0 15:0 16:1(n
9) cis-16:1(n
7) trans-16:1(n
7) 16:0 18:2(n
6) 18:1(n
9) 18:1(n
7) 18:0 20:0 Degree of unsaturation UFA/SFA Average chain length

1.22970.034 0.10570.010 0.38170.008 21.68370.200 0.10170.004 16.11370.144 26.92170.276 30.60670.076 0.57870.025 2.10870.029 0.17470.003 1.07270.004 4.0770.04 17.18670.007

Cold selected line Hardening (N ¼ 18) 1.17870.033* 0.09970.006 0.38570.006 21.34770.155 0.09170.003 15.95170.144 27.80970.222* 30.32170.081* 0.52870.029* 2.12170.017 0.16970.003 1.08370.004* 4.1370.05 17.19870.005

No hardening (N ¼ 15) y

1.31170.027 0.10470.007 0.39270.008 21.78570.109y 0.09270.003 16.05370.145 26.96670.226 30.60070.108 0.50170.018y 2.01770.020y 0.17770.003 1.07370.004 4.0970.04 17.18270.004

Hardening (N ¼ 18) 1.23170.031* 0.09170.007 0.39170.006 21.92170.170y 0.09370.003 15.84570.172 27.39570.250* 30.39670.107* 0.46170.013*y 2.00770.021y 0.16870.005 1.08170.004* 4.1870.05 17.18670.006

PLFAs are presented as molar percentages and are shown for both rapid cold hardened and untreated flies from both selection lines. Significant effects of rapid cold hardening are indicated by an asterisk and boldface numbers. Significant effects of cold tolerance selection are indicated by a cross and italicized numbers. All values are mean7SEM. Note: UFA/SFA: sum of unsaturated fatty acids/sum of saturated fatty acids.

ARTICLE IN PRESS J. Overgaard et al. / Journal of Insect Physiology 51 (2005) 1173–1182

4.1. Effects of rapid cold hardening and cold selection on survival after cold shock Since the RCH response of insects was first described by Lee et al. (1987) a growing number of studies have confirmed that RCH markedly improves cold shock tolerance in different insect species including D. melanogaster (see Kelty and Lee 1999, 2001; Shreve et al., 2004). In D. melanogaster the effect of rapid cold hardening is already evident after a few degrees of cooling below the acclimation temperature. The effect is, however, enhanced by decreasing the rate of cooling rates, as well as by lowering of the target hardening temperature (Kelty and Lee 1999, 2001). The RCH protocol used in the present study lowered the tolerable cold shock temperature by 2–4 1C, which is consistent with previous studies on D. melanogaster using a similar RCH protocol (Kelty and Lee, 1999). We tested the effects of RCH on flies from two different selection regimes using three independent lines in each selection regime to avoid effects of random genetic drift between selection regimes. Our hypothesis was that selection for cold tolerance would either improve cold shock survival per se and/or improve the ability to rapidly cold harden. The results from the first experimental run did not support this hypothesis. However, we found an improved survival in the hardened flies from the cold selected regime in the second experimental run. Thus, it appears that the cold selection protocol used here does not improve cold shock tolerance in unhardened flies, whereas it is possible that cold selection improves the responsiveness to RCH. The absent or marginal effects of cold selection in the present study may relate to the selection protocol used. Cold selected flies were not selected for the ability to survive short subzero cold shocks. Rather, they were selected for long term cold resistance at 0 1C, and selection clearly improved their ability to survive such exposures (Bubliy and Loeschcke, 2005). Thus, the traits important during RCH may not necessarily be the same as those responding to long-term exposure to 0 1C. The delay of mortality following cold shock presented in Fig. 3 is not a novel observation in insects (see Baust and Rojas, 1985). This delay may indicate that the injury inflicted by cold shock disrupts systems that are not immediately necessary for coordinated movement. Instead, we suggest the possibility that the affected systems are important for sustained water and energy balance. 4.2. Effects of rapid cold hardening and cold selection on the expression level of Hsp70 Previous studies have demonstrated that ‘‘coldinduced’’ Hsp70 expression is only initiated after the return to ‘‘control’’ temperature (Burton et al., 1988; Goto and Kimura, 1998; Sejerkilde et al., 2003). This is

1179

consistent with the lack of Hsp70 induction in D. melanogaster during mild RCH as shown by Kelty and Lee (2001). In the present study we examined if selection for cold tolerance or RCH affected the dynamics of the post-cold-shock expression of Hsp70. We did, however, not find any induction of Hsp70 in any of the experimental groups following a 1 h cold shock at
6 1C. This result suggests that Hsp70 induction plays no significant role for survival after short cold stress exposures. Furthermore, we have recently shown by use of a heat shock factor mutant that this most likely also holds true for other inducible Hsps (Nielsen et al., in press). These results are consistent with the finding that RCH is generally independent of protein synthesis as RCH was equally effective in D. melanogaster treated with cycloheximide (a protein synthesis inhibitor) as in untreated controls (Misener et al., 2001). Whereas Hsps generally are induced by heat stress in a variety of species (Feder and Hofmann, 1999; Sørensen et al., 2003), the pattern is less clear for cold stress. For example, a cold shock exposure of more than 8 h at 0 1C is needed to induce Hsp70 in larvae or adult D. melanogaster (Burton et al., 1988; Goto and Kimura, 1998; Sejerkilde et al., 2003). We suggest that protein denaturation or aggregation, and hence Hsp70 expression is unlikely to be a direct effect of the relatively mild cold exposures used in these studies. Instead, Hsp70 expression may result from the heating during the return to ‘‘normal’’ temperature (Hayward et al., 2004), or non-native proteins may arise as a secondary effect of other physiological disorders, such as membrane phase transitions during cold.

4.3. Effects of rapid cold hardening and cold selection on composition of PLFA Cell membranes function as selective barriers between the intra- and extracellular compartments. At ‘‘normal’’ temperature they are in a liquid-crystalline phase, but when biological membranes are cooled sufficiently they gradually go from the liquid crystalline phase to the gel phase, whereby the membranes partly lose their selective properties (Cossins and Raynard, 1987; Hazel, 1995). Such phase transition can lead to loss of intracellular lipids, proteins and ions, as well as to an undesirable uptake of sodium and calcium to the intracellular compartment (Watson and Morris, 1987). The physical structure of biological membranes is governed by several properties including phospholipid headgroups, cholesterol content and the composition of the fatty acyl sidechains (Cossins and Raynard, 1987; Hazel, 1995). It has been proposed that ‘‘the particular distribution of fatty acyl residues present in a membrane provides the appropriate membrane fluidity at a particular environmental temperature to match the diffusion rate or rate

ARTICLE IN PRESS 1180

J. Overgaard et al. / Journal of Insect Physiology 51 (2005) 1173–1182

of metabolic processes required for the tissue’’ (Lee and Chapman, 1987). In terms of cold adaptation in ectothermic animals the changes in PLFAs are typically associated with an increase in the degree of unsaturation and, in particular, an increased proportion of polyunsaturated long-chain fatty acids leading to a more disordered membrane which is less likely to undergo phase transition at low temperature (Lee and Chapman, 1987; Hazel, 1995; Cossins et al., 2002). In accordance with this, previous studies have shown that seasonal changes in cold tolerance of insects were associated with alterations of the PLFA composition (Bennett et al., 1997; Kostal and Simek, 1998; Ohtsu et al., 1998). In insects the changes in PLFA composition during cold acclimation seem to be smaller than in many other ectothermic organisms (cf. Hazel and Landrey, 1988; Suutari et al., 1997; Hatab and Gaugler, 1997; Petersen and Holmstrup, 2000). Furthermore, the adjustments in PLFA composition following lowered acclimation temperature varies between different tissues (Baldus and Mutchmore, 1988; Kostal and Simek, 1998), phospholipids headgroups (Kostal et al., 2003) and PLFA composition is also strongly influenced by photoperiod (Ohtsu et al., 1998; Hodkova et al., 2002). Despite these sources of variation, the general trend in previous studies of insect PLFAs is that the degree of unsaturation increases by 0.002–0.011 per degree of lowered acclimation temperature (Keith, 1966; Bridges and Watts, 1975; Rapport, 1986; Kostal and Simek, 1998; Kostal et al., 2003). Similarly, the ratio unsaturated and saturated PLFA increase by 0.01–0.09 per degree of lowered acclimation temperature. The time needed to optimize membrane viscosity after a change in growth temperature (homeoviscous adaptation) differs between species and tissue types ranging from 30 to 40 days in the brain of goldfish to less than 4 h in unicellular organisms (Cossins and Raynard, 1987). In this study we show that a 5 h and 10 min RCH protocol is sufficient to significantly change the PLFA composition in the direction that would be expected to support an increased cold tolerance. Thus, RCH was associated with an increase in polyunsaturated fatty acids, which lead to a slightly increased degree of unsaturation. Moreover, there was also a trend towards an increase in the ratio of UFA/SFA. The changes in PLFA composition were small, but when compared to those observed in insects (including Drosophila) exposed to lowered acclimation temperature the increase in degree of unsaturation and UFA/SFA is equivalent to what would be expected from a 1 to 5 1C lowering of acclimation temperature (Keith, 1966; Bridges and Watts, 1975; Rapport, 1986; Kostal and Simek, 1998; Kostal et al., 2003). The magnitude of the changes in PLFA composition is, therefore, in accordance with the increased cold tolerance provided by RCH. In this study

the RCH protocol was sufficient to lower the absolute cold tolerance by 2–4 1C, and in a previous study (using a similar protocol) the RCH treatment lowered the critical thermal minimum by 2–3 1C (Kelty and Lee, 1999). In the present study, the major effect of RCH on PLFA composition was that RCH caused a reduction in monounsaturated C18 fatty acids and an increase in the polyunsaturated linoleic acid, 18:2(n
6). Furthermore, there was a significant reduction in 14:0. Changes in the composition of phospholipid fatty acids can arise from selective incorporation of fatty acids from the pool of free fatty acids (addition synthesis). This pool, in turn, is influenced by the dietary intake and subsequently subject to modifications by desaturases and chain elongation enzymes. A second possibility for changes in the PLFA composition is via direct modifications of existing membrane lipids by desaturases or the deacylation–reacylation cycle (Cossins and Raynard, 1987; Hazel, 1995). Some insect species are able to synthesize 18:2(n
6) de novo (Blomquist et al., 1982, 1991; Buckner and Hagen, 2003). Rapport et al. (1984) suggested that this might also be the case for D. melanogaster, but other studies have found that high levels of 18:2(n
6) rely on availability of this fatty acid in the diet (Stanley-Samuelson et al., 1985). We propose that the changes in PLFA composition observed following RCH are due to a selective incorporation of phospholipids with 18:2(n
6) at the expense of saturated and monounsaturated acids. Overall these changes lead to an increase in the degree of unsaturation following RCH. Even though the observed changes in the proportions of individual fatty acids were small, there was a striking similarity in the response to RCH between control and cold selected flies (each of which consisted of three independent replicate lines). This may reflect that fatty acid composition is under tight regulation at a given temperature regime. A number of differences in PLFA composition were observed between controls and cold selected flies. Cold selection resulted in significant decreases in 18:0 and 18:1(n
7) while 14:0 and cis-16:1(n
7) increased. These changes were, however, not large enough to significantly decrease the average length of the PLFAs (Table 1). These differences may be related to the improved ability of cold selected flies to survive long periods at 0 1C (Bubliy and Loeschcke, 2005) but, as discussed previously, they do not seem to confer a markedly improved cold shock tolerance in unhardened flies. 4.4. Conclusions The main finding of this study was that RCH was associated with selective changes in the phospholipid composition of D. melanogaster and these changes were, furthermore, similar in flies with different selection

ARTICLE IN PRESS J. Overgaard et al. / Journal of Insect Physiology 51 (2005) 1173–1182

backgrounds. These changes were, as hypothesized, related to an increased degree of unsaturation and an increase in polyunsaturated PLFAs. Such changes may counteract detrimental phase transitions in membranes during cold periods and we argue that these changes may be sufficient to account for the increased cold tolerance observed following RCH. In contrast, we found that neither RCH nor cold shock induced expression of Hsp70. While we recognize that other physiological mechanisms may also be involved, we hypothesize that the PLFA changes served to diminish phase transition during cold shock treatments. This is to our knowledge the first study to present a possible functional mechanism underlying RCH in insects. It is possible that the observed compositional changes arise from a mixture of larger changes in some tissues or organs and less in others. Future studies including measures of tissue specific changes in PLFAs could be rewarding and so also will be investigations of the phase transition behaviour of membranes with and without RCH.

Acknowledgements We are grateful to Doth Andersen for laboratory assistance, M. B. Evgenev and S. Lindquist for providing 7FB antibody. The work was supported by centre and frame grants of the Danish Natural Sciences Research Council.

References Baldus, T.J., Mutchmore, J.A., 1988. The effects of temperature acclimation on the fatty acid composition of nervecord and fatbody of the American cockroach, Periplaneta Americana. Comparative Biochemistry and Physiology 89A, 141–147. Baust, J.G., Rojas, R.R., 1985. Review—insect cold hardiness: facts and fancy. Journal of Insect Physiology 31, 755–759. Bennett, V.A., Pruitt, N.L., Lee, R.E., 1997. Seasonal changes in fatty acid composition associated with cold-hardening in third instar larvae of Eurosta solidaginis. Journal of Comparative Physiology B 167, 249–255. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37, 911–917. Blomquist, G.J., Dwyer, L.A., Chu, A.J., Ryan, R.O., Renobales, M., 1982. Biosynthesis of linoleic acid in a termite, cockroach and cricket. Insect Biochemistry 12, 349–353. Blomquist, G.J., Borgeson, C.E., Vundla, M., 1991. Polyunsaturated fatty acids and eicosanoids in insects. Insect Biochemistry 21, 99–106. Bridges, R.G., Watts, S.G., 1975. Changes in fatty-acid composition of phospholipids and triglycerides of Musca domestica resulting from choline deficiency. Journal of Insect Physiology 21, 861–871. Bubliy, O.A., Loeschcke, V., 2005. Correlated responses to selection for stress resistance and longevity in a laboratory population of Drosophila melanogaster. Journal of Evolutionary Biology 18, 789–803.

1181

Buckner, J.S., Hagen, M.M., 2003. Triacylglycerol and phospholipid fatty acids of the silverleaf whitefly: composition and biosynthesis. Archives of Insect Biochemistry and Physiology 53, 66–79. Burks, C.S., Hagstrum, D.W., 1999. Rapid cold hardening capacity in five species of coleopteran pests of stored grain. Journal of Stored Products Research 35, 65–75. Burton, V., Mitchell, H.K., Young, P., Petersen, N.S., 1988. Heat shock protection against cold stress of Drosophila melanogaster. Molecular and Cellular Biology 8, 3550–3552. Chen, C.P., Denlinger, D.L., Lee, R.E., 1987. Cold-shock injury and rapid cold hardening in the flesh fly Sarcophaga crassipalpis. Physiological Zoology 60, 297–304. Cossins, A.R., Raynard, R.S., 1987. Adaptive responses of animal cell membranes to temperature. In: Bowler, K., Fuller, B.J. (Eds.), Temperature and Animal Cells. The Company of Biologist Limited, Cambridge, pp. 91–112. Cossins, A.R., Murray, P.A., Gracey, A.Y., Logue, J., Polley, S., Caddick, M., Brooks, S., Postle, T., Maclean, N., 2002. The role of desaturases in cold-induced lipid restructuring. Biochemical Society Transactions 30, 1082–1086. Czajka, M.C., Lee, R.E., 1990. A rapid cold-hardening response protecting against cold shock injury in Drosophila melanogaster. Journal of Experimental Biology 148, 245–254. Dowling, N.J.E., Widdel, F., White, D.C., 1986. Phospholipid esterlinked fatty acid biomarkers of acetate oxidizing sulphate reducers and other sulphide-forming bacteria. Journal of Genetic Bacteriology 132, 1815–1825. Feder, M.E., Hofmann, G.E., 1999. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annual Review of Physiology 61, 243–282. Frostegard, A., Tunlid, A., Baath, E., 1993. Phospholipid fatty-acid composition, biomass, and activity of microbial communities from 2 soil types experimentally exposed to different heavy-metals. Applied Environmental Microbiology 59, 3605–3617. Goto, S.G., Kimura, M.T., 1998. Heat- and cold-shock responses and temperature adaptations in subtropical and temperate species of Drosophila. Journal of Insect Physiology 44, 1233–1239. Hatab, M.A.A., Gaugler, R., 1997. Influence of growth temperature on fatty acids and phospholipids of Steinernema riobravis infective juveniles. Journal of Thermal Biology 22, 237–244. Hayward, S.A.L., Rinehart, J.P., Denlinger, D.L., 2004. Desiccation and rehydration elicit distinct heat shock protein transcript responses in flesh fly pupae. Journal of Experimental Biology 207, 963–971. Hazel, J.R., 1995. Thermal adaptation in biological membranes: is homeoviscous adaptation the explanation? Annual Review of Physiology 57, 19–42. Hazel, J.R., Landrey, S.R., 1988. Time course of thermal adaptation in plasma membranes of trout kidney. II. Molecular species composition. American Journal of Physiology 255, R628–R634. Hodkova, M., Berkova, P., Zahradnickova, H., 2002. Photoperiodic regulation of the phospholipid molecular species composition in thoracic muscles and fat body of Pyrrhocoris apterus (Heteroptera) via an endocrine gland, corpus allatum. Journal of Insect Physiology 48, 1009–1019. Kates, M., 1986. Techniques of lipidology. isolation, analysis and identification of lipids, second revised ed. Elsevier, Amsterdam. Keith, A.D., 1966. Analysis of lipids in Drosophila melanogaster. Comparative Biochemistry and Physiology 17, 1127–1136. Kelty, J.D., Lee, R.E., 1999. Induction of rapid cold hardening by cooling at ecologically relevant rates in Drosophila melanogaster. Journal of Insect Physiology 45, 719–726. Kelty, J.D., Lee, R.E., 2001. Rapid cold-hardening of Drosophila melanogaster (Diptera: Drosophilidae) during ecologically based thermoperiodic cycles. Journal of Experimental Biology 204, 1659–1666.

ARTICLE IN PRESS 1182

J. Overgaard et al. / Journal of Insect Physiology 51 (2005) 1173–1182

Kelty, J.D., Killian, K.A., Lee, R.E., 1996. Cold shock and rapid coldhardening of pharate adult flesh flies (Sarcophaga crassipalpis)— effects on behaviour and neuromuscular function following eclosion. Physiological Entomology 21, 283–288. Knight, J.D., Bale, J.S., Franks, F., Mathias, S.F., Baust, J.G., 1986. Insect cold hardiness—supercooling points and prefreeze mortality. Cryo-Letters 7, 194–203. Kostal, V., Simek, P., 1998. Changes in fatty acid composition of phospholipids and triacylglycerols after cold-acclimation of an aestivating insect prepupa. Journal of Comparative Physiology B 168, 453–460. Kostal, V., Berkova, P., Simek, P., 2003. Remodelling of membrane phospholipids during transition to diapause and cold-acclimation in the larvae of Chymomyza costata (Drosophilidae). Comparative Biochemistry and Physiology 135B, 407–419. Kostal, V., Vambera, J., Bastl, J., 2004. On the nature of pre-freeze mortality in insects: water balance, ion homeostasis and energy charge in the adults of Pyrrhocoris apterus. Journal of Experimental Biology 207, 1509–1521. Larsen, K.J., Lee, R.E., 1994. Cold tolerance including rapid cold-hardening and inoculative freezing of fall migrant monarch butterflies in Ohio. Journal of Insect Physiology 40, 859–864. Lee, D.C., Chapman, D., 1987. The effects of temperature on biological membranes and their models. In: Bowler, K., Fuller, B.J. (Eds.), Temperature and Animal Cells. The Company of Biologist Limited, Cambridge, pp. 35–52. Lee, R.E., 1991. Principles of insect low temperature tolerance. In: Lee, L.E., Denlinger, D.L. (Eds.), Insects at Low Temperature. Chapman and Hall, New York, pp. 17–47. Lee, R.E., Denlinger, D.L., 1985. Cold tolerance in diapausing and non-diapausing stages of the flesh fly, Sarcophaga crassipalpis. Physiological Entomology 10, 309–315. Lee, R.E., Chen, C.P., Denlinger, D.L., 1987. A rapid cold-hardening process in insects. Science 238, 1415–1417. Misener, S.R., Chen, C.P., Walker, V.K., 2001. Cold tolerance and proline metabolic gene expression in Drosophila melanogaster. Journal of Insect Physiology 47, 393–400. Nielsen, M.M., Overgaard, J., Sørensen, J.G., Holmstrup, M., Justesen, J., Loeschcke, V., in press. Role of HSF activation during cold hardening and for high temperature knock-down resistance. Journal of Insect Physiology. Ohtsu, T., Kimura, M.T., Katagiri, C., 1998. How Drosophila species acquire cold tolerance—qualitative changes of phospholipids. European Journal of Biochemistry 252, 608–611. Powell, S.J., Bale, J.S., 2004. Cold shock injury and ecological costs of rapid cold hardening in the grain aphid Sitobion avenae (Hemiptera: Aphididae). Journal of Insect Physiology 50, 277–284. Petersen, S.O., Klug, M.J., 1994. Effects of sieving, storage, and incubation temperature on the phospholipid fatty acid profile of a soil microbial community. Applied Environmental Microbiology 60, 2421–2430.

Petersen, S.O., Holmstrup, M., 2000. Temperature effects on lipid composition of the earthworms Lumbricus rubellus and Eisenia nordenskioeldi. Soil Biology and Biochemistry 32, 1787–1791. Rapport, E.W., 1986. Fatty acid composition of wild type and mutant Drosophila. Insect Biochemistry 16, 653–657. Rapport, E.W., Stanley-Samuelson, D., Dadd, R.H., 1984. Ten generations of Drosophila melanogaster reared axenically on fatty acid-free holidic diet. Archives of Insect Biochemistry and Physiology 1, 243–250. Sejerkilde, M., Sørensen, J.G., Loeschcke, V., 2003. Effects of coldand heat hardening on thermal resistance in Drosophila melanogaster. Journal of Insect Physiology 49, 719–726. Shreve, S.M., Kelty, J.D., Lee, R.E., 2004. Preservation of reproductive behaviors during modest cooling: rapid cold-hardening finetunes organismal response. Journal of Experimental Biology 207, 1797–1802. Sinclair, B.J., Vernon, P., Klok, C.J., Chown, S.L., 2003. Insects at low temperatures: an ecological perspective. Trends in Ecology and Evolution 18, 257–262. Sokal, R.R., Rohlf, F.J., 1995. Biometry, third ed. W.H. Freeman and Company, New York. Stanley-Samuelson, D.W., Rapport, E.W., Dadd, R.H., 1985. Effects of dietary polyunsaturated fatty acids on tissue monounsaturated and saturate proportions in two insect species. Comparative Biochemistry and Physiology B 81, 749–754. Suutari, M., Rintamaki, A., Laakso, S., 1997. Membrane phospholipids in temperature adaptation of Candida utilis: alterations in fatty acid chain length and unsaturation. Journal of Lipid Research 38, 790–794. Sørensen, J.G., Michalak, P., Justesen, J., Loeschcke, V., 1999. Expression of the heat-shock protein HSP70 in Drosophila buzzatii lines selected for thermal resistance. Hereditas 131, 155–164. Sørensen, J.G., Kristensen, T.N., Loeschcke, V., 2003. The evolutionary and ecological role of heat shock proteins. Ecology Letters 6, 1025–1037. Watson, M.J.O., Hoffmann, A.A., 1995. Cross-generation effects for cold resistance in tropical populations of Drosophila melanogaster and D. simulans. Australian Journal of Zoology 43, 51–58. Watson, P.F., Morris, G.J., 1987. Cold shock injury in animals. In: Bowler, K., Fuller, B.J. (Eds.), Temperature and Animal Cells. The Company of Biologist Limited, Cambridge, pp. 311–340. Welte, M.A., Tetrault, J.M., Dellavalle, R.P., Lindquist, S.L., 1993. A new method for manipulating transgenes: engineering heat tolerance in a complex, multicellular organism. Current Biology 3, 842–853. White, D.C., Davis, W.M., Nickels, J.S., King, J.D., Bobbie, R.J., 1979. Determination of the sedimentary microbial biomass by extractible lipid phosphate. Oecologia 40, 51–62. Yi, S-X., Lee, R.E., 2004. In vivo and in vitro rapid cold-hardening protects cells from cold shock injury in the flesh fly, Sarcophaga crassipalpis. Journal of Comparative Biology B 174, 611–615. Zachariassen, K.E., 1985. Physiology of cold tolerance in insects. Physiological Reviews 65, 799–832.