Oleic acid is elevated in membranes during rapid cold-hardening and pupal in the flesh fly, Sarcophaga crassipalpis. M. Robert Michaud and D.L. Denlinger Department of Entomology, Ohio State University, 318 W. 12th Avenue, Columbus, OH 43210-1242, USA. [email protected]

How does low temperature damage cell membranes? 60 12 B A FA's of greatest FA's of intermediate abundance abundance 50 10 25oC Fluid membrane – Under homeostatic 16:0 Abstract 40 8 18: 0 Conclusions conditions, the membrane is in a liquid- 16:1n7 20:4n6 crystalline state, allowing to 18:1n9 The integrity of cellular membranes is critical to the survival of 30 6 20:5n3 function normally due to proper 18:2n6 • Our experiments provide strong evidence for homeoviscous at low temperatures, thus there is tremendous advantage positioning in the membrane matrix. 20 4 conferred to insects that can adjust their composition of active 10 2 in flesh fly cell membranes during RCH and active membrane fatty acids (FA’s). Such changes, known as 0 0 diapause. This is evidenced by an increase in the overall homeoviscous adaptation, allow cellular membranes to maintain a 01248 01248 0.7 C 1.2 D degree of unsaturation of fatty acids as well as changes in liquid-crystalline state at temperatures that are normally low FA's of low abundance FA's of low abundance Gel phase membrane – Low 0.6 short chain 1 long chain temperature causes membrane phospholipid head groups. Both promote membrane fluidity enough to cause the membrane to enter the gel state and lose the -8oC 0.5 14:0 phospholipids to move closer in percentagepercentageFAFA of of signal signal 0.8 18:3n3 relation with each other (“packing”), 0.4 14:0(m) ability to maintain homeostasis. 18:3n6 at low temperatures. which shifts the position of proteins in 15:0 0.6 0.3 20: 0 relation to the membrane. Many of 16:0(m) Flesh flies (Sarcophaga crassipalpis) were subjected to two 0.4 20:2n6 these proteins will lose function as a 0.2 16:1n9 20:3n9 • change due to RCH and diapause both feature a result. 0.2 experimental conditions that elicit low temperature tolerance: rapid 0.1 cold-hardening and diapause. FA’s were isolated and analyzed 0 0 14-17% increase in oleic acid, a monoene with a single 01248 01248 using gas chromatography-mass spectrometry. FA’s changed in inactive inactive time exposed to 4oC (h) double bond located in the middle of the acyl chain. This response to both rapid cold-hardening and diapause. In response Homeoviscous adaptation counters this effect by altering the particular fatty acid possesses the unique ability to promote a to rapid cold-hardening, the proportion of oleic acid (18:1n-9) in composition of membrane phospholipids to maintain constant Figure 1. Change in individual fatty acids (FA) as a result of chilling consistent with membrane fluidity even though the ambient temperature is wide widow of fluidity rather than the shift of a thinner fluidity pharate adults increased from 30% to 47% of the total fatty acid flesh fly rapid cold-hardening induction. Sarcophaga crassipalpis pupae were subjected dropping. pool. The proportion of almost every other FA was reduced. to 4oC for a period of 0-8 hrs and their fatty acids were analyzed with GC-MS. Each window to a lower temperature (McIlhaney 1974). In this Diapausing pupae experienced an even greater increase in oleic data point represents the mean ± standard error (n=5). Each point that bears an manner, the flesh fly can maintain membrane fluidity for low acid proportion to 58% of the total FA pool. Oleic acid not only asterisk is considered statistically significant with respect to the zero-hr treatment group (one-way ANOVA with Tukey’s post-test). It is clear from the above data that oleic acid temperatures as well as warmer temperatures. increases membrane fluidity at low temperature, but also allows (18:1n9) is dramatically increased (17.6% total increase) between 4 and 8 hrs at the the to maintain a liquid crystalline state should the Methods expense of many other fatty acids. • Increase in the proportion of phosphatidylethanolamine at temperature increase. This is the first demonstration of the expense of phosphatidylcholine aids homeoviscous homeoviscous adaptation in a cold-hardy with a pupal adaptation by reducing torsional strain on cell membranes as Experimental Design diapause. temperatures drop. Rapid cold-hardening (RCH) 70 A Major peaks a b b Fatty acid analysis 60 • The “kinks” in the acyl chains of fatty acids with double 0 hr 1 hr 2 hr ND 50 Fig. 1 & 2 D bonds promote membrane fluidity (and homeoviscous 40 a b b D+C 4 hr 8 hr Introduction 30 adapatation) by increasing overall molecular disorder as Homogenize flies and Expose nondiapausing 20 a a a Preventing cellular damage due to low extract whole-body a b b lowering temperatures increase molecular order (e.g. pharate adults to RCH percentagepercentage FA FA of of signal signal 10 inducing conditions at packing). temperatures (cold shock) is a major challenge for 4oC 0 16: 0 16:1 18:1 18:2 insects. The flesh fly, Sarcophaga crassipalpis, Minor peaks Solid phase extraction a b b Diapause 1. 6 B a a a A. B. of phospholipids a a b possesses two known mechanisms by which cold 1. 4 Gas chromatography – mass spectrometry shock can be prevented or attenuated: by entering diapausing pupae signal signal FA FA of of percentage percentage 1. 2 a b c Whole-body phospholipids 1 into a cold-hardy pupal diapause and by rapid cold- nondiapausing pupae 0. 8 a b a a a b hardening (RCH). Diapause for S. crassipalpis is a 0. 6 a a b Sample 30 day diapausing pupae a a a 0. 4 a b a and non-diapausing pupae in the percentagepercentage of of FA signal FA signal a b a photoperiod-induced developmental arrest that 0. 2 a b a same developmental stage a a a 4 0 2 0: 0: 0:3 2 20:5 features physiological changes that contribute to low 2 2 :0 16:1 18:0 4 15:0 18:3n3 18:3n6 1 Phospholipid head group 16:0(m) temperature survival such as upregulation of heat analysis 14:0(m) Derivativization into fatty fatty acid A. Fluid membrane –Contains many B. Ordered membrane – Contains many shock proteins and (Lee et al. 1987, Hayward Fig. 3 acid methyl esterases unsaturated fatty acids. Maintains fluid saturated fatty acids (shown here) or Thin-layer chromatography state as low temperature “packs” the results from low temperature packing. If et al. 2005). RCH for the flesh fly is induced by short Figure 2. Change in major (A) and minor (B) fatty acid (FA) proportions due to membrane. “Kinks” in acyl chains from low temperature causes this, the exposures (hrs) to temperatures between 0oC and diapause and chilling in the flesh fly, Sarcophaga crassiplapis. Fatty acid methyl double bonds maintain distance between membrane transitions to gel state and esterases from non-diapausing pupae (ND) were compared with 30 day diapausing adjacent phospholipids. homeostasis is lost. 10oC and is characterized by a three-fold increase in pupae (D) and 30 day diapausing pupae that had been chilled at 4oC for 8 hrs Results (D+C). Bars respresent means ± standard error (n=5). Significance for each fatty whole body glycerol content. Glycerol alone probably acid peak was determined through a one-way ANOVA, and significant peaks were does not fully explain the protection imparted from • Oleic acid (18:1n-9) levels increased by assigned statistical groupings (a,b,c) by Tukey’s post-ANOVA t-tests. RCH because glycerol concentrations never reach a 17% in response to rapid cold-hardening References level shown to protect proteins and membranes (Fig 1A). All other fatty acids were Macartney, A., B. Maresca, A.R. Cossins. 1994. Acyl-CoA desaturases and the adaptive (Macartney et al. 1994), and other insects that 80 decreased or remained the same. c regulation of membrane composition. Temperature adaptation of biological possess RCH ability do not have any detectable c membranes, ed. by [A.R. Cossins], Portland Press, London. pps.129-139. a b upregulation of polyols (Kelty and Lee 2001). • Diapause also increased oleic acid levels 60 Kelty, J.D. and R.E. Lee, Jr. 2001. Rapid cold-hardening of Homeoviscous adaptation, the ability to alter the by 14% of the fatty acid pool (Fig. 2). All (Diptera: Drosophilidae) during ecologically based thermoperiodic cycles. Journal of Experimental Biology 204.1659-1666. composition of membrane phospholipids to maintain other fatty acids were reduced or 40 PhoEtn McElhaney, R.N., 1974. The effect of alterations in the physical state of the membrane fluidity during temperature changes (Sinensky 1974), remained the same. Unlike rapid cold- PhoCho lipids on the ability of Acholeplasma laidlawii B to grow at various temperatures. Journal of has been linked to cold adaptation in a number of hardening, the change in oleic acid 20 Molecular Biology 84.145-157. , but has not been investigated extensively increased the unsaturation index of the percentage of phospholipids Sinensky, M. 1974. Homeoviscous adaptation – A homeostatic process that regulates the 0 viscosity of membrane lipids in Escherichia coli. Proceedings of the National Academy of in insects. In the present study we investigate the fatty acid pool. ND ND D D Sciences, U.S.A. 71.522-525. +4°C 8h +4°C 8h hypothesis that homeoviscous adaptation in S. • Both diapause and rapid cold-hardening Lee, R.E., C.P. Chen, M.H. Meacham, and D.L. Denlinger. 1987a. Ontogenic patterns of crassipalpis is involved in the RCH and diapause cold-hardiness and glycerol production in Sarcophaga crassipalpis. Journal of Insect cause phopholipid head group changes Figure 3. Change in phospholipid class due to rapid cold-hardening and diapause in the 33.587-592. responses of this flesh fly by chromatographic flesh fly, Sarcophaga crassipalpis. Phosphatidylethanolamines (PhoEtn) are upregulated at with an increase in the proportion of the expense of phosphatidycholines (PhoCho) due to rapid cold hardening (p<0.05) and Hayward, S. A. L., S.C Pavlides, S.P. Tammariello, J.P. Rinehart, and D.L. Denlinger. 2005. Temporal expression patterns of diapause-associated in flesh fly pupae from analysis of fatty acid constituents of phospholipids as phosphatidylethanolamine in relation to diapause (p<0.01), but chilling a fly in dipause does not enhance this response beyond that well as by thin-layer chromatographic analysis of induced by diapause alone. Letters above the bars (mean ± standard error, n=5) represent the onset of diapause through post-diapause quiescence. Journal of Insect Physiology phosphatidylcholine (Fig. 3). statistical groupings (Tukey’s). D is diapause and ND is nondiapause. Chilling was carried 51.631-640. phospholipid head groups.. out by exposing the fly to 4oC for 8 hrs