Journal of Insect Physiology 47 (2001) 393–400 www.elsevier.com/locate/jinsphys Cold tolerance and proline metabolic gene expression in Drosophila melanogaster Stephen R. Misener, Cheng-Ping Chen, Virginia K. Walker * Department of Biology, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Received 3 May 2000; accepted 2 October 2000 Abstract Treatment of Drosophila melanogaster adults with an inhibitor of protein synthesis led to a decrease in intrinsic cold-shock tolerance, but no difference in the rapid cold hardening response, which is apparent only if a period at 4°C precedes the cold stress. Increases in energy reserves, including proline, were found in lines of flies selected for resistance to chilling injury. Since an increase in proline levels has been associated with overwintering in insects, and for salt and cold tolerance in plants, an RNase protection assay was developed to assess changes in transcript abundance for two genes encoding enzymes important for proline metabolism, pyrroline 5-carboxylate reductase and proline oxidase. The mRNA levels did not change in response to low temperature, but the high level of pyrroline 5-carboxylate reductase transcript is consistent with the interpretation that a large proline pool is important for Drosophila metabolism and survival during cold stress.  2001 Elsevier Science Ltd. All rights reserved. Keywords: Pyrroline 5-carboxylate reductase; Proline oxidase; Cold tolerance; Salt resistance; RNase protection assay 1. Introduction Drosophila melanogaster is a species that has been extensively studied because of the wealth of known gen- etic mutations, its short generation time, its easy domes- ticity, the active genome project, and a reliable gene transfer system. The genus has also been studied with respect to general cold tolerance in relation to field ecol- ogy, evolution and biochemistry (David et al., 1983; Yamanoto and Ohba, 1984; Kimura, 1988; Parsons, 1990; Ohtsu et al., 1993, 1998; Gilchrist et al., 1997; Kelty and Lee, 1999). Ironically, however, few experi- ments have focused on this insect’s physiological adap- tations to the rigors of its natural habitat and the cold- hardening response. It is vulnerable to cold conditions: it is freeze-intolerant and is susceptible to cold-shock, or non-freeze injury (Czajka and Lee, 1990). Multiple genes presumably contribute to cold-shock survival as marked cold-shock tolerance could be readily selected, even in standard laboratory strains (Chen and Walker, * Corresponding author. Tel.: + 1-613-533-6123; fax: + 1-613-533- 6617. E-mail address: walkervk@biology.queensu.ca (V.K. Walker). 0022-1910/01/$ - see front matter  2001 Elsevier Science Ltd. All rights reserved. PII:S0022-1910(00)00141-4 1993). Energy reserves, including glycogen, triacylgly- cerols and total proteins, were higher in these selected lines and were correlated with the observed elevated chilling tolerance at acclimation temperatures (Chen and Walker, 1994). Proline, too, is an important metabolic energy source in many insects, and in dipterans this amino acid appears to be preferentially oxidized for energy production in flight muscle (Sacktor and Childress, 1967; Bursell and Slack, 1976). In the freeze-intolerant gall moth, Epib- lema scudderiara, alanine levels increase after long exposure to cold (Churchill and Storey, 1989) and oxi- dation of proline to alanine can theoretically provide 14 ATP molecules of metabolic energy. Indeed, in Epib- lema solidaginis, mitochondria from cold acclimated lar- vae show a preference for proline oxidation, suggesting that proline may be an important metabolic fuel for over- wintering (Storey and Storey, 1992). In addition, in spec- ies as divergent as flies, beetles and crickets, cold acclimation and cold tolerance are correlated with sig- nificant increases in proline levels (Shimada and Riihi- maa, 1990; Fields et al., 1998; Ramlov, 1999). The majority of investigations on stress induction of proline synthesis, however, have not been in insects, but in plants, where it has become a major focus for genetic