Cold tolerance and cold-induced modulation of gene expression in two Drosophila virilis group species with different distributions L. Vesala*, T. S. Salminen*, A. Laiho†, A. Hoikkala* and M. Kankare* *Department of Biological and Environmental Science, Centre of Excellence in Evolutionary Research, University of Jyväskylä, Finland; and †Turku Centre for Biotechnology, The Finnish Microarray and Sequencing Centre, Tykistökatu, Turku, Finland Abstract The importance of high and low temperature tolerance in adaptation to changing environmental conditions has evoked new interest in modulations in gene expression and metabolism linked with stress tolerance. We investigated the effects of rapid cold hardening and cold acclimatization on the chill coma recovery times of two Drosophila virilis group species, Drosophila montana and D. virilis, with dif- ferent distributions and utilized a candidate gene approach to trace changes in their gene expression during and after the cold treatments. The study showed that cold acclimatization clearly decreases chill coma recovery times in both species, whereas rapid cold hardening did not have a significant effect. Microarray analysis revealed several genes showing expression changes during different stages of cold response. Amongst the 219 genes studied, two genes showed rather consistent expression changes: hsr- omega, which was up-regulated in both study species during cold acclimatization, and Eip71CD, which was down-regulated in nearly all of the cold treatments. In addition, 29 genes showed expression changes that were more treatment- and/or species specific. Overall, different stages of cold response elicited changes mainly in genes involved in heat shock response, cir- cadian rhythm and metabolism. Keywords: DNA microarray, candidate genes, chill coma recovery, Drosophila montana, Drosophila virilis. Introduction Adaptation of insects to fluctuating temperature conditions prevailing at high latitudes includes a capability to survive and remain active at lower than optimal temperatures. This has evoked interest in the physiological and genetic mechanisms helping the insects to cope with low tempera- tures (Hoffmann et al., 2003; Sinclair et al., 2003; Danks, 2005). Most insect species are able to adjust their toler- ance levels according to ambient temperature, which is important especially in fluctuating temperature conditions. Short-term exposure to nonlethal low temperatures pre- ceding a more severe cold period has been found to sustain insects’ courtship and mating activity (Shevre et al., 2004) and to improve their survival (Czajka & Lee, 1990) at low temperatures. This phenomenon, termed rapid cold hardening, has been suggested to be at least partly based on the mechanisms that prevent phase tran- sitions in the membrane phospholipids and help to pre- serve membrane fluidity (Overgaard et al., 2005). Yi et al. (2007) found rapid cold hardening to decrease the inci- dence of apoptosis by 38% after a cold shock, when compared to a nonhardened control group in Drosophila melanogaster. Cold acclimatization, by contrast, usually refers to a process that occurs over a longer time span (days or weeks). It is accompanied with qualitative and quantitative changes in sugars, polyols and amino acids, which act as cryoprotectants shielding an organism against injurious effects of low temperature (reviewed in Denlinger & Lee, 1998). Many insect species exhibit chill coma when the tem- perature decreases below a certain point, but still remains above the lethal temperature. In chill coma the electrical activity of muscles is disrupted, which leads to immobili- zation (Goller & Esch, 1990). This is a reversible state, if not prolonged, and an insect regains its ability to move First published online 28 November 2011. Correspondence: Laura Vesala, Department of Biological and Environ- mental Science, P.O. Box 35, FI-40014 University of Jyväskylä, Finland. E-mail: laura.vesala@jyu.fi Insect Molecular Biology Insect Molecular Biology (2012) 21(1), 107–118 doi: 10.1111/j.1365-2583.2011.01119.x © 2011 The Authors Insect Molecular Biology © 2011 The Royal Entomological Society 107