1. Genetic control of plastic and evolutionary responses to the environment in Drosophila melanogaster Bregje Wertheim, Patrick Goymer, Alex Kraaijeveld, Meirion Hopkins, Charles Godfray, and Linda Partridge Centre for Evolutionary Genomics, University College London, Gower Street, London WC1E 6BT NERC Centre for Population Biology, Imperial College London, Silwood Park Campus, Ascot, Berks SL5 7PY Introduction Our aim is to identify and compare the genes involved in phenotypic plasticity and those that change during evolutionary adaptation. Drosophila melanogaster is an ideal model organism for these studies, because of its sequenced genome and because we have substantial knowledge of its ecology. Our approach is to expose Drosophila to a biotic and an abiotic stress factor, respectively attack by a parasitoid and temperature stress. We monitor changes in gene expression after inducing a plastic response and compare gene expression among strains that differ in their ability to cope with the specific environmental stress. Clinal Variation due to Temperature Latitudinal variation in temperature has resulted in clines of various phenotypic traits in Drosophila melanogaster. These include developmental time and body size. One particularly well characterised body size cline is that along the eastern coast of Australia. From our knowledge of the genetics of this cline and the mechanisms of body size determination we have identified several candidate genes. We are using real-time PCR to look for heritable differences in expression level of these genes between northern and southern cline-end populations. Acute Temperature Stress The ability to withstand heat shock is very important to flies from tropical environments that are frequently exposed to direct sunlight. We tested for heritable differences between Northern and Southern Australian populations in the time taken to be knocked down by a 38ºC heat shock. Though reared in the lab at the same temperature, the populations of tropical origin have a greater resistance to heat shock. This characterised phenotype will form the basis of a microarray experiment. Chronic Temperature Stress It is well known that lifespan in insects is inversely related to ambient temperature. We tested whether this was due to increased accumulation of age-related damage at high temperature, or merely an increased instantaneous risk of death. Flies were cultured at 18ºC and 27ºC and deaths recorded in order to calculate age-specific mortality, a measure of the instantaneous risk of death. We found that switching flies from high to low temperature reduced the rate of increase in mortality but did not reduce the absolute risk to that of the 18ºC control. Likewise, switching flies from low to high temperature increased the mortality gradient but did not increase risk to the level of the 27ºC control. These results suggest that high temperature reduces lifespan by increasing the rate of living and hence the accumulation of age-related damage. Wing area of lab-reared (upper line) and wild- caught (lower line) male flies from the Australian cline. The differing slopes illustrate that there are both genetic and environmental contributions to body size. Figure adapted from James et al (1997). Parasitoid attack In the ecology of many insects, parasitoids pose a formidable threat. Larvae of D. melanogaster are doomed after an attack by the parasitoid Asobara tabida, unless they launch a rapid immune response to encapsulate the parasitoid egg and hence prevent it from developing. The capsule consists of specialised blood cells that melanise. Inducible immune response Using whole genome micro-arrays (Affymetrix), we measured gene expression during the 3 days spanning the immune response. At least 100 genes show different behaviour in control and parasitised larvae (2-way Anova with Bonferroni correction). These genes comprise many of unknown molecular function, and are largely different from those expressed after bacterial and fungal challenge. The genes are involved in the recognition of non-self, production of specialised blood cells, and encapsulation of the foreign body. t = 0 ……… 72h0 ……… 72h parasitised larvae control larvae Asobara tabida, parasitoids that attack larvae of D. melanogaster encapsulated parasitoid egg inside a larva of D. melanogaster Evolution for parasitoid resistance To compare genes for the induced responses with genes that changed during evolution for parasitoid resistance, we study various geographical strains and sets of selection & control lines. These differ in encapsulation ability, and gene expression is profiled both before parasitoid attack and during the induced immune response. And next… We obtained significant differences in selection and control lines after 5 generations of artificial selection. In our next experiment, one of these lines will be profiled on whole genome micro-arrays. Subsequently, candidate genes can be quantified with real-time PCR in the other selection and control lines and in geographical strains. Geographical variation in parasitoid resistance in larvae of D. melanogaster Kraaijeveld & Van Alphen (1995). Evol.Ecol., 9: 10-17. Percentage of larvae with successfully encapsulated parastoid egg in selection lines (coloured) and control lines (white) of D. melanogaster. encapsulation (%) Gene expression in parasitised and controlD. melanogaster larvae across the 3 days spanning the immune response. Each line represents a gene that is up-regulated or down- regulated in parasitised larvae.