Fiona Cockerell (PhD Candidate) , Louise Toft, Travis Johnson (PhD Candidate)

To help elucidate any affects of rates of hsp70 production immediately following heat shock on heat resistance a sensitive Western blot assay was developed for hsp70 quantification and then characterised the time course of hsp70 build up. In a large association study she then applied this technique to a set of 40 single-pair mating lines that were also characterised for basal and hardened heat knockdown tolerance, as well as for rates of general protein synthesis and heat-shocked protein synthesis. No association whatsoever was detected, a result consistent with a concurrent effort by using ELISA assays and a set of 20 isofemale lines. These data make it clear that natural variation in hsp70 expression is not a major determinant of knockdown heat resistance of adults. The data suggest that effects of such hsp70 variation on thermal fitness variation is likely to subtle and/or involve changes in survival or reproductive success. This set of lines was also characterised for resting and heat-shocked level of both the cytoplasmic and nuclear transcripts of the hsr-omega heat stress gene, since variation in this gene is a candidate for influencing knockdown heat tolerance and rates of protein synthesis following heat shock. Interestingly, comparison of hsp70 data with the hsr-omega data indicated a positive association between the extent to which the hsr-omega cytoplasmic transcript increased following heat shock and the rate of hsp70 synthesis. We will investigate if this result holds up in future experiments since it is consistent with a current general model about the function of the cytoplasmic omega transcript in binding to ribosomes and ‘monitoring’ rates of protein synthesis. This is an exciting possibility since it would begin to provide meaningful insight into how the cellular heat stress response is controlled and provide a deeper understanding of a key mechanism controlling heat resistance variation. It would allow us to investigate specific ideas about adaptive, climatically-variable, heat resistance mechanisms.

Our earlier work demonstrated that a SNP site in the promoter region of the Hsp68 gene changed in frequency in populations selected for high resistance to heat knockdown when the flies had been heat hardened.  Follow up experiments with single-pair-mating lines from a climatically ‘central’ population confirmed this result, although not in all experiments. Other than the constitutive expression of Hsp68 in testies this gene is only expressed in other tissues after a mild heat shock. While no latitudinal cline in frequency variation was detected for the Hsp68 SNP we continued to work with this gene in 2007 since field release-recapture data suggested that recaptured SNP genotypes were not random, but related to time after release on hot days. This year we carried out an experiment to examine any effects of under or over-expression of Hsp68 on male fertility following a heat stress, making use of a GAL4-UAS-Hsp68 strain and of a Hsp68–RNAi strain. While no obvious effects were detected we are in the process of repeating these crosses since we need to also assess whether changes in expression of Hsp68 alone, when no heat hardening is experienced, result in altered level of heat knockdown resistance, and we need to test harsher heat treatments for males to see if Hsp68 over- or under-expression influence fertility.