This is a proposal to investigate the molecular population genetics of bacteriocidal genes in Drosophila melanogaster. There are three components to the research plan. First, nucleotide sequence variation will be determined for genes that encode and regulate antimicrobial proteins in D. melanogaster and related species. Sequence data will be obtained for the coding, intronic, and 5' regulatory regions in 6 genes and 1 gene cluster (7 Cecropin genes) encoding antimicrobial peptides, and 6 immune regulatory genes, in 48 lines of D. melanogaster, plus homologues of each gene in five related Drosophila species. A total of 16 D. melanogaster alleles will be sampled from each of three populations, one from North American, one from Europe, and Zimbabwe, Africa. (The latter population is partly reproductively isolated from cosmopolitan populations.) Population genetic analyses will be used to infer the evolutionary forces acting on these genes. Second, molecular variation at the 19 candidate genes will be associated with phenotypic variation in microbial defenses within D. melanogaster. The sample of 16 alleles of each candidate gene will be expanded to 100 from each of the three populations. (Note: this number is based on the addendum to the proposal.) The alleles will be maintained as extracted chromosome lines (100 alleles, 3 populations, plus loci on the second and third chromosomes = 600 extracted chromosomes). Variation among the isochromosomal lines will be measured for four components of resistance to infection: survival time; total pathogen load at several time points post-infection; the level of induction of immunity genes (determined by quantitative RT-PCR); and bactericidal activity of extracted hemolymph (determined by inhibition of growth and respiration of E. coli). Molecular variation at the candidate gene alleles that have not been sequenced will be evaluated by denaturing HPLC. These data will be used to assess the associations between polymorphisms in the candidate loci and variation in these phenotypes. The full sequence of alleles in phenotypically extreme lines will also be determined. The third component is a theoretical study of the coevolution of bacterial pathogens and insect hosts. Systems of simultaneous differential equations will be solved numerically to determine the constraints on genetic and epidemiological parameters that enable the continued survival of insects that have a defense system with no memory, when challenged by rapidly diversifying pathogens.
