Insulin-like Signaling in Parasitic Nematode Development

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DESCRIPTION: (provided by the applicant): Parasitic nematodes sicken or debilitate millions of persons worldwide. In the vast majority of these nematodes the third larval stage (L3) constitutes the infective stage for the vertebrate host. Regardless of how these L3 are acquired during the infection process, they may be viewed as transitional stages, in a state of developmental arrest, which are reactivated only when exposed to cues present in the definitive host. The mechanisms by which these parasites regulate development in the L3 remain unclear, studies in this area having been hampered by the lack of a molecular genetic system involving a parasitic nematode. By contrast, the developmental biology of L3, including the switch between continuous and arrested (dauer) development, has been under active investigation in the free-living nematode Caenorhabditis elegans, resulting in a wealth of relevant molecular genetic information on that organism. The overall goal of the proposed study is to ascertain whether mechanisms similar to those acting in C. elegans also regulate development in the parasite Strongyloides stercoralis. S. stercoralis was chosen as a model because, among numerous other functional and morphological similarities, this worm has an alternate free-living cycle reminiscent of the continuous developmental cycle of C. elegans. The specific aims of this proposal are, first, to ascertain the existence in S. stercoralis of orthologs to four key genes on the insulin-like branch of the daf pathway, which controls development in C. elegans L3. Work toward this aim will stress a PCR approach involving primers based on published C. elegans gene sequences. Genes targeted for study are orthologs of C. elegans daf-2, age-1, daf-18 and daf-16. Second, we will investigate the function of the putative S. stercoralis daf orthologs. Functional homology of putative dauer inducing genes will be ascertained by methods such as inducing targeted mutations with chimeric RNA/DNA oligonucleotides and ablating specific transcripts with specific double stranded RNA. Homology of dauer inducers and putative dauer suppressing genes will be investigated by complementation and other transgenesis studies in appropriate C. elegans strains. Finally, we will endeavor to develop methods for germ line transformation of S. stercoralis in order to assess function of putative regulatory genes. Methods widely used for DNA transformation of C. elegans via microinjection into gonadal syncytia will be adapted.