Lagenidium giganteum (Oomycetes: Lagenidiales), a facultative parasite of mosquito larva, is the only biological control agent (this category does not include the microbial insecticides produced by various Bacillus spp.) that is approaching operational use for mosquito control. This is significant because the use of chemicals commonly applied for mosquito control is being increasingly restricted due to regulatory action or development of resistance. The infective stage of the fungus is a motile zoospore, produced by either sexual or asexual reproduction, which specifically attaches to, encysts, and penetrates the cuticle of mosquito larvae. Use of L. giganteum is limited, therefore, to mosquito habitats in which sporulation, host recognition by the zoospore, and subsequent events in the infection process can occur. The long-term objective of the proposed research is to identify the molecular bases for host recognition by the fungus, so that selection for more virulent isolates can proceed systematically. This work will provide basic information (protoplast generation, proteins involved in host infection) for genetic engineering should this be necessary to develop more virulent strains or to modify host range. The project encompasses two main areas; 1) Elucidation of the chemical components on the cuticular surface of larvae and pupae of Culex tarsalis which trigger L. giganteum zoospore attachment, encystment and penetration; and 2) Identification of the putative proteins on the zoospore anterior flagellum and cyst cell wall which are involved in the recognition process. Investigations of cuticular chemistry will entail isolation and structural identification of lipids, (poly)phenols, chitin and its derivatives, and other components using high pressure liquid chromatography, gas chromatography - mass spectrometry, nuclear magnetic resonance and other standard spectroscopic techniques. Selective modification or removal of cuticular components will be followed by assaying the effects of these treatments on zoospore behavior. Zoospore and cyst proteins, and peroxidase isozymes will be purified and characterized. Monoclonal antibodies will be made to these proteins, and fluorescence microscopy used to confirm their spatial distribution on the zoospore/cyst surface. Documentation of zoospore/cyst behavior following incubation with the various monoclonals will confirm a role for the complementary proteins in host recognition.
