The broad objectives are to further illucidate the life cycle of Lambornella clarki, (Tetrahymenidae: Ciliphora) and to assess its potential as a manipulated biological control agent of the western treehole mosquito, Aedes sierrensis, and related mosquitoes. We also plan to establish L. clarki in known L. clarki-negative treeholes and to determine factors influencing infection rates and persistence in mosquito breeding sites. Specific laboratory objectives include determining environmental and physiological cues that stimulate encystation and excystation, determining how long cysts remain viable, and studying L. clarki and its pathogenesis for Ae. sierrensis and other mosquito species. The long-term objectives include development of an in vitro culture system for L. clarki, and ultimately development of techniques in laboratory mass-production, storage, and field inoculation of treeholes. Dispersal of the parasite by infected adults will be quantified under field conditions. Laboratory colonies will be used to study interrelationships between L. clarki and other pathogens. Laboratory mass production and field application of this mosquito pathogen could result in the successful use of a biological control agent with excellent potential for persistence in aquatic habitats of container-breeding mosquitoes. Container-type mosquito habitas are breeding sites for some of the most effective mosquito vectors of important human pathogens (e.g. Ae. aegypti and Yellow Fever and Dengue Fever, Ae. polynesiensis and Filariasis, Ae. albopictus and Dengue, and Ae. triseriatus and LaCrosse Virus). In California, Ae. sierrensis is a major biting pest, and it is a known vector of dog heartworm (Dirofilaria immitis) and the deer body worm (Setaria yehi), both of which occasionally infect humans.