This Small Business Innovation Research Phase I project develops a more efficient and environmentally safe method to produce and deliver Bacillus thuringiensis israelensis (Bti) toxins and BtBoosters (BtB) for mosquito control. Bti toxins have strong mosquito species- specific activities, and are safer than chemical insecticides. BtB proteins enhance Bti toxicity by increasing toxin uptake. A novel and inexpensive system for Bti and BtB production and delivery is needed to reach mosquitoes in various aquatic environments. This project's biotechnical hypothesis is: Bti and BtB coupled to oleosins on the surface of seed oil bodies of oil-rich crops can be delivered effectively as milled Bt-flour that will float at various levels in the water column and kill feeding mosquito larvae. This Phase I application will test this hypothesis by coupling Bti and BtB to oleosins in the oil-rich seeds of Arabidopsis thaliana. Seeds will be milled into Bt-flour, with a particle size approximating that of food consumed by filter-feeding larvae. The Bt-flour will be dispersed in water and fed to larvae of three mosquito species that act as disease vectors and insect mortality quantified. This will demonstrate the synergism between BtB and Bti can lead to increased control of disease vectoring mosquito species. Mosquitoes are the insect vectors for numerous blood-borne human diseases including malaria, West Nile fever, yellow fever, and dengue that cause millions of deaths each year worldwide. Mosquito larvae are filter-feeders that inhabit quiet open-water environments, and depending upon species, their larvae live at various depths in the water column. Of all vectors for zoonotic transmission of infectious agents, mosquitoes are one of the more difficult to control. This is possible, but only by adding large amounts of chemical or biological insecticides to the aquatic environment. This project will demonstrate that the synergy between BtB and Bti can lead to substantially improved control of disease vectoring mosquito species by biological pesticides, lowering the amount of chemical pesticides entering aquatic environments. The U.S. market for mosquito larvicide control agents is about $45 million per year, with a much larger worldwide market. The market opportunity will be from both the traditional segment of mosquito control and also the emerging regulatory segment. A phase II application is planned in which oleosin coupled Bti and BtB will be expressed in canola (Brassica napus). The Bti/BtB expressing canola seeds can be produced and milled into Bt-flour at relatively low cost. PUBLIC HEALTH RELEVANCE: Mosquitoes are vectors for numerous blood-borne diseases, including malaria, yellow fever, West Nile virus, filariasis, Japanese encephalitis, and dengue fever and cause millions of deaths worldwide annually. This project is designed to develop a novel, more efficient, inexpensive, and environmentally safe solution to the production and delivery of Bti-related toxins and BtBoosters for mosquito control at the larval stage. The synergy between Bti and BtBooster as milled flours that float at various levels in the water column will lead to substantially improved control of disease-vectoring mosquito species by biological pesticides, lowering the amount of chemical pesticides entering aquatic environments and thereby producing significant public health-related benefits through better control of vector-borne diseases.