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The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.

Sporulation of Bacillus sphaericus strain 2297 in a synchronous liquid culture was studied by electron microscopy. The t0 of sporulation occurred 7 h after the beginning of the lag phase. Crystal-like inclusions first appeared at t2 and reached their final size between t5 and t6. The release of the spore/inclusion complex occurred at about t15 (22 h after inoculation). Toxicity against Culex pipiens larvae was related to sporulation and appeared during the early stages of sporulation. The LC50 (24 h) decreased about 10(5)-fold between t0-2 and t7, in correlation with the formation of crystalline inclusions. Heat resistance of spores appeared later than toxicity.

A lethal fraction for Anopheles stephensi larvae has been isolated from Bacillus sphaericus strain 1593 spores by various methods The best results have been obtained by freezing and thawing which give an extract made in majority of protein with high molecular weight and causing 100% of mortality in 24 hours. Because of its easy extracction and its lack of diaminopimelic acid, the toxic fraction is thought to be located outside the spore coats.

Asporogenous mutants from Bacillus sphaericus strains 2297 and 1593-4, blocked at different stages of the sporulation process, were isolated. Two mutants (2297 Aspo30A and 2297 Aspo34) which were blocked early in sporulation did not possess any crystalline inclusions and were poorly toxic to Culex pipiens mosquito larvae. Other mutants (2297 Aspo115, 2297 Aspo24 and 1593-4 Aspo12) which were blocked at later stages synthesized crystal-like inclusions next to the forespores, and were highly toxic to mosquito larvae. Electrophoretic protein analysis of alkali extracts from mutants and wild type strains confirmed the absence of toxic crystal-related proteins in early-blocked mutants and their presence in later ones. Western blots with antisera directed against the crystal proteins confirmed those observations.