Sporulation of Clostridium bifermentans serovar malaysia, which has a larvicidal activity towards mosquitoes, was examined by electron microscopy. Parasporal inclusion bodies lacking a crystalline structure were first detected at t5 (5 h after the end of exponentional growth). Also, the presence of "brush-bottle"-like appendages appearing first at t5 was noted; these remained attached to the spores when released after sporangium lysis. Larvicidal activity assayed on Anopheles stephensi larvae appeared at t0 and increased rapidly to a maximum between t5 and t8. However, a decrease in bacterial toxicity occurred with sporangium lysis.
A strain of Clostridium bifermentans individualized as serovar malaysia (C.b.m.) according to its specific H antigen is toxic to mosquito and blackfly larvae when given orally. The toxicity occurs in sporulated cells which contain, in addition to spores, proteinic parasporal inclusion bodies and feather-like appendages; the amino acid content of the inclusion bodies is similar to that of Bacillus thuringiensis serovar israelensis (B.t.i.) and B. sphaericus crystals. The toxicity to Anopheles stephensi is as high as that of B.t.i. and the best strains of B. sphaericus. Culex pipiens is somewhat less susceptible, and Aedes aegypti much less. Pure parasporal inclusion bodies, isolated by ultracentrifugation on sucrose gradients, are highly toxic to mosquito larvae. The larvicidal power is destroyed by heating at 80 degrees C or by treatment with 50 mM NaOH. It is preserved by freeze-drying. The innocuity to mice of the sporulated cells is shown by different routes of administration: force-feeding, percutaneous, subcutaneous, intraperitoneal or intravenous injections. The potential for the biological control of mosquito and blackfly larvae is suggested.