The toxicity of Clostridium bifermentans serovar malaysia to mosquito larvae is due to protein toxins, belonging to a novel class of insecticidal toxins. Toxic extracts contains three major proteins of 66, 18 and 16 kDa. The 18-kDa and 16-kDa proteins are probably involved in toxicity. They are synthesised during sporulation, concomitant with activity. They are absent from non-toxic strains of C. bifermentans and are present at very low levels in non-toxic C. bifermentans serovar malaysia cultures produced at 42 degrees C. The 66-kDa protein is present throughout the growth phases of C. bifermentans serovar malaysia, and an immunologically related 66-kDa protein is present in non-toxic C. bifermentans strains.
Clostridium bifermentans serovar. malaysia (C.b.m.) is toxic to mosquito larvae. In this study, we quantified its toxicity to the mosquitoes, Aedes aegypti, Ae. albopictus, Ae. caspius, Ae. detritus, Anopheles stephensi, An. gambiae, Culex pipiens and Cx. quinquefasciatus. Anopheles larvae are the most susceptible, followed by Ae. detritus and Ae. caspius, then Culex and other Aedes larvae. According to mosquito species, the LC50 varies from 7 x 10(3) to 1.3 x 10(6) cells/ml. Three concentrations (10(7), 10(6) and 10(5) cells/ml) of C.b.m., Bacillus thuringiensis var. israelensis (B.t.i.) and Bacillus sphaericus were tested on Ae. aegypti, An. stephensi and Cx. pipiens larvae in order to determine the time necessary for each concentration to kill 50 and 90% of the population. Ninety percent of the 3 mosquito populations are killed within 4-15 h by the C.b.m. concentrations. Whatever the concentrations, C.b.m. kills at least 10 times less rapidly than B.t.i. but always quicker than B. sphaericus. Bioassays of C.b.m. bacterial cells or final whole culture were not toxic to Musca domestica and Drosophila melanogaster (Diptera) as well as to Phaedon cochleariae (Coleoptera) and Spodoptera littoralis (Lepidoptera).
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.
The safety of bacterial cells of Clostridium bifermentans serovar malaysia, which is highly toxic to mosquito larvae, was tested on mice, guinea pigs, rabbits, and goldfish. Inoculations of at least 1 x 10(8) cells per animal by routes recommended by World Health Organization (subcutaneous, percutaneous, inhalation, force-feeding, intraperitoneal, intravenous) and tests of subacute toxicity, anaphylactic shock, persistence in heart blood, and virulence by successive passages, were performed on mice, guinea pigs, or both. Growth was monitored for 1 mo before necropsy. Ocular irritation and skin scarification were tested with rabbits. C. bifermentans serovar malaysia did not induce any mortality or abnormal reactions in mammals at a dose 1,000 times higher than the level established by W.H.O. for the demonstration of safety. Bacterial cells are not toxic to goldfish at a dose 1,000 times higher than the LD50 for the target-mosquito larvae. We conclude that C. bifermentans serovar malaysia bacterial cells are safe for laboratory mammals and goldfish.
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.