H. de Barjac

Essai de classification biochimique et sérologique de 24 souches deBacillus du typeB. Thuringiensis

Summary24 strains ofBacillus thuringiensis type are studied by the determination of 35 cultural and biochemical characters. About 10 different biochmeical characters make it possible to classify these strains into six major biochemical groups represented by one or two type-strains (biotypes). On the other hand, the study of these 24 strains by the flagellate agglutination reaction also leads to six serological groups, each one provided with a different H antigen, and which fit perfectly the biochemical classification. This fact emphasizes the validity of this latter classification and, for the first time, brings in a double biochemicals-serological basis for the classification of theB. thuringiensis type strains.

Growth, sporulation and larvicidal activity of Bacillus sphaericus

SummaryA new medium (MBS) for optimal sporulation of Bacillus sphaericus was defined. With the two main mosquito pathogenic strains grown in this medium, 1593-4 and 2297, highest cell and spore yields were obtained, concomitantly with an highest larvicidal activity against Culex pipiens. Study of both strains asporulated mutants showed a decrease in larvicidal power. After plasmid curing treatments, toxicity of strain 1593-4 did not decrease, neither toxic parasporal inclusion bodies of strain 2297 disappear.

Serological classification of Bacillus sphaericus strains on the basis of toxicity to mosquito larvae

SummaryA toxicity study of 54 Bacillus sphaericus strains isolated from vectors or breeding sites has led to a relatively homogeneous grouping of mosquito pathogenic strains into five H-serotypes among the nine serotypes determined. Each serotype seems to be characterized by a different level of toxicity and a classification of these five serotypes can be made on the basis of this toxicity. Within these serotypes, a scale of toxicity has been tentatively fixed and an arbitrary limit of toxicity suggested.

Sporulation of Bacillus sphaericus 2297: an Electron Microscope Study of Crystal-like Inclusion Biogenesis and Toxicity to Mosquito 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.

Action des cristauxde Bacillus thuringiensis var Israelensis sur l'intestin moyen des larves de Aedes aegypti L., en microscopie électronique

Summary Ingestion of Bacillus thuringiensis var. israelensis crystals by Aedesaegypti larvae is followed by midgut epithelium disruption. Earliest ultrastructural changes consist of an enlargement of intra- and intercellular spaces in the basal region of the cell. Endoplasmic reticula disintegrate by forming spherical structures which increase in size during intoxication. Mitochondria are transformed at first into a condensed form, then become swollen with the disappearance of internal cristae. In the cardia cells, which secrete the peritrophic membrane, the Golgi apparatus may produce electron-dense secretion vesicles; in this event, the peritrophic membrane assumes an abnormal configuration. Before complete breakdown, a cellular hypertrophy is observed: few cells become balloon-like. At the same time, the microvilli decrease in size, widen and then disappear after a few hours exposure to crystal δ-endotoxin.

Selection of the most potent Bacillus sphaericus strains based on activity ratios determined on three mosquito species

SummaryThe larvicidal power of more than 180 Bacillus sphaericus strains belonging to six H serotypes has been assayed on Culex pipiens, Anopheles stephensi and Aedes aegypti under standardized conditions. The most potent strains are distributed into serotype H5a5b, generally toxic to the three mosquito species, and serotypes H6 and H25, toxic to C. pipiens and A. stephensi. Strains of serotypes 26a26b and H2a2b are much less toxic and most often only on C. pipiens. The relative potency of each strain can be expressed by specific titres on the different mosquito species and by activity ratios derived from such titres.

Phage typing of Bacillus subtilis and B. thuringiensis

Phage typing schemes for Bacillus subtilis and B. thuringiensis were constructed using 98 phages and 743 bacterial strains. Most phages were host-species-specific. Phages were classified by electron microscopy. The B. subtilis scheme includes 10 phages and 29 phage types. The B. thuringiensis scheme comprises 8 phages and 25 phage types and can be applied to B. cereus. There is no correlation between H antigen serotypes and phagovars in B. thuringiensis. Characteristics of typing phages are described for identity control.

Another Bacillus sphaericus serotype harbouring strains very toxic to mosquito larvae: Serotype H6

Ten isolates of Bacillus sphaericus from Ghana, very toxic to mosquito larvae, have been identified as belonging to serotype H6. These isolates can be represented by the head-group strain IAB59. They form crystals at the sporulation stage. Their larvicidal effect on Culex pipiens and Anopheles stephensi larvae is as high as that of the most toxic strains already known, e.g. 1593 and 2362 (serotype H5a,5b) and 2297 (serotype H25). Spore-crystal extracts of all these strains contain a 43-Kd polypeptide immunologically related to the 43-Kd polypeptide from strain 2362 described by other authors.

Bacillus sphaericus asporogenous mutants: morphology, protein pattern and larvicidal activity

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.

Efficacy of Dry Powders from Bacillus sphaericus: RB 80, a Potent Reference Preparation for Biological Titration

Abstract Larvicidal potency of three primary powders based on Bacillus sphaericus strains 1593 and 1881 was studied on mosquito larvae. Two acetone powders, P 1593 and P 1881, were very toxic for Anopheles stephensi larvae. The potency of a third lyophilized powder RB 80 made from 1593 strain compared even better when tested against Anopheles stephensi and Culex pipiens pipiens larvae. LC 50 s after 48 hr were 0.15 and 0.003 mg/ml, respectively. After storage of RB 80 aqueous suspensions over 2 years or after heat exposure of RB 80 powder, larvicidal potency was still high, indicating an excellent stability. The use of RB 80, because of all its qualities, is suggested as a first experimental standard for titration of B. sphaericus preparations.