Keio Aizawa

Distribution of Bacillus thuringiensis in soils of Japan

Abstract A total of 136 soil samples collected from nonsericultural areas of 18 prefectures of Japan were examined for the distribution of Bacillus thuringiensis . Of 6910 isolates referable to the Bacillus cereus-B. thuringiensis group, 189 (2.7%) produced parasporal inclusions (PIs). Among these PI-forming isolates, only 66 (34.9%) were identified as B. thuringiensis H serotypes 3a, 4a:4c, 5a:5c, 8a:8c, 10, 11a:11c, and 17. Of the remaining 123 isolates, 84 were well-motile but untypeable by H antisera against the known B. thuringiensis serotypes 1–21, and 39 were untestable because of nonmotility or marked autoagglutination. The results suggested that B. thuringiensis flora in soils was remarkably different from that in the sericultural environment.

Insect toxicity of Bacillus thuringiensis isolated from soils of Japan

Abstract A total of 189 isolates of Bacillus thuringiensis producing parasporal inclusions (PIs), obtained from soils of Japan, were examined for their oral toxicity against larvae of the silkworm, Bombyx mori , and the mosquito, Aedes aegypti , and adults of a chrysomelid coleopteran, Pyrrhalta tibialis . Of these isolates, 68 (36.0%) were toxic to insects tested: 48 were Lepidoptera-toxic and 20 were mosquito-toxic. The Lepidoptera-specific isolates were referable to B. thuringiensis serotypes 3a (2 isolates), 5a:5c (1 solate), and 10 (44 isolates), while one isolate was untypable by the known B. thuringiensis H antisera. Unlike PIs of the known Lepidoptera-toxic B. thuringiensis strains, PIs produced by these untypable and serotype 10 isolates were spherical in shape. Of 20 mosquitotoxic isolates, 7 were referable to B. thuringiensis serotype 11a:11c, and 13 were untypable. Although 121 isolates (64%) produced PIs with various morphologies including typically bipyramidal ones, they did not demonstrate any toxicity against insects of three orders, suggesting that nontoxic PI-forming bacteria predominate in natural environments rather than toxic ones.

Deoxyribonucleic Acid Relatedness between Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis

proved as distinct taxa (1, 2), as they are different in pathogenicity and in some other properties. However it is also widely accepted that they are quite similar. In fact some pathogens may produce non-pathogenic offsprings that are hardly distinguishable from normal B. cereus. Some B. cereus strains share some flagella antigens with B. thuringiensis (6; Ohba and Aizawa, unpublished data), and the formation of intracellular protein crystals, which has been considered as another characteristic of the latter, is now known in Bacillus subtilis (4) too. In their comprehensive studies Smith et al (9, 10) and Gordon et al (3) emphasized their similarities rather than difference and took a view that they are from academic viewpoint the different varieties of B. cereus. Since DNA homology was introduced as a new taxonomic criterion, Somerville and Jones (11) made some DNA-DNA competition experiments and suggested close interrelatendness of the three, though their data do not appear very clearcut. DNADNA hybridisation studies by Azuma (personal communication) and by Seki et al

Isolation of a Bacillus thuringiensis strain (serotype 8a:8b) highly and selectively toxic against mosquito larvae

Abstract An isolate of Bacillus thuringiensis designated as PG-14 obtained from the Philippines was highly toxic to the mosquitoes Aedes aegypti and Culex molestus but nontoxic to the silkworm, Bombyx mori , and adults of a daphnid. The degree of toxicity to mosquito larvae was the same as that of the reference strain of Bacillus thuringiensis subsp. israelensis (serotype 14). Parasporal inclusion produced by the isolate PG-14 was spherical or irregular in shape and morphologically similar to that produced by the reference strain of subsp. israelensis . The H antigenic structure of the isolate was identical to that of the reference strain of B. thuringiensis subsp. morrisoni (serotype 8a:8b). Differences were shown in the O antigenic structures and in the production of lecithinase. Thermostable exotoxin was not produced by the isolate PG-14. The results indicate the isolation of a B. thuringiensis strain which shows the same toxicity as that of subsp. israelensis .

Serological identification of Bacillus thuringiensis and related bacteria isolated in Japan

Abstract Distribution of Bacillus thuringiensis and related sporeforming bacteria in Japan was investigated and it was found that most of the crystalliferous isolates belonged to B. thuringiensis serotypes 3a, 4a:4b, 7, and 8. Serotypes 1, 3a:3b, 4a:4c, and 11 were rarely isolated. H antigens of 189 isolates of acrystalliferous sporeformers were analyzed and 26 isolates were agglutinated by B. thuringiensis H antisera against serotypes 3a, 4a:4b, 5a:5c, 6, 7, 8, 10, 11, and 12 at high serum dilutions. Heat-stable somatic antigens of these isolates differed significantly from that of reference B. thuringiensis .

The isolates of Bacillus thuringiensis serotype 10 with a highly preferential toxicity to mosquito larvae.

Abstract Comparative bacteriological and serological studies of three isolates and the reference strain of Bacillus thuringiensis subsp. darmstadiensis (serotype 10) were conducted. No difference was shown in the flagellar antigenic structure between the three isolates and the reference strain. Differences were observed in the O antigenic structures and in the following biochemical properties: lecithinase production, DNase production, arginine decarboxylase production, acid production from inulin, and malonate utilization. β-Exotoxin production was not detected in these three isolates. The reference strain produced parasporal inclusions toxic to the lepidopterous larvae but nontoxic to mosquito larvae. On the contrary, two among the three isolates, which produced spherical parasporal inclusions, were not toxic to the lepidopterous larvae but highly toxic to larvae of the mosquitoes, Culex tritaenlorhynchus, Culex molestus , and Aedes aegypti . Another isolate produced large irregular-shaped inclusions nontoxic to the insects of both orders. Accordingly, B. thuringiensis serotype 10 was divided into three groups from the viewpoint of toxicity against lepidopterous and mosquito larvae.

Production of heat-stable exotoxin by Bacillus thuringiensis and related bacteria

Abstract Heat-stable exotoxin production by 740 strains of Bacillus thuringiensis and related bacteria was investigated using the housefly, Musca domestica , from the following viewpoints: (1) the relation-ship between B. thuringiensis flagellar (H) serotypes and exotoxin production and (2) the exotoxin production by Bacillus species other than B. thuringiensis . Of 437 isolates belonging to 11 serotypes of B. thuringiensis which had been confirmed to produce parasporal inclusions, 35 isolates belonging to serotypes 1, 3a:3b, 4a:4c, and 10 produced heat-stable exotoxin. Exotoxin was not detected in the isolates of serotypes 3a, 4a:4b, 5a:5b, 5a:5c, 6, 7, and 8a:8b. No heat-stable exotoxin was demonstrated in 28 acrystalliferous isolates which possessed H antigens of B. thuringiensis serotypes 1, 3a, 4a:4b, 4a:4c, 5a:5c, 6, 7, 10, 11a:11c, and 12. A total of 270 B. cereus isolates which did not possess B. thuringiensis H antigen were examined and three isolates were found to produce heat-stable exotoxin. No heat-stable exotoxin was produced by B. subtilis (two strains), B. natto (one strain), and B. megaterium (two strains). These results indicate that the heat-stable exotoxin production in B. thuringiensis is a strain-specific property rather than a serotype(subspecies)-specific property.

Occurrence of Non-Insecticidal Bacillus thuringiensis Flagellar Serotype 14 in the Soil of Japan

Summary Three spore-forming isolates obtained from a soil sample of Hokkaido Island, Japan, were referable to Bacillus thuringiensis flagellar (H) serotype 14 (subsp. israelensis). Parasporal inclusions of these isolates were morphologically very similar to those produced by the type strain of B. thuringiensis H serotype 14. The type strain of H serotype 14 was highly virulent to aedine and culicine mosquito larvae, while the three isolates were avirulent to mosquito larvae. Immunologically, there was no relationship between parasporal inclusion proteins of the type strain and the three avirulent isolates. A great difference was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of parasporal inclusion proteins. Parasporal inclusions of the type strain contained proteins with molecular masses of 130 kD, 70 kD, 38 kD, and 24 kD, while those of the avirulent isolates contained proteins of 47 kD, 36 kD, 30 kD, and 24 kD and a few minor proteins. The results suggested that the avirulent property of the H serotype 14 isolates was attributable to the lack of 130-kD protoxin in their parasporal inclusions.

Purification of the toxic protein from Bacillus thuringiensis serotype 10 isolate demonstrating a preferential larvicidal activity to the mosquito

Abstract The protein demonstrating larvicidal activity to the mosquito Aedes aegypti was purified from the alkali extract of the spore-parasporal inclusion complex of the isolate, 73-E-10-2, belonging to Bacillus thuringiensis serotype 10. By Sepharose CL-4B gel filtration and DEAE-cellulose column chromatography, a toxic protein was obtained, and its homogeneity was confirmed by Sephadex G-150 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the toxic protein was 67,000, when estimated by SDS-PAGE. The LC 50 of the toxic protein against 4-day-old larvae of A. aegypti was 16.8 μg/ml. There was no serological relationship between the toxic protein from the isolate 73-E-10-2 and that ( M r 67,000) from the type strain of B. thuringiensis subsp. israelensis .

Multiplication of Chilo iridescent virus in noninsect arthropods

Abstract Attempts to infect noninsect arthropods with Chilo iridescent virus (CIV) originally isolated from Lepidoptera were made by using eight species belonging to four classes. Multiplication of CIV was demonstrated in two species of terrestrial Crustacea (the pill bug, Armadillidium vulgare , and the slater, Porcello scaber ) and one species of Chilopoda, the house centipede, Thereuonema higendorfi . The lethality experiment of CIV for A. vulgare suggested that chronic infection is a characteristic of the CIV infection in both classes, Crustacea and Insecta. Neither iridescence nor recovery of virus infectivity was demonstrated in the following arthropod species: the sea slater, Ligia exotica (Crustacea: Isopoda), the grapsid crab, Sesarma haematocheir (Crustacea: Decapoda), the millipede, Oxidus gracillis (Diplopoda: Polydesmoidea), Rhysodesmus semicirculatus (Diplopoda: Polydesmoidea), and the giant crab spider, Heteropoda venatoria (Arachnida: Araneae).