Potential of the mosquito pathogen Bacillus sphaericus for recycling and gene transfer in larval cadavers

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Virginia Tech


The ability of spores to germinate, vegetatively multiply and produce new spores and toxin in the larval cadaver is known as recycling. The ability to recycle is an important characteristic since it may enhance effectiveness and persistence of the microbial insecticides in the larval habitat.

The ability of Bacillus sphaericus to recycle has only been examined in the low toxicity strain SSII-1 and in the two highly toxic strains 2362 and 1593, both belonging to serotype 5a5ab. This study was expanded and the ability to germinate and recycle of several B. sphaericus strains was investigated. Strains tested represented different serological and DNA homology groups, and expressed either or both toxins (the binary toxin, proteins of 51 and 42 kDa or the 100-kDa toxin). Nontoxic strains were also tested as recombinants expressing the toxin genes or with soluble binary toxin.

Results of this study showed that only spores of the highly toxic B. sphaericus strains, which normally produce both, the binary toxin and the 100-Kda toxins, were able to germinate in high percentage and to recycle.

The ability of conjugal transfer of plasmids from B. sphaericus to other bacteria was also investigated. The most likely place for conjugation to occur is in the larval cadaver. where spores of B. sphaericus germinate and grow vegetatively and interact with bacteria present in the larval cadaver. Bacillus sphaericus 2362 carrying the broad host range plasmid pAM8&1, was used as donor in filter mating experiments with other B. sphaericus strains, a restrictionless B. subtilis strain and bacteria isolated from field collected larvae. Conjugal transfer of pAMB1 was observed with strains of the same serotype as the donor and two other serotypes.

The possibility that the large cryptic plasmid present in B. sphaericus 2362 (180 kb), could promote its own transfer and mobilize the small nonconjugative plasmids pUB110 to other 8B. sphaericus strains was also tested. No transfer was detected.

Conjugation experiments in vivo (the larval cadaver) were done by feeding Culex quinquefasciatus larvae the spores of donor 2362 (pAM81) along with spores of the recipient strains, 2362a or 1593-P51. No transconjugants were detected in cadavers 72 hours after feeding the spores.



recycling larva