| dc.description.abstract | The use of biological agents is gaining prominence over conventional chemical methods for the control of vector mosquitoes. Two species of Bacilli have proved to be satisfactory as biocides with potential for exploitation in mosquito eradication. Bacillus sphaericus is a specific agent that can be used in combination with Bacillus thuringiensis var. israelensis for successful mosquito control. It has no side effects and is non toxic to other predaceous insects, fish, and higher vertebrates.
The information currently available on the nature and mode of action of the larvicidal toxin of B. sphaericus 1593 is still incomplete and is not nearly as extensive as in the case of the endotoxin of B. thuringiensis. Although the genes of B. sphaericus encoding the synthesis of the larvicidal component have been cloned and expressed in Escherichia coli, complete purification of the toxin has not been achieved to date. The present study is an attempt to isolate the larvicidal toxin from B. sphaericus 1593.
Larvicidally active proteins were extracted from purified spores using a freeze–thaw lysis method. Further fractionation was attempted using different chromatographic columns. The larvicidal proteins were also isolated by spore coat protein extraction and fractionated. All conventional chromatographic procedures failed to achieve separation due to the inherent tendency of the toxin to aggregate.
A bioassay was standardized using early III instar larvae of the mosquito Culex pipiens var. quinquefasciatus. A 12 fold increase in specific biological activity was achieved despite the failure to purify the toxin to homogeneity. More than one protein band was found to exhibit larvicidal activity when individual bands eluted from preparative polyacrylamide gel electrophoresis were tested. The toxic proteins were also shown to be glycosylated.
Since conventional column procedures failed to purify the larvicidal protein to homogeneity, an immunological approach was attempted. The antigenicity of the larvicidal protein was first established by raising antibodies to the (NH ) SO fraction of spore coat proteins. After detecting the antibodies by Ouchterlony immunodiffusion analysis, the neutralizing activity of the antiserum was tested. The antibodies raised against the (NH ) SO fraction were able to neutralize and precipitate the soluble toxin.
As the next step, a single protein band eluted from polyacrylamide gel electrophoresis of the (NH ) SO fraction exhibiting toxicity was used for immunization in rabbits. The IgG fraction from the antiserum was purified and coupled to CNBr activated Sepharose 4B. This immunoaffinity matrix was used for isolation of the larvicidal principle.
The immunoaffinity purified protein was found to be electrophoretically homogeneous on 7.5% polyacrylamide gels under non denaturing conditions. This protein produced a single immunoprecipitin band in Ouchterlony immunodiffusion analysis. The protein contained 12% carbohydrates. On SDS–polyacrylamide gels, this protein resolved into four bands of molecular weights 42.6, 44.1, 50.7, and 51.3 kDa, with a possible fifth band at 52 kDa. By this method, a fifteen fold purification of the toxic protein was achieved.
Western blotting using crude protein and the antiserum (raised against a single toxic band) demonstrated the presence of more than one immunologically related protein in spores of B. sphaericus 1593.
The mechanism of action of the larvicidal principle was also studied. The toxin significantly inhibited the activity of choline acetyltransferase from Culex pipiens var. quinquefasciatus larvae. The effect of the toxin on mitochondrial oxidative phosphorylation was also examined. It inhibited the state 3 oxygen uptake of mitochondria prepared from larvae of Culex pipiens var. quinquefasciatus both in vitro and in vivo. These studies were extended to rat liver mitochondria, where the toxin inhibited state 3 oxygen uptake, stimulated state 4 oxygen uptake, and caused loss of respiratory control, thereby uncoupling intact mitochondria.
The presence of a plasmid or its relevance to toxin production in B. sphaericus 1593 had not been unequivocally established earlier. In the present studies, the presence of a plasmid was demonstrated using a rapid lysis method. The plasmid could be restricted by several restriction enzymes. Plasmid curing studies confirmed that the genes for larvicidal activity are chromosomal, since no loss of toxicity occurred after plasmid curing. | |
