Synergistic Activity Of Chitinase And Insecticidal Crystal Proteins Against Mosquito Larvae

Bacillus sphaericus and Bacillus thuringiensis subsp, israelensis are gram-positive, spore-forming, aerobic mosquito-larvaecidal bacteria that have been successfully used for biological control of mosquitos worldwide. Their activities against the target mosquito larvae axe attributed to the protoxins synthesized and assembled into parasporal crystals during bacterial sporulation. The toxin of B. sphaericus consists of two equimolar amounts of proteins acting as a binary toxin, which might induce high level of resistance in mosquitoes after long-term continuous application in the field. Meanwhile, the toxin of B. thuringiensis subsp, israelensis consists of at least four proteins, which act synergistically to kill mosquito larvae.Chitinases, which are found in a variety of organisms, can hydrolyze chitin in the peritrophic membrane and then cause pore formation or delay the formation of peritrophic membrane, thus make insect larvae more susceptible to other toxins. To investigate the potential value of chitinase in mosquito control, the activity of chitinase against mosquitos, cloning and expression of chitinse gene and the synergistic activity of chitinase and crystal proteins to mosquitos were studied in this research.Bioassays demonstrated that chitinase from Serretia marcescens had low mortality against susceptible and B. sphaericus-resistant Culex quinquefasciatus larvae, with the LC₅₀ values of 8.99 and 11.9μg/ml, compared to the values of 6.36 and 19,500 ng/ml of B. sphaericus C3-41 binary toxin to the targets, respectively. Additionally, it has been proved that the chitinase not only had a delayed effect on susceptible C quinquefasciatus larvae after the treatment, but also enhanced the larvicidal activity of binary toxin against susceptible mosquito.Furthermore, the presence of chitinase gene and the chitinolytic activity among Bacillus spp. have been detected by PCR and chitinase activity assays. It was showed that no chitinase gene and chitinase production has been observed in 1 of 26 Bacillus cereus strains, 4 of 62 B. thuringiensis strains and all 21 B. sphaericus strains. All other tested strains could produce chitinolytic activity during their growth and several high chitinase production strains were selected for further study. Three chitinase genes were cloned and sequenced from B. thuringiensis strains T04A001, T13003 and Gr-9, respectiely,. Sequence analysis revealed that all three genes shared high similarity (over 98%) and their encoding protein showed a high degree of identity to other chitinases from B. thuringiensis strains. All chitinase proteins were composed of four conserved domains: signal peptide, catalytic domain, Fibronectin typeâ…¢like domain and chitin-binding domain.A chitinase gene chiAC of B. thuringiensis strains T04A001 was expressed in Escherchia coli under the control of T7 promoter. A 70-kDa peptide corresponding to the mature ChiAC protein and two peptides with molecular weights about 40 kDa and 29 kDa were detected in the recombinant E. coli strain E-pETC21 by SDS-PAGE and Western blot analysis.The expressions of chiAC under the control of a vegetative and a spore-dependent promoter in Bti IPS78 and Bs 2297 were investigated. The results indicated that all reconbinants could express ChiAC during their vegetative and sporulation stages and that the vegetative expression of ChiAC in Bti-pBC resulted in reduction of bacteria sporulation, thus causing a partial loss of toxicity of the recombinant to mosquito larvae. And expression of ChiAC in vegetative and sporular stage in Bti-pBbC sporulated like the wide-type strain and showed similar toxicity against mosquito larvae in turn. However, no deletious effect on bacterial growth and development in two 2297 recombinants have been noticed. Bioassays showed that the expression of ChiAC in B. sphaericus strain 2297-pBbC increased the toxicity against resistant C. quinquefasciatus by a factor of 4,700 compared with the parental strain 2297. This is the first report of coexpression of ChiAC and Binary toxin in B. sphaericus, and the results might provide information for further understanding the synergistic effect of chitinase and other mosquitocidal toxins for resistant mosquito control.