Bacillus thuringiensis subsp. israelensis producing endochitinase ChiA74Δsp inclusions and its improved activity against Aedes aegypti

Aims: The objective of this study was to produce stable inclusions of chitinase ChiA74Δsp in Bacillus thuringiensis subsp. israelensis (Bti) and to assay its insecticidal activity against Aedes aegypti larvae. Methods and Results: Bti was transformed with chiA74Δsp regulated by its own promoter or by the strong chimeric cytAp/STAB-SD promoter system to generate two recombinant Bti strains. These recombinants produced their native parasporal bodies composed of Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa and ChiA74Δsp inclusions, and showed a approx. threefold increase in both endochitinase activity and viable spore count when compared with the parental strain. Both recombinants were approximately twofold more toxic (LC_50s 8·02, 9·6 ng ml^-1) than parental Bti (19·8 ng ml^-1) against 4^th instars of A. aegypti larvae. Conclusions: ChiA74Δsp inclusions, together with the insecticidal crystals and spores of Bti increased the toxicity against A. aegypti larvae by at least twofold. Significance and Impact of the Study: We report for the first time the engineering of Bti to produce spore-parasporal body-ChiA74 Δsp inclusions in the same sporangium, which are released together following autolysis. Our work lays a foundation for engineering Bti to produce more efficacious combinations of Cry4Aa, Cry4Ba, Cry11Aa, Cyt1Aa and chitinase inclusions.