Response of three mosquito species to recombinant bacterial toxins from bacillus thuringiensis subsp. Israelensis expressed in two model systems

Abstract

Toxicity of lyophilized powders prepared prepared 16 combinations of four genes, cry4Aa, cry11Aa, cyt1Aa and p20 from Bacillus thuringiensis subsp.israelensis (Bti) expressed in Escherichia coli were examined against three mosquito vectors of diseases, Culex quinquefasciatus Say, Anopheles arabiensis Patton, and Aedes aegypti Linnaeus, followed by simulated studies using transgenic Anabaena PCC7120 expressing the most toxic combination of genes. The following clones were the most toxic to the three mosquito species: pVE4- ADRC expressing all four genes (LC50s of 0.59, 3.2, and 0.68 μg ml-1); pVE4-ARC expressing cry4Aa, cyt1Aa, p20 (LC50s of 0.93, 6.2 and 0.87 μg ml-1), and pVE4-AD expressing cry4Aa and cry11Aa (LC50s of 1.51, 7.5 and 1.3 μg ml-1). The clone pVE4-ADRC was undoubtedly the most effective. The role of appropriate promoter(s) in enhancing toxicity was demonstrated by comparing expression of the same gene combination under a strong E. coli promoter (PA1) either singly, in pVRE4-DRC or two (the second preceding cyt1Aa in pVE4- DRC). The latter produced more Cyt1Aa, which is less toxic, at the expense of the more toxic Cry11Aa, thus reducing toxicity. On the other hand, the combination of toxins under pVRE4-DRC control had an enhanced toxicity. This observation implies that further toxicity fine-tuning could be reached by manipulating promoters to enhance toxicity in recombinant systems. In simulated semi-field experiments, transgenic Anabaena PCC 7120 protected the toxins from premature degradation and better delivered the toxins to the larvae compared to commercial Bti preparations.