Functional Bacillus thuringiensis Cyt1Aa is necessary to synergize Lysinibacillus sphaericus Binary toxin against Bin-resistant and refractory mosquito species

The Binary (Bin) toxin from Lysinibacillus sphaericus is effective to mosquito larvae but its utilization is threatened by the development of insect resistance. Bin toxin is composed by the BinB subunit required for binding to midgut receptors, and the BinA subunit that causes toxicity after cell internalization, mediated by BinB. Culex quinquefasciatus resistance to this toxin is caused by mutations that prevent expression of Bin toxin-receptors in the midgut. Previously, it was shown that the Cyt1Aa toxin, from Bacillus thuringiensis subsp. israelensis (Bti), restores Bin toxicity to Bin-resistant Cx. quinquefasciatus and to Aedes aegypti larvae, which is naturally devoid of functional Bin receptors. Our goal was to elucidate the mechanism involved in Cyt1Aa synergism with Bin in such larvae. In vivo assays showed that the mixture of Bin toxin, or its BinA subunit, with Cyt1Aa was effective to kill resistant larvae. However, no specific binding interaction between Cyt1Aa and the Bin toxin, or its subunits, was observed. The synergy between Cyt1Aa and Bin toxins is dependent on functional Cyt1Aa, as demonstrated by using the non-toxic Cyt1AaV122E mutant toxin affected in oligomerization and membrane insertion, which was unable to synergize Bin toxicity in resistant larvae. The synergism correlated with the internalization of Bin or BinA into anterior and medium midgut epithelial cells, which occurred only in larvae treated with wild-type Cyt1Aa toxin. This toxin is able to overcome failures in the binding step involving BinB-receptor, by allowing the internalization of Bin toxin, or its BinA subunit, into the midgut cells. IMPORTANCE One promising management strategy for mosquito control is the utilization of a mixture of L. sphaericus and Bti insecticidal toxins. From this set, Bin and Cyt1Aa toxins synergize and display toxicity to resistant Culex quinquefasciatus and to Aedes aegypti larvae, whose midgut cells lack Bin toxin receptors. Our dataset provide evidence that functional Cyt1Aa is essential for internalization of Bin or its BinA subunit into such cells but binding interaction between Bin and Cyt1Aa is not observed. Thus, this mechanism contrasts with that for the synergy between Cyt1Aa and the Bti Cry toxins where active Cyt1Aa is not necessary but a specific binding between Cry and Cyt1Aa is required. Our study established the initial molecular basis of the synergy between Bin-Cyt1Aa and these findings enlarge our knowledge of their mode of action that could help to develop improved strategies to cope with insect resistance.