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 of 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 restores Bin toxicity to Bin-resistant C. quinquefasciatus and to Aedes aegypti larvae, which are 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 nontoxic 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 B. thuringiensis subsp. israelensis insecticidal toxins. From this set, Bin and Cyt1Aa toxins synergize and display toxicity to resistant C. quinquefasciatus and to A. aegypti larvae, whose midgut cells lack Bin toxin receptors. Our data set provides 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 B. thuringiensis subsp. israelensis 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 and Cyt1Aa, and these findings enlarge our knowledge of their mode of action, which could help to develop improved strategies to cope with insect resistance.

球形芽孢杆菌的二元（Bin）毒素对蚊虫幼虫有效，但其利用受到昆虫抗药性发展的威胁。Bin毒素由结合中肠受体所需的BinB亚基和由BinB介导的细胞内化后引起毒性的BinA亚基组成。库克斯对这种毒素的抗性是由阻止中肠中Bin毒素受体表达的突变引起的。以前显示苏云金芽孢杆菌亚种的Cyt1Aa毒素。以色列将Bin毒性恢复为对Bin耐药的quinquefasciatus和埃及伊蚊幼虫，自然缺乏功能性Bin受体。我们的目标是阐明在此类幼虫中与Cyt1Aa协同作用的机制。体内分析表明，Bin毒素或其BinA亚基与Cyt1Aa的混合物可有效杀死抗药性幼虫。但是，没有观察到Cyt1Aa与Bin毒素或其亚基之间的特异性结合相互作用。Cyt1Aa和Bin毒素之间的协同作用取决于功能性Cyt1Aa，如通过使用受寡聚和膜插入影响的无毒Cyt1AaV122E突变毒素所证明的那样，该毒素无法协同抗性幼虫的Bin毒性。协同作用与Bin或BinA内化到前肠和中肠上皮细胞有关，仅在用野生型Cyt1Aa毒素处理的幼虫中发生。通过允许Bin毒素或其BinA亚基内化到中肠细胞中，该毒素能够克服涉及BinB受体的结合步骤中的失败。

重要事项一种有希望的灭蚊管理策略是利用球形乳杆菌和苏云金芽孢杆菌亚种的混合物。以色列杀虫毒素。从这一组中，Bin和Cyt1Aa毒素协同作用，并显示出对抗性西洋参线虫和埃及中提琴虫幼虫的毒性，后者的中肠细胞缺乏Bin毒素受体。我们的数据集提供了功能性Cyt1Aa对于Bin或其BinA亚基内化到此类细胞中必不可少的证据，但未观察到Bin与Cyt1Aa之间的结合相互作用。因此，该机制与Cyt1Aa和苏云金芽孢杆菌亚种之间的协同作用相反。以色列Cry毒素，不需要活性Cyt1Aa，但Cry和Cyt1Aa之间需要特异性结合。我们的研究建立了Bin和Cyt1Aa协同作用的最初分子基础，这些发现扩大了我们对它们的作用方式的了解，这可能有助于制定改进的策略来应对昆虫的抗药性。