Identification of Cry48Aa/Cry49Aa toxin ligands in the midgut of Culex quinquefasciatus larvae

- Overlay and pull-down assays were performed to identify ligands of the Cry48Aa/Cry49Aa toxin in C. quinquefasciatus midgut.
- A mix of Cry48Aa/Cry49Aa activated toxins binds to proteins from midgut brush border membranes.
- Aminopeptidases, maltases and alkaline phosphatases, among other proteins, were identified Cry48Aa/Cry49Aa ligands.
- Some of them are orthologs of other Cry's receptors and are potential receptors of Cry48Aa/Cry49Aa in C. quinquefasciatus.

A binary mosquitocidal toxin composed of a three-domain Cry-like toxin (Cry48Aa) and a binary-like toxin (Cry49Aa) was identified in Lysinibacillus sphaericus. Cry48Aa/Cry49Aa has action on Culex quinquefasciatus larvae, in particular, to those that are resistant to the Bin Binary toxin, which is the major insecticidal factor from L. sphaericus-based biolarvicides, indicating that Cry48Aa/Cry49Aa interacts with distinct target sites in the midgut and can overcome Bin toxin resistance. This study aimed to identify Cry48Aa/Cry49Aa ligands in C. quinquefasciatus midgut through binding assays and mass spectrometry. Several proteins, mostly from 50 to 120 kDa, bound to the Cry48Aa/Cry49Aa toxin were revealed by toxin overlay and pull-down assays. These proteins were identified against the C. quinquefasciatus genome and after analysis a set of 49 proteins were selected which includes midgut bound proteins such as aminopeptidases, amylases, alkaline phosphatases in addition to molecules from other classes that can be potentially involved in this toxin's mode of action. Among these, some proteins are orthologs of Cry receptors previously identified in mosquito larvae, as candidate receptors for Cry48Aa/Cry49Aa toxin. Further investigation is needed to evaluate the specificity of their interactions and their possible role as receptors.

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