Functional characterization of Aedes aegypti alkaline phosphatase ALP1 involved in the toxicity of Cry toxins from Bacillus thuringiensis subsp israelensis and jegathesan

Presently three major groups of proteins from Aedes aegypti, cadherin, alkaline phosphatases (ALP) and aminopeptidases N (APN), have been identified as Cry1 1Aa toxin receptors. To further characterize their role on toxicity, transgenic mosquitoes with silenced Aedes cadherin expression were previously generated and the role of cadherin in mediating the toxicity of four different mosquitocidal toxins (Cry1 1Aa, Cry1 1Ba, Cry4Aa and Cry4Ba) was demonstrated. Here, we investigated the role of another reported Cry1 1Aa receptor, ALP1. As with Aedes cadherin, this protein is localized in the apical cell membrane of distal and proximal gastric caecae and the posterior midgut. We also successfully generated transgenic mosquitoes that knockdowned ALP1 transcript levels using an inducible Aedes heat shock promoter, Hsp70A driving dsALP1RNA. Four different mosquitocidal toxins were used for larval bioassays against this transgenic mosquito. Bioassay results show thatCry1 1Aa toxicity to these transgenic larvae following a heat shock decreased (4.4 fold) and Cry1 1Ba toxicity is slightly attenuated. But Cry4Aa and Cry4Ba toxicity to ALP1 silenced larvae is unchanged. Without heat shock, toxicity of all four toxins does not change, suggesting this heat shock promoter is heat-inducible. Notably, transgenic mosquitoes with ALP1 knockdown are about 3.7 times less resistant to Cry1 1Aa toxin than those with Aedes cadherin knockdown. These results demonstrate that the ALP1 is an important secondary receptor for Cry1 1Aa and Cry1 1Ba, but it might not be involved in Cry4Aa and Cry4Ba toxicity.