Characterization and functional analyses of midgut proteins involved in Bacillus thuringiensis subsp. jegathesan Cry11Ba toxicity to the malaria mosquito Anopheles gambiae
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Cry11Ba, a crystal component produced by Bacillus thuringiensis subsp. jegathesan, is highly toxic to a broad spectrum of mosquito larvae. The toxicity of Cry11Ba correlates with its functional binding to receptors on mosquito larval midgut epithelial cells. Receptors identified as mediating Cry11Ba toxicity include cadherin, aminopeptidase N and alkaline phosphatase in An. gambiae larvae. The objectives of this dissertation research are to investigate the functional roles of receptors in mediating Cry11Ba toxin that lead to its high mosquitocidal potency and to further understand the mechanism of Cry toxin action in mosquito larvae. In the first part of my dissertation, I describe two glycophosphatidylinositol (GPI)-anchored proteins, α-amylase (AgAmy1) and α-glucosidase (Agm3) acting as putative binding receptors of Cry11Ba. Immunohistochemistry revealed their different locations in An. gambiae larvae. Enzyme-linked immunosorbent assay showed that AgAmy1 and Agm3 proteins bound to Cry11Ba with high affinities (37.6 nM and 21.1 nM) through shared binding sites on Cry11Ba. AgAmy1 and Agm3 neutralized Cry11Ba toxicity against An. gambiae larvae. The results provide evidence that AgAmy1 and Agm3 are putative receptors of Cry11Ba in An. gambiae larvae. Cadherins are essential receptors of Cry toxins in several Orders of insects, and loss of a midgut cadherin by mutation or gene silencing can cause resistance. In the second part of my dissertation, I establish the in vivo role of cadherin AgCad1 as a binding receptor of Cry11Ba using chitosan/DsiRNA nanoparticle-mediated RNAi in An. gambiae larvae. Larvae became more tolerant when cadherin AgCad1 was silenced. Because AgCad1 was co-suppressed by AgCad2 DsiRNA, the role of AgCad2 in Cry11Ba toxicity could not be determined. We conclude that AgCad1 and possibly AgCad2 are involved in Cry11Ba toxin in vivo in An. gambiae larvae.
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