Over 180 holotype forms of crystal proteins have been endogenously expressed by cry genes of Bacillus thuringiensis (Bt). Although a variety of Cry proteins exist, they generally demonstrate activity that is limited to a particular order of insects. Uniquely, Cry2Aa has been reported as exhibiting dual larvicidal activity to Lepidoptera (caterpillar and moths) and Diptera (mosquitoes and flies) classified insects. Prior studies suggest that loop regions within domain II contribute to this phenomenon. While Cry2Ab protein differs in amino acid sequence from Cry2Aa by only 13%, insect activity is reportedly limited to Lepidoptera. A putative receptor binding epitope has been suggested, highlighting twenty-three residues within the lepidopteran (L) and dipteran (D) blocks that contribute to Cry2Aa specificity. Nine residues are play a role in conferring Cry2Aa dipteran specificity. Cry2Aa L block residue 350 was investigated for conferring dipteran specificity. We observed an enhancement in toxicity to Anopheles gambiae in the presence of L block (supposed Lepidoptera-specific region) mutant, L350D, as compared to wild-type. Mutagenesis of Cry2Aa loop 1 residues was carried out using L350D as a scaffold. To explore Cry2Ab dipteran toxicity, site-directed mutagenesis was employed to exchange Cry2Ab residues with Cry2Aa D (dipteran) block residues. Cry2Ab wild-type demonstrated high toxicity (LC50 of 540ng/mL) to Anopheles gambiae, but not Aedes or Culex, within a twenty-four hour time period. Cry2Ab should be reclassified as a dual active Cry toxin. Cry2Ab mutagenesis revealed critical residues for Cry2Ab protein function, as well as enhanced activity against the malarial mosquito, Anopheles gambiae.
Currently, field-evolved resistance by insects has been observed for transgenic crops, such as Bt cotton. Approximately 80 subspecies of Bt have been identified, but the cry gene content of various subspecies has not been extensively explored. The identification and classification of cry genes maybe a beneficial approach toward thwarting resistance development efforts. To further investigate the presence of novel cry genes, unique primers were designed to replicate and identify cry genes and PCR products were subsequently enzyme restricted and/or DNA sequenced. Immunoblot analysis was carried out to identify protein expression in partially characterized and novel Bt isolates, to confirm the absence or presence of cryptic genes.