, 2008). Incorporating a hydroxyl group at position 334 enhanced toxicity and may be attributed to its participation in hydrogen bonding. Cry2Ab mutants, V324G and L336N, both exhibited a marked decrease in toxicity to Anopheles. CD spectrum for L336N confirmed that structurally, integrity was not compromised, demonstrating the alpha-helical structure commonly seen in Cry proteins (Liu & Dean, 2006). Loss of Anopheles toxicity in the altered toxin, L336N, revealed that a hydrophobic interaction may be essential at residue 336. Conformational changes may have also contributed
to this decline in toxicity, as L336 is positioned within a packed cluster (Foote & Winter, 1992; Morse et al., 2001). When solvent-exposed D block residue, V324, was modified to Gly, a considerable loss of Anopheles toxicity was seen, similar to that of L336N mutant. Residue 324 is located in a domain II region of the this website protein that has been implicated in dipteran receptor interactions (Morse et al., 2001). Previous studies have described Cry2AaWT (Gly324) having activity against An. gambiae (Ahmad et al., 1989) within a bioassay time period > 30 h. Cry2Ab substitution of Val to the isosteric Gly leads to abolishing wild-type Anopheles toxicity. Proteolysis of V324G mutant lead to extensive degradation. The Gly substitution at solvent-accessible position 324 possibly contributed to a change in protein structure, exposing chymotrypsin-sensitive
sites, thus leading check details to protein instability. While Cry2AbWT is generally considered Protein kinase N1 to be solely Lepidoptera active (Hofte & Whiteley, 1989; Widner & Whiteley, 1989; Dankocsik et al., 1990; Morse et al., 2001), Nicholls et al. (1989) reported an LC50 of 100 000 ng mL−1
to An. gambiae in a 48-h period, a negligible level of toxicity. We observed that Cry2AbWT has an LC50 of 540 ng mL−1 in a 24-h period, which is a significant level of toxicity, comparable to that of Cry2Aa (Table 2). There are several reasons why our results differ from those of Nicholls et al. We used third instar larvae, while Nicholls et al. used 4- to 6-day-old larvae, which are likely to be fourth instar. The Cry2Ab protein used by Nicholls et al. was from B. thuringiensis sp. galleriae, while the cry2Ab gene we used was from B. thuringiensis sp. kurstaki (Morse et al., 2001). There may differences in amino acid sequences between the two Cry2Ab proteins, which may affect toxicity. Reclassification of Cry2Ab is warranted to reflect its dipteran-specific nature and binary dipteran/lepidopteran specificity, like that of Aedes-specific Cry2Aa (Morse et al., 2001). The in vivo analyses across three different genera of mosquitoes and their susceptibility to Cry2Ab, reveal a specific cellular requirement for toxicity. We report that while Aedes and Culex were not sensitive to Cry2AbWT, toxicity to Anopheles was observed. It is probable that the toxicity demonstrated was more likely due to receptor interaction, which is species specific (Hua et al., 2008).