Simple nonradioactive DNA hybridization method for identification of sibling species of Anopheles dirus (Diptera: Culicidae) complex.

A simple method was developed for species identification of mosquitoes in the Anopheles dirus complex found in Thailand using horseradish peroxidase-labeled DNA probes and a chemiluminescent detection system. Species-specific DNA probes for Anopheles dirus, species B, C, and D detected 1-5 ng of target DNA, a sensitivity that was comparable with the radioisotopic detection system. Identification of individual mosquitoes was performed by dot-blot analysis of crude mosquito DNA. The method allowed identification using third or fourth instars as well as the adult head, thorax, or abdomen. This technique successfully identified the sibling species of the A. dirus complex in field collections.

Production of chymotrypsin-resistant Bacillus thuringiensis Cry2Aa1 delta-endotoxin by protein engineering.

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when (125)I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.