Insecticide resistance spectra and resistance mechanisms in populations of Japanese encephalitis vector mosquitoes, Culex tritaeniorhynchus and Cx. gelidus, in Sri Lanka.

Culex tritaeniorhynchus Giles and Cx. gelidus Theobald (Diptera: Culicidae), both vectors of Japanese encephalitis, were collected in 1984 and 1998 from two disease endemic localities in Sri Lanka: Anaradhapura and Kandy. Using wild-caught adult mosquitoes from light traps, log dosage-probit mortality curves for insecticide bioassays were obtained for three insecticides: malathion (organophosphate), propoxur (carbamate) and permethrin (pyrethroid). LD50 values showed that, in 1998, Cx. tritaeniorhynchus was -100-fold more resistant to malathion and 10-fold more resistant to propoxur than was Cx. gelidus. This difference was attributed to Cx. tritaeniorhynchus breeding mostly in irrigated rice paddy fields, where it would have been exposed to pesticide selection pressure, whereas Cx. gelidus breeds in other types of aquatic habitats less prone to pesticide applications. Resistance in Cx. tritaeniorhynchus increased between 1984 and 1998, whereas Cx. gelidus remained predominantly susceptible. Propoxur inhibition of acetylcholinesterase (AChE) activity (the target site of organophosphates and carbamates) indicated that in 1998, frequencies of insensitive AChE-based resistance were 9% in Cx. gelidus and 2-23% in Cx. tritaeniorhynchus, whereas in 1984 this resistance mechanism was detected only in 2% of the latter species from Anaradhapura. The AChE inhibition coefficient (ki) with propoxur was 1.86+/-0.24 x 10(5) M(-)1 min(-1) for Cx. tritaeniorhynchus from Anaradhapura in 1998. Both species were tested for activity levels of detoxifying glutathione S-transferases (GSTs) and malathion-specific as well as general carboxylesterases. High activities of GSTs and carboxylesterases were detected in Cx. tritaeniorhynchus but not Cx. gelidus. Malathion-specific carboxylesterase was absent from both species. Native polyacrylamide gel electrophoresis resolved two elevated general carboxylesterases, CtrEstbeta1 and CtrEstalpha1, from Cx. tritaeniorhynchus and none from Cx. gelidus. CtrEstbeta1 was the most intensely staining band. Gel inhibition experiments showed that both elevated esterases were inhibited by organophosphates and carbamates but not by pyrethroids. The major elevated esterase CtrEstbeta1 was partially purified (15-fold) by sequential Q-Sepharose and phenyl Sepharose column chromatography. The bimolecular rate constant (ka) and the deacylation rate constant (k3) for the malaoxon/enzyme interaction were 9.9+/-1.1 x 10(3) M(-1) min(-1) and 3.5+/-0.05 x 10(-4) M(-1) min(-1), respectively, demonstrating that the role of this enzyme in organophosphorus insecticide resistance is sequestration.