Insecticide resistance spectra and resistance mechanisms in populations ofJapanese 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 fr om two disease endemic localities in Sri Lanka: Anaradhapura and Kandy. Usi ng wild-caught adult mosquitoes from light traps, log dosage-probit mortali ty curves for insecticide bioassays were obtained for three insecticides: m alathion (organophosphate), propoxur (carbamate) and permethrin (pyrethroid ). LD50 values showed that, in 1998, Cx. tritaeniorhynchus was similar to 1 00-fold more resistant to malathion and 10-fold more resistant to propoxur than was Cx. gelidus. This difference was attributed to Cx. tritaeniorhynch us breeding mostly in irrigated rice paddy fields, where it would have been exposed to pesticide selection pressure, whereas Cx. gelidus breeds in oth er types of aquatic habitats less prone to pesticide applications. Resistan ce in Cx. tritaeniorhynchus increased between 1984 and 1998, whereas Cx. ge lidus remained predominantly susceptible. Propoxur inhibition of acetylcholinesterase (AChE) activity (the target sit e of organophosphates and carbamates) indicated that in 1998, frequencies o f 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 coe fficient (k(i)) with propoxur was 1.86 +/- 0.24 x 10(5) m(-1) min(-1) for C x. tritaeniorhynchus from Anaradhapura in 1998. Both species were tested for activity levels of detoxifying glutathione S-t rans- ferases (GSTs) and malathion-specific as well as general carboxyleste rases. High activities of GSTs and carboxylesterases were detected in Cx. t ritaeniorhynchus but not Cx. gelidus. Malathion-specific carboxylesterase w as absent from both species. Native polyacrylamide gel electrophoresis resolved two elevated general car boxylesterases, CtrEst beta1 and CtrEst alpha1, from Cx. tritaeniorhynchus and none from Cx. gelidus. CtrEst beta1 was the most intensely staining ban d. Gel inhibition experiments showed that both elevated esterases were inhi bited by organophosphates and carbamates but not by pyrethroids. The major elevated esterase CtrEst beta1 was partially purified (15-fold) b y sequential Q-Sepharose and phenyl Sepharose column chromatography. The bi molecular rate constant (k(a)) and the deacylation rate constant (k(3)) 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 sequestra tion.