Nivel de resistencia a tres formulaciones de insecticidas en Aedes aegypti de la región occidental de Cuba Translated title: Resistance of western Cuba Aedes aegypti mosquitoes to three insecticide formulations

Background Aedes aegypti is an important vector for dengue virus and thus has been targeted with pyrethroid insecticides in many areas of the world. As such, resistance has been detected to several of these insecticides, including in China, but the mechanisms of the resistance are not well understood in this country. Methods Using the World Health Organization larval mosquito bioassay, five field populations of Aedes aegypti from Southern China were characterized for their resistance to cypermethrin and cyhalothrin. RNA extraction with PCR amplification, cloning and sequencing of the sodium channel gene was followed by comparisons of susceptible and wild mosquito strains Additionally, genomic DNA was used for Allele-specific PCR (AS-PCR) genotyping of the sodium channel genes to detect S989P, V1016G and F1534C mutations and allow for correlation analysis of resistance expression for the different mutations. Results All wild strains expressed resistance to cypermethrin and cyhalothrin and the resistance expression between the two insecticides was highly correlated suggesting cross-resistance between these two pyrethroids. The AS-PCR technique effectively distinguished individual genotypes for all three mutations. Among the five wild strains tested, two strains carried all three mutations. Although the S989P and V1016G mutations were positively correlated to resistance expression of both pyrethroids, the F1534C mutation was negatively correlated. Conclusions Our methodology proved highly reliable and will aid future detection of kdr mutations. The three sodium channel mutations were common in the Ae. aegypti strains sampled from Southern China. The V1016G mutation appears to be the most important kdr mutation in Ae. aegypti strains in Southern China.

Dengue (family Flaviridae, genus Flavivirus, DENV) and dengue hemorrhagic fever (DHF) are presently important public health problems in Costa Rica. The primary strategy for disease control is based on reducing population densities of the main mosquito vector Aedes aegypti (L.) (Diptera: Culicidae). This is heavily dependent on use of chemical insecticides, thus the development of resistance is a frequent threat to control program effectiveness. The objective of this study was to determine the levels of insecticide resistance and the metabolic resistance mechanisms involved in two Ae. aegypti strains collected from two provinces (Puntarenas and Limon) in Costa Rica. Bioassays with larvae were performed according to World Health Organization guidelines and resistance in adults was measured through standard bottle assays. The activities of beta-esterases, cytochrome P450 monooxygenases, and glutathione S-transferases (GST), were assayed through synergists and biochemical tests, wherein the threshold criteria for each enzyme was established using the susceptible Rockefeller strain. The results showed higher resistance levels to the organophosphate (OP) temephos and the pyrethroid deltamethrin in larvae. The efficacy of commercial formulations of temephos in controlling Ae. aegypti populations was 100% mortality up to 11 and 12 d posttreatment with daily water replacements in test containers. Temephos and deltamethrin resistance in larvae were associated with high esterase activity, but not to cytochrome P450 monooxygenase or GST activities. Adult mosquitoes were resistant to deltamethrin, and susceptible to bendiocarb, chlorpyrifos, and cypermethrin. Because temephos and deltamethrin resistance are emerging at the studied sites, alternative insecticides should be considered. The insecticides chlorpyrifos and cypermethrin could be good candidates to use as alternatives for Ae. aegypti control.

A sample of Aedes aegypti L. from Santiago de Cuba with a high level of deltamethrin resistance (113.7 x at the 50% lethal concentration [LC50]), was subjected to deltamethrin selection to determine the capacity of this population to evolve higher resistance under intensive laboratory selection pressure, to characterize that resistance, to attempt to identify some of the mechanisms involved, and to use it as a reference strain for future molecular research. High resistance developed after 12 generations of selection (1,425 x). After selection for 12 generations with deltamethrin, the Santiago de Cuba colony (SAN-F12) showed little or no cross-resistance to the organophosphates evaluated, but high cross-resistance was observed for all the pyrethroids in larvae from this strain: lambdacyhalothrin (197.5 x), cypermethrin (45 x), and cyfluthrin (41.2 x). Adult bioassays reveal that a SAN-F12 strain was resistant to the pyrethroid and the organochlorine dichlorodiphenyltrichloroethane (DDT). Synergism tests implicated detoxifying esterase or glutathione S-transferase (GST) and monooxygenase in pyrethroid resistance. Biochemical tests reveal that acetylcholinesterase was not involved in deltamethrin resistance. The frequency of GST enzyme increased from 0.43 in Santiago de Cuba to 0.88 in SAN-F12. Esterase frequency increased from 0.12 in Santiago de Cuba to 0.63 in SAN-F6 and it diminished to 0.38 in SAN-F12. The polyacrylamide gel electrophoresis and inhibition study suggests the presence of elevated esterase activity not associated with pyrethroid resistance. The presence of both DDT and pyrethroid resistance in the SAN-F12 strain suggests the presence of a knockdown (Kdr)-type resistance mechanism, although the frequency of this mechanism was low. Resistance to deltamethrin could be associated with esterase or GST mechanisms, and more investigation is required. This information contributes to the improvement of resistance management strategies in the Cuban Ae. aegypti control program.