Identification of resistant carboxylesterase alleles in Culex pipiens complex via PCR-RFLP

Hanying Zhang,Chuanling Qiao,Fengxia Meng,Feng Cui

doi:10.1186/1756-3305-5-209

Hanying Zhang, Chuanling Qiao + Show 2 more

Open Access

https://doi.org/10.1186/1756-3305-5-209

Copy DOI

Abstract

BackgroundCarboxylesterase overproduction is a frequently observed resistance mechanism of insects to organophosphate insecticides. As a major transmitter of human diseases, mosquitoes in the Culex pipiens complex have evolved 13 carboxylesterase alleles (Ester) that confer organophosphate resistance. Six alleles, EsterB1, Ester2, Ester8, Ester9, EsterB10, and Ester11, have been observed in field populations in China, sometimes co-existing in one population. To differentiate the carboxylesterase alleles found in these field populations, PCR-RFLP was designed for use in resistance monitoring.ResultsBased on the DNA sequences of resistant and nonresistant carboxylesterase alleles, Ester B alleles were first amplified with PCR-specific primers and then digested with the restriction enzyme DraI. In this step, Ester2 and Ester11 were differentiated from the other Ester alleles. When the other Ester B alleles were digested with the restriction enzyme XbaI, EsterB1 and the susceptible C. p. pallens Ester were screened out. Ester8 and Ester9 were differentiated from EsterB10 and the susceptible C. p. quinquefasciatus esterase allele, respectively, by amplifying and digesting the Ester A alleles with the restriction enzyme ApaLI. The effectiveness of the custom-designed PCR-RFLP was verified in two field mosquito populations.ConclusionsA PCR-RFLP based approach was developed to differentiate carboxylesterase alleles in Culex pipiens complex mosquitoes. These processes may be useful in monitoring the evolutionary dynamics of known carboxylesterase alleles as well as in the identification of new alleles in field populations.

Highlights

Carboxylesterase overproduction is a frequently observed resistance mechanism of insects to organophosphate insecticides
Mosquitoes Eight standard C. pipiens complex strains were used. These strains are S-LAB, which is an OP-susceptible C. p. quinquefasciatus strain without increased esterase activity [20]; BJSU, an insecticide-susceptible C. p. pallens strain collected from Beijing in the 1970s and laboratory-reared for over 40 years without insecticide exposure; and SB1, SA2, MAO2, LING, KARA2, and WU, which are homozygous C. p. quinquefasciatus for EsterB1, Ester2, Ester8, Ester9, EsterB10, and Ester11, respectively [13,18,21]
Discrimination of carboxylesterase alleles in standard strains via polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) To discriminate the carboxylesterase alleles, the Ester B allele fragments were first amplified by PCR

Summary

Introduction

Carboxylesterase overproduction is a frequently observed resistance mechanism of insects to organophosphate insecticides. As a major transmitter of human diseases, mosquitoes in the Culex pipiens complex have evolved 13 carboxylesterase alleles (Ester) that confer organophosphate resistance. Results: Based on the DNA sequences of resistant and nonresistant carboxylesterase alleles, Ester B alleles were first amplified with PCR-specific primers and digested with the restriction enzyme DraI. In this step, Ester and Ester were differentiated from the other Ester alleles. Mosquitoes in the Culex pipiens complex (Diptera: Culicidae) are common in temperate and tropical countries These insects have been subjected to insecticide control in many places around the world [2].

Methods

Results

Conclusion