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

Abstract

Background: 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. Six alleles, Ester(B1), Ester², Ester⁸, Ester⁹, Ester(B10), 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.

Results: Based 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, Ester(B1) and the susceptible C. p. pallens Ester were screened out. Ester⁸ and Ester⁹ were differentiated from Ester(B10) 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.

Conclusions: A 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.

Figure 1

Agarose gel electrophoresis for PCR products of (a) esterase B and (b) esterase A from standard Culex pipiens complex strains. M, Marker D2000; B1 from the SB1 strain; A2-B2 from the SA2 strain; A8-B8 from the MAO2 strain; A9-B9 from the LING strain; A11-B11 from the WU strain; B10 from the KARA2 strain; Sp, susceptible esterases of C. p. pallens from the BJSU strain; Sq, susceptible esterases of C. p. quinquefasciatus from the S-LAB strain.

Figure 2

(a)DraI and (b)XbaI PCR-RFLP profiles of esterase B and (c)ApaLI PCR-RFLP profiles of esterase A from standard C. pipiens complex strains. M1, Marker I. M2, Marker D2000. The standard strains are the same as those described in Figure 1.

Figure 3

Diagrammatic summary of PCR-RFLP discrimination of esterase alleles from C. pipiens complex mosquitoes.

Figure 4

High-activity esterases of single adults from the R-SG and TAA field-collected populations, as determined via starch gel electrophoresis. (a) R-SG population: 1, B1 from the SB1 strain as control; 2–5 and 7–10, R-SG field samples; 6, A2-B2 from the SA2 strain as control. (b) TAA population: 1, A2-B2 from the SA2 strain as the control; 2–9, TAA field samples; 10, A11-B11 from the WU strain as control.

Figure 5

Agarose gel electrophoresis of the PCR products of (a) esterase B and (b) esterase A from the R-SG population. M, Marker D2000; 1, B1 from the SB1 strain as control; 6, A2-B2 from the SA2 strain as control; 2–5 and 7–10, R-SG field samples

Figure 6

(a)DraI and (b)XbaI PCR-RFLP profiles of esterase B and (c)ApaLI PCR-RFLP profiles of esterase A from the R-SG population. M1, Marker I; M2, Marker D2000; 1–10, as described in Figure 5

Figure 7

Agarose gel electrophoresis of the PCR products of (a) esterase B and (b) esterase A from the TAA population. M, Marker D2000; 1, A2-B2 from the SA2 strain as control; 2–9, TAA field samples; 10, A11-B11 from the WU strain as control

Figure 8

(a)DraI and (b)XbaI PCR-RFLP profiles of esterase B and (c)ApaLI PCR-RFLP profiles of esterase A from the TAA population. M1, Marker I; M2, Marker D2000; 1–10, as described in Figure 7.