Identification of resistant carboxylesterase alleles in Culex pipiens complex via PCR-RFLP
© Zhang et al.; licensee BioMed Central Ltd. 2012
Received: 8 August 2012
Accepted: 13 September 2012
Published: 24 September 2012
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 , Ester 2 , Ester 8 , Ester 9 , Ester B10 , and Ester 11 , 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.
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 Dra I. In this step, Ester 2 and Ester 11 were differentiated from the other Ester alleles. When the other Ester B alleles were digested with the restriction enzyme Xba I, Ester B1 and the susceptible C. p. pallens Ester were screened out. Ester 8 and Ester 9 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 Apa LI. The effectiveness of the custom-designed PCR-RFLP was verified in two field mosquito populations.
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.
Insecticides are vital to agricultural production and public health, particularly in countries with huge human populations, such as China. In China, large quantities of chemical insecticides have been used to control mosquitoes since the mid-1950s. Consequently, resistance has developed in vector mosquitoes, which makes their control increasingly difficult . 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 . Four subspecies comprise the complex: C. p. quinquefasciatus, C. p. pallens, C. p. pipiens, and C. p. molestus. C. p. quinquefasciatus and C. p. pallens are prevalent in South China and North China, respectively .
Global surveys of insecticide resistance have indicated that one of the major mechanisms of resistance is the increased detoxification in resistant individuals . Three primary detoxifying enzymes involved in insecticide resistance are carboxylesterase (or esterases), glutathione-S-transferases, and P450 monooxygenases, which are qualitatively or quantitatively changed to confer resistance . Esterase overproduction is a common resistance mechanism of C. pipiens complex mosquitoes to organophosphate (OP) insecticides. This process is achieved mainly by gene amplification or occasionally by gene up regulation [6,, 7]. Some studies indicated that gene duplication or amplification may be a more common adaptive evolutionary mechanism in arthropods, and that certain genomic loci may be “hot spots” for gene duplication, as evidenced by parallel evolution in several arthropod species .
In the C. pipiens complex, two carboxylesterase loci, Est-3 (encoding esterase A) and Est-2 (encoding esterase B), are amplified in the genome and subsequently confer resistance to insecticides [9,, 10]. Est-3 and Est-2 are usually in complete linkage disequilibrium when amplified and are thus referred to as the Ester superloci . To date, 13 alleles that confer insecticide resistance have been identified at the Ester superloci in the C. pipiens complex. These alleles (with the corresponding overproduced esterases indicated in parentheses) are Ester A1 (A1), Ester 2 (A2-B2), Ester 4 (A4-B4), Ester 5 (A5-B5), Ester 8 (A8-B8), Ester 9 (A9-B9), Ester B1 (B1), Ester B6 (B6), Ester B7 (B7), Ester B10 (B10), Ester 11 (A11-B11), Ester B12 (B12), and Ester A13 (A13) [2,, 7,, 12, 13, 14, 15]. Some resistant alleles are distributed globally. For instance, Ester 2 is found in Africa, Asia, Europe, North America, and the Caribbean [12,, 16]. Meanwhile, some resistant alleles are found in restricted geographic areas. For example, Ester 8 , Ester 9 , Ester B10 , and Ester 11 are endemic to China [14,, 17, 18, 19]. Thus, an unusual diversity of Ester alleles is observed in field populations in China, where Ester B1 , Ester 2 , Ester 8 , Ester 9 , Ester B10 , and Ester 11 have been reported to coexist in one population [13,, 14]. The polymorphism of resistant esterase alleles may be the result of changes in insecticide use and/or of a recent contact between relatively isolated treated areas through migration .
The identification and monitoring of the frequencies of these resistant Ester alleles in field populations of mosquitoes are vital not only to the control of vector mosquitoes and the diseases they transmit, but also to field evolutionary studies of these alleles. Current methods of genotyping or allele identification include restriction fragment length polymorphism (RFLP) identification, random amplified polymorphic detection (RAPD), amplified fragment length polymorphism detection (AFLPD), polymerase chain reaction (PCR), DNA sequencing, allele specific oligonucleotide (ASO) probes, and hybridization to DNA microarrays or beads. The most applied method in studies of Culex Ester alleles is the starch gel electrophoresis, which only reveals the phenotype of Ester alleles instead of the genotype. In this paper, we designed specific primers and selected the appropriate restriction endonucleases for use in PCR-RFLP to differentiate between resistant and nonresistant Ester alleles in C. pipiens complex mosquitoes. The effectiveness of the PCR-RFLP system was verified in field-collected 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 ; 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 Ester B1 , Ester 2 , Ester 8 , Ester 9 , Ester B10 , and Ester 11 , respectively [13,, 18,, 21]. Two field populations were also obtained: R-SG, which was C. p. quinquefasciatus and collected from Foshan in Guangdong Province (South China) in July 2007, and TAA, which was C. p. pallens and collected from Tai’an in Shandong Province (North China) in July 2010 .
DNA isolation and PCR-RFLP
PCR and RFLP products of esterase B alleles
DraI digestion (bp)
XbaI digestion (bp)
512, 369, 64
951, 888, 424, 89, 69, 22
951, 424, 180
402, 292, 242
402, 292, 242
519, 423, 742, 423
742, 519, 423
951, 888, 424, 89, 68, 22
951, 424, 180
519, 375, 48
402, 292, 242
402, 292, 242
PCR and RFLP products of esterase A alleles
ApaLI digestion (bp)
98, 223, 392
98, 223, 409
Starch gel electrophoresis
Single adult mosquitoes from the R-SG and TAA field populations were cut into two parts: the head-thorax and the abdomen. The abdomen of each mosquito was homogenized in 10 μl distilled water using a pestle. The homogenate was spread onto a Whatman Grade No. 3 filter paper (3 mm × 8 mm) and analyzed via starch gel electrophoresis to identify the carboxylesterase phenotypes . The head-thorax was used in PCR-RFLP for carboxylesterase genotype identification. At least 10 individuals from each population were analyzed.
Results and discussion
Discrimination of carboxylesterase alleles in standard strains via PCR-RFLP
The PCR products of the three pairs of Ester B alleles were digested by the restriction enzyme Xba I (Figure. 2b) to allow differentiation among members of the same pair. The pair consisting of Ester B1 (lane 1) and the susceptible C. p. pallens Ester B (lane 7) displayed different digestion profiles, which allowed the differentiation of Ester B1 from the susceptible C. p. pallens. However, the XbaI enzyme could not discriminate Ester B8 (lane 3) from Ester B10 (lane 6), or Ester B9 (lane 4) from the susceptible C. p. quinquefasciatus Ester B (lane 8). At this step, four Ester alleles had already been distinguished from one another: Ester 2 , Ester 11 , Ester B1 , and the susceptible C. p. pallens.
Ester A1 , Ester 4 , Ester 5 , Ester B6 , Ester B7 , Ester B12 , and Ester A13 are found in other places in the world. However, given that no sequence information on the association of Ester B6 , Ester B7 , and Ester B to Ester A1 and Ester A13 is available, and that only a partial sequence of Ester B12 is currently known, the designed PCR-RFLP cannot be used to differentiate these alleles. The predicted PCR product sizes for Ester B4 and Ester B5 are 1289 and 1337 bp according to the Ester 4 and Ester 5 sequences, respectively; these values deviate from those of other Ester B alleles (Table 1). However, the Dra I digestion profiles of Ester B4 and Ester B5 (764 and 467 bp for Ester B4, and 770 and 518 bp for Ester 5 ) may be difficult to distinguish in 2% agarose gel electrophoresis. No Xba I cut site exists in Ester B4 and Ester B5. The predicted PCR products of Ester A4 and Ester A5 are 714 and 713 bp, respectively. Only Ester A5 can be cut by Apa LI to produce 321 and 392 bp bands. Thus, the designed PCR-RFLP can identify eight resistant Ester alleles (Ester B1 , Ester 2 , Ester 4 , Ester 5 , Ester 8 , Ester 9 , Ester B10 , and Ester 11 ) and two susceptible alleles.
Identification of carboxylesterase alleles in mosquitoes from field populations
The custom-designed PCR-RFLP method can be used to differentiate the insecticide-resistant esterase alleles existing in China. The method can help monitor the evolutionary dynamics of these esterase alleles and identify new esterase alleles in field populations of C. pipiens complex.
This work was supported by grants from the Major State Basic Research Development Program of China (973 Program) (No. 2012CB114102) and the Chinese Academy of Sciences (No. KSCX2-EW-N-5).
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