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

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, 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. 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, 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. 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.


Background
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 [1]. 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]. 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 [3].
Global surveys of insecticide resistance have indicated that one of the major mechanisms of resistance is the increased detoxification in resistant individuals [4]. 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 [5]. 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 [8].
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.

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 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 [22].

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 [24]. 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 To discriminate the carboxylesterase alleles, the Ester B allele fragments were first amplified by PCR. Most PCR products of the Ester B alleles were approximately 900 bp long, whereas those of Ester B8 and Ester B10 were approximately 2400 bp in length ( Figure. 1a and Table 1). Meanwhile, the PCR product of Ester B11 showed two predominant bands that correspond to 942 and 1165 bp fragments. Sequencing data show that the 942 bp band is Ester B11, whereas the 1165 bp band had a 223 bp insertion in one Ester B11 intron. This insertion may have originated from gene recombination or transposition in the genome. Whether this insertion affects the insecticide resistance in mosquitoes remains to be determined. After the PCR products were digested by the restriction enzyme DraI, the Ester B alleles showed various digestion patterns. Given the resolution limit of the 2% agarose gel electrophoresis, some Ester B allele digestion patterns in the agarose gel electrophoresis differed from the predicted theoretical patterns (Table 1). In the electrophoresis graph ( Figure.    Ester 8 and Ester 9 were differentiated from Ester B10 and the susceptible C. p. quinquefasciatus, respectively, by amplifying an approximately 700 bp fragment of their Ester A via PCR using specific primers ( Figure. 1b and Table 2) and then digesting the fragment with the restriction enzyme ApaLI. Ester A8 (lane 3) displayed digestion profiles that differ from that of the Ester B10 Ester A (lane 6) (Figure. 2c). In addition, Ester A9 (lane 4) displayed profiles that differ from those of the susceptible C. p. quinquefasciatus Ester A (lane 8) (Figure. 2c). Thus, the six resistant Ester alleles Ester B1 , Ester 2 , Ester 8 , Ester 9 , Ester B10 , and Ester 11 , as well as the susceptible alleles, were correctly identified ( Figure. 3).
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 DraI 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 XbaI 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 ApaLI 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 phenotype and genotype of carboxylesterase alleles in mosquitoes from two field populations, R-SG (C. p. quinquefasciatus) and TAA (C. p. pallens), were identified through starch gel electrophoresis and PCR-RFLP. Of the eight mosquitoes from the R-SG population, Nos. 4, 5, 7, and 9 showed the A8-B8 phenotype in starch gel electrophoresis ( Figure. 4a). PCR-RFLP results confirm that these mosquitoes were homozygous with  Ester 8 ( Figure 5 and Figure 6). Starch gel electrophoresis results suggest that the Nos. 3 and 8 mosquitoes are heterozygous to A2-B2 and A9-B9 or A9-B10 and A9-B9 because of the similar mobilities of A2-B2 and A9-B10 [14]. However, PCR-RFLP results indicate that these mosquitoes are actually heterozygous to A9-B10 and B2 ( Figure 5 and Figure 6). Three esterase Bs and two esterase As were found in the starch gel electrophoresis profile of Nos. 2 and 10 mosquitoes (Figure. 4a). The whole set of PCR-RFLP shows that the No. 2 mosquito expressed A8-B8, A9-B9, and B2, whereas the No. 10 mosquito expressed A8-B8, A9-B10, and B2 ( Figure 5 and Figure 6). The fewer than expected DraI digestion bands of esterase B alleles in mosquito No. 2 ( Figure 6A, lane 2) could be due to the higher gene copy number of B2 in the genome over B8 and B9. Without the PCR-RFLP results, the differentiation of the phenotypes of the two mosquito types would have been difficult.
Most of the eight mosquito types from the TAA population ( Figure. 4b) overproduced esterase B1 (No. 2, 4, 6, 7, 8, and 9 mosquitoes). The No. 3 mosquito was heterozygous to B1 and A2-B2. These phenotypes were confirmed by PCR-RFLP (Figure 7 and Figure 8). The No. 5 individual did not appear to have overproduced esterases based on the light-colored band in the starch gel electrophoresis. However, PCR-RFLP results indicate the presence of A2-B2 and A9 (Figure 7 and Figure 8). The results for the two field populations show that the A2-B2 and A9-B9 linkages are not always established. This result deviates from the usual observations and theories [7]. By now the recombination event leading to the existence of three esterase B alleles in one individual 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. mosquito has only been observed in field populations of China [13 and this study]. The dislinkage between A2 and B2 or A9 and B9 probably results from the rare recombination event among these esterase alleles.

Conclusions
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.  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.