Identification of protease m1 zinc metalloprotease conferring resistance to deltamethrin by characterization of an AFLP marker in Culex pipiens pallens

Abstract

BackgroundContinuous and excessive application of deltamethrin (DM) has resulted in the rapid development of insecticide resistance in Culex pipiens pallens. The quantitative trait loci (QTL) responsible for resistance to DM had previously been detected in Cx. pipiens pallens. But locating the QTLs on the chromosomes remained difficult. An available approach is to first characterize DNA molecular markers linked with the phenotype, and then identify candidate genes.MethodsIn this study, the amplified fragment length polymorphism (AFLP) marker L3A8.177 associated with the QTL, was characterized. We searched for potential candidate genes in the flank region of L3A8.177 in the genome sequence of the closely related Cx. pipiens quinquefasciatus and conducted mRNA expression analysis of the candidate gene via quantitative real-time PCR. Then the relationship between DM resistance and the candidate gene was identified using RNAi and American CDC Bottle Bioassay in vivo. We also cloned the ORF sequences of the candidate gene from both susceptible and resistant mosquitoes.ResultsThe genes CYP6CP1 and protease m1 zinc metalloprotease were in the flank region of L3A8.177 and had significantly different expression levels between susceptible and resistant strains. Protease m1 zinc metalloprotease was significantly up-regulated in the susceptible strains compared with the resistant and remained over-expressed in the susceptible field-collected strains. For deduced amino acid sequences of protease m1 zinc metalloprotease, there was no difference between susceptible and resistant mosquitoes. Knockdown of protease m1 zinc metalloprotease not only decreased the sensitivity of mosquitoes to DM in the susceptible strain but also increased the expression of CYP6CP1, suggesting the role of protease m1 zinc metalloprotease in resistance may be involved in the regulation of the P450 gene expression.ConclusionOur study represents an example of candidate genes derived from the AFLP marker associated with the QTL and provides the first evidence that protease m1 zinc metalloprotease may play a role in the regulation of DM resistance in Cx. pipiens pallens.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1450-4) contains supplementary material, which is available to authorized users.

A cluster of CYP6 gene family associated with the major quantitative trait locus is responsible for the pyrethroid resistance in Culex pipiens pallen

“…With the increase of resistance levels in the strains, the CYP6BB4 expression levels were 1.26, 2.07, 4.83 and 7.46‐fold higher in the Lab‐DR1, Lab‐DR2, Lab‐DR3 and Lab‐DR4 strains respectively, compared with those in the Lab‐DS, and the CYP6AA9 levels were 20.10, 45.37, 66.20 and 74.45‐fold higher respectively. Correlation analysis was applied to investigate the relationship between relative quantification of the gene ( Y ) and the LC 50 ( X ) (Zhou et al ., ; Zou et al ., ). The correlation coefficient r ( xy ) of CYP6BB4 and CYP6AA9 was 0.95 ( P < 0.01) and 0.97 ( P < 0.01) respectively (Figs B and B), which indicates a significant positive correlation between CYP6BB4 and CYP6AA9 expression and resistance.…”

“…The quantitative real time (qRT)‐PCR primers were designed using primer 5.0 (File S2), based on the corresponding cDNA sequences obtained from VectorBase. The qRT‐PCR analysis was performed using the method described previously (Fang et al ., ; Zou et al ., ). The β‐actin and RsP7 genes were used as the standard for expression normalization for each gene because of their stable expressions (Canales et al ., ; Leal et al ., ; Zou et al ., ).…”

“…The qRT‐PCR analysis was performed using the method described previously (Fang et al ., ; Zou et al ., ). The β‐actin and RsP7 genes were used as the standard for expression normalization for each gene because of their stable expressions (Canales et al ., ; Leal et al ., ; Zou et al ., ). We assessed the statistical significance of the gene expression analysis using equal or unequal t ‐tests, depending on the homoscedasticity results of F ‐tests.…”

“…One‐day‐old Lab‐DR4 mosquitoes were used to investigate the role of CYP6BB4 in DM resistance in vivo . The procedures of RNAi analysis and CDC bottle bioassay have previously been described (Zou et al ., ).…”

“…With the C. pipiens complex genomes having not been assembled into chromosomes, this is a significant impediment to clone the DR‐6 interval (Bartholomay et al ., ). Since locating QTLs on the chromosomes has remained difficult in C. pipiens , an alternative approach could be used to solve the problem: cloning the markers in the flank of the QTL, and then isolating and characterizing the flanking sequence in the genome to search for the genes associated with the QTL (Lyons et al ., ; Podio et al ., ; Zou et al ., ).…”