The continuous and widespread use of insecticides results in the development of insecticide resistance, which is an ongoing challenge in the prevention of insect-borne diseases [32
]. Identifying novel resistance genes and revealing the potential resistance mechanisms have become focal issues in the field of insect vector resistance. In this study, the full cDNA of arrestin from Cx. pipiens pallens
was obtained at 1,650
bps. The putative amino acid sequence shares 99%, 92%, and 71% relevance with arrestin genes from Cx. quinquefasciatusAnopheles gambiae
, and Drosophila melanogaster,
respectively. Meanwhile, two domains have been observed within this protein, namely, pfam: Arrestin_N domain from 17 to 174 and pfam: Arrestin_C domain from 193 to 351. Despite the absence of similarity in any sequence, the two domains shared a very similar fold, known as the “arrestin fold” [33
]. Arrestins are 48 kD to 55 kD proteins that have highly conserved structure (70% identity). Arrestins have a common constant amino acid sequence in the N-terminus, including the activate identification zone, phosphorylation recognition region, and the second hydrophobic interaction district, which enhances the affinity between arrestins and GPCRs [34
]. All these data confirm that the gene obtained from Cx. pipiens pallens
The understanding of how resistance evolves at the molecular level is known predominantly to be involved in amplification, over-expression, and coding sequence variation of genes related to mechanisms of insecticide resistance [35
]. Over expression of genes possibly accounts for the relative importance of conferring resistance [37
]. Daborn reported that the up-regulation of CYP6Z1 gene led to DM resistance in A. gambiae
] and the up-regulation of CYP6G1 gene resulted in DDT resistance in D. melanogaster
CYP6A1 gene was over-expressed 12 and 10 fold in adults and larvae of diazinon-resistant M. domestica
]. Insecticide resistance is a complicated genetic phenomenon and is involved in the multi-mechanism or interaction of several genes [41
]. In this study, arrestin gene was up-regulated in the DR strain at the egg, pupae, male, and female stages. Over-expression of arrestin in mosquito cells increased their resistance to DM. Meanwhile, arrestin knockdown by siArrestin transfection decreased DM resistance. The result that arrestin can affect cell sensitivity to DM indicates that arrestin probably is a DM resistance related gene. Arrestin can regulate the expression levels of some genes in at least two ways: 1) Arrestin transfers from the cytoplasm to the nucleus and provides a scaffold for the transcription factor in the promoter region of target genes, which is called direct regulation; 2) Arrestin combines transcription regulation factors to change their activity and sub-cellular distribution, which is called indirect regulation [43
]. Therefore, arrestin possibly affects some insecticide-resistant genes by regulating gene transcription.
The transcriptional levels of CYP6A1 in the corresponding cells were examined to further investigate the mechanism of arrestin in DM resistance. CYP6A1 is one of the cytochrome P450 family members, which plays an important role in insecticide resistance and was first isolated from diazinon-resistant M. domestica
]. Some researchers found that CYP6A1 was expressed significantly higher in pyrethroid-resistant strains than in susceptible strains from other insects [40
]. In our previous study on the relationship of ribosomal protein L22 and DM resistance, CYP6A1 was also found to be up-regulated in mosquito cells resistant to DM [45
]. In this study, the expression levels of CYP6A1 were up-regulated and down-regulated when arrestin was over-expressed and knocked down. These changes were positively correlated with cell viability variations. Therefore, arrestin is possibly related to DM resistance by affecting the transcriptional levels of CYP6A1.
Some reports revealed that arrestins contribute to morphine tolerance through desensitization and internalization of opioid receptor (one member of GPCRs) [46
]. Opsin, another member of GPCRs, was found to be over expressed in DDT-resistant D. melanogaster
and several pyrethroid-resistant mosquitoes [21
]. Opsin is a DM resistance related gene identified in our previous study [20
]. In Classic GPCR signaling pathway, opsin and arrestin change their conformations, bind or separate with each other, and affect the downstream signaling [51
]. In this study, the expression levels of opsin were up-regulated and down-regulated when arrestin was over expressed and knocked down in mosquito cells. These changes were positively correlated with cell viability variations. Therefore, arrestin and opsin are essential in DM resistance through the GPCR pathway.
In summary, our results provided evidence, for the first time, that arrestin might be a resistance relative gene. In addition, whether arrestin participates in resistance through the opsin GPCR pathway or by regulating the transcriptional levels of CYP6A1, which finally leads to resistance, still needs in-depth research.
In this study, the arrestin gene was expressed at every developmental stage in Cx. pipiens pallens
and varied in different developmental stages. This observation suggests that arrestin is important to cell growth and development. The expression levels of arrestin gene were significantly different between DM-resistant and DM-susceptible strains at egg, pupae, male, and female stages, but not in larval stages, implying that arrestin possibly plays a role in DM resistance at all stages except larval stages. Interestingly, the expression level of arrestin in males was higher than that in female adults of DS strain. This result coincides with the results obtained from Anopheles gambiae
]. Therefore, arrestin displays preferential function involved in male-specific activities. Moreover, arrestin, as a multifunctional protein, performs different functions at different stages. However, the specific mechanisms need to be further elucidated.