Functional characterization of an arrestin gene on insecticide resistance of Culex pipiens pallens

Abstract

Background: Continuous and excessive application of insecticides has resulted in the rapid development of insecticide resistance in several mosquito species, including Culex pipiens pallens. Previous studies in our laboratory found that arrestin gene expression was higher in the deltamethrin-resistant (DR) strain than in the deltamethrin-susceptible (DS) strain of Cx. pipiens pallens. Similarly, other studies reported that arrestin was highly expressed in permethrin-resistant Cx. quinquefasciatus and in dichlorodiphenyltrichloroethane (DDT)-resistant Drosophila melanogaster.

Methods: Full-length cDNAs of an arrestin gene were cloned from Cx. pipiens pallens via polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE). The mRNA levels of the arrestin gene in the whole life cycle of DR and DS strains of Cx. pipiens pallens were investigated via quantitative real-time PCR. In addition, the relationship between arrestin and deltamethrin (DM) resistance were identified using genetic overexpression strategies and arrestin RNAi in mosquito cells. Cell viability was analyzed with cholecystokinin octapeptide after DM treatment. Moreover, the mRNA levels of cytochrome P450 6A1 (CYP6A1) and opsin in the transfected cells and controls were analyzed.

Results: Complete arrestin gene sequence was cloned and expressed throughout the life cycle of Cx. pipiens pallens. Moreover, arrestin was significantly upregulated in the DR strain, compared with that in the DS strain at the egg, pupae, and adult stages. Arrestin overexpression comparably increased the mosquito cell viability, whereas arrestin knockdown by siRNA decreased mosquito cell viability with deltamethrin (DM) treatment. Meanwhile, the mRNA levels of CYP6A1 and opsin were upregulated in mosquito cells transfected with arrestin and downregulated in mosquito cells with arrestin knockdown.

Conclusion: This study presented the first evidence that arrestin might be associated with insecticide resistance in Cx. pipiens pallens.

Figure 1

Quantitative real-time PCR analysis of arrestin at different developmental stages ofCx. pipiens pallens. The raw threshold cycle (Ct) values were normalized against standard β-actin to obtain normalized Ct values. The expression level of arrestin in susceptible strain eggs was considered as background 1. The data are means ± SD of three independent experiments. *p < 0.05,**p < 0.01.

Figure 2

Quantitative real-time PCR analysis of the overexpression efficiency of arrestin in cells. Total RNAs (1.0 μg) from pIB/Arrestin cells and pIB/Ctrl cells were analyzed to detect arrestin expression. β-actin was used as internal control. n = 3; **p < 0.01. The experiment was repeated three times.

Figure 3

Western blot analysis of arrestin expression in cells transfected with pIB/Arrestin and pIB/Ctrl. 1: pIB/Arrestin; 2: pIB/Ctrl.

Figure 4

Quantitative real-time PCR analysis of the knockdown efficiency of arrestin in cells. Total RNAs (1.0 μg) from siCtrl transfected and siArrestin transfected cells were analyzed. β-actin was used as internal control. n = 3; ***p < 0.001. The experiment was repeated three times.

Figure 5

Western blot analysis of arrestin expression in cells transfected with siCtrl and siArrestin. 1: siCtrl; 2: siArrestin.

Figure 6

Overexpression of arrestin enhances DM resistance in mosquito cells. Transfected cells of pIB/Arrestin and pIB/Ctrl were treated with DM at indicated concentrations. Viable cells were measured by CCK-8 after 72 h of treatment. The percentage of viable cells is shown relative to the control (absorbance value of 0 μM). Results were expressed as mean ± standard deviation (SD) of triplicate wells from one out of three representative experiments. *p < 0.05 compared with pIB/Ctrl.

Figure 7

siRNA mediated arrestin silencing reduces DM resistance in mosquito cells. Transfected cells of siArrestin and siCtrl were treated with DM at indicated concentrations, and viable cells were measured by CCK-8 after 72 h of treatment. The percentage of viable cells is shown relative to the control (absorbance value of 0 mg/l). Results were expressed as mean ± standard deviation (SD) of triplicate wells from one representative experiment out of three. *p < 0.05 compared with siCtrl.

Figure 8

Real-time quantitative PCR assay of CYP6A1 and opsin in pIB/Arrestin and pIB/Ctrl cells. The relative expression of CYP6A1 or opsin in pIB/Ctrl cells was considered as background. Results were expressed as mean ± standard error. β-actin was used as internal control. n = 3; *p < 0.05, **p < 0.01. The experiment was repeated three times. A: CYP6A1 expression level; B: opsin expression level.

Figure 9

Real-time quantitative PCR assay of CYP6A1 and opsin in siCtrl and siArrestin cells. Results were expressed as mean ± standard error. The relative expression of CYP6A1 in siCtrl cells was considered as background level or 1. β-actin was used as internal control. n = 3; *p < 0.05, **p < 0.01. The experiment was repeated three times. A: CYP6A1 expression level; B: opsin expression level.