CPR63 promotes pyrethroid resistance by increasing cuticle thickness in Culex pipiens pallens

Abstract

The cuticle protein (CP) encoded by CPR63 plays a role in deltamethrin resistance in Culex pipiens pallens. Herein, we investigated the distribution of CPR63 transcripts in this organism and observed high expression levels in legs and wings. Furthermore, expression of CPR63 in the legs of deltamethrin-resistant (DR) strains was 2.17-fold higher than in deltamethrin-susceptible (DS) strains. Cuticle analysis of small interfering RNA (siRNA) groups by scanning electron microscopy (SEM) revealed a significantly thinner cuticle of the tarsi in the siCPR63 group than in the siNC (negative control siRNA) group. Transmission electron microscopy (TEM) revealed that the exocuticle and endocuticle thickness of the tarsi were significantly thinner, which contributes the thinner procuticle of tarsi in the siCPR63 group than in the siNC group. Our results suggested that CPR63 might contribute to the resistance phenotype by thickening the cuticle and thereby possibly increasing the tolerance of mosquitoes to deltamethrin.

Keywords: CPRs; Culex pipiens pallens; Cuticle thickening; Cuticular resistance; Deltamethrin; Mosquito.

Fig. 1

Expression profiles of CPR63 in different mosquito tissues. Constitutive expression of CPR63 in DS and DR strains; mRNA expression levels were measured in the head, thorax, abdomen, legs and wings in DS and DR strain mosquitos. Head of DS strain was ascribed an arbitrary value of 1. Results are presented as the mean ± standard deviation (SD) of three biological replicates. ****p ≤ 0.0001; ns, not signifcant, p > 0.05

Fig. 2

Relative expression levels of CPR63 after RNAi silencing. Levels of CPR63 expression in whole mosquito bodies (a) and legs (b) after silencing of CPR63 were measured by qPCR. Results are shown as the mean ± SD of three biological replicates. *p ≤ 0.05; **p ≤ 0.01

Fig. 3

SEM analysis of the effects of siNC and siCPR63. a Illustration of the position of sectioning on the Cx. pipiens pallens tarsomere 1 (t1–t5 = five tarsal segments) [20]. The red line indicates in which leg part the sections were taken. The SEM images show a front view of a sectioned leg for the siNC group (b) and the siCPR63 group (c)

Fig. 4

SEM analysis of cuticle thickness. Measurements were made at 23 different points per individual, allowing for the calculation of mean cuticle thickness. Results are shown as the mean ± SD; n = the number of measurements carried out on each group of 11 mosquitoes. **p = 0.0011

Fig. 5

TEM analysis of the effects of siNC and siCPR63. SEM images show a front view of a sectioned leg for siNC (a) and siCPR63 (b) groups. The curve represents the chitin parallel laminae, and the circle represents the pores

Fig. 6

TEM analysis of cuticle thickness. Measurements were performed at 58 points per individual, allowing for the calculation of mean cuticle thickness. Results are shown as the mean ± SD; n = the number of measurements carried out on each batch of six mosquitoes. ****p ≤ 0.0001; not signifcant, p > 0.05