Cuticle proteins play an important role in insect cuticle resistance, by thickening the cuticle to prevent the penetration of insecticides, and changing the density, thickness and insect morphological development of the cuticle. There is increasing evidence that alteration of the cuticle plays a role in insecticide resistance, based on analysis of CP transcripts and measurement of cuticle thickness [19, 21, 22, 23, 24, 25]. For example, CYP4G16, CPLCG3, CPLCG5 and CPLC8 have been implicated in insecticide resistance by contributing to a thicker cuticle and thereby slowing penetration of insecticides [21, 25, 26]. Three CPR genes (CPR124, CPR129 and CPR127) were found to be constitutively overexpressed in resistant Anopheles gambiae [25]. In another study, 31 cuticle proteins were differentially regulated in the leg proteome, of which 29 including CPR106, CPR126, CPR121 and CPR151 were overexpressed, and only two were downregulated [22]. Furthermore, more than 65% of differentially expressed CPs belonged to the CPR family. Strong overexpression of cuticle protein CPR31 was also reported in multi-insecticide-resistant A. gambiae [27], and CPR63, CPR47, CPR48, CPR45 and CPR44 are highly expressed in DR strains of Cx. pipiens pallens [13].
Overexpression of CPRs in resistant mosquitoes has been widely reported, but their cuticle resistance mechanisms main poorly understood. Our previous study found that silencing the CPR63 gene made mosquitoes more susceptible to deltamethrin, suggesting that CPR63 participates in pyrethroid resistance [13]. In the present study, our results illuminate the resistance mechanism by which CPR63 contributes to the resistance phenotype; CPR63 is involved in thickening the cuticle, substantially reducing uptake of insecticides.
Insect CPs are diverse and expressed in the head, thorax and abdomen. Some CPs are also highly expressed in insect legs. For example, CPLCG5 is highly expressed in the legs of Cx. pipiens pallens [14], and members of the CPCFC CP family in A. gambiae are mainly distributed in legs [28]. Noh et al. found that cuticle protein TcCPR4 in Tribolium castaneum was mainly enriched in the legs, and participated in the formation of pore canals in the rigid cuticle [8]. CPF3, CPLCG3, CPLCG4 and CPLCG5 mRNA transcripts were mainly located in appendages (legs and wings) [14, 24]. Similarly, in the present study, CPR63 mRNAs were mainly located in mosquito legs and wings. Since these appendages are associated with motion, CPR63 might be related to flight. Additionally, CPR63 was expressed more highly in the legs of DR strains, indicating that it might help mosquitoes avoid areas treated with insecticides, but this hypothesis requires further exploration.
Different CPs play different roles in cuticular resistance. Huang et al. reported that CPLCG5 acts as a major CP and is highly expressed in the legs in Cx. pipiens pallens. Our current results showed that expression of CPR63 was increased in insecticide-resistant Cx. pipiens pallens, and also highly expressed in the legs. In addition, silencing of CPLCG5 resulted in larger pore canals, indistinct chitinous parallel laminae, and thinner endocuticle in the leg structures. Specifically, silencing of CPR63 resulted in thinner endocuticle and exocuticle, but the chitin content and number and size of pores as not significantly altered, indicating that different CPs perform distinct functions to contribute to cuticular resistance, and CPR63 participates in cuticular resistance mainly by increasing the cuticle thickness.
Early studies suggest that RR-1 and RR-2 proteins are present in different regions within the cuticle itself; RR-2 proteins contribute to exocuticle and RR-1 proteins are be found in the endocuticle [29, 30]. However, a more recent study showed that the location of RR-1s and RR-2s depends more on the properties of individual proteins [12]. Our current study showed that silencing CPR63 led to thinner endocuticle and exocuticle. We therefore speculate that CPR63 may be distributed in both the endocuticle and exocuticle, but this hypothesis needs further verification.
In summary, our results revealed that CPR63 participates in pyrethroid resistance by increasing the thickness of the cuticle, which might be beneficial for the penetration of insecticides, thereby increasing the sensitivity of mosquitoes to deltamethrin. This is the first report linking CPRs to insecticide resistance in mosquito legs.