Gene silencing through RNAi and antisense Vivo-Morpholino increases the efficacy of pyrethroids on larvae of Anopheles stephensi

Abstract

Background Insecticides are still at the core of insect pest and vector control programmes. Several lines of evidence indicate that ABC transporters are involved in detoxification processes against insecticides, including permethrin and other pyrethroids. In particular, the ABCG4 gene, a member of the G subfamily, has consistently been shown to be up-regulated in response to insecticide treatments in the mosquito malaria vector Anopheles stephensi (both adults and larvae). Methods To verify the actual involvement of this transmembrane protein in the detoxification process of permethrin, bioassays on larvae of An. stephensi, combining the insecticide with a siRNA, specifically designed for the inhibition of ABCG4 gene expression were performed. Administration to larvae of the same siRNA, labeled with a fluorescent molecule, was effected to investigate the systemic distribution of the inhibitory RNA into the larval bodies. Based on siRNA results, similar experiments using antisense Vivo-Morpholinos (Vivo-MOs) were effected. These molecules, compared to siRNA, are expected to guarantee a higher stability in environmental conditions and in the insect gut, and present thus a higher potential for future in-field applications. Results Bioassays using two different concentrations of siRNA, associated with permethrin, led to an increase of larval mortality, compared with results with permethrin alone. These outcomes confirm that ABCG4 transporter plays a role in the detoxification process against the selected insecticide. Moreover, after fluorescent labelling, it was shown the systemic dissemination of siRNA in different body districts of An. stephensi larvae, which suggest a potential systemic effect of the molecule. At the same time, results of Vivo-MO experiments were congruent with those obtained using siRNA, thus confirming the potential of ABCG4 inhibition as a strategy to increase permethrin susceptibility in mosquitoes. For the first time, Vivo-MOs were administered in water to larvae, with evidence for a biological effect. Conclusions Targeting ABCG4 gene for silencing through both techniques resulted in an increased pyrethroid efficacy. These results open the way toward the possibility to exploit ABCG4 inhibition in the context of integrated programmes for the control An. stephensi mosquitoes and malaria transmission.

Deltamethrin interacts with Culex quinquefasciatus odorant-binding protein: a novel potential resistance mechanism

“…Singh et al [ 44 ] used a dipping method to interfere with the β-tubulin gene in Aedes aegypti and showed that 7 days of interference was required to reach the peak effect. A feeding method was used to silence the topoisomerase inhibitor-suppressed gene in Anopheles stephensi [ 45 ]; the interference effect was best at 3 h, plateaued at 6 h, and had decreased significantly by 24 h. The best interference time in our study may have been different from the latter’s due to the different dsRNA concentrations, insect stages, interference methods and target genes examined [ 45 ]. The results of our RNAi experiments provided further evidence of a negative relationship between OBP28 gene expression levels and deltamethrin resistance in mosquitoes.…”

Knockout of ABC Transporter ABCG4 Gene Confers Resistance to Cry1 Proteins in Ostrinia furnacalis

“…Moreover, Shan et al [39] showed that ABCG was related to chlorantraniliprole resistance in P. xylostella. Nrgri et al [40] showed that silencing the ABCG4 gene resulted in an increased pyrethroid efficacy. However, little research has been reported on how ABCG is related to Bt resistance.…”

Delivery Methods for RNAi in Mosquito Larvae

“…gambiae ( Mysore et al 2017 ) and An. stephensi ( Negri et al 2019 ). Larval soaking is a simple and easy technique but not applicable in the field as it needs the application of huge amounts of dsRNA to ensure delivery.…”