Field populations of Aphis gossypii (SDR) have evolved high resistance to neonicotinoids, including thiamethoxam and imidacloprid. Synergism bioassays and transcriptomic comparison of the SDR and susceptible (SS) strains revealed that the cytochrome P450s may contribute to the neonicotinoid resistance evolution. The transcripts of some P450s were constitutively overexpressed in the SDR strain, and many genes showed expression plasticity under insecticide exposure. Drosophila that ectopically expressed CYPC6Y9, CYP4CK1, CYP6DB1, and CYP6CZ1 showed greater resistance (>8.0-fold) to thiamethoxam, and Drosophila that expressed CYPC6Y9, CYP6CY22, CYP6CY18, and CYP6D subfamily genes showed greater resistance (>5-fold) to imidacloprid. Five P450 genes that caused thiamethoxam resistance also conferred resistance to α-cypermethrin. Furthermore, the knockdown of CYP4CK1, CYP6CY9, CYP6CY18, CYPC6Y22, CYP6CZ1, and CYP6DB1 dramatically increased the sensitivity of the SDR strain to thiamethoxam or imidacloprid. These results indicate the involvement of multiple P450 genes, rather than one key gene, in neonicotinoid resistance in field populations.

Chemosensory proteins (CSPs) are a class of transporters in arthropods. Deeper research on CSPs showed that CSPs may be involved in some physiological processes beyond chemoreception, such as insect resistance to pesticides. We identified two upregulated CSPs in two resistant strains of Aphis gossypii Glover. To understand their role in the resistance of aphids to pesticides, we performed the functional verification of CSP1 and CSP4 in vivo and in vitro. Results showed that the sensitivity of the thiamethoxam-resistant strain to thiamethoxam increased significantly with the silencing of CSP1 and CSP4 by RNAi (RNA interference), and the sensitivity of the spirotetramat-resistant strain to spirotetramat increased significantly with the silencing of CSP4. Transgenic Drosophila melanogaster expressing CSPs exhibited stronger resistance to thiamethoxam, spirotetramat, and alpha-cypermethrin than the control did. In the bioassay of transgenic Drosophila, CSPs showed different tolerance mechanisms for different pesticides, and the overexpressed CSPs may play a role in processes other than resistance to pesticides. In brief, the present results prove that CSPs are related to the resistance of cotton aphids to insecticides.

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ATP-binding cassette (ABC) transporters regulate the efflux of a broad spectrum of substrates to extracellular transporting, which play an important role in the detoxification process in arthropods. Here, we described a comprehensive approach to explore the involvement of ABC transporters in spirotetramat resistance in cotton aphids. In this study, synergism bioassays showed 17.05% and 35.42% increases in the toxicity to spirotetramat with the ABC inhibitor verapamil in adult and 3rd instar nymph aphids of the SR strain, respectively. In a competitive assay based on the microinjection of a fluorescent ABC transporter substrate, verapamil (a general ABC inhibitor) and spirotetramat significantly inhibited the elimination of Texas Red. Based on transcriptome data of midguts of spirotetramat-susceptible (SS) and -resistant (SR) strains, the expression levels of ABCB4, ABCB5, ABCF2, MRP11, and MRP12 were significantly upregulated in the SR strain midgut compared to that of the SS strain. Gene functional analysis based on ectopic expression and RNA interference (RNAi) proved that ABCB4, ABCB5, ABCF2, MRP11, and MRP12 were involved in the tolerance of cotton aphids to spirotetramat. Moreover, the upregulated ABCF2, ABCB4, and ABCB5 in the midgut of the SR strain contributed more to the resistance of spirotetramat in in vitro functional analysis. In summary, these results demonstrate that candidate ABC transporter genes in the midgut tissue were involved in spirotetramat resistance, which will help reveal the relationship between ABC transporters and the development of spirotetramat resistance in field populations.