The cytochrome P450 CYP6ER1 has been reported to play an important role in imidacloprid resistance of the brown planthopper (BPH), Nilaparvata lugens , and is overexpressed in most resistant populations. In the present study, we confirmed that CYP6ER1 expression can be induced by certain levels of imidacloprid. Developmental expression analysis revealed that CYP6ER1 was expressed highly in the adult stage, and tissue distribution analysis showed that CYP6ER1 was expressed mainly in the fat body and midgut. RNA interference (RNAi) of CYP6ER1 and transgenic expression of CYP6ER1 in Drosophila melanogaster both suggested that the expression of CYP6ER1 is sufficient to confer imidacloprid resistance. Furthermore, we analyzed the interaction of imidacloprid and CYP6ER1 monooxygenase by using dynamic simulations and molecular docking. We found that Nitrogen atoms in the heterocycle of the imidacloprid molecule may bind to iron atoms in the center of the homology model of CYP6ER1 via 4,5-dihedro-1H-imidazole. This finding contributes to a better understanding of how CYP6ER1 takes part in the insecticide metabolism.

Chitin synthases are very important enzymes for chitin synthesis in various species, which makes them a specific target of insecticides. In the present study, the function of the chitin synthase A (CHSA) gene isolated from Mythimna separate is investigated. The majority of dsMysCHSA treated larvae (89.50%) exhibit lethal phenotypes, including three phenotypes with severe cuticle deformations. The dsMysCHSA treatment in adult females affects oogenesis, and significantly reduce the ovary size and the oviposition number compared with controls. To determine how MysCHSA affects female fecundity, combined analyses of RNA-sequencing (RNA-Seq) transcriptome and TMT proteome (tandem mass tags) data in M. separata after treatment with MysCHSA-RNAi is performed. The differentially expressed proteins and genes affect fecundity-related proteins, energy metabolism, fatty acid metabolism, amino sugars, and nucleotide sugar metabolism pathways. Taken together, these results suggest that MysCHSA acts on M. separata ecdysis and fecundity, and has the potential as a target gene for pest control.

Chemical insecticides have been heavily employed as the most effective measure for control of agricultural and medical pests, but evolution of resistance by pests threatens the sustainability of this approach. Resistance-conferring mutations sometimes impose fitness costs, which may drive subsequent evolution of compensatory modifier mutations alleviating the costs of resistance. However, how modifier mutations evolve and function to overcome the fitness cost of resistance still remains unknown. Here we show that overexpression of P450s not only confers imidacloprid resistance in the brown planthopper, Nilaparvata lugens, the most voracious pest of rice, but also leads to elevated production of reactive oxygen species (ROS) through metabolism of imidacloprid and host plant compounds. The inevitable production of ROS incurs a fitness cost to the pest, which drives the increase or fixation of the compensatory modifier allele T65549 within the promoter region of N. lugens peroxiredoxin (NlPrx) in the pest populations. T65549 allele in turn upregulates the expression of NlPrx and thus increases resistant individuals’ ability to clear the cost-incurring ROS of any source. The frequent involvement of P450s in insecticide resistance and their capacity to produce ROS while metabolizing their substrates suggest that peroxiredoxin or other ROS-scavenging genes may be among the common modifier genes for alleviating the fitness cost of insecticide resistance.

The tracheal apical extracellular matrix (aECM) is vital for expansion of the tracheal lumen and supports the normal structure of the lumen to guarantee air entry and circulation in insects. Although it has been found that some cuticular proteins are involved in the organization of the aECM, unidentified factors still exist. Here, we found that mind the gap (Mtg), a predicted chitin-binding protein, is required for the normal formation of the apical chitin matrix of airway tubes in the model holometabolous insect Drosophila melanogaster. Similar to chitin, the Mtg protein was linearly arranged in the tracheal dorsal trunk of the tracheae in Drosophila. Decreased mtg expression in the tracheae seriously affected the viability of larvae and caused tracheal chitin spiral defects in some larvae. Analysis of mtg mutant showed that mtg was required for normal development of tracheae in embryos. Irregular taenidial folds of some mtg mutant embryos were found on either lateral view of tracheal dorsal trunk or internal view of transmission electron microscopy analysis. These abnormal tracheae were not fully filled with gas and accompanied by a reduction in tracheal width, which are characteristic phenotypes of tracheal aECM defects. Furthermore, in the hemimetabolous brown planthopper (BPH) Nilaparvata lugens, downregulation of NlCPAP1-N (a homolog of mtg) also led to the formation of abnormal tracheal chitin spirals and death. These results suggest that mtg and its homolog are involved in the proper organization of the tracheal aECMs in flies and BPH, and that this function may be conserved in insects.

Krotzkopf verkehrt (kkv) is a key enzyme that catalyzes the synthesis of chitin, an important component of the Drosophila epidermis, trachea, and other tissues. Here, we report the use of comprehensive RNA interference (RNAi) analyses to search for kkv transcriptional regulators. A cell‐based RNAi screen identified 537 candidate kkv regulators on a genome‐wide scale. Subsequent use of transgenic Drosophila lines expressing RNAi constructs enabled in vivo validation, and we identified six genes as potential kkv transcriptional regulators. Weakening of the kkvDsRed signal, an in vivo reporter indicating kkv promoter activity, was observed when the expression of Akirin, NFAT, 48 related 3 (Fer3), or Autophagy‐related 101(Atg101) was knocked down in Drosophila at the 3rd‐instar larval stage; whereas we observed disoriented taenidial folds on larval tracheae when Lines (lin) or Autophagy‐related 3 (Atg3) was knocked down in the tracheae. Fer3, in particular, has been shown to be an important factor in the activation of kkv transcription via specific binding with the kkv promoter. The genes involved in the chitin synthesis pathway were widely affected by the downregulation of Fer3. Furthermore, Atg101, Atg3, Akirin, Lin, NFAT, Pnr, and Abd‐A showed that the potential complex mechanism of kkv transcription is regulated by an interaction network with bithorax complex components. Our study revealed the hitherto unappreciated diversity of modulators impinging on kkv transcription and opens new avenues in the study of kkv regulation and chitin biosynthesis.