摘要: The insulin and 20-hydroxyecdysone (20E) pathways coordinately regulate insect vitellogenesis and ovarian development. However, the detailed molecular mechanisms such as the genes mediating the cooperation of the interaction of these 2 pathways in regulating insect reproductive development are not well understood. In the present study, a small GTPase, Rab40C, was identified from the notorious agricultural pest Bactrocera dorsalis. In addition to the well-known RAB domain, it also has a unique SOCS-box domain, which is different from other Rab-GTPases. Moreover, we found that Rab40C was enriched in the ovaries of sexually mature females. RNA interference (RNAi)-mediated knockdown of BdRab40C resulted in a decrease in vitellogenin synthesis, underdeveloped ovaries, and low fertility. Furthermore, depletion of insulin receptor InR or the heterodimer receptor of 20E (EcR or USP) by RNAi significantly decreased the transcription of BdRab40C and resulted in lower fecundity. Further studies revealed that the transcription of BdRab40C could be upregulated by the injection of insulin or 20E. These results indicate that Rab40C participates in the insulin and 20E pathways to coordinately regulate reproduction in B. dorsalis. Our results not only provide new insights into the insulin- and 20E-stimulated regulatory pathways controlling female reproduction in insects but also contribute to the development of potential eco-friendly strategies for pest control.

摘要: 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.

摘要: The sigma glutathione S-transferases (GSTSs) are a class of cytosolic glutathione S transferases (GSTs) that play important roles in antioxidant defense in insects, but the mechanisms by which GSTSs contribute to antioxidant activity remain unclear. Here, we isolated a GSTS (GSTS6) from Tribolium castaneum and explored its function. Homology and phylogenetic analysis revealed that TcGSTS6 shared high identity with other evolutionarily conserved GSTSs. The recombinant TcGSTS6 protein had strong activity toward cumene hydroperoxide and 4-hydroxynonenal but low activity toward the universal substrate 1-chloro-2,4-dinitrobenzene. Exposure to various types of oxidative stress, including heat, cold, UV and pathogenic microbes, significantly induced TcGSTs6 expression, which indicates that it is involved in antioxidant defense. Knockdown TcGSTs6 by using RNA interference (RNAi) caused reduced antioxidant capacity, which was accomplished by cooperating with other antioxidant genes. Moreover, treatment with various insecticides such as phoxim, lambda-cyhalothrin, dichlorvos and carbofuran revealed that TcGSTS6 plays an important role in insecticide detoxification. The RNAi results showed that TcGSTS6 is essential for embryogenesis in T. castaneum. Our study elucidates the mechanism by which a GSTS contributes to antioxidant activity and enhances our understanding of the functional diversity of GSTSs in insects.

摘要: The evolution of nitenpyram resistance has been confirmed to be related to overexpression of two key metabolic enzyme genes, CYP6ER1 and CarE1, in Nilaparvata lugens, a highly destructive rice pest that causes substantial economic losses and has developed insecticide resistance. As microRNAs (miRNAs) are important post-transcriptional regulators of gene expression, whether they are involved in nitenpyram resistance is poorly understood in N. lugens. In this study, knockdown of key genes in the miRNA biogenesis pathway (Dicer1, Drosha, and Argonaute1) changed CYP6ER1 and CarE1 abundance, which confirmed the importance of miRNAs in nitenpyram resistance. Furthermore, global screening of miRNAs associated with nitenpyram resistance in N. lugens was performed, and a total of 42 known and 178 novel miRNAs were identified; of these, 57 were differentially expressed between the susceptible and resistant strains, and two (novel_85 and novel_191) were predicted to target CYP6ER1 and CarE1, respectively. Luciferase reporter assays demonstrated that novel_85 and novel_191 bind to the CYP6ER1 and CarE1 coding regions, respectively, and downregulate their expression. Moreover, modulating novel_85 and novel_191 expression by injection of miRNA inhibitors and mimics significantly altered N. lugens nitenpyram susceptibility. This is the first study to systematically screen and identify miRNAs associated with N. lugens nitenpyram resistance, and provides important information that can be used to develop new miRNA-based targets in insecticide resistance management.

摘要: Olfaction plays an essential role in insect behavior such as host location, foraging, mating, and oviposition. The odorant receptor co-receptor (Orco) is an obligatory odorant receptor and indispensable in odor perception. Here, we characterized the Orco gene from the oriental fruit fly, Bactrocera dorsalis (Hendel), a notorious agriculture pest. The olfactory deficiency mutants were generated by editing the BdorOrco gene using the CRISPR/Cas9 system. Electroantennograms (EAG) and olfactory preference assays confirmed that BdorOrco(-/-) mutant flies had reduced perception of methyl eugenol, beta-caryophyllene, and ethyl acetate. Oviposition bioassays showed that the eggs laid by BdorOrco(-/-) females mediated by benzothiazole and 1-octen-3-ol were significantly decreased. In addition, BdorOrco(-/-) mutant flies took a significantly longer time to locate the food source compared with wild type (WT) flies. Altogether, our data indicated that Orco is essential for multiple physiological processes in B. dorsalis, and it expands our understanding of the function of insect Orco.