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Background: Difference in physiology level between the immature and mature stages of insects likely contribute to different mechanisms of insecticide resistance. It is well acknowledged that insect 20‐hydroxyecdysone (20E) plays an important role in many biological processes in the immature stage, whether 20E confers insecticide resistance at this specific stage is still poorly understood. By gene cloning, reverse transcription quantitative real‐time PCR, RNA interference (RNAi) and in vitro metabolism experiments, this study aimed to investigate the potential role of 20E‐related genes in conferring imidacloprid (IMD) resistance in the immature stage of the whitefly Bemisia tabaci Mediterranean. Results: After identification of low to moderate IMD resistance in the whitefly, we found CYP306A1 of the six 20E‐related genes was overexpressed in the nymph stage of the three resistant strains compared to a laboratory reference susceptible strain, but not in the adult stage. Further exposure to IMD resulted in an increase in CYP306A1 expression in the nymph stage. These results together imply that CYP306A1 may be implicated in IMD resistance in the nymph stage of the whitefly. RNAi knockdown of CYP306A1 increased the mortality of nymphs after treatment with IMD in bioassay, suggesting a pivotal role of CYP306A1 in conferring IMD resistance in the nymph stage. Additionally, our metabolism experiments in vivo showed that the content of IMD reduced by 20% along with cytochrome P450 reductase and heterologously expressed CYP306A1, which provides additional evidence for the important function of CYP306A1 in metabolizing IMD that leads to the resistance. Conclusion: This study uncovers a novel function of the 20E biosynthesis gene CYP306A1 in metabolizing imidacloprid, thus contributing to such resistance in the immature stage of the insect. These findings not only advance our understanding of 20E‐mediated insecticide resistance, but also provide a new target for sustainable pest control of global insect pests such as whitefly. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The superfamily cytochrome P450s is involved in the evolution of insecticide resistance. However, whether CYP4G68, a differentially expressed gene identified from our transcriptomics analysis, confers resistance to the world's heavily used insecticide class neonicotinoids is unknown. Hence, we explored the role of CYP4G68 in conferring imidacloprid and thiamethoxam resistance in Bemisia tabaci. The species B. tabaci MED developed low-to-high resistance to imidacloprid and thiamethoxam. Exposure to imidacloprid and thiamethoxam significantly increased the expression of CYP4G68. Moreover, quantitative real-time PCR analysis demonstrated that CYP4G68 was remarkably overexpressed in imidacloprid-resistant and thiamethoxam-resistant strains compared to susceptible strains. Further correlation analysis showed that CYP4G68 expression was significantly positively correlated with the associated resistance level in various strains of B. tabaci. These results suggest that the enhanced expression of CYP4G68 appears to mediate imidacloprid and thiamethoxam resistance in B. tabaci. Additionally, silencing CYP4G68 via RNA interference strongly increased the susceptibility of B. tabaci MED to imidacloprid and thiamethoxam. Collectively, this work revealed that CYP4G68 plays a vital role in imidacloprid and thiamethoxam resistance in B. tabaci MED. These findings will not only advance our understanding of the role of P450s in insecticide resistance but also provide a great potential target for the sustainable control of destructive insect pests such as whiteflies. [ABSTRACT FROM AUTHOR]

NADPH cytochrome P450 reductase (CPR) is an integral component of cytochrome P450-mediated biological reactions, such as the metabolism of xenobiotics, including insecticides. CPR has been reported to be associated with insecticide tolerance in several insects. However, the biochemical characteristics and biological function of CPR in Bemisia tabaci Q (BtCPR) remain undefined. In this study, BtCPR was cloned, and bioinformatic analysis showed that BtCPR is a transmembrane protein with a molecular weight (MW) of 76.73 kDa and contains conserved binding domains (FMN, FAD, and NADPH). Tissue- and developmental stage-dependent expression indicated that the highest expression levels of BtCPR occurred in head tissue and in male adults. Transcripts of BtCPR in the field B. tabaci Q strain were 1.62-fold higher than those of the laboratory B. tabaci Q strain. In both field and laboratory adults, the susceptibility of BtCPR -knockdown B. tabaci Q to imidacloprid substantially increased compared to that of the B. tabaci Q control group. Furthermore, the heterologous expression of BtCPR in Sf9 cells exhibited catalytic activity for cytochrome c reduction, following Michaelis-Menten kinetics. Sf9 cells overexpressing BtCPR had greater viability than the control cells when treated with imidacloprid. The results suggest that BtCPR could affect the susceptibility of B. tabaci Q to imidacloprid and could also be considered a novel target for pest control. Unlabelled Image • Full-length cDNA of BtCPR gene were cloned and characterized in Bemisia tabaci Q • In both field and laboratory adults, the susceptibility of BtCPR-knockdown B. tabaci Q to imidacloprid substantially increased compared to that of the B. tabaci Q control group. • Heterologous expression of BtCPR in Sf9 cells exhibited catalytic activity for cytochrome c reduction and had greater viability than the control cells when treated with imidacloprid [ABSTRACT FROM AUTHOR]

Abstract Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), a globally invasive species complex that causes serious damage to field crops, has developed resistance to imidacloprid and many other pesticides. Insect detoxify to pesticides may partially depend on ABC transporters, which contribute to the detoxification of xenobiotics. To determine whether genes in the ABCG subfamily are involved in imidacloprid detoxification in B. tabaci Q, we cloned four ABCG subfamily genes based on the published MED/Q genome and on our previous study of the transcriptional response of ABC transporters in B. tabaci Q adults to imidacloprid. As indicated by the quantification of mRNA levels after a 6-h exposure, the expression level of ABCG3 was 3.3-fold higher in B. tabaci Q adults exposed to 100 μg/mL imidacloprid rather than to the buffer control. The expression level of ABCG3 was higher in females than in males but did not significantly differ among eggs or nymphal stages and did not significantly differ among head, thorax, and abdomen tissues of adults. Knockdown of ABCG3 via RNA interference significantly increased the mortality of imidacloprid-treated laboratory and field-collected adults of B. tabaci Q. These results indicate that the ABCG3 gene may be involved in imidacloprid detoxification by B. tabaci Q. Graphical abstract Unlabelled Image Highlights • Full-length cDNA of four ABCG genes were cloned and characterized in Bemisia tabaci Q • The transcript of ABCG3 was up-regulated after Imidacloprid treatment. • Suppression of ABCG3 increased the sensitivity of Bemisia tabaci Q to Imidacloprid [ABSTRACT FROM AUTHOR]

High level resistance for a variety of insecticides has emerged in Bemisia tabaci , a globally notorious insect. Neonicotinoid insecticides have been applied widely to control B. tabaci. Whether a differentially expressed gene CYP6DB3 discovered from transcriptome data of B. tabaci is involved in the resistance to neonicotinoid insecticides remains unclear. In the study, CYP6DB3 expression was significantly up-regulated in both thiamethoxam- and imidacloprid-resistant strains relative to the susceptive strains. We also found that CYP6DB3 expression was up-regulated after B. tabaci adults were exposed to thiamethoxam and imidacloprid. Moreover, knocking down CYP6DB3 expression via feeding corresponding dsRNA significantly reduced CYP6DB3 mRNA levels by 34.1%. Silencing CYP6DB3 expression increased the sensitivity of B. tabaci Q adults against both thiamethoxam and imidacloprid. Overexpression of CYP6DB3 gene reduced the toxicity of imidacloprid and thiamethoxam to transgenic D. melanogaster. In addition, metabolic studies showed that CYP6DB3 can metabolize 24.41% imidacloprid in vitro. Collectively, these results strongly support that CYP6DB3 plays an important role in the resistance of B. tabaci Q to imidacloprid and thiamethoxam. This work will facilitate a deeper insight into the part of cytochrome P450s in the evolution of insecticide resistance and provide a theoretical basis for the development of new integrated pest resistance management. [Display omitted] • Silencing CYP6DB3 enhanced thiamethoxam and imidacloprid toxicity to B. tabaci. • CYP6DB3 overexpression reduced thiamethoxam and imidacloprid toxicity to transgenic D. melanogaster. • Recombinant CYP6DB3 protein can metabolize imidacloprid in vitro. [ABSTRACT FROM AUTHOR]

Cuticular proteins (CPs) play an important role in protecting insects from adverse environmental conditions, like neonicotinoid insecticides, which are heavily used for numerous pests and caused environmental problems and public health concerns worldwide. However, the relationship between CPs and insecticides resistance in Bemisia tabaci , a serious and developed high insecticide resistance, is lacking. In this study, 125 CPs genes were identified in B. tabaci. Further phylogenetic tree showed the RR-2-type genes formed large gene groups in B. tabaci. Transcriptional expression levels of CPs genes at different developmental stages revealed that some CPs genes may play a specific role in insect development. The TEM results indicated that the cuticle thickness of susceptible strain was thinner than imidacloprid-resistance strain. Furthermore, 16 CPs genes (5 in RR-1 subfamily, 7 in RR-2 subfamily, 3 in CPAP3 subfamily and 1 in CPCFC subfamily) were activated in response to imidacloprid. And RNAi results indicated that CP9 and CP83 involved in imidacloprid resistance. In conclusion, this study was the first time to establish a basic information framework and evolutionary relationship between CPs and imidacloprid resistance in B. tabaci , which provides a basis for proposing integrated pest management strategies. [ABSTRACT FROM AUTHOR]