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BACKGROUND Cyflumetofen is an outstanding acaricide with a novel mode of action. Tetranychus cinnabarinus, an important agricultural pest, is notorious for developing resistance to most classes of acaricides rapidly and results in enormous loss for the economy. Our previous study had pointed out glutathione S‐transferase (GSTs) significantly contributed to the cyflumetofen‐resistance formation in T. cinnabarinus, but the more specific mechanism needed to be further investigated. RESULTS The unstable resistance was observed in cyflumetofen‐resistant strain (CyR)under acaricide‐free condition. The activity of GSTs increased along with the development of resistance. The expressions of 13 GST genes were detected in CyR and susceptible strain (SS), of which six genes were overexpressed in CyR and the TcGSTm02 was selected as the representative for functional study. The expression of TcGSTm02 changed along with the resistant level of CyR with the same trend. Recombinant protein of TcGSTm02 with high activity was successfully obtained by E. coli expression system, whose activity could be inhibited by cyflumetofen (IC50 = 0.23 mM). Recombinant TcGSTm02 could effectively decompose cyflumetofen, and catalyze GS− to conjugate with cyflumetofen. CONCLUSION All clues confirmed that GSTs strongly associated with cyflumetofen‐resistance and a representative gene, TcGSTm02, showed function on contributing the evolution of cyflumetofen‐resistance in T. cinnabarinus. © 2019 Society of Chemical Industry

BACKGROUND: Acaricide resistance is a serious problem in spider mites. Cyflumetofen is a new complex II inhibitor, whereas pyridaben acts at complex I and has been used for decades. Although cross-resistance between cyflumetofen and pyridaben has been observed in Tetranychus cinnabarinus, the specific mechanisms at play have not yet been investigated. -resistant strain. In addition, recombinant CYP389C16 (40 pmol) effectively metabolized 25.0 ± 0.7% of cyflumetofen, 39.7 ± 1.0% of pyridaben, and 69.3 ± 3.3% of AB-1 (active de-esterified metabolite of cyflumetofen) within 2 h. In addition, hydroxylation metabolite of AB-1 was identified by HPLC-MS/MS. RESULTS: Investigation into the cross-resistance mechanisms identified five P450s, among which CYP389C16 was evaluated as the most likely candidate conferring cross-resistance. Knockdown of CYP389C16 expression via RNA interference diminished the level of cross-resistance in the cyflumetofen CONCLUSIONS:The study reveals that overexpressed CYP389C16 is involved in the cross-resistance between cyflumetofen and pyridaben in T. cinnabarinus.

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Pesticide resistance and resurgence are serious problems often occurring simultaneously in the field. In our long‐term study of a fenpropathrin‐resistant strain of Tetranychus cinnabaribus, enhancement of detoxification and modified fecundity mechanisms were both observed. Here we investigate the network across these two mechanisms and find a key node between resistance and resurgence. We show that the ecdysone pathway is involved in regulating the fecundity of T. cinnabaribus. The concentration change of ecdysone is consistent with the fecundity curve; the concentration of ecdysone is higher in the fenpropathrin‐resistant strain which has stronger fecundity. The enhancement of ecdysone is due to overexpression of two P450 genes (CYP314A1 and CYP315A1) in the ecdysone synthesis pathway. Silencing expression of these CYP genes resulted in lower concentration of ecdysone, reduced expression of vitellogenin, and reduced fecundity of T. cinnabaribus. The expression of CYP315A1 is regulated by transcription factors Cap‐n‐collar isoform C (CncC) and Musculoaponeurotic fibrosarcoma protein (Maf), which are involved in regulating other P450 genes functioning in detoxification of fenpropathrin in T. cinnabaribus. A similar regulation is established in citrus pest mite Panonychus citri showing that the CncC pathway regulates expression of PcCYP315A1, which affects mite fecundity. Transcription factors are activated to upregulate detoxification genes facilitating pesticide resistance, while the “one to multiple” regulation mode of transcription factors simultaneously increases expression of metabolic enzyme genes in hormone pathways and alters the physiology of pests. This is an important response of arthropods to pesticides which leads to resistance and population resurgence.