Functional analysis of the regulation of beta-cypermethrin resistance by Deg-CYP-3 in Dermanyssus gallinae

Dermanyssus gallinae is a significant haematophagous ectoparasite of laying hens. Despite the worldwide application of beta-cypermethrin for mite control, resistance to this compound has been developed by D. gallinae. P450s, as key detoxification enzymes involved in the metabolism of exogenous and endogenous chemical substances, have not been functionally characterized in D. gallinae. Here, the role of the P450s gene (Deg-CYP-3) of D. gallinae in acaricide resistance was studied. Firstly, P450s activities of the five beta-cypermethrin resistant strains (RR) against p-nitroanisole were significantly higher than that of the susceptible strain (RS), suggesting enhanced enzymatic activities might contribute to mite beta-cypermethrin resistance. Both mRNA expression level and DNA copy number of Deg-CYP-3 were significantly higher in RR than in RS, except for DK RR strain. Moreover, recombinant Deg-CYP-3 (rCYP-3) expressed in Escherichia coli displayed high enzymatic activity against p-nitroanisole, while Qulaton-100, acetone, isoacetone, avermectin, and ivermectin were found to inhibit this activity. Immunohistochemical analysis revealed the presence of Deg-CYP-3 protein in the digestive tract and Malpighian tubules. Finally, silencing of Deg-CYP-3 via RNAi resulted in decreased enzyme activity and increased susceptibility of PG RR strain to beta-cypermethrin, confirming that Deg-CYP-3 is crucial for beta-cypermethrin detoxification. RNAi also resulted in enhanced mortality, as well as reduced oviposition and fecundity in the PG RR strain. Collectively, these findings indicate that resistance of D. gallinae to beta-cypermethrin is associated with elevated P450s protein activity and increased Deg-CYP-3 expression levels. The results provide insights into the metabolic resistance mechanisms of D. gallinae and offer scientific guidance for the management and control of mite.