Functional analysis of novel cystatins from Haemaphysalis doenitzi and evaluation of their roles in cypermethrin and λ-cyhalothrin resistance

Currently, the primary strategy for tick control relies on chemical agents. Pyrethrins, which are botanically derived compounds, have demonstrated efficacy in controlling ticks without posing a risk to human or animal health. However, research into pyrethrins' metabolic mechanisms remains sparse. Cystatin, as a reversible binding inhibitor of cysteine protease, may be involved in the initiation of pyrethrin detoxification of Haemaphysalis doenitzi. In this study, two novel cystatins were cloned, HDcyst-3 and HDcyst-4, the relative expression of which was highest in the Malpighian tubules compared with the tick midguts, salivary glands, and ovaries. Prokaryotic expression and in vitro studies revealed that cystatins effectively inhibit the enzymatic activities of cathepsins B and S. RNAi results showed that the reduction of cystatins significantly decreased the engorgement weight, egg mass weight, and egg hatching rate of adult female ticks, and prolonged feeding time by two days. The control rate of rHDcyst-3 and rHDcyst-4 protein vaccination against female adults were 55.9% and 63.2%, respectively. In addition, the tick immersion test showed that cypermethrin and λ-cyhalothrin had significant acaricidal effects against adult unfed H. doenitzi. The qPCR result indicated that compared with the control group, the expression of HDcyst-3 and HDcyst-4 was markedly decreased in the sublethal cypermethrin and λ-cyhalothrin group at LC₅₀. Enzyme activity showed that cypermethrin and λ-cyhalothrin could significantly induce the activities of glutathione S-transferase (GST), carboxylesterase (CarE), and acetylcholinesterase (AchE). The aforementioned results provided indirect evidence that cystatin plays an important role in pyrethrin detoxification and provides a theoretical basis for future acaricide experiments and pest management.