Cystatin antibodies interfere with ovary development in Haemaphysalis doenitzi (Acari: Ixodidae)

Anti-tick vaccines are gaining attention as a strategy to prevent tick infestations by activating the immune response of the host. Antibodies produced by the host inhibit tick growth and reproduction, but the molecular mechanism remains to be clarified. In this study, we investigated the effects of cystatin antibodies on the ovarian function of Haemaphysalis doenitzi. Histological analysis revealed that exposure to cystatin antibodies resulted in a significant reduction in the number of eggs produced and caused severe damage to the ovarian tissue structure. Immunofluorescence experiments confirmed the significant expression of cystatin within the ovary. Proteomics and phosphoproteomics identified 31 and 10 differentially expressed proteins in the relevant pathways, respectively. These changes in protein levels were found to be regulated by various mechanisms, including ribosomes activity, regulation of actin cytoskeleton, RNA transport, the TCA cycle, drug metabolism, and mTOR signaling pathways. Notably, there was high expression of tropomyosin and low expression of glutathione S-transferase (GST) during ovarian detoxification. Enzyme activity assays indicated a significant down-regulation of GST enzyme activity in the immunized group, suggesting that cystatin antibodies impaired the detoxification capacity of the ticks. Both tropomyosin and GST were successfully cloned and designated as HD-TPMa and HD-GSTa, respectively. RNA interference (RNAi) successfully knocked down the target gene. Ticks subjected to immersion in cystatin antibodies exhibited a significantly increased mortality rate after 72 hours. This study elucidated the molecular mechanism by which cystatin antibodies inhibit the growth and development of tick ovaries, providing an important scientific basis for the development of effective tick ovary control strategies.