The potential of type 2 cystatin in blood feeding and innate immunity of the tick Haemaphysalis doenitzi (Acari: Ixodidae)

Abstract

Abstract Ticks, which are obligate blood-sucking parasites, transmit a wide range of pathogens during their blood-feeding process. Enzymes and macromolecules that inhibit enzymes play a crucial role in tick physiology. In the present study, genes encoding type 2 cystatin were cloned and characterized from Haemaphysalis doenitzi, and their functions in innate immunity and tick blood feeding were further assessed. Four cystatin genes, namely HDcyst-1, HDcyst-2, HDcyst-3, and HDcyst-4, were successfully cloned from the tick. Their open reading frames vary in length, with sizes of 390, 426, 426, and 426 base pairs (bp), respectively. Proteinase inhibition assays indicated that 0.2 µM of cystatin displayed a minimum of 50% inhibitory activity against cathepsin B and S. The qPCR results imply that HDcyst-2 could play a pivotal role in tick hemophagia, whereas HDcyst-1, HDcyst-3, and HDcyst-4 may be integral to the regulation of blood digestion. Our investigation unveiled that adult ticks subjected to LPS injection or exposed to low-temperature stress exhibited elevated expression levels of type 2 cystatin compared to the control group. RNA interference experiments demonstrated that the expression of type 2 cystatins had a significant impact on engorged weight, the number of eggs laid, hatching rate, and mortality of H. doenitzi, along with an extension of blood-feeding duration by 2 days. These discoveries suggest the involvement of type 2 cystatin in tick innate immunity and blood-feeding processes. Consequently, type 2 cystatin emerges as a promising candidate for tick control and the development of novel immunosuppressive drugs in the future.