A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of pathenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to a Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delays bovine plasma clotting time and inhibits both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene expresses at all stages of the tick except for the egg stage and mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene led to a 2-day extension of the tick blood feeding period, and 27.7% of the ticks did not successfully complete the blood feeding. These findings indicate that the newly indentified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.

Blood feeding tightly regulates the reproductive cycles in ticks. Vitellogenesis and nutritional signaling are a key event in the tick reproductive cycle. Here we report the identification of a Haemaphysalis longicornis GATA factor, (HlGATA), which is synthesized after a blood meal and acts as a transcriptional activator of vitellogenin (Vg). HlGATA shares structural similarity with other GATA factors of invertebrates and vertebrates. Tick GATA mRNA accumulated in the fat body and midgut prior to blood feeding. However, translation of GATA was activated by blood feeding because the GATA protein increased dramatically in engorged females. RNA interference (RNAi)-mediated knock down of GATA transcript resulted in a significant inhibition of Vg expression and effectively disrupts egg development after blood meal in engorged tick. In addition, HlGATA translation was inhibited by RNAi-mediated knock down of S6 kinase. These experiments have revealed that the GATA factor, which is the specific transcriptional activator of Vg gene, represents important molecule for tick reproduction.

The life cycle of ticks is characterized by alternate off-host and on-host conditions. The life span is estimated at several years and most ixodid ticks spend more than 95% of their life off the host. They seem to have a unique strategy to endure the off-host state for a long period. By electron microscopy, isolation membrane-, autophagosome- and autolysosome-like structures were found in the midgut epithelial cells of unfed ticks. Therefore, we focused on autophagy which is well-conserved from yeast to higher eukaryotes and induced by starvation. We have identified homologues of autophagy-related (ATG) genes (ATG3, ATG4 and ATG8) from cDNA libraries of the 3-host tick Haemaphysalis longicornis. Each expression profile of H. longicornis ATG (HlATG) genes and HlAtg proteins at the stages of nymph and adult were examined by real-time PCR and immunoblotting. Moreover, autophagy is known to be induced by inactivation of target of rapamycin (TOR), a phosphatidylinositol kinase. To examine the effect of TOR function on the expression of HlAtg protein(s), rapamycin, a specific inhibitor of the signal transduction mediated by TOR, was injected to unfed adults. It was revealed that the expression of HlAtg protein(s) was enhanced in response to the rapamycin. This result indicates that tick have the nutrient-sensitive TOR signaling pathway which regulate autophagy.