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.

• Min Liao(Laboratory of Emerging Infectious Diseases, Department of Frontier Veterinary Medicine, Faculty of Agriculture, Kagoshima University, Japan)
• Jinlin Zhou(National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Japan)
• Haiyan Gong(National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Japan)
• Damdinsuren Boldbaatar(Laboratory of Emerging Infectious Diseases, Department of Frontier Veterinary Medicine, Faculty of Agriculture, Kagoshima University, Japan)
• Rika Umemiya-Shirafuji(Laboratory of Emerging Infectious Diseases, Department of Frontier Veterinary Medicine, Faculty of Agriculture, Kagoshima University, Japan)
• Banzragch Battur(Laboratory of Emerging Infectious Diseases, Department of Frontier Veterinary Medicine, Faculty of Agriculture, Kagoshima University, Japan)
• Jianbao Dong(Laboratory of Emerging Infectious Diseases, Department of Frontier Veterinary Medicine, Faculty of Agriculture, Kagoshima University, Japan)
• Yoshifumi Nishikawa(National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Japan)
• Myungjo You(Department of Veterinary Parasitology, College of Veterinary Medicine, Chonbuk National University)
• Kozo Fujisaki(Laboratory of Emerging Infectious Diseases, Department of Frontier Veterinary Medicine, Faculty of Agriculture, Kagoshima University, Japan)