The hard tick, Haemaphysalis longicornis (Ixodida: Ixodidae) is one of the vectors of severe fever with thrombocytopenia syndrome virus (SFTSV) causing in humans, however little consideration was given to the biological control of the pest. Therefore, this study provides a screening method to select entomopathogenic fungi, having high virulence against H. longicornis. The virulence test was conducted by dipping method with a conidial suspension (1×107 conidia/ml). As a result, cadavers of H. longicornis infected with entomopathogenic fungi were obtained by this method. Based on this results, we selected several isolates having high virulence to H. longicornis. We suggest that, selected isolates in this study can be used for the control of H. longicornis.

The hard tick, Haemaphysalis longicornis (Ixodida: Ixodidae) is one of the vectors of severe fever with thrombocytopeniasyndrome virus (SFTSV) causing in humans, however little consideration was given to the biological control of the pest.We evaluated three bioassay methods, such as spray, dipping and dropping. As a result, the dipping method was themost efficient way to measure the fungal virulence. Finally, we constructed a Haemaphysalis longicornis pathogenic fungallibrary to further facilitate the resources to be used as potential biological control agents. A conidial suspension of 1×107conidia/ml was exposed to the ticks by dipping. We confirmed that some isolates were highly virulent to the ticks. Thisresults indicated that some fungi could be used to effectively control the Haemaphysalis longicornis.

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.