Haemaphysalis longicornis (H. longicornis) is the most dominant species that accounts for more than 90% of domestic ticks in Korea. It is also a main mediator and propagator of Severe Fever with Thrombocytopenia Syndrome virus. In order to efficient control of H. longicornis, it is essential to obtain the genetic information of pesticide target genes and determine the mutations putatively involved in insecticide resistance. Acetylcholinesterase (Ace) is a target gene of organophosphorus and carbamate insecticides, but no sequence information of Ace is available to date in H. longicornis. Therefore, in this study, we analyzed the transcriptome of H. longicornis and obtained the sequence information of three Ace genes (HlAce1, HlAce2 and HlAce3). Based on the comparison analysis with sequences of multiple ace genes from other hard ticks, mites and insects, three HlAces were grouped in Acari Ace1, Ace2 and Ace3 clade, respectively. Among three HlAces, HlAce1 seems to be major enzyme because of its conserved amino acids involved in catalytic function.

The longhorned tick, Haemaphysalis longicornis, is one of the vectors of severe fever with thrombocytopenia syndrome virus (SFTSV) in human. The use of pyrethroid insecticides induced pest resistance and environmental residual toxicity. Here in this work, our interest was given to the selection of highly virulent fungi against longhorned tick. A total of 101 fungal pathogens were assayed by dipping the nymph stage of ticks into a conidial suspension. Interestingly of the several species, one species showed high virulence and mycosis were observed in 7-15 days. Highly virulent strains were selected, and semi-field experiments were conducted. As a result, the control efficacy of the isolate was over 80% at 30 days of treatment. This work suggests that entomopathogenic fungi could be used to effectively control longhorend ticks.

Acaricidal activities of 63 commecial pesticides against the longhorned tick, H. logicornis were investigated. Twenty-two pesticides (4 cabamates, 5 organophophates, 9 pyrethroids, 1 octopamine receptor agonists, 1 diamides, and 1 unknown) against adults H. logicornis showed >80% mortalities after 72 h of treatment. The residual effect of 22 pesticides was investigated at 1, 2, 3, 7, and 10 day intervals on grass for H. logicornis adults and nymphs. Eight and twenty pesticides in H. logicornis adults and nymphs showed >80% acaricidal activity at 3 days after the treatment, respectively. 7 days after the treatment, H. logicornis adults showed <60% acaricidal activity in all pesticides, but 7 pesticides (benfuracarb, gamma cyhalothrin, cypermethrin, lammda cyhalothrin, fenvalerate, cyantraniliprole, fluxametamide) showed >80% acaricidal activity in nymphs. all pesticides exhibited <60% acaricidal activity after 10 days of treatment both adults and mymphs, except benfuracarb showed 96.7% mortality in H. logicornis nymph.

Although understanding dispersal ecology is important of Haemaphysalis longiconirs (Acari: Ixodidae) to control severe fever with thrombocytopenia syndrome, the small size of H. longicornis makes it challenging to study their behaviors. We examined the potential of fluorescent marking system (FMS) paired with an ultraviolet laser, an effective insect detection method, in tracking of H. longicornis. When we assessed potential effects of FMS on the survivorship and behavioral parameters including horizontal movement, vertical climbing height, and response to CO₂ of all development stages (larva, nymph, and adult) of H. longicornis, it did not induce any adverse effects on the hard ticks. Furthermore, we measured the detectable distance and detection rate of larvae, nymphs and adults to evaluate the detection efficacy of FMS. Using an ultraviolet laser, operators could detect the hard ticks from a distance ranging from 12 m (larva) to 29 m (adult). When the detection rate was measured in a grassland both during day and night, it was higher at night compared to day and increased with the body size. The detection rate was over 90% for all stages of the hard ticks at night, whereas larvae and nymphs showed that of 23% and 34% respectively during the day. However, the detection rate of adults was higher compared to other development stages regardless of time of experiment. Our results indicate that FMS can serve as a promising tool to track the hard ticks by providing reliable detection efficacy with no adverse effects on the marked individuals.

Longhorned tick, Haemaphysalis longicornis (Ixodida: Ixodidae) is one of the vectors of severe fever with thrombocytopenia syndrome virus (SFTSV) in human. The tick occurs in mostly grass fields, and the use of pyrethroid insecticides induced pest resistance and environmental residual toxicity. Particularly the use of chemicals near residential areas where persons live become a big issue, so environmentally safe control agents need to be considered. Here in this work, our interest was given to the selection of highly virulent fungi against H. longicornis. A total of 101 fungal pathogens collected from mountainous areas were assayed by a dipping the nymph stage of ticks into a conidial suspension (1×107conidia/ml). Interestingly among several species, one species showed high virulence and mycosis were observed in 7-15 days. Most of the selected isolates produced a large amount of conidia in Italian millet, rice and millet with thermotolerance at 40-45°C. Based on these results, we selected a couple of isolates with high virulence against H. longicornis and they could be used for the control of the ticks after the set-up of a practical application strategy in fields by optimizing fungal colonization in soil and phyllosphere.

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.

Haemaphysalis longicornis (Acari: Ixodidae) is a medically important arthropod vector of tick-borne diseases, especially the severe fever with thrombocytopenia syndrome (SFTS). To establish a management strategy against tick-borne diseases, it is fundamental to understand the basic biology of vector species. Therefore under laboratory conditions, we evaluated the horizontal movement of adult and nymph stages of H. longicornis to better understand their dispersal behaviors. In the experiments, field populations were subjected to video recording of the horizontal movement of the ticks in a petri-dish arena for 3 hours. Our results indicate that nymphs showed a significantly higher mobility than adults, whereas there was no significant difference in angular velocity. Nymphs moved 3.74 ± 0.26 m at an angular velocity (AV) of 1.60 ± 0.07 deg/s. and adults moved 2.91 ± 0.26 m at an AV of 1.61 ± 0.14 deg/s.

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.

The activities of three class, five acaricides (Permethrin, Deltamethrin, Fenitrothion, Chlorpyrifos, Imidacloprid) againstHaemaphysalis longicornis nymph, a major vector for Severe Fever with Thrombo cytopenia Syndrome (SFTS) and Lymedisease. The ticks distribute at vast open fields and various kind of habitats. Five acaricides were applied to ticks collectedform four areas(Gapyeong, Paju, Chuncheon, Wonju) to confirm the effect of acaricide. This areas was the area whereSFTS disease occurred and a large nember of ticks were collected. As a result, All areas is the most acaricidal effectto Deltamethrin. And two areas(Gapyeong, Paju) has the lowest acaricidal effect to Imidacloprid and two area(Chuncheon,Wonju) has the lowest acaricidal effect to Permethrin. Futher study will need to compare the acaricidal efficacy of theremaining seven province.

The Ixodid ticks has been suspected to be the vector of severe fever with thrombocytopenia syndrome virus (SFTSV) in Korea. In order to confirm transovarial transmission of Ixodid ticks, a total of blood fed 284 ticks were collected from six host animals. They were identified as 3 genera and 4 species, Haemaphysalis longicornis (97.2%), H. flava (0.7%), Ixodes nipponensis (1.8%) and Amblyomma testudinarium (0.4%). Among them, 192 female adults hatched their eggs successfully. Our results showed a high prevalence of SFTSV among collected ticks (3.1%, 6/192 ticks), and we detected SFTSV from their larvae. This result suggests that the parent generation of H. longicornis can transmit SFTSV transovarially into their F1 progeny and H. longicornis might be one of major vectors of SFTSV in Korea.

Haemaphysalis longicornis is a major vector for Severe fever with thrombocytopenia syndrome (SFTS) throughout Republic of Korea (ROK). The ticks distribute at vast open fields and various kind of habitate. Control of the ticks with pesticides is almost impossible and personal protection such as use of commercial repellents is very critical for control. However occasional toxic effects of the commercial repellents have been reported that include central nervous system depression and urticaria. In our studies, repellency of three plant essential oils was assessed and was compared with two commercial repellents. Lavendual angustifolia demonstrated the highest repellency and higher repellency than DEET when was individually tested, but binary mixture of Cinnamomum zeylinicam and Juniperus verginiana showed significantly higher repellency with synergic effects than mixture of Lavendula angustifolia and Juniperus verginiana. Further study on repellency of the C. zelinicam in field need.

Previous laboratory repellent assessment with three plant essential oils against Haemaphysalis longicornis, a major vector for Severe fever with thrombocytopenia syndrome, revealed high repellency of Lavendual angustifolia and high synergic effects of Cinnamomum zeylinicam and Juniperus verginiana. Based on the previous laboratory repellent assessment field studies on repellency of the three plant oils were carried out with formulations such as cream type and spray type. 10% spray type of Cinnamomum zeylinicam demonstrated > 70% of repellency against H. longicornis. 10% spray type of Lavendual angustifolia showed > 80% of repellency. 10% spray type of binary mixture containing Cinnnamomum zeylinicam and Juniperus verginiana showed > 98% repellency against H. longicornis. Further studies on repellency of cream type of the plant oils might be carried out and compared with two commercial repellents.

Ixodid ticks are notorious as the vector of severe fever with thrombocytopenia syndrome virus (SFTSV) in Korea. To confirm the transovarial transmission of SFTSV, we collected blood-fed adult ticks from animals in SFTS case reported area of Jeju island in 2015. A total of 476 ticks were collected from four host animal species, horse (81.3%), dog (9.0%), roe deer (7.1%) and wild boar (2.5%). They were identified as 1 genus and 2 species, Haemaphysalis longicornis (87.8%) and H. flava (12.2%). Among them, 131 adults hatched their eggs successfully. The body weight of females was highly correlated with the number of egg masses laid (P < 0.05). We confirmed that one adult H. flava was infected with SFTSV and its infection rate was 0.77%. The eggs of the SFTSV-infected adults also were infected with SFTSV and its transovarial transmission was confirmed. This result suggests that the parent generation of H. flava could transmit SFTSV transovarially into their F1 progeny and H. flava might be one of major vectors of SFTSV in Korea.

During tick infestation, the tick secretes bioactive substances that modify the host’s physiological and immunological reactions. The study of tick saliva is important to understand tick biology as tick saliva plays a special physiological role in pathogen transmission. The average salivary protein concentration was found to be 0.169 μg/μl/tick and saliva secretion decreased with increased time of tick detachment from the host. Saliva secretion volume increased to 3.56 μl in the group of ticks with a body weight between 301–350 mg as compared to higher and lower body weight groups. On-chip-electrophoresis results show 13 distinct bands ranging from 9.9 to 294 kDa. For salivary protein LC-MS/MS was performed. A total of 135 tick salivary proteins were identified of which 30 proteins were found exclusively in fully engorged nymph saliva, 74 in fully engorged adult females, and 31 were detected in both stages. Results of this may help researchers to identify tick proteins as potential candidates for further studies aimed to develop novel tick control strategies to affect both the ticks and the pathogens transmitted by them.

We investigated the distribution of ixodid ticks in severe fever with thrombocytopenia syndrome (SFTS) case reported areas using flagging method from May to November 2014 in Korea. We collected 13,471 ixodid ticks with 2 genera and 3 species. Among them, Genus Haemaphysalis and Ixodes accounted for 99.9% (13,455) and 0.1% (16), respectively. Genus Haemaphysalis consisted of 2 species, H. longicornis (96.2%) and H. flava (3.7%). I. nipponensis was only collected in Genus Ixodes. H. longicornis was the dominant species (over 90%) in most study areas. Ixodid ticks were evenly distributed at various environmental sites, such as residences, hills, grassfields, fields, graves, copses, other sites. The further study on SFTSV infection in ixodid ticks will be needed.

Haemaphysalis longicornis (Hl) as members of the ixodid tick inhabits lots of grass thicket of field and mountain. Ticks are blood-feeding ectoparasites that can mediate a variety of diseases to human and animals, causing Lyme disease, Rocky Mountain spotted fever, and human monocytic ehrlichiosis. Particularly, ticks can trigger an inflammatory response representing symptoms about swelling and itching in human. The aim of this study is to investigate the effect of H. longicornis extract (HlE) on production of inflammatory cytokines and their mRNA in human monocytic THP-1 cells. In a time- and dose-dependent manner, human monocytic THP-1 cells was treated with HlE. Supernatants were analyzed for the production of cytokines using enzyme-linked immunosorbent assay (ELISA). mRNA level in the culture cells was measured by a reverse transcriptase-polymerase chain reaction (RT-PCR). As a result of this study, HlE significantly induced secretion of IL-6, IL-8, and MCP-1 in THP-1 cells. These results suggest that HlE increase the release of proteins and mRNAs level of inflammatory cytokines in THP-1 cells. HlE may play a role in contributing to inflammatory diseases through stimulation of immune cells. Further research of H. longicornis is needed to better understand the elucidation of the pathogenic mechanism.

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.

Antimicrobial peptides represent an essential alternative first line of defense. Naturally occurring molecules associated with the innate immune system in disease-bearing vectors such as mosquito, tick could be the target for searching more potent and effective agents to combat against the pathogens resistant to conventionally used antibiotics. Recently, we explored expression of a defensinlike peptide, longicin from the hard tick Haemaphysalis longicornis. Longicin and one of its synthetic partial analog (P4) displayed antimicrobial/fungicidal/parasiticidal activity and, therefore, proposed to be a chemotherapeutic compounds against tick-borne disease organisms. Structural characterization of antimicrobial peptides is very important to understand the peptide activity. In addition, harmful side effects such as lysis of red blood cells or cytotoxicity towards mammalian host cells commonly associated with antimicrobial peptides as potential therapeutic agent should also be elucidated. In this study, we analyzed some structural features using bioinformatics tool, CD Spectroscopy, and also determined cytolytic activity of P4 peptide. According to the chemicophysical characteristics, the P4 is suggested to be a cationic peptide with hydrophobic and amphipathic character. The predicted secondary structure indicated the existence of β-sheet which was also observed in modelled tertiary structure. CD spectroscopy results also revealed the existence of a β-sheet and changes of helical content in the presence of membrane-mimic condition. These structural observations on P4 suggest that the antimicrobial activity could be due to the well developed β-sheet. In addition, sequence homology search showed that antimicrobial molecule identified in other ticks and in organisms have the P4 analogous domain at their C-terminal, which indicates P4 as a conserved antimicrobial domain. The peptide P4 also showed less cytolytic activity against various cell lines or erythrocytes of various species. The data presented here strongly suggests that the peptide P4 could be developed as future therapeutic agent against tick-borne microorganisms.

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.