Phenology and habitat associations of the invasive Asian longhorned tick from Ohio, USA.

Geographically expanding and invading ticks are a global concern. The Asian longhorned tick (ALT, Haemaphysalis longicornis) was introduced to the mid-Atlantic US between 2010 and 2017 and recently invaded Ohio, an inland state. To date, ALTs in the US have been associated with livestock exsanguination and transmission of the agent of bovine theileriosis. To inform management, studies describing tick ecology and epidemiology of associated disease agents are critical. In this study, we described phenology, habitat and host associations, and tested for agents of medical and veterinary concern at the site of the first known established ALT population in Ohio, where pesticide treatment was applied in early fall 2021. In spring-fall 2022, we sampled wildlife (small mammals) and collected ticks from forest, edge, and grassland habitats. We also opportunistically sampled harvested white-tailed deer at nearby processing stations and fresh wildlife carcasses found near roads. Field-collected ALTs were tested for five agents using real-time PCR. We found that ALT nymphs emerged in June, followed by adults, and concluded with larvae in the fall. ALTs were detected in all habitats but not in wildlife. We also found a 4.88% (2/41) prevalence of Anaplasma phagocytophilum across ALT adults and nymphs. Host and habitat associations were similar to other studies in the eastern United States, but two potential differences in phenology were identified. Whether ALTs will acquire more endemic disease agents requires further investigations. Our findings provide the first evidence regarding ALT life history from the Midwest region of the United States and can inform exposure risk and guide integrated management.

The contact toxicity and toxic mechanism of essential oils from Pimenta racemosa and Eugenia caryophyllata against Haemaphysalis longicornis (Acari: Ixodidae).

Haemaphysalis longicornis, which is widely distributed in China, can transmit various tick-borne diseases such as severe fever with thrombocytopenia syndrome, babesiosis, rickettsia disease and so on, and do great harm to human health and the development of animal husbandry. Chemical acaricides are the most traditional tick control method, but because of its many shortcomings, there is an urgent need to find a substitute with high efficiency, environmental protection and low toxicity. It has been found that some plant essential oils (EOs) have good insecticidal activity and environmental safety. In this study, the components of EOs from Pimenta racemosa and Eugenia caryophyllata were analyzed by gas chromatography-mass spectrometry (GC-MS), and their potential for application in the control of Haemaphysalis longicornis were studied. Gas chromatography-mass spectrometry analysis showed that the main components of P. racemosa EO were eugenol (64.07%), those of E. caryophyllata EO were Hexadecanoic acid, 2-methylpropyl ester (51.84%) and eugenol (39.76%). Larval packet test showed that the EOs of P. racemosa and E. caryophyllata had significant acaricidal activity against unfed larvae of H. longicornis, with LC50 values of 1.20 mg/mL and 0.47 mg/mL and LC90 values of 8.76 mg/mL and 2.91 mg/mL, respectively. The P. racemosa EO, E. caryophyllata EO and eugenol showed significant acaricidal activity against unfed nymph H. longicornis, with LC50 values of 1.65 mg/mL, 2.29 mg/mL and 0.93 mg/mL and LC90 values of 5.03 mg/mL, 11.01 mg/mL and 4.77 mg/mL, respectively. The P. racemosa EO, E. caryophyllata EO and eugenol showed significant acaricidal activity against unfed adults H. longicornis, with LC50 values of 0.51 mg/mL, 2.57 mg/mL and 1.83 mg/mL and LC90 values of 2.44 mg/mL, 11.44 mg/mL and 2.54 mg/mL, respectively. Enzyme assays revealed that the E. caryophyllata EO and eugenol significantly inhibited the activity of carboxylesterase (CarE), eugenol significantly inhibited the activity of catalase (CAT), and two EOs and eugenol had no significant effect on acetylcholinesterase (AchE) (p < 0.05). The above results suggest that the essential oils from P. racemosa and E. caryophyllata have great potential for use as alternatives to synthetic acaricides for tick control.

Distribution characteristics and influencing factors of Haemaphysalis longicornis around goat sheds in Jinan city, East China.

As one of the most important disease vectors worldwide, ticks can transmit a number of pathogenic organisms to humans and domestic animals and cause a variety of important natural focal diseases and zoonoses. Domestic livestock play a vital role in the dispersal of ticks from the field environment to the human settlement, contributing to the prevalence of tick-borne diseases. Identification of the tick control region could contribute a vital role in strategic planning and cost-effective tick control measures. However, little is known about the spatial distribution characteristics of ticks around livestock sheds, which will lead to abusage and overuse of insecticides. Therefore, this study aimed to explore spatial distribution characteristics and correlation factors of ticks around goat sheds. A total of 3898 ticks were collected from eight goat sheds from April to June in Jinan city. All the sampled ticks belonged to the same species, namely Haemaphysalis longicornis, and 88.8% of them were nymphs. A significant positive correlation was noted between free-living ticks and parasitic ticks (r = 0.411, P < 0.001). However, there was a significant negative correlation between number of free-living ticks and distance from the goat sheds (r = -0.622, P < 0.001). Within 20 m from the goat sheds, 2211 ticks were collected respectively, representing 56.7% of the total free-living ticks. At a distance of 30 m, 57.6% decline in the tick density was found with a significant difference (q = 5.534, P < 0.001). In conclusion, focusing control efforts near the goat sheds should be recommend for tick prevention and control.

Projecting spatiotemporal dynamics of severe fever with thrombocytopenia syndrome in the mainland of China.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with increasing incidence and geographic extent. The extent to which global climate change affects the incidence of SFTS disease remains obscure. We use an integrated multi-model, multi-scenario framework to assess the impact of global climate change on SFTS disease in China. The spatial distribution of habitat suitability for the tick Haemaphysalis longicornis was predicted by applying a boosted regression tree model under four alternative climate change scenarios (RCP2.6, RCP4.5, RCP6.0, and RCP8.5) for the periods 2030-2039, 2050-2059, and 2080-2089. We incorporate the SFTS cases in the mainland of China from 2010 to 2019 with environmental variables and the projected distribution of H. longicornis into a generalized additive model to explore the current and future spatiotemporal dynamics of SFTS. Our results demonstrate an expanded geographic distribution of H. longicornis toward Northern and Northwestern China, showing a more pronounced change under the RCP8.5 scenario. In contrast, the environmental suitability of H. longicornis is predicted to be reduced in Central and Eastern China. The SFTS incidence in three time periods (2030-2039, 2050-2059, and 2080-2089) is predicted to be increased as compared to the 2010s in the context of various RCPs. A heterogeneous trend across provinces, however, was observed, when an increased incidence in Liaoning and Shandong provinces, while decreased incidence in Henan province is predicted. Notably, we predict possible outbreaks in Xinjiang and Yunnan in the future, where only sporadic cases have been reported previously. These findings highlight the need for tick control and population awareness of SFTS in endemic regions, and enhanced monitoring in potential risk areas.

Epidemiological study of ticks harbouring Aeromonas hydrophila in areas endemic and non-endemic to Japanese-spotted fever.

OBJECTIVES: Aeromonas spp. often cause life-threatening diseases, including necrotizing fasciitis, which may lead to septic shock and ultimately death. Aeromonas infections are believed to be transmitted via minor wounds or the consumption of fresh fish. However, after the detection of Aeromonas hydrophila in ticks in areas endemic to Japanese-spotted fever (JSF), a novel transmission route of A. hydrophila (i.e., via tick bites) has been proposed. We investigated the prevalence of A. hydrophila in ticks in areas endemic and not endemic to JSF in the Mie Prefecture, Japan. METHODS: We collected ticks from endemic and nonendemic areas in summer and winter and assessed them for presence of A. hydrophila using polymerase chain reaction. RESULTS: Six A. hydrophila isolates were obtained from 95 ticks in endemic areas, whereas one A. hydrophila isolate was obtained from 142 ticks in non-endemic areas, in summer. All ticks that harboured A. hydrophila were Haemaphysalis longicornis (H.L); these ticks were almost at the larval stage and also carried Rickettsia spp. in the endemic area. In contrast, 51 and 41 ticks in the endemic and non-endemic areas were captured in winter, respectively; A. hydrophila was not detected in these. CONCLUSIONS: This study revealed the prevalence of tick-borne A. hydrophila. Therefore, the risk of transmission of A. hydrophila via a tick bite should be considered in the following conditions: areas abundant in H. L. harbouring Rickettsia spp., in areas endemic for JSF, presence of ticks in the larval stage and during the summer season.

Detection of viral genes in Metarhizium anisopliae JEF-290-infected longhorned tick, Haemaphysalis longicornis using transcriptome analysis.

Ticks are carriers of viruses that can cause disease in humans and animals. The longhorned ticks (Haemaphysalis longicornis; LHT), for example, mediates the severe fever with thrombocytopenia syndrome virus (SFTSV) in humans, and the population of ticks is growing due to increases in temperature caused by climate change. As ticks carry primarily RNA viruses, there is a need to study the possibility of detecting new viruses through tick virome analysis. In this study, viruses in LHTs collected in Korea were investigated and virus titers in ticks exposed to the entomopathogenic fungus Metarhizium anisopliae JEF-290 were analyzed. Total RNA was extracted from the collected ticks, and short reads were obtained from Illumina sequencing. A total of 50,024 contigs with coding capacity were obtained after de novo assembly of the reads in the metaSPAdes genome assembler. A series of BLAST-based analyses using the GenBank database was performed to screen viral contigs, and three putative virus species were identified from the tick meta-transcriptome, such as Alongshan virus (ALSV), Denso virus and Taggert virus. Measurements of virus-expression levels of infected and non-infected LHTs failed to detect substantial differences in expression levels. However, we suggest that LHT can spread not only SFTSV, but also various other disease-causing viruses over large areas of the world. From the phylogenetic analysis of ALSV glycoproteins, genetic differences in the ALSV could be due to host differences as well as regional differences. Viral metagenome analysis can be used as a tool to manage future outbreaks of disease caused by ticks by detecting unknown viruses.

The genome-wide characterization and associated cold-tolerance function of the superoxide dismutase in the cold response of the tick Haemaphysalis longicornis.

Accumulating evidence suggests that superoxide dismutase (SOD) is the first line of antioxidant defense in organisms and plays an important role in scavenging reactive oxygen species produced during environmental stress. However, limited information is available regarding the response of SOD genes to cold stress in ticks. Therefore, in the present study, SOD genes were cloned and identified from the genome of Haemaphysalis longicornis, and the function of SOD during the cold response was further explored. Seven SOD genes were characterized: HlCCS1, HlCCS2, HlMSD, HlCSD1, HlCSD2, HlCSD3, and HlCSD4. Bioinformatics analysis showed that HlCCS1 and HlCCS2 are copper chaperones of SODs. HlCSD1-HlCSD4 belong to the Cu/Zn SOD, whereas HlMSD belongs to the Mn SOD gene family. Fluorescence quantitative PCR showed that the expression of HlCCS2, HlMSD, and HlCSD1-3 was upregulated, whereas HlCCS1 and HlCSD4 were downregulated during the cold response of H. longicornis. Western blotting confirmed changes in the relative expression of HlCSD3 and HlMSD in H. longicornis after cold treatment. Mortality of H. longicornis increased significantly after dsRNA injection of HlCCS2, HlMSD, HlCSD1, and HlCSD3. The above results show that SODs have different regulatory functions during the cold response in H. longicornis, and there might be an interaction between treatment temperature and duration. Furthermore, the results lay a foundation for subsequent research on the molecular mechanism of cold tolerance in H. longicornis and shed light on the population distribution and diffusion limit of ticks.

The new Haemaphysalis longicornis genome provides insights into its requisite biological traits.

Ticks are a large group of blood-feeding arthropods that transmit multiple human and animal pathogens and are hence of importance to public health. The tick Haemaphysalis longicornis is associated with the transmission of multiple human pathogens in Asia, and recently found invading to the United States. Here, we report the sequencing, assembly and annotation of the 3.16 gigabase genome of this species, which is larger than the previous assembled one. The present Haemaphysalis longicornis genome was characterized by 6519 scaffolds, 24,189 protein-coding genes and a high proportion of simple sequence repeats (54.72%). By genomic assembly and comparative genomic analysis, we characterized the key genes that play essential roles in iron metabolism, detoxification, and freeze tolerance of H. longicornis. Furthermore, a total of 79 endogenous viral elements were identified within the genome, which might have had a considerable impact on its evolution. Decoding the H. longicornis genome not only provides insight into the genetic underpinnings of specific biological processes but also offers the basis for the subsequent integrated control of ticks and tick-borne diseases.

Population dynamics of hard ticks (Acari: Ixodidae) and their harboring rates of Severe Fever with Thrombocytopenia Syndrome (SFTS) virus in four landscapes of Gyeonggi Province, South Korea.

Population dynamics of hard ticks and their harboring rates of fatal severe fever with thrombocytopenia syndrome (SFTS) were monitored from 2021 to 2022 in Gyeonggi-do, South Korea. Hard ticks were surveyed monthly using CO2-bait traps in four vegetation types, including grassland, grave, mountain trail, and shrub. From the 2-year monitoring, totals of 5,737 and 14,298 hard ticks were collected in 2021 and 2022, respectively, all of which belonged to the genus Haemaphysalis. Of these collected ticks, 97.9 and 98.3% of adults and nymphs were identified as Haemaphysalis longicornis. Generally, density peaks of H. longicornis nymphs and adults were observed from April to May and from June to July, respectively. For Haemaphysalis flava, adults showed density peaks in September, whereas no obvious seasonal patterns were observed for nymphs. The density peak of Haemaphysalis larvae was observed in August and September, followed by a density peak of adults. There was a large variation in the number of hard ticks collected among the four vegetation types, yielding no significant difference among them over the 2-year monitoring. Half of the collected ticks from each vegetation type were pooled into groups by species and developmental stage and subjected to analysis of SFTS virus harboring rates, which yielded no SFTS positive pool detected over the 2-year monitoring.

Characterization of bacterial communities in ticks parasitizing cattle in a touristic location in southwestern China.

The purpose of this study was to investigate tick species around Mount Fanjing and analyze bacterial communities in two species - Rhipicephalus microplus and Haemaphysalis longicornis - parasitizing cattle in Tongren, Guizhou province, Southwest China, using high-throughput sequencing methods. In April 2019, ticks were collected from five sites in Jiangkou County, Yinjiang County, and Songtao County. In total, 296 ticks were collected, comprising two genera and three species: H. longicornis, Haemaphysalis flava, and R. microplus. Rhipicephalus microplus was the most representative species (57.4%) within the collected group, being the dominant species in Tongren City, followed by H. longicornis (39.5%) and H. flava (3.0%). Beta-diversity analysis revealed differences in bacterial community composition among the tick species. The bacterial community structure of R. microplus collected in the three counties was highly similar. Chlorella and Bacillus were highly abundant in H. longicornis. Rickettsia was detected at high relative abundance in R. microplus but in low relative abundance in H. longicornis, suggesting that Rickettsia is more associated with R. microplus than with H. longicornis. More in-depth investigations are needed to determine the pathogenic risk of Rickettsia and its relationship with the host. This is the first survey on tick-borne bacterial communities in this area, which is of great significance for the prevention and control of tick-borne diseases locally.

Effects of temperature on cathepsin B, cathepsin D and acid phosphatase during embryo development of the hard tick Haemaphysalis longicornis.

The effects of temperature on the expression patterns and enzyme activity of cathepsin B (HlCatB), cathepsin D (HlCatD) and acid phosphatase (HlACP) during the embryo development of Haemaphysalis longicornis (bisexual population) were investigated in this study. Eggs were exposed to 20 °C (low temperature), 26 °C (normal temperature), and 30 °C (high temperature) immediately after laying, and collected on odd days of embryo development to measure HlCatB, HlCatD and HlACP gene expression using quantitative real-time PCR, as well as three enzyme activities using spectrophotometry. Then the associations between mRNA expression levels of three enzymes and their enzyme activities were assessed. Compared with normal temperature, the mRNA expression peaks of HlCatB were higher and appeared later at low and high temperatures and the activity of HlCatB increased on most days of embryonic development at high temperature. As for HlCatD, the expression peak appeared later at low temperature, but earlier at high temperature. The activity peaks of HlCatD were lower and appeared earlier at low and high temperatures. As for HlACP, the expression peak was higher and appeared later at low temperature, whereas it formed no prominent peak at high temperature. The activity peak of HlACP was higher at low temperature, but lower at high temperature. The linear regression analysis showed that activities of three enzymes were associated with their mRNA expression levels (P < 0.05). Three enzymes are involved in the embryo adaptation to temperature stress. Moreover, the mRNA expression level may be another factor affecting its enzyme activity.

Severe fever with thrombocytopenia syndrome virus from ticks: a molecular epidemiological study of a patient in the Republic of Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease caused by Dabie bandavirus, commonly called SFTS virus (SFTSV). In the Republic of Korea (ROK), 1,504 cases of SFTS have been reported since the first human case was identified in 2013 until 2021. However, no case exists to provide molecular evidence between questing tick and patients with confirmed SFTS in the same living environment. In this study, we investigated the presence of ticks near the area of a patient infected with SFTSV. Ticks were collected by flagging and dry ice-baited traps at three spots in the vegetation around the patients' residence in Chuncheon City, Gangwon Province (ROK). Among the tick samples collected, the presence of SFTSV was genetically determined using reverse transcription PCR, followed by the phylogenetic analysis of the tick virus sequences and SFTSV found in the patient. In total 1,212 Haemaphysalis longicornis ticks were collected, and SFTSV was detected at a minimum infection rate of 5.3% (33 pools/618 tested ticks). The sequences of SFTSV in ticks were 99.6-100% identical with the patient's SFTSV in the M segment. To the best of our knowledge, this study is the first case to provide a molecular correlation between SFTSV in questing ticks collected from residence and patient with SFTS in the ROK. The present results provide useful information for the epidemiological investigation of patients with SFTS using ticks as vectors of SFTSV.

Mechanism of the toxic effects of tetracycline on blood meal digestion in Haemaphysalis longicornis.

The extensive utilization of antibiotics in the field of animal husbandry gives rise to various concerns pertaining to the environment and human health. Here, we demonstrate that the administration of tetracycline impedes blood meal digestion in the tick Haemaphysalis longicornis. Tissue sectioning, 16S rRNA high-throughput sequencing, and transcriptome sequencing of the midgut were employed to elucidate the mechanism underlying tetracycline toxicity. The treatment group consisted of engorged female ticks that were subjected to tetracycline microinjections (75 µg per tick), whereas the control group received sterile water injections. On days 2 and 4 following the injections, the tick body weight changes were assessed and the midguts were dissected and processed. Change in tick body weight in tetracycline-treated group was less than in the control group. In tetracycline-treated ticks, midgut epithelial cells were loosely connected and blood meal digestion was impaired compared to the control group. There was no significant change in midgut bacterial diversity after tetracycline treatment. On day 2 following treatment, the relative abundance of Escherichia-Shigella was significantly decreased, whereas the relative abundance of Allorhizobium was significantly increased compared to the control group. On day 4 following treatment, the relative abundance of Escherichia-Shigella, Allorhizobium, Ochrobactrum, and Acidibacter decreased significantly, whereas the relative abundance of Paraburkholderia and Pelomonas increased significantly. Tetracycline treatment also affected midgut gene expression, producing a cumulative effect wherein the differentially expressed genes (DEGs) were mostly down-regulated. KEGG enrichment pathway analysis revealed that on day 2 the up-regulated DEGs were significantly enriched in 21 pathways, including apoptosis and phagosome. Comparatively, the down-regulated DEGs were significantly enriched in 26 pathways, including N-glycan biosynthesis, lysosome, and autophagy. In contrast, on day 4 the up-regulated DEGs were significantly enriched in 10 pathways including aminoacyl-tRNA biosynthesis, ribosome biogenesis, RNA transport, and DNA replication, whereas the down-regulated differential genes were significantly enriched in 11 pathways including lysosome, peroxisome, N-glycan biosynthesis, and fatty acid synthesis. This indicates that tetracycline injection inhibited blood meal digestion by affecting midgut digestive cells, gut flora diversity, and gene expression. These findings could contribute to tick control by inhibiting blood meal digestion.

Transovarial Transmission of Heartland Virus by Invasive Asian Longhorned Ticks under Laboratory Conditions.

We demonstrated experimental acquisition and transmission of Heartland bandavirus by Haemaphysalis longicornis ticks. Virus was detected in tick salivary gland and midgut tissues. A total of 80% of mice exposed to 1 infected tick seroconverted, suggesting horizontal transmission. H. longicornis ticks can transmit the virus in the transovarial mode.

Hedgehogs as Amplifying Hosts of Severe Fever with Thrombocytopenia Syndrome Virus, China.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tickborne bandavirus mainly transmitted by Haemaphysalis longicornis ticks in East Asia, mostly in rural areas. As of April 2022, the amplifying host involved in the natural transmission of SFTSV remained unidentified. Our epidemiologic field survey conducted in endemic areas in China showed that hedgehogs were widely distributed, had heavy tick infestations, and had high SFTSV seroprevalence and RNA prevalence. After experimental infection of Erinaceus amurensis and Atelerix albiventris hedgehogs with SFTSV, we detected robust but transitory viremias that lasted for 9-11 days. We completed the SFTSV transmission cycle between hedgehogs and nymph and adult H. longicornis ticks under laboratory conditions with 100% efficiency. Furthermore, naive H. longicornis ticks could be infected by SFTSV-positive ticks co-feeding on naive hedgehogs; we confirmed transstadial transmission of SFTSV. Our study suggests that the hedgehogs are a notable wildlife amplifying host of SFTSV in China.

Molecular detection of haemophilic pathogens reveals evidence of Candidatus Mycoplasma haemobos in dogs and parasitic ticks in central China.

BACKGROUND: In addition to Mycoplasma haemocanis and Candidatus Mycoplasma haematoparvum, a few hemoplasma species that mainly infect other livestock have been detected in dogs. 'Candidatus Mycoplasma haemobos' (Ca. M. haemobos) has been found in a variety of animals in China. The present study was aimed to investigate the occurrence of 'Ca. M. haemobos' infections in dogs and ticks collected from the Henan province, China. RESULTS: Overall, 55 dog blood samples and 378 ticks on skins were collected from anemic and healthy dogs, and these samples were subjected to PCR, sequence analysis, and identification. The results showed that Haemaphysalis longicornis (266) and Rhipicephalus (Boophilus) microplus (112) were the only two parasitic ticks on dogs. Molecular detection revealed that 163 M. haemocanis, 88 'Ca. M. haemobos' and 32 Anaplasma platys positive amplicons could be amplified from dogs, H. longicornis and R. (B.) microplus. In addition, co-infections (M. haemocanis + A. platys and 'Ca. M. haemobos'+ A. platys) could be also detected. CONCLUSIONS: To the best of our knowledge, this is the first molecular evidence of 'Ca. M. haemobos' natural infection in dogs and tick species identified as H. longicornis and R. (B.) microplus from China.

Evaluation of anti-tick efficiency in rabbits induced by DNA vaccines encoding Haemaphysalis longicornis lipocalin homologue.

Haemaphysalis longicornis is an obligate haematophagous ectoparasite, transmitting a variety of pathogens, which brings great damage to human health and animal husbandry development. Lipocalins (LIP) are a family of proteins that transport small hydrophobic molecules and also involve in immune regulation, such as the regulation of cell homeostasis, inhibiting the host's inflammatory response and resisting the contractile responses in host blood vessels. Therefore, it is one of the candidate antigens for vaccines. Based on previous studies, we constructed the recombinant plasmid pcDNA3.1-HlLIP of LIP homologue from H. longicornis (HlLIP). ELISA results showed that rabbits immunized with pcDNA3.1-HlLIP produced higher anti-rHlLIP antibody levels compared with the pcDNA3.1 group, indicating that pcDNA3.1-HlLIP induced the humoral immune response of host. Adult H. longicornis infestation trial in rabbits demonstrated that the engorgement weight, oviposition and hatchability of H. longicornis fed on rabbits immunized with pcDNA3.1-HlLIP decreased by 7.07%, 14.30% and 11.70% respectively, compared with that of the pcDNA3.1 group. In brief, DNA vaccine of pcDNA3.1-HlLIP provided immune protection efficiency of 30% in rabbits. This study demonstrated that pcDNA3.1-HlLIP can partially protect rabbits against H. longicornis, and it is possible to develop a new candidate antigen against ticks.

DNA Methylation Variation Is a Possible Mechanism in the Response of Haemaphysalis longicornis to Low-Temperature Stress.

Ticks are notorious ectoparasites and transmit the greatest variety of pathogens than any other arthropods. Cold tolerance is a key determinant of tick abundance and distribution. While studies have shown that DNA methylation is one of the important epigenetic regulations found across many species and plays a significant role in their response to low-temperature stress, its role in the response of ticks to low-temperature stress remains unexplored. Herein, we explored the DNA methylation profile of the tick, Haemaphysalis longicornis, exposed to low-temperature stress (4 °C) using whole-genome bisulfite sequencing (WGBS). We found that approximately 0.95% and 0.94% of the genomic C sites were methylated in the control and low-temperature groups, respectively. Moreover, the methylation level under the CG context was about 3.86% and 3.85% in the control and low-temperature groups, respectively. In addition, a total of 6087 differentially methylated regions (DMRs) were identified between the low-temperature and control groups, including 3288 hypermethylated and 2799 hypomethylated DMRs. Further, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially methylated genes revealed that most of the DMGs were significantly enriched in binding and RNA transport pathways. Taken together, this research confirmed, for the first time, the whole genome DNA methylation profile of H. longicornis and provided new insights into the DNA methylation changes relating to low-temperature stress in H. longicornis, as well as provided a foundation for future studies on the epigenetic mechanism underlying the responses of ticks to abiotic stress.

Acaricidal potential of active components derived from Alpinia galanga rhizome oils and their derivatives against Haemaphysalis longicornis (Acari: Ixodidae).

Acaricidal activities and acetylcholinesterase (AChE) inhibitory activities were evaluated of active constituents of the essential oil extracted from Alpinia galanga rhizomes cultivated from India and their derivatives against Haemaphysalis longicornis nymphs. In addition, the effect was investigated of active components of A. galanga oil on egg laying of adult females of H. longicornis and egg hatchability. Of the volatile components identified in A. galanga oil, ethyl cinnamate, ethyl methoxycinnamate, and methyl cinnamate at 0.32 mg/cm2 resulted in 100% mortality, respectively, indicating that the acaricidal activity of the A. galanga oil against H. longicornis nymphs could be attributed to these compounds. To evaluate the structure-activity relationship between cinnamate derivatives and their acaricidal activities, allyl cinnamate, benzyl cinnamate, isopropyl cinnamate, isobutyl cinnamate, and isoamyl cinnamate were selected. Among cinnamate derivatives tested, allyl cinnamate exhibited the most potent toxicity (LC50 = 0.055 mg/cm2) against H. longicornis nymphs. The allyl cinnamate was also tested for AChE activity in vivo in H. longicornis nymphs and was found to affect the AChE activity. Allyl cinnamate at 10-50 mg/mL inhibited egg laying of adult females of H. longicornis by 10-43%. Egg hatching was suppressed completely by treatment with allyl cinnamate at 50 mg/mL, whereas allyl cinnamate was minimally toxic against non-target earthworms, Eisenia fetida. These results suggest that allyl cinnamate can be used as an active ingredient for the development of eco-friendly tick acaricides against H. longicornis, a vector for Sever fever with thrombocytopenia syndrome (SFTS) virus.

Identification and expression of potential olfactory-related genes related to Niemann-Pick C2 protein and ionotropic receptors in Haemaphysalis longicornis.

Studies have shown that the main pathway for tick host localization and perception of mating information may be chemosensory. However, chemical communication in ticks is poorly understood, especially in those other than the Ixodes ticks. Niemann-Pick C2 (NPC2) protein and ionotropic receptors (IRs) are considered to be closely related to the perception of infochemicals in arthropods. Through bioinformatic analysis, eight NPC2 and four IR candidate genes were identified through screening and identification of the transcriptome sequencing database of Haemaphysalis longicornis. Phylogenetic tree analysis indicated that H. longicornis possesses similar homology to the genus Ixodes. A comparison of the expression of NPC2 and IR in tick forelegs (first pair of legs), hind legs (fourth pair of legs), and capitula using RT-PCR revealed that, barring HlonNPC2-8, 11 candidate genes were highly expressed in the foreleg and capitulum, which are the main sensory organs of ticks. They were also expressed in the hind legs, except for six genes that were not expressed in the males. RT-qPCR analysis showed upregulation and higher relative expression of HlonNPC2-1, HlonNPC2-3, HlonNPC2-6, and HlonNPC2-8 when stimulated by ammonium hydroxide, whereas the others were downregulated and demonstrated lower relative expression. These results further support the putative role of NPC2s as a new odorant carrier in ticks and present 12 promising candidate genes for understanding tick olfactory communication, enriching the data on these genes, especially outside the genus Ixodes.