The Asian longhorned tick (Haemaphysalis longicornis) was first reported in the United States in 2017 and has since been detected in at least 17 states. This tick infests cattle and can produce large populations quickly due to its parthenogenetic nature, leading to significant livestock mortalities and economic losses. While H. longicornis has not been detected in Texas, species distribution models have identified southern Texas as a possible hospitable region for this tick. Southern Texas is currently home to the southern cattle tick (Rhipicephalus microplus), which can transmit the causative agent of cattle fever (Babesia bovis). With the potential for H. longicornis and B. bovis to overlap in southern Texas and their potential to negatively impact the national and global livestock industry, it is imperative to identify the role H. longicornis may play in the cattle fever disease system. A controlled acquisition and transmission experiment tested whether H. longicornis is a vector for B. bovis, with the R. microplus-B. bovis system used as a positive control. Transstadial (nymphs to adults) and transovarial (adults to larvae) transmission and subsequent transstadial maintenance (nymphs and adults) routes were tested in this study. Acquisition-fed, splenectomized animals were used to increase the probability of tick infection. Acquisition nymphs were macerated whole and acquisition adults were dissected to remove midguts and ovaries at five time points (4, 6, 8, 10, and 12 days post-repletion), with 40 ticks processed per time point and life stage. The greatest percentage of nymphs with detectable B. bovis DNA occurred six days post-repletion (20.0 %). For adults, the percentage of positive midguts and ovaries increased as days post-repletion progressed, with day 12 having the highest percentage of positive samples (67.5 % and 60.0 %, respectively). When egg batches were tested in triplicate, all H. longicornis egg batches were negative for B. bovis, while all R. microplus egg batches were positive for B. bovis. During the transmission phase, the subsequent life stages for transstadial (adults) and transovarial transmission/transstadial maintenance (larvae, nymphs, and adults) were fed on naïve, splenectomized calves. All life stages of H. longicornis ticks tested during transmission were negative for B. bovis. Furthermore, the transmission fed animals were also negative for B. bovis and did not show signs of bovine babesiosis during the 45-day post tick transmission period. Given the lack of successful transstadial or transovarial transmission, it is unlikely that H. longicornis is a vector for B. bovis.

The present study is focused on evaluating acaricidal activity and chemical compositions of Erigeron acer root, which was identified as a promising candidate among fifteen Mongolian plant extracts tested for acaricidal activity. The acaricidal effect was evaluated against Haemaphysalis longicornis, assessed for toxicity to normal human skin fibroblast, and analyzed for its chemical constituents. The acetone extract of E. acer root showed significant activity against H. longicornis, with a lethal concentration (LC50) of 5.31 mg/mL and low toxicity, evidenced by a cytotoxic concentration (CC50) of 267.00 µg/mL. Using liquid chromatography-tandem mass spectrometry and molecular networking, thirteen natural compounds were identified, including pyrrolidines, alkaloids, fatty acids, and flavonoids, highlighting the efficacy of E. acer root extract as an effective acaricide against H. longicornis and offering insights for developing new tick control solutions.

Haemaphysalis longicornis is a common tick species that carries several pathogens. There are few reports on the influence of different hosts on the structure of midgut microflora in H. longicornis. In this study, midgut contents of fully engorged female H. longicornis were collected from the surface of tiger (Panthera tigris) and deer (Dama dama). The bacterial genomic DNA of each sample was extracted, and the V3-V4 regions of the bacterial 16S rRNA were sequenced using the Illumina NovaSeq sequencing. The diversity of the bacterial community of the fully engorged female H. longicornis on the surface of tiger was higher than that of deer. In total, 8 phyla and 73 genera of bacteria annotations were detected in the two groups. At the phylum level, the bacterial phyla common to the two groups were Proteobacteria, Firmicutes, and Actinobacteriota. At the genus level, there were 20 common bacterial genera, among which the relative abundances of Coxiella, Morganella, Diplorickettsia, and Acinetobacter were high. The Morganella species was further identified to be Morganella morganii. The alpha diversity index indicated that the bacterial diversity of the tiger group was higher than that of the deer group. Bacteroidota, Patescibacteria, Desulfobacterota, Verrucomicrobiota, and Cyanobacteria were solely detected in the tiger group. A total of 52 bacterial genera were unique in the tiger group, while one bacterial genus was unique in the deer group. This study indicates that there are differences in the structure of the gut bacteria of the same tick species among different hosts. Further culture-based methods are needed to provide a more comprehensive understanding of the tick microbiota parasitizing different hosts.

BACKGROUND: Powassan virus, a North American tick-borne flavivirus, can cause severe neuroinvasive disease in humans. While Ixodes scapularis are the primary vectors of Powassan virus lineage II (POWV II), also known as deer tick virus, recent laboratory vector competence studies showed that other genera of ticks can horizontally and vertically transmit POWV II. One such tick is the Haemaphysalis longicornis, an invasive species from East Asia that recently established populations in the eastern USA and already shares overlapping geographic range with native vector species such as I. scapularis. Reports of invasive H. longicornis feeding concurrently with native I. scapularis on multiple sampled hosts highlight the potential for interspecies co-feeding transmission of POWV II. Given the absence of a clearly defined vertebrate reservoir host for POWV II, it is possible that this virus is sustained in transmission foci via nonviremic transmission between ticks co-feeding on the same vertebrate host. The objective of this study was to evaluate whether uninfected H. longicornis co-feeding in close proximity to POWV II-infected I. scapularis can acquire POWV independent of host viremia.
METHODS: Using an in vivo tick transmission model, I. scapularis females infected with POWV II (\"donors\") were co-fed on mice with uninfected H. longicornis larvae and nymphs (\"recipients\"). The donor and recipient ticks were infested on mice in various sequences, and mouse infection status was monitored by temporal screening of blood for POWV II RNA via quantitative reverse transcription polymerase chain reaction (q-RT-PCR).
RESULTS: The prevalence of POWV II RNA was highest in recipient H. longicornis that fed on viremic mice. However, nonviremic mice were also able to support co-feeding transmission of POWV, as demonstrated by the detection of viral RNA in multiple H. longicornis dispersed across different mice. Detection of viral RNA at the skin site of tick feeding but not at distal skin sites indicates that a localized skin infection facilitates transmission of POWV between donor and recipient ticks co-feeding in close proximity.
CONCLUSIONS: This is the first report examining transmission of POWV between co-feeding ticks. Against the backdrop of multiple unknowns related to POWV ecology, findings from this study provide insight on possible mechanisms by which POWV could be maintained in nature.

Ticks are notorious blood-sucking ectoparasites that affect both humans and animals. They serve as a unique vector of various deadly diseases. Here, we have shown the roles of the receptor for advanced glycation end products (RAGE) during repeated infestations by the tick Haemaphysalis longicornis using RAGE-/- mice. In primary infestation, a large blood pool developed, which was flooded with numerous RBCs, especially during the rapid feeding phase of the tick both in wild-type (wt) and RAGE-/- mice. Very few inflammatory cells were detected around the zones of haemorrhage in the primary infestations. However, the number of inflammatory cells gradually increased in the subsequent tick infestations, and during the third infestations, the number of inflammatory cells reached to the highest level (350.3 ± 16.8 cells/focus). The site of attachment was totally occupied by the inflammatory cells in wt mice, whereas very few cells were detected at the ticks\' biting sites in RAGE-/- mice. RAGE was highly expressed during the third infestation in wt mice. In the third infestation, infiltration of CD44+ lymphocytes, eosinophils and expression of S100A8 and S100B significantly increased at the biting sites of ticks in wt, but not in RAGE-/- mice. In addition, peripheral eosinophil counts significantly increased in wt but not in RAGE-/- mice. Taken together, our study revealed that RAGE-mediated inflammation and eosinophils played crucial roles in the tick-induced inflammatory reactions.

Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase,s role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.

This study evaluated the potential repellent and acaricidal effects of 4 essential oils (clove, eucalyptus, lavender, and mint) against the Asian longhorned tick Haemaphysalis longicornis, a vector of various tick-borne diseases in medical and veterinary contexts. Selected for their potential repellent and acaricidal properties, the 4 essential oils were tested on adult and nymph H. longicornis ticks at different concentrations. The experiment assessed mortality rates and repellency, particularly during tick attachment to host skin. There was a significant increase (p<0.05) in tick mortality and repellency scores across all groups. At a 1% concentration, adult tick mortality ranged from 36% to 86%, while nymph mortality ranged from 6% to 97%. Clove oil exhibited notable efficacy, demonstrating high mortality rates of nymphs and adults. Clove oil also displayed strong repellency properties, with a repellency index of 0.05, surpassing those of mint, eucalyptus, and lavender oils. Clove oil showed the highest effectiveness in deterring nonattached adult ticks (90%) and nymphs (95%) when applied to skin. Clove oil was the most effective against adult and nymph ticks, achieving mortality rates of 86% and 97%, respectively, and led to the highest nonattachment rates when applied to skin. In conclusion, essential oils such as clove, eucalyptus, lavender, and mint oils present promising results for tick population control.

OBJECTIVE: Haemaphysalis longicornis is an obligate blood-sucking ectoparasite that has gained attention due its role of transmitting medically and veterinary significant pathogens and it is the most common tick species in Republic of Korea. The preferred strategy for controlling ticks is a multi-antigenic vaccination. Testing the efficiency of a combination antigen is a promising method for creating a tick vaccine.
OBJECTIVE: The aim of the current research was to analyze the role of subolesin and enolase in feeding and reproduction of H. longicornis by gene silencing.
METHODS: In this study, we used RNA interference to silence salivary enolase and subolesin in H. longicornis. Unfed female ticks injected with double-stranded RNA targeting subolesin and enolase were attached and fed normally on the rabbit\'s ear. Real-time polymerase chain reaction was used to confirm the extent of knockdown.
RESULTS: Ticks in the subolesin or enolase dsRNA groups showed knockdown rates of 80% and 60% respectively. Ticks in the combination dsRNA (subolesin and enolase) group showed an 80% knockdown. Knockdown of subolesin and enolase resulted in significant depletion in feeding, blood engorgement weight, attachment rate, and egg laying. Silencing of both resulted in a significant (p < 0.05) reduction in tick engorgement, egg laying, egg hatching (15%), and reproduction.
CONCLUSIONS: Our results suggest that subolesin and enolase are an exciting target for future tick control strategies.

BACKGROUND: Epigenetic modifications of histones play important roles in the response of eukaryotic organisms to environmental stress. However, many histone acetyltransferases (HATs), which are responsible for histone acetylation, and their roles in mediating the tick response to cold stress have yet to be identified. In the present study, HATs were molecularly characterized and their associations with the cold response of the tick Haemaphysalis longicornis explored.
METHODS: HATs were characterized by using polymerase chain reaction (PCR) based on published genome sequences, followed by multiple bioinformatic analyses. The differential expression of genes in H. longicornis under different cold treatment conditions was evaluated using reverse transcription quantitative PCR (RT-qPCR). RNA interference was used to explore the association of HATs with the cold response of H. longicornis.
RESULTS: Two HAT genes were identified in H. longicornis (Hl), a GCN5-related N-acetyltransferase (henceforth HlGNAT) and a type B histone acetyltransferase (henceforth HlHAT-B), which are respectively 960 base pairs (bp) and 1239 bp in length. Bioinformatics analysis revealed that HlGNAT and HlHAT-B are unstable hydrophilic proteins characterized by the presence of the acetyltransferase 16 domain and Hat1_N domain, respectively. RT-qPCR revealed that the expression of HlGNAT and HlHAT-B decreased after 3 days of cold treatment, but gradually increased with a longer period of cold treatment. The mortality rate following knockdown of HlGNAT or HlHAT-B by RNA interference, which was confirmed by RT-qPCR, significantly increased (P < 0.05) when H. longicornis was treated at the lowest lethal temperature (- 14 °C) for 2 h.
CONCLUSIONS: The findings demonstrate that HATs may play a crucial role in the cold response of H. longicornis. Thus further research is warranted to explore the mechanisms underlying the epigenetic regulation of the cold response in ticks.

The invasion of the Asian longhorned tick, Haemaphysalis longicornis Neumann, into the United States poses a significant ecological, veterinarian, and public health threat. We conducted a comprehensive tick survey using standard tick flagging protocol for collection over 3 field seasons, March-August, and 56 surveyed sites to identify the presence of H. longicornis in the native ecosystem and prove its establishment according to USDA criteria. Of the total 56 state parks and wildlife management areas (WMA) surveyed, only one was found to be invaded by H. longicornis; detection of H. longicornis occurred at Buck Shoals Wildlife Management area in White County, GA. This site is maintained by the state of Georgia, has no agricultural animals present, and hosts a large white-tailed deer population. After the initial detection of H. longicornis in 2022, an additional field season occurred in 2023, where H. longicornis was confirmed as established based on USDA criteria. The increase in H. longicornis populations from 2021 to 2023 at Buck Shoals WMA points to the rapid spread of this tick within the environment. Our findings provide evidence of the rapid establishment of H. longicornis in the southern edge of suitability for this tick and within the native ecosystem beyond farmlands and private land.