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We report the discovery of large numbers of Haemaphysalis longicornis Neumann (Ixodida: Ixodidae) infesting a sheep in Hunterdon County, New Jersey, United States. All life stages were found on the sheep, which had no history of travel outside the country. H. longicornis is native to East Asia, and there are invasive populations in Australia, New Zealand and several Pacific islands, where this tick is a major livestock pest. It is currently unknown whether the New Jersey collections represent a limited or established population, but because this species could present a significant threat to human and animal health in the United States, vigilance is encouraged.

Many animals are dependent on microbial partners that provide essential nutrients lacking from their diet. Ticks, whose diet consists exclusively on vertebrate blood, rely on maternally inherited bacterial symbionts to supply B vitamins. While previously studied tick species consistently harbor a single lineage of those nutritional symbionts, we evidence here that the invasive tick Hyalomma marginatum harbors a unique dual-partner nutritional system between an ancestral symbiont, Francisella , and a more recently acquired symbiont, Midichloria . Using metagenomics, we show that Francisella exhibits extensive genome erosion that endangers the nutritional symbiotic interactions. Its genome includes folate and riboflavin biosynthesis pathways but deprived functional biotin biosynthesis on account of massive pseudogenization. Co-symbiosis compensates this deficiency since the Midichloria genome encompasses an intact biotin operon, which was primarily acquired via lateral gene transfer from unrelated intracellular bacteria commonly infecting arthropods. Thus, in H. marginatum , a mosaic of co-evolved symbionts incorporating gene combinations of distant phylogenetic origins emerged to prevent the collapse of an ancestral nutritional symbiosis. Such dual endosymbiosis was never reported in other blood feeders but was recently documented in agricultural pests feeding on plant sap, suggesting that it may be a key mechanism for advanced adaptation of arthropods to specialized diets.

Background Tick infestation is the major problem for animal health that causes substantial economic losses, particularly in tropical and subtropical countries. To better understand the spatial distribution of tick species and risk factors associated with tick prevalence in livestock in Pakistan, ticks were counted and collected from 471 animals, including 179 cattle, 194 buffaloes, 80 goats and 18 sheep, on 108 livestock farms in nine districts, covering both semi-arid and arid agro-ecological zones. Results In total, 3,807 ticks representing four species were collected: Hyalomma anatolicum ( n  = 3,021), Rhipicephalus microplus ( n  = 715), Hyalomma dromedarii ( n  = 41) and Rhipicephalus turanicus ( n  = 30). The latter species is reported for the first time from the study area. Rhipicephalus microplus was the predominant species in the semi-arid zone, whereas H. anatolicum was the most abundant species in the arid zone. The overall proportion of tick-infested ruminants was 78.3% (369/471). It was highest in cattle (89.9%), followed by buffaloes (81.4%), goats (60.0%) and sheep (11.1%). The median tick burden significantly differed among animal species and was highest in cattle (median 58), followed by buffaloes (median 38), goats (median 19) and sheep (median 4.5). Female animals had significantly higher tick burdens than males and, in large ruminants, older animals carried more ticks than younger animals. The intensity of infestation was significantly lower in indigenous animals compared to exotic and crossbred cows. Analysis of questionnaire data revealed that the absence of rural poultry, not using any acaricides, traditional rural housing systems and grazing were potential risk factors associated with a higher tick prevalence in livestock farms. Conclusion Absence of rural poultry, not performing acaricide treatments, traditional rural housing systems and grazing were important risk factors associated with higher tick prevalence in livestock farms. Age, gender, breed and animal species significantly affected the intensity of tick infestation. This report also describes the presence of R. turanicus in the Punjab Province of Pakistan for the first time. The outcomes of this study will be useful in the planning of integrated control strategies for ticks and tick-borne diseases in Pakistan.

The states of Kansas and Oklahoma, in the central Great Plains, lie at the western periphery of the geographic distributions of several tick species. As the focus of most research on ticks and tick-borne diseases has been on Lyme disease which commonly occurs in areas to the north and east, the ticks of this region have seen little research attention. Here, we report on the phenology and activity patterns shown by tick species observed at 10 sites across the two states and explore factors associated with abundance of all and life specific individuals of the dominant species. Ticks were collected in 2020–2022 using dragging, flagging and carbon-dioxide trapping techniques, designed to detect questing ticks. The dominant species was A . americanum (24098, 97%) followed by Dermacentor variabilis (370, 2%), D . albipictus (271, 1%), Ixodes scapularis (91, <1%) and A . maculatum (38, <1%). Amblyomma americanum , A . maculatum and D . variabilis were active in Spring and Summer, while D . albipictus and I . scapularis were active in Fall and Winter. Factors associated with numbers of individuals of A . americanum included day of year, habitat, and latitude. Similar associations were observed when abundance was examined by life-stage. Overall, the picture is one of broadly distributed tick species that shows seasonal limitations in the timing of their questing activity.

Chemical repellents against arthropods have limitations in terms of toxicity and resistance. Natural plant compounds can be utilized as alternatives for developing environmentally friendly repellents for humans and animals. A variety of plant essential oils exhibit strong repellent effects against ticks; however, the mechanisms of action against ticks remain unknown. Here, we investigated the repellency of cinnamaldehyde, a primary compound found in cinnamon oil, and demonstrated that it affected the electrophysiological responses on Haller’s organs of parthenogenetic Haemaphysalis longicornis . Transcriptome data indicated that the cinnamaldehyde response was linked to ionotropic receptor (HL-IR) at various tick developmental stages. HL-IR was widely expressed in a variety of tissues and developmental stages of ticks according to RT-qPCR. In situ hybridization results showed that HL-IR was highly expressed on Haller’s organs of the ticks. Microinjection of HL-IR double-stranded RNA (dsRNA) showed that reduced transcript levels led to significant decreases in the tick repellency rate from cinnamaldehyde and the EAG response of Haller’s organ. Experiments using competitive fluorescence binding and mutation sites showed that 218ASN was the critical binding site for cinnamaldehyde and HL-IR. We conclude that Haller’s organ of ticks expresses HL-IR, and that this interaction mediates tick-repellent behavior by binding to cinnamaldehyde.

Background The goal of this study was to reassess the taxonomic status of A. maculatum , A. triste and A. tigrinum by phylogenetic analysis of five molecular markers [four mitochondrial: 12S rDNA, 16S rDNA, the control region (DL) and cytochrome c oxidase 1 ( cox 1), and one nuclear: ribosomal intergenic transcribed spacer 2 (ITS2)]. In addition, the phenotypic diversity of adult ticks identified as A. maculatum and A. triste from geographically distinct populations was thoroughly re-examined. Results Microscopic examination identified four putative morphotypes distinguishable by disjunct geographical ranges, but very scant fixed characters. Analysis of the separated mitochondrial datasets mostly resulted in conflicting tree topologies. Nuclear gene sequences were almost identical throughout the geographical ranges of the two species, suggesting a very recent, almost explosive radiation of the terminal operational taxonomic units. Analysis of concatenated molecular datasets was more informative and indicated that, although genetically very close to the A. maculatum - A. triste lineage, A. tigrinum was a monophyletic separate entity. Within the A. maculatum - A. triste cluster, three main clades were supported. The two morphotypes, corresponding to the western North American and eastern North American populations, consistently grouped in a single monophyletic clade with many shared mitochondrial sequences among ticks of the two areas. Ticks from the two remaining morphotypes, south-eastern South America and Peruvian, corresponded to two distinct clades. Conclusions Given the paucity of morphological characters, the minimal genetic distance separating morphotypes, and more importantly the fact that two morphotypes are genetically indistinguishable, our data suggest that A. maculatum and A. triste should be synonymized and that morphological differences merely reflect very recent local adaptation to distinct environments in taxa that might be undergoing the first steps of speciation but have yet to complete lineage sorting. Nonetheless, future investigations using more sensitive nuclear markers and/or crossbreeding experiments might reveal the occurrence of very rapid speciation events in this group of taxa. Tentative node dating revealed that the A. tigrinum and A. maculatum - A. triste clades split about 2 Mya, while the A. maculatum - A.triste cluster radiated no earlier than 700,000 years ago.

The Asian long-horned tick, Haemaphysalis longicornis Neumann, 1901, is the competent vector for severe fever with thrombocytopenia syndrome virus (SFTSV). Haemaphysalis longicornis originated mainly in eastern Asia and invaded many areas like Australia, New Zealand, and the Pacific islands, and was recently introduced to eastern parts of the USA. This species is characterized by high adaptability to a wide range of temperatures and can reproduce parthenogenically under stressful conditions. Migratory birds are important hosts of H. longicornis and are thought to be responsible for its unexpected invasion and introduction into new areas worldwide. This study predicted the historical (near current) global environmental suitability and the possible shifts in environmental suitability for H. longicornis under the ongoing climate change between 2021 and 2100. The results demonstrated that Europe is at potential of high environmental suitability for H. longicornis invasion although this species has not been recorded in any regions of Europe yet. Our model also anticipated the environmental suitability for H. longicornis in eastern parts of the USA, although the recently recorded occurrences there were not used in the model calibration. Climate change is thought to affect and increase the range of suitable environments for H. longicornis. The different maps introduced in this study may help improve understanding of the global environmental suitability for this invasive disease vector and predict the areas at high environmental suitability for possible invasion to prioritize the control programs and enhance quarantine procedures in these areas.

Amblyomma americanum ticks transmit more than a third of human tick-borne disease (TBD) agents in the United States. Tick saliva proteins are critical to success of ticks as vectors of TBD agents, and thus might serve as targets in tick antigen-based vaccines to prevent TBD infections. We describe a systems biology approach to identify, by LC-MS/MS, saliva proteins (tick = 1182, rabbit = 335) that A. americanum ticks likely inject into the host every 24 h during the first 8 days of feeding, and towards the end of feeding. Searching against entries in GenBank grouped tick and rabbit proteins into 27 and 25 functional categories. Aside from housekeeping-like proteins, majority of tick saliva proteins belong to the tick-specific (no homology to non-tick organisms: 32%), protease inhibitors (13%), proteases (8%), glycine-rich proteins (6%) and lipocalins (4%) categories. Global secretion dynamics analysis suggests that majority (74%) of proteins in this study are associated with regulating initial tick feeding functions and transmission of pathogens as they are secreted within 24-48 h of tick attachment. Comparative analysis of the A. americanum tick saliva proteome to five other tick saliva proteomes identified 284 conserved tick saliva proteins: we speculate that these regulate critical tick feeding functions and might serve as tick vaccine antigens. We discuss our findings in the context of understanding A. americanum tick feeding physiology as a means through which we can find effective targets for a vaccine against tick feeding.

Ticks are the most common arthropod vector, after mosquitoes, and are capable of transmitting the greatest variety of pathogens. For both humans and animals, the worldwide emergence or re-emergence of tick-borne disease is becoming increasingly problematic. Despite being such an important issue, our knowledge of pathogen transmission by ticks is incomplete. Several recent studies, reviewed here, have reported that the expression of some tick factors can be modulated in response to pathogen infection, and that some of these factors can impact on the pathogenic life cycle. Delineating the specific tick factors required for tick-borne pathogen transmission should lead to new strategies in the disruption of pathogen life cycles to combat emerging tick-borne disease.

Vector ticks' perception of characteristic odors emitted by infected hosts is key to understand tick's foraging behavior for infected host and design odor-based control strategies for tick-borne diseases. Laboratory mice knocked out for type I interferon (IFN) receptors (Ifnar-/-) were used to develop a simulated host by intraperitoneal infection with Bandavirus dabieense (SFTSV). Urine and fecal samples were collected 4 days post-infection and analyzed to detect differential volatile metabolites (DVMs) during infection. Next, the two salient odor cues among the SFTSV-induced host DVMs, indole and 3-methylindole, were used to test the olfactory response of Haemaphysalis. longicornis by electroantennographic detection (EAD) and Y-tube olfactometry, respectively. To gain insight into the potential olfactory mechanism, two olfactory-associated proteins, Niemann-Pick type C2 (NPC2) and Odor Binding Protein-like (OBPL) proteins were annotated from the transcriptomic data derived from H. longicornis forelegs. Online tools were used to predict the ligand binding properties of the two proteins to the two indole candidates. Simultaneously, quantitative RT-PCR using β-actin as an internal reference gene was used to monitor the relative transcript levels of NPC2 and OBPL proteins under the stimulation of two indole candidates. The significantly regulated proteins were cloned and expressed with the vector plasmid pET-28b in vitro. The purified proteins were tested for the binding properties to the two indole candidates. SFTSV-infected Ifnar-/- mice upregulated 11 DVMs in fecal samples, mostly indoles and phenols, along with indole biosynthesis and related metabolic processes. In the urine samples, 29 DVMs were downregulated in the infected host, with eucalyptol and phenylalanine acid being the most altered. We test the olfactory responses of H. longicornis to indole and 3-methylindole, which influence tick foraging behavior. The olfactometers showed that the tick preferred both indole and 3-methylindole. EAD tests showed that stimulation of the olfactory receptor neuron in Haller's organ produced significant active potential in response to indoles. Two olfactory proteins, NPC2 and OBPL, were successfully annotated from H. longicornis foreleg transcriptomic data. NPC2 has a β-barrel structure that binds signal chemicals, while OBPL is a classical OBP with a hydrophobic binding cavity. When monitoring the transcript levels of NPC2 and OBPL in the tick forelegs, the increased transcript level (1.2-1.4 folds change) of OBPL was observed following indoles stimulation, compared to the downregulated level (0.6-0.8 folds change) of NPC2 under the same circumstances. The OBPL and NPC2 gene from H. longicornis were successfully cloned and expressed as inclusion proteins respectively. The purified OBPL (20.28 kDa) showed higher affinity for both indole (Ki 2.256μM) and 3-methylindole (Ki 4.191μM) than NPC2 in the competitive fluorescence binding assays with 1-NPN as a competitor. Facilitated by the olfactory OBPL protein in Haller's organ, H. longicornis smells and is attracted to the characteristic indolic scents of hosts induced by SFTSV infection. Olfactory associations between infected hosts and vector arthropods could provide a new perspective to understand host foraging behavior and design novel control strategies for tick-borne diseases based on pathogen-induced scent according to chemical ecology theory.