Ticks are major vectors of zoonotic diseases, harboring and transmitting various bacteria, viruses and parasites. In mainland China, the rapidly growing population of companion animals has rendered pets important tick hosts and a critical bridge for tick-borne pathogens to infiltrate domestic environments, yet no systematic investigations have been conducted on tick species parasitizing pets and the pathogens they carry.This study was conducted from May 2024 to August 2025. We collected ticks from the body surfaces of outpatient dogs and cats at 231 pet hospitals across 27 Chinese provinces and municipalities. Tick species were identified, and four key tick-borne pathogens (Babesia spp., Anaplasma phagocytophilum, Ehrlichia spp., and Lyme disease spirochetes) were detected via polymerase chain reaction (PCR); positive samples were further sequenced and subjected to phylogenetic analysis.A total of 2158 ticks were collected, 1942 (90.0%) from dogs and 216 (10.0%) from cats, belonging to 16 species of four genera. Haemaphysalis longicornis (40.6%) and Rhipicephalus sanguineus sensu lato (35.5%) were the dominant species. A. phagocytophilum was not detected in any samples. Babesia spp. had the highest infection rate (1.44%), including B. vogeli and B. gibsoni; Ehrlichia spp. and Lyme disease spirochetes were detected at 0.37% and 0.28%, respectively, with multiple species/genotypes identified for both. Pathogen distribution exhibited distinct tick-species specificity and spatial clustering.This is the first systematic survey of the diversity of pet-parasitizing ticks and the prevalence of four key tick-borne pathogens in mainland China, confirming that pets face persistent and diverse threats from tick-borne diseases. Our findings highlight the need for regionally differentiated surveillance, year-round tick control measures, and enhanced prevention and control awareness among veterinarians and pet owners, providing a scientific basis for clinical diagnosis, treatment and public health prevention and control.

Ticks are ectoparasites that can transmit zoonotic pathogens, causing significant economic losses worldwide. RNA interference (RNAi) offers a gene-specific alternative to chemical acaricides; however, its application is limited by double-stranded RNA (dsRNA) instability and poor cuticle penetration. Using Haemaphysalis longicornis (H. longicornis) as a model, we identified the ecdysone receptor gene (ECR) as an effective RNAi target. Microinjection of dsECR completely abolished engorgement rate (0%) and reduced body weight by ∼90%. To enable practical delivery, three nanocarrier systems-chitosan (CS), star polycation (SPc), and disulfide-crosslinked chitosan (CS-ss) were developed to improve the stability and uptake of dsRNA. All three nanocarrier systems enhanced dsRNA resistance to ribonuclease (RNase) degradation while exhibiting distinct physicochemical properties: CS-ss formed the smallest nanoparticles (∼175 nm), whereas SPc showed superior surface wettability. All nanocarrier-dsRNA complexes achieved comparable RNAi efficiency under microinjection. In contrast, immersion delivery of naked dsECR was ineffective, whereas SPc-dsECR and CS-ss-dsECR enabled significant ECR silencing, reduced engorgement rate, and decreased body weight. Similar RNAi efficiency levels were validated in Hyalomma asiaticum, supporting the cross-species applicability of this strategy. Notably, SPc-dsRNA exhibited lower contact angles on the tick cuticle, indicating enhanced spreading and suggesting improved penetration efficiency. Collectively, the results of this study demonstrate that nanocarrier encapsulation is effective for immersion-based RNAi control of ticks. SPc provides superior dispersion and surface interaction for consistent delivery, while CS-ss offers the potential for redox-responsive release. The findings establish a scalable and non-invasive RNAi strategy for sustainable tick control.

This study was conducted to investigate tick species that may harbour severe fever with thrombocytopenia syndrome virus (SFTSV) and Babesia microti in the provinces of Henan, Anhui, and Zhejiang, as well as in Shanghai in the central and eastern parts of China. Between March and September 2023, 721 pools of ticks were collected belonging to three genera and five species: Haemaphysalis longicornis (n = 612; 84.9%), Haemaphysalis fusca (n = 94; 13.0%), Rhipicephalus microplus (n = 10; 1.4%), Amblyomma testudinarium (n = 3; 0.4%), and Haemaphysalis wellingtoni (n = 2; 0.3%). The SFTSV-positive pool rate was 20.0%, 13.0%, 5.8%, and 4.1% in Xinyang, Henan; Songjiang, Shanghai; Lu’an, Anhui; and Zhoushan, Zhejiang, respectively. SFTSV was detected in all five tick species collected. Among the SFTSV-positive pools, H. longicornis constituted the highest proportion (83.9%, 78/93), whereas pools containing R. microplus and H. wellingtoni each represented the lowest proportion (1.1%, 1/93). Babesia microti was assayed only in these SFTSV-positive tick pools, and co-infection was found in both H. longicornis and H. wellingtoni, though it was most frequent in H. longicornis.

BackgroundTicks are major vectors of human and animal pathogens, and the development of novel, species-specific control strategies is urgently needed. RNA interference (RNAi) holds promise as a targeted approach for tick control; however, its practical application has been limited by variable efficiency and an incomplete understanding of the underlying regulatory mechanisms in these arthropods.MethodsUsing transcriptomic screening, molecular cloning, and functional genomics approaches, we identified and characterized Hl48, a previously uncharacterized ~48 kDa protein in Haemaphysalis longicornis (H. longicornis). Loss-of-function (RNAi) and gain-of-function (RNA activation, RNAa) strategies were employed to assess the role of Hl48 in RNAi regulation in vivo. Mechanistic investigations included dual-luciferase reporter assays, co-immunoprecipitation (Co-IP), molecular docking, site-directed mutagenesis, and surface plasmon resonance (SPR) analysis.ResultsHl48 expression was significantly upregulated in response to RNA virus infection or exogenous dsRNA stimulation, but not to protozoan infection, functionally linking it to the RNAi pathway. Silencing of Hl48 substantially impaired RNAi efficiency against multiple target genes, whereas its activation enhanced RNAi responsiveness both in vivo and ex vivo. Mechanistically, Hl48 regulates the RNAi pathway through a dual mode of action: it directly interacts with the PIWI domain of Argonaute 2 (AGO2), the catalytic core of the RNA-induced silencing complex (RISC), and concurrently promotes transcriptional upregulation of ago2. Key interaction residues (Thr28 and Glu93) were identified and validated by mutagenesis.ConclusionThese findings establish Hl48 as a key positive regulator of the RNAi pathway in ticks, revealing a previously unrecognized layer of RNAi control in arthropods. This work provides a mechanistic foundation for the development of enhanced RNA-based strategies for tick-borne disease control.

Background:Alongshan virus (ALSV) is an emerging tick-borne segmented flavivirus associated with human febrile illness, belonging to Jingmenvirus group in the family Flaviviridae. Ixodes persulcatus (I. persulcatus) has been considered as the competent vector of ALSV in the field studies; however, no experimental study has yet evaluated the vector competence of I. persulcatus in the maintenance and transmission of ALSV. Methods:I. persulcatus adult ticks were infected with ALSV via anal pore microinjection. salivary glands (SG), midgut (MG), and ovaries (OV) were collected and subjected to quantitative real-time polymerase chain reaction (RT-qPCR) for ALSV RNA detection. Ticks fed on Kunming (KM) or NOD-SCID IL2rg-/- (NTG) mice, and their eggs, molted progeny, and mice tissues were collected for ALSV RNA detection to evaluate vector competence in maintaining and transmitting ALSV. Results:Viral RNA levels in ALSV-microinjected ticks increased significantly, peaking at 7 d.p.i. Viral RNA in SG, MG, and OV tissues showed a significant upward trend over time, with higher copy numbers in MG than in SG and OV. Eggs, hatched larvae, engorged larvae, molted nymphs, engorged nymphs, and adults all exhibited high levels of viral RNA copies, ranging from approximately 106 to 109 copies/μL. Viral RNA was detected in the blood and tissues of mice bitten by ALSV-infected adult, larval, and nymphal ticks, with lower RNA copies in adult-bitten mice than in larvae- or nymph-bitten mice. Notably, viral RNA copy numbers were significantly higher in the blood and tissues of mice inoculated with ALSV and then bitten by ALSV-free immature ticks, compared to those receiving ALSV alone. Conclusion:The results demonstrate that I. persulcatus ticks are competent vectors for ALSV, capable of transmitting the virus both vertically and horizontally with high efficiency, with tick bite-induced modulation of viral levels in mice varying according to the developmental stage of the tick.

IntroductionToxoplasma gondii can cause toxoplasmosis. It is an important type of pathogen within the broad category of emerging and re-emerging zoonoses. As an infectious disease featuring a complex multi-host transmission cycle, it poses an increasingly severe threat to global public health. No licensed vaccines are currently available for pets and humans, and thus a novel high-efficiency vaccine is urgently required.MethodsSix antigens (GRA1, MIC17A, OWP2, LEA880, LEA870, and a hypothetical protein LEA530) representing different stages of the parasite lifecycle were selected from ToxoDB. T-cell and B-cell epitopes were predicted using immunoinformatics tools and screened based on antigenicity, allergenicity, and toxicity. The multi-epitope peptide (MEP1) was evaluated using molecular docking with Toll-like receptor 4 (TLR4) and immune simulation. The optimized sequence was expressed in HEK293T cells as a recombinant plasmid (MEP1-pcDNA3.1) and further evaluated in BALB/c mice.ResultsMEP1 contained 13 cytotoxic T lymphocyte epitopes, 16 helper T lymphocyte epitopes, and 12 B-cell epitopes, with a length of 732 amino acids and a predicted molecular weight of 75.73 kDa. The antigenicity score was 0.7343, and structural modeling indicated stable secondary and tertiary conformations. Molecular docking suggested strong binding affinity to TLR4. Immune simulation predicted increased B-cell and T-cell responses following vaccination. In vivo, MEP1-pcDNA3.1 immunization significantly increased serum IFN-γ levels (526.81 pg/mL) compared with PBS and pcDNA3.1 controls. Splenocyte proliferation was significantly enhanced in the MEP1-pcDNA3.1 group (SI = 1.58 ± 0.21) compared with PBS (1.10 ± 0.09) and pcDNA3.1 (1.12 ± 0.04) groups (P < 0.01). Following challenge with 5 × 10³ tachyzoites of the PLK strain, survival was markedly prolonged in vaccinated mice, whereas all control mice died within 2–4 days.ConclusionThis study demonstrates an immunoinformatics-guided multi-epitope vaccine strategy against T. gondii, supported by in vivo immunogenicity and partial protective efficacy in a mouse model.

Haemaphysalis (Acari: Ixodidae) ticks remain poorly studied in Mongolia. Here, we report the first confirmed record of Haemaphysalis concinna Koch, 1844 in the Numrug Strictly Protected Area, Dornod Province, eastern Mongolia, based on both morphological characteristics and molecular analyses. This region lies along the Mongolia-China border within the forest-steppe and steppe zones of the western Greater Khingan Mountains. Tick sampling was conducted at six sites (lakeshores, riverbanks, and marshy forests) between early April and mid-August over a three-year period.A total of 1014 H concinna specimens were collected, of which 148 (including two nymphs) were selected for DNA extraction. PCR amplifications targeting the mitochondrial 16S rRNA (∼455 bp) and Cox1 (∼820 bp) genes were performed. Eight samples were sequenced, with three 16S and two Cox1 sequences meeting quality thresholds. The sequences of both genes exhibited 99.8 % and 100 % identity, respectively, to H. concinna sequences from Russia (Cox1: PP851089.1, PP851095.1; 16S rRNA: KP866207.1) and China (Cox1: KR108863.1; 16S rRNA: ON097130.1) available in GenBank. This high level of sequence identity strongly supports the identification of the Mongolian samples as H. concinna. Species identification was further supported by morphological characteristics consistent with published descriptions of adult ticks. This record from the Khalkh Numrug basin represents the first verified occurrence of H. concinna in eastern Mongolia. These findings underscore the need for further research on the species’ ecology, host range, and potential public health significance in the region.

Ticks (Haemaphysalis longicornis) transmit pathogens to their hosts through their salivary glands during blood-feeding. The salivary glands of adult parthenogenetic H. longicornis undergo degeneration post-engorgement. Clarifying the molecular mechanisms underlying salivary gland degeneration of H. longicornis is conducive to identifying novel targets for preventing and controlling these widespread vectors. In this study, we investigated the salivary glands of adult parthenogenetic H. longicornis to elucidate the relationship between ferroptosis, iron-dependent cell death, H. longicornis ferritin 1 (HlFer1) and salivary gland degeneration post-attachment and post-engorgement. Fluorescence microscopy, revealed increased iron accumulation, reactive oxygen species, lipid peroxidation, and decreased mitochondrial cristae in the granular acini of H. longicornis salivary glands post-engorgement. The results of a qPCR analysis indicated that HlFer1, glutathione peroxidase 4 (GPX4), transferrin (TRF), and high mobility group protein B1 (HMGB1) expression elevated in H. longicornis salivary glands post-attachment and post-engorgement. In vitro culture of H. longicornis salivary glands showed that erastin promotes ferroptosis, while ferrostatin-1 blocks this process. RNA interference (RNAi) targeting HlFer1 promoted ferroptosis in salivary gland granular acini. In conclusion, we demonstrated that HlFer1-induced ferroptosis is a key molecular mechanism underlying the salivary gland granular acini degeneration of H. longicornis. Our findings are important for developing novel preventive measures against H. longicornis as a disease vector.

B. microti is a tick-transmitted zoonotic erythrocytic intracellular parasite. Ferroptosis is an iron-dependent form of programmed cell death that affects pathogen replication in the host. Currently, there is limited research concerning the effect of tick ferroptosis on Babesia infection and the underlying mechanism of action. The present study used a B. microti -mouse- Haemaphysalis longicornis infection model in which nymphs fed on the blood of B. microti-infected mice. The midgut divalent iron (p<0.01) and reactive oxygen species (ROS) (p<0.05) levels were significantly elevated in infected ticks, and transmission electron microscopy (TEM) showed that mitochondrial ridges were absent or decreased in size. Downregulation of ferritin 1 and glutathione peroxidase 4 (GPX4) in ticks infected with B. microti suggests that these changes promote ferroptosis. In vivo studies demonstrated that the ferroptosis promoter Erastin increased B. microti load (p<0.05), while the inhibitor Ferrostatin-1 effectively decreased load (p<0.01). Tick histamine-releasing factor (HRF), a protein related to the antioxidant system, was downregulated in infected nymphs compared with uninfected nymphs (p<0.05), and interference with HRF promoted tick acquisition of B. microti (p<0.001). Transcriptomic analyses showed that HRF interference promotes tick ferroptosis by downregulating ferritin 1 and GPX4. Meanwhile, interference with tick HRF molecules showed increased divalent iron and ROS and decreased mitochondrial ridges compared with controls. These findings highlight the critical role of tick HRF molecules in regulating ferroptosis and acquisition of B. microti, thereby providing important insights for a deeper understanding of the tick-Babesia interaction.

IntroductionTicks are the primary vectors of Babesia sp, with the midgut as the initial site of pathogen invasion following blood feeding. Elucidating the molecular interactions between tick midguts and Babesia is essential for developing targeted strategies to control tick-borne babesiosis. However, studies in this field remain limited.MethodsTo investigate tick-pathogen interactions, we employed RNA-seq to profile gene expression, and qRT-PCR served to validate key findings. Apoptosis and autophagy were assessed via TUNEL staining and Transmission Electron Microscopy (TEM). Furthermore, RNA interference (RNAi) and pharmacological modulation were employed to evaluate the impact of ticks on pathogen load.ResultsOur RNA-seq analysis identified 540 and 569 Differentially Expressed Genes (DEGs) in infected midguts at 0 and 4 d post-engorgement, respectively. These DEGs were enriched in pathways related to metabolic processes, immunity, and cellular processes. To clarify the functional relevance of these findings, the roles of apoptosis and autophagy during infection were further evaluated. Quantitative Real-Time PCR (qRT-PCR) analysis revealed significant upregulation of apoptosis-related genes (caspase-7, caspase-8, and caspase-9) and autophagy genes (ATG5, ATG8, and ATG12) in response to B. microti infection. TUNEL assay and Transmission Electron Microscopy (TEM) analysis demonstrated that B. microti infection significantly induced apoptosis and autophagosome formation in tick midgut tissues. Functional assays demonstrated that RNA interference (RNAi)-mediated knockdown of caspase-7, caspase-9, and ATG5 significantly reduced the burden of B. microti. Conversely, pharmacological induction of autophagy using rapamycin increased B. microti load, whereas inhibition with 3-methyladenine (3-MA) decreased B. microti load.DiscussionThese findings underscore the critical roles of apoptosis and autophagy in facilitating B. microti infection within tick midguts, highlighting these pathways as potential molecular targets for disrupting the transmission of tick-borne Babesia infections.

Ticks can transmit a wide range of pathogens that endanger human and animal health. Although repellents are commonly used for tick control, understanding their mechanisms aren't complete. The repellent effects of N, N-diethyl-meta-toluamide (DEET); sec-butyl 2-(2-hydroxyethyl) piperidine-1-carboxylate (icaridin); N, N-diethyl-3-methylbenzamide (IR3535); and cinnamaldehyde on the parthenogenetic tick Haemaphysalis longicornis at the nymph stage were assessed using Y-tubes. The involvement of transient receptor potential (HL-TRP) channel molecules in the repellent mechanism was investigated through in situ hybridization, subcellular localization, real-time fluorescence quantitative polymerase chain reaction (PCR), RNA interference, and electroantennography. In addition, the binding affinity of HL-TRP molecules to repellents was predicted using AlphaFold3. DEET, icaridin, IR3535, and cinnamaldehyde have been shown to effectively repel nymphs. HL-TRP channel is shared among various arthropods, particularly several species of ticks. It is localized to the cell membrane and Haller’s organ. Moreover, microinjection of double-stranded RNA elicited tick repellency behavior, and the electroantennogram responses to those repellents were significantly decreased. The TYR783 site was proposed as an essential binding site to establish hydrogen bonds with icaridin, DEET, and cinnamaldehyde. This exploration of ticks and repellents found that HL-TRP channel functions as a chemosensory receptor for repellents and, thereby, mediates avoidance behavior.

Background: The host skin is the first line of defense against most microorganisms and parasites such as bacteria, fungi and ticks. The immune system present in skin takes part of a sophisticate defense mechanism, firstly as physical, cellular and chemical barriers, followed by a wide range of antimicrobial molecules and specialized immune cells. These cells are responsible for inflammatory processes, antigen uptake and presentation, allergic responses that untimely could control the pathogens. Review: Concerning tick parasitism, skin immunity has a paramount role during tick attachment and blood feeding through both the innate and adaptive responses. In recent years, an increasing number of discoveries in tick physiology revealed a more detailed picture of the role of immune cells and their mediators against tick parasitism. Therefore, a systematic review and summarization of this information can give a more comprehensive understanding of the orchestration of the diverse and complex host immune response mechanisms that reject at least part of infesting ticks and give clues to suggest potential applications to develop better methods for tick control.Conclusion: The local skin immune response to tick and other ectoparasite infestations is intricately influenced by the microenvironment created by parasite attachment components and secreted proteins, attracting and engaging local immune cells. Host immune status further contributes to this dynamic. This review discusses the major cellular responses, functional diversity, and host skin immunity mechanisms stimulated by ticks. However, more research is needed to fill existing gaps and fully understand how the skin responds to ticks and other parasites. For example, studying B-cell responses, their diversity, and exploring the full Th2 immune response could provide valuable insights for improving tick control strategies.Keywords: tick, skin, immunity, parasite, vaccine

The salivary glands of female ticks rapidly degenerate after feeding via programmed cell death mediated by an ecdysteroid receptor (ECR). The degeneration includes both apoptosis and autophagy. The process of degeneration can also be regulated by microRNAs (miRNAs), but the underlying mechanism of miRNA involvement in salivary gland degeneration remains incompletely understood. Here, we demonstrate that microRNA34-5p (miR-34-5p) regulates the process of salivary gland degeneration in Rhipicephalus haemaphysaloides by modulating the target gene RhECR. Dual luciferase reporter assays and phenotypic rescue experiments identified RhECR as a direct target of miR-34-5p. The overexpression and inhibition of miR-34-5p were quantified by hematoxylin and eosin (H E) and Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) staining. The results showed that miR-34-5p inhibited the expression of RhECR to retard apoptosis in salivary gland acini. The study identified the roles of miR-34-5p and RhECR and their interactions in tick salivary gland degeneration. The findings will aid in the application of ECR genes for tick control.

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.

IntroductionThe salivary glands of female ticks rapidly degenerate after feeding. The mechanism involves programmed cell death mediated by an ecdysteroid receptor. A competing endogenous RNA (ceRNA) network has been established using miRNA and the competitive binding of three types of RNA (lncRNA, circRNA, and mRNA), that were demonstrated to be involved in the regulation of biological processes. However, the comprehensive expression profile and competing endogenous RNA (ceRNA) regulatory network between mRNAs and ncRNAs involved in salivary gland development remain unclear.MethodsIn the current study, we employed whole-transcriptome sequencing (RNA sequencing) at various stages of feeding to identify differentially expressed lncRNAs, circRNAs, miRNAs, and mRNAs. The ceRNA networks combining lncRNAs, circRNAs, miRNAs, and mRNAs were predicted and constructed based on the miRanda and TargetScan databases. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed for target mRNAs with significantly different expression levels.ResultsWe identified several pathways related to organ growth and development: Insulin secretion, the Hippo signaling pathway, the Pl3K-Akt signaling pathway, the FoxO signaling pathway, and the Ferroptosis pathway in the lncRNA-miRNA-mRNA network, and Steroid biosynthesis, Cholesterol metabolism, the FoxO signaling pathway, and the Ferroptosis pathway in the circRNA-miRNA-mRNA network, each of which involved insulin and ecdysteroid regulation.DiscussionOur findings have advanced our understanding of the underlying mechanisms of salivary gland development and degeneration.

Download This Paper Open PDF in Browser Add Paper to My Library Share: Permalink Using these links will ensure access to this page indefinitely Copy URL Copy DOI

The study of tick olfaction is relatively new compared to that of insects, and the molecular mechanisms involved remain poorly understood. Despite several potential chemosensory genes identified in multiple tick species, these are yet to be validated through independent functional experiments. In this research, we cloned and analyzed a microplusin-like gene, HlonML-1, and investigated its role in the chemosensory activities of H. longicornis. The results showed that this gene’s amino acid sequences lack histidine residues essential for antimicrobial activity, and it is evolutionarily linked to putative chemosensory microplusins in ticks. Gene expression analyses indicated that HlonML-1 was significantly more abundant in ticks exposed to potential attractants and in the forelegs of H. longicornis than in non-exposed ticks and the hindlegs, respectively. Tick forelegs support the Haller’s organ, which is a sensory structure mostly specialized for chemosensation. Furthermore, Y-tube olfactometer assays indicated that silencing HlonML-1 significantly impaired adult ticks’ ability to detect selected odors, while their gustatory-related behavior remained unaffected compared to the control groups. Given its unique sequences, relative abundance in chemosensory tissues, and impact on odor detection, HlonML-1 is likely involved in the olfactory chemosensation of H. longicornis. Future research validating putative chemosensory microplusins in the genomes of various tick species may enhance our understanding of their olfactory functions in tick and lead to the identification of new molecular targets for developing tick repellents.

Most tick-borne viruses (TBVs) are highly pathogenic and require high biosecurity, which severely limits their study. We found that Sindbis virus (SINV), predominantly transmitted by mosquitoes, can replicate in ticks and be subsequently transmitted, with the potential to serve as a model for studying tick-virus interactions. We found that both larval and nymphal stages of Rhipicephalus haemaphysaloides can be infected with SINV-wild-type (WT) when feeding on infected mice. SINV replicated in two species of ticks (R. haemaphysaloides and Hyalomma asiaticum) after infecting them by microinjection. Injection of ticks with SINV expressing enhanced Green Fluorescent Protein (eGFP) revealed that SINV-eGFP specifically aggregated in the tick midguts for replication. During blood-feeding, SINV-eGFP migrated from the midguts to the salivary glands and was transmitted to a new host. SINV infection caused changes in expression levels of tick genes related to immune responses, substance transport and metabolism, cell growth and death. SINV mainly induced autophagy during the early stage of infection; with increasing time of infection, the level of autophagy decreased, while the level of apoptosis increased. During the early stages of infection, the transcript levels of immune-related genes were significantly upregulated, and then decreased. In addition, SINV induced changes in the transcription levels of some functional genes that play important roles in the interactions between ticks and tick-borne pathogens. These results confirm that the SINV-based transmission model between ticks, viruses, and mammals can be widely used to unravel the interactions between ticks and viruses.

The control and prevention of ticks and tick-borne diseases rely on chemical insecticides and repellents. Plantderived compounds potentially represent new and safer repellents. Cinnamaldehyde, a component of cinnamon oil, exhibits antibacterial, anti-inflammatory, acaricidal, and repellent activity against ticks. Here we studied the molecular mechanism of the repellent effect of cinnamaldehyde on Haemaphysalis longicornis. A 2 % cinnamaldehyde treatment resulted in >90 % nymph repellency within 6 h. Nymphs were exposed to cinnamaldehyde for 30 min, and subsequent transcriptome and metabolome analyses revealed the involvement of H. longicornis Acetylcholinesterases (HL-AchEs) in the response process. HL-AchEs was transcribed in all tick developmental stages and tissues. Following cinnamaldehyde treatment, the transcript and specific activity of the enzyme of AchE were significantly altered. Following RNAi, electroantennography (EAG) tests demonstrated a significant decrease in response to various repellents as well as a significant decrease in repellency. Our findings have revealed that HL-AchEs mediates cinnamaldehyde-induced tick repellency, and the results provide insights into the mechanism of plant-derived tick repellents.

Tick infestations transmit various infectious agents and result in significant socioeconomic consequences. Currently, the primary focus of tick control efforts is identifying potential targets for immune intervention. In a previous study, we identified a highly conserved protein abundant in tick haemolymph extracellular vesicles (EVs) known as translationally controlled tumour protein (TCTP). We have found that native TCTP is present in various tissues of the Rhipicephalus haemaphysaloides tick, including salivary glands, midgut, ovary, and fat body. Notably, TCTP is particularly abundant in the tick ovary and its levels increase progressively from the blood-feeding stage to engorgement. When the TCTP gene was knocked down by RNAi, there was a noticeable delay in ovarian development, and the reproductive performance, in terms of egg quantity and survival, was also hindered. Our investigations have revealed that the observed effects in ovary and eggs in dsRNA-treated ticks are not attributable to cell death mechanisms like apoptosis and autophagy but rather to the reduction in the expression of vitellogenin (Vg1, Vg2, and Vg3) and ferritin (ferritin 1 and ferritin 2) proteins crucial for ovarian development and embryo survival in ticks. Additionally, phylogenetic analysis and structural comparisons of RhTCTP and its orthologues across various tick species, vertebrate hosts, and humans have shown that TCTP is conserved in ticks but differs significantly between ticks and their hosts, particularly in the TCTP_1 and TCTP_2 domains. Overall, TCTP plays a vital role in tick reproductive development and presents itself as a potential target for tick control in both humans and animals.