RNA activation in ticks.

RNA activation (RNAa) is a burgeoning area of research in which double-stranded RNAs (dsRNAs) or small activating RNAs mediate the upregulation of specific genes by targeting the promoter sequence and/or AU-rich elements in the 3'- untranslated region (3'-UTR) of mRNA molecules. So far, studies on the phenomenon have been limited to mammals, plants, bacteria, Caenorhabditis elegans, and recently, Aedes aegypti. However, it is yet to be applied in other arthropods, including ticks, despite the ubiquitous presence of argonaute 2 protein, which is an indispensable requirement for the formation of RNA-induced transcriptional activation complex to enable a dsRNA-mediated gene activation. In this study, we demonstrated for the first time the possible presence of RNAa phenomenon in the tick vector, Haemaphysalis longicornis (Asian longhorned tick). We targeted the 3'-UTR of a novel endochitinase-like gene (HlemCHT) identified previously in H. longicornis eggs for dsRNA-mediated gene activation. Our results showed an increased gene expression in eggs of H. longicornis endochitinase-dsRNA-injected (dsHlemCHT) ticks on day-13 post-oviposition. Furthermore, we observed that eggs of dsHlemCHT ticks exhibited relatively early egg development and hatching, suggesting a dsRNA-mediated activation of the HlemCHT gene in the eggs. This is the first attempt to provide evidence of RNAa in ticks. Although further studies are required to elucidate the detailed mechanism by which RNAa occurs in ticks, the outcome of this study provides new opportunities for the use of RNAa as a gene overexpression tool in future studies on tick biology, to reduce the global burden of ticks and tick-borne diseases.

Localization of secreted ferritin (FER2) in the embryos of the tick Haemaphysalis longicornis.

Despite the absence of a blood meal, embryogenesis involves many processes that require nutrients and other essential elements, including iron. Due to the lack of an external source of these nutrients, these requirements are acquired maternally. Because of the toxic nature of iron, they are transferred through iron transport molecules such as secreted ferritin (FER2). Here we tried to follow the trail of the FER2 through indirect immunofluorescence, and we observed an apparent shift of FER2 from the germ layer at the early part of development to the appendages during the late stage of embryogenesis. FER2 is also found in the middle part of the legs of the embryo. The apparent movement not only sheds light on iron processing events during embryogenesis but also indirectly guides organogenesis in the tick.

Ambivalent Roles of Oxidative Stress in Triangular Relationships among Arthropod Vectors, Pathogens and Hosts.

Blood-feeding arthropods, particularly ticks and mosquitoes are considered the most important vectors of arthropod-borne diseases affecting humans and animals. While feeding on blood meals, arthropods are exposed to high levels of reactive oxygen species (ROS) since heme and other blood components can induce oxidative stress. Different ROS have important roles in interactions among the pathogens, vectors, and hosts. ROS influence various metabolic processes of the arthropods and some have detrimental effects. In this review, we investigate the various roles of ROS in these arthropods, including their innate immunity and the homeostasis of their microbiomes, that is, how ROS are utilized to maintain the balance between the natural microbiota and potential pathogens. We elucidate the mechanism of how ROS are utilized to fight off invading pathogens and how the arthropod-borne pathogens use the arthropods' antioxidant mechanism to defend against these ROS attacks and their possible impact on their vector potentials or their ability to acquire and transmit pathogens. In addition, we describe the possible roles of ROS in chemical insecticide/acaricide activity and/or in the development of resistance. Overall, this underscores the importance of the antioxidant system as a potential target for the control of arthropod and arthropod-borne pathogens.

Relationship between hepatic grayish-white solid nodules in horses imported from Canada and larval Echinococcus multilocularis infection.

Histopathological and genetic examinations were conducted on grayish-white solid hepatic nodules in 150 horses imported from Canada, in order to investigate larval Echinococcus multilocularis infection. Ten of the 150 horses (6.7%) were diagnosed with alveolar hydatid disease. The sequences of the mitochondrial cytochrome b genes obtained from all 10 polymerase chain reaction positive samples had 99 to 100% identity with the European haplotype E1 of E. multilocularis. Therefore, we concluded that the infections likely originated in Canada.

Relation entre les nodules hépatiques solides blanc-grisâtre trouvés chez des chevaux importés du Canada et l'infection larvaire à Echinococcus multilocularis . Des examens histopathologiques et génétiques ont été effectués sur des nodules hépatiques solides blanc-grisâtre observés chez 150 chevaux importés du Canada afin d'étudier l'infection larvaire à Echinococcus multilocularis. Dix des 150 chevaux (6,7 %) ont reçu un diagnostic de maladie hydatique alvéolaire. Les séquences des gènes mitochondriaux du cytochrome b obtenus à partir des 10 échantillons positifs par réaction d'amplification en chaîne par la polymérase ont montré une identité de 99 à 100 % avec l'haplotype européen E1 d'E. multilocularis. L'haplotype d'E. multilocularis obtenu à partir de cette étude suggère que les infections sont probablement originaires du Canada.(Traduit par Dr Serge Messier).

The antiviral immunity of ticks against transmitted viral pathogens.

Ticks, being obligate hematophagous arthropods, are exposed to various blood-borne pathogens, including arboviruses. Consequently, their feeding behavior can readily transmit economically important viral pathogens to humans and animals. With this tightly knit vector and pathogen interaction, the replication and transmission of tick-borne viruses (TBVs) must be highly regulated by their respective tick vectors to avoid any adverse effect on the ticks' biological development and viability. Knowledge about the tick-virus interface, although gaining relevant advances in recent years, is advancing at a slower pace than the scientific developments related to mosquito-virus interactions. The unique and complicated feeding behavior of ticks, compared to that of other blood-feeding arthropods, also limits the studies that would further elaborate the antiviral immunity of ticks against TBVs. Hence, knowledge of molecular and cellular immune mechanisms at the tick-virus interface, will further elucidate the successful viral replication of TBVs in ticks and their effective transmission to human and animal hosts.

An Ixodes scapularis glutathione S-transferase plays a role in cell survival and viability during Langat virus infection of a tick cell line.

Ticks are important vectors of diseases affecting both humans and animals. To be an efficient vector, ticks have to survive infection by pathogens such as the Langat virus (LGTV). One method utilized by ticks is their complex antioxidant mechanism. Included in the vast antioxidant processes are several enzymes involved in redox homeostasis. The ubiquitous glutathione S-transferases (GSTs) belong to the antioxidant family of enzymes. In this study, we evaluated the role of a GST during LGTV infection. ISE6 cells were infected with LGTV with a multiplicity of infection (MOI) of 0.01 and observed daily. The infection success was monitored via indirect immunofluorescent antibody test (IFAT) for LGTV for up to 4 days. The gene expression of IsGST1 was determined by real-time polymerase chain reaction (PCR) using IsGST1 gene-specific primers. Knockdown of the IsGST1 gene with subsequent LGTV infection was also performed. Afterward, ISE6 cell mortality and viability were checked daily until the fourth day. The virus titer from supernatants of IsGST1-knockdown cells was quantified using a focus-formation assay. IFAT data showed that LGTV infects ISE6 cells in a time-dependent manner with increasing infection from day 0 to day 4. The IsGST1 genes showed an increasing expression until day 2 of infection, while decreased expression was observed from day 3 to day 4 post-infection. Knockdown of the IsGST1 resulted in increased mortality on the third day of infection, while the cell viability was also negatively affected by the knockdown of the IsGST1 genes from day 0 to day 4 post-infection. Knockdown of the IsGST1 genes also resulted in a decreased viral titer from the supernatants of the ISE6 cells infected with LGTV. Based on the results, GSTs are possibly utilized both by cells and the virus for mutual survival and proliferation.

Expression analysis of glutathione S-transferases and ferritins during the embryogenesis of the tick Haemaphysalis longicornis.

In the tick life cycle, embryogenesis is the only stage of development wherein no blood meal is required. Nevertheless, even in the absence of a blood meal, which is the source of nutrients as well as the ferrous iron and heme that could cause oxidative stress in ticks, malondialdehyde (MDA) has been reported to increase during this period. Additionally, the knockdown of some oxidative stress-related molecules such as ferritin has resulted in abnormal eggs and embryonic death. Here, we investigate the gene and protein expression profiles of the identified glutathione S-transferases (GSTs) and ferritins (Fers) of the tick H. longicornis during embryogenesis through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting, respectively. We also confirm the lipid peroxidation and ferrous iron concentration level using a thiobarbituric acid reactive substances (TBARS) assay. Finally, we attempt to correlate these findings with the events occurring by establishing a staging process in H. longicornis embryos. Lipid peroxidation increased during the course of embryogenesis, as does the amount of GST proteins. On the other hand, the GST genes have high expression at the 1st day post-oviposition, during the early stage of embryogenesis and at day 10 during the period wherein the germ band is observable. Fer gene expression also starts to increase at day 10 and peaks at day 15. In the ferritin proteins, only the secretory ferritin (Fer2) is detected and constitutively expressed during embryogenesis. Events occurring during embryogenesis, such as energy production and iron metabolism for cellular proliferation and differentiation cause oxidative stress in the embryo. To counteract oxidative stress, it is possible that the embryo may utilize oxidative stress-related molecules such as GSTs and Fer2, which could be either maternally or embryo-derived.

A Peroxiredoxin From the Haemaphysalis longicornis Tick Affects Langat Virus Replication in a Hamster Cell Line.

Ticks are hematophagous arthropods, and their blood feeding on vertebrate hosts is essential for their development. The vertebrate blood contains high levels of free iron that can react with oxygen in ticks, resulting in the production of hydrogen peroxide (H2O2), one of the reactive oxygen species. Peroxiredoxins (Prxs), H2O2-scavenging enzymes, take on an important role in the ticks' oxidative stress coping mechanism. Ticks also transmit several disease-causing pathogens, including tick-borne encephalitis virus (TBEV), in animals and humans. Therefore, the control of ticks and tick-borne pathogens is a key issue that needs to be addressed. Infection with an arthropod-borne flavivirus is known to induce oxidative stress in insect cells. We hypothesize that vector-derived Prxs could have an effect on the infection and/or replication of flaviviruses in the hosts, since ticks Prxs are possibly transmitted from ticks to their hosts. In this study, we established stable strains of baby hamster kidney (BHK) cells expressing two types of H2O2-scavenging Prxs from the hard tick Haemaphysalis longicornis (BHK-HlPrx and BHK-HlPrx2 cells). Although the infection of TBEV surrogate Langat virus (LGTV) did not induce H2O2 production in normal BHK cells, the mortality rate and the virus titer of LGTV infected BHK-HlPrx cells increased. In addition, HlPrx proteins in BHK cells can facilitate LGTV replication in cells, while HlPrx2 proteins in BHK cells cannot. The results also demonstrated that this facilitation of LGTV replication by the 1-Cys Prx in the BHK cells is not by scavenging H2O2 but by an unknown mechanism. In order to understand this mechanism, more studies using tick-derived cells and ticks are necessary.

Characterization of an iron-inducible Haemaphysalis longicornis tick-derived promoter in an Ixodes scapularis-derived tick cell line (ISE6).

BACKGROUND: Ticks are important vectors of disease-causing pathogens. With the rise of resistance to chemical acaricides, alternative methods in tick control are warranted. Gene manipulation has been successful in controlling mosquitoes and mosquito-borne diseases and is now looked upon as a candidate method to control ticks and tick-borne pathogens. Our previous study has identified the actin and ferritin promoter regions in the Haemaphysalis longicornis tick. RESULTS: Here, the ferritin-derived promoter from the H. longicornis tick was characterized in silico, and the core promoter sequences and some of its important components were identified. Several truncations of the promoter region were created and inserted to a reporter plasmid to determine the important components for its activity. The activities of the truncated promoters on the Ixodes scapularis tick cell line (ISE6) were measured via a dual luciferase assay using experimental and control reporter genes. To induce the promoter's activity, transfected ISE6 cells were exposed to ferrous sulfate. The 639 nucleotides truncated promoter showed the highest activity on ISE6 cells when exposed to 1 mM ferrous sulfate. CONCLUSION: In this study, we characterized an iron-inducible tick promoter that could be a valuable tool in the development of a gene-manipulation system to control ticks and tick-borne pathogens.

The case for oxidative stress molecule involvement in the tick-pathogen interactions -an omics approach.

The blood-feeding behavior of ticks has resulted in them becoming one of the most important vectors of disease-causing pathogens. Ticks possess a well-developed innate immune system to counter invading pathogens. However, the coevolution of ticks with tick-borne pathogens has adapted these pathogens to the tick's physiology and immune response through several mechanisms including transcriptional regulation. The recent development in tick and tick-borne disease research greatly involved the "omics" approach. The omics approach takes a look en masse at the different genes, proteins, metabolomes, and the microbiome of the ticks that could be differentiated during pathogen infection. Data from this approach revealed that oxidative stress-related molecules in ticks are differentiated and possibly being exploited by the pathogens to evade the tick's immune response. In this study, we review and discuss transcriptomic and proteomic data for some oxidative stress molecules differentially expressed during pathogen infection. We also discuss metabolomics and microbiome data as well as functional genomics in order to provide insight into the tick-pathogen interaction.

Induction of intracellular ferritin expression in embryo-derived Ixodes scapularis cell line (ISE6).

Iron is a very important nutrient for cells; however, it could also cause fatal effects because of its capability to trigger oxidative stress. Due to high exposure to iron from their blood diet, ticks make use of several mechanisms to cope up with oxidative stress. One mechanism is iron sequestration by ferritin and its control protein (IRP). Since the IRP activity is dependent on the ferrous iron concentration, we tried to induce intracellular ferritin (FER1) protein expression by exposing Ixodes scapularis embryo-derived cell line (ISE6) to different concentrations of ferrous sulphate at different time points. We were able to induce FER1 protein after exposure to 2 mM of ferrous sulphate for 48 h, as observed in both Western blotting and indirect immunofluorescent antibody tests. This could indicate that the FER1 produced could be a product of the release of IRPs from the FER1 mRNA leading to its translation. The RNA interference of FER1, through the transfection of dsRNA, led to an increase in mortality and decrease in the cellular proliferation of ISE6 cells. Overall, ISE6 cells could be a good tool in further understanding the mechanism of FER1 action, not just in Ixodes ticks but in other tick species as well.

Glutathione S-transferases play a role in the detoxification of flumethrin and chlorpyrifos in Haemaphysalis longicornis.

BACKGROUND: Haemaphysalis longicornis is a tick of importance to health, as it serves as a vector of several pathogens, including Theileria orientalis, Babesia ovata, Rickettsia japonica and the severe fever with thrombocytopenia syndrome virus (SFTSV). Presently, the major method of control for this tick is the use of chemical acaricides. The glutathione S-transferase (GST) system is one mechanism through which the tick metabolizes these acaricides. Two GSTs from H. longicornis (HlGST and HlGST2) have been previously identified. RESULTS: Enzyme kinetic studies were performed to determine the interaction of acaricides with recombinant H. longicornis GSTs. Recombinant HlGST activity was inhibited by flumethrin and cypermethrin, while recombinant HlGST2 activity was inhibited by chlorpyrifos and cypermethrin. Using real-time RT-PCR, the upregulation of the HlGST gene was observed upon exposure to sublethal doses of flumethrin, while the HlGST2 gene was upregulated when exposed to sublethal doses of chlorpyrifos. Sex and strain dependencies in the induction of GST gene expression by flumethrin were also observed. Knockdown of the HlGST gene resulted in the increased susceptibility of larvae and adult male ticks to sublethal doses of flumethrin and the susceptibility of larvae against sublethal doses of chlorpyrifos was increased upon knockdown of HlGST2. CONCLUSIONS: HlGST could be vital for the metabolism of flumethrin in larvae and adult male ticks, while HlGST2 is important in the detoxification of chlorpyrifos in larval ticks.

Hemolymph defensin from the hard tick Haemaphysalis longicornis attacks Gram-positive bacteria.

Ticks are key vectors of some important diseases of humans and animals. Although they are carriers of disease agents, the viability and development of ticks are not harmed by the infectious agents due to their innate immunity. Antimicrobial peptides directly protect hosts against pathogenic agents such as viruses, bacteria, and parasites. Among the identified and characterized antimicrobial peptides, defensins have been considerably well studied. Defensins are commonly found among fungi, plants, invertebrates, and vertebrates. The sequence of the tick hemolymph defensin (HEdefensin) gene from the hard tick Haemaphysalis longicornis was analyzed after identification and cloning from a cDNA library. HEdefensin has a predicted molecular mass of 8.15 kDa including signal peptides and a theoretical isoelectric point of 9.48. Six cysteine residues were also identified in the amino acids. The synthetic HEdefensin peptide only showed antibacterial activity against Gram-positive bacteria such as Micrococcus luteus. A fluorescence propidium iodide exclusion assay also showed that HEdefensin increased the membrane permeability of M. luteus. Additionally, an indirect fluorescent antibody test showed that HEdefensin binds to M. luteus. These results suggested that HEdefensin strongly affects the innate immunity of ticks against Gram-positive bacteria.

Vector competence of Haemaphysalis longicornis ticks for a Japanese isolate of the Thogoto virus.

Thogoto virus (THOV), a tick-borne arbovirus not previously reported in East Asia, was recently isolated from Haemaphysalis longicornis in Kyoto, Japan. In this study, we investigated the vector competence of H. longicornis ticks for a Japanese isolate of the Thogoto virus using anal pore microinjection and experimental virus acquisition. Our results showed that anal pore microinjection can readily infect adult ticks, and THOV-infected ticks can successfully transmit the virus to mice. Blood feeding was also critical in the distribution of the virus in tick organs, most especially in the salivary glands. Furthermore, co-feeding between an infected adult and naïve nymphs can also produce infected molted adults that can horizontally transmit THOV to mice. Altogether, our results suggest that H. longicornis is a competent vector for the Japanese THOV isolate and could be the primary tick vector of the virus in Japan.

Peroxiredoxins are important for the regulation of hydrogen peroxide concentrations in ticks and tick cell line.

Ticks are obligate hematophagous ectoparasites, as they need to feed blood from vertebrate hosts for development. Host blood contains high levels of iron. Host-derived iron may lead to high levels of reactive oxygen species (ROS), including hydrogen peroxide (H2O2). Since a high concentration of H2O2 causes serious damage to organisms, this molecule is known to be a harmful chemical compound for aerobic organisms. On the other hand, the transparent method is compatible with chemical fluorescent probes. Therefore, we tried to establish the visualizing method for H2O2 in unfed tick tissues. The combination method of a chemical fluorescent probe (BES-H2O2-Ac) with the transparent method, Scale, demonstrated in unfed tick tissues that H2O2 and paraquat could induce oxidative stress in the tissues, such as the midgut and ovary. In addition, an H2O2 detection method using BES-H2O2-Ac was established in Ixodes scapularis embryo-derived cell line (ISE6) in vitro to evaluate the antioxidant activity of peroxiredoxins (PRXs), H2O2 scavenging enzymes, against H2O2 in the cells. The effects of paraquat in ISE6 cells were also observed in the PRXs gene-silenced ISE6 cells. A high intensity of H2O2 fluorescence induced by paraquat was observed in the PRX gene-knockdowned cells. These results suggest that H2O2 and paraquat act as an H2O2 inducer, and PRX genes are important for the regulation of the H2O2 concentration in unfed ticks and ISE6 cells. Therefore, this study contributes to the search for H2O2 visualization in ticks and tick cell line and furthers understanding of the tick's oxidative stress induced by H2O2.

Characterization and expression analysis of a newly identified glutathione S-transferase of the hard tick Haemaphysalis longicornis during blood-feeding.

BACKGROUND: Ticks are obligate hematophagous parasites important economically and to health. Ticks consume large amounts of blood for their survival and reproduction; however, large amounts of iron in blood could lead to oxidative stress. Ticks use several molecules such as glutathione S-transferases (GSTs), ferritins, and peroxiredoxins to cope with oxidative stress. This study aimed to identify and characterize the GSTs of the hard tick Haemaphysalis longicornis in order to determine if they have a role in coping with oxidative stress. METHODS: Genes encoding GSTs of H. longicornis were isolated from the midgut CDNA library. Genes have been cloned and recombinant GSTs have been expressed. The enzymatic activities, enzyme kinetic constants, and optimal pH of the recombinant GSTs toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined. The gene transcription and protein expression profiles were determined in the whole ticks and internal organs, and developmental stages using real time RT-PCR and Western blotting during blood feeding. The localization of GST proteins in organs was also observed using immunofluorescent antibody test (IFAT). RESULTS: We have isolated two genes encoding GSTs (HlGST and HlGST2). The enzymatic activity toward CDNB is 9.75 ± 3.04 units/mg protein for recombinant HlGST and 11.63 ± 4.08 units/mg protein for recombinant HlGST2. Kinetic analysis of recombinant HlGST showed K m values of 0.82 ± 0.14 mM and 0.64 ± 0.32 mM for the function of CDNB and GSH, respectively. Meanwhile, recombinant HlGST2 has K m values of 0.61 ± 0.20 mM and 0.53 ± 0.02 mM for the function of CDNB and GSH, respectively. The optimum pH of recombinant HlGST and recombinant HlGST2 activity was 7.5-8.0. Transcription of both GSTs increases in different developmental stages and organs during blood-feeding. GST proteins are upregulated during blood-feeding but decreased upon engorgement in whole ticks and in some organs, such as the midgut and hemocytes. Interestingly, salivary glands, ovaries, and fat bodies showed decreasing protein expression during blood-feeding to engorgement. Varying localization of GSTs in the midgut, salivary glands, fat bodies, ovaries, and hemocytes was observed depending on the feeding state, especially in the midgut and salivary glands. CONCLUSIONS: In summary, a novel GST of H. longicornis has been identified. Characterization of the GSTs showed that GSTs have positive correlation with the degree and localization of oxidative stress during blood-feeding. This could indicate their protective role during oxidative stress.

Evaluation of vaccine potential of 2-Cys peroxiredoxin from the hard tick Haemaphysalis longicornis.

Ticks require blood feeding on vertebrate animals throughout their life cycle, and also concentrate the iron-containing blood, resulting in a high concentration of hydrogen peroxide (H2O2). High concentrations of H2O2 are harmful to organisms, due to their serious damage of macromolecules. Ticks have antioxidant enzymes, such as peroxiredoxins (Prxs), that scavenge H2O2. Prxs may have important roles in regulating the H2O2 concentration in ticks during blood feeding and oviposition. Moreover, Prxs are considered potential vaccine candidates in other parasites, such as Leishmania and Fasciola. In the present study, the efficacy of a tick Prx (HlPrx2) as a vaccine candidate antigen was evaluated. First, recombinant HlPrx2 (rHlPrx2) was expressed in Escherichia coli, and then, its purity and endotoxin levels were confirmed prior to administration. The rHlPrx2 proteins were of high purity with acceptably low endotoxin levels. Second, the ability of rHlPrx2 administration to stimulate mouse immunity was evaluated. The rHlPrx2 protein, with or without an adjuvant, could stimulate immunity in mice, especially the IgG1 of Th2 immune response. Using Western blot analysis, we also observed whether rHlPrx2-immunized mice sera could recognize native HlPrx2 protein in crude tick midgut proteins. Western blot analysis demonstrated that rHlPrx2-administrated mouse sera could detect the native HlPrx2. Finally, the effects of rHlPrx2 immunization in mice were studied using nymphal ticks. Although the challenged ticks were not affected by rHlPrx2 immunization, rHlPrx2 still might be considered as a vaccine candidate against ticks because of its high immunogenicity.

Immunofluorescent detection in the ovary of host antibodies against a secretory ferritin injected into female Haemaphysalis longicornis ticks.

Due to the continuous threat of ticks and tick-borne diseases to human and animal health worldwide, and the drawbacks of chemical acaricide application, many researchers are exploring vaccination as an alternative tick control method. Earlier studies have shown that host antibodies can circulate in the ticks, but it has not been confirmed whether these antibodies can be passed on to the eggs. We previously reported that ticks infesting rabbits immunized with a recombinant secretory ferritin of Haemaphysalis longicornis (HlFER2) had reduced egg production and hatching. Here we attempted to detect the presence of antibodies against HlFER2 in the ovary and eggs of female ticks through immunofluorescent visualization. Purified anti-HlFER2 antibodies or rabbit IgG for control was directly injected to engorged female H. longicornis. Ovaries and eggs after oviposition were collected and prepared for an indirect immunofluorescent antibody test. Positive fluorescence was detected in ovaries one day post-injection of anti-HlFER2 antibodies. Through silencing of Hlfer2 gene, we also determined whether the injected antibodies can specifically bind to native HlFER2. Immunofluorescence was observed in the oocytes of dsLuciferase control ticks injected with anti-HlFER2 antibodies, but not in the oocytes of Hlfer2-silenced ticks also injected with anti-HlFER2 antibodies. Our current findings suggest that host antibodies can be passed on to the oocytes, which is significant in formulating a vaccine that can disrupt tick reproduction.

Synchronous Langat Virus Infection of Haemaphysalis longicornis Using Anal Pore Microinjection.

The tick-borne encephalitis virus (TBEV) serocomplex of flaviviruses consists of arboviruses that cause important diseases in animals and humans. The transmission of this group of viruses is commonly associated with tick species such as Ixodes spp., Dermacentor spp., and Hyalomma spp. In the case of Haemaphysalis longicornis, the detection and isolation of flaviviruses have been previously reported. However, studies showing survival dynamics of any tick-borne flavivirus in H. longicornis are still lacking. In this study, an anal pore microinjection method was used to infect adult H. longicornis with Langat virus (LGTV), a naturally attenuated member of the TBEV serocomplex. LGTV detection in ticks was done by real-time PCR, virus isolation, and indirect immunofluorescent antibody test. The maximum viral titer was recorded at 28 days post-inoculation, and midgut cells were shown to be the primary replication site. The tick can also harbor the virus for at least 120 days and can successfully transmit LGTV to susceptible mice as confirmed by detection of LGTV antibodies. However, no transovarial transmission was observed from the egg and larval samples. Taken together, our results highly suggest that anal pore microinjection can be an effective method in infecting adult H. longicornis, which can greatly assist in our efforts to study tick and virus interactions.

Characterization and antiviral activity of a newly identified defensin-like peptide, HEdefensin, in the hard tick Haemaphysalis longicornis.

Tick defensins are antimicrobial peptides that play a major role in the innate immunity of ticks by providing a direct antimicrobial defense. In this study, we identified and characterized a defensin-like encoding gene, HEdefensin, from the expressed sequence tags (EST) database of hemolymph from the hard tick Haemaphysalis longicornis. Expression of the gene in whole adult ticks and in different organs was upregulated during blood feeding, though not after Langat virus (LGTV) challenge. A synthetic HEdefensin peptide demonstrated significant virucidal activity against LGTV but not against an adenovirus in co-incubation virucidal assays. Moreover, the RNAi-mediated gene silencing of HEdefensin did not significantly affect the virus titer as compared to the control group. The data reported here have established the in vitro virucidal activity of the peptide against LGTV. However, its role in the innate antiviral immunity of H. longicornis remains to be explored, and further studies are needed to fully evaluate the potential biological activities of the peptide against bacteria, fungi or parasites.