Tick Haemaphysalis Longicornis Research Articles

The Complete Mitochondrial Genome and Expression Profile of Mitochondrial Protein-Coding Genes in the Bisexual and Parthenogenetic Haemaphysalis longicornis.

The tick Haemaphysalis longicornis is widely distributed in eastern Asia, New Zealand and Australia, and is well-known as a vector of multiple zoonotic pathogens. This species exhibits two reproductive strategies, bisexual and obligate parthenogenetic reproduction. Hence, in the current study, the complete mitochondrial genomes of the bisexual and parthenogenetic populations were assembled and analyzed, and the expression of the mitochondrial protein-coding genes was evaluated and compared between the two reproductive populations. The results indicated that the length of the mitochondrial genomes of the two reproductive populations is 14,694 and 14,693 bp in the bisexual and parthenogenetic populations, respectively. The AT content in the mitochondrial genome of the bisexual and obligate parthenogenetic population reached 77.22 and 77.34%, respectively. The phylogenetic tree was constructed combining 13 protein-coding genes, which showed that the genetic distance between the bisexual and parthenogenetic populations was less than that between the subspecies. The expression of the mitochondrial protein-coding genes was quantitatively analyzed at different feeding status for the bisexual and parthenogenetic populations, and the results showed significant differences in the expression patterns of these genes, suggesting that they might trigger specific energy utilization mechanisms due to their different reproductive strategies and environmental pressures.

First Recognized Human Bite in the United States by the Asian Longhorned Tick, Haemaphysalis longicornis.

We present the case summary of the first human recognized to have been bitten by the Haemaphysalis longicornis tick in the United States, which occurred in New York State. Subsequent field studies confirmed that this tick was present in multiple geographic locations near the patient's residence, including on manicured lawns.

Vitellogenin Receptor as a Target for Tick Control: A Mini-Review.

While much effort has been put into understanding vitellogenesis in insects and other organisms, much less is known of this process in ticks. There are several steps that facilitate yolk formation in developing oocytes of which the vitellogenin receptor (VgR) is a key component. The tick VgR binds vitellogenin (Vg) circulating in the hemolymph to initiate receptor-mediated endocytosis and its transformation into vitellin (Vn). The conversion of Vg into Vn, the final form of the yolk protein, occurs inside oocytes of the female tick ovary. Vn is critical to tick embryos since it serves as the nutritional source for their development, survival, and reproduction. Recent studies also suggest that pathogenic microbes, i.e., Babesia spp., that rely on ticks for propagation and dissemination likely “hitchhike” onto Vg molecules as they enter developing oocytes through the VgR. Suppressing VgR messenger RNA synthesis via RNA interference (RNAi) completely blocked Babesia spp. transmission into developing tick oocytes, thereby inhibiting vertical transmission of these pathogenic microbes from female to eggs. To date, VgRs from only four tick species, Dermacentor variabilis, Rhipicephalus microplus, Amblyomma hebraeum, and Haemaphysalis longicornis, have been fully sequenced and characterized. In contrast, many more VgRs have been described in various insect species. VgR is a critical component in egg formation and maturation that can serve as a precise target for tick control. However, additional research will help identify unique residues within the receptor that are specific to ticks or other arthropod disease vectors while avoiding cross-reactivity with non-target species. Detailed knowledge of the molecular structure and functional role of tick VgRs will enable development of novel vaccines to control ticks and tick-borne diseases.

A 4-Yr Survey of the Range of Ticks and Tick-Borne Pathogens in the Lehigh Valley Region of Eastern Pennsylvania.

Questing ticks were surveyed by dragging in forested habitats within the Lehigh Valley region of eastern Pennsylvania for four consecutive summers (2015–2018). A high level of inter-annual variation was found in the density of blacklegged tick nymphs, Ixodes scapularis Say, with a high density of host-seeking nymphs (DON) in summer 2015 and 2017 and a relatively low DON in summer 2016 and 2018. Very few American dog ticks (Dermacentor variabilis Say) and Ixodes cookei Packard were collected. Lone star ticks (Amblyomma americanum L.) and longhorned ticks (Haemaphysalis longicornis Neumann) were not represented among the 6,398 ticks collected. For tick-borne pathogen surveillance, DNA samples from 1,721 I. scapularis nymphs were prepared from specimens collected in summers 2015–2017 and screened using qPCR, high resolution melting analysis, and DNA sequencing when necessary. The overall 3-yr nymphal infection prevalence of Borrelia burgdorferi was 24.8%, Borrelia miyamotoi was 0.3%, Anaplasma phagocytophilum variant-ha was 0.8%, and Babesia microti was 2.8%. Prevalence of coinfection with B. burgdorferi and B. microti as well as B. burgdorferi and A. phagocytophilum variant-ha were significantly higher than would be expected by independent infection. B. burgdorferi nymphal infection prevalence is similar to what other studies have found in the Hudson Valley region of New York, but levels of B. microti and A. phagocytophilum variant-ha nymphal infection prevalence are relatively lower. This study reinforces the urgent need for continued tick and pathogen surveillance in the Lehigh Valley region.

Multistate Infestation With the Exotic Disease-vector Tick Haemaphysalis longicornis—United States, August 2017 to September 2018

The Pediatric Infectious Disease Journal 38(4):p 418, April 2019. | DOI: 10.1097/INF.0000000000002282

The mRNA expression and enzymatic activity of three enzymes during embryonic development of the hard tick Haemaphysalis longicornis

BackgroundThree main enzymes including cathepsin B, cathepsin D and acid phosphatase are involved in vitellin degradation, which is a major biochemical event of the embryonic development and can provide nutrients and metabolites for tick embryos. In the present study, the mRNA expression profiles and enzymatic activity of cathepsin B, cathepsin D and acid phosphatase were investigated during embryonic development in the tick Haemaphysalis longicornis.ResultsThe results revealed that all three enzymes were expressed throughout embryonic development. Both cathepsin B and acid phosphatase transcripts were accumulated during the first four days. Cathepsin B reached its highest expression on day 5, whereas the peak expression of acid phosphatase and cathepsin D occurred on day 11. The highest activity of cathepsin B was observed on the first day of egg development, whereas cathepsin D reached its highest activity on day 13. Acid phosphatase activity increased gradually during the first five days and then remained stable until the end of egg development.ConclusionsThree enzymes were expressed and activated in eggs, and also presented different dynamic changes with the development of embryos. The profiles of both mRNA expression and enzymatic activity of these enzymes indicate that they are controlled orderly and play multiple roles during embryonic development in ticks.

NewsCAP: Haemaphysalis longicornis tick reported in nine states.

NewsCAP: Haemaphysalis longicornis tick reported in nine states.

A microRNA profile of saliva and the role of miR-375 in Haemaphysalis longicornis (Ixodida: Ixodidae)

BackgroundTick saliva contains many bioactive molecules that are involved in attachment to the host, blood-feeding and transmission of pathogens. MicroRNAs (miRNAs) are a class of short non-coding RNAs with a length of 19–24 nucleotides. They act as regulators of gene expression by binding to their target mRNA at the post-transcriptional level and control a variety of cellular functions, including regulation of growth, metabolism and development. The detection and characterizations of miRNAs from tick saliva may help explain the molecular mechanisms involved in the interaction between ticks, pathogens and hosts. They may also contribute to the discovery of vaccines, which can control ticks and the pathogens they transmit.ResultsAn RNA library was generated from the saliva of fed adult Haemaphysalis longicornis ticks, containing 17.4 million clean reads of 18–30 nucleotides. Overall, 319 known miRNAs and 1 novel miRNA were found. The 10 most abundantly expressed miRNAs present in tick saliva were miR-100_2, miR-315, miR-184_1, miR-100-5p_2, miR-5307, miR-184-3p_3, Let-7-5p_6, miR-71_5, miR-1-3p_6 and miR-10-5p_2. miR-375, one of the abundantly expressed, was subjected to quantitative real-time PCR analysis (qRT-PCR) in various tick developmental stages, as well as in different tissues isolated from adult ticks. The expression of miR-375 in different tick development stages was highest in unfed nymphs and lowest in the egg stage. In the tissues of adult ticks, miR-375 was most highly expressed in the salivary gland. To investigate the possible role of miR-375, Ant-375 was used to inhibit the miR-375. The treated group (Ant-375) had a reduced number of eggs (t(10) = 2.652, P = 0.0242), eggs that were partially desiccated, and reduced egg hatchability (t(10) = 2.272, P = 0.044) compared to Ms-Ant and the non-injected control.ConclusionsThis is the first study to investigate the miRNA profile in tick saliva and the role of miR-375 in H. longicornis. The identification and characterization of miRNA in tick saliva may help to reveal the molecular mechanisms of interactions among ticks, pathogens and hosts, and suggest new vaccine strategies to control tick-borne diseases.

Human Tick-Borne Diseases in Australia.

There are 17 human-biting ticks known in Australia. The bites of Ixodes holocyclus, Ornithodoros capensis, and Ornithodoros gurneyi can cause paralysis, inflammation, and severe local and systemic reactions in humans, respectively. Six ticks, including Amblyomma triguttatum, Bothriocroton hydrosauri, Haemaphysalis novaeguineae, Ixodes cornuatus, Ixodes holocyclus, and Ixodes tasmani may transmit Coxiella burnetii, Rickettsia australis, Rickettsia honei, or Rickettsia honei subsp. marmionii. These bacterial pathogens cause Q fever, Queensland tick typhus (QTT), Flinders Island spotted fever (FISF), and Australian spotted fever (ASF). It is also believed that babesiosis can be transmitted by ticks to humans in Australia. In addition, Argas robertsi, Haemaphysalis bancrofti, Haemaphysalis longicornis, Ixodes hirsti, Rhipicephalus australis, and Rhipicephalus sanguineus ticks may play active roles in transmission of other pathogens that already exist or could potentially be introduced into Australia. These pathogens include Anaplasma spp., Bartonella spp., Burkholderia spp., Francisella spp., Dera Ghazi Khan virus (DGKV), tick-borne encephalitis virus (TBEV), Lake Clarendon virus (LCV), Saumarez Reef virus (SREV), Upolu virus (UPOV), or Vinegar Hill virus (VINHV). It is important to regularly update clinicians' knowledge about tick-borne infections because these bacteria and arboviruses are pathogens of humans that may cause fatal illness. An increase in the incidence of tick-borne infections of human may be observed in the future due to changes in demography, climate change, and increase in travel and shipments and even migratory patterns of birds or other animals. Moreover, the geographical conditions of Australia are favorable for many exotic ticks, which may become endemic to Australia given an opportunity. There are some human pathogens, such as Rickettsia conorii and Rickettsia rickettsii that are not currently present in Australia, but can be transmitted by some human-biting ticks found in Australia, such as Rhipicephalus sanguineus, if they enter and establish in this country. Despite these threats, our knowledge of Australian ticks and tick-borne diseases is in its infancy.

Comprehensive Analysis of the Global Protein Changes That Occur During Salivary Gland Degeneration in Female Ixodid Ticks Haemaphysalis longicornis.

Ticks are notorious blood-sucking arthropods that can spread a variety of pathogens and cause great harm to the health of humans, wildlife and domestic animals. The salivary glands of female ticks degenerate rapidly when the ticks reach critical weight or become engorged, which can be caused by hormones and by the synergistic effects of multiple proteins. To explore the complex molecular mechanisms of salivary gland degeneration in ticks, this study applies iTRAQ quantitative proteomic technology for the first time to study changes in protein expression in the salivary glands of female Haemaphysalis longicornis during the process of degeneration and to search for proteins that play an important role in salivary gland degeneration. It was found that the expression of some proteins associated with energy production was continuously down-regulated during salivary gland degeneration, while some proteins associated with DNA or protein degradation were consistently up-regulated. Furthermore, the expression of some proteins related to cell apoptosis or autophagy was also changed. These proteins were knocked down by RNAi to observe the phenotypic and physiological changes in female ticks. The results showed that the time required for engorgement and the mortality rates of the female ticks increased after RNAi of F0F1-type ATP synthase, NADH-ubiquinone oxidoreductase, cytochrome C, or apoptosis-inducing factor (AIF). The corresponding engorged weights, oviposition amounts, and egg hatching rates of the female ticks decreased after RNAi. Interference of the expression of AIF in engorged ticks by RNAi showed that the degeneration of salivary glands of female ticks was slowed down.

Efficacy of orally administered fluralaner in dogs against laboratory challenge with Haemaphysalis longicornis ticks

BackgroundHaemaphysalis longicornis ticks represent an ectoparasitic health threat to dogs. This study evaluated the immediate and persistent efficacy of orally administered fluralaner for control of this tick.MethodsTwenty-four dogs were sorted into 4 groups based on their tick carrying capacity measured in a preliminary challenge. Two days before treatment, dogs were challenged with Haemaphysalis longicornis and then at the time of treatment dogs received with oral fluralaner at 10, 25 or 50 mg/kg respectively to 3 of the groups, while the remaining group was sham treated. Ticks were counted and categorized on all dogs 2 days after treatment (4 days after challenge). Tick challenges were repeated at 28, 56, 84 and 112 days following treatment with tick counts 48 hours following each challenge. Tick control efficacy was evaluated by comparing the mean (geometric) total live attached and dead engorged ticks on each fluralaner treated group with the sham treated dogs.ResultsOral fluralaner is highly acaricidal for H. longicornis that feed on treated dogs. The mean efficacy rate in dogs treated with fluralaner at the commercial dose range of 25 to 50 mg/kg was greater than 90% at 114 days after treatment, whereas efficacy at this time in dogs treated at 10 mg/kg was 79%.ConclusionsFluralaner administered orally to dogs within the commercial dose range at 25 to 50 mg/kg is effective for up to 114 days against laboratory challenge with H. longicornis ticks.

Molecular Detection and Characterization of p44/msp2 Multigene Family of Anaplasma phagocytophilum from Haemaphysalis longicornis in Mie Prefecture, Japan.

Anaplasma phagocytophilum is an obligate intracellular bacterium that causes human granulocytic anaplasmosis (HGA), an emerging tick-borne infectious disease. This bacterium expresses various 44-kDa major outer membrane proteins encoded by the p44/msp2 multigene family to avoid the host immune system. We previously detected A. phagocytophilum p44/msp2 from the tick Haemaphysalis longicornis in Mie Prefecture, Japan in 2008. In this study, we further investigated a total of 483 H. longicornis ticks (220 adults and 263 nymphs) collected from the Mie Prefecture by PCR targeting p44/msp2 to characterize the p44/msp2 multigene family of A. phagocytophilum. Six of the 483 ticks tested were PCR-positive for A. phagocytophilum p44/msp2, and these positive individuals were at the nymph stage of the tick life cycle. Cloning, sequencing, and phylogenetic analyses of the amplicons revealed that the 11 p44/msp2 clones obtained from the positive ticks shared a 54.9%-99.3% amino acid sequence similarity with the 27 previously identified clones from HGA patients in Japan. In particular, 6 p44/msp2 clones displayed the highest similarities (97.2%-99.3%) with 3 previously identified clones (FJ417343, FJ417345, FJ417357). Thus, the data from this study provide important public health information regarding A. phagocytophilum infection transmitted by H. longicornis ticks, especially at the nymph stage.

Modeling the Asian Longhorned Tick (Acari: Ixodidae) Suitable Habitat in North America.

Asian longhorned or bush tick (Haemaphysalis longicornis Neumann) is a vector species of considerable medical and veterinary importance within its native range in East Asia, and in introduced areas of Australia and Oceania. Recently, this tick species was detected in several regions of the United States. This study aimed at modeling areas suitable for H. longicornis in North America using maximum entropy distribution modeling or Maxent. Occurrence records of H. longicornis within its present range were obtained from published literature. The Maxent model contained a small number of a priori climatic and ecological variables. Annual temperature, precipitation, and ecological zones were found the most important in creating sensitive and specific model (success rate = 91.8%) that had a good fit to the existing data. The model predicted suitable H. longicornis habitat in most of eastern North America from southern Canada to the Gulf Coast, and in a small temperate area on the West Coast. Coastal areas were among the highest ranked suitable habitat. Another highly suitable region was identified in mid-western and southern United States where Heartland virus transmission takes place. This finding is of concern, since H. longicornis ticks vector a closely related virus in East Asia. Delineation of areas suitable for H. longicornis can facilitate detection, prepare public health authorities, and inform the general public about this potentially pestiferous and medically important species.

High genetic diversity in hard ticks from a China-Myanmar border county

BackgroundMany tick species have great morphological similarity and are thus grouped into species complexes. Molecular methods are therefore useful in the classification and identification of ticks. However, little is known about the genetic diversity of hard ticks in China, especially at the subspecies level. Tengchong is one of the epidemic foci of tick-borne diseases in China, but the tick species inhabiting the local area are still unknown.MethodsEighteen villages in Tengchong County, China, were selected for sampling carried out from September to October 2014. Infesting hard ticks were removed from the body surface of domestic animals and questing ticks were collected from grazing fields. After morphological identification, molecular characteristics of each tick species were analyzed based on both 16S rRNA and cytochrome c oxidase subunit 1 (cox1) gene fragments.ResultsSix tick species were identified based on morphology: Rhipicephalus microplus, R. haemaphysaloides, Ixodes ovatus, Haemaphysalis longicornis, H. shimoga and H. kitaokai. Phylogenetic analysis using the cox1 gene revealed that R. microplus ticks from the present study belong to clade C. For tick samples of both R. haemaphysaloides and I. ovatus, three phylogenetic groups were recognized, and the intergroup genetic distances exceeded the usual tick species boundaries. Haemaphysalis longicornis ticks were clustered into two separate clades based on the cox1 gene. For ticks from both H. shimoga and H. kitaokai, two phylogenetic groups were recognized based on the phylogenetic analysis of the 16S rRNA gene, and the intergroup genetic distances also exceeded the known boundaries for closely related tick species.ConclusionsAccording to molecular analyses, new species or subspecies closely related to R. haemaphysaloides, I. ovatus, H. shimoga and H. kitaokai probably exist in the China-Myanmar border Tengchong County, or these ticks form species complexes with highly divergent mitochondrial lineages. Morphological comparisons are warranted to further confirm the taxonomic status of these tick species.

The efficacy of propylene glycol alginate (PGA), a food additive, in controlling Haemaphysalis longicornis ticks

Ticks and tick-borne pathogens threaten the health of both domestic animals and humans, and are associated with a high economic burden in many countries. Tick control can be achieved with chemical acaricides, but issues remain regarding their safety as well as emerging tick resistance. Propylene glycol alginate (PGA) is a food additive commonly used in China. It has been used to kill whiteflies in agriculture as an environmentally friendly insecticide. The aims of this study were to (i) explore the efficacy of PGA to kill Haemaphysalis longicornis ticks and (ii) assess the potential to develop a new tick control acaricide containing PGA and a reduced amount of synthetic pyrethroid. Beta-cypermethrin was chosen as the reference pyrethroid in this study. PGA, beta-cypermethrin and mixes (PGA and beta-cypermethrin formulated in proportions of A = 2:1, B = 1:1, and C = 1:2) were compared for efficacy to kill larval and adult H. longicornis ticks. Overall, we found no statistically significant differences in the killing efficacy of PGA as compared to beta-cypermethrin across examined time-points post-tick exposure. At 24 h post-tick exposure, similar killing efficacy for H. longicornis larvae was recorded for beta-cypermethrin alone, PGA alone, and mixed formulations B and C. Mixed formulation C had the strongest killing effect when compared to PGA alone or mixed formulation B. Similar outcomes were observed in experiments with adult H. longicornis ticks. Based on these findings, we propose that PGA can be useful as a tick control acaricide, either as a single active ingredient or formulated together with a pyrethroid.

Histamine Released From Skin-Infiltrating Basophils but Not Mast Cells Is Crucial for Acquired Tick Resistance in Mice.

Ticks are blood-feeding arthropods that can transmit pathogens to humans and animals, leading to serious infectious diseases such as Lyme disease. After single or multiple tick infestation, some animal species develop resistance to tick feeding, leading to reduced risk of pathogen transmission. In mice infested with larval Haemaphysalis longicornis ticks, both mast cells and basophils reportedly play key roles in the manifestation of acquired tick resistance (ATR), but it remains ill-defined how they contribute to it. Here, we investigated their products responsible for ATR. Treatment of mice with antihistamine abolished the ATR while histamine or histamine H1 receptor agonist reduced tick-feeding even in the first infestation. In accordance with these, mice deficient for histamine production showed little or no ATR, indicating the crucial role for histamine in the expression of ATR. Adoptive transfer of mast cells and basophils derived from histamine-sufficient or deficient mice to recipient mice lacking mast cells and basophils, respectively, revealed that histamine produced by basophils but not mast cells is essential for the manifestation of ATR, in contrast to the case of local and systemic anaphylaxis where mast cell-derived histamine is the major player. During the second but not first tick infestation, basophils accumulated and made a cluster, surrounding a tick mouthpart, in the epidermis whereas mast cells were scattered and localized mainly in the dermis, more distantly from a tick mouthpart. This appears to explain why basophil-derived histamine is much more effective than mast cell-derived one. Histamine-sufficient, but not -deficient mice showed the thickened epidermis at the second tick-feeding site. Taken together, histamine released from skin-infiltrating basophils rather than skin-resident mast cells plays a crucial role in the manifestation of ATR, perhaps through promotion of epidermal hyperplasia that may inhibit tick feeding.

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.

Molecular surveillance of piroplasms in ticks from small and medium-sized urban and peri-urban mammals in Australia.

Natural landscape alterations as a consequence of urbanisation are one of the main drivers in the movements of wildlife into metropolitan and peri-urban areas. Worldwide, these wildlife species are highly adaptable and may be responsible for the transmission of tick-borne pathogens including piroplasms (Babesia, Theileria and Cytauxzoon spp.) that cause piroplasmosis in animals and occasionally in humans. Little is known about piroplasms in the ticks of urban wildlife in Australia. Ticks from long-nosed bandicoots (Perameles nasuta; n = 71), eastern-barred bandicoots (Perameles gunnii; n = 41), northern-brown bandicoots (Isoodon macrourus; n = 19), southern-brown bandicoots (Isoodon obesulus; n = 4), bandicoot sp. (n = 2), flying foxes (Pteropus sp.; n = 3), black rats (Rattus rattus; n = 7), bush rats (Rattus fuscipes; n = 4), brushtail possums (Trichosurus vulpecula; n = 19), ringtail possums (Pseudocheirus peregrinus; n = 12), short-eared possums (Trichosurus caninus; n = 6), possum sp. (Trichosurus sp.; n = 8), and red foxes (Vulpes vulpes; n = 12) were analysed using piroplasm-specific 18S primers and Sanger sequencing. Seven Ixodes tasmani ticks from long-nosed bandicoots and bandicoots sp., three I. tasmani ticks and one Ixodes holocyclus tick from brushtail possums, and one Haemaphysalis longicornis tick from a red fox were positive for piroplasms. New genotypes, with sequences sharing 98% nucleotide similarities with Theileria sp. K1 detected in a burrowing bettong (Bettongia lesueur), were identified from bandicoot ticks. New genotypes were detected in ticks from brushtail possums, which shared 98% similarity with a Babesia sp. (JQ682877) previously identified in marsupials. Theileria orientalis was identified in the H. longicornis tick from the red fox. Babesia and Theileria spp. in the ticks parasitizing bandicoots and brushtail possums clustered closely with respective Babesia and Theileria clades derived from Australian marsupials. This represents the first detection of piroplasms in ticks parasitizing brushtail possums and a red fox in Australia.

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

BackgroundTicks 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.MethodsGenes 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).ResultsWe 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 Km 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 Km 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.ConclusionsIn 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.

Expression Patterns of Host Inflammatory Cytokine Genes during Infestation with Haemaphysalis longicornis, a Zoonotic Vector, in Blood Sucking Periods

Tick saliva is critically important for continuous attachment to the host, blood feeding for days, and transmission of tick-borne pathogens. To characterize the patterns of inflammatory cytokine gene expression during its attachment and blood sucking time, peripheral blood samples of rabbits infested with Haemaphysalis longicornis ticks were collected at different intervals. Blood histamine concentration was evaluated as well as gene encoding IFN-γ, TNF-α, IL-2, IL-6, IL-4, and IL-10 were compared with non-infested rabbits. Blood histamine concentration of tick-infested rabbits during fast feeding time was significantly higher than that of non-infested rabbits. In both nymph and adult tick infested rabbits, expression of TNF-α and IFN-γ genes were decreased significantly (P<0.05), while expression of IL-4, IL-6, and IL-10 were increased 1.3 to 7 folds in adult infested rabbits with the exception of IL-6 that was significantly (P<0.05) decreased in nymph infested rabbits. IL-2 was not expressed in either nymph or adult infestation. H. longicornis saliva is capable of modulate host responses through a complex correlation with histamine and Th1, Th2 mediated cytokines that suppress the inflammatory responses directed toward inflammatory mediators introduced into the host during tick feeding.