Tick Haemaphysalis Longicornis Research Articles

Characterization of acid phosphatase from the tick Haemaphysalis longicornis

The full-length cDNA encoding acid phosphatase (HL-3) from Haemaphysalis longicornis was obtained by 5′ rapid amplification of cDNA ends (RACE). The cDNA contained a 1137bp open reading frame (ORF) coding for 356 amino acids with a predicted theoretical isoelectric point (pI) of 6.35 and molecular weight of 41.0kDa. The recombinant protein was expressed in Escherichia coli. The enzyme could hydrolyze para-nitrophenyl phosphate (pNPP) substrate at an optimum pH of 5.0. Real-time RT-PCR analysis showed that the HL-3 transcripts were expressed in various stages of unfed ticks and were significantly induced by blood feeding. Furthermore, the expression of HL-3 in midguts was significantly higher than in other tested tissues of partially fed adult ticks. The transcripts of the HL-3 mRNA in lipopolysaccharide (LPS)-injected ticks were 1.75 times of the PBS-injected control; Theileria sergenti infected larvae expressed 3.86 more times than that of uninfected ones. Western blot analysis showed that rabbit antiserum against the recombinant rHL-3 could recognize a native protein of approximately 41.0kDa in the lysates from different stages of ticks. Vaccination of rabbits with the rHL-3 conferred partial protective immunity against ticks, resulting in 28% mortality and 10.6% reduction in engorgement weight of adult ticks, respectively. These results suggested that the HL-3 was involved in tick innate immunity and could be used as a potential candidate antigen for the development of anti-tick vaccines.

High Incidence of Rickettsiosis Correlated to Prevalence of Rickettsia japonica among Haemaphysalis longicornis Tick

Endemic spotted fever group rickettsiosis was reported in Shimane Prefecture, Japan. From an analysis of 14 clinical cases found in the endemic area, the infectious agent of spotted fever group rickettsiosis was identified as Rickettsia japonica. In this study, we also found that Rickettsia japonica was highly infected with the vector tick, Haemaphysalis longicornis, in the endemic area. These findings suggest that the high incidence of rickettsiosis in Shimane Prefecture can be explained by the high prevalence of Rickettsia japonica among Haemaphysalis longicornis ticks.

Bacterial Endosymbiont Localization in Hyalesthes obsoletus , the Insect Vector of Bois Noir in Vitis vinifera

One emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by "Candidatus Phytoplasma solani" and spread in vineyards by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the "Candidatus Sulcia muelleri" cluster in the Bacteroidetes and in the "Candidatus Purcelliella pentastirinorum" group in the Gammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to the Betaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tick Haemaphysalis longicornis, we propose the new name "Candidatus Vidania fulgoroideae." Other bacterial endosymbionts identified in H. obsoletus were related to the intracellular bacteria Wolbachia pipientis, Rickettsia sp., and "Candidatus Cardinium hertigii." Fluorescent in situ hybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As "Ca. Sulcia" is usually reported in association with other symbiotic bacteria, we propose that in H. obsoletus, it may occur in a bipartite or even tripartite relationship between "Ca. Sulcia" and "Ca. Purcelliella," "Ca. Vidania," or both.

The identification and characterization of lysozyme from the hard tick Haemaphysalis longicornis

A full-length cDNA-encoding lysozyme was obtained from cDNA libraries of salivary glands of the hard tick Haemaphysalis longicornis and designated as HlLysozyme. The HlLysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 121 amino acids. The calculated molecular weight of the protein is 13.7 kDa, and the theoretical isoelectric point is 9.85. HlLysozyme shares 41–79% amino acid sequence identity with the lysozymes of other organisms. The activity of recombinant HlLysozyme expressed in Escherichia coli was confirmed by a lytic zone assay using lyophilized Micrococcus lysodeikticus. The HlLysozyme activity decreased at 70 °C and was demonstrated at acidic side and neutral in a pH range. Elevated gene expression of HlLysozyme was observed when female ticks were challenged with bacteria, suggesting possible roles of lysozyme as an innate immunity of ticks against microorganisms.

Multiple vitellogenins from the Haemaphysalis longicornis tick are crucial for ovarian development

Ovarian development and egg maturation are crucial processes for the success of reproduction in ticks. Three full-length cDNAs encoding the precursor of major yolk protein, vitellogenin, were obtained from cDNA libraries of the Haemaphysalis longicornis tick and designated as HlVg-1, HlVg-2 and HlVg-3. The HlVg mRNAs were found in fed females with major expression sites in the midgut, fat body and ovary. Native PAGE and Western blot demonstrated that HlVgs in the hemolymph, fat body and ovary of fed females consisted of four major polypeptides. RNAi results showed that HlVg dsRNA-injected ticks obtained lower body weight, egg weight and showed higher mortality of engorged females after blood sucking than control groups. Our results indicate that all HlVgs are essential for egg development and oviposition.

Increased expression of ATG genes during nonfeeding periods in the tick Haemaphysalis longicornis

Ticks are long-lived hematophagous arthropods and have tolerance to starvation. They can survive without food during the host-seeking period for several months to years. To understand how ticks obtain energy over a long period of non-feeding (starvation), we focused on autophagy, a crucial proteolysis system via the lysosomes for various cellular processes that is induced during starvation in eukaryotes. In the present study, EST databases for several organs of the tick Haemaphysalis longicornis led to the identification of HlATG3, HlATG4 and HlATG8, homologues of 3 autophagy-related (ATG) genes, ATG3, ATG4 and ATG8/LC3/GABARAP, respectively, which are essential for the Atg8 conjugation system in model animals. Real-time PCR results revealed that the expression of HlATG3, HlATG4 and HlATG8 in the tick showed higher levels during the non-feeding period than the feeding period, suggesting that the Atg8 conjugation system is at work in unfed ticks. Notably, their expression levels were higher in the midgut, a digestive organ, of unfed than fed adults. Histological analysis demonstrated that lipids and glycogen accumulated within the epithelial cells of the midgut in unfed ticks, implying that the midgut of unfed ticks serves as storage of those components as nutrients during non-feeding. Furthermore, autophagic organelles were found in the midgut undifferentiated cells of unfed ticks. The starved condition appears to be associated with the increased expression of HlATG genes in the midgut of unfed ticks. Tick autophagy might help compensate for the loss of nutrients derived from host blood components during the non-feeding period.

Molecular Detection ofEhrlichia chaffeensisandAnaplasma bovisin the Salivary Glands fromHaemaphysalis longicornisTicks

The salivary gland (SG) of tick plays an important role as a route in the dissemination of tick-borne pathogens to their hosts. We evaluated the presence of these pathogens in the SGs of Haemaphysalis longicornis ticks, and these ticks were collected from grazing cattle in Jeju Island, Korea. Of total 463 one-side SGs, 56 (12.1%) SGs were positive for Ehrlichia chaffeensis and 11 (2.4%) were positive for Anaplasma bovis. In addition, two (0.4%) SGs were co-infected with both E. chaffeensis and A. bovis. In conclusion, we specifically describe the presence of E. chaffeensis and A. bovis in the SGs of H. longicornis ticks in Korea.

Leucine aminopeptidase, HlLAP, from the ixodid tick Haemaphysalis longicornis, plays vital roles in the development of oocytes

Female ixodid ticks are amazing invertebrate animals which efficiently convert a large amount of nutrients derived from their ingested blood meals into eggs. Although oocyte development (vitellogenesis) in ticks is triggered by a blood meal and is assumed to be supported by nutrition derived from ovarian cells connecting oocytes, little is known about the ovarian molecules processing nutrient materials for the vitellogenesis. In this study, we have suggested a putative function of leucine aminopeptidase (HlLAP) in the ovary of parthenogenetic adult ixodid tick Haemaphysalis longicornis regarding a negative output of reproduction following disruption of HlLAP gene by RNA interference. Endogenous HlLAP was shown to be localized in the ovarian cells, including ovarian epithelial and pedicel cells which were assumed to provide nutrients for the developing oocytes. Histological studies demonstrated that a majority of immature oocytes in HlLAP gene knockdown ticks were transformed into abnormal morpho-histological oocytes with vacuolated cytoplasm and/or condensed nucleus. Taken together, a reduction of the numbers of laid eggs in the HlLAP gene knockdown ticks may be due to the degeneration of immature oocytes following deprivation of nutrients such as amino acids supplied not only by midgut HlLAP but also by the ovarian HlLAP. Regulation of the tick molecules involved in nutrient metabolism for the reproduction, including blood digestion and vitellogenesis, would help in controlling the tick population and tick-borne pathogens.

Characterization of Hlcyst-3 as a member of cystatins from the tick Haemaphysalis longicornis

The gene encoding cystatin from the tick Haemaphysalis longicornis has been reported previously. In the study reported here, we characterized a member of cystatins and designated it as Hlcyst-3 (H. longicornis cystatin-3). Its full-length cDNA is 602 bp, and it encodes a putative 129 amino acid protein with an obvious signal peptide. Sequence analysis revealed that it has significant homology with the known secreted cystatin. The recombinant protein was expressed in a GST-fused soluble form in Escherichia coli and was purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain and cathepsin L was identified by fluorogenic substrate analysis. Real-time PCR revealed that Hlcyst-3 was mostly expressed in the tick midgut.

Hlcyst-1 and Hlcyst-2 are Potential Inhibitors of HlCPL-A in the Midgut of the Ixodid Tick Haemaphysalis longicornis

Although the actions of cysteine proteases are controlled in part by endogenous tight-binding cysteine protease inhibitors from the cystatin superfamily, regulatory mechanisms used by ticks to control protease activities are unknown. We report here the interaction of 2 endogenous midgut cysteine protease inhibitors, Hlcyst-1 and Hlcyst-2, with an endogenous midgut cysteine protease, HlCPL-A in Haemaphysalis longicornis. In vitro inhibition assays demonstrated that the hydrolytic activity of HlCPL-A was inhibited by Hlcyst-1 and Hlcyst-2 in dose dependent manner. Immunofluorescent studies revealed that Hlcyst-1 and Hlcyst-2 are co-localized with HlCPL-A in the epithelial cells of the midgut. The hemoglobin degradation activity of HlCPL-A was dose-dependently inhibited by Hlcyst-1 and Hlcyst-2. These results strongly indicate that, Hlcyst-1 and Hlcyst-2 are possible inhibitor of HlCPL-A and play a key role in regulatory mechanisms of hemoglobin degradation process in ticks.

Structural Characterization and Cytolytic Activity of a Potent Antimicrobial Motif in Longicin, a Defensin-Like Peptide in the Tick Haemaphysalis longicornis

Longicin, a defensin-like peptide, was recently identified in the hard tick Haemaphysalis longicornis. Longicin and one of its synthetic partial analogs (P4) displayed antimicrobial/fungicidal/parasiticidal activity. In the present study, we compared longicin-derived synthetic analogs in order to characterize the antimicrobial motif (P4) by analyzing some structural features using various bioinformatic tools and/or CD spectroscopy. According to the chemicophysical characteristics, P4 is suggested to be a cationic peptide with hydrophobic and amphipathic character. The predicted secondary structure indicated the existence of a beta-sheet, which was also observed in the modeled tertiary structure. CD spectroscopic results also showed the existence of a beta-sheet and transition to a helical conformation in the presence of membrane-mimicking conditions. These structural observations on P4 suggested that the antimicrobial activity could be due to the beta-sheet as well as the alpha-helix. In addition, a sequence homology search showed that molecules identified in other ticks and organisms also have the P4 analogous domain at their C-terminal, which indicates P4 as a conserved domain. The peptide P4 also showed low cytolytic activity. Based on the present result and previously reported studies, the peptide P4 could be suggested as a novel antimicrobial domain indicating future therapeutic agent against bacteria.

GATA transcription, translation and regulation in Haemaphysalis longicornis tick: Analysis of the cDNA and an essential role for vitellogenesis

Blood feeding tightly regulates the reproductive cycles of ticks. Vitellogenesis and nutritional signaling are key events in the tick reproductive cycle. Here we report the identification of a GATA factor that is synthesized after a blood meal and acts as a transcriptional activator of vitellogenin (Vg), and the identification of an S6 kinase that is a transcription regulator of the amino acid signaling pathway. Tick GATA mRNA accumulated in the midgut prior to blood feeding. However, translation of GATA was activated by blood feeding because the GATA protein dramatically increased in the fat body of engorged females. RNA interference-mediated knockdown of S6 kinase and GATA factor revealed the involvements of S6 kinase in GATA activation and resulted in a significant inhibition of the major yolk protein vitellogenin in engorged ticks and effectively disrupting egg development after a blood meal. These results indicate that the GATA factor, a specific transcriptional activator of Vg gene, represents an important molecule for the regulation of tick vitellogenesis and reproduction.

Longistatin, a novel EF-hand protein from the ixodid tick Haemaphysalis longicornis, is required for acquisition of host blood-meals

Calcium and the EF-hand Ca++-binding proteins have been undisputedly recognised as the key players in almost all aspect of cell functions, starting from the cell’s birth, during mitosis to its end with apoptosis. But in a few exceptional cases the EF-hand proteins are secreted from the cells and play their crucial roles extracellularly. Here, to our knowledge for the first time, we have identified and characterised an EF-hand Ca++-binding protein from the salivary glands of the ixodid tick, Haemaphysalis longicornis, herein called longistatin. Longistatin possesses two EF-hand domains which conserve canonical structure and bind with Ca++. Both the recombinant and endogenous proteins were stained with Rutheninum red. Reverse-transcription PCR data showed that longistatin-specific transcript was expressed in all life-cycle stages of H. longicornis and was up-regulated only in blood-fed ticks. Organ-specific transcription analysis revealed a salivary gland-specific expression of the gene which peaked at 96–120h of feeding when ticks acquired full blood-meals and become engorged but its expression declined sharply as they detached and dropped off the host. Consistently, endogenous protein was localised in the salivary glands of adult ticks and in the lumen of the functional acini of the salivary glands. Furthermore, longistatin was detected in feeding lesions at the site of attachment of ticks on the host. These results suggest that longistatin is synthesised in, and is secreted from, the salivary glands and may have functional roles in the feeding process of ixodid ticks.

A salivary cystatin, HlSC-1, from the ixodid tick Haemaphysalis longicornis play roles in the blood-feeding processes

Ticks feed exclusively on blood to obtain their nutrients, but the gene products that mediate blood-sucking processes in ticks are still unknown. We report here the molecular characterization and possible biological function of a cysteine protease inhibitor (HlSC-1) identified in the salivary gland of the ixodid tick Haemaphysalis longicornis. The HlSC-1 cDNA contains 423 bp that code for 140 amino acids with a predictable molecular weight of 12 kDa. The recombinant HlSC-1 expressed in Escherichia coli was shown to inhibit the activity of papain and cathepsin L, while cathepsin B activity was unaffected. Immunolocalization studies detected the endogenous enzyme in the salivary gland type II acini of an adult tick. Furthermore, quantitative RT-PCR analysis showed that the expression of HlSC-1 transcripts was associated with blood-feeding processes and was highly up-regulated in the early phase of feeding. Our results strongly suggest that HlSC-1 may play pivotal roles in the blood-feeding processes.

Comparative immunogenicity of Haemaphysalis longicornis and Rhipicephalus ( Boophilus) microplus calreticulins

The ticks Rhipicephalus ( Boophilus) microplus and Haemaphysalis longicornis are blood-sucking ectoparasites of bovines, causing serious damages to the livestock production. The main control method for these ticks is based on acaricides. However, the use of vaccines has been studied as a promising control strategy. Calreticulin (CRT) is a multifunctional, predominantly intracellular protein present in almost all cells of animals. The secretion of CRT during feeding might be linked to the modulation of the parasite–host interaction. In the present study, recombinant CRTs of R. microplus (rBmCRT) and H. longicornis (rHlCRT) were expressed in Escherichia coli and purified by ion exchange chromatography and used for the immunization of bovines and mouse. ELISA demonstrated that both rCRTs are recognized by the sera of immunized bovines. In silico, despite the difference in amino acid sequences, antigenic index analysis of HlCRT and BmCRT using the Jameson–Wolf algorithm indicated that both proteins were very similar in antigenicity index, although six different epitopes between the tick CRTs have been inferred. These data were corroborated by competitive ELISA analyses, which suggest the presence of different epitopes within the proteins. Western blot analyses showed that anti-rBmCRT and anti-rHlCRT bovine sera also recognized the native proteins in larvae extracts and, moreover, sera of bovines immunized with saliva and extract of salivary glands recognized both recombinant CRTs. Thus, mouse and bovine immune system recognized rCRTs, resulting in the production of antibodies with similar specificity for both recombinant proteins, although different epitopes could be distinguished between rBmCRT and rHlCRT.

Hemoglobinase activity of a cysteine protease from the ixodid tick Haemaphysalis longicornis

We report here the molecular characterization and possible function of a cysteine protease (termed HlCPL-A) identified in the midgut of the hard tick Haemaphysalis longicornis. HlCPL-A is a 333 amino acid protein belonging to the papain family of the cysteine protease. A construct encoding proHlCPL-A was expressed in Escherichia coli and purified as both procathepsin L and active processed cathepsin L forms. The HlCPL-A gene expression was up-regulated by blood-feeding process. HlCPL-A exhibited substrate specificity against synthetic peptidyl substrates (Z-Phe-Arg-MCA and Z-Arg-Arg-MCA; k cat / K m = 0.19 and 0.0023 M − 1 S − 1 , respectively). The proteolytic activity of HlCPL-A was inhibited by leupeptin, antipain and E-64 but was unaffected by pepstatin. HlCPL-A was capable of degrading bovine hemoglobin at pH 3.2 to 5.6. These results suggest that HlCPL-A may play important roles in the digestion of host hemoglobin in ticks.

Cloning and molecular characterization of tick kynurenine aminotransferase (HlKAT) from Haemaphysalis longicornis (Acari: Ixodidae)

A complementary DNA coding a novel kynurenine aminotransferase (KAT) molecule from Haemaphysalis longicornis tick embryo was cloned and characterized. The transcription of the HlKAT occurs at all stages during tick development as well as in the midgut, salivary glands, ovary, and synganglion of adult ticks, and protein expression levels increased during the blood-feeding course. The HlKAT gene without signal peptide was successfully expressed as a glutathione S-transferase fusion protein in soluble form, which is capable of catalyzing the transamination of kynurenine and 3-hydroxykynurenine to kynurenic acid and xanthurenic acid, respectively. The purified recombinant HlKAT showed dose-dependent inhibition effect on the growth of equine babesial parasite, Babesia caballi, in in vitro culture. All results suggested that a specific HlKAT is present in tick and HlKAT may play an important physiological role in H. longicornis. This is the first report of a member enzyme of tryptophan pathway in Chelicerata.

Blocking the secretion of saliva by silencing the HlYkt6 gene in the tick Haemaphysalis longicornis

Soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) have been identified as the key components of the protein complexes that facilitate vesicle traffic, of which Ykt6 (from Saccharomyces cerevisiae, v-SNARE) is proved to be a multifunctional protein in the membrane fusion. In the present study, a tick homologue of Ykt6 (HlYkt6, predicted 22.6 kDa), was isolated from the ixodid tick Haemaphysalis longicornis. RT-PCR and Western blot analysis indicated that the gene and the encoded protein were expressed ubiquitously in different tissues of the partially fed adult tick. Silencing of the HlYkt6 gene resulted in a significant decrease of the engorged body weight (82.9 ± 26.8 mg vs. 232.17 ± 59.1 mg in the PBS-injected control group and 178.7 ± 57.0 mg in the GFP dsRNA-injected control group) and high mortality of replete ticks (100% in tested group vs. 4.8% in the PBS and 20.4% in GFP dsRNA-injected control groups). Disruption of HlYkt6 mRNA led to the suppression of saliva secretion, and a lower anticoagulant activity of the released liquid from the glands (APTT time: 25.25 ± 1.50 s) than that of the control groups (39.25 ± 0.50 s in the PBS-treated group and 40.0 ± 1.41 s in the GFP dsRNA-treated group). These results suggest the vital role of the HlYkt6 protein in the exocytosis of saliva proteins, the feeding and survival of ticks.

Leucine Aminopeptidase in the Ixodid Tick Haemaphysalis longicornis: Endogenous Expression Profiles in Midgut

We previously identified a cDNA from the ixodid tick Haemaphysalis longicornis that encodes leucine aminopeptidase, HlLAP. Functionally, recombinant HlLAP effectively hydrolyzed synthetic amino acid derivatives. Here, we investigated the temporal expression profiles of midgut HlLAP in adult H. longicornis parthenogenetic ticks from the starting of blood feeding until just before the onset of oviposition. Midgut HlLAP transcript expression level was higher during post-engorgement period than that during feeding period. Endogenous HlLAP in the midgut was also observed with higher expression level during post-engorgement period. Histological localization of HlLAP was in the cytosol of midgut epithelial cells, notably the newly differentiated basophilic cells at post-engorgement. Our data suggested that HlLAP was dominantly localized in basophilic cells, where it may play regulatory roles in protein biosynthesis and degradation.

Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus ( Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.