Data from expressed sequence tags from the organs and embryos of parthenogenetic Haemaphysalis longicornis.

OBJECTIVES: Haemaphysalis longicornis is the most important tick species in Japan and has a wide range of vector capacity. Due to its veterinary and medical importance, this tick species has been used as a model for tick/vector biological studies. To identify the key molecules associated with physiological processes during blood feeding and embryogenesis, full-length cDNA libraries were constructed using the fat body, hemocytes-containing hemolymph, midgut, ovary and salivary glands of fed females and embryos of the laboratory colony of parthenogenetic H. longicornis. The sequences of cDNA from the salivary glands had been already released. However, the related information is still poor, and the other expressed sequence tags have not yet been deposited. DATA DESCRIPTION: A total of 39,113 expressed sequence tags were obtained and deposited at the DNA DataBank of Japan. There were 7745 sequences from embryos, 7385 from the fat body, 8303 from the hemolymph including hemocytes, 7385 from the midgut, and 8295 from the ovary. The data, including expressed sequence tags from the salivary glands was summarized into Microsoft Excel files. Sharing this data resource with the tick research community will be valuable for the identification of novel genes and advance the progress of tick research.

Establishment of a novel tick-Babesia experimental infection model.

Ticks are potent vectors of many deadly human and animal pathogens. Tick-borne babesiosis is a well-recognized malaria-like disease that occurs worldwide and recently has attracted increased attention as an emerging zoonosis. Although the proliferation of Babesia organisms is essential in the vectors, their detailed lifecycle with time information for migration in ticks remains unknown. A novel study model for the elucidation of the migration speed of Babesia parasites in their vector tick, Haemaphysalis longicornis, has been developed using an artificial feeding system with quantitative PCR method. The detectable DNA of Babesia parasites gradually disappeared in the tick midgut at 1 day post engorgement (DPE), and in contrary increased in other organs. The results indicated that the Babesia parasite passed the H. longicornis midgut within 24 hours post engorgement, migrated to the hemolymph, and then proliferated in the organs except the midgut. This time point may be an important curfew for Babesia parasites to migrate in the tick lumen. We also visualized the Babesia parasites in the experimentally infected ticks and in their eggs using IFAT for detecting their cytoskeletal structure, which suggested the successful tick infection and transovarial transmission of the parasite. This model will shed light on the further understanding of tick-Babesia interactions.

Transcriptional profiles of virulent and precocious strains of Eimeria tenella at sporozoite stage; novel biological insight into attenuated asexual development.

Chicken coccidiosis is caused by Eimeria spp., particularly Eimeria tenella, and is characterized by watery or hemorrhagic diarrhea, resulting in death in severe cases. Precociously attenuated live vaccines are widely used to control the disease, and these are produced by serially passaging virulent strains through chickens, and the collection of oocysts from feces at progressively earlier time points during oocyst shedding. Sporozoites of the precocious strain rapidly enter the intestinal mucosa, and their subsequent asexual development reduces their growth. However, there have been few detailed genetic or transcriptional analyses of the strains. Here, we used RNA sequencing to gain novel biological insight into the pathogenicity and precocity of E. tenella. We compared the differential transcription in the sporozoites (the initial stage of endogenous development) of virulent and precocious strains by mapping the sequence reads onto the draft genome of E. tenella. About 90% of the reads from both strains were mapped to the genome, and 16,630 estimated transcript regions were identified. Using Gene Ontology slim and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and the annotation of the estimated transcripts with Blastx, we found that the expression of some genes involved in carbohydrate metabolism were expressed two-fold more strongly in the virulent strain than in the precocious strain. Characteristically, genes related to proteins secreted from the apical complex, proteases, cell attachment proteins, mitochondrial proteins, and transporters were most strongly upregulated in the virulent strain. Interestingly, the expression of genes associated with cell survival, development, or proliferation was strongly upregulated in the precocious strain. These findings suggest that virulent strains survive long before invasion and invade actively/successfully into host cells, whereas proliferative processes appear to affect precocity.

Longistatin in tick saliva blocks advanced glycation end-product receptor activation.

Ticks are notorious hematophagous ectoparasites and vectors of many deadly pathogens. As an effective vector, ticks must break the strong barrier provided by the skin of their host during feeding, and their saliva contains a complex mixture of bioactive molecules that paralyze host defenses. The receptor for advanced glycation end products (RAGE) mediates immune cell activation at inflammatory sites and is constitutively and highly expressed in skin. Here, we demonstrate that longistatin secreted with saliva of the tick Haemaphysalis longicornis binds RAGE and modulates the host immune response. Similar to other RAGE ligands, longistatin specifically bound the RAGE V domain, and stimulated cultured HUVECs adhered to a longistatin-coated surface; this binding was dramatically inhibited by soluble RAGE or RAGE siRNA. Treatment of HUVECs with longistatin prior to stimulation substantially attenuated cellular oxidative stress and prevented NF-κB translocation, thereby reducing adhesion molecule and cytokine production. Recombinant longistatin inhibited RAGE-mediated migration of mouse peritoneal resident cells (mPRCs) and ameliorated inflammation in mouse footpad edema and pneumonia models. Importantly, tick bite upregulated RAGE ligands in skin, and endogenous longistatin attenuated RAGE-mediated inflammation during tick feeding. Our results suggest that longistatin is a RAGE antagonist that suppresses tick bite-associated inflammation, allowing successful blood-meal acquisition from hosts.

Localization of eimeripain, an Eimeria tenella cathepsin B-like cysteine protease, during asexual and sexual intracellular development in chicken ceca.

Hemorrhagic diarrhea in poultry is caused by Eimeria tenella, the most pathogenic avian coccidian parasite, and new approaches to treat the disease are continually being sought. Although eimeripain, a cathepsin B-like cysteine protease from E. tenella, has recently been identified as a novel anticoccidial drug target, its localization during the intracellular development of parasites remains unclear. Here, we demonstrate the expression of eimeripain during asexual and sexual development of E. tenella in vivo. Promature eimeripain was detected only in the early immature second generation of schizonts. In contrast, the mature eimeripain was most strongly detected in the middle-sized immature second generation of schizonts. Both promature and mature eimeripain disappeared depending on the maturation level of second generation of schizonts, but were strongly expressed again in the third generation of schizonts. In the sexual stage, both promature and mature eimeripain were detected in the cytoplasm of micro- and macro-gametocytes and zygotes, but expression became weak in zoites forming oocysts. Collectively, our findings suggest that eimeripain might play a key role in the differentiation of intracellular zoites in the ceca and could be an interesting candidate to develop a novel, effective anti-coccidian drug.

Actin-mediated transovarial transmission of a yeastlike symbiont in the brown planthopper.

The brown planthopper Nilaparvata lugens (Hemiptera, Delphacidae) harbors an obligate endosymbiont called the yeastlike symbiont (YLS) in their abdominal fat body. YLS, a filamentous ascomycete belonging to the family Clavicipitaceae, does not spend any part of its life cycle outside the planthopper's body. The YLS is transferred to the next generation via transovarial transmission; it enters the epithelial plug at the posterior end of the host female's ovariole and is transferred to her offspring. In the present study, microscopic examination revealed that actin filaments play an important role in the transmission of YLS. An irregular cell protrusion on the surface of the epithelial plug facilitated the uptake of the YLS, which was then incorporated into the epithelial plug cell. Actin assembly apparently produces the protrusion and actin appears to participate in almost every stage of the process, from the entry of the YLS into the epithelial plug to its delivery to the oocyte. The epithelial plug employs a recognition system for YLS, which drastically changes the cell surface structure to enable the YLS to enter the ovariole.

High-throughput RNA sequencing profiles and transcriptional evidence of aerobic respiratory enzymes in sporulating oocysts and sporozoites of Eimeria tenella.

Seven species of Eimeria are responsible for coccidiosis in chickens. Eimeria tenella is one of the most pathogenic parasites since it is associated with high mortality and great economic impact. The life cycle of the parasite includes development in the environment and in the intestinal tract. We conducted RNA sequencing using a next generation sequencer to obtain transcriptome information from the sporulating oocysts, and sporozoites. We collected 2.8 million 75 bp reads of a short-tag sequence, and 25,880 contigs were generated by the Oases assembler. A Blastx search of GenBank databases revealed that 7780 contigs (30.1%) had significant homology with deposited sequence data (E-value <1e-6); among these contigs, 6051 contigs were similar to those of Toxoplasma gondii while only 513 contigs (6.6%) were similar to those of E. tenella. After an orthological analysis conducted with the UniProt database of T. gondii, 6661 contigs were distributed within the categories of cellular components (1528 gene categories), biological processes (861 gene categories), and molecular functions (241 gene categories). The significantly matched contigs contained high numbers of enzymes associated with glycolysis, TCA, and the pentose-phosphate pathway. Most of the enzymes, measured by quantitative reverse transcription-PCR, were up-regulated in sporulating stage. These results suggest that the intracellular carbohydrate amylopectin could be used as an energy source for ATP production including glycolysis and the pentose-phosphate pathway, which generates NADPH and pentoses. Our data also suggest that Eimeria might possess a partial or similar pathway to the TCA cycle essential for aerobic respiration. Furthermore, the newly annotated and non-annotated contigs might contain E. tenella-specific or novel sequences.

HlCPL-A, a cathepsin L-like cysteine protease from the ixodid tick Haemaphysalis longicornis, modulated midgut proteolytic enzymes and their inhibitors during blood meal digestion.

Ticks employ a battery of proteases to digest the contents of host blood meals. Host hemoglobin degradation is facilitated by proteolytic networks in the midgut, the first major region of the body where ingested blood comes into contact with the tick's internal tissues. Our previous studies indicated that HlCPL-A, a cathepsin L-like cysteine protease isolated from the midgut of the ixodid tick Haemaphysalis longicornis, is a potent hemoglobinase, and plays important roles in the digestion of blood acquired from a host. In this paper, we report the effects of silencing of the HlCPL-A gene in H. longicornis using RNA interference (RNAi). We observed that the survival of HlCPL-A-silenced ticks was reduced compared with that of controls during blood digestion, most likely due to the compromised ability of ticks to digest blood. The morphological analysis results of midgut lumen were different between HlCPL-A-silenced ticks and controls, indicating that HlCPL-A plays a crucial role in hemolysis in the midgut of ticks. The expression level was analyzed using quantitative RT-PCR-based endogenous expression approach. Compared to that in malE double stranded RNA (dsRNA)-treated ticks, in the midgut of HlCPL-A dsRNA-treated ticks, some proteases and inhibitors related to the hemoglobin digestive cascade were up-regulated while the others were down-regulated. These results suggest that HlCPL-A is related to the multi-enzyme cascade and protease network for hemoglobin digestion. These findings suggest that the hemoglobin digestive cascade may assemble in the midgut of ticks.

Quantitative PCR-based parasite burden estimation of Babesia gibsoni in the vector tick, Haemaphysalis longicornis (Acari: Ixodidae), fed on an experimentally infected dog.

Most causative agents of babesiosis, Babesia parasites, are transmitted transovarially in ixodid ticks. In this study, B. gibsoni, the causative agent of canine babesiosis which has transovarial transmission, was detected in tissues of the vector tick, Haemaphysalis longicornis using a modified quantitative PCR assay. Conventional PCR results showed that the newly designed primer set, which amplifies a 143-bp fragment of rhoptry-associated protein-1 (BgRAP-1) gene in B. gibsoni, was 100 times more sensitive than primers targeting P18 gene encoding 18 kDa protein of B. gibsoni, which was recently renamed as thrombospondin related adhesive protein (BgTRAP) gene, in an artificially generated sample solution containing metagenomic DNA (B. gibsoni DNA extracted from infected dog blood mixed with tick DNA). The TaqMan probe-based quantitative PCR (qPCR) for BgRAP-1 could also detect infected RBCs (iRBCs) at levels of 3.5 × 10(5) to 3.5 × 10(1)/µl, a range that is broader than that of a past SYBR Green-based qPCR method for P18/BgTRAP, which had a detection limit of 3.5 × 10(3) iRBCs/µl. Using this qPCR assay, we attempted to quantify the B. gibsoni burden in tick ovaries and embryonated eggs. Levels of infection were normalized to the copy number of tick's genomic DNA fragment of ribosomal DNA internal transcribed spacer region 2 (ITS2) for the standardization. According to this, low levels of parasite burden were quantified in ovaries and eggs. This detection system is sensitive and is recommended as a tool for elucidating the biological interactions between the vector tick H. longicornis and the parasite, B. gibsoni.

Semi-artificial mouse skin membrane feeding technique for adult tick, Haemaphysalis longicornis.

BACKGROUND: An in vitro artificial feeding technique for hard ticks is quite useful for studying the tick-pathogen interactions. Here, we report a novel semi-artificial feeding technique for the adult parthenogenetic tick, Haemaphysalis longicornis, using mouse skin membrane. FINDINGS: Skin with attached adult ticks was removed from the mouse body at 4 to 5 days post-infestation for the construction of the feeding system. This system supplied with rabbit blood was kept in >95% relative humidity at 30°C during the feeding, and ticks were fully engorged (artificially engorged, AE) within 12 to 48 h. For comparison, ticks were fed to engorgement solely on rabbit or mouse for 5 days as controls (naturally engorged on rabbit, NEr, or mouse, NEm). Blood digestion-related gene expression in the midgut and reproductive fitness were compared. Body weight, egg mass weight, egg conversion ratio, and hatchability of eggs did not show any significant differences. We analyzed transcription profiles of selected genes assayed by quantitative RT-PCR and revealed similar patterns of expression between NEr and AE but some differences between NEm and AE or NEm and NEr. CONCLUSIONS: Our results demonstrate that this semi-artificial feeding technique mimics natural feeding processes of ticks and can be utilized as a standardized method to inoculate pathogens, especially Babesia protozoa, into H. longicornis and possibly other tick species as well.

A hemocyte-derived Kunitz-BPTI-type chymotrypsin inhibitor, HlChI, from the ixodid tick Haemaphysalis longicornis, plays regulatory functions in tick blood-feeding processes.

Inhibitors of proteases play key roles in the biological processes of vertebrate and invertebrate animals, including arthropod parasites. Here, we describe a cDNA that encodes a functionally active chymotrypsin inhibitor of the BPTI/Kunitz family of serine protease inhibitors from the hemocytes of the ixodid tick, Haemaphysalis longicornis, herein called HlChI. HlChI sequence is evolutionarily conserved and contains six cysteine residues and three disulfide bonds with a calculated molecular weight of 9.1 kDa. HlChI-specific mRNA was expressed in all developmental stages of ticks and the expression was up-regulated by host's blood-feeding processes. Endogenous HlChI was localized mainly in the hemocytes. HlChI potently inhibited bovine pancreatic α-chymotrypsin for hydrolyzing the fluorogenic substrate (IC(50) 8.32 nM, K(d) 5.35 ± 1.01 nM) and bovine casein digestion. However, HlChI weakly inhibited bovine pancreatic trypsin and could not affect the porcine elastase activity, suggesting its narrow specificity to chymotrypsin. HlChI was stable over the pH range 2-11 and heating up to 70 °C at pH 8. HlChI was highly stable to 8 M urea and 2% SDS at pH 8.0, when treated for 24 h at 37 °C. However, 0.2 M 2-mercaptoethanol caused complete but reversible inactivation of HlChI. Knockdown of HlChI gene by RNA interference (RNAi) caused death of the feeding ticks, failure of ticks to engorge and significantly reduced body weight gain. RNAi also resulted in significantly decreased egg conversion ratio and fecundity. These results suggest that HlChI is a chymotrypsin-specific inhibitor with high stability and may play regulatory functions in host's blood-feeding processes and tick reproduction.

Longistatin is an unconventional serine protease and induces protective immunity against tick infestation.

Classical serine proteases use the conserved Ser/His/Asp catalytic triad to hydrolyze substrates. Here, we show that longistatin, a salivary gland protein with two EF-hand domains from the vector tick Haemaphysalis longicornis, does not have the conserved catalytic triad, but still functions as a serine protease. Longistatin was synthesized in and secreted from the salivary glands of ticks, and is injected into host tissues during the acquisition of blood-meals. Longistatin hydrolyzed fibrinogen, an essential plasma protein in the coagulation cascade, and activated plasminogen, into its active form plasmin, a serine protease that dissolves fibrin clots. Longistatin efficiently hydrolyzed several serine protease-specific substrates showing its specificity to the amide bond of Arg. Longistatin did not hydrolyze synthetic substrates specific for other groups of proteases. The enzyme was active at a wide range of temperatures and pHs, with the optimum at 37°C and pH 7. Its activity was efficiently inhibited by various serine protease inhibitors such as phenylmethanesulfonyl fluoride (PMSF), aprotinin, antipain, and leupeptin with the estimated IC(50) of 278.57 µM, 0.35 µM, 41.56 µM and 198.86 µM, respectively. In addition, longistatin was also potently inhibited by Zinc (Zn(2+)) in a concentration-dependent manner with an IC(50) value of 275 µM, and the inhibitory effect of Zn(2+) was revived by ethylenediaminetetra acetic acid (EDTA). Immunization studies revealed that longistatin sharply induced high levels of protective IgG antibodies against ticks. Immunization with longistatin reduced repletion of ticks by about 54%, post engorgement body weight by >11% and molting of nymphs by approximately 34%; thus, the vaccination trial was approximately 73% effective against tick infestation. Taken together, our results suggest that longistatin is a new potent atypical serine protease, and may be an interesting candidate for the development of anti-tick vaccines.

Longistatin, a novel plasminogen activator from vector ticks, is resistant to plasminogen activator inhibitor-1.

Thrombo-occlusive diseases are major causes of morbidity and mortality, and tissue-type plasminogen activator (t-PA) is recommended for the treatment of the maladies. However, both t-PA and u-PA are rapidly inactivated by plasminogen activator inhibitor-1 (PAI-1). Here, we show that longistatin, a novel plasminogen activator isolated from the ixodid tick, Haemaphysalis longicornis is resistant to PAI-1. Longistatin was relatively less susceptible to the inhibitory effect of SDS-treated platelet lysate than physiologic PAs. Platelet lysate inhibited t-PA and tcu-PA with the IC(50) of 7.7 and 9.1 µg/ml, respectively, whereas for longistatin inhibition IC(50) was 20.1 µg/ml (p<0.01). Similarly, activated PAI-1 (20 nM) inhibited only 21.47% activity of longistatin but almost completely inhibited t-PA (99.17%) and tcu-PA (96.84%). Interestingly, longistatin retained 76.73% initial activity even after 3h of incubation with 20 nM of PAI-1. IC(50) of PAI-1 during longistatin inhibition was 88.3 nM while it was 3.9 and 3.2 nM in t-PA and tcu-PA inhibition, respectively. Longistatin completely hydrolyzed fibrin clot by activating plasminogen efficiently in the presence of 20 nM of PAI-1. Importantly, unlike t-PA, longistatin did not form complex with PAI-1. Collectively, our results suggest that longistatin is resistant to PAI-1 and maybe an interesting tool for the development of a PAI-1 resistant effective thrombolytic agent.

Longistatin, a plasminogen activator, is key to the availability of blood-meals for ixodid ticks.

Ixodid ticks are notorious blood-sucking ectoparasites and are completely dependent on blood-meals from hosts. In addition to the direct severe effects on health and productivity, ixodid ticks transmit various deadly diseases to humans and animals. Unlike rapidly feeding vessel-feeder hematophagous insects, the hard ticks feed on hosts for a long time (5-10 days or more), making a large blood pool beneath the skin. Tick's salivary glands produce a vast array of bio-molecules that modulate their complex and persistent feeding processes. However, the specific molecule that functions in the development and maintenance of a blood pool is yet to be identified. Recently, we have reported on longistatin, a 17.8-kDa protein with two functional EF-hand Ca(++)-binding domains, from the salivary glands of the disease vector, Haemaphysalis longicornis, that has been shown to be linked to blood-feeding processes. Here, we show that longistatin plays vital roles in the formation of a blood pool and in the acquisition of blood-meals. Data clearly revealed that post-transcriptional silencing of the longistatin-specific gene disrupted ticks' unique ability to create a blood pool, and they consequently failed to feed and replete on blood-meals from hosts. Longistatin completely hydrolyzed α, ß and γ chains of fibrinogen and delayed fibrin clot formation. Longistatin was able to bind with fibrin meshwork, and activated fibrin clot-bound plasminogen into its active form plasmin, as comparable to that of tissue-type plasminogen activator (t-PA), and induced lysis of fibrin clot and platelet-rich thrombi. Plasminogen activation potentiality of longistatin was increased up to 4 times by soluble fibrin. Taken together, our results suggest that longistatin may exert potent functions both as a plasminogen activator and as an anticoagulant in the complex scenario of blood pool formation; the latter is critical to the feeding success and survival of ixodid ticks.

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.

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.

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 96-120 h 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 Kunitz-type proteinase inhibitor from the midgut of the ixodid tick, Haemaphysalis longicornis, and its endogenous target serine proteinase.

Although previous studies strongly suggested the involvement of serine proteases in blood digestion in the midgut of ticks, the regulating molecules of these proteinases are still unidentified. A novel Haemaphysalis longicornis Kunitz-type serine proteinase inhibitor with a single Kunitz-domain (HlMKI) has been identified and its co-localization with a midgut-derived serine proteinase (HlSP) within the epithelial cells has been demonstrated. Recombinant HlMKI inhibited the hydrolytic activity of HlSP, suggesting that HlMKI is a possible inhibitor of HlSP and may be part of a regulatory system of midgut serine proteinases.

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.

The Kunitz-like modulatory protein haemangin is vital for hard tick blood-feeding success.

Ticks are serious haematophagus arthropod pests and are only second to mosquitoes as vectors of diseases of humans and animals. The salivary glands of the slower feeding hard ticks such as Haemaphysalis longicornis are a rich source of bioactive molecules and are critical to their biologic success, yet distinct molecules that help prolong parasitism on robust mammalian hosts and achieve blood-meals remain unidentified. Here, we report on the molecular and biochemical features and precise functions of a novel Kunitz inhibitor from H. longicornis salivary glands, termed Haemangin, in the modulation of angiogenesis and in persistent blood-feeding. Haemangin was shown to disrupt angiogenesis and wound healing via inhibition of vascular endothelial cell proliferation and induction of apoptosis. Further, this compound potently inactivated trypsin, chymotrypsin, and plasmin, indicating its antiproteolytic potential on angiogenic cascades. Analysis of Haemangin-specific gene expression kinetics at different blood-feeding stages of adult ticks revealed a dramatic up-regulation prior to complete feeding, which appears to be functionally linked to the acquisition of blood-meals. Notably, disruption of Haemangin-specific mRNA by a reverse genetic tool significantly diminished engorgement of adult H. longicornis, while the knock-down ticks failed to impair angiogenesis in vivo. To our knowledge, we have provided the first insights into transcriptional responses of human microvascular endothelial cells to Haemangin. DNA microarray data revealed that Haemangin altered the expression of 3,267 genes, including those of angiogenic significance, further substantiating the antiangiogenic function of Haemangin. We establish the vital roles of Haemangin in the hard tick blood-feeding process. Moreover, our results provide novel insights into the blood-feeding strategies that enable hard ticks to persistently feed and ensure full blood-meals through the modulation of angiogenesis and wound healing processes.