Inhibitory effect of cyclophilin A from the hard tick Haemaphysalis longicornis on the growth of Babesia bovis and Babesia bigemina.

Haemaphysalis longicornis is known as one of the most important ticks transmitting Babesia parasites in East Asian countries, including Babesia ovata and Babesia gibsoni, as well as Theileria parasites. H. longicornis is not the natural vector of Babesia bovis and Babesia bigemina. Vector ticks and transmitted parasites are thought to have established unique host-parasite interaction for their survival, meaning that vector ticks may have defensive molecules for the growth control of parasites in their bodies. However, the precise adaptation mechanism of tick-Babesia parasites is still unknown. Recently, cyclophilin A (CyPA) was reported to be important for the development of Babesia parasites in ticks. To reveal a part of their adaptation mechanism, the current study was conducted. An injection of B. bovis-infected RBCs into adult female H. longicornis ticks was found to upregulate the expression profiles of the gene and protein of CyPA in H. longicornis (HlCyPA). In addition, recombinant HlCyPA (rHlCyPA) purified from Escherichia coli exhibited significant inhibitory growth effects on B. bovis and B. bigemina cultivated in vitro, without any hemolytic effect on bovine RBCs at all concentrations used. In conclusion, our results suggest that HlCyPA might play an important role in the growth regulation of Babesia parasites in H. longicornis ticks, during natural acquisition from an infected host. Furthermore, rHlCyPA may be a potential alternative chemotherapeutic agent against babesiosis.

RNAi of the translation inhibition gene 4E-BP identified from the hard tick, Haemaphysalis longicornis, affects lipid storage during the off-host starvation period of ticks.

4E-BP, an eIF4E-binding protein, is well known as a cap-dependent translation inhibitor. Here, the 4E-BP homolog, Hl4E-BP, was isolated and identified from the hard tick Haemaphysalis longicornis. Hl4E-BP transcripts were ubiquitously expressed in the active stages, including the larvae, nymphs, and female adults, and the transcription levels were found to be higher in unfed than engorged ticks. In contrast, the expression levels of non-phosphorylated Hl4E-BP, which is a 13.4-kDa protein detected by the anti-recombinant Hl4E-BP antibody, were the highest in engorged ticks and significantly decreased progressively during the unfed starvation period of ticks. The functional role of Hl4E-BP as a metabolic brake was verified by histochemical observations on the lipid storage in midguts and fat bodies during the starvation period using ticks injected with dsHl4E-BP. The results indicate that Hl4E-BP is highly relevant to the lipid storage of ticks during the non-feeding starvation period. Our results suggest, for the first time, that Hl4E-BP may have a crucial role in the starvation resistance of ticks in an off-host condition via lipid metabolism control, although it was unclear whether Hl4E-BP might be involved in lipid synthesis regulation and/or lipid consumption inhibition.

Cloning and characterization of the autophagy-related gene 6 from the hard tick, Haemaphysalis longicornis.

Autophagy is the intracellular protein degradation process which is induced by starvation. Ticks have a unique tolerance for starvation, and it is possible that this tolerance is associated with their longevity. Previously, we isolated the homologues of four autophagy-related (ATG) genes in the hard tick, Haemaphysalis longicornis, suggesting that autophagy appeared to play an important role in tolerance for starvation as well as the development of ticks. In this study, the homologue of ATG6 was isolated from H. longicornis (HlATG6). HlATG6 mRNA expression was higher in the egg and unfed larval stages than in other stages and upregulated in ovaries during the blood-feeding period. Moreover, HlATG6-knockdowned ticks laid a few and poorly developed eggs that were white brown in color and not well surface-coated with wax. However, the expression of vitellogenin (Vg)-2, HlVg-2, in the fat body of HlATG6-knockdowned ticks was significantly upregulated. In addition, hemolymph had a deep brown color in HlATG6-knockdowned ticks on day 21 after engorgement and drop-off, indicating that the Vgs synthesized by the fat body and midgut are retained and accumulated in the hemolymph of HlATG6-knockdowned ticks, probably due to the downregulation of the Vg uptake capability of oocytes. Interestingly, HlATG6 knockdown provided non-significant influences on the expression of the Vg receptor (HlVgR) at oocytes, suggesting a non-significant depression of VgR-mediated endocytosis in the oocytes of HlATG6-knockdowned ticks. Therefore, it was interpreted that the repression of Vg uptake in the oocytes of HlATG6-knockdowned ticks may be involved in endocytic processes other than the receptor recognition of Vgs in oocytes.

Identification and characterization of class B scavenger receptor CD36 from the hard tick, Haemaphysalis longicornis.

Scavenger receptors (SRs) are cell-surface proteins and exhibit distinctive ligand-binding properties, recognizing a wide range of ligands that include microbial surface constituents and intact microbes. The class B scavenger receptor CD36 (SRB) is predominantly expressed by macrophages and is considered important in innate immunity. We here show the identification and characterization of SRB from the hard ixodid tick, Haemaphysalis longicornis (HlSRB). The full-length cDNA was 2,908 bp, including an ORF encoding of 1,518 amino acids with a pI value of 5.83. H. longicornis SRB contains a hydrophobic SRB domain and four centrally clustered cysteine residues for arrangement of disulfide bridges. Deduced amino acid sequence has an identity of 30-38% with the SRB of other organisms. RT-PCR analysis showed that mRNA transcripts were expressed in multiple organs of adult ticks but with a different transcript level in the developmental stages of H. longicornis ticks. His-tagged recombinant HlSRB was expressed in Escherichia coli with an expected molecular mass of 50 kDa. In Western blot analysis, mouse anti-rHlSRB serum recognized a strong reaction with a 50 kDa protein band in lysates prepared from egg and adult tick but showed a weak reaction with lysates of larva and nymph. In an indirect immunofluorescent antibody test, HlSRB antiserum recognized the protein located on the midgut, salivary glands, and ovary of partially fed H. longicornis females. Silencing of the HlSRB gene by RNAi led to a significant reduction in the engorged female body weight. It is noteworthy that more than a dozen SRB orthologs have been identified in the genomes of insect species with functions related to pheromone signaling, innate immunity, phagocytic clearance of apoptotic cells, and various aspects of the fatty acid metabolism. This is the first report of the identification and characterization of the SRB homologue in Chelicerata, including ticks, horseshoe crabs, scorpions, spiders, and mites.

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.

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.

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.

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.

Evaluation of Babesia bigemina 200 kDa recombinant antigen in enzyme-linked immunosorbent assay.

A truncated fragment of the gene encoding the 200-kDa protein (P200) of Babesia bigemina was cloned into a plasmid vector, pGEX-4 T-1 and expressed in Escherichia coli as a glutathione-S-transferase fused protein. An indirect enzyme-linked immunosorbent assay (ELISA) using the rp200/CT detected specific antibodies in cattle experimentally infected with B. bigemina. Furthermore, the antigen did not cross-react with antibodies to Babesia bovis, a closely related Babesia parasite indicating that rp200/CT is a specific antigen for the diagnosis of B. bigemina infection. Additionally, ELISA using p200/CT and polymerase chain reaction were conducted on serum and corresponding DNA samples obtained from field cattle to evaluate the diagnostic utility of the p200/CT antigen. Results from the current study suggest that p200/CT ELISA is a sensitive and specific method for improved serodiagnosis of B. bigemina infection.

Differential diagnosis and molecular characterization of Theileria spp. in sika deer (Cervus nippon) in Hokkaido, Japan.

Sika deer (Cervus nippon) is widely distributed in Asian countries and is one of the most common wildlife animals in Hokkaido, Japan. Previous studies identified Theileria spp. in sika deer in Japan including Theileria sp. Thrivae belonging to T. cervi group and Theileria sp. sola belonging to T. capreoli group. However, the studies failed to differentiate these two species without sequencing. Therefore, epidemiological information on cervine theileriosis in Hokkaido, Japan is limited. This study differentiated the two Theileria spp. using restriction fragments length polymorphism (RFLP). Based on the PCR-RFLP, Theileria spp. were identified in 103 (88.0%) of 117 samples, and the prevalence of each parasites were 86.3% (n = 101) and 57.3% (n = 67) for Theileria sp. Thrivae and T. capreoli-like, respectively. Phylogenetic analysis based on the 18S rRNA showed a close relationship between Theileria sp. Thrivae and T. cervi in China. In addition, phylogenetic analysis of internal transcribed spacer regions also showed a close relationship between Theileria sp. Thrivae and T. cervi.

First molecular detection and characterization of tick-borne pathogens in water buffaloes in Bohol, Philippines.

The water buffalo industry is a vital part of the Philippine livestock economy and is an essential contributor to the developing local dairy industry. Although relatively less susceptible to diseases, water buffaloes can still be infected and can act as reservoirs of tick-borne pathogens (TBPs). However, limited information is available regarding the prevalence of tick-borne infections in water buffaloes in the Philippines. This study was conducted to identify TBPs harbored by water buffaloes and to characterize these pathogens molecularly. One hundred water buffalo blood samples collected from three areas in Bohol, Visayas region, Philippines were screened for various TBPs using pathogen-specific PCR assays. TBPs were detected in 46% of the samples (39% singly infected, 7% coinfected). The pathogens detected were Anaplasma marginale (29%), Babesia bovis (21%), and B. bigemina (3%). None of the blood samples were positive for Theileria annulata, T. orientalis, and B. ovata. A. marginale infection rates were significantly higher (37.5%) among water buffaloes aged ≤6 years (P = 0.046) than those >6 years old (18.2%) and was detected only in Bulgarian Murrah (36.1%) and US Murrah (25.9%) breeds. Phylogenetic analyses revealed that groEL sequences of A. marginale were 100% identical with isolates from the Philippines (Batangas and Cebu) and China. Two B. bigemina RAP-1a gene sequences were identical to each other and were homologous with previous isolates from Thailand, Indonesia, Uruguay, and the Philippines. Moreover, four B. bovis SBP-2 partial sequences obtained in this study had 92.4-99.7% identities. This study is the first molecular detection and characterization of A. marginale, B. bigemina and B. bovis in water buffaloes in the Visayas region, and the first molecular confirmation of B. bovis infection in water buffaloes in the country. The findings presented in this study may serve as baseline data for crafting effective tick-borne disease surveillance and prevention programs in Bohol and in the Philippines.

Functional analysis of protein disulfide isomerases in blood feeding, viability and oocyte development in Haemaphysalis longicornis ticks.

Three protein disulfide isomerases from Haemaphysalis longicornis ticks (designated as HlPDI-1, HlPDI-2, and HlPDI-3) were previously identified. In order to further analyze their biological functions, the dsRNA of each HlPDI gene and one dsRNA combination of HlPDI-1/HlPDI-3 were separately injected into female ticks. Reduction of gene and protein expression of HlPDIs by RNA interference (RNAi) was demonstrated by real-time PCR, RT-PCR and Western blot analysis. In single dsRNA-injected groups, HlPDI-1 RNAi impacted tick blood feeding and oviposition, HlPDI-2 RNAi impacted tick viability and HlPDI-3 RNAi had no significant impact by itself. However, the injection of a combination of HlPDI-1/HlPDI-3 dsRNA had synergistic effects on tick viability. Furthermore, the midgut and cuticle were severely damaged in HlPDI-2 dsRNA-injected ticks and HlPDI-1/HlPDI-3 dsRNA-injected ticks, respectively, and disruption of HlPDI genes led to a significant reduction of disulfide bond-containing vitellogenin (Vg) expression in ticks. These results indicate that PDIs from H. longicornis are involved in blood feeding, viability and oocyte development, probably by mediating the formation of disulfide bond-containing proteins of the ticks and the formation of basement membrane and cuticle components such as extracellular matrix (ECM). This is the first report on the functional analysis of PDI family molecules as well as the interactions of PDI and other molecules in blood-feeding arthropods.

Cloning and characterization of an autophagy-related gene, ATG12, from the three-host tick Haemaphysalis longicornis.

Ticks are obligate hematophagous ectoparasites with a life cycle characterized by a period of starvation; many ticks spend more than 95% of their life off the host. Autophagy, which is the process of bulk cytoplasmic degradation in eukaryotic cells, is induced by starvation and is essential for extension of the lifespan. Therefore, we hypothesized that autophagy also occurs in ticks; however, there has been no report on autophagy-related (ATG) genes in ticks. Here, we show the homologue of an ATG gene, ATG12, and its expression pattern from the nymphal to adult stages in the three-host tick Haemaphysalis longicornis. The sequence analysis showed that H. longicornis ATG12 (HlATG12) cDNA is 649bp, has a 411bp ORF coding for a 136-amino acid polypeptide with the carboxy-terminal glycine residue, and has a predicted molecular mass of 15.2kDa. Moreover, RT-PCR revealed that HlATG12 was downregulated at the beginning of feeding, upregulated after engorgement, and downregulated again after molting. The expression level of HlATG12 was highest at 3 months after engorgement. By immuno-electron microscopy, it was demonstrated that HlAtg12 was localized to the region around granule-like structures within midgut cells of unfed adults. In conclusion, HlATG12 might function during unfed and molting stages.

Molecular cloning and functional characterization of an aspartic protease from the hard tick Haemaphysalis longicornis.

Haemaphysalis longicornis cDNA encoding an aspartic protease (longepsin) was identified from a midgut cDNA library. The longepsin cDNA contains 1176bp that code for 392 amino acid residues with a predictable molecular weight of 39.3kDa. The cDNA has a signal peptide sequence associated with the N-terminal domains and domain structure analysis revealed that the deduced protein has two aspartic acid residues that are characteristic of a single active site for aspartic proteases. This novel longepsin cDNA exhibits 57% identity to the lysosomal aspartic protease of Aedes aegypti, 52% to Bombyx mori cathepsin D, 38% to Ancylostoma caninum, 44% to Schistosoma mansoni and 28% to Boophilus microplus aspartic proteases. The DNA fragment coding for longepsin was cloned into a pGEX-4T-3 vector and expressed in Escherichia coli. The recombinant longepsin, once activated was able to hydrolyze casein substrate as well as hemoglobin (Hb) under acidic conditions (pH 3.5). RT-PCR analysis showed that the longepsin mRNA transcripts were expressed in salivary glands and midgut and not in the ovary. Northern blot analysis revealed that longepsin (1.5kb) was expressed in unfed and partially fed ticks and expression levels increased during feeding. The finding that longepsin is expressed in the midgut and salivary glands, proteolytic activity occurs under acidic conditions and longepsin can be gene silenced of longepsin provides compelling support for the hypothesis that longepsin plays an integral role in the proteolysis of erythrocyte Hb obtained from a host blood meal.

A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity.

Proteins capable of selective and specific inhibition of cysteine protease have been identified as cystatins and are isolated from a variety of microbes and tissues of animals and plants. The physiological function of these proteins has been proposed to be the regulation of protein turnover and defense against pathogens as well as the balance of the host-parasite immune relationship. Genes encoding cystatins have been found in several species of ticks, but the function of cystatin in ticks is not understood. We cloned a gene encoding cystatin from tick H. longicornis and designated it as Hlcyst-2 (H. longicornis cystatin-2). Its full-length cDNA is 569 bp, and it encodes a putative 133 amino acid protein with an obvious signal peptide. Sequence analysis demonstrated that it has significant homology with the known cystatin. The recombinant protein was expressed in a GST-fused soluble form in Escherichia coli and purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain, cathepsin L, and cathepsin B was identified by fluorogenic substrate analysis. Cystatin was mostly expressed in the tick midgut and hemocyte. Blood feeding induced significantly increased expression in the midgut. Real-time PCR confirmed that LPS-injected adult ticks expressed Hlcyst-2 1.6 more times than the PBS-injected control; Babesia gibsoni-infected larvae ticks expressed Hlcyst-2 1.8 more times than normal larvae ticks. The recombinant protein also showed a significant growth-inhibitory effect on Babesia bovis cultured in vitro. These results indicated this cystatin Hlcyst-2 is involved in tick innate immunity.

Epidemiological study of equine piroplasmosis in Mongolia.

The purpose of this study was to demonstrate the occurrence of equine piroplasmosis in Mongolia, a country in which the disease occurs epidemically in different climatic conditions. Antibodies to Babesia equi and B. caballi were determined in serum samples of 254 pastured horses in different locations of Mongolia using an enzyme-linked immunosorbent assay with recombinant antigens. One hundred and eighty-five (72.8%) and 102 (40.1%) of all serum samples were positive for B. equi and B. caballi infections, respectively. In addition, 78 (30.7%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread in Mongolia. To our knowledge, this is the first report describing an epidemiological study on equine piroplasmosis in different geographic regions in Mongolia.

Detection of equine Babesia spp. gene fragments in Dermacentor nuttalli Olenev 1929 infesting mongolian horses, and their amplification in egg and larval progenies.

Babesia equi (EMA-1) and Babesia caballi (BC48) gene fragments were amplified by polymerase chain reaction (PCR), in blood samples, and partially fed-females and egg and larval progenies of Dermacentor nuttalli, collected from horses in Altanbulag, Tuv Province, Mongolia. While Babesia parasite DNA was detected in some horse blood samples during the first PCR, all positive cases in partially fed-female ticks, eggs and larvae were confirmed by nested PCR. Present study reinforces earlier similar findings in unfed D. nuttalli ticks collected from an open space vegetation in Bayanonjuul, Tuv Province in Central Mongolia, pointing to the most likely important role of D. nuttalli in the transmission of equine babesiosis in Mongolia. The detection of parasite DNA in eggs and larval progenies is likewise suggestive of transovarial parasite transmission in this tick species.

Akt is an essential player in regulating cell/organ growth at the adult stage in the hard tick Haemaphysalis longicornis.

Ticks grow rapidly during blood feeding, and their body weight may ultimately increase 100-fold more than that before feeding. The molecular mechanisms controlling growth during blood feeding in ticks remain largely unknown. The conserved insulin/PI3K/Akt signaling pathway regulates growth and metabolism in eukaryotes. Here, we show evidence for the involvement of Akt in growth during blood feeding in the parthenogenetic strain of the hard tick Haemaphysalis longicornis. We identified a homolog of the Ser/Thr kinase Akt (HlAkt) from the EST database of the H. longicornis embryo. HlAkt cDNA had a 1,590 bp ORF that encodes 529 amino acids with a predicted molecular weight of 60 kDa. HlAkt possesses a PH domain, a Ser/Thr kinase domain, a hydrophobic motif, and dual phosphorylation residues (Thr 338 and Ser 503) that are essential for kinase activation. Knockdown of HlAkt by RNA interference caused inhibition of blood feeding in female ticks. Histological observation demonstrated that HlAkt knockdown led to the arrest of growth in internal organs. HlAkt knockdown also affected the expressions of blood meal-induced genes that are essential for blood digestion, development, and reproduction in the female tick. These results strongly indicate that HlAkt is essential to complete the blood feeding process accompanied by the growth of internal organs in adult ticks. This is the first report of identification and characterization of Akt in Chelicerata, including ticks.

Target of rapamycin (TOR) controls vitellogenesis via activation of the S6 kinase in the fat body of the tick, Haemaphysalis longicornis.

Vitellogenin (Vg) synthesis, vitellogenesis, is an essential process for the development and reproduction of ticks. Our previous finding led to the hypothesis that target of rapamycin (TOR) pathway is important for vitellogenesis in the hard tick, Haemaphysalis longicornis. The TOR pathway controls cellular activity according to nutrient availability in eukaryotes. TOR, a member of the phosphatidylinositol 3-kinase family, is a central player in this pathway. Here, we present preliminary evidence that H. longicornis TOR (HlTOR) controls vitellogenesis via activation of S6 kinase (S6K) in the fat body. RNA interference (RNAi)-mediated gene silencing of HlTOR was undertaken to elucidate the involvement of HlTOR in the vitellogenesis of the tick. HlTOR-RNAi caused inhibition of S6K phosphorylation in the fat body. HlTOR-RNAi also altered not only the expression levels of GATA mRNA and protein but also the intracellular localisation of GATA in the fat body. The expression levels of Vg mRNA and protein in the fat body of HlTOR-RNAi ticks were significantly lower than those in control ticks. In the pre-ovipositional stage, the ovaries of control ticks had brown oocytes developing, but those of HlTOR-RNAi ticks were white and immature. The haemolymph colour indicated that the amount of Vg was lower in HlTOR-RNAi ticks than in the controls. Furthermore, rapamycin inhibited S6K phosphorylation and reduced the expression levels of Vg mRNA and protein in the fat bodies. Vg proteins were not detected in rapamycin-treated fat bodies in the presence of 20-hydroxyecdysone. These results suggest that HlTOR activity is critical for vitellogenesis stimulated by 20-hydroxyecdysone.

HlSRB, a Class B scavenger receptor, is key to the granulocyte-mediated microbial phagocytosis in ticks.

Ixodid ticks transmit various pathogens of deadly diseases to humans and animals. However, the specific molecule that functions in the recognition and control of pathogens inside ticks is not yet to be identified. Class B scavenger receptor CD36 (SRB) participates in internalization of apoptotic cells, certain bacterial and fungal pathogens, and modified low-density lipoproteins. Recently, we have reported on recombinant HlSRB, a 50-kDa protein with one hydrophobic SRB domain from the hard tick, Haemaphysalis longicornis. Here, we show that HlSRB plays vital roles in granulocyte-mediated phagocytosis to invading Escherichia coli and contributes to the first-line host defense against various pathogens. Data clearly revealed that granulocytes that up-regulated the expression of cell surface HlSRB are almost exclusively involved in hemocyte-mediated phagocytosis for E. coli in ticks, and post-transcriptional silencing of the HlSRB-specific gene ablated the granulocytes' ability to phagocytose E. coli and resulted in the mortality of ticks due to high bacteremia. This is the first report demonstrating that a scavenger receptor molecule contributes to hemocyte-mediated phagocytosis against exogenous pathogens, isolated and characterized from hematophagous arthropods.