Novel symbionts and potential human pathogens excavated from argasid tick microbiomes that are shaped by dual or single symbiosis.

Research on vector-associated microbiomes has been expanding due to increasing emergence of vector-borne pathogens and awareness of the importance of symbionts in the vector physiology. However, little is known about microbiomes of argasid (or soft-bodied) ticks due to limited access to specimens. We collected four argasid species (Argas japonicus, Carios vespertilionis, Ornithodoros capensis, and Ornithodoros sawaii) from the nests or burrows of their vertebrate hosts. One laboratory-reared argasid species (Ornithodoros moubata) was also included. Attempts were then made to isolate and characterize potential symbionts/pathogens using arthropod cell lines. Microbial community structure was distinct for each tick species. Coxiella was detected as the predominant symbiont in four tick species where dual symbiosis between Coxiella and Rickettsia or Coxiella and Francisella was observed in C. vespertilionis and O. moubata, respectively. Of note, A. japonicus lacked Coxiella and instead had Occidentia massiliensis and Thiotrichales as alternative symbionts. Our study found strong correlation between tick species and life stage. We successfully isolated Oc. massiliensis and characterized potential pathogens of genera Ehrlichia and Borrelia. The results suggest that there is no consistent trend of microbiomes in relation to tick life stage that fit all tick species and that the final interpretation should be related to the balance between environmental bacterial exposure and endosymbiont ecology. Nevertheless, our findings provide insights on the ecology of tick microbiomes and basis for future investigations on the capacity of argasid ticks to carry novel pathogens with public health importance.

Production of the yolk protein precursor vitellogenin is mediated by target of rapamycin (TOR) in the soft tick Ornithodoros moubata (Acari: Argasidae).

Initiation of vitellogenesis by blood feeding is essential for egg maturation in ticks. Nutrients derived from the blood meal are utilized by female ticks to synthesize the yolk protein precursor vitellogenin (Vg). Engorged Ornithodoros moubata ticks can synthesize Vg whether mated or virgin, thus O. moubata is an excellent model for studying the relative roles of blood feeding and mating in tick vitellogenesis. Injection of rapamycin into engorged O. moubata resulted in a reduction of ovarian growth and yolk accumulation in the oocytes of mated females. OmVg expression in the midgut and fat body and protein concentrations in the hemolymph significantly decreased in mated ticks after injection with rapamycin, indicating that inhibition of the nutrient-sensing target of rapamycin (TOR) pathway disrupts egg maturation at the levels of Vg expression and synthesis. These results suggest that the TOR-signaling pathway induces vitellogenesis in response to nutritional stimulation after a blood meal in O. moubata and is functionally independent of the mating-induced pathway.

Identification and expression of nuclear receptor genes and ecdysteroid titers during nymphal development in the spider Agelena silvatica.

Ecdysteroids play an essential role in the regulation of the molting processes of arthropods. Nuclear receptors of the spider Agelena silvatica that showed high homology with other arthropods especially in the functional domains were identified, two isoforms of ecdysone receptor (AsEcRA, AsEcRB), retinoid X receptor (AsRXR) and two isoforms of E75 (AsE75A, AsE75D). AsEcR and AsRXR mRNA did not show major changes in expression but occurred throughout the third instar nymphal stage. AsE75DBD was low or non-existent at first then showed a sudden increase from D7 to D10. On the other hand, AsE75D was expressed in the first half and decreased from D6 to D10. Ecdysteroid titers showed a peak on D6 in A. silvatica third instar nymphs. LC-MS/MS analysis of the ecdysteroid peak revealed only 20-hydroxyecdysone (20E) was present. The 20E peak on D6 and increase in AsE75DBD from D7 is likely a result of ecdysteroids binding to the heterodimer formed with constant expression of the AsEcR and AsRXR receptors. These findings indicate the mechanisms regulating molting widely conserved in insects and other arthropods also similarly function in spiders.

Ovarian Ecdysteroidogenesis in Both Immature and Mature Stages of an Acari, Ornithodoros moubata.

Ecdysteroidogenesis is essential for arthropod development and reproduction. Although the importance of ecdysteroids has been demonstrated, there is little information on the sites and enzymes for synthesis of ecdysteroids from Chelicerates. Ecdysteroid functions have been well studied in the soft tick Ornithodoros moubata, making this species an excellent candidate for elucidating ecdysteroidogenesis in Chelicerates. Results showed that O. moubata has at least two ecdysteroidogenic enzymes, Spook (OmSpo) and Shade (OmShd). RNAi showed both enzymes were required for ecdysteroidogenesis. Enzymatic assays demonstrated OmShd has the conserved functions of ecdysone 20-hydroxylase. OmSpo showed specific expression in the ovaries of final nymphal and adult stages, indicating O. moubata utilizes the ovary as an ecdysteroidogenic tissue instead of specific tissues as seen in other arthropods. On the other hand, OmShd expression was observed in various tissues including the midgut, indicating functional ecdysteroids can be produced in these tissues. In nymphal stages, expression of both OmSpo and OmShd peaked before molting corresponding with high ecdysteroid titers in the hemolymph. In fed adult females, OmSpo expression peaked at 8-10 days after engorgement, while OmShd expression peaked immediately after engorgement. Mated females showed more frequent surges of OmShd than virgin females. These results indicate that the regulation of synthesis of ecdysteroids differs in nymphs and adult females, and mating modifies adult female ecdysteroidogenesis. This is the first report to focus on synthesis of ecdysteroids in ticks and provides essential knowledge for understanding the evolution of ecdysteroidogenesis in arthropods.

Inhibitory effect of gut bacteria from the Japanese honey bee, Apis cerana japonica, against Melissococcus plutonius, the causal agent of European foulbrood disease.

European foulbrood is a contagious bacterial disease of honey bee larvae. Studies have shown that the intestinal bacteria of insects, including honey bees, act as probiotic organisms. Microbial flora from the gut of the Japanese honey bee, Apis cerana japonica F. (Hymenoptera: Apidae), were characterized and evaluated for their potential to inhibit the growth of Melissococcus plutonius corrig. (ex White) Bailey and Collins (Lactobacillales: Enterococcaceae), the causative agent of European foulbrood. Analysis of 16S rRNA gene sequences from 17 bacterial strains isolated by using a culture-dependent method revealed that most isolates belonged to Bacillus, Staphylococcus, and Pantoea. The isolates were screened against the pathogenic bacterium M. plutonius by using an in vitro growth inhibition assay, and one isolate (Acja3) belonging to the genus Bacillus exhibited inhibitory activity against M. plutonius. In addition, in vivo feeding assays revealed that isolate Acja3 decreased the mortality of honey bee larvae infected with M plutonius, suggesting that this bacterial strain could potentially be used as a probiotic agent against European foulbrood.

Characterization of bifidobacteria in the digestive tract of the Japanese honeybee, Apis cerana japonica.

Bifidobacteria were isolated from the intestinal tract of the Japanese honeybee, Apis cerana japonica, and investigated for potential application as a probiotic agent against Melissococcus plutonius, the causal agent of European foulbrood (EFB), based on the findings of in vitro inhibition assays. A total of 11 bifidobacteria strains (designated as AcjBF1-AcjBF11) were isolated using a culture-dependent method and their 16S rRNA gene sequences were analyzed. The AcjBF isolates belonged to three distinct bifidobacterial phylotypes that were similar to those found in the European honeybee, Apis mellifera. Although the Japanese and European honeybees are distinct species with different traits and habits, the observation that they share highly similar bifidobacterial phylotypes suggests that bifidobacteria are conserved among honeybee species. Despite having extremely high 16S rRNA gene sequence similarities, the AcjBF isolates had markedly different carbohydrate fermentation profiles. In addition, in vitro growth inhibition assays revealed that the cell-free supernatants of all AcjBF isolates exhibited antagonistic effects on M. plutonius growth. These results indicate that the bifidobacteria isolated from the gut of Japanese honeybee could potentially be employed as a new biological agent to control EFB.

LKR/SDH plays important roles throughout the tick life cycle including a long starvation period.

BACKGROUND: Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) is a bifunctional enzyme catalyzing the first two steps of lysine catabolism in plants and mammals. However, to date, the properties of the lysine degradation pathway and biological functions of LKR/SDH have been very little described in arthropods such as ticks. METHODOLOGY/PRINCIPAL FINDINGS: We isolated and characterized the gene encoding lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) from a tick, Haemaphysalis longicornis, cDNA library that encodes a bifunctional polypeptide bearing domains similar to the plant and mammalian LKR/SDH enzymes. Expression of LKR/SDH was detected in all developmental stages, indicating an important role throughout the tick life cycle, including a long period of starvation after detachment from the host. The LKR/SDH mRNA transcripts were more abundant in unfed and starved ticks than in fed and engorged ticks, suggesting that tick LKR/SDH are important for the starved tick. Gene silencing of LKR/SDH by RNAi indicated that the tick LKR/SDH plays an integral role in the osmotic regulation of water balance and development of eggs in ovary of engorged females. CONCLUSIONS/SIGNIFICANCE: Transcription analysis and gene silencing of LKR/SDH indicated that tick LKR/SDH enzyme plays not only important roles in egg production, reproduction and development of the tick, but also in carbon, nitrogen and water balance, crucial physiological processes for the survival of ticks. This is the first report on the role of LKR/SDH in osmotic regulation in animals including vertebrate and arthropods.

Multiple functions of short synthetic enantiomeric peptides based on beetle defensins.

Four enantiomeric 9-mer peptides, D-peptides A (RLYLRIGRR-NH(2)), B (RLRLRIGRR-NH(2)), C (ALYLAIRRR-NH(2)), and D (RLLLRIGRR-NH(2)), were designed and synthesized on the basis of a beetle defensin antimicrobial peptide. These D-9-mer peptides have been reported to exhibit multiple functions, including antimicrobial and antiprotozoan activity, without cytotoxicity on normal fibroblasts and leukocyte cells. In this study, we found that the D-9-mer peptides inhibited telomerase activity (IC(100) = 40 microM). A new peptide, D-peptide C2 (ALYLAIRRRRRRRR-NH(2)), designed from D-peptide C to translocate into the cytoplasm by a penetrating sequence (octa-arginine), showed extremely strong telomerase inhibitory activity (IC(100) = 0.1 microM). D-Peptide C2 exhibited a great increase in cytotoxicity against various cancer cell lines (IC(50) = 3.4-26.4 microM). However, the immediate death of the cells suggested that the high cytotoxicity was not an effect of telomerase inhibitory activity. Mitochondrial swelling assay and microscopical observations of mitochondria indicated that the major target of the D-peptide C2 was the mitochondrial membrane.

Selective cancer cell cytotoxicity of enantiomeric 9-mer peptides derived from beetle defensins depends on negatively charged phosphatidylserine on the cell surface.

Four enantiomeric 9-mer peptides named d-peptide A, B, C and D were designed and synthesized on the basis of 43-mer insect defensins from two beetles. The d-9-mer peptides maintained bacterial membrane disruptive activity similar to the original peptides and also showed various extents of growth inhibitory activity against different cancer cell lines. Of these peptides, d-peptide B exhibited the highest selective cancer cell cytotoxicity against the mouse myeloma cell line, P3-X63-Ag8.653. Flow cytometric and scanning electron microscopic analysis revealed d-peptide B disrupts mouse myeloma membrane construction, whereas no cytotoxic effect on normal leukocytes was observed. Moreover, a strong correlation between negatively charged phosphatidylserine (PS) density in cancer cell membrane surface and sensitivity to d-9-mer peptides were observed in various cancer cell lines. These results suggest that d-9-mer peptides have negative charge-dependent selective cancer cell cytotoxicity targeting PS in the cancer cell membrane. In addition, synergic growth inhibitory activity against mouse myeloma was observed in combinations of d-peptide B and dexamethasone. These results suggest d-9-mer peptides are promising candidates for novel anticancer drugs.

Isolation and expression of the retinoid X receptor from last instar nymphs and adult females of the soft tick Ornithodoros moubata (Acari: Argasidae).

Retinoid X receptors (RXR) exist broadly from invertebrates to vertebrates, and play essential roles in physiological processes of these organisms. In arthropods, RXRs form a complex with the ecdysteroid receptor (EcR) and ecdysteroids to mediate the regulation of ecdysis and reproduction. Compared to EcR, RXR and its homologue ultraspiracle (USP) are much less well understood. Therefore, we identified RXR of the soft tick Ornithodoros moubata (OmRXR) and used real-time PCR to examine the expression of OmRXR. This is the first report of RXR from a soft tick. OmRXR showed higher homology to hard tick, crustacean and vertebrate RXRs than insect RXRs and USPs. OmRXR expression was observed during molting in the last instar nymphs coinciding with EcR expression and increases in ecdysteroid titers. Tick vitellogenesis normally occurs soon after engorgement and OmRXR expression coinciding with EcR expression and ecdysteroid titers in engorged females occurred before vitellogenin (Vg) synthesis and egg maturation. The ecdysteroid/EcR/RXR complex appears to be important in the regulation of molting and vitellogenesis of soft ticks.

Immunohistochemical examination of PDGF-AB, TGF-beta and their receptors in the hemocytes of a tick, Ornithodoros moubata (Acari: Argasidae).

Growth factors, Platelet Derived Growth Factor (PDGF) and Transforming Growth Factor (TGF)-beta, were demonstrated in vertebrate and invertebrate immmunocytes. It is generally known that the growth factors are important in various biological processes, such as the regulation of cell differentiation, development and wound healing. In the present study, the presence of TGF-beta1 and PDGF-receptor-alpha in plasmatocytes and PDGF-AB in granulocytes of a soft tick, Ornithodoros moubata, was confirmed immunohistochemically. The tick midgut might be damaged by intracellular digestion and penetration of protozoa. Therefore, it is considered that PDGF from granulocytes may affect the PDGF-receptor-alpha in plasmatocytes and TGF-beta from plasmatocytes may function to repair the midgut. The results obtained here add to the elucidation of the functions of tick hemocytes.

Identification and expression analysis of an actin gene from the soft tick, Ornithodoros moubata (Acari: Argasidae).

Actin genes are found in all living organisms and highly conserved in various animals as shown by numerous studies on actin gene expression and function. Because of this ubiquitous nature of actin, it is often used as an internal control in gene expression studies. To clarify the suitability of actin gene as an internal control in soft ticks, isolation and expression analyses of an actin gene from Ornithodoros moubata was performed. An actin gene of Ornithodoros moubata (OmAct2, GenBank accession no. AB208021) with 1,131 bp and 376 amino acid residues was identified. The homology of OmAct2 with other arthropod actin genes was greater than 80% in nucleotides and 99% in amino acids. OmAct2 gene was classified as a cytoskeletal actin type by absence of muscle-specific amino acids commonly found in insects and ubiquitous expression in all stages and both sexes. Southern blot revealed that O. moubata has four to seven actin genes. In addition, actin expression analyzed by real-time PCR before and after blood feeding was not significantly different indicating OmAct2 is an appropriate internal control for the analysis of gene expression in these ticks.

RNA interference of cytosolic leucine aminopeptidase reduces fecundity in the hard tick, Haemaphysalis longicornis.

Ticks are effective vectors of pathogens because of their blood feeding and high fecundity. This high fecundity is related to the size of the blood meal. Therefore, knowledge of how blood proteins are degraded and converted to proteins, including yolk protein, is important for the development of ways to inhibit the utilization of blood proteins by ticks. RNA interference (RNAi) is becoming a powerful post-transcriptional gene silencing technique that provides insight into gene function. We constructed a double-stranded RNA (dsRNA) based on a previously cloned Haemaphysalis longicornis leucine aminopeptidase (HlLAP) gene to reevaluate the biological role in tick blood digestion. Gene specific transcriptional, translational, and functional disruptions were achieved by the introduction of dsRNA into the ticks. Significantly delayed onset of egg-laying and reduced egg oviposition resulted from the RNAi for the HlLAP gene. These results suggest that HlLAP actually works as a blood digestive enzyme and affects tick fecundity via unknown mechanisms. The reduction of egg oviposition may be caused by a decrease in nutrients, especially free amino acids generated by HlLAP, from the blood meal. This is the first report of an impact on tick reproduction caused by gene silencing of a blood digestion-related molecule.

Ecdysteroid hormone titer and its relationship to vitellogenesis in the soft tick, Ornithodoros moubata (Acari: Argasidae).

A blood meal is required for reproduction in most argasid female ticks. The blood meal appears to stimulate an organ in the posterior end to produce a fat body stimulating factor (FSF), which is thought to be an ecdysteroid, to induce vitellogenin (Vg) synthesis. In this study, the relationship of vitellogenesis and ecdysteroids was investigated by measuring Vg and ecdysteroid titers while observing oocyte development and oviposition in mated and virgin females. Oviposition occurred from day 10 after engorgement in mated females and continued up to 40-50 days, whereas egg maturation and oviposition did not occur in virgin females. Vg titers in the hemolymph peaked on day 6 after engorgement and subsequently declined in mated females. Interestingly, Vg synthesis occurred and ovarian development progressed to the development of early vitellogenic oocytes in virgin females but oocyte maturation and oviposition did not occur. Topical application of ecdysteroids induced oviposition in fed virgin females indicating that ecdysteroids may induce oviposition. Concentrations of ecdysteroids for 20 days after engorgement revealed several peaks in mated female whole body extracts, but no peaks in virgin female extracts. In the hemolymph of only mated females, ecdysteroid titers showed two peaks that followed the early peak of ecdysteroids in the whole body on day 4 and 6 after engorgement. In addition, ecdysteroids in the reproductive tissues increased with the development of the ovary in mated females and this increase coincided with the latter peaks of the whole body. These observations indicate that physiological elevation of ecdysteroids accelerate Vg synthesis, and may induce egg maturation and stimulate oviposition in fed mated Ornithodoros moubata females.

Fate of GFP-expressing Escherichia coli in the midgut and response to ingestion in a tick, Ornithodoros moubata (Acari: Argasidae).

Ticks are well-known vectors of various pathogens but migration of the pathogens in the tick midgut is not fully understood. In the present study, the fate of microbes in the midgut of Ornithodoros moubata was observed using green fluorescent protein (GFP)-expressing Escherichia coli. Fluctuations in the percentage of hemocytes in the hemolymph (Hc) and expression of an antimicrobial peptide, defensin, in the midgut was also investigated. Most E. coli gradually disappeared in the midgut after ingestion fluctuations in Hc coincided with the changes. Expression of defensin was also confirmed and slightly up-regulated after E. coli ingestion. Moreover, it was demonstrated that E. coli can not pass through the tick midgut epithelium after ingestion by the hemolymph cultures. It is known that various pathogens and host immunoglobulins ingested with a blood meal can enter into the hemocoel, which suggests the presence of unique and complex passage mechanisms for each molecule and organism. The results obtained here help to clarify that digestion enzymes is an important function of the tick midgut to protect against invading molecules and organisms.

Tickcidal effect of monoclonal antibodies against hemocytes, Om21, in an adult female tick, Ornithodoros moubata (Acari: Argasidae).

In the present study, monoclonal antibodies (mAbs) against adult Ornithodoros moubata hemocytes were established. Afterward, artificial feeding was performed to assess the tickcidal effect of fetal bovine serum meal containing each mAb. As a result, Om21 showed the strongest tickcidal effect on adult female O. moubata. The reactivity of various tick cells and organs, including the hemocyte, midgut, trachea, ovary, fat body, and muscle, to Om21 was then examined by an indirect immunofluorescent antibody test and by immunoelectron microscopy. Om21 reacted with not only hemocytes but also with fat body cells, epidermis, cuticle of the trachea, connective tissue of the muscle, and the basement membrane of the midgut, trachea, fat body, oocyte, and epidermis. These results suggest that Om21 passing through the midgut epithelium induced a tickcidal effect on hemocytes or various organs. However, the target of Om21 could not be identified in the present study. The antihemocyte mAb produced in this study, Om21, may be useful for the immunological control of ticks.

Antibacterial activity and mechanism of action of tick defensin against Gram-positive bacteria.

Defensins are a major group of antimicrobial peptides and are found widely in vertebrates, invertebrates and plants. Invertebrate defensins have been identified from insects, scorpions, mussels and ticks. In this study, chemically synthesized tick defensin was used to further investigate the activity spectrum and mode of action of natural tick defensin. Synthetic tick defensin showed antibacterial activity against many Gram-positive bacteria but not Gram-negative bacteria and low hemolytic activity, characteristic of invertebrate defensins. Furthermore, bactericidal activity against pathogenic Gram-positive bacteria including Bacillus cereus, Enterococcus faecalis and methicillin-resistant Staphylococcus aureus was observed. However, more than 30 min was necessary for tick defensin to completely kill bacteria. The interaction of tick defensin with the bacterial cytoplasmic membrane and its ability to disrupt the membrane potential was analyzed. Tick defensin was able to disrupt the membrane potential over a period of 30-60 min consistent with its relatively slow killing. Transmission electron microscopy of Micrococcus luteus treated with tick defensin showed lysis of the cytoplasmic membrane and leakage of cellular cytoplasmic contents. These findings suggest that the primary mechanism of action of tick defensin is bacterial cytoplasmic membrane lysis. In addition, incomplete cell division with multiple cross-wall formation was occasionally seen in tick defensin-treated bacteria showing pleiotropic secondary effects of tick defensin.

Up-regulated humoral immune response in the soft tick, Ornithodoros moubata (Acari: Argasidae).

Ticks have an efficient defense system for preventing microbial infection. The antimicrobial peptide defensin is one effective molecule in this system. Here we investigated immune competence and the involvement of defensin in the humoral defense of the soft tick, Ornithodoros moubata. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that gene expression of all four defensin isoforms was up-regulated by bacteria or bacterial components. Defensin gene up-regulation by hemocoelic inoculation of bacteria involves the midgut and granulocytes. In immunodetection analysis, immunization by bacterial injection increases the relative concentration of defensin-like material in the hemolymph plasma. Furthermore, elevated antibacterial activity against Gram-positive bacteria but not against Gram-negative bacteria was observed after immunization by a liquid growth inhibition assay. Therefore, enhanced anti-Gram-positive bacterial activity appears to be partially dependent on the release of defensin into the hemolymph. These findings demonstrate that defensin plays an important role in the up-regulated humoral response of O. moubata.

Antibacterial hemoglobin fragments from the midgut of the soft tick, Ornithodoros moubata (Acari: Argasidae).

Midgut contents of Ornithodoros moubata showed strong antibacterial activity against Staphylococcus aureus. A combination of reversed-phase chromatography and mass spectrometric analysis was used to isolate two antibacterial peptides from the tick midgut lumen. Partial amino acid sequences by Edman degradation of these two peptides showed they are identical with the 1-11 and 3-19 portions of rabbit a hemoglobin. Host rabbit a hemoglobin appears to be cleaved between Met32 and Phe33 to produce these two antibacterial peptides. Isolation of a host bovine hemoglobin fragment with antimicrobial activity has been demonstrated in the Ixodid tick, Boophilus microplus (Fogaca et al. 1999). Similar immune mechanisms in the two major families of ticks, Ixodidae and Argasidae, appear to use the hemoglobin of the host as an antimicrobial agent in midgut defense.

Identification and molecular characterization of a chitinase from the hard tick Haemaphysalis longicornis.

A cDNA encoding tick chitinase was cloned from a cDNA library of mRNA from Haemaphysalis longicornis eggs and designated as CHT1 cDNA. The CHT1 cDNA contains an open reading frame of 2790 bp that codes for 930 amino acid residues with a coding capacity of 104 kDa. The deduced amino acid sequence shows a 31% amino acid homology to Aedes aegypti chitinase and a multidomain structure containing one chitin binding peritrophin A domain and two glycosyl hydrolase family 18 chitin binding domains. The endogenous chitinase of H. longicornis was identified by a two-dimensional immunoblot analysis with mouse anti-rCHT1 serum and shown to have a molecular mass of 108 kDa with a pI of 5.0. A recombinant baculovirus AcMNPV.CHT1-expressed rCHT1 is glycosylated and able to degrade chitin. Chitin degradation was ablated by allosamidin in a dose-dependent manner. The optimal temperature and pH for activity of the purified chitinase were 45 degrees C and pH 5-7. The CHT1 cDNA has an ELR motif for chemokine-mediated angiogenesis and appears to be a chitinase of the chemokine family. Localization analysis using mouse anti-rCHT1 serum revealed that native chitinase is highly expressed in the epidermis and midgut of the tick. AcMNPV.CHT1 topically applied to H. longicornis ticks exhibited replication. This is the first report of insect baculovirus infection of ticks. The importance of AcMNPV.CHT1 as a novel bio-acaricide for tick control is discussed.