Cathepsin L-a novel cysteine protease from Haemaphysalis flava Neumann, 1897.

Ixodid ticks are ectoparasites responsible for the transmission of a large number of bacterial, viral, and protozoan pathogens to animals and humans. As long-term blood-pool feeders, the digestion of host blood is critical to their development as well as to the establishment of the sexual cycle of hemoparasites such as Babesia parasites, the agents of human and animal babesiosis. Previous studies have demonstrated that cysteine proteases are involved in blood digestion, embryogenesis, and pathogen transmission in other species of ticks, but their characteristics and functions are still unidentified in Haemaphysalis flava. Here, we describe the characterization of a cysteine protease HfCL from H. flava. We show that HfCL belongs to the L-like papain family of proteases, exhibits high expression in nymphs and adults, and localizes to both the midgut and salivary glands. Biochemical assays using purified recombinant enzyme reveal that rHfCL can hydrolyze the fluorogenic substrate Z-phe-Arg-MCA with optimal activity detected at pH 6. Furthermore, the short-term growth assay indicates that rHfCL can inhibit the intraerythrocytic development of Babesia microti and Babesia gibsoni in vitro.

Catalase is a determinant of the colonization and transovarial transmission of Rickettsia parkeri in the Gulf Coast tick Amblyomma maculatum.

The Gulf Coast tick (Amblyomma maculatum) has evolved as a competent vector of the spotted-fever group rickettsia, Rickettsia parkeri. In this study, the functional role of catalase, an enzyme responsible for the degradation of toxic hydrogen peroxide, in the colonization of the tick vector by R. parkeri and transovarial transmission of this pathogen to the next tick generation, was investigated. Catalase gene (CAT) expression in midgut, salivary glands and ovarian tissues exhibited a 2-11-fold increase in transcription level upon R. parkeri infection. Depletion of CAT transcripts using an RNA-interference approach significantly reduced R. parkeri infection levels in midgut and salivary gland tissues by 53-63%. The role of CAT in transovarial transmission of R. parkeri was confirmed by simultaneously blocking the transcript and the enzyme by injecting double-stranded RNA for CAT and a catalase inhibitor (3-amino-1,2,4-triazole) into gravid females. Simultaneous inhibition of the CAT transcript and the enzyme significantly reduced the egg conversion ratio with a 44% reduction of R. parkeri transovarial transmission. These data suggest that catalase is required for rickettsial colonization of the tick vector and transovarial transmission to the next generation.

Comparative study of esterases in deltamethrin and diazinon resistant Rhipicephalus microplus and Hyalomma anatolicum ticks collected from the Trans-Gangetic plains of India.

A comparative analysis of esterases in susceptible and resistant ticks revealed six types of esterases (EST-1b, EST-2b, EST-3b, EST-4b, EST-5b and EST-6b) in Rhipicephalus microplus and four types (EST-1h, EST-2h, EST-3h, EST-4h) in Hyalomma anatolicum using α-naphthyl acetate substrate. Inhibition studies with eserine sulfate, p-chloromercuribenzoate, copper sulphate and phenylmethylsulfonyl fluoride revealed a marked variation in band intensity between susceptible and resistant ticks, with the latter being more intense. Qualitative expression of EST-4b along with an extra band of EST-5b and EST-6b were indicative of deltamethrin and diazinon resistance in R. microplus, whereas qualitative expression of EST-4h was probably responsible for diazinon resistance in H. anatolicum. The data suggest that increased esterase activity may represent a detoxification strategy leading to the development of resistance in these tick populations.

Functional analysis of recombinant 2-Cys peroxiredoxin from the hard tick Haemaphysalis longicornis.

Ticks are obligate haematophagous arthropods that feed on vertebrate blood containing high levels of iron. The host-derived iron reacts to oxygen in the tick's body, and then high levels of reactive oxygen species, including hydrogen peroxide (H(2)O(2)), may be generated. High levels of H(2)O(2) cause oxidative stress to aerobic organisms. Therefore, antioxidant responses are necessary to control H(2)O(2). We focused on peroxiredoxins (Prxs), H(2)O(2) -scavenging enzymes. The sequence of Haemaphysalis longicornis 2-Cys Prx (HlPrx2) was identified from fat body cDNA libraries of this tick and recombinant HlPrx2 was then prepared using Escherichia coli. By comparison with the 2-Cys Prxs of other organisms, we found two conserved cysteines in HlPrx2, Cys51 and Cys172. We examined the antioxidant activity of HlPrx2 and mutant proteins produced by a single base substitution, converting one or both of these cysteines into serines. The assays revealed that proteins containing Cys51 showed antioxidant activity when H(2)O(2) was removed. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and size-exclusion chromatography demonstrated that only the wild-type HlPrx2 formed homodimers and that all of the proteins that we made had a high molecular weight peak. These results indicate that both Cys51 and Cys172 are essential for the dimerization of HlPrx2, whereas only the Cys51 residue is necessary for antioxidant activity.

Enolase, a plasminogen receptor isolated from salivary gland transcriptome of the ixodid tick Haemaphysalis flava.

Enolase, a multifunctional protein, is shown to act as a plasminogen receptor that contributes to fibrinolysis, which plays an important role in preventing the formation of blood clots during tick feeding. The study of enolase genes provides opportunities to develop a potential antigen target for tick control. So far, enolase has been identified in only a few species of ticks. Knowledge of the exact mechanisms of plasminogen activation and fibrinolysis by enolase as a plasminogen receptor is limited. Here, we cloned the enolase full-length complementary DNA (cDNA) from the salivary glands of Haemaphysalis flava, expressed it, and analyzed the function of the recombinant H. flava enolase. The enolase cDNA was 1988 bp in length and encoded 433 amino acid residues. It contained two domains and some highly conserved functional motifs including an assumed membrane re-association region "AAVPSGASTGI." The enolase exhibited 83.3 % amino acid similarity to that of the putative enolase of Ixodes ricinus, and 85 % to that of Ornithodoros moubata enolase. After eukaryotic expression in insect cells, Western blot analysis showed that the mouse antiserum against the hexahistidine-tagged recombinant enolase protein recognized a band of approximately 48 kDa. The recombinant enolase bound human plasminogen in a dose-dependent manner and enhanced plasminogen activation in the presence of host tissue plasminogen activator (t-PA), most probably to promote fibrinolysis and maintain blood flow at the host-tick interface. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that the expression level of enolase in salivary glands was significantly higher than in other tested tissues. Although the enolase was expressed in all developmental stages, it had the highest expression in the rapid blood feeding period of ticks. These findings indicate that the enolase might play an important role in blood feeding of H. flava.

An insight into the functional role of thioredoxin reductase, a selenoprotein, in maintaining normal native microbiota in the Gulf Coast tick (Amblyomma maculatum).

Tick selenoproteins have been associated with antioxidant activity in ticks. Thioredoxin reductase (TrxR), also a selenoprotein, belongs to the pyridine nucleotide-disulphide oxidoreductase family of proteins and is an important antioxidant. Molecular interactions between native microbiota and tick hosts have barely been investigated to date. In this study, we determined the functional role of TrxR in tick feeding and in maintenance of the native microbial community. TrxR transcript levels remained high and microbial load was reduced throughout tick attachment to the vertebrate host. RNA interference (RNAi) showed that depletion of TrxR activity did not interfere with tick haematophagy or phenotype but did reduce the viability of the microbiome within the tick tissues, presumably by perturbing redox homeostasis. The transcriptional activity of various antioxidant genes remained unaffected whereas the antioxidant genes Manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismutase (Cu/Zn SOD) and selenoprotein M (SelM) were significantly down-regulated in salivary glands of the ticks subjected to RNAi. The perturbed TrxR enzymatic activity in the knocked-down tick tissues negatively affected the bacterial load as well. Furthermore, we observed the altered bacterial profiles in TrxR-silenced tick tissues. Taken together, these results indicate an essential functional role for TrxR in maintaining the bacterial community associated with ticks.

Esterase and glutathione S-transferase levels associated with synthetic pyrethroid resistance in Hyalomma anatolicum and Rhipicephalus microplus ticks from Punjab, India.

Larval packet test was used for assessment of resistance status against cypermethrin and deltamethrin in Hyalomma anatolicum and Rhipicephalus microplus from various districts of Punjab (India). Among the various field isolates of H. anatolicum susceptible status was recorded against cypermethrin in all isolates, whereas against deltamethrin resistance status (level I-III) was recorded. In R. microplus lower resistance levels (I-II) were recorded against cypermethrin in comparison to deltamethrin (level I-IV). Quantitative analysis of general esterase activity revealed a range of 4.21 ± 0.46 to 6.05 ± 0.55 and 2.23 ± 0.23 to 2.66 ± 0.24 µmol/min/mg protein for α- and ß-esterase activity, respectively, in different field isolates of H. anatolicum and the increase in comparison to susceptible was not significant (P > 0.05). In contrast to H. anatolicum, the α- and ß-esterase activity in all field isolates (except Jalandhar) of R. microplus was higher (range of 3.89 ± 0.26 to 10.85 ± 0.47 and 1.75 ± 0.08 to 5.87 ± 0.29 µmol/min/mg protein, respectively) (P < 0.001). The glutathione-S-transferase (GST) activity in field isolates of H. anatolicum and R. microplus was in the range of 0.01 ± 0.001 to 0.03 ± 0.001 and 0.02 ± 0.0003 to 0.03 ± 0.001 mM/mg/min. The enzyme ratios (α-and ß-esterase and GST) and RR95 against deltamethrin of H. anatolicum isolates were correlated (P < 0.05), whereas in R. microplus only α-and ß-esterase and RR50 against deltamethrin were correlated (P < 0.05).

Dual silencing of long and short Amblyomma americanum acidic chitinase forms weakens the tick cement cone stability.

This study demonstrates that Amblyomma americanum (Aam) constitutively and ubiquitously expresses the long (L) and short (S) putative acidic chitinases (Ach) that are distinguished by a 210 base pair (bp) deletion in AamAch-S. Full-length AamAch-L and AamAch-S cDNA are 1959 and 1718 bp long, containing 1332 and 1104 bp open reading frames that code for 443 and 367 amino acid residues proteins with the former predicted to be extracellular and the latter intracellular. Both AamAch-L and AamAch-S mRNA are expressed in multiple organs as revealed by qualitative RT-PCR analysis. Furthermore, quantitative reverse transcription polymerase chain reaction analysis revealed that AamAch-L mRNA was downregulated in the mid-gut, but was unchanged in the salivary gland and in other organs in response to feeding. Of significant interest, AamAch-L and/or AamAch-S functions are probably associated with formation and/or maintenance of stability of A. americanum tick cement cone. Dual RNA interference silencing of AamAch-L and/or AamAch-S mRNA caused ticks to loosely attach onto host skin as suggested by bleeding around tick mouthparts and ticks detaching off host skin with a light touch. AamAch-L may apparently encode an inactive chitinase as indicated by Pichia pastoris-expressed recombinant AamAch-L failing to hydrolyse chitinase substrates. Unpublished related work in our laboratory, and published work by others that found AamAch-L in tick saliva, suggest that native AamAch-L is a non-specific immunoglobulin binding tick saliva protein in that rAamAch-L non-specifically bound rabbit, bovine and chicken non-immune sera. We discuss findings in this study with reference to advancing knowledge on tick feeding physiology.

Reprolysin metalloproteases from Ixodes persulcatus, Rhipicephalus sanguineus and Rhipicephalus microplus ticks.

Metalloproteases (MPs) have been considered essential for blood feeding and other physiological functions in several hematophagous animals, including ticks. We report the characterization of MP sequences of three important ticks from Asia, Africa and America: Ixodes persulcatus (Ip-MPs), Rhipicephalus sanguineus (Rs-MPs) and R. microplus (BrRm-MPs). Amino acid sequence identity between R. microplus and R. sanguineus MPs ranged from 76 to 100 %, and identities among I. persulcatus, I. ricinus and I. scapularis MP sequences ranged from 88 to 97 %. This high sequence identity and typical functional motifs show that all sequences are MPs. The presence of a zinc binding site, a Met-turn and cysteine rich domain at the C-terminal region indicates that these proteins belong to the reproplysin family of MPs. Differences in amino acid sequences of BrRm-MP1, BrRm-MP2, BrRm-MP4 and BrRm-MP5 (from Porto Alegre strain ticks) were 6, 2, 7 and 5 %, respectively, when compared with sequences deposited in GenBank for the same genes from other R. microplus isolates. Analyses of MPs predicted that they have various highly antigenic regions. Semi-quantitative RT-PCR analysis revealed the presence of transcripts in salivary glands of partially and fully fed female ticks. None of these transcripts were observed in males (except BrRm-MP4) and eggs. These enzymes may be functional components required during tick feeding to manipulate host defenses and support tick hematophagy.

Molecular characterization and induced changes of histone acetyltransferases in the tick Haemaphysalis longicornis in response to cold stress.

BACKGROUND: Epigenetic modifications of histones play important roles in the response of eukaryotic organisms to environmental stress. However, many histone acetyltransferases (HATs), which are responsible for histone acetylation, and their roles in mediating the tick response to cold stress have yet to be identified. In the present study, HATs were molecularly characterized and their associations with the cold response of the tick Haemaphysalis longicornis explored. METHODS: HATs were characterized by using polymerase chain reaction (PCR) based on published genome sequences, followed by multiple bioinformatic analyses. The differential expression of genes in H. longicornis under different cold treatment conditions was evaluated using reverse transcription quantitative PCR (RT-qPCR). RNA interference was used to explore the association of HATs with the cold response of H. longicornis. RESULTS: Two HAT genes were identified in H. longicornis (Hl), a GCN5-related N-acetyltransferase (henceforth HlGNAT) and a type B histone acetyltransferase (henceforth HlHAT-B), which are respectively 960 base pairs (bp) and 1239 bp in length. Bioinformatics analysis revealed that HlGNAT and HlHAT-B are unstable hydrophilic proteins characterized by the presence of the acetyltransferase 16 domain and Hat1_N domain, respectively. RT-qPCR revealed that the expression of HlGNAT and HlHAT-B decreased after 3 days of cold treatment, but gradually increased with a longer period of cold treatment. The mortality rate following knockdown of HlGNAT or HlHAT-B by RNA interference, which was confirmed by RT-qPCR, significantly increased (P < 0.05) when H. longicornis was treated at the lowest lethal temperature (- 14 °C) for 2 h. CONCLUSIONS: The findings demonstrate that HATs may play a crucial role in the cold response of H. longicornis. Thus further research is warranted to explore the mechanisms underlying the epigenetic regulation of the cold response in ticks.

Molecular cloning, identification, transcriptional analysis, and silencing of enolase on the life cycle of Haemaphysalis longicornis (Acari, Ixodidae) tick.

Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase,s role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.

Cloning and catalytic profile of Hyalomma dromedarii leucine aminopeptidase.

Ticks have harmful impacts on both human and animal health and cause considerable economic losses. Leucine aminopeptidase enzymes (LAP) play important roles during tick infestation to liberate vital amino acids necessary for growth. The aim of the current study is to identify, express and characterize the LAP from the hard tick Hyalomma dromedarii and elucidate its biochemical characteristics. We cloned an open reading frame of 1560 bp encoding a protein of 519 amino acids. The LAP full-length was expressed in Escherichia coli BL21 (DE3) and purified. The recombinant enzyme (H.d rLAP- 6×His) had a predicted molecular mass of approximately 55 kDa. Purification and the enzymatic characteristics of H.d rLAP- 6×His were studied. The purified enzyme showed maximum activity at 37 °C and pH 8.0-8.5 using Leu-p-nitroanilide as a substrate. The activity of H.d rLAP- 6×His was sensitive to ß-mercaptoethanol, dl-dithiothreitol, 1,10- phenanthroline, bestatin HCl, and EDTA and completely abolished by 0.05 % SDS. In parallel, the enzymatic activity was enhanced by Ni2+, Mn2+ and Mg2+, partially inhibited by Na+, Cu2+, Ca2+ and completely inhibited by Zn2+.

Identification and characterization of sialidase-like activity in the developmental stages of Amblyomma variegatum.

Amblyomma variegatum F. are obligate hematophagous ectoparasites of livestock that serve as the vectors of Ehrlichia ruminantium (formerly known as Cowdria ruminantium), the causative agent of heartwater disease. In the light of the fact that they are blood-feeding, their salivary glands play prominent role in their acquisition of nutrients from the bloodmeal. Sialic acids are a major component of glycoprotein in mammalian blood fluid and cells. Sialome of hard ticks is still sparse. Here, for the first time, the possible expression of sialidase in A. variegatum was investigated. Our finding established the presence of type II sialidase-like activity in the three stages (larva, nymph, and adult) of the fed and unfed tick. There was no statistically significant difference in sialidase activity in the various stages of this ectoparasite (P > 0.05). The enzyme was purified by combination of salting out and ion exchange chromatography on DEAE--cellulose and hydroxylapatite columns. Characterization of the enzyme revealed that it is optimally active at 40 degrees C and pH 5.5, and is activated by bivalent cations Zn2+ or Fe2+. The enzyme has a Km of 0.023 mM and Vmax of 0.16 millimol/min with Fetuin as the substrate. To assess the susceptibility of some mammalian cells to the tick sialidase, we prepared erythrocyte ghost cells from different animals, which were incubated with the enzyme. Results revealed that the ruminant cells were better substrates. Our work and findings contribute to the preliminary characterization of the A. variegatum salivary proteome, and may pave way to the development of new acaricides.

Acaricide resistance status in Indian isolates of Hyalomma anatolicum.

The multi host tick, Hyalomma anatolicum, is the commonest Hyalomma species in India and cattle serves as the main host of this species. A study to evaluate the acaricide resistance of H. anatolicum to deltamethrin, cypermethrin and diazinon was conducted in 20 areas located in three agro climatic regions known to have abundance of the species. Results obtained by the "larval packet test" (LPT) showed a low grade resistance (level-I, RF <5) in the tick species to both deltamethrin and cypermethrin in 10 areas and higher grade resistance (level-II, RF <25) to deltamethrin in one area, where intensive use of synthetic pyrethroids are practiced for tick control. Low grade resistance to diazinon (level I) was recorded in six areas where organophosphates compounds are extensively used for agricultural practices allowing increased exposure of the moulting instars of the ticks to these chemicals. Biochemical analysis of the samples suggested involvement of esterase and alterations of acetylcholinesterase in the resistance mechanisms.

Cross immunity with Haemaphysalis longicornis glutathione S-transferase reduces an experimental Rhipicephalus (Boophilus) microplus infestation.

Recombinant Glutathione S-transferase of Haemaphysalis longicornis (rGST-Hl) was expressed in Escherichia coli, purified by affinity chromatography and used in the immunization of cattle. Western blot analysis showed positive antibody response in cattle immunized with rGST-Hl. The tests also showed that immunized bovine sera recognize native Rhipicephalus microplus proteins in different tissue extracts. Furthermore, the vaccine potential of rGST-Hl was investigated against infestation of Hereford cattle by R. microplus. Vaccination of cattle with rGST-Hl conferred partial cross-protection immunity against R. microplus. Considering the effect on number of engorged ticks, egg laying capacity and egg fertility, the overall efficacy of vaccination was of 57%, as compared with control group.

Recombinant interferon-gamma promotes immunoglobulin G and cytokine memory responses to cathepsin L-like cysteine proteinase of Hyalomma asiaticum and the efficacy of anti-tick.

Among bloodsucking arthropods, hard tick is a vector of transmitting the most diverse human and animal pathogens, leading to an increasing number of manifestations worldwide. The development of the anti-tick vaccine has the potential to be an environmentally friendly and cost-effective option for tick management. We have previously demonstrated the induction of both humoral and cellular response against Hyalomma asiaticum (H. asiaticum) following immunization with recombinant cathepsin L-like cysteine protease from H. asiaticum tick (rHasCPL), and could control tick infestations. Interferon-gamma (IFN-γ), is an immunomodulatory factor that plays an important role in the regulation of adaptive immunity against infection. In the present study, recombinant BALB/c mouse IFN-γ (rMus-IFN-γ) was cloned and expressed using a prokaryotic expression system, and verified by Western blotting and IFN-γ-ELISA kit analysis. Female BALB/c mice (n = 12) were used for immunization using rHasCPL (100 µg) plus IFN-γ as adjuvant (10 µg). In immunized female BALB/c mice, the levels of anti-CPL antibodies as well as cytokines were determined using ELISA analysis. Protective efficacy of immunization was evaluated by larvae H. asiaticum challenge of immunized female BALB/c mice. Using rMus-IFN-γ as an adjuvant to rHasCPL vaccine (CPL + IFN-γ) promoted specific antibody IgG (IgG1 > IgG2a) and increased production of IFN-γ and IL-4 compared to immune rHasCPL group (CPL). The protected rate of immunized mice from tick challenge was significantly higher after immunization with CPL + IFN-γ (85.11 %) than with CPL (63.28 %). Immunization using CPL + IFN-γ promoted the activation of anti-HasCPL humoral and cellular immune responses, and could provide better protection against H. asiaticum infestation. This approach may could help develop a candidate vaccine for control tick infestations.

Prediction, mapping and validation of tick glutathione S-transferase B-cell epitopes.

In search of ways to address the increasing incidence of global acaricide resistance, tick control through vaccination is regarded as a sustainable alternative approach. Recently, a novel cocktail antigen tick-vaccine was developed based on the recombinant glutathione S-transferase (rGST) anti-sera cross-reaction to glutathione S-transferases of Rhipicephalus appendiculatus (GST-Ra), Amblyomma variegatum (GST-Av), Haemaphysalis longicornis (GST-Hl), Rhipicephalus decoloratus (GST-Rd) and Rhipicephalus microplus (GST-Rm). Therefore, the current study aimed to predict the shared B-cell epitopes within the GST sequences of these tick species. Prediction of B-cell epitopes and proteasomal cleavage sites were performed using immunoinformatics algorithms. The conserved epitopes predicted within the sequences were mapped on the homodimers of the respective tick GSTs, and the corresponding peptides were independently used for rabbit immunization experiments. Based on the dot blot assay, the immunogenicity of the peptides and their potential to be recognized by corresponding rGST anti-sera raised by rabbit immunization in a previous work were investigated. This study revealed that the predicted conserved B-cell epitopes within the five tick GST sequences were localized on the surface of the respective GST homodimers. The epitopes of GST-Ra, GST-Rd, GST-Av, and GST-Hl were also shown to contain a seven residue-long peptide sequence with no proteasomal cleavage sites, whereas proteasomal digestion of GST-Rm was predicted to yield a 4-residue fragment. Given that a few proteasomal cleavage sites were found within the conserved epitope sequences of the four GSTs, the sequences could also contain a T-cell epitope. Finally, the peptide and rGST anti-sera reacted against the corresponding peptide, confirming their immunogenicity. These data support the claim that the rGSTs, used in the previous study, contain conserved B-cell epitopes, which elucidates why the rGST anti-sera cross-reacted to non-homologous tick GSTs. Taken together, the data suggest that the B-cell epitopes predicted in this study could be useful for constituting epitope-based GST tick vaccines.

A Peroxiredoxin From the Haemaphysalis longicornis Tick Affects Langat Virus Replication in a Hamster Cell Line.

Ticks are hematophagous arthropods, and their blood feeding on vertebrate hosts is essential for their development. The vertebrate blood contains high levels of free iron that can react with oxygen in ticks, resulting in the production of hydrogen peroxide (H2O2), one of the reactive oxygen species. Peroxiredoxins (Prxs), H2O2-scavenging enzymes, take on an important role in the ticks' oxidative stress coping mechanism. Ticks also transmit several disease-causing pathogens, including tick-borne encephalitis virus (TBEV), in animals and humans. Therefore, the control of ticks and tick-borne pathogens is a key issue that needs to be addressed. Infection with an arthropod-borne flavivirus is known to induce oxidative stress in insect cells. We hypothesize that vector-derived Prxs could have an effect on the infection and/or replication of flaviviruses in the hosts, since ticks Prxs are possibly transmitted from ticks to their hosts. In this study, we established stable strains of baby hamster kidney (BHK) cells expressing two types of H2O2-scavenging Prxs from the hard tick Haemaphysalis longicornis (BHK-HlPrx and BHK-HlPrx2 cells). Although the infection of TBEV surrogate Langat virus (LGTV) did not induce H2O2 production in normal BHK cells, the mortality rate and the virus titer of LGTV infected BHK-HlPrx cells increased. In addition, HlPrx proteins in BHK cells can facilitate LGTV replication in cells, while HlPrx2 proteins in BHK cells cannot. The results also demonstrated that this facilitation of LGTV replication by the 1-Cys Prx in the BHK cells is not by scavenging H2O2 but by an unknown mechanism. In order to understand this mechanism, more studies using tick-derived cells and ticks are necessary.

Investigation of three enzymes and their roles in the embryonic development of parthenogenetic Haemaphysalis longicornis.

BACKGROUND: The tick Haemaphysalis longicornis exhibits two separate reproductive populations: bisexual and parthenogenetic, which have diploid and triploid karyotypes, respectively. The parthenogenetic population can undergo engorgement without copulation and produce viable female-only offspring with a longer incubation period than the bisexual population. Three enzymes, cathepsin B, cathepsin D and acid phosphatase, were found to be involved in vitellin degradation during the embryonic development of bisexual H. longicornis. However, the expression and activity profiles of these enzymes during the embryonic development of parthenogenetic ticks remain unknown. In the present study, the transcriptional expression profile, enzyme activity and roles in embryogenesis of the three enzymes during the embryonic development of parthenogenetic H. longicornis were investigated. METHODS: Quantitative real-time polymerase chain reaction (qPCR) and fluorescence detection were used to analyze the dynamic changes in the three enzymes during embryogenesis. The roles of the three enzymes during embryogenesis were also explored using RNA interference (RNAi). RESULTS: The three enzymes were all expressed during embryonic development in parthenogenetic H. longicornis. The expression of cathepsin B was highest on day 15, whereas that of cathepsin D was highest on day 3 and the peak of acid phosphatase expression occurred on day 9. The activity of cathepsin B was highest on day 3 and lowest on day 5, then gradually increased and remained stable. Cathepsin D activity was highest on day 1 and showed a gradually decreasing trend, whereas acid phosphatase showed the opposite trend and reached a peak on day 23. RNA interference experiments in engorged female ticks revealed that there was no significant difference in the number of eggs laid, but the hatching rate of the eggs was significantly decreased. CONCLUSION: The three enzymes all play important roles in embryonic development of H. longicornis, but the expression patterns and changes in the activity of the enzymes in the bisexual and parthenogenetic populations are different. The results will help a better understanding of the similarities and differences underlying embryonic development in the bisexual and parthenogenetic populations and contribute to the future exploration of the development of the parthenogenetic population of H. longicornis.

A borreliacidal factor in Amblyomma americanum saliva is associated with phospholipase A2 activity.

Previous work in our laboratory described the in vitro killing of Borrelia burgdorferi when co-cultured with saliva from adult Amblyomma americanum. Borreliacidal activity was not evident using Ixodes scapularis saliva. Mixing trypsin with saliva eliminated the borreliacidal activity of A. americanum saliva, while incorporating a trypsin inhibitor restored all borreliacidal activity, indicating this factor was of protein or peptide origin. One-dimensional PAGE indicated at least 7 major protein differences between I. scapularis and A. americanum saliva. To determine the borreliacidal factor, A. americanum saliva was fractionated by gel filtration and subsequent killing of B. burgdorferi was associated with a single fraction. Two-dimensional gel analysis indicated protein and/or peptide(s) in borreliacidal fractions running between 38 and 64 kDa. Finally, admixing saliva with the phospholipase A2 inhibitor oleyloxyethyl phosphorylcholine completely eliminated the ability of A. americanum saliva to kill B. burgdorferi. These studies indicate the borreliacidal activity found in A. americanum saliva is likely due to phospholipase A2 enzymatic activity.