Comparative analysis of the mitochondrial genome of Dermacentor steini from different regions in China.

Ticks are a group of blood-sucking ectoparasites that play an important role in human health and livestock production development as vectors of zoonotic diseases. The phylogenetic tree of single genes cannot accurately reflect the true kinship between species. Based on the complete mitochondrial genome analysis one can help to elucidate the phylogenetic relationships among species. In this study, the complete mitochondrial genome of Dermacentor steini (isolate Longyan) was sequenced and compared with the mitochondrial genes of 3 other Chinese isolates (Nanchang, Jinhua and Yingtan). In Dermacentor steini 4 isolates had identical or similar mitochondrial genome lengths and an overall variation of 0.76% between sequences. All nucleotide compositions showed a distinct AT preference. The most common initiation and stop codons were ATG and TAA, respectively. Fewer base mismatches were found in the tRNA gene of D. steini (isolate Longyan), and the vicinity of the control region and tRNA gene was a hot rearrangement region of the genus Dermacentor. Maximum likelihood trees and Bayesian trees indicate that D. steini is most closely related to Dermacentor auratus. The results enrich the mitochondrial genomic data of species in the genus Dermacentor and provide novel insights for further studies on the phylogeographic classification and molecular evolution of ticks.

Iron Reduction in Dermacentor andersoni Tick Cells Inhibits Anaplasma marginale Replication.

Anaplasma spp. are obligate intracellular, tick-borne, bacterial pathogens that cause bovine and human anaplasmosis. We lack tools to prevent these diseases in part due to major knowledge gaps in our fundamental understanding of the tick-pathogen interface, including the requirement for and molecules involved in iron transport during tick colonization. We determine that iron is required for the pathogen Anaplasma marginale, which causes bovine anaplasmosis, to replicate in Dermacentor andersoni tick cells. Using bioinformatics and protein modeling, we identified three orthologs of the Gram-negative siderophore-independent iron uptake system, FbpABC. Am069, the A. marginale ortholog of FbpA, lacks predicted iron-binding residues according to the NCBI conserved domain database. However, according to protein modeling, the best structural orthologs of Am069 are iron transport proteins from Cyanobacteria and Campylobacterjejuni. We then determined that all three A. marginale genes are modestly differentially expressed in response to altered host cell iron levels, despite the lack of a Ferric uptake regulator or operon structure. This work is foundational for building a mechanistic understanding of iron uptake, which could lead to interventions to prevent bovine and human anaplasmosis.

Precise annotation of tick mitochondrial genomes reveals multiple copy number variation of short tandem repeats and one transposon-like element.

BACKGROUND: In the present study, we used long-PCR amplification coupled with Next-Generation Sequencing (NGS) to obtain complete mitochondrial (mt) genomes of individual ticks and unprecedently performed precise annotation of these mt genomes. We aimed to: (1) develop a simple, cost-effective and accurate method for the study of extremely high AT-content mt genomes within an individual animal (e.g. Dermacentor silvarum) containing miniscule DNA; (2) provide a high-quality reference genome for D. silvarum with precise annotation and also for future studies of other tick mt genomes; and (3) detect and analyze mt DNA variation within an individual tick. RESULTS: These annotations were confirmed by the PacBio full-length transcriptome data to cover both entire strands of the mitochondrial genomes without any gaps or overlaps. Moreover, two new and important findings were reported for the first time, contributing fundamental knowledge to mt biology. The first was the discovery of a transposon-like element that may eventually reveal much about mechanisms of gene rearrangements in mt genomes. Another finding was that Copy Number Variation (CNV) of Short Tandem Repeats (STRs) account for mitochondrial sequence diversity (heterogeneity) within an individual tick, insect, mouse or human, whereas SNPs were not detected. The CNV of STRs in the protein-coding genes resulted in frameshift mutations in the proteins, which can cause deleterious effects. Mitochondria containing these deleterious STR mutations accumulate in cells and can produce deleterious proteins. CONCLUSIONS: We proposed that the accumulation of CNV of STRs in mitochondria may cause aging or diseases. Future tests of the CNV of STRs hypothesis help to ultimately reveal the genetic basis of mitochondrial DNA variation and its consequences (e.g., aging and diseases) in animals. Our study will lead to the reconsideration of the importance of STRs and a unified study of CNV of STRs with longer and shorter repeat units (particularly polynucleotides) in both nuclear and mt genomes.

Sequence identification and expression profile of seven Dermacentor marginatus glutathione S-transferase genes.

Dermacentor marginatus is a widespread tick species and a vector of many pathogens in Eurasia. Due to the medical importance of D. marginatus, control measures are needed for this tick species. Currently tick control approaches rely mostly on acaricide application, whereas wrong and irrational acaricide use may result in drug resistance and residue problems. Vaccination as an alternative approach for tick control has been proven to be effective towards some tick species. However, immunization against D. marginatus has not yet reached satisfactory protection. The effort of in silico based analysis could predict antigenicity and identify candidates for anti-tick vaccine development. We carried out an in silico analysis of D. marginatus glutathione S-transferases (DmGSTs) in order to identify blood-feeding induced GSTs as antigens that can be used in anti-tick vaccine development. Phylogenetic analysis, linear B-cell epitope prediction, homology modeling, and conformational B-cell epitope mapping on the GST models were performed to identify highly antigenic DmGSTs. Relative gene expressions of the seven GSTs were profiled through real-time quantitative PCR (RT-qPCR) to outline GSTs up-regulated during blood feeding. The phylogenetic analysis indicated that the seven GSTs belonged to four classes of GST, including one in epsilon-class, one in zeta-class, one in omega-class, and four in mu-class. Linear B-cell epitope prediction revealed mu-class GSTs share similar conserved antigenic regions. The conformational B-cell epitope mapped on the homology model of the GSTs displayed that GSTs of mu-class showed stronger antigenicity than that of other classes. RT-qPCR revealed DmGSTM1 and DmGSTM2 were positively related to blood feeding. In sum, the data suggest that DmGSTM1 and DmGSTM2 could be tested for potential anti-tick vaccine trials.

Progressive behavioural, physiological and transcriptomic shifts over the course of prolonged starvation in ticks.

Ticks are obligatorily hematophagous but spend the majority of their lives off host in an unfed state where they must resist starvation between bouts of blood feeding. Survival during these extended off-host periods is critical to the success of these arthropods as vectors of disease; however, little is known about the underlying physiological and molecular mechanisms of starvation tolerance in ticks. We examined the bioenergetic, transcriptomic and behavioural changes of female American dog ticks, Dermacentor variabilis, throughout starvation (up to nine months post-bloodmeal). As starvation progressed, ticks utilized glycogen and lipid, and later protein as energy reserves with proteolysis and autophagy facilitating the mobilization of endogenous nutrients. The metabolic rate of the ticks was expectedly low, but showed a slight increase as starvation progressed possibly reflecting the upregulation of several energetically costly processes such as transcription/translation and/or increases in host-seeking behaviours. Starved ticks had higher activity levels, increased questing behaviour and augmented expression of genes related to chemosensing, immunity and salivary gland proteins. The shifts in gene expression and associated behavioural and physiological processes are critical to allowing these parasites to exploit their ecological niche as extreme sit-and-wait parasites. The overall responses of ticks to starvation were similar to other blood-feeding arthropods, but we identified unique responses that could have epidemiological and ecological significance for ticks as ectoparasites that must be tolerant of sporadic feeding.

When Do We Eat? The Clock is Ticking.

Ticks are simultaneously fascinating and disgusting. Anyone who has removed a bloated blood-filled tick from themselves or a pet understands the "yuck" factor they arouse. But ticks are also fascinating from a physiological perspective. Ticks are the ultimate sit-and-wait predators. Female ixodid ticks (hard ticks) consume a single meal during each life stage (larva, nymph and adult), which means only three lifetime meals over a 1- to 3-year lifespan. Most males do not feed as adults, so they only feed twice. Thus, prolonged starvation is a quintessential aspect of tick life history. Although ticks have been widely studied for their importance as disease vectors, the vast majority of research has focused on tick-host interactions. Ixodid ticks spend the overwhelming majority of their lives off their hosts, but little is known about these periods. A new study begins to fill in some of these knowledge gaps. In this issue of Molecular Ecology, Rosendale, Dunlevy, Marshall, and Benoit examine physiological, behavioural and transcriptomic changes occurring during long-term starvation of the American dog tick, Dermacentor variabilis. Their work provides insights into how ticks are able to go so long between meals and how they prepare for their next meal.

The mitochondrial genome of the ornate sheep tick, Dermacentor marginatus.

The ornate sheep tick, Dermacentor marginatus, is widespread in Europe. Its vector role of various zoonotic pathogens received much attention in these regions. However, the genomic resources of the ticks are limited. In this study, the complete mitochondrial genome of a single female D. marginatus collected in Slovakia was sequenced through the Illumina HiSeq sequencing platform. The mitochondrial genome is 15,067 bp long and contains 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes. The overall G+C content is 21.6%. The gene order is identical to that of Metastriata ticks. The codon usage pattern is similar with that of other tick species. As in other ticks, two truncated tRNA genes were observed. Two control regions were found between tRNA-Leu and tRNA-Cys, tRNA-Ile and rrnS, respectively. The mitochondrial genome contains three noncoding regions, which is similar to that in D. nitens. The noncoding region located between rrnS and tRNA-Val is shorter than that of other Dermacentor species. Phylogenetic analyses indicate that D. marginatus is clustered with other Dermacentor species. These findings are helpful for exploring the systematics and evolution of ticks in the future.

Mechanistic underpinnings of dehydration stress in the American dog tick revealed through RNA-Seq and metabolomics.

Ticks are obligate blood feeders but spend the majority of their lifetime off-host where they must contend with a multitude of environmental stresses. Survival under desiccating conditions is a determinant for habitats where ticks can become established, and water-balance characteristics of ticks have been extensively studied. However, little is known about the underlying aspects associated with dehydration stress in ticks. In this study, we examined the response of male American dog ticks, Dermacentor variabilis, to dehydration using a combined transcriptomics and metabolomics approach. During dehydration, 497 genes were differentially expressed, including an up-regulation of stress-response and protein-catabolism genes and concurrent down-regulation of several energetically expensive biological processes. Accumulation of several metabolites, including specific amino acids, glycerol and gamma aminobutyric acid (GABA), and transcript shifts in the associated pathways for generating these metabolites indicated congruence between changes in the metabolome and gene expression. Ticks treated with exogenous glycerol and GABA demonstrated altered water-balance characteristics; specifically, increased water absorption at high relative humidity. Finally, we observed changes in locomotor activity in response to dehydration, but this change was not influenced by the accumulation of GABA. Overall, the responses to dehydration by these ticks were similar to those observed in other dehydration-tolerant arthropods, but several molecular and behavioral responses are distinct from those associated with other taxa.

Complete mitochondrial genomes of Dermacentor silvarum and comparative analyses with another hard tick Dermacentor nitens.

Ticks are obligate blood-sucking ectoparasites of a wide range of vertebrates. They can transmit a range of pathogens that cause economic losses to livestock production as well as human disease. In the present study, the complete mitochondrial (mt) genome of Dermacentor silvarum was determined. The mt genome is 14,945 bp in length contains 37 genes, including 13 are protein-coding genes (cox1-3, nad1-6, nad4L, cytb, atp6 and atp8), two ribosomal RNA genes and 22 transfer RNA genes. The nucleotide composition of the D. silvarum mt genome was A + T biased at 78.78%; T was the most abundant nucleotide and G the least abundant. The mt genome of D. silvarum was 106 bp longer than that of Dermacentor nitens and the arrangements of two genomes were identical. For the 13 protein-coding genes, comparison between D. silvarum and D. nitens revealed sequence divergence at both the nucleotide (15.46-35.14%) and amino acid (6.05-48.98%) levels. Among them, cox1 was the most conserved gene, while atp8 was the least conserved. The lengths of the 13 protein-coding genes were the same or similar, except for cytb which was significantly longer in D. silvarum than in D. nitens. The mtDNA contained a variable repeat region consisting of a "similar to nad1" motif that was repeated three times, and the "Tick-box" motifs were also found. The overall difference between the nucleotide sequences of the two complete mt genomes was 21.4%. The mtDNA data presented in this study provide a rich resource for further studies on the phylogenetics, population genetics, and molecular epidemiology of ticks.

Expression pattern of subA in different tissues and blood-feeding status in Haemaphysalis flava.

Tick-borne-diseases (TBD) pose a huge threat to the health of both humans and animals worldwide. Tick vaccines constitute an attractive alternative for tick control, due to their cost-efficiency and environmental-friendliness. Subolesin, a protective antigen against ticks, is reported to be a promising candidate for the development of broad-spectrum vaccines. However, the entire length of its gene, subA, and its gene expression pattern in different tissues and blood-feeding status (or different levels of engorgement) have not been studied extensively. In our study, the full-length of subA in Haemaphysalis flava, Rhipicephalus haemaphysaloides, Rhipicephalus microplus, and Dermacentor sinicus was cloned by RACE-PCR. The subA expression pattern was analyzed further in H. flava in different tissues and blood-feeding status by RT-PCR. We found that the full-length of subA in H. flava, R. haemaphysaloides, R. microplus, and D. sinicus was 1318, 1498, 1316, and 1769 bp, respectively, with encoded proteins at 180, 162, 162, and 166 aa in length, respectively. The primary structure of subolesin in H. flava included three conserved regions and two hypervariable regions, with no signal peptide. SubA expression in female H. flava of different blood-feeding status was in the order of the fasted < the 1/4-engorged < the half-engorged < the fully-engorged (p < 0.01). Tissue expression of subA was in the order of salivary gland > midgut > integument (p < 0.01), but its expression in salivary glands was not statistically different from that in ovaries. We concluded that subolesin was a conserved antigen and that subA was expressed differentially in H. flava in different tissues and blood-feeding status. Those features made subolesin feasible as a potential target antigen for development of a universal vaccine for the control of tick infestations and a reduction in TBD.

Identification of host proteins involved in rickettsial invasion of tick cells.

Tick-borne spotted fever group (SFG) Rickettsia species are obligate intracellular bacteria capable of infecting both vertebrate and invertebrate host cells, an essential process for subsequent bacterial survival in distinct hosts. The host cell signaling molecules involved in the uptake of Rickettsia into mammalian and Drosophila cells have been identified; however, invasion into tick cells is understudied. Considering the movement of SFG Rickettsia between vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for host cell invasion. The current study employed biochemical inhibition assays to determine the tick proteins involved in Rickettsia montanensis infection of tick-derived cells from a natural host, Dermacentor variabilis. The results revealed several tick proteins important for rickettsial invasion, including actin filaments, actin-related protein 2/3 complex, phosphatidylinositol-3'-kinase, protein tyrosine kinases (PTKs), Src family PTK, focal adhesion kinase, Rho GTPase Rac1, and neural Wiskott-Aldrich syndrome protein. Delineating the molecular mechanisms of rickettsial infection is critical to a thorough understanding of rickettsial transmission in tick populations and the ecology of tick-borne rickettsial diseases.

Virome analysis of Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis ticks reveals novel highly divergent vertebrate and invertebrate viruses.

UNLABELLED: A wide range of bacterial pathogens have been identified in ticks, yet the diversity of viruses in ticks is largely unexplored. In the United States, Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis are among the principal tick species associated with pathogen transmission. We used high-throughput sequencing to characterize the viromes of these tick species and identified the presence of Powassan virus and eight novel viruses. These included the most divergent nairovirus described to date, two new clades of tick-borne phleboviruses, a mononegavirus, and viruses with similarity to plant and insect viruses. Our analysis revealed that ticks are reservoirs for a wide range of viruses and suggests that discovery and characterization of tick-borne viruses will have implications for viral taxonomy and may provide insight into tick-transmitted diseases. IMPORTANCE: Ticks are implicated as vectors of a wide array of human and animal pathogens. To better understand the extent of tick-borne diseases, it is crucial to uncover the full range of microbial agents associated with ticks. Our current knowledge of the diversity of tick-associated viruses is limited, in part due to the lack of investigation of tick viromes. In this study, we examined the viromes of three tick species from the United States. We found that ticks are hosts to highly divergent viruses across several taxa, including ones previously associated with human disease. Our data underscore the diversity of tick-associated viruses and provide the foundation for further studies into viral etiology of tick-borne diseases.

Babesia spp. in questing ticks from eastern Poland: prevalence and species diversity.

A total of 853 questing Ixodes ricinus males, females, and nymphs and of 582 questing Dermacentor reticulatus males and females were collected from vegetation on the territory of the Lublin province (eastern Poland). The ticks were examined for the presence of Babesia by PCR detecting part of 18S ribosomal RNA (rRNA) gene and nuclear small subunit rRNA (SS-rDNA) for determining of Babesia spp. and Babesia microti, respectively. The overall incidence of Babesia strains in I. ricinus ticks was 4.6%. Three species of Babesia were identified. The prevalent species was B. microti which occurred in 2.8% of ticks, while Babesia venatorum, Babesia divergens, and unidentified Babesia species were found at the frequency of 1.2, 0.2, and 0.3%, respectively. Altogether, B. microti constituted 61.5% of the total strains detected in I. ricinus, B. venatorum-25.7%, B. divergens-5.1%, and unidentified Babesia species-7.7%. The prevalence of Babesia species in I. ricinus did not depend significantly on locality (χ(2) = 1.885, P = 0.390) nor on the tick stage (χ(2) = 4.874, P = 0.087). The incidence of Babesia strains in D. reticulatus ticks was 2.7%. Two species of Babesia were identified. Again, the prevalent species was B. microti which occurred in 2.1% of ticks, while B. canis was found in 0.7% of ticks. In one D. reticulatus female, B. canis and B. microti co-infection was found. Altogether, B. microti constituted 75% of the total strains detected in D. reticulatus while B. canis formed 25% of the total strains. The frequency of the occurrence of Babesia species in D. reticulatus did not depend significantly on locality (χ(2) = 0.463, P = 0.793). The difference between the prevalence of Babesia in males and females of D. reticulatus was insignificant (P = 0.0954); nymphs were not found. The dominance of B. microti in the species composition of tick-borne Babesia found in this study was typical for eastern Europe. In conclusion, the results revealed that the population inhabiting the forested area of eastern Poland could be exposed to Babesia parasites, especially to those from the species B. microti, by a bite of I. ricinus, a competent vector of human babesiosis, and probably also by a bite of D. reticulatus whose role in the transmission of human babesiosis needs to be clarified.

Consequences of in vitro host shift for St. Louis encephalitis virus.

Understanding the potential for host range shifts and expansions of RNA viruses is critical to predicting the evolutionary and epidemiological paths of these pathogens. As arthropod-borne viruses (arboviruses) experience frequent spillover from their amplification cycles and are generalists by nature, they are likely to experience a relatively high frequency of success in a range of host environments. Despite this, the potential for host expansion, the genetic correlates of adaptation to novel environments and the costs of such adaptations in originally competent hosts are still not characterized fully for arboviruses. In the studies presented here, we utilized experimental evolution of St. Louis encephalitis virus (SLEV; family Flaviviridae, genus Flavivirus) in vitro in the Dermacentor andersoni line of tick cells to model adaptation to a novel invertebrate host. Our results demonstrated that levels of adaptation and costs in alternate hosts are highly variable among lineages, but also that significant fitness increases in tick cells are achievable with only modest change in consensus genetic sequence. In addition, although accumulation of diversity may at times buffer against phenotypic costs within the SLEV swarm, an increased proportion of variants with an impaired capacity to infect and spread on vertebrate cell culture accumulated with tick cell passage. Isolation and characterization of a subset of these variants implicates the NS3 gene as an important host range determinant for SLEV.

Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA.

Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.

Molecular and functional characterization of vacuolar-ATPase from the American dog tick Dermacentor variabilis.

Vacuolar (V)-ATPase is a proton-translocating enzyme that acidifies cellular compartments for various functions such as receptor-mediated endocytosis, intracellular trafficking and protein degradation. Previous studies in Dermacentor variabilis chronically infected with Rickettsia montanensis have identified V-ATPase as one of the tick-derived molecules transcribed in response to rickettsial infection. To examine the role of the tick V-ATPase in tick-Rickettsia interactions, a full-length 2887-bp cDNA (2532-bp open reading frame) clone corresponding to the transcript of the V0 domain subunit a of D. variabilis V-ATPase (DvVATPaseV0a) gene encoding an 843 amino acid protein with an estimated molecular weight of ~96 kDa was isolated from D. variabilis. Amino acid sequence analysis of DvVATPaseV0a showed the highest similarity to VATPaseV0a from Ixodes scapularis. A potential N-glycosylation site and eight putative transmembrane segments were identified in the sequence. Western blot analysis of tick tissues probed with polyclonal antibody raised against recombinant DvVATPaseV0a revealed the expression of V-ATPase in the tick ovary. Transcriptional profiles of DvVATPaseV0a demonstrated a greater mRNA expression in the tick ovary, compared with the midgut and salivary glands; however, the mRNA level in each of these tick tissues remained unchanged after infection with R. montanensis for 1 h. V-ATPase inhibition bioassays resulted in a significant decrease in the ability of R. montanensis to invade tick cells in vitro, suggesting a role of V-ATPase in rickettsial infection of tick cells. Characterization of tick-derived molecules involved in rickettsial infection is essential for a thorough understanding of rickettsial transmission within tick populations and the ecology of tick-borne rickettsial diseases.

Molecular identification and phylogeny of Dermacentor nuttalli (Acari: Ixodidae).

Dermacentor nuttalli is an epidemiologically important tick in Palearctic Asia which transmits several infectious diseases including tularemia, North Asian tick-borne rickettsiosis, Lyme disease and tick-borne encephalitis. The genetic specificity and phylogeny of D. nuttalli from four geographic localities in Eastern Siberia were characterized using the mitochondrial (mt) 16S ribosomal RNA (rRNA) gene and internal transcribed spacer 2 (ITS2). Low genetic diversity was observed in the populations of ticks distributed from South Siberia to North China. From 11 detected mt 16S haplotypes, one was found in all populations, whereas the others were restricted to specific localities. These results suggested that the genetic structure of D. nuttalli represents integrated populations with no geographic isolation across the distribution area. The phylogenetic reconstructions inferred from the mt 16S rRNA gene and ITS2 were in agreement and showed a distinct D. nuttalli clade within a monophyletic Eurasian lineage of Dermacentor sp.

Dermacentor everestianus Hirst, 1926 (Acari: Ixodidae): phylogenetic status inferred from molecular characteristics.

Dermacentor everestianus Hirst, 1926, is only reported in Northwestern China and Nepal. Few researches about this species have been involved, especially for molecular characteristics. The taxonomy studies of D.everestianus are mainly based on morphological features, and its taxonomic status is an ongoing controversy. To clarify the molecular characteristics and phylogenetic status of D.everestianus and other related species, the sequences of mitochondrial 16S ribosomal DNA (rDNA) and cox1 fragments were analyzed in the present study. Analysis of 16S rDNA and cox1 sequences showed 99.3-100% identity within D.everestianus individuals, with the genetic divergence among them was 0-0.0086. The interspecific distance of 16S rDNA and cox1 between D.everestianus and some other Palaearctic species including D. silvarum, D. nuttalli, and D. marginatus was much smaller than that between D.everestianus and Nearctic Dermacentor ticks (D.albipictus, D.nitens, and D.variabilis). Such relationships of these ticks were also verified in the phylogenetic analysis. Two major clades were recovered within Dermacentor spp. with more than 90% bootstrap support in the phylogenetic trees. D.everestianus together with D.silvarum, D.nuttalli, and D.marginatus were included in the clade I (Eurasia lineage). Other analyzed tick species including D.variabilis, D.nitens, and D.albipictus formed clade II, which are distributed in Nearctic realm. These indicated that the genus Dermacentor was at least composed of two lineages. Thus, further researches including additionally molecular markers on all Dermacentor species globally should be taken to precisely resolve relationships within Dermacentor.

Gene cloning, expression and immunogenicity of the protective antigen subolesin in Dermacentor silvarum.

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl ß-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.

[Construction of suppression subtractive hybridization cDNA library of half-blood males of Dermacentor silvarum and analysis of differentially expressed genes].

OBJECTIVE: To construct a suppression subtractive hybridization (SSH) cDNA library of half-blood males of Dermacentor silvarum, and analyze the differentially expressed genes. METHODS: Total RNA was extracted from the half-blood males and unfed males of D. silvarum. cDNA was synthesized following the protocol of SMARTER cDNA synthesis kit. After Rsa I digestion, cDNA was ligated to adaptors. The cDNA from the half-blood males was used as the tester, and unfed males as the driver. The SSH library was constructed using TaKaRa PCR-select cDNA subtraction kit. Differentially expressed cDNAs were amplified by nested PCR, cloned into PMD-18T vector, transformed into E. coli DH5alpha, and the white-blue plaque selection was used to get the positive clones. The titer of SSH library and the recombination efficiency were calculated. Individual colonies were randomly selected from library. Subtractive efficiency of the subtracted cDNA library was examined by reverse Northern blotting and RT-PCR. Positive clones with differentially expressed genes were sequenced. Homology comparison and function prediction were performed by Blastn and Blastx. RESULTS: The bands of double-stranded cDNAs from half-blood males and unfed males of D. silvarum were dispersed and longer than 500 bp. After Rsa I digestion, the ds cDNA-fragments were 100-1000 bp. The ligation reaction efficiency of adaptor was more than 25%. Nested PCR showed that the bands of subtracted ds cDNA were gathered, ranging from 250 to 500 bp. The titer of SSH library was 700,000 pfu/ml, and the recombination efficiency was 88.5% (239/270). Reverse Northern hybridization revealed that the clones showed stronger signals in half-blood males cDNA probes than in unfed males cDNA probes. RT-PCR showed that among the eight random selected positive clones, 5 clones were up-expressed under half-blood condition. A total of 87 differentially expressed sequence tags (ESTs, 200-800 bp) were obtained from 115 positive clones. Among the 87 ESTs, 53 ESTs showed sequence similarities to genes from other tick species, and 34 were homologous with genes from other insects. The main biological function of obtained ESTs were related to blood sucking and digestion, such as energy metabolism, signal transduction, and transcription regulation. CONCLUSION: The SSH cDNA library of half-blood male Dermacentor silvarum is constructed. The differential expressed genes are related to blood sucking and digestion.