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Abstract Background Histone acetylation is involved in the regulation of stress responses in multiple organisms. Dermacentor silvarum is an important vector tick species widely distributed in China, and low temperature is a crucial factor restricting the development of its population. However, knowledge of the histone acetyltransferases and epigenetic mechanisms underlying cold-stress responses in this tick species is limited. Methods Histone acetyltransferase genes were characterized in D. silvarum, and their relative expressions were determined using qPCR during cold stress. The association and modulation of histone acetyltransferase genes were further explored using RNA interference, and both the H3K9 acetylation level and relative expression of KAT5 protein were evaluated using western blotting. Results Three histone acetyltransferase genes were identified and named as DsCREBBP, DsKAT6B, and DsKAT5. Bioinformatics analysis showed that they were unstable hydrophilic proteins, characterized by the conserved structures of CBP (ZnF_TAZ), PHA03247 super family, Creb_binding, and MYST(PLN00104) super family. Fluorescence quantitative PCR showed that the expression of DsCREBBP, DsKAT6B, and DsKAT5 increased after 3 days of cold treatment, with subsequent gradual decreases, and was lowest on day 9. Western blotting showed that both the H3K9 acetylation level and relative expression of KAT5 in D. silvarum increased after treatment at − 4, 4, and 8 °C for 3 and 6 days, whereas they decreased significantly after a 9-day treatment. RNA interference induced significant gene silencing, and the mortality rate of D. silvarum significantly increased at the respective semi-lethal temperatures. Conclusion These results imply that histone acetyltransferases play an important role in tick adaptation to low temperatures and lay a foundation for further understanding of the epigenetic regulation of histone acetylation in cold-stressed ticks. Further research is needed to elucidate the mechanisms underlying histone acetylation during cold stress in ticks. Graphical Abstract

Background: Histone acetylation is involved in the regulation of stress responses in multiple organisms. Dermacentor silvarum is an important vector tick species widely distributed in China, and low temperature is a crucial factor restricting the development of its population. However, knowledge of the histone acetyltransferases and epigenetic mechanisms underlying cold-stress responses in this tick species is limited. Methods: Histone acetyltransferase genes were characterized in D. silvarum, and their relative expressions were determined using qPCR during cold stress. The association and modulation of histone acetyltransferase genes were further explored using RNA interference, and both the H3K9 acetylation level and relative expression of KAT5 protein were evaluated using western blotting. Results: Three histone acetyltransferase genes were identified and named as DsCREBBP, DsKAT6B, and DsKAT5. Bioinformatics analysis showed that they were unstable hydrophilic proteins, characterized by the conserved structures of CBP (ZnF_TAZ), PHA03247 super family, Creb_binding, and MYST(PLN00104) super family. Fluorescence quantitative PCR showed that the expression of DsCREBBP, DsKAT6B, and DsKAT5 increased after 3 days of cold treatment, with subsequent gradual decreases, and was lowest on day 9. Western blotting showed that both the H3K9 acetylation level and relative expression of KAT5 in D. silvarum increased after treatment at − 4, 4, and 8 °C for 3 and 6 days, whereas they decreased significantly after a 9-day treatment. RNA interference induced significant gene silencing, and the mortality rate of D. silvarum significantly increased at the respective semi-lethal temperatures. Conclusion: These results imply that histone acetyltransferases play an important role in tick adaptation to low temperatures and lay a foundation for further understanding of the epigenetic regulation of histone acetylation in cold-stressed ticks. Further research is needed to elucidate the mechanisms underlying histone acetylation during cold stress in ticks. [ABSTRACT FROM AUTHOR]

Background: Jingmen tick virus (JMTV) has attracted great attention due to its potential pathogenicity in humans and its transmission by ticks. Dermacentor silvarum (D. silvarum) is one of the dominant tick species in northeastern China, and can transmit many pathogens to humans and animals. However, there have been no report of transmission of JMTV by D. silvarum. Materials and Methods: Ticks were collected from vegetation at the Aershan Port in Inner Mongolia in April 2019. And we do attempt to infect D. silvarum with JMTV by the immersion technique in laboratory conditions. The transmission of JMTV was examined by reverse transcriptase PCR, fluorescence in situ hybridization, and indirect immunofluorescence assay. Statistical analysis was performed using SPSS 24.0. Results: We found that JMTV may only be maintained in the tick without replication, and could not be transmitted to a host following transstadial transmission. Moreover, no virus colonization was found in the midgut or salivary glands of unfed D. silvarum; therefore, D. silvarum may not be susceptible to JMTV infection and therefore unlikely to carry and transmit JMTV. Conclusion: Our study has to some extent filled the knowledge gap regarding the possibility of JMTV transmission by a medically important tick vector, D. silvarum. [ABSTRACT FROM AUTHOR]

Fructose-1, 6-bisphosphate aldolase (FBA) catalyses the conversion of fructose-1, 6-diphosphatein to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. It is an important enzyme for both glycolysis and gluconeogenesis in parasites. In this study, we cloned the open reading frame of the FBA gene from Dermacentor silvarum, which was 1095 bp and encoded a protein of 364 amino acids. The deduced amino acid sequence of D. silvarum FBA had high similarity with FBA homologues from other ticks. Phylogenetic analysis of FBA showed that D. silvarum and Amblyomma variegatum belonged to the same clade. The recombinant FBA of D. silvarum (rDsFBA) was expressed in Escherichia coli and the molecular weight was about 62 kDa, which was abundantly expressed in the supernatant of E. coli following induction by 0.5 mM IPTG for 8 h at 25°C. The activity of rDsFBA was characterized using a coupled enzymatic assay. The rDsFBA had a Km of 21.22 μM and a Vmax of 1.57 U/mg. Western blot analysis demonstrated that rDsFBA could be recognized by rabbit anti-D. silvarum polyclonal antibodies, suggesting it probably owned immunogenicity. The data reported here contribute to future research into the role of FBA in D. silvarum, and provides a new candidate antigen for controlling tick. [ABSTRACT FROM AUTHOR]

Abstract Background Anaplasma ovis is a gram-negative, tick-borne obligate intraerythrocytic pathogen, which causes ovine anaplasmosis in small ruminants worldwide. VirB10 of A. ovis is an integral component of the Type IV Secretion System (T4SS). The T4SS is used by bacteria to transfer DNA and/or proteins undeviatingly into the host cell to increase their virulence. To more thoroughly understand the interaction between A. ovis and Dermacentor silvarum, a vector containing the virb10 gene of A. ovis was used as a bait plasmid to screen interacting proteins from the cDNA library of the D. silvarum salivary gland using the yeast two-hybrid system. Methods The cDNA of the D. silvarum salivary gland was cloned into the pGADT7-SmaI vector (prey plasmid) to construct the yeast two-hybrid cDNA library. The virb10 gene was cloned into the pGBKT7 vector to generate a bait plasmid. Any gene auto-activation or toxicity effects in the yeast strain Y2HGold were excluded. The screening was performed by combining the bait and prey plasmids in yeast strains to identify positive preys. The positive preys were then sequenced, and the obtained sequences were subjected to further analyses using Gene Ontology, UniProt, SMART, and STRING. Additionally, the interaction between the bait and the prey was evaluated using the glutathione S-transferase (GST) pull-down assay. Results A total of two clones were obtained from the cDNA library using the yeast two-hybrid system, and the sequence analysis showed that both clones encoded the same large tegument protein, UL36. Furthermore, the proteins GST-UL36 and His-VirB10 were successfully expressed in vitro and the interaction between the two proteins was successfully demonstrated by the GST pull-down assay. Conclusions To our knowledge, this study is the first to screen for D. silvarum salivary gland proteins that interact with A. ovis VirB10. The resulting candidate, UL36, is a multi-functional protein. Further investigations into the functionality of UL36 should be carried out, which might help in identifying novel prevention and treatment strategies for A. ovis infection. The present study provides a base for exploring and further understanding the interactions between A. ovis and D. silvarum.

DNA methylation, mediated by DNA methyltransferases (Dnmts), is a typical epigenetic process that plays an important role in affecting organism acclimatization and adaptation to environmental changes. However, information about Dnmts and their associations with the cold tolerance of ticks remains meager. Hence, in the present study, the Dnmts in important vector ticks Dermacentor silvarum and Haemaphysalis longicornis were cloned and identified, and their functions in cold response were further explored. Results showed that the length of DsDnmt and DsDnmt1 in D. silvarum, and HlDnmt1 and HlDnmt in H. longicornis were 1,284, 549, 1,500, and 1,613 bp, respectively. Bioinformatics in protein analysis revealed that they were all unstable hydrophilic proteins and were mainly characterized with Dcm (DNA cytosine methyltransferase domain), Dnmt1-RFD (DNA methyltransferase replication foci domain), zf-CXXC (zinc finger-CXXC domain), and BAH (Bromo adjacent homology domain). The relative expression of these Dnmts was reduced after cold treatment for 3 days (P < 0.05), and increased with the extension of treatment. Western blot revealed that Dnmt1 decreased first and then increased significantly (P < 0.05) in both tick species, whereas other Dnmts fluctuated at varying degrees. RNA interference significantly silenced the genes Dnmts (P < 0.01), and mortality increased significantly (P < 0.05), when exposed to sub-lethal temperature, underscoring the important roles of Dnmts during the cold response of D. silvarum and H. longicornis. The above results lay the foundation for further understanding of the epigenetic regulation of DNA methylation in cold acclimatization and adaptation of ticks.

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Defensins are the most diverse groups of antimicrobial peptides in invertebrate animals. In ticks, defensins show great potential as targets for tick control, and display future prospect for therapeutic drug development. In the present study, a novel defensin-like gene (Ds-defensin) contributing to the antimicrobial and antioxidant capacity of the tick Dermacentor silvarum was characterized. The full-length of the Ds-defensin gene was 382 bp, which displayed tissue-specific expression and was highly abundant in the salivary glands and carcasses of the adults. It encodes a 71-amino acid defensin-like protein, and the protein precursor is characterized by a 22-amino acid signal peptide and a 34-amino acid mature peptide. The peptide displayed potent activity against most of the tested gram-positive bacteria, including Staphylococcus aureus, S. carnosus and Nocardia asteroides, and one tested gram-negative bacterium, Psychrobacter faecalis. Scanning electron microscopy revealed that the cell wall and surface of treated bacteria became rough and gradually formed pores after a 30-min exposure to the Ds-defensin peptide. Additionally, the peptide also showed significant antioxidant capacity. The above results implied that the defensin-like peptide may play an important role in tick defense and the interaction with microorganisms. [ABSTRACT FROM AUTHOR]

Dermacentor silvarum is widely distributed in northern China and transmits several pathogens that cause diseases in humans and domestic animals. We analysed the comprehensive bacterial community of the saliva and midgut from partially and fully engorged female adult D. silvarum. Dermacentor silvarum samples were collected from Guyuan, China. Bacterial DNA was extracted from the saliva and midgut contents of partially or fully engorged female adult D. silvarum. Sequencing of the V3–V4 hypervariable regions of the 16S rRNA genes was performed using the IonS5TMXL platform. The bacterial diversity in saliva was higher than in the midgut. The bacterial diversity of saliva from fully engorged ticks was greater than in partially engorged tick saliva. The bacterial diversity in midguts from partially engorged ticks was greater than in fully engorged tick midguts. Proteobacteria was the most dominant bacterial phylum in all of the samples. Twenty-nine bacterial genera were detected in all of the samples. Rickettsia, Anaplasma, and Stenotrophomonas were the main genera. The symbionts Coxiella, Arsenophonus, and Wolbachia were also detected in all of the samples. Eight bacterial species were identified in all of the experimental samples. Anaplasma marginale was reported for the first time in D. silvarum. [ABSTRACT FROM AUTHOR]

Background: Anaplasma ovis is a gram-negative, tick-borne obligate intraerythrocytic pathogen, which causes ovine anaplasmosis in small ruminants worldwide. VirB10 of A. ovis is an integral component of the Type IV Secretion System (T4SS). The T4SS is used by bacteria to transfer DNA and/or proteins undeviatingly into the host cell to increase their virulence. To more thoroughly understand the interaction between A. ovis and Dermacentor silvarum, a vector containing the virb10 gene of A. ovis was used as a bait plasmid to screen interacting proteins from the cDNA library of the D. silvarum salivary gland using the yeast two-hybrid system. Methods: The cDNA of the D. silvarum salivary gland was cloned into the pGADT7-SmaI vector (prey plasmid) to construct the yeast two-hybrid cDNA library. The virb10 gene was cloned into the pGBKT7 vector to generate a bait plasmid. Any gene auto-activation or toxicity effects in the yeast strain Y2HGold were excluded. The screening was performed by combining the bait and prey plasmids in yeast strains to identify positive preys. The positive preys were then sequenced, and the obtained sequences were subjected to further analyses using Gene Ontology, UniProt, SMART, and STRING. Additionally, the interaction between the bait and the prey was evaluated using the glutathione S-transferase (GST) pull-down assay. Results: A total of two clones were obtained from the cDNA library using the yeast two-hybrid system, and the sequence analysis showed that both clones encoded the same large tegument protein, UL36. Furthermore, the proteins GST-UL36 and His-VirB10 were successfully expressed in vitro and the interaction between the two proteins was successfully demonstrated by the GST pull-down assay. Conclusions: To our knowledge, this study is the first to screen for D. silvarum salivary gland proteins that interact with A. ovis VirB10. The resulting candidate, UL36, is a multi-functional protein. Further investigations into the functionality of UL36 should be carried out, which might help in identifying novel prevention and treatment strategies for A. ovis infection. The present study provides a base for exploring and further understanding the interactions between A. ovis and D. silvarum. [ABSTRACT FROM AUTHOR]

The tick Dermacentor silvarum mainly overwinters in northern China as an unfed adult entering diapause; however, little is known about the involvement of genes and pathways in summer diapause and overwintering adults of this species. Hence, we investigated the differential transcriptome profile of D. silvarum ticks' diapause and cold-responsive genes and pathways during its summer diapause, overwintering, and questing/ active stages. Through transcriptomic sequencing, a total of 136,740 unigenes were assembled, and 1,549, 2,196, and 2,125 differentially expressed genes (DEG) were identified in active and diapause, diapause and overwintering, and active and overwintering ticks, respectively. Between active and diapause ticks, 875 genes were up-regulated, while 674 genes were found down-regulated in diapause ticks. When compared with active ticks, overwintering ticks had 993 up-regulated genes and 1,132 down-regulated genes. Comparison between diapause and overwintering ticks revealed 745 up-regulated and 1,451 down-regulated genes in overwintering ticks. Gene Ontology analysis revealed that, among the active, diapause, and overwintering ticks, most of the DEGs in molecular function were enriched in catalytic activity and hydrolase activity. In the cellular component, most DEGs were assigned to the integral component of the cell membrane, whereas the oxidation-reduction process was the most enriched among biological processes. In addition, the lysosome pathway was the most enriched pathway identified in KEGG pathway analysis. The above results deepened our understanding of the mechanism underlying diapause and overwintering adaptation of ticks, which is important for integrative prevention and control of ticks and tick-borne diseases. [ABSTRACT FROM AUTHOR]

Lymphocytic choriomeningitis virus (LCMV) is a rodent‐borne arenavirus that is considered a neglected cause of neurologic diseases in humans. In this study, we described genomic characterization of newly isolated LCMVs in Haemaphysalis longicornis, Dermacentor nuttalli, Dermacentor silvarum and Ixodes persulcatus in Jilin Province, northeastern China. The complete sequences of the small (S) and large (L) segments of LCMVs in ticks contained 3,375 and 7,235–7,241 nucleotides, respectively. Sequence comparison showed 82.1%–86.0% identity of S segment with other lineage I strains at the nucleotide level and 91.2%–97.5% at the deduced amino acid level, while a lower identity was observed in the L segment at both nucleotide (75.4%–82.2%) and amino acid (82.4%–93.4%) levels. Phylogenetic analysis grouped the tick LCMVs together with the lineage I strains, but in an isolated cluster with a high bootstrap value. Bayesian analysis indicated that the molecular evolutionary rate was estimated to be 3.3 × 10−4 substitutions/site/year for the S segment and 6.3 × 10−4 substitutions/site/year for the L segment, and the time to most recent common ancestor was 1980 and 1970 years ago, respectively, showing that tick LCMVs were predicted to originate between 1970s and 1980s. A long evolutionary history and high prevalence of LCMV in H. longicornis were found compared to other tick species. This study represented the first report on isolation of LCMV in China, showing that LCMV is circulating among ticks in Jilin Province, but the role of ticks in the epidemiology of LCMV remains to be explored. [ABSTRACT FROM AUTHOR]

Abstract Background Ticks are distributed worldwide and considered as vectors of many human diseases. Tick defensins, a family of antimicrobial peptides, form the first line of defense against pathogens. Findings A defensin-like gene, named Ds-defensin, was identified from a cDNA library of the hard tick Dermacentor silvarum collected from northeast China. The full-length cDNA of Ds-defensin was 225 bp, encoding a 74 amino acid peptide. The nucleotide sequence of Ds-defensin shared 98.2% similarity to putative defensin from Dermacentor marginatus. RT-PCR results suggested that Ds-defensin was extensively expressed in tick salivary gland and midgut, with a higher expression level in midgut. Ds-defensin showed broad antimicrobial activity against various Gram-positive and Gram-negative bacteria, as well as the fungus Candida albicans. Conclusions We characterized a functional defensin from D. silvarum of China. Ds-defensin showed bactericidal activity against various Gram-positive and Gram-negative bacteria. Ds-defensin can be expected to be introduced to the medical field as a new molecule with antibacterial activity.

Highlights • Rickettsia raoultii was found mainly in Dermacentor silvarum ticks from the Far East. • Rickettsia heilongjiangensis was found mainly in Haemaphysalis concinna ticks. • Rickettsia canadensis was found for the first time in a H. japonica tick. • R. aeschlimannii was first found in H. concinna and H. japonica from the Far East. • Candidatus R. principis and Candidatus R. rara were genotyped at several genes. Abstract The Russian Far East is an endemic region for tick-borne rickettsioses. However, the prevalence and genetic variability of Rickettsia species in this region have not been extensively investigated. In this study, 188 Dermacentor silvarum , 439 Haemaphysalis concinna , and 374 Haemaphysalis japonica adult ticks were collected from four locations in Khabarovsk Province and three locations in Amur Province in the Russian Far East. These ticks were examined for the presence of Rickettsia spp. by amplifying a fragment of the gltA gene. Identified rickettsiae were genotyped by sequencing of the gltA , 16S rRNA, ompA , ompB , and sca4 genes. In the examined ticks, Rickettsia heilongjiangensis , the causative agent of Far-Eastern tick-borne rickettsiosis, was found in 10.5% of H. concinna and in 1.9% of H. japonica ticks, while Rickettsia sibirica, the agent of Siberian tick typhus, was detected in only one H. concinna tick. In addition, Rickettsia raoultii was found predominantly in D. silvarum (>70%) and significantly less frequently in Haemaphysalis ticks (<3%). “ Candidatus Rickettsia tarasevichiae” was found in all examined tick species (1.6–5.3% in different species). Notably, this study is the first observation of “ Candidatus R. tarasevichiae” in D. silvarum ticks. Moreover, DNA of Rickettsia canadensis was found for the first time in a H. japonica tick; DNA of Rickettsia aeschlimannii was revealed for the first time in H. concinna and H. japonica ticks. “ Candidatus Rickettsia principis” and “ Candidatus Rickettsia rara” were found in Haemaphysalis spp. ticks. “ Candidatus R. principis” was associated with H. japonica and identified in 5.6% of the examined ticks, while “ Candidatus R. rara” was found more frequently in H. concinna (3.0%) compared to H. japonica ticks (1.1%). In this study, “ Candidatus R. principis” and “ Candidatus R. rara” were characterized for the first time by the 16S rRNA, ompA , ompB , and sca4 genes. [ABSTRACT FROM AUTHOR]

The important pathogen vector Dermacentor silvarum is widely distributed in China. However, the tick’s adaptation to low winter temperatures remains poorly understood. We therefore investigated the cold hardiness and physiological and biochemical responses of D. silvarum ticks exposed to low temperatures. The results indicated that the lower lethal temperatures (LT50s) for larvae, nymphs, females and males were −16.9 °C, −15.8 °C, −20.0 °C and −20.1 °C, respectively. The discriminating temperatures (resulting in 20% survival) for larvae, nymphs, females and males were −18.5 °C, −20.0 °C, −21.7 °C and −22.6 °C, respectively. The supercooling temperature points (at which body fluids spontaneously freeze) of larvae, nymphs, females and males averaged −20.0 °C, −23.5 °C, −24.2 °C and −23.9 °C, respectively. These results indicate that adult ticks are more tolerant to cold than the immatures. Low-temperature stress can enhance adult cold hardiness and trigger decreases in glycogen and protein in both females and males, whereas nymphs displayed different biochemical responses, including an increase in water and total fat content. An increase of glycerol observed in nymphs and females suggests that glycerol is important for cold hardiness. The findings of this study will help to define the dispersal limits for D. silvarum and thus inform the need for tick control efforts. [ABSTRACT FROM AUTHOR]

We used molecular methods to identify Rickettsia raoultii infections in 2 persons in China. These persons had localized rashes around sites of tick bites. R. raoultii DNA was detected in 4% of Dermacentor silvarum ticks collected in the same area of China and in 1 feeding tick detached from 1 patient.

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 ( cox 1–3, nad 1–6, nad 4L, cyt b, atp 6 and atp 8), 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, cox 1 was the most conserved gene, while atp 8 was the least conserved. The lengths of the 13 protein-coding genes were the same or similar, except for cyt b which was significantly longer in D . silvarum than in D . nitens . The mtDNA contained a variable repeat region consisting of a “similar to nad 1” 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. [ABSTRACT FROM AUTHOR]

In order to explore tick proteins as potential targets for further developing vaccine against ticks, the total proteins of unfed female Dermacentor silvarum were screened with anti-D. silvarum serum produced from rabbits. The results of western blot showed that 3 antigenic proteins of about 100, 68, and 52 kDa were detected by polyclonal antibodies, which means that they probably have immunogenicity. Then, unfed female tick proteins were separated by 12% SDS-PAGE, and target proteins (100, 68, and 52 kDa) were cut and analyzed by LC-MS/MS, respectively. The comparative results of peptide sequences showed that they might be vitellogenin (Vg), heat shock protein 60 (Hsp60), and fructose- 1, 6-bisphosphate aldolase (FBA), respectively. These data will lay the foundation for the further validation of antigenic proteins to prevent infestation and diseases transmitted by D. silvarum. [ABSTRACT FROM AUTHOR]

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 β-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. [ABSTRACT FROM AUTHOR]

We determined the mortality of adult tickDermacentor silvarumOlenev after it was exposed to five entomopathogenic nematode species:Steinernema carpocapsaeBJ (Sc BJ),S. glaseriNC32 (Sg NC32),S. ceratophorumD43 (Sc D43),S. feltiaeOtio (Sf Otio) andHeterorhabditis bacteriophoraE-6-7 (Hb E-6-7). All five species had lethal effects on adult ticks, and caused significant decreases in the amount of eggs laid by engorged female ticks (P< 0.05). Among them, Hb E-6-7 and Sc BJ were the most pathogenic to female adult ticks, with mortalities ranging from 70 to 90% (P< 0.05). Engorged females were more susceptible than unfed and partially fed females. The cumulative mortalities of engorged females exposed to Hb E-6-7 and Sc BJ for 14 days were 90%, with LT50at 6.3 and 7.0 days, respectively. Dose-dependent effect was observed in the oviposition of the ticks after they were exposed to nematodes at concentrations of 1000–5000 IJs/dish. When the concentration was higher than 5000 IJs/dish, dose dependent effects disappeared (P> 0.05). All five nematode species were lethal to unfed male ticks, and there was significant difference in mortality when compared to the control group (P< 0.05); however, the pathogenicity varied among them. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we investigated the life cycle and biological characteristics of all developmental stages of Dermacentor silvarum under field conditions in Hebei province in North China. Larvae, nymphs and adults were fed on rabbits, and maintained under natural lighting and climate conditions. Ticks were released into a confined plot located in natural tick habitat, and we observed the host questing behavior and seasonal occurrence. Due to a behavioral diapause in females and the synchronization of oviposition and larval hatching, D. silvarum only active from late February to early September under field conditions. The duration of the life cycle of D. silvarum was highly variable due to different preovipositional and egg hatching periods for ticks engorging in March, April or May. The developmental periods of larvae (18 days) and nymphs (29 days) under field conditions were similar to those under laboratory conditions. Moreover, there was a highly significant correlation between engorged body weight of females and egg masses laid (r =0.76, p <0.001). The female reproductive efficiency index (REI) in March, April and May was 5.7, 7.1 and 6.2, respectively. Finally, we also observed different ovipositional patterns between females that engorged in the field and those that engorged under laboratory conditions. [Copyright &y& Elsevier]

Here we present the results of the analysis of mt16S ribosomal RNA gene of two species of ticks from Irkutsk region - Dermacentor nuttalli and D. silvarum.. The ticks from are closely related to each other with 99-100 % identity of nucleotide sequence of mt16S rRNA gene. The phylogenetic analysis has shown that both species are closely related and. formed the separate clade together with specimens of D. nuttalli and. D. silvarum. from China.

• Identification of proteins from the midgut of Dermacentor silvarum interacting with VirD4 of Anaplasma ovis. • Evaluation of the interaction between Ribosomal protein L12 from the midgut of D. silvarum and the VirD4 of A. ovis. • Mechanism of molecular interaction between D. silvarum and A. ovis may have an influential role on the life cycle of A. ovis. Anaplasma ovis , a tick-borne intra-erythrocytic Gram-negative bacterium, is a causative agent of ovine anaplasmosis. It is known that Dermacentor ticks act as biological vectors for A. ovis. VirD4 is the machine component of Type IV Secretion System of A. ovis. To better understand the pathogen-vector interaction, VirD4 was used as a bait protein for screening midgut proteins of Dermacentor silvarum via yeast two-hybrid mating assay. As a result, a ribosomal protein RL12 was identified from the midgut cDNA library of D. silvarum. For further validation, using in vitro Glutathione S-transferase (GST) pull-down assay, interaction between the proteins, GST-RL12 and HIS-VirD4, was observed in Western blot analysis. The study is first of its kind reporting a D. silvarum midgut protein interaction with VirD4 from A. ovis. Functional annotations showed some important cellular processes are attributed to the protein, particularly in the stringent response and biogenesis. The results of the study suggest the involvement of the VirD4-RL12 interaction in the regulation of signaling pathways, which is a tool for understanding the pathogen-vector interaction. [ABSTRACT FROM AUTHOR]

DNA methylation, mediated by DNA methyltransferases (Dnmts), is a typical epigenetic process that plays an important role in affecting organism acclimatization and adaptation to environmental changes. However, information about Dnmts and their associations with the cold tolerance of ticks remains meager. Hence, in the present study, the Dnmts in important vector ticks Dermacentor silvarum and Haemaphysalis longicornis were cloned and identified, and their functions in cold response were further explored. Results showed that the length of DsDnmt and DsDnmt1 in D. silvarum , and HlDnmt1 and HlDnmt in H. longicornis were 1,284, 549, 1,500, and 1,613 bp, respectively. Bioinformatics in protein analysis revealed that they were all unstable hydrophilic proteins and were mainly characterized with Dcm (DNA cytosine methyltransferase domain), Dnmt1-RFD (DNA methyltransferase replication foci domain), zf-CXXC (zinc finger-CXXC domain), and BAH (Bromo adjacent homology domain). The relative expression of these Dnmts was reduced after cold treatment for 3 days ( P < 0.05), and increased with the extension of treatment. Western blot revealed that Dnmt1 decreased first and then increased significantly ( P < 0.05) in both tick species, whereas other Dnmts fluctuated at varying degrees. RNA interference significantly silenced the genes Dnmts ( P < 0.01), and mortality increased significantly ( P < 0.05), when exposed to sub-lethal temperature, underscoring the important roles of Dnmts during the cold response of D. silvarum and H. longicornis . The above results lay the foundation for further understanding of the epigenetic regulation of DNA methylation in cold acclimatization and adaptation of ticks., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Agwunobi, Zhang, Shi, Zhang, Zhang, Wang, Masoudi, Yu and Liu.)

Dermacentor silvarum is an obligate blood sucking arthropod and transmits various pathogens to humans and domestic animals. Recently several new viruses were detected in D. silvarum as an emerging disease threat. In this study, we aimed to analyze its geographical distribution and associated pathogens. Data were collected from multiple sources, including a field survey, reference book, and literature review. We searched various electronic databases with the terms " Dermacentor silvarum " OR " D. silvarum " for studies published since 1963 and the positive rates for Dermacentor silvarum -associated pathogens were estimated by meta-analysis. D. silvarum was found only in four countries in Eurasia, ranging from 22° N to 57° N latitude. At least 20 human pathogens were associated with D. silvarum , including five species of spotted fever group rickettsiae, three species in the family of Anaplasmataceae, three genospecies in the complex Borrelia burgdorferi sensu lato, Francisella tularensis, Babesia venatorum, Coxiella buenetii, Borrelia miyamotoi, and five species of virus. Among them, Rickettsia raoultii was widely detected in D. silvarum , showing the highest pooled positive rate (25.15%; 95% CI 13.31-39.27). Our work presents the most comprehensive data and analysis (to our knowledge) for the geographical distribution of D. silvarum and associated pathogens, revealing an emerging threat to public health and stocking farming. Continued surveillance and further investigations should be enhanced.

We used molecular methods to identify Rickettsia raoultii infections in 2 persons in China. These persons had localized rashes around sites of tick bites. R. raoultii DNA was detected in 4% of Dermacentor silvarum ticks collected in the same area of China and in 1 feeding tick detached from 1 patient.

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 β-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.