Tick Genes Research Articles

Tick-Borne pathogens and defensin genes expression: A closer look at Ixodes ricinus and Dermacentor reticulatus

The immune system of ticks, along with that of other invertebrates, is comparatively simpler than that of vertebrates, relying solely on innate immune responses. Direct antimicrobial defence is provided by the synthesis of antimicrobial peptides (AMPs), including defensins. The aim of this study was to investigate the differences in defensin genes expression between questing and engorged Ixodes ricinus (def1 and def2) and Dermacentor reticulatus (defDr) ticks, in the presence of selected pathogens: Borrelia spp., Rickettsia spp., Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis in the natural environment. After pathogen screening by PCR/qPCR, the expression of defensin genes in pathogen positive ticks and ticks without any of the tested pathogens, was analysed by reverse transcription qPCR. The results showed an increased expression of defensin genes in I. ricinus ticks after blood feeding and I. ricinus and D. reticulatus ticks during in cases of co-infection. In particular, the expression of defensins genes was higher in questing D. reticulatus than in questing and engorged I. ricinus ticks, when borreliae were detected. This study contributes to uncovering the expression patterns of defensin genes in the presence of several known tick pathogens, the occurrence of these pathogens and possible regulatory mechanisms of defensins in tick vector competence.

Towards modelling tick-virus interactions using the weakly pathogenic Sindbis virus: Evidence that ticks are competent vectors.

Most tick-borne viruses (TBVs) are highly pathogenic and require high biosecurity, which severely limits their study. We found that Sindbis virus (SINV), predominantly transmitted by mosquitoes, can replicate in ticks and be subsequently transmitted, with the potential to serve as a model for studying tick-virus interactions. We found that both larval and nymphal stages of Rhipicephalus haemaphysaloides can be infected with SINV-wild-type (WT) when feeding on infected mice. SINV replicated in two species of ticks (R. haemaphysaloides and Hyalomma asiaticum) after infecting them by microinjection. Injection of ticks with SINV expressing enhanced Green Fluorescent Protein (eGFP) revealed that SINV-eGFP specifically aggregated in the tick midguts for replication. During blood-feeding, SINV-eGFP migrated from the midguts to the salivary glands and was transmitted to a new host. SINV infection caused changes in expression levels of tick genes related to immune responses, substance transport and metabolism, cell growth and death. SINV mainly induced autophagy during the early stage of infection; with increasing time of infection, the level of autophagy decreased, while the level of apoptosis increased. During the early stages of infection, the transcript levels of immune-related genes were significantly upregulated, and then decreased. In addition, SINV induced changes in the transcription levels of some functional genes that play important roles in the interactions between ticks and tick-borne pathogens. These results confirm that the SINV-based transmission model between ticks, viruses, and mammals can be widely used to unravel the interactions between ticks and viruses.

Nanopore-based metagenomics reveal a new Rickettsia in Europe

Accurate identification of tick-borne bacteria, including those associated with rickettsioses, pose significant challenges due to the polymicrobial and polyvectoral nature of the infections. We aimed to carry out a comparative evaluation of a non-targeted metagenomic approach by nanopore sequencing (NS) and commonly used PCR assays amplifying Rickettsia genes in field-collected ticks. The study included a total of 310 ticks, originating from Poland (44.2 %) and Bulgaria (55.8 %). Samples comprised 7 species, the majority of which were Ixodes ricinus (62.9 %), followed by Dermacentor reticulatus (21.2 %). Screening was carried out in 55 pools, using total nucleic acid extractions from individual ticks. NS and ompA/gltA PCRs identified Rickettsia species in 47.3 % and 54.5 % of the pools, respectively. The most frequently detected species were Rickettsia asiatica (27.2 %) and Rickettsia raoultii (21.8 %), followed by Rickettsia monacensis (3.6 %), Rickettsia helvetica (1.8 %), Rickettsia massiliae (1.8 %) and Rickettsia tillamookensis (1.8 %). Phylogeny construction on mutS, uvrD, argS and virB4 sequences and a follow-up deep sequencing further supported R. asiatica identification, documented in Europe for the first time. NS further enabled detection of Anaplasma phagocytophilum (9.1 %), Coxiella burnetii (5.4 %) and Neoehrlichia mikurensis (1.8 %), as well as various endosymbionts of Rickettsia and Coxiella. Co-detection of multiple rickettsial and non-rickettsial bacteria were observed in 16.4 % of the pools with chromosome and plasmid-based contigs. In conclusion, non-targeted metagenomic sequencing was documented as a robust strategy capable of providing a broader view of the tick-borne bacterial pathogen spectrum.

Tetracycline inhibits tick host reproduction by modulating bacterial microbiota, gene expression and metabolism levels.

Ticks are disease vectors that are a matter of worldwide concern. Antibiotic treatments have been used to explore the interactions between ticks and their symbiotic microorganisms. In addition to altering the host microbial community, antibiotics can have toxic effects on the host. In the tick Haemaphysalis longicornis, engorged females showed reproductive disruption after microinjection of tetracycline. Multi-omics approaches were implemented to unravel the mechanisms of tick reproductive inhibition in this study. There were no significant changes in bacterial density in the whole ticks on Day (D)2 or D4 after tetracycline treatment, whereas the bacterial microbial community was significantly altered, especially on D4. The relative abundances of the bacteria Staphylococcus, Bacillus and Pseudomonas decreased after tetracycline treatment, whereas the relative abundances of Coxiella and Rhodococcus increased. Ovarian transcriptional analysis revealed a cumulative effect of tetracycline treatment, as there was a significant increase in the number of differentially expressed genes with treatment time and a higher number of downregulated genes. The tick physiological pathways including lysosome, extracellular matrix (ECM)-receptor interaction, biosynthesis of ubiquinone and other terpenoids-quinones, insect hormone biosynthesis, and focal adhesion were significantly inhibited after 4 days of tetracycline treatment. Metabolite levels were altered after tetracycline treatment and the differences increased with treatment time. The differential metabolites were involved in a variety of physiological pathways; the downregulated metabolites were significantly enriched in the nicotinate and nicotinamide metabolism, galactose metabolism, and ether lipid metabolism pathways. These findings indicate that tetracycline inhibits tick reproduction through the regulation of tick bacterial communities, gene expression and metabolic levels. The results may provide new strategies for tick control. © 2023 Society of Chemical Industry.

De novo assembled transcriptomics assisted label-free quantitative proteomics analysis reveals sex-specific proteins in the intestinal tissue of Haemaphysalis qinghaiensis.

The hard tick Haemaphysalis qinghaiensis is the vector of a wide variety of infectious agents, such as spirochetes and other bacteria as well as viruses in the western plateau of China. Tick midgut is the key tissue involved in the host-pathogen-vector interface. Multiple midgut proteins are related to key functions in blood digestion, tick survival, and tick-borne pathogen transmission. However, information on the sex-specific proteins expressed in the midgut tissue of H. qinghaiensis for which the genome has not been sequenced is limited. Hence, we assembled and characterized the transcriptome of the H. qinghaiensis midgut and identified the differentially expressed genes (DEGs) in female and male ticks. The sequencing of the mRNA for this nonmodel species is essential for producing a protein database for mass spectrometry-based identification. Here, we combined high-throughput parallel sequencing and label-free quantitative proteomics analysis to extensively characterize the tick midgut using massive RNA sequencing and mass spectrometry, which allowed the detection of genes and proteins. A total of 279,186 transcripts were annotated into 125,790 coding sequences (CDSs), which were manually curated into 96 different gene families. A total of 12,837 DEGs between the two sexes were found by RNA-seq analysis. Of these, 5401 were upregulated genes, while 7436 were downregulated genes. The most common molecular functions were those related to the endocrine system, translation, signal transduction, transport, and catabolism. Meanwhile, the most common biological processes were related to cellular processes, metabolic processes, cellular anatomical entities, and cargo receptor activities. An analysis of the label-free protein quantitation dataset showed 272 upregulated proteins and 46 downregulated proteins when the fold-change was >2.0 (LC-MS/MS). Association analysis of the transcriptome and proteome with GO functional enrichment showed that the majority of the genes (proteins) were those related to catalytic activity, binding, cellular processes, metabolic processes, and responses to stimuli. This study aims to elucidate the digestive physiology of H. qinghaiensis as well as its physiological sexual dimorphism. This will allow the identification of protein candidates with physiological importance that could be used as targets to control the vector as well as the transmission of tick-borne pathogens to humans and animals.

The Impact of RNA Interference in Tick Research.

Over the past two decades, RNA interference (RNAi) in ticks, in combination with omics technologies, have greatly advanced the discovery of tick gene and molecular function. While mechanisms of RNAi were initially elucidated in plants, fungi, and nematodes, the classic 2002 study by Aljamali et al. was the first to demonstrate RNAi gene silencing in ticks. Subsequently, applications of RNAi have led to the discovery of genes that impact tick function and tick-host-pathogen interactions. RNAi will continue to lead to the discovery of an array of tick genes and molecules suitable for the development of vaccines and/or pharmacologic approaches for tick control and the prevention of pathogen transmission.

Rickettsial Pathogen Perturbs Tick Circadian Gene to Infect the Vertebrate Host.

Ixodes scapularis is a medically important tick that transmits several microbes to humans, including rickettsial pathogen Anaplasma phagocytophilum. In nature, these ticks encounter several abiotic factors including changes in temperature, humidity, and light. Many organisms use endogenously generated circadian pathways to encounter abiotic factors. In this study, we provide evidence for the first time to show that A. phagocytophilum modulates the arthropod circadian gene for its transmission to the vertebrate host. We noted a circadian oscillation in the expression of arthropod clock, bmal1, period and timeless genes when ticks or tick cells were exposed to alternate 12 h light: 12 h dark conditions. Moreover, A. phagocytophilum significantly modulates the oscillation pattern of expression of these genes. In addition, increased levels of clock and bmal1 and decreased expression of Toll and JAK/STAT pathway immune genes such as pelle and jak, respectively, were noted during A. phagocytophilum transmission from ticks to the vertebrate host. RNAi-mediated knockdown of clock gene expression in ticks resulted in the reduced expression of jak and pelle that increased bacterial transmission from ticks to the murine host. Furthermore, clock-deficient ticks fed late and had less engorgement weights. These results indicate an important role for circadian modulation of tick gene expression that is critical for arthropod blood feeding and transmission of pathogens from vector to the vertebrate host.

The ecdysteroid receptor regulates salivary gland degeneration through apoptosis in Rhipicephalus haemaphysaloides

BackgroundIt is well established that ecdysteroid hormones play an important role in arthropod development and reproduction, mediated by ecdysteroid receptors. Ticks are obligate hematophagous arthropods and vectors of pathogens. The salivary gland plays an essential role in tick growth and reproduction and in the transmission of pathogens to vertebrate hosts. During tick development, the salivary gland undergoes degeneration triggered by ecdysteroid hormones and activated by apoptosis. However, it is unknown how the ecdysteroid receptor and apoptosis regulate salivary gland degeneration. Here, we report the functional ecdysteroid receptor (a heterodimer of the ecdysone receptor [EcR] and ultraspiracle [USP]) isolated from the salivary gland of the tick Rhipicephalus haemaphysaloides and explore the molecular mechanism of ecdysteroid receptor regulation of salivary gland degeneration.MethodsThe full length of RhEcR and RhUSP open reading frames (ORFs) was obtained from the transcriptome. The RhEcR and RhUSP proteins were expressed in a bacterial heterologous system, Escherichia coli. Polyclonal antibodies were produced against synthetic peptides and were able to recognize recombinant and native proteins. Quantitative real-time PCR and western blot were used to detect the distribution of RhEcR, RhUSP, and RhCaspases in the R. haemaphysaloides organs. A proteomics approach was used to analyze the expression profiles of the ecdysteroid receptors, RhCaspases, and other proteins. To analyze the function of the ecdysteroid receptor, RNA interference (RNAi) was used to silence the genes in adult female ticks. Finally, the interaction of RhEcR and RhUSP was identified by heterologous co-expression assays in HEK293T cells.ResultsWe identified the functional ecdysone receptor (RhEcR/RhUSP) of 20-hydroxyecdysone from the salivary gland of the tick R. haemaphysaloides. The RhEcR and RhUSP genes have three and two isoforms, respectively, and belong to a nuclear receptor family but with variable N-terminal A/B domains. The RhEcR gene silencing inhibited blood-feeding, blocked engorgement, and restrained salivary gland degeneration, showing the biological role of the RhEcR gene in ticks. In the ecdysteroid signaling pathway, RhEcR silencing inhibited salivary gland degeneration by suppressing caspase-dependent apoptosis. The heterologous expression in mammalian HEK293T cells showed that RhEcR1 interacts with RhUSP1 and induces caspase-dependent apoptosis.ConclusionsThese data show that RhEcR has an essential role in tick physiology and represents a putative target for the control of ticks and tick-borne diseases.Graphical

Tick immunity using mRNA, DNA and protein-based Salp14 delivery strategies

Guinea pigs exposed to multiple infestations with Ixodes scapularis ticks develop acquired resistance to ticks, which is also known as tick immunity. The I. scapularis salivary components that contribute to tick immunity are likely multifactorial. An anticoagulant that inhibits factor Xa, named Salp14, is present in tick saliva and is associated with partial tick immunity. A tick bite naturally releases tick saliva proteins into the vertebrate host for several days, which suggests that the mode of antigen delivery may influence the genesis of tick immunity. We therefore utilized Salp14 as a model antigen to examine tick immunity using mRNA lipid nanoparticles (LNPs), plasmid DNA, or recombinant protein platforms. salp14 containing mRNA-LNPs vaccination elicited erythema at the tick bite site after tick challenge that occurred earlier, and that was more pronounced, compared with DNA or protein immunizations. Humoral and cellular responses associated with tick immunity were directed towards a 25 amino acid region of Salp14 at the carboxy terminus of the protein, as determined by antibody responses and skin-testing assays. This study demonstrates that the model of antigen delivery, also known as the vaccine platform, can influence the genesis of tick immunity in guinea pigs. mRNA-LNPs may be useful in helping to elicit erythema at the tick bite site, one of the most important early hallmarks of acquired tick resistance. mRNA-LNPs containing tick genes is a useful platform for the development of vaccines that can potentially prevent selected tick-borne diseases.

Screening for the "Achilles Heel" of Hyalomma anatolicum Ticks by RNA Interference Technology and an Update on Anti-Tick Vaccine Design.

Over the years, RNA interference (RNAi) has evolved as a valuable tool to study the tick gene function, screening and preliminary characterization of tick-protective antigens in a relatively short time, with a minimal use of laboratory animals before conducting expensive vaccine trials for the development of improved vaccine composition. In this process, a double-stranded RNA (dsRNA) of gene of interest is introduced into the tick system which specifically suppresses expression of a target gene. The results of RNAi-based gene silencing were interpreted by reduction in targeted gene transcript, changes in phenotypic data and anatomical/ biochemical changes in ticks; thereby, providing a clue to the probable role played by the gene in the tick biological system. Across the globe, various tick research groups applied RNAi techniquefor characterization and identification of new anti-tick vaccine targets. Herein, we used the RNAi tool in Hyalomma anatolicum ticks for identification and characterization of vaccine candidates.

Interactive Gene Expression Between Metarhizium anisopliae JEF-290 and Longhorned Tick Haemaphysalis longicornis at Early Stage of Infection.

The longhorned tick, Haemaphysalis longicornis (Acari: Ixodidae), is a hard tick and a vector for severe fever with thrombocytopenia syndrome (SFTS) virus. The number of patients infected with SFTS is rapidly increasing. Recently, the invertebrate pathogen Metarhizium anisopliae JEF-290 was reported to be useful to control the tick as an alternative to chemical acaricides, which are not easily applicable in human living areas where the tick is widely spread. In this study, we analyzed how the tick and the fungal pathogen interact at the transcriptional level. Field-collected tick nymphs were treated with JEF-290 conidia at 1 × 108 conidia/ml. In the early stage of infection with 2.5% mortality, the infected ticks were subjected to RNA sequencing, and non-infected ticks and fungal masses served as controls. Fungus and tick genes were mostly up-regulated at the early stage of infection. In the gene set enrichment analysis of the infecting fungus, catabolic processes that included lipids, phospholipids, and detoxification processes, the response to oxidative stress, and toxic substances were significantly up-regulated. In this fungal up-regulation, various lipase, antioxidant enzyme, and hydrolase genes were highly transcribed. The gene set enrichment analysis of the infected tick showed that many peptide synthesis processes including translation, peptide metabolism, ribonucleotide metabolism, and energy production processes that included ATP generation and ADP metabolism were significantly up-regulated. Structurally, mitochondria and ribosome subunit genes in ticks were highly transcribed to upregulate these processes. Together these results indicate that JEF-290 initiates process that infects the tick while the tick actively defends against the fungal attack. This work provides background to improve our understanding of the early stage of fungal infection in longhorned tick.

Identification of Tick Ixodes ricinus Midgut Genes Differentially Expressed During the Transmission of Borrelia afzelii Spirochetes Using a Transcriptomic Approach.

Lyme borreliosis is an emerging tick-borne disease caused by spirochetes Borrelia burgdorferi sensu lato. In Europe, Lyme borreliosis is predominantly caused by Borrelia afzelii and transmitted by Ixodes ricinus. Although Borrelia behavior throughout tick development is quite well documented, specific molecular interactions between Borrelia and the tick have not been satisfactorily examined. Here, we present the first transcriptomic study focused on the expression of tick midgut genes regulated by Borrelia. By using massive analysis of cDNA ends (MACE), we searched for tick transcripts expressed differentially in the midgut of unfed, 24h-fed, and fully fed I. ricinus nymphs infected with B. afzelii. In total, we identified 553 upregulated and 530 downregulated tick genes and demonstrated that B. afzelii interacts intensively with the tick. Technical and biological validations confirmed the accuracy of the transcriptome. The expression of five validated tick genes was silenced by RNA interference. Silencing of the uncharacterized protein (GXP_Contig_30818) delayed the infection progress and decreased infection prevalence in the target mice tissues. Silencing of other genes did not significantly affect tick feeding nor the transmission of B. afzelii, suggesting a possible role of these genes rather in Borrelia acquisition or persistence in ticks. Identification of genes and proteins exploited by Borrelia during transmission and establishment in a tick could help the development of novel preventive strategies for Lyme borreliosis.

Predicted iron metabolism genes in hard ticks and their response to iron reduction in Dermacentor andersoni cells

For most organisms, iron is an essential nutrient due to its role in fundamental cellular processes. Insufficient iron causes sub-optimal metabolism with potential effects on viability, while high levels of iron are toxic due to the formation of oxidative radicals, which damage cellular components. Many molecules and processes employed in iron uptake, storage, transport and metabolism are conserved, however significant knowledge gaps remain regarding these processes in ticks due to their unique physiology. In this study, we first identified and sequenced 13 genes likely to be involved in iron metabolism in Dermacentor andersoni cells. We then developed a method to reduce iron levels in D. andersoni cells using the iron chelator 2,2′-bipyridyl and measured the transcriptional response of these genes to iron reduction. The genes include a putative transferrin receptor, divalent metal transporter 1, duodenal cytochrome b, zinc/iron transporters zip7, zip13, zip14, mitoferrin, ferrochelatase, iron regulatory protein 1, ferritin1, ferritin2, transferrin and poly r(C)-binding protein. Overall, the transcriptional response of the target genes to iron reduction was modest. The most marked changes were a decrease in ferritin2, which transports iron through the tick hemolymph, the mitochondrial iron transporter mitoferrin, and the mitochondrial enzyme ferrochelatase. Iron regulatory protein1 was the only gene with an overall increase in transcript in response to reduced iron levels. This work lays the foundation for an improved understanding of iron metabolism in ticks which may provide molecular targets for the development of novel tick control methods and aid in the understanding of tick-pathogen interactions.

A family of serine protease inhibitors (serpins) and its expression profiles in the ovaries of Rhipicephalus haemaphysaloides

Serpins are evolutionarily conserved serine protease inhibitors found in many organisms. In arthropods, serpins are involved in feeding, development, oviposition, anti-coagulation and innate immune responses. We characterized of 11 serpins in the tick Rhipicephalus haemaphysaloides. These serpins have orthologous genes in other ticks, as indicated by phylogenetic analysis. Analysis of the reactive center loop and hinge regions of the protein sequences indicated that RHS7 encodes proteins that may lack proteinase inhibitor activity. All R. haemaphysaloides serpins had high amino acid sequence identities to Rhipicephalus microplus serpins. Tissue and temporal transcriptional profiling of eight R. haemaphysaloides serpins located in the ovaries demonstrated that they are transcribed during feeding and oviposition. These suggested their participation in the regulation of tick physiology. Immune serum from rabbits repeatedly infested with larvae, nymphs and adults of R. haemaphysaloides can recognize multiple recombinant serpins, respectively. After gene silencing, the blood feeding to repletion time was significantly longer and the 24 h attachment rate was significantly lower in the RHS3 and RHS7 knock down groups. The RHS9 and RHS11 silenced ticks had significant reduction in repletion time and egg-laying rate. Egg hatchability was significantly decreased in RHS4, RHS5 and RHS9 silenced ticks. All groups had significant reductions in engorged body weight. This study increases information on the serpins of R. haemaphysaloides and suggests that some RHSs are potential targets for development of tick vaccines.

Identification and validation of internal reference genes for real-time quantitative polymerase chain reaction-based studies in Hyalomma anatolicum ticks

Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne viral disease caused by the orthonairovirus CCHF virus (CCHFV). Ticks of the genus Hyalomma are the viral reservoir and they represent the main vector transmitting the virus to their hosts during blood feeding. However, how CCHFV replicates in its natural arthropod host cells and the nature of virus/host interactions are still largely unknown. With the aim of developing tools for use in this field, we identified and validated expression of four candidate endogenous control tick genes in a Hyalomma anatolicum-derived cell line. These genes will be useful for normalization of viral/cellular transcripts in infection/expression studies or as internal controls in molecular epidemiology surveys of pathogens transmitted by Hyalomma ticks.

A high gene flow in populations of Amblyomma ovale ticks found in distinct fragments of Brazilian Atlantic rainforest.

The genetic structure of populations of the tick Amblyomma ovale from five distinct areas of the Brazilian Atlantic rainforest was evaluated via DNA sequencing and associated with the presence of domestic dogs acting as hosts at the edge of forest fragments. Ticks were collected from domestic dogs and from the environment between 2015 and 2017. Four collection areas were located in the surroundings and within the Serra do Mar State Park, São Paulo State (23°37'21"S, 45°24'43"W), where dogs were bimonthly monitored along 2 years using camera traps and GSM trackers. To determine the spatial limits of genetic structure, ticks collected upon dogs living near the Serra do Baturié, Ceará State (4°15'40"S, 38°55'54"W) were included as well. A total of 39 haplotypes of 16S rRNA and Cox 1 mitochondrial genes sequences were observed, with 27 of them coming from areas within the Serra do Mar State Park. No haplotype was shared between the Serra do Mar and the Serra do Baturié indicating isolation of tick populations at the scale of 2000km. Although three different haplotype lineages of A. ovale occurred within the Serra do Mar State Park, no genetic structure was found across the study sites within this park, suggesting high tick gene flow across a range of 45km. Monitoring data from domestic dogs and wild carnivores showed that these species share the same habitats at the forest edge, with dogs playing a likely limited role in tick dispersal. Our findings have important implications for understanding the genetic structure of wide spread A. ovale along Brazilian rainforest remnants, which can further be associated to tick-borne infectious agents, such as Rickettsia parkeri, and used for predicting future patterns of tick diversity in the Brazilian Atlantic rainforest.

The tick endosymbiont Candidatus Midichloria mitochondrii and selenoproteins are essential for the growth of Rickettsia parkeri in the Gulf Coast tick vector

BackgroundPathogen colonization inside tick tissues is a significant aspect of the overall competence of a vector. Amblyomma maculatum is a competent vector of the spotted fever group rickettsiae, Rickettsia parkeri. When R. parkeri colonizes its tick host, it has the opportunity to dynamically interact with not just its host but with the endosymbionts living within it, and this enables it to modulate the tick’s defenses by regulating tick gene expression. The microbiome in A. maculatum is dominated by two endosymbiont microbes: a Francisella-like endosymbiont (FLE) and Candidatus Midichloria mitochondrii (CMM). A range of selenium-containing proteins (selenoproteins) in A. maculatum ticks protects them from oxidative stress during blood feeding and pathogen infections. Here, we investigated rickettsial multiplication in the presence of tick endosymbionts and characterized the functional significance of selenoproteins during R. parkeri replication in the tick.ResultsFLE and CMM were quantified throughout the tick life stages by quantitative PCR in R. parkeri-infected and uninfected ticks. R. parkeri infection was found to decrease the FLE numbers but CMM thrived across the tick life cycle. Our qRT-PCR analysis indicated that the transcripts of genes with functions related to redox (selenogenes) were upregulated in ticks infected with R. parkeri. Three differentially expressed proteins, selenoprotein M, selenoprotein O, and selenoprotein S were silenced to examine their functional significance during rickettsial replication within the tick tissues. Gene silencing of the target genes was found to impair R. parkeri colonization in the tick vector. Knockdown of the selenogenes triggered a compensatory response from other selenogenes, as observed by changes in gene expression, but oxidative stress levels and endoplasmic reticulum stress inside the ticks were also found to have heightened.ConclusionsThis study illustrates the potential of this new research model for augmenting our understanding of the pathogen interactions occurring within tick hosts and the important roles that symbionts and various tick factors play in regulating pathogen growth.

Arthropod transcriptional activator protein-1 (AP-1) aids tick-rickettsial pathogen survival in the cold

Ixodes scapularis ticks transmit several pathogens to humans including rickettsial bacterium, Anaplasma phagocytophilum. Here, we report that A. phagocytophilum uses tick transcriptional activator protein-1 (AP-1) as a molecular switch in the regulation of arthropod antifreeze gene, iafgp. RNAi-mediated silencing of ap-1 expression significantly affected iafgp gene expression and A. phagocytophilum burden in ticks upon acquisition from the murine host. Gel shift assays provide evidence that both the bacterium and AP-1 influences iafgp promoter and expression. The luciferase assays revealed that a region of approximately 700 bp upstream of the antifreeze gene is sufficient for AP-1 binding to promote iafgp gene expression. Furthermore, survival assays revealed that AP-1-deficient ticks were more susceptible to cold in comparison to the mock controls. In addition, this study also indicates arthropod AP-1 as a global regulator for some of the tick genes critical for A. phagocytophilum survival in the vector. In summary, our study defines a novel mode of arthropod signaling for the survival of both rickettsial pathogen and its medically important vector in the cold.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization.

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Contributions to the knowledge of hard ticks (Acari: Ixodidae) in Colombia

The known tick fauna of Colombia includes 58 species (15 Argasidae and 43 Ixodidae). To add to the knowledge of the biology of ticks in Colombia, hard ticks (Ixodidae) were collected from domestic animals or vegetation during 2014–2016 in 10 of Colombia’s Departments. Ticks were identified to species through morphological examinations. Taxonomic identification was confirmed for some specimens by molecular methods, including phylogenetic analyses inferred from three tick genes (cytochrome c oxidase, 16S rDNA, second internal transcribed spacer). A total of 1745 tick specimens encompassing 8 species were collected. Overall, 5 tick species were recorded on cattle [Amblyomma dissimile, Amblyomma mixtum, Dermacentor nitens, Rhipicephalus microplus, Rhipicephalus sanguineus sensu lato (s.l.)], 5 on dogs (Amblyomma maculatum, Amblyomma ovale, Amblyomma varium, R. microplus, R. sanguineus s.l.), 3 on horses (A. mixtum, D. nitens, R. sanguineus s.l.), 3 on donkeys (A. mixtum, D. nitens, R. microplus), 1 on pig (D. nitens), and 2 from vegetation (A. mixtum, A. dissimile). This included the first records of A. mixtum from two Colombian Departments, indicating that the distribution of this tick in Colombia may be broader than currently known. Phylogenetic analyses confirmed that R. sanguineus s.l. specimens from 8 Departments belong to the “tropical species”. Moreover, Colombian specimens of A. maculatum formed a large clade with GenBank sequences of A. maculatum and A. triste, although some Colombian specimens grouped with A. maculatum from the United States while others grouped with A. triste from Brazil. Significant polymorphisms were observed between specimens of A. ovale or D. nitens; for the former species, it is noteworthy that two distinct clades were observed. Our study provides new records for 8 tick species parasitizing domestic animals in Colombia, including species with veterinary and medical importance in the Neotropical region, such as R. microplus, R. sanguineus, D. nitens, A. mixtum, and A. maculatum. Noteworthy, we provide the first record of A. varium infesting a domestic mammal.