Diversity and host specificity of Borrelia burgdorferi's outer surface protein C (ospC) alleles in synanthropic mammals, with a notable ospC allele U absence from mixed infections.

Interactions among pathogen genotypes that vary in host specificity may affect overall transmission dynamics in multi-host systems. Borrelia burgdorferi, a bacterium that causes Lyme disease, is typically transmitted among wildlife by Ixodes ticks. Despite the existence of many alleles of B. burgdorferi's sensu stricto outer surface protein C (ospC) gene, most human infections are caused by a small number of ospC alleles ["human infectious alleles" (HIAs)], suggesting variation in host specificity associated with ospC. To characterize the wildlife host association of B. burgdorferi's ospC alleles, we used metagenomics to sequence ospC alleles from 68 infected individuals belonging to eight mammalian species trapped at three sites in suburban New Brunswick, New Jersey (USA). We found that multiple allele ("mixed") infections were common. HIAs were most common in mice (Peromyscus spp.) and only one HIA was detected at a site where mice were rarely captured. ospC allele U was exclusively found in chipmunks (Tamias striatus), and although a significant number of different alleles were observed in chipmunks, including HIAs, allele U never co-occurred with other alleles in mixed infections. Our results suggest that allele U may be excluding other alleles, thereby reducing the capacity of chipmunks to act as reservoirs for HIAs.

The tick Ixodes scapularis has five different GPCRs specifically activated by ACP (adipokinetic hormone/corazonin-related peptide).

Insects have about 50 neuropeptide genes and about 70 genes, coding for neuropeptide G protein-coupled receptors (GPCRs). An important, but small family of evolutionarily related insect neuropeptides consists of adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP). Normally, insects have one specific GPCR for each of these neuropeptides. The tick Ixodes scapularis is not an insect, but belongs to the subphylum Chelicerata, which comprises ticks, scorpions, mites, spiders, and horseshoe crabs. Many of the neuropeptides and neuropeptide GPCRs occurring in insects, also occur in chelicerates, illustrating that insects and chelicerates are evolutionarily closely related. The tick I. scapularis is an ectoparasite and health risk for humans, because it infects its human host with dangerous pathogens during a blood meal. Understanding the biology of ticks will help researchers to prevent tick-borne diseases. By annotating the I. scapularis genome sequence, we previously found that ticks contain as many as five genes, coding for presumed ACP receptors. In the current paper, we cloned these receptors and expressed each of them in Chinese Hamster Ovary (CHO) cells. Each expressed receptor was activated by nanomolar concentrations of ACP, demonstrating that all five receptors were functional ACP receptors. Phylogenetic tree analyses showed that the cloned tick ACP receptors were mostly related to insect ACP receptors and, next, to insect AKH receptors, suggesting that ACP receptor genes and AKH receptor genes originated by gene duplications from a common ancestor. Similar duplications have probably occurred for the ligand genes, during a process of ligand/receptor co-evolution. Interestingly, chelicerates, in contrast to all other arthropods, do not have AKH or AKH receptor genes. Therefore, the ancestor of chelicerates might have lost AKH and AKH receptor genes and functionally replaced them by ACP and ACP receptor genes. For the small family of AKH, ACP, and corazonin receptors and their ligands, gene losses and gene gains occur frequently between the various ecdysozoan clades. Tardigrades, for example, which are well known for their survival in extreme environments, have as many as ten corazonin receptor genes and six corazonin peptide genes, while insects only have one of each, or none.

Genome resequencing reveals population divergence and local adaptation of blacklegged ticks in the United States.

Tick vectors and tick-borne disease are increasingly impacting human populations globally. An important challenge is to understand tick movement patterns, as this information can be used to improve management and predictive modelling of tick population dynamics. Evolutionary analysis of genetic divergence, gene flow and local adaptation provides insight on movement patterns at large spatiotemporal scales. We develop low coverage, whole genome resequencing data for 92 blacklegged ticks, Ixodes scapularis, representing range-wide variation across the United States. Through analysis of population genomic data, we find that tick populations are structured geographically, with gradual isolation by distance separating three population clusters in the northern United States, southeastern United States and a unique cluster represented by a sample from Tennessee. Populations in the northern United States underwent population contractions during the last glacial period and diverged from southern populations at least 50 thousand years ago. Genome scans of selection provide strong evidence of local adaptation at genes responding to host defences, blood-feeding and environmental variation. In addition, we explore the potential of low coverage genome sequencing of whole-tick samples for documenting the diversity of microbial pathogens and recover important tick-borne pathogens such as Borrelia burgdorferi. The combination of isolation by distance and local adaptation in blacklegged ticks demonstrates that gene flow, including recent expansion, is limited to geographical scales of a few hundred kilometres.

First detection and a new avian host of the tick Ixodes ventalloi Gil Collado, 1936, in Slovakia.

This study describes the first detection of Ixodes ventalloi in Slovakia. Two engorged females of I. ventalloi were collected from Dunnocks (Prunella modularis) captured in eastern Slovakia. The identification of females was based on morphological and molecular 16S rRNA gene features. Phylogenetic analysis revealed a classification of the females into distinct genogroups. Moreover, comparative morphological analysis highlighted variations between the two females, particularly in the curvature of the auriculae, the shape of coxa I, and the internal spur. These findings suggest the potential for varied phenotypes of I. ventalloi correlated with their genogroups. Nonetheless, I. ventalloi population establishment within Slovakia necessitates further investigation through flagging or drag sampling.

Anthropogenic electromagnetic radiation alters the transcription levels of the genes encoding the SIFamide and myoinhibitory peptide and their receptors in Ixodes ricinus synganglion.

The research of the influences of man-made electromagnetic fields on tick physiology has been very sparse and long neglected since the pioneer studies published in 1996 and 2000. Once multiple behavioral tests confirmed an attraction and possible perception of electromagnetic fields in ticks, a new interest in this topic erupted in recent years. In this study, qRT-PCR is utilized to determine the changes in the mRNA transcript levels of neuropeptides SIFamide and myoinhibitory peptide (mip and sifa) and their representative receptors (mip-r1 and sifa-r1) in the synganglia of the tick Ixodes ricinus irradiated by 900 MHz radiofrequency electromagnetic field. It was determined that 40 V/m intensity has a significant suppressory effect on the transcript levels of all genes after at least 60 minutes of constant exposure in both sexes. Commonly occurring intensity of radiation in urban areas (2 V/m) produced an elevation in mRNA levels after various timespans in every gene. A significant decrease of transcript abundances was detected in females after one hour of exposure to 2 V/m. Results of this study widen the knowledge of EMF-induced alterations in the neurophysiology of I. ricinus, the most commonly distributed hard tick in Europe.

Age- and Sex-Associated Pathogenesis of Cell Culture-Passaged Kemerovo Virus in IFNAR(-/-) Mice.

Kemerovo virus (KEMV) is a tick-borne orbivirus transmitted by ticks of the genus Ixodes. Previous animal experimentation studies with orbiviruses, in particular the interferon receptor double knock-out (IFNAR(-/-)) mouse model, did not indicate bias that is related to age or sex. We endeavoured to assess the effect of serial and alternated passages of KEMV in mammalian or Ixodes cells on virus replication and potential virulence in male or female IFNAR(-/-) mice, with important age differences: younger males (4-5 months old), older males (14-15 months old), and old females (14-15 months old). After 30 serial passages in mammalian or tick cells, or alternated passages in the two cell types, older female mice which were inoculated with the resulting virus strains were the first to show clinical signs and die. Younger males behaved differently from older males whether they were inoculated with the parental strain of KEMV or with any of the cell culture-passaged strains. The groups of male and female mice inoculated with the mammalian cell culture-adapted KEMV showed the lowest viraemia. While older female and younger male mice died by day 6 post-inoculation, surprisingly, the older males survived until the end of the experiment, which lasted 10 days. RNA extracted from blood and organs of the various mice was tested by probe-based KEMV real-time RT-PCR. Ct values of the RNA extracts were comparable between older females and younger males, while the values for older males were >5 Ct units higher for the various organs, indicating lower levels of replication. It is noteworthy that the hearts of the old males were the only organs that were negative for KEMV RNA. These results suggest, for the first time, an intriguing age- and sex-related bias for an orbivirus in this animal model. Changes in the amino acid sequence of the RNA-dependent RNA polymerase of Kemerovo virus, derived from the first serial passage in Ixodes cells (KEMV Ps.IRE1), were identified in the vicinity of the active polymerase site. This finding suggests that selection of a subpopulation of KEMV with better replication fitness in tick cells occurred.

Bulk and single-nucleus RNA sequencing highlight immune pathways induced in individuals during an Ixodes scapularis tick bite.

Ticks are hematophagous arthropods that use a complex mixture of salivary proteins to evade host defenses while taking a blood meal. Little is known about the immunological and physiological consequences of tick feeding on humans. Here, we performed the first bulk and single-nucleus RNA sequencing (snRNA-seq) of skin and blood of four persons presenting with naturally acquired, attached Ixodes scapularis ticks. Pathways and individual genes associated with innate and adaptive immunity were identified based on bulk RNA sequencing, including interleukin-17 signaling and platelet activation pathways at the site of tick attachment or in peripheral blood. snRNA-seq further revealed that the Hippo signaling, cell adhesion, and axon guidance pathways were involved in the response to an I. scapularis bite in humans. Features of the host response in these individuals also overlapped with that of laboratory guinea pigs exposed to I. scapularis and which acquired resistance to ticks. These findings offer novel insights for the development of new biomarkers for I. scapularis exposure and anti-tick vaccines for human use.

A corazonin G protein-coupled receptor gene in the tick Ixodes scapularis yields two splice variants, each coding for a specific corazonin receptor.

We have identified a corazonin G protein-coupled receptor (GPCR) gene in the tick Ixodes scapularis, which likely plays a central role in the physiology and behavior of this ectoparasite. This receptor gene is unusually large (1.133 Mb) and yields two corazonin (CRZ) receptor splice variants, where nearly half of the coding regions are exchanged: CRZ-Ra (containing exon 2, exon 3, and exon 4 of the gene) and CRZ-Rb (containing exon 1, exon 3, and exon 4 of the gene). CRZ-Ra codes for a GPCR with a canonical DRF sequence at the border of the third transmembrane helix and the second intracellular loop. The positively-charged R residue from the DRF sequence is important for coupling of G proteins after activation of a GPCR. CRZ-Rb, in contrast, codes for a GPCR with an unusual DQL sequence at this position, still retaining a negatively-charged D residue, but lacking a positively-charged R residue, suggesting different G protein coupling. Another difference between the two splice variants is that exon 2 from CRZ-Ra codes for an N-terminal signal sequence. Normally, GPCRs do not have N-terminal signal sequences, although a few mammalian GPCRs have. In the tick CRZ-Ra, the signal sequence probably assists with inserting the receptor correctly into the RER membrane. We stably transfected Chinese Hamster Ovary cells with each of the two splice variants and carried out bioluminescence bioassays that also included the use of the human promiscuous G protein G16. CRZ-Ra turned out to be selective for I. scapularis corazonin (EC50 = 10-8 M) and could not be activated by related neuropeptides like adipokinetic hormone (AKH) and AKH/corazonin-related peptide (ACP). Similarly, also CRZ-Rb could only be activated by corazonin, although about 4-fold higher concentrations were needed to activate it (EC50 = 4 x 10-8 M). The genomic organization of the tick corazonin GPCR gene is similar to that of the insect AKH and ACP receptor genes. This similar genomic organization can also be found in the human gonadotropin-releasing hormone (GnRH) receptor gene, confirming previous conclusions that the corazonin, AKH, and ACP receptor genes are the true arthropod orthologues of the human GnRH receptor gene.

Phylogeography of the blacklegged tick (Ixodes scapularis) throughout the USA identifies candidate loci for differences in vectorial capacity.

The blacklegged tick (Ixodes scapularis (Journal of the Academy of Natural Sciences of Philadelphia, 1821, 2, 59)) is a vector of Borrelia burgdorferi sensu stricto (s.s.) (International Journal of Systematic Bacteriology, 1984, 34, 496), the causative bacterial agent of Lyme disease, part of a slow-moving epidemic of Lyme borreliosis spreading across the northern hemisphere. Well-known geographical differences in the vectorial capacity of these ticks are associated with genetic variation. Despite the need for detailed genetic information in this disease system, previous phylogeographical studies of these ticks have been restricted to relatively few populations or few genetic loci. Here we present the most comprehensive phylogeographical study of genome-wide markers in I. scapularis, conducted by using 3RAD (triple-enzyme restriction-site associated sequencing) and surveying 353 ticks from 33 counties throughout the species' range. We found limited genetic variation among populations from the Northeast and Upper Midwest, where Lyme disease is most common, and higher genetic variation among populations from the South. We identify five spatially associated genetic clusters of I. scapularis. In regions where Lyme disease is increasing in frequency, the I. scapularis populations genetically group with ticks from historically highly Lyme-endemic regions. Finally, we identify 10 variable DNA sites that contribute the most to population differentiation. These variable sites cluster on one of the chromosome-scale scaffolds for I. scapularis and are within identified genes. Our findings illuminate the need for additional research to identify loci causing variation in the vectorial capacity of I. scapularis and where additional tick sampling would be most valuable to further understand disease trends caused by pathogens transmitted by I. scapularis.

Function and evolution of the aquaporin IsAQP1 in the Lyme disease vector Ixodes scapularis.

Ticks are important vectors of pathogenic viruses, bacteria, and protozoans to humans, wildlife, and domestic animals. Due to their life cycles, ticks face significant challenges related to water homeostasis. When blood-feeding, they must excrete water and ions, but when off-host (for stretches lasting several months), they must conserve water to avoid desiccation. Aquaporins (AQPs), a family of membrane-bound water channels, are key players in osmoregulation in many animals but remain poorly characterized in ticks. Here, we bioinformatically identified AQP-like genes from the deer tick Ixodes scapularis and used phylogenetic approaches to map the evolution of the aquaporin gene family in arthropods. Most arachnid AQP-like sequences (including those of I. scapularis) formed a monophyletic group clustered within aquaglycerolporins (GLPs) from bacteria to vertebrates. This gene family is absent from insects, revealing divergent evolutionary paths for AQPs in different hematophagous arthropods. Next, we sequenced the full-length cDNA of I. scapularis aquaporin 1 (IsAQP1) and expressed it heterologously in Xenopus oocytes to functionally characterize its permeability to water and solutes. Additionally, we examined IsAQP1 expression across different life stages and adult female organs. We found IsAQP1 is an efficient water channel with high expression in salivary glands prior to feeding, suggesting it plays a role in osmoregulation before or during blood feeding. Its functional properties are unique: unlike most GLPs, IsAQP1 has low glycerol permeability, and unlike most AQPs, it is insensitive to mercury. Together, our results suggest IsAQP1 plays an important role in tick water balance physiology and that it may hold promise as a target of novel vector control efforts.

Climate change in the Arctic: Testing the poleward expansion of ticks and tick-borne diseases.

Climate change is most strongly felt in the polar regions of the world, with significant impacts on the species that live there. The arrival of parasites and pathogens from more temperate areas may become a significant problem for these populations, but current observations of parasite presence often lack a historical reference of prior absence. Observations in the high Arctic of the seabird tick Ixodes uriae suggested that this species expanded poleward in the last two decades in relation to climate change. As this tick can have a direct impact on the breeding success of its seabird hosts and vectors several pathogens, including Lyme disease spirochaetes, understanding its invasion dynamics is essential for predicting its impact on polar seabird populations. Here, we use population genetic data and host serology to test the hypothesis that I. uriae recently expanded into Svalbard. Both black-legged kittiwakes (Rissa tridactyla) and thick-billed murres (Uria lomvia) were sampled for ticks and blood in Kongsfjorden, Spitsbergen. Ticks were genotyped using microsatellite markers and population genetic analyses were performed using data from 14 reference populations from across the tick's northern distribution. In contrast to predictions, the Spitsbergen population showed high genetic diversity and significant differentiation from reference populations, suggesting long-term isolation. Host serology also demonstrated a high exposure rate to Lyme disease spirochaetes (Bbsl). Targeted PCR and sequencing confirmed the presence of Borrelia garinii in a Spitsbergen tick, demonstrating the presence of Lyme disease bacteria in the high Arctic for the first time. Taken together, results contradict the notion that I. uriae has recently expanded into the high Arctic. Rather, this tick has likely been present for some time, maintaining relatively high population sizes and an endemic transmission cycle of Bbsl. Close future observations of population infestation/infection rates will now be necessary to relate epidemiological changes to ongoing climate modifications.

Repression of tick microRNA-133 induces organic anion transporting polypeptide expression critical for Anaplasma phagocytophilum survival in the vector and transmission to the vertebrate host.

The microRNAs (miRNAs) are important regulators of gene expression. In this study, we provide evidence for the first time to show that rickettsial pathogen Anaplasma phagocytophilum infection results in the down-regulation of tick microRNA-133 (miR-133), to induce Ixodes scapularis organic anion transporting polypeptide (isoatp4056) gene expression critical for this bacterial survival in the vector and for its transmission to the vertebrate host. Transfection studies with recombinant constructs containing transcriptional fusions confirmed binding of miR-133 to isoatp4056 mRNA. Treatment with miR-133 inhibitor resulted in increased bacterial burden and isoatp4056 expression in ticks and tick cells. In contrast, treatment with miR-133 mimic or pre-mir-133 resulted in dramatic reduction in isoatp4056 expression and bacterial burden in ticks and tick cells. Moreover, treatment of ticks with pre-mir-133 affected vector-mediated A. phagocytophilum infection of murine host. These results provide novel insights to understand impact of modulation of tick miRNAs on pathogen colonization in the vector and their transmission to infect the vertebrate host.

Characterization of the complete mitochondrial genomes of five hard ticks and phylogenetic implications.

Ticks are blood-sucking ectoparasites with significant medical and veterinary importance, capable of transmitting bacteria, protozoa, fungi, and viruses that cause a variety of human and animal diseases worldwide. In the present study, we sequenced the complete mitochondrial (mt) genomes of five hard tick species and analyzed features of their gene contents and genome organizations. The complete mt genomes of Haemaphysalis verticalis, H. flava, H. longicornis, Rhipicephalus sanguineus and Hyalomma asiaticum were 14855 bp, 14689 bp, 14693 bp, 14715 bp and 14722 bp in size, respectively. Their gene contents and arrangements are the same as those of most species of metastriate Ixodida, but distinct from species of genus Ixodes. Phylogenetic analyses using concatenated amino acid sequences of 13 protein-coding genes with two different computational algorithms (Bayesian inference and maximum likelihood) revealed the monophylies of the genera Rhipicephalus, Ixodes and Amblyomma, however, rejected the monophyly of the genus Haemaphysalis. To our knowledge, this is the first report of the complete mt genome of H. verticalis. These datasets provide useful mtDNA markers for further studies of the identification and classification of hard ticks.

Revealing the Tick Microbiome: Insights into Midgut and Salivary Gland Microbiota of Female Ixodes ricinus Ticks.

The ectoparasite Ixodes ricinus is an important vector for many tick-borne diseases (TBD) in the northern hemisphere, such as Lyme borreliosis, rickettsiosis, human granulocytic anaplasmosis, or tick-borne encephalitis virus. As climate change will lead to rising temperatures in the next years, we expect an increase in tick activity, tick population, and thus in the spread of TBD. Consequently, it has never been more critical to understand relationships within the microbial communities in ticks that might contribute to the tick's fitness and the occurrence of TBD. Therefore, we analyzed the microbiota in different tick tissues such as midgut, salivary glands, and residual tick material, as well as the microbiota in complete Ixodes ricinus ticks using 16S rRNA gene amplicon sequencing. By using a newly developed DNA extraction protocol for tick tissue samples and a self-designed mock community, we were able to detect endosymbionts and pathogens that have been described in the literature previously. Further, this study displayed the usefulness of including a mock community during bioinformatic analysis to identify essential bacteria within the tick.

Transcriptome of the synganglion in the tick Ixodes ricinus and evolution of the cys-loop ligand-gated ion channel family in ticks.

BACKGROUND: Ticks represent a major health issue for humans and domesticated animals. Exploring the expression landscape of the tick's central nervous system (CNS), known as the synganglion, would be an important step in understanding tick physiology and in managing tick-borne diseases, but studies on that topic are still relatively scarce. Neuron-specific genes like the cys-loop ligand-gated ion channels (cys-loop LGICs, or cysLGICs) are important pharmacological targets of acaricides. To date their sequence have not been well catalogued for ticks, and their phylogeny has not been fully studied. RESULTS: We carried out the sequencing of transcriptomes of the I. ricinus synganglion, for adult ticks in different conditions (unfed males, unfed females, and partially-fed females). The de novo assembly of these transcriptomes allowed us to obtain a large collection of cys-loop LGICs sequences. A reference meta-transcriptome based on synganglion and whole body transcriptomes was then produced, showing high completeness and allowing differential expression analyses between synganglion and whole body. Many of the genes upregulated in the synganglion were associated with neurotransmission and/or localized in neurons or the synaptic membrane. As the first step of a functional study of cysLGICs, we cloned the predicted sequence of the resistance to dieldrin (RDL) subunit homolog, and functionally reconstituted the first GABA-gated receptor of Ixodes ricinus. A phylogenetic study was performed for the nicotinic acetylcholine receptors (nAChRs) and other cys-loop LGICs respectively, revealing tick-specific expansions of some types of receptors (especially for Histamine-like subunits and GluCls). CONCLUSIONS: We established a large catalogue of genes preferentially expressed in the tick CNS, including the cysLGICs. We discovered tick-specific gene family expansion of some types of cysLGIC receptors, and a case of intragenic duplication, suggesting a complex pattern of gene expression among different copies or different alternative transcripts of tick neuro-receptors.

Host blood meal identity modifies vector gene expression and competency.

A vector's susceptibility and ability to transmit a pathogen-termed vector competency-determines disease outcomes, yet the ecological factors influencing tick vector competency remain largely unknown. Ixodes pacificus, the tick vector of Borrelia burgdorferi (Bb) in the western U.S., feeds on rodents, birds, and lizards. Rodents and birds are reservoirs for Bb and infect juvenile ticks, while lizards are refractory to Bb and cannot infect feeding ticks. Additionally, the lizard bloodmeal contains borreliacidal properties, clearing previously infected feeding ticks of their Bb infection. Despite I. pacificus feeding on a range of hosts, it is undetermined how the host identity of the larval bloodmeal affects future nymphal vector competency. We experimentally evaluate the influence of larval host bloodmeal on Bb acquisition by nymphal I. pacificus. Larval I. pacificus were fed on either lizards or mice and after molting, nymphs were fed on Bb-infected mice. We found that lizard-fed larvae were significantly more likely to become infected with Bb during their next bloodmeal than mouse-fed larvae. We also conducted the first RNA-seq analysis on whole-bodied I. pacificus and found significant upregulation of tick antioxidants and antimicrobial peptides in the lizard-fed group. Our results indicate that the lizard bloodmeal significantly alters vector competency and gene regulation in ticks, highlighting the importance of host bloodmeal identity in vector-borne disease transmission and upends prior notions about the role of lizards in Lyme disease community ecology.

Quantitative proteomics analysis reveals core and variable tick salivary proteins at the tick-vertebrate host interface.

Few studies have examined tick proteomes, how they adapt to their environment, and their roles in the parasite-host interactions that drive tick infestation and pathogen transmission. Here we used a proteomics approach to screen for biologically and immunologically relevant proteins acting at the tick-host interface during tick feeding and, as proof of principle, measured host antibody responses to some of the discovered candidates. We used a label-free quantitative proteomic workflow to study salivary proteomes of (i) wild Ixodes ricinus ticks fed on different hosts, (ii) wild or laboratory ticks fed on the same host, and (iii) adult ticks cofed with nymphs. Our results reveal high and stable expression of several protease inhibitors and other tick-specific proteins under different feeding conditions. Most pathways functionally enriched in sialoproteomes were related to proteolysis, endopeptidase, and amine-binding activities. The generated catalogue of tick salivary proteins enabled the selection of six candidate secreted immunogenic peptides for rabbit immunizations, three of which induced strong and durable antigen-specific antibody responses in rabbits. Furthermore, rabbits exposed to ticks mounted immune responses against the candidate peptides/proteins, confirming their expression at the tick-vertebrate interface. Our approach provides insights into tick adaptation strategies to different feeding conditions and promising candidates for developing antitick vaccines or markers of exposure of vertebrate hosts to tick bites.

Cationic Glycopolyelectrolytes for RNA Interference in Tick Cells.

The black-legged tick (Ixodes scapularis) is the primary vector for bacteria that cause Lyme disease (Borrelia burgdorferi), where numerous glycosylated tick proteins are involved at the interface of vector-host-pathogen interactions. Reducing the expression of key tick proteins, such as selenoprotein K (SelK), through RNA interference is a promising approach to reduce pathogen transmission, but efficient delivery of nucleic acids to arthropods has proven challenging. While cationic glycopolymers have been used as nonviral gene delivery vehicles in mammalian cells, their use in arthropod or insect gene transfection has not been established. In this study, statistical acrylamide-based cationic glycopolymers with glucose or galactose pendant groups were synthesized by reversible addition-fragmentation chain transfer polymerization, and the effects of the saccharide pendant group and cationic monomer loading on polymer cytotoxicity, RNA complexation, and SelK gene knockdown in ISE6 cells were evaluated. All polymers exhibited low cytotoxicity, yet RNA/copolymer complex cell uptake and gene knockdown were highly dependent on the saccharide structure and the N:P (amino to phosphate groups) ratio.

Characterization of the complete mitochondrial genome of Ixodes granulatus (Ixodidae) and its phylogenetic implications.

Ticks are deemed to be second only to mosquitoes as the most common vector of human infectious diseases worldwide that give rise to human and animal diseases and economic losses to livestock production. Our understanding of the phylogenetic analysis between tick lineages has been restricted by the phylogenetic markers of individual genes. Genomic data research could help advance our understanding of phylogenetic analysis and molecular evolution. Mitochondrial genomic DNA facilitated the phylogenetic analysis of eukaryotes containing ticks. In this study, we sequenced and assembled the circular complete mitogenome information of Ixodes granulatus. The 14,540-bp mitogenome consists of 37 genes, including 13 protein-coding genes (PCGs), two genes for ribosomal RNA (rRNAs), and 22 genes for transfer RNA (tRNAs), and the origin of the L-strand replication region. The directions of the coding strand and component genes in the non-Australasian Ixodes mitochondrial genome were similar to those found in most other Australasian Ixodes, except for the loss of a lengthy control region. The phylogenetic tree based on maximum likelihood (ML) and Bayesian inference (BI) computational algorithms showed that I. granulatus exhibits a close relationship with I. hexagonus and I. ricinus. To our knowledge, this is the first study exploring the complete mitogenome for the species I. granulatus. Our results provide new insights for further research on the evolution, population genetics, systematics, and molecular ecology of ticks.

Detection of Leishmania sp. kDNA in questing Ixodes ricinus (Acari, Ixodidae) from the Emilia-Romagna Region in northeastern Italy.

To date, sand flies (Phlebotominae) are the only recognized biological vectors of Leishmania infantum, the causative agent of human visceral leishmaniasis, which is endemic in the Mediterranean basin and also widespread in Central and South America, the Middle East, and Central Asia. Dogs are the main domestic reservoir of zoonotic visceral leishmaniasis, and the role of secondary vectors such as ticks and fleas and particularly Rhipicephalus sanguineus (the brown dog tick) in transmitting L. infantum has been investigated. In the present paper, the presence of Leishmania DNA was investigated in questing Ixodes ricinus ticks collected from 4 rural areas included in three parks of the Emilia-Romagna Region (north-eastern Italy), where active foci of human visceral leishmaniasis have been identified. The analyses were performed on 236 DNA extracts from 7 females, 6 males, 72 nymph pools, and 151 larvae pools. Four samples (1.7%) (i.e., one larva pool, 2 nymph pools, and one adult male) tested positive for Leishmania kDNA. To the best of our knowledge, this is the first report of the presence of Leishmania kDNA in questing I. ricinus ticks collected from a rural environment. This finding in unfed larvae, nymphs, and adult male ticks supports the hypothesis that L. infantum can have both transstadial and transovarial passage in I. ricinus ticks. The potential role of I. ricinus ticks in the sylvatic cycle of leishmaniasis should be further investigated.