Co-infection dynamics of B. afzelii and TBEV in C3H mice: insights and implications for future research.

Ticks are important vectors of disease, particularly in the context of One Health, where tick-borne diseases (TBDs) are increasingly prevalent worldwide. TBDs often involve co-infections, where multiple pathogens co-exist in a single host. Patients with chronic Lyme disease often have co-infections with other bacteria or parasites. This study aimed to create a co-infection model with Borrelia afzelii and tick-borne encephalitis virus (TBEV) in C3H mice and to evaluate symptoms, mortality, and pathogen level compared to single infections. Successful co-infection of C3H mice with B. afzelii and TBEV was achieved. Outcomes varied, depending on the timing of infection. When TBEV infection followed B. afzelii infection by 9 days, TBEV symptoms worsened and virus levels increased. Conversely, mice infected 21 days apart with TBEV showed milder symptoms and lower mortality. Simultaneous infection resulted in mild symptoms and no deaths. However, our model did not effectively infect ticks with TBEV, possibly due to suboptimal dosing, highlighting the challenges of replicating natural conditions. Understanding the consequences of co-infection is crucial, given the increasing prevalence of TBD. Co-infected individuals may experience exacerbated symptoms, highlighting the need for a comprehensive understanding through refined animal models. This study advances knowledge of TBD and highlights the importance of exploring co-infection dynamics in host-pathogen interactions.

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.

Neural and endocrine regulation of osmoregulatory organs in tick: Recent discoveries and implications.

Ticks can survive in harsh and fluctuating vegetated environments for long durations between blood feedings with highly developed osmoregulatory mechanisms. Like the unique life history of hematophagous ticks, osmoregulatory organs and their regulatory mechanisms are significantly different from those in the closely related insect taxa. Over the last ten years, research has uncovered several neuropeptidergic innervations of the primary osmoregulatory organ, the salivary glands: myoinhibitory peptide (MIP), SIFamide, and elevenin. These neuropeptides are thought to be modulators of dopamine's autocrine or paracrine actions controlling the salivary glands, including the activation of fluid transport into the lumen of salivary acini and the pumping and gating action of salivary acini for expelling fluids out into salivary ducts. These actions are through two different dopamine receptors, D1 receptor and invertebrate D1-like dopamine receptor, respectively. Interestingly, MIP and SIFamide are also involved in the control of another important excretory/osmoregulatory organ, the hindgut, where SIFamide is myostimulatory, with MIP having antagonistic effects. FGLamide related allatostatin is also found to have axonal projections located on the surface of the rectum. Investigations of the osmoregulatory mechanisms of these critical vector species will potentially lead to the development of a measure to control tick species.

Natalisin, a tachykinin-like signaling system, regulates sexual activity and fecundity in insects.

An arthropod-specific peptidergic system, the neuropeptide designated here as natalisin and its receptor, was identified and investigated in three holometabolous insect species: Drosophila melanogaster, Tribolium castaneum, and Bombyx mori. In all three species, natalisin expression was observed in 3-4 pairs of the brain neurons: the anterior dorso-lateral interneurons, inferior contralateral interneurons, and small pars intercerebralis neurons. In B. mori, natalisin also was expressed in two additional pairs of contralateral interneurons in the subesophageal ganglion. Natalisin-RNAi and the activation or silencing of the neural activities in the natalisin-specific cells in D. melanogaster induced significant defects in the mating behaviors of both males and females. Knockdown of natalisin expression in T. castaneum resulted in significant reduction in the fecundity. The similarity of the natalisin C-terminal motifs to those of vertebrate tachykinins and of tachykinin-related peptides in arthropods led us to identify the natalisin receptor. A G protein-coupled receptor, previously known as tachykinin receptor 86C (also known as the neurokinin K receptor of D. melanogaster), now has been recognized as a bona fide natalisin receptor. Taken together, the taxonomic distribution pattern of the natalisin gene and the phylogeny of the receptor suggest that natalisin is an ancestral sibling of tachykinin that evolved only in the arthropod lineage.

Branched amphiphilic cationic oligopeptides form peptiplexes with DNA: a study of their biophysical properties and transfection efficiency.

Over the past decade, peptides have emerged as a new family of potential carriers in gene therapy. Peptides are easy to synthesize and quite stable. Additionally, sequences shared by the host proteome are not expected to be immunogenic or trigger inflammatory responses, which are commonly observed with viral approaches. We recently reported on a new class of branched amphiphilic peptide capsules (BAPCs) that self-assemble into extremely stable nanospheres. These capsules are capable of retaining and delivering alpha-emitting radionuclides to cells. Here we report that, in the presence of double stranded plasmid DNA, BAPCs are unable to form. Instead, depending of the peptide/DNA ratios, the peptides either coat the plasmid surface forming nanofibers (high peptide to DNA ratio) or condense the plasmid into nanometer-sized compacted structures (at low peptide to DNA ratios). Different gene delivery efficiencies are observed for the two types of assemblies. The compacted nanometer-sized structures display much higher transfection efficiencies in HeLa cells. This level of transfection is greater than that observed for a lipid-based reagent when the total number of viable transfected cells is taken into account.

Orchestration of salivary secretion mediated by two different dopamine receptors in the blacklegged tick Ixodes scapularis.

Salivary secretion is crucial for successful tick feeding, and it is the mediator of pathogen transmission. Salivation functions to inhibit various components of the host immune system and remove excess water and ions during the ingestion of large blood meals. Control of salivary glands involves autocrine/paracrine dopamine, which is the most potent inducer of tick salivation. Previously, we reported the presence of two dopamine receptors in the salivary glands of the blacklegged tick (Ixodes scapularis): dopamine receptor (D1) and invertebrate specific D1-like dopamine receptor (InvD1L). Here, we investigated the different physiological roles of the dopamine receptors in tick salivary glands by using pharmacological tools that discriminate between the two distinct receptors. Heterologous expressions followed by reporter assays of the dopamine receptors identified receptor-specific antagonists and agonists. These pharmacological tools were further used to discriminate the physiological role of each receptor by using in vitro assays: measuring salivary secretions of isolated salivary glands and monitoring dynamic changes in the size of individual salivary gland acini. We propose that the D1 receptor acts on salivary gland acini epithelial cells for inward fluid transport. InvD1L controls (or modulates) each acinus for expelling saliva from the acini to the salivary ducts, presumably through the actions of myoepithelial cells and valves for pumping/gating. We conclude that dopamine acts on the D1 and the InvD1L receptors and leads different physiological actions to orchestrate tick salivary secretion.

Validation of internal reference genes for real-time quantitative polymerase chain reaction studies in the tick, Ixodes scapularis (Acari: Ixodidae).

Obtaining reliable gene expression data using real-time quantitative polymerase chain reaction (qPCR) is highly dependent on the choice of normalization method. We tested the expression stability of multiple candidate genes in the salivary glands (SG) and synganglia (SYN) of female Ixodes scapularis (Say) ticks in multiple blood-feeding phases. We found that the amount of total RNA in both the SG and SYN increases dramatically during tick feeding, with 34x and 5.8x increases from 62 and 7.1 ng of unfed tick, respectively. We tested candidate genes that were predicted from I. scapularis genome data to encode glyceraldehyde 3-phosphate dehydrogenase (gapdh), ribosomal protein L13A (l13a), TATA box-binding protein (tbp), ribosomal protein S4 (rps4), glucose 6-phosphate dehydrogenase (gpdh), and beta-glucuronidase (gusb). The geNorm and NormFinder algorithms were used to analyze data from different feeding phases (i.e., daily samples from unfed to fully engorged females over a 7-d period in three replicate experiments). We found that the rps4 and l13a genes showed highly stable expression patterns over the feeding duration in both the SG and SYN. Furthermore, the highly expressed rps4 gene makes it useful as a normalization factor when we perform studies using minute amounts of dissected tissue for qPCR. We conclude that rps4 and l13a, whether individually or as a pair, serve as suitable internal reference genes for qRT-PCR studies in the SG and SYN of I. scapularis.

50 Years since Kaufman and Phillips' Groundbreaking Trilogy Elucidating Ion and Water Homeostasis in Ixodid Ticks.

The enormous volume of blood ingested by hard ticks during their long attachment period is without a doubt the hallmark of their biology. Maintaining a homeostatic balance between ion and water intake and loss during their feeding is critical to preventing osmotic stress and death. Exactly 50 years ago, Kaufman and Phillips published a series of three consecutive papers on "Ion and water balance in the ixodid tick Dermacentor andersoni", Journal of Experimental Biology (1973): I. Routes of ion and water excretion, 58: 523-36; II. Mechanism and control of salivary secretion 58: 537-547; and III. Influence of monovalent ions and osmotic pressure on salivary secretion 58: 549-564. This classic series significantly expanded our knowledge of the unique regulatory processes governing ion and water balance in fed ixodid ticks, highlighting its uniqueness among the blood-feeding arthropods. Their pioneer work had an enormous impact on understanding the vital role of salivary glands in these actions, and ultimately provided a consequential stepping stone for a new era of hard tick salivary gland physiological research.

Functional characterization of three G protein-coupled acetylcholine receptors in parasitic nematode Trichinella spiralis.

The physiological significance of metabotropic acetylcholine receptors in parasitic nematodes remains largely unexplored. Here, three different Trichinella spiralis G protein-coupled acetylcholine receptors (TsGAR-1, -2, and -3) were identified in the genome of T. spiralis. The phylogenetic analyses showed that TsGAR-1 and -2 receptors belong to a distinct clade specific to invertebrates, while TsGAR-3 is closest to the cluster of mammalian-type muscarinic acetylcholine receptors (mAChR). The mRNA of TsGAR-1, -2, and -3 was detected in muscle larvae, newborn larvae, and adults. The functional aequorin-based assay in Chinese hamster ovary cells revealed that all three types of T. spiralis GARs trigger the Gq/11 pathway upon activation of the receptor with the acetylcholine ligand. TsGAR-1 and TsGAR-2 showed atypical affinity with classical muscarinic agonists, while TsGAR-3 was sensitive to all muscarinic agonists tested. High concentrations of propiverine antagonist blocked the activities of all three TsGARs, while atropine and scopolamine antagonists effectively inhibited only TsGAR-3. Our data indicate that the distinct pharmacological profile of TsGAR-1 and -2 receptors, as well as the phylogenetic distance between them and their mammalian orthologs, place them as attractive targets for the development of selective anthelmintic drugs interfering with nematodes' cholinergic system.

Dual SIFamide receptors in Ixodes salivary glands.

Salivary glands are vital to tick feeding success and also play a crucial role in tick-borne pathogen transmission. In previous studies of Ixodes scapularis salivary glands, we demonstrated that saliva-producing type II and III acini are innervated by neuropeptidergic axons which release different classes of neuropeptides via their terminals (Simo et al., 2009b, 2013). Among these, the neuropeptide SIFamide-along with its cognate receptor-were postulated to control the basally located acinar valve via basal epithelial and myoepithelial cells (Vancová et al., 2019). Here, we functionally characterized a second SIFamide receptor (SIFa_R2) from the I. scapularis genome and proved that it senses a low nanomolar level of its corresponding ligand. Insect SIFamide paralogs, SMYamides, also activated the receptor but less effectively compared to SIFamide. Bioinformatic and molecular dynamic analyses suggested that I. scapularis SIFamide receptors are class A GPCRs where the peptide amidated carboxy-terminus is oriented within the receptor binding cavity. The receptor was found to be expressed in Ixodes ricinus salivary glands, synganglia, midguts, trachea, and ovaries, but not in Malpighian tubules. Investigation of the temporal expression patterns suggests that the receptor transcript is highly expressed in unfed I. ricinus female salivary glands and then decreases during feeding. In synganglia, a significant transcript increase was detected in replete ticks. In salivary gland acini, an antibody targeting the SIFa_R2 recognized basal epithelial cells, myoepithelial cells, and basal granular cells in close proximity to the SIFamide-releasing axon terminals. Immunoreactivity was also detected in specific neurons distributed throughout various I. ricinus synganglion locations. The current findings, alongside previous reports from our group, indicate that the neuropeptide SIFamide acts via two different receptors that regulate distinct or common cell types in the basal region of type II and III acini in I. ricinus salivary glands. Our study investigates the peptidergic regulation of the I. ricinus salivary gland in detail, emphasizing the complexity of this system.

Isolation and electrophysiological recording of Ixodes ricinus synganglion neurons.

The central nervous system of hard ticks (Ixodidae) consists of a concentrated merged nerve mass known as the synganglion. Although knowledge of tick neurobiology has dramatically improved over the last two decades, this is the first time that isolation and electrophysiological recordings have been carried out on tick neurons from the synganglion. Method: We developed a simple protocol for synganglion neuron isolation and used a whole-cell patch clamp to measure ionic currents induced by acetylcholine, nicotine and muscarine. Relatively large neurons (∼ 25 µm and âˆ¼ 35 µm) were isolated and 1 mM acetylcholine was used to induce strong inward currents of -0.38 ± 0.1 nA and - 1.04 ± 0.1 nA, respectively, with the corresponding cell capacitances being at around 142 pF and 188 pF. In addition, successive application of 1 mM acetylcholine through ∼25 µm and âˆ¼ 35 µm cells for increasing amounts of time resulted in a rapid reduction in current amplitudes. We also found that acetylcholine-evoked currents were associated with a reversible increase in intracellular calcium levels for each neuronal type. In contrast, 1 mM muscarine and nicotine induced a strong and non-reversible increase in intracellular calcium levels. This study serves as a proof of concept for the mechanical isolation of tick synganglion neurons followed by their electrophysiological recording. This approach will aid investigations into the pharmacological properties of tick neurons and provides the tools needed for the identification of drug-targeted sites and effective tick control measures.

Effects of Electromagnetic Radiation on Neuropeptide Transcript Levels in the Synganglion of Ixodes ricinus.

Anthropogenic electromagnetic radiation is an important environmental factor affecting the functionality of biological systems. Sensitivity to various frequencies of electromagnetic radiation has been detected in ixodid ticks in the past. However, the physiological aspects of radiation effects have not yet been studied in ticks. In the presented experiment, 360 Ixodes ricinus ticks, 180 males and 180 females, were divided into 16 irradiated and 8 control groups. The irradiated groups were exposed to two different intensities of electromagnetic radiation with a frequency of 900 MHz at different lengths of exposure time. RT-PCR was utilized to determine the changes in mRNA levels in tick synganglia after irradiation. Four randomly selected neuropeptide genes were tested-allatotropin (at), FGLa-related allatostatins (fgla/ast), kinin, and arginine-vasopressin-like peptide (avpl). A significant decrease in transcript levels in all female groups exposed to higher intensity radiofrequency radiation for 1 to 3 h was found. After one hour of radiofrequency exposure, a significant downregulation in allatotropin expression in males was detected. A consistent downregulation of the at gene was detected in males irradiated with at a higher intensity. Unfortunately, the specific functions of the studied neuropeptides in ticks are not known yet, so a more comprehensive study is necessary to describe the effects of EMF on observed neuropeptides. This study represents the first report on the effects of the abiotic environment on tick neurophysiology.

Frankenbacteriosis targeting interactions between pathogen and symbiont to control infection in the tick vector.

Tick microbiota can be targeted for the control of tick-borne diseases such as human granulocytic anaplasmosis (HGA) caused by model pathogen, Anaplasma phagocytophilum. Frankenbacteriosis is inspired by Frankenstein and defined here as paratransgenesis of tick symbiotic/commensal bacteria to mimic and compete with tick-borne pathogens. Interactions between A. phagocytophilum and symbiotic Sphingomonas identified by metaproteomics analysis in Ixodes scapularis midgut showed competition between both bacteria. Consequently, Sphingomonas was selected for frankenbacteriosis for the control of A. phagocytophilum infection and transmission. The results showed that Franken Sphingomonas producing A. phagocytophilum major surface protein 4 (MSP4) mimic pathogen and reduce infection in ticks by competition and interaction with cell receptor components of infection. Franken Sphingomonas-MSP4 transovarial and trans-stadial transmission suggests that tick larvae with genetically modified Franken Sphingomonas-MSP4 could be produced in the laboratory and released in the field to compete and replace the wildtype populations with associated reduction in pathogen infection/transmission and HGA disease risks.

High-Throughput Microfluidic Real-Time PCR for the Detection of Multiple Microorganisms in Ixodid Cattle Ticks in Northeast Algeria.

Ixodid ticks are hematophagous arthropods considered to be prominent ectoparasite vectors that have a negative impact on cattle, either through direct injury or via the transmission of several pathogens. In this study, we investigated the molecular infection rates of numerous tick-borne pathogens in ticks sampled on cattle from the Kabylia region, northeastern Algeria, using a high-throughput microfluidic real-time PCR system. A total of 235 ticks belonging to seven species of the genera Rhipicephalus, Hyalomma, and Ixodes were sampled on cattle and then screened for the presence of 36 different species of bacteria and protozoans. The most prevalent tick-borne microorganisms were Rickettsia spp. at 79.1%, followed by Francisella-like endosymbionts (62.9%), Theileria spp. (17.8%), Anaplasma spp. (14.4%), Bartonella spp. (6.8%), Borrelia spp. (6.8%), and Babesia spp. (2.5%). Among the 80.4% of ticks bearing microorganisms, 20%, 36.6%, 21.7%, and 2.1% were positive for one, two, three, and four different microorganisms, respectively. Rickettsia aeschlimannii was detected in Hyalomma marginatum, Hyalomma detritum, and Rhipicephalus bursa ticks. Rickettsia massiliae was found in Rhipicephalus sanguineus, and Rickettsiamonacensis and Rickettsia helvetica were detected in Ixodesricinus. Anaplasma marginale was found in all identified tick genera, but Anaplasma centrale was detected exclusively in Rhipicephalus spp. ticks. The DNA of Borrelia spp. and Bartonella spp. was identified in several tick species. Theileria orientalis was found in R. bursa, R. sanguineus, H. detritum, H. marginatum, and I. ricinus and Babesia bigemina was found in Rhipicephalus annulatus and R. sanguineus. Our study highlights the importance of tick-borne pathogens in cattle in Algeria.

Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick-Pathogen Interactions.

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick-pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick-pathogen interactions.

Anti-Microbiota Vaccines Modulate the Tick Microbiome in a Taxon-Specific Manner.

The lack of tools for the precise manipulation of the tick microbiome is currently a major limitation to achieve mechanistic insights into the tick microbiome. Anti-tick microbiota vaccines targeting keystone bacteria of the tick microbiota alter tick feeding, but their impact on the taxonomic and functional profiles of the tick microbiome has not been tested. In this study, we immunized a vertebrate host model (Mus musculus) with live bacteria vaccines targeting keystone (i.e., Escherichia-Shigella) or non-keystone (i.e., Leuconostoc) taxa of tick microbiota and tested the impact of bacterial-specific antibodies (Abs) on the structure and function of tick microbiota. We also investigated the effect of these anti-microbiota vaccines on mice gut microbiota composition. Our results showed that the tick microbiota of ticks fed on Escherichia coli-immunized mice had reduced Escherichia-Shigella abundance and lower species diversity compared to ticks fed on control mice immunized with a mock vaccine. Immunization against keystone bacteria restructured the hierarchy of nodes in co-occurrence networks and reduced the resistance of the bacterial network to taxa removal. High levels of E. coli-specific IgM and IgG were negatively correlated with the abundance of Escherichia-Shigella in tick microbiota. These effects were not observed when Leuconostoc was targeted with vaccination against Leuconostoc mesenteroides. Prediction of functional pathways in the tick microbiome using PICRUSt2 revealed that E. coli vaccination reduced the abundance of lysine degradation pathway in tick microbiome, a result validated by qPCR. In contrast, the gut microbiome of immunized mice showed no significant alterations in the diversity, composition and abundance of bacterial taxa. Our results demonstrated that anti-tick microbiota vaccines are a safe, specific and an easy-to-use tool for manipulation of vector microbiome. These results guide interventions for the control of tick infestations and pathogen infection/transmission.

The TRH-ortholog EFLamide in the migratory locust.

Arthropod EFLamide genes in chelicerates, myriapods, decapods and non pterygote hexapods encode various EFLamide paracopies on a single precursor. However, in more advanced insect species such multiple EFLamide paracopies encoding genes are absent. In some Hemiptera putative exons of an EFLamide gene coding for a single EFLamide have been identified, while in the migratory locust a similar exon could potentially code for two EFLamide peptides. The recent identification of an EFLGamide from Platynereis dumerilii as the ligand for an ortholog of the TRH GPCR, suggested that the arthropod EFLamides might similarly activate TRH GPCR orthologs. We here identify the TRH GPCR ortholog from Locusta migratoria and show that it is activated in nanomolar concentrations by the two EFLamides previously predicted from this species. We also show that in the central nervous system there seems to be only a single bilateral neuron in the protocerebrum expressing this peptide. Given this very limited expression of EFLamide in locusts, it is perhaps not surprising that this gene and its receptor have been lost in many other insect species. This shows again that although neuropeptides and their receptors may persist in different evoltionary lineages, their functions can change dramatically.

A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice.

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of mouse models in tick research is critical for understanding their biology and tick-host-pathogen interactions. Here we demonstrate a non-laborious technique for the feeding of immature stages of hard ticks on laboratory mice. The benefit of the method is its simplicity, short duration, and the ability to monitor or collect ticks at different time points of an experiment. In addition, the technique allows attachment of two individual capsules on the same mouse, which is beneficial for a variety of experiments where two different groups of ticks are required to feed on the same animal. The non-irritating and flexible capsule is made from easily accessible materials and minimizes the discomfort of the experimental animals. Furthermore, euthanasia is not necessary, mice recover completely after the experiment and are available for re-use.

Alpha-Gal and Cross-Reactive Carbohydrate Determinants in the N-Glycans of Salivary Glands in the Lone Star Tick, Amblyomma americanum.

Ticks are important ectoparasites and vectors of numerous human and animal pathogens. Ticks secrete saliva that contains various bioactive materials to evade the host defense system, and often facilitates the pathogen transmission. In addition, the Lone star tick saliva is thought to be the sensitizer in red meat allergy that is characterized by an allergic reaction to glycan moieties carrying terminal galactose-alpha-1,3-galactose (aGal). To assess N-glycome of Amblyomma americanum, we examined the N-glycan structures in male and female salivary glands at three different feeding stages and in carcasses of partially fed lone star ticks. We also surveyed the genes involved in the N-glycosylation in the tick species. The aGal epitopes and cross-reactive carbohydrate determinants (CCD) increases over time after the onset of blood feeding in both male and female A. americanum. These CCDs include xylosylation of the core mannose, 1,3-mono and 1,3- and 1,6-difucosylations of the basal GlcNac and mono- or diantennary aGal. Combinations of both xylosylation and aGal and fucosylation and aGal were also found on the N-glycan structures. While the enzymes required for the early steps of the N-glycosylation pathway are quite conserved, the enzymes involved in the later stages of N-glycan maturation in the Golgi apparatus are highly diverged from those of insects. Most of all, we propose that the aGal serves as a molecular mimicry of bioactive proteins during tick feedings on mammalian hosts, while it contributes as a sensitizer of allergy in atypical host human.

Cholinergic axons regulate type I acini in salivary glands of Ixodes ricinus and Ixodes scapularis ticks.

Regulatory factors controlling tick salivary glands (SGs) are direct upstream neural signaling pathways arising from the tick's central nervous system. Here we investigated the cholinergic signaling pathway in the SG of two hard tick species. We reconstructed the organization of the cholinergic gene locus, and then used in situ hybridization to localize mRNA encoding choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) in specific neural cells in the Ixodes synganglion. Immunohistochemical staining revealed that cholinergic axonal projections exclusively reached type I acini in the SG of both Ixodes species. In type I acini, the rich network of cholinergic axons terminate within the basolateral infoldings of the lamellate cells. We also characterized two types (A and B) of muscarinic acetylcholine receptors (mAChRs), which were expressed in Ixodes SG. We pharmacologically assessed mAChR-A to monitor intracellular calcium mobilization upon receptor activation. In vivo injection of vesamicol-a VAChT blocker-at the cholinergic synapse, suppressed forced water uptake by desiccated ticks, while injection of atropine, an mAChR-A antagonist, did not show any effect on water volume uptake. This study has uncovered a novel neurotransmitter signaling pathway in Ixodes SG, and suggests its role in water uptake by type I acini in desiccated ticks.