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The control of ticks through vaccination offers a sustainable alternative to the use of chemicals that cause contamination and the selection of resistant tick strains. However, only a limited number of anti-tick vaccines have reached commercial realization. In this sense, an antigen effective against different tick species is a desirable target for developing such vaccines. A peptide derived from the tick P0 protein (pP0) conjugated to a carrier protein has been demonstrated to be effective against the Rhipicephalus microplus, Rhipicephalus sanguineus, and Amblyomma mixtum tick species. The aim of this work was to assess the efficacy of this peptide when conjugated to the Bm86 protein against Dermacentor nitens and Ixodes ricinus ticks. An RNAi experiment using P0 dsRNA from I. ricinus showed a dramatic reduction in the feeding of injected female ticks on guinea pigs. In the follow-up vaccination experiments, rabbits were immunized with the pP0-Bm86 conjugate and challenged simultaneously with larvae, nymphs, and the adults of I. ricinus ticks. In the same way, horses were immunized with the pP0-Bm86 conjugate and challenged with D. nitens larva. The pP0-Bm86 conjugate showed efficacies of 63% and 55% against I. ricinus and D. nitens ticks, respectively. These results, combined with previous reports of efficacy for this conjugate, show the promising potential for its development as a broad-spectrum anti-tick vaccine. [ABSTRACT FROM AUTHOR]

In addition to being vectors of pathogenic bacteria, ticks also harbor intracellular bacteria that associate with ticks over generations, aka symbionts. The biological significance of such bacterial symbiosis has been described in several tick species but its function in Ixodes ricinus is not understood. We have previously shown that I. ricinus ticks are primarily inhabited by a single species of symbiont, Midichloria mitochondrii, an intracellular bacterium that resides and reproduces mainly in the mitochondria of ovaries of fully engorged I. ricinus females. To study the functional integration of M. mitochondrii into the biology of I. ricinus, an M. mitochondriidepleted model of I. ricinus ticks was sought. Various techniques have been described in the literature to achieve dysbiosed or apo-symbiotic ticks with various degrees of success. To address the lack of a standardized experimental procedure for the production of apo-symbiotic ticks, we present here an approach utilizing the ex vivo membrane blood feeding system. In order to deplete M. mitochondrii from ovaries, we supplemented dietary blood with tetracycline. We noted, however, that the use of tetracycline caused immediate toxicity in ticks, caused by impairment of mitochondrial proteosynthesis. To overcome the tetracyclinemediated off-target effect, we established a protocol that leads to the production of an apo-symbiotic strain of I. ricinus, which can be sustained in subsequent generations. In two generations following tetracycline administration and tetracycline-mediated symbiont reduction, M. mitochondrii was gradually eliminated from the lineage. Larvae hatched from eggs laid by such M. mitochondrii-free females repeatedly performed poorly during blood-feeding, while the nymphs and adults performed similarly to controls. These data indicate that M. mitochondrii represents an integral component of tick ovarian tissue, and when absent, results in the formation of substandard larvae with reduced capacity to blood-feed. [ABSTRACT FROM AUTHOR]

During feeding on vertebrate hosts, ticks secrete saliva composed of a rich cocktail of bioactive molecules modulating host immune responses. Although most of the proteinaceous fraction of tick saliva is of little immunogenicity, repeated feeding of ticks on mammalian hosts may lead to impairment of tick feeding, preventing full engorgement. Here, we challenged rabbits with repeated feeding of both Ixodes ricinus nymphs and adults and observed the formation of specific antibodies against several tick salivary proteins. Repeated feeding of both I. ricinus stages led to a gradual decrease in engorged weights. To identify the salivary antigens, isolated immunoglobulins from repeatedly infested rabbits were utilized for a protein pull-down from the saliva of pilocarpine-treated ticks. Eluted antigens were first identified by peptide mass fingerprinting with the aid of available I. ricinus salivary gland transcriptomes originating from early phases of tick feeding. To increase the authenticity of immunogens identified, we also performed, for the first time, de novo assembly of the sialome from I. ricinus females fed for six days, a timepoint used for pilocarpine-salivation. The most dominant I. ricinus salivary immunogens identified in our study were zinc-dependent metalloproteases of three different families. To corroborate the role of metalloproteases at the tick/host interface, we fed ticks micro-injected with a zinc metalloprotease inhibitor, phosphoramidon, on a rabbit. These ticks clearly failed to initiate feeding and to engorge. However, neither feeding to ticks immune blood of repeatedly infested rabbits, nor phosphoramidon injection into ticks, prevented their engorgement when fed in vitro on an artificial membrane system. These data show that Zn metalloproteases play a decisive role in the success of tick feeding, mediated by complex molecular interactions between the host immune, inflammatory, and hemostatic processes, which are absent in in vitro feeding. This basic concept warrants further investigation and reconsideration of the current strategies towards the development of an effective "anti-tick" vaccine. [ABSTRACT FROM AUTHOR]

Culture-independent metagenomic methodologies have enabled detection and identification of microorganisms in various biological systems and often revealed complex and unknown microbiomes. In many organisms, the microbiome outnumbers the host cells and greatly affects the host biology and fitness. Ticks are hematophagous ectoparasites with a wide host range. They vector a number of human and animal pathogens and also directly cause major economic losses in livestock. Although several reports on a tick midgut microbiota show a diverse bacterial community, in most cases the size of the bacterial population has not been determined. In this study, the microbiome was quantified in the midgut and ovaries of the ticks Ixodes ricinus and Rhipicephalus microplus before, during, and after blood feeding. Although the size of bacterial community in the midgut fluctuated with blood feeding, it was overall extremely low in comparison to that of other hematophagous arthropods. In addition, the tick ovarian microbiome of both tick species exceeded the midgut 16S rDNA copy numbers by several orders of magnitude. This indicates that the ratio of a tick midgut/ovary microbiome represents an exception to the general biology of other metazoans. In addition to the very low abundance, the tick midgut diversity in I. ricinus was variable and that is in contrast to that found in the tick ovary. The ovary of I. ricinus had a very low bacterial diversity and a very high and stable bacterial abundance with the dominant endosymbiont, Midichloria sp. The elucidation of this aspect of tick biology highlights a unique tissue-specific microbial-invertebrate host interaction. [ABSTRACT FROM AUTHOR]

Blood-feeding parasites are inadvertently exposed to high doses of potentially cytotoxic haem liberated upon host blood digestion. Detoxification of free haem is a special challenge for ticks, which digest haemoglobin intracellularly. Ticks lack a haem catabolic mechanism, mediated by haem oxygenase, and need to dispose of vast majority of acquired haem via its accumulation in haemosomes. The knowledge of individual molecules involved in the maintenance of haem homeostasis in ticks is still rather limited. RNA-seq analyses of the Ixodes ricinus midguts from blood- and serum-fed females identified an abundant transcript of glutathione S-transferase ( gst ) to be substantially up-regulated in the presence of red blood cells in the diet. Here, we have determined the full sequence of this encoding gene, ir-gst1 , and found that it is homologous to the delta-/epsilon-class of GSTs. Phylogenetic analyses across related chelicerates revealed that only one clear Ir GST1 orthologue could be found in each available transcriptome from hard and soft ticks. These orthologues create a well-supported clade clearly separated from other ticks' or mites’ delta-/epsilon-class GSTs and most likely evolved as an adaptation to tick blood-feeding life style. We have confirmed that Ir GST1 expression is induced by dietary haem(oglobin), and not by iron or other components of host blood. Kinetic properties of recombinant Ir GST1 were evaluated by model and natural GST substrates. The enzyme was also shown to bind haemin in vitro as evidenced by inhibition assay, VIS spectrophotometry, gel filtration, and affinity chromatography. In the native state, Ir GST1 forms a dimer which further polymerises upon binding of excessive amount of haemin molecules. Due to susceptibility of ticks to haem as a signalling molecule, we speculate that the expression of Ir GST1 in tick midgut functions as intracellular buffer of labile haem pool to ameliorate its cytotoxic effects upon haemoglobin intracellular hydrolysis. [ABSTRACT FROM AUTHOR]

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 (Šimo 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. [Display omitted] • Ixodes scapularis SIFamide receptor 2 (SIFa_R2) displays high affinity for SIFamide as well as to its insect paralogs. • I. scapularis SIFa_R1 displays exclusive affinity for SIFamide and not to its insect paralogs. • SIFa_R2 is expressed in I. ricinus salivary glands, synganglion, midguts, trachea, and ovaries. • SIFa_R2 is localized in distinct basal cell types in both type II and III salivary gland acini. [ABSTRACT FROM AUTHOR]