Ticks are ectoparasites that feed on blood and have an impressive ability to consume and process enormous amounts of host blood, allowing extremely long periods of starvation between blood meals. The central role in the parasitic lifestyle of ticks is played by the midgut. This organ efficiently stores and digests ingested blood and serves as the primary interface for the transmission of tick-borne pathogens. In this study, we used a label-free quantitative approach to perform a novel dynamic proteomic analysis of the midgut of Ixodesricinus nymphs, covering their development from unfed to pre-molt stages. We identified 1534 I. ricinus-specific proteins with a relatively low proportion of host proteins. This proteome dataset, which was carefully examined by manual scrutiny, allowed precise annotation of proteins important for blood meal processing and their dynamic changes during nymphal ontogeny. We focused on midgut molecules related to lipid hydrolysis, storage, and transport, opening a yet unexplored avenue for studying lipid metabolism in ticks. Further dynamic profiling of the tick's multi-enzyme digestive network, protease inhibitors, enzymes involved in redox homeostasis and detoxification, antimicrobial peptides, and proteins responsible for midgut colonization by Borrelia spirochetes promises to uncover new targets for targeting tick nymphs, the most critical life stage for transmission the pathogens that cause tick-borne diseases.

• Klíčová slova
• Borrelia, Ixodes, antimicrobial peptides, label-free quantification, lipid metabolism, midgut, protease inhibitors, proteases, proteome, ticks,
• MeSH
• klíště * parazitologie   MeSH
• proteom MeSH
• proteomika MeSH
• trávicí systém MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteom MeSH

It has been demonstrated that impairing protein synthesis using drugs targeted against tRNA amino acid synthetases presents a promising strategy for the treatment of a wide variety of parasitic diseases, including malaria and toxoplasmosis. This is the first study evaluating tRNA synthetases as potential drug targets in ticks. RNAi knock-down of all tested tRNA synthetases had a strong deleterious phenotype on Ixodes ricinus feeding. Our data indicate that tRNA synthetases represent attractive, anti-tick targets warranting the design of selective inhibitors. Further, we tested whether these severely impaired ticks were capable of transmitting Borrelia afzelii spirochaetes. Interestingly, biologically handicapped I. ricinus nymphs transmitted B. afzelii in a manner quantitatively sufficient to develop a systemic infection in mice. These data suggest that initial blood-feeding, despite the incapability of ticks to fully feed and salivate, is sufficient for activating B. afzelii from a dormant to an infectious mode, enabling transmission and dissemination in host tissues.

• Klíčová slova
• Borrelia, Lyme disease, borreliosis, tRNA synthetase, tick, transmission,
• MeSH
• akaricidy farmakologie   MeSH
• aminoacyl-tRNA-synthetasy antagonisté a inhibitory   genetika   MeSH
• Borrelia burgdorferi komplex MeSH
• klíšťata účinky léků   mikrobiologie   MeSH
• lidé MeSH
• lymeská nemoc farmakoterapie   mikrobiologie   přenos   MeSH
• proteosyntéza účinky léků   MeSH
• vyvíjení léků MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akaricidy MeSH
• aminoacyl-tRNA-synthetasy MeSH

In Europe, Ixodes ricinus is the most important vector of human infectious diseases, most notably Lyme borreliosis and tick-borne encephalitis virus. Multiple non-natural hosts of I. ricinus have shown to develop immunity after repeated tick bites. Tick immunity has also been shown to impair B. burgdorferi transmission. Most interestingly, multiple tick bites reduced the likelihood of contracting Lyme borreliosis in humans. A vaccine that mimics tick immunity could therefore potentially prevent Lyme borreliosis in humans. A yeast surface display library (YSD) of nymphal I. ricinus salivary gland genes expressed at 24, 48 and 72 h into tick feeding was constructed and probed with antibodies from humans repeatedly bitten by ticks, identifying twelve immunoreactive tick salivary gland proteins (TSGPs). From these, three proteins were selected for vaccination studies. An exploratory vaccination study in cattle showed an anti-tick effect when all three antigens were combined. However, immunization of rabbits did not provide equivalent levels of protection. Our results show that YSD is a powerful tool to identify immunodominant antigens in humans exposed to tick bites, yet vaccination with the three selected TSGPs did not provide protection in the present form. Future efforts will focus on exploring the biological functions of these proteins, consider alternative systems for recombinant protein generation and vaccination platforms and assess the potential of the other identified immunogenic TSGPs.

• MeSH
• antigeny krev   imunologie   izolace a purifikace   MeSH
• Borrelia burgdorferi izolace a purifikace   MeSH
• imunizace MeSH
• infestace klíšťaty imunologie   parazitologie   MeSH
• klíště imunologie   MeSH
• kousnutí klíštětem imunologie   MeSH
• králíci MeSH
• lidé MeSH
• lymeská nemoc krev   parazitologie   přenos   MeSH
• metody zobrazení buněčného povrchu metody   MeSH
• peptidová knihovna MeSH
• peptidové fragmenty imunologie   MeSH
• Saccharomyces cerevisiae MeSH
• skot MeSH
• slinné proteiny a peptidy imunologie   MeSH
• slinné žlázy imunologie   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• mužské pohlaví MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny MeSH
• peptidová knihovna MeSH
• peptidové fragmenty MeSH
• slinné proteiny a peptidy MeSH

Ixodes ricinus is the vector for Borrelia afzelii, the predominant cause of Lyme borreliosis in Europe, whereas Ixodes scapularis is the vector for Borrelia burgdorferi in the USA. Transcription of several I. scapularis genes changes in the presence of B. burgdorferi and contributes to successful infection. To what extend B. afzelii influences gene expression in I. ricinus salivary glands is largely unknown. Therefore, we measured expression of uninfected vs. infected tick salivary gland genes during tick feeding using Massive Analysis of cDNA Ends (MACE) and RNAseq, quantifying 26.179 unique transcripts. While tick feeding was the main differentiator, B. afzelii infection significantly affected expression of hundreds of transcripts, including 465 transcripts after 24 h of tick feeding. Validation of the top-20 B. afzelii-upregulated transcripts at 24 h of tick feeding in ten biological genetic distinct replicates showed that expression varied extensively. Three transcripts could be validated, a basic tail protein, a lipocalin and an ixodegrin, and might be involved in B. afzelii transmission. However, vaccination with recombinant forms of these proteins only marginally altered B. afzelii infection in I. ricinus-challenged mice for one of the proteins. Collectively, our data show that identification of tick salivary genes upregulated in the presence of pathogens could serve to identify potential pathogen-blocking vaccine candidates.

• MeSH
• arachnida jako vektory mikrobiologie   MeSH
• bakteriální vakcíny aplikace a dávkování   MeSH
• Borrelia burgdorferi komplex účinky léků   MeSH
• infestace klíšťaty genetika   mikrobiologie   prevence a kontrola   přenos   MeSH
• klíště účinky léků   MeSH
• lymeská nemoc genetika   mikrobiologie   prevence a kontrola   přenos   MeSH
• myši MeSH
• proteiny členovců genetika   MeSH
• slinné žlázy mikrobiologie   MeSH
• transkriptom * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální vakcíny MeSH
• proteiny členovců MeSH

BACKGROUND: The castor bean tick Ixodes ricinus is an important vector of several clinically important diseases, whose prevalence increases with accelerating global climate changes. Characterization of a tick life-cycle is thus of great importance. However, researchers mainly focus on specific organs of fed life stages, while early development of this tick species is largely neglected. METHODS: In an attempt to better understand the life-cycle of this widespread arthropod parasite, we sequenced the transcriptomes of four life stages (egg, larva, nymph and adult female), including unfed and partially blood-fed individuals. To enable a more reliable identification of transcripts and their comparison in all five transcriptome libraries, we validated an improved-fit set of five I. ricinus-specific reference genes for internal standard normalization of our transcriptomes. Then, we mapped biological functions to transcripts identified in different life stages (clusters) to elucidate life stage-specific processes. Finally, we drew conclusions from the functional enrichment of these clusters specifically assigned to each transcriptome, also in the context of recently published transcriptomic studies in ticks. RESULTS: We found that reproduction-related transcripts are present in both fed nymphs and fed females, underlining the poorly documented importance of ovaries as moulting regulators in ticks. Additionally, we identified transposase transcripts in tick eggs suggesting elevated transposition during embryogenesis, co-activated with factors driving developmental regulation of gene expression. Our findings also highlight the importance of the regulation of energetic metabolism in tick eggs during embryonic development and glutamate metabolism in nymphs. CONCLUSIONS: Our study presents novel insights into stage-specific transcriptomes of I. ricinus and extends the current knowledge of this medically important pathogen, especially in the early phases of its development.

• Klíčová slova
• Ixodes ricinus, Life stage, Reference gene validation, Tick development, Transcriptome assembly,
• MeSH
• klíště genetika   růst a vývoj   MeSH
• nymfa růst a vývoj   MeSH
• rozmnožování genetika   MeSH
• stadia vývoje MeSH
• stanovení celkové genové exprese * MeSH
• stravovací zvyklosti MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Anti-tick vaccines have the potential to be an environmentally friendly and cost-effective option for tick control. In vaccine development, the identification of efficacious antigens forms the major bottleneck. In this study, the efficacy of immunization with recombinant ferritin 2 and native tick protein extracts (TPEs) against Ixodes ricinus infestations in calves was assessed in two immunization experiments. In the first experiment, each calf (n = 3) was immunized twice with recombinant ferritin 2 from I. ricinus (IrFER2), TPE consisting of soluble proteins from the internal organs of partially fed I. ricinus females, or adjuvant, respectively. In the second experiment, each calf (n = 4) was immunized with protein extracts from the midgut (ME) of partially fed females, the salivary glands (SGE) of partially fed females, a combination of ME and SGE, or adjuvant, respectively. Two weeks after the booster immunization, calves were challenged with 100 females and 200 nymphs. Blood was collected from the calves before the first and after the second immunization and fed to I. ricinus females and nymphs using an in vitro artificial tick feeding system. The two calves vaccinated with whole TPE and midgut extract (ME) showed hyperemia on tick bite sites 2 days post tick infestation and exudative blisters were observed in the ME-vaccinated animal, signs that were suggestive of a delayed type hypersensitivity (DTH) reaction. Significantly fewer ticks successfully fed on the three animals vaccinated with TPE, SGE, or ME. Adults fed on the TPE and ME vaccinated animals weighed significantly less. Tick feeding on the IrFER2 vaccinated calf was not impaired. The in vitro feeding of serum or fresh whole blood collected from the vaccinated animals did not significantly affect tick feeding success. Immunization with native I. ricinus TPEs thus conferred a strong immune response in calves and significantly reduced the feeding success of both nymphs and adults. In vitro feeding of serum or blood collected from vaccinated animals to ticks did not affect tick feeding, indicating that antibodies alone were not responsible for the observed vaccine immunity.

• Klíčová slova
• Ixodes ricinus, anti-tick vaccine, artificial tick feeding, ferritin, midgut extract, salivary glands extract,
• Publikační typ
• časopisecké články MeSH

Ticks salivate while feeding on their hosts. Saliva helps blood feeding through host anti-hemostatic and immunomodulatory components. Previous transcriptomic and proteomic studies revealed the complexity of tick saliva, comprising hundreds of polypeptides grouped in several multi-genic families such as lipocalins, Kunitz-domain containing peptides, metalloproteases, basic tail secreted proteins, and several other families uniquely found in ticks. These studies also revealed that the composition of saliva changes with time; expression of transcripts from the same family wax and wane as a function of feeding time. Here, we examined whether host immune factors could influence sialome switching by comparing sialomes of ticks fed naturally on a rabbit, to ticks artificially fed on defibrinated blood depleted of immune components. Previous studies were based on transcriptomes derived from pools of several individuals. To get an insight into the uniqueness of tick sialomes, we performed transcriptomic analyses of single salivary glands dissected from individual adult female I. ricinus ticks. Multivariate analysis identified 1,279 contigs differentially expressed as a function of time and/or feeding mode. Cluster analysis of these contigs revealed nine clusters of differentially expressed genes, four of which appeared consistently across several replicates, but five clusters were idiosyncratic, pointing to the uniqueness of sialomes in individual ticks. The disclosure of tick quantum sialomes reveals the unique salivary composition produced by individual ticks as they switch their sialomes throughout the blood meal, a possible mechanism of immune evasion.

• MeSH
• klíště genetika   metabolismus   MeSH
• králíci MeSH
• lidé MeSH
• sekvenční analýza RNA MeSH
• slinné žlázy metabolismus   MeSH
• sliny metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom * MeSH
• výpočetní biologie MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The hard tick Ixodes ricinus is an important disease vector whose salivary secretions mediate blood-feeding success on vertebrate hosts, including humans. Here we describe the expression profiles and downstream analysis of de novo-discovered microRNAs (miRNAs) expressed in I. ricinus salivary glands and saliva. Eleven tick-derived libraries were sequenced to produce 67,375,557 Illumina reads. De novo prediction yielded 67 bona fide miRNAs out of which 35 are currently not present in miRBase. We report for the first time the presence of microRNAs in tick saliva, obtaining furthermore molecular indicators that those might be of exosomal origin. Ten out of these microRNAs are at least 100 times more represented in saliva. For the four most expressed microRNAs from this subset, we analyzed their combinatorial effects upon their host transcriptome using a novel in silico target network approach. We show that only the inclusion of combinatorial effects reveals the functions in important pathways related to inflammation and pain sensing. A control set of highly abundant microRNAs in both saliva and salivary glands indicates no significant pathways and a far lower number of shared target genes. Therefore, the analysis of miRNAs from pure tick saliva strongly supports the hypothesis that tick saliva miRNAs can modulate vertebrate host homeostasis and represents the first direct evidence of tick miRNA-mediated regulation of vertebrate host gene expression at the tick-host interface. As such, the herein described miRNAs may support future drug discovery and development projects that will also experimentally question their predicted molecular targets in the vertebrate host.

• Klíčová slova
• deep-sequencing, disease biology, gene target prediction, interactomes/systems biology, microRNA, tick–vertebrate host interaction,
• MeSH
• genové regulační sítě * MeSH
• infestace klíšťaty genetika   parazitologie   MeSH
• interakce hostitele a parazita genetika   MeSH
• klíště genetika   MeSH
• mikro RNA analýza   genetika   MeSH
• obratlovci parazitologie   MeSH
• počítačová simulace MeSH
• slinné žlázy metabolismus   MeSH
• sliny chemie   metabolismus   MeSH
• transkriptom * MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA MeSH

Adult females of the genus Ixodes imbibe blood meals exceeding about 100 times their own weight within 7‒9 days. During this period, ticks internalise components of host blood by endocytic digest cells that line the tick midgut epithelium. Using RNA-seq, we aimed to characterise the midgut transcriptome composition in adult Ixodes ricinus females during early and late phase of engorgement. To address specific adaptations to the haemoglobin-rich diet, we compared the midgut transcriptomes of genetically homogenous female siblings fed either bovine blood or haemoglobin-depleted serum. We noted that tick gut transcriptomes are subject to substantial temporal-dependent expression changes between day 3 and day 8 of feeding. In contrast, the number of transcripts significantly affected by the presence or absence of host red blood cells was low. Transcripts relevant to the processes associated with blood-meal digestion were analysed and involvement of selected encoded proteins in the tick midgut physiology discussed. A total of 7215 novel sequences from I. ricinus were deposited in public databases as an additional outcome of this study. Our results broaden the current knowledge of tick digestive system and may lead to the discovery of potential molecular targets for efficient tick control.

• MeSH
• klíště genetika   metabolismus   MeSH
• sekvenční analýza RNA * MeSH
• skot MeSH
• stanovení celkové genové exprese * MeSH
• střeva patologie   MeSH
• střevní sliznice metabolismus   MeSH
• transkriptom fyziologie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

The saliva of ixodid ticks contains a mixture of bioactive molecules that target a wide spectrum of host defense mechanisms to allow ticks to feed on the vertebrate host for several days. Tick salivary proteins cluster in multigenic protein families, and individual family members display redundancy and pluripotency in their action to ameliorate or evade host immune responses. It is now clear that members of different protein families can target the same cellular or molecular pathway of the host physiological response to tick feeding. We present and discuss our hypothesis that redundancy and pluripotency evolved in tick salivary immunomodulators to evade immune recognition by the host while retaining the immunomodulatory potential of their saliva.

• Klíčová slova
• immunomodulation, multigenic protein families, pluripotency, redundancy, silent antigens, tick salivary proteins,
• MeSH
• arachnida jako vektory imunologie   parazitologie   MeSH
• imunitní únik imunologie   MeSH
• interakce hostitele a parazita imunologie   MeSH
• Ixodidae imunologie   parazitologie   MeSH
• lidé MeSH
• parazitární nemoci imunologie   přenos   MeSH
• proteiny členovců imunologie   MeSH
• slinné proteiny a peptidy imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• proteiny členovců MeSH
• slinné proteiny a peptidy MeSH