Decontamination protocols affect the internal microbiota of ticks.

Studies on the microbiota of ticks have promoted hypotheses about the combined effects of the bacterial community, its functional contributions to the tick's physiology or probable competition effects with some tick-borne pathogens. However, knowledge on the origin of the microbiota of newly hatched larvae is missing. This study aimed to elucidate the source(s) of the microbiota in unfed tick larvae, addressing the composition of the "core microbiota" and the best ways to decontaminate eggs for microbiota studies. We applied laboratory degree bleach washes and/or ultraviolet light treatments on engorged Rhipicephalus australis females and/or their eggs. No significant effects of these treatments on the reproductive parameters of females and the hatching rates of eggs were observed. However, the different treatments did show striking effects on the composition of the microbiota. The results indicated that bleach washes disrupted the internal tick microbiota in females, implying that bleach may have entered the tick and subsequently affected the microbiota. Furthermore, the analyses of results demonstrated that the ovary is a main source of tick microbiota, while the contribution of Gené's organ (a part of the female reproductive system that secretes a protective wax coat onto tick eggs) or the male's spermatophore requires further investigation. Further studies are needed to identify best practice protocols for the decontamination of ticks for microbiota studies.

Changes in the Ixodes ricinus microbiome associated with artificial tick feeding.

Artificial tick feeding systems (ATFS) can be used to study tick biology and tick-pathogen interactions. Due to the long feeding duration of hard ticks, antibiotics are commonly added to the in vitro blood meal to prevent the blood from decaying. This may affect the ticks' microbiome, including mutualistic bacteria that play an important role in tick biology. This effect was examined by the consecutive feeding of Ixodes ricinus larvae, nymphs, and adults in vitro with and without the supplementation of gentamicin and in parallel on calves. DNA extracted from unfed females was analyzed by 16S rRNA sequencing. The abundance of Candidatus Midichloria mitochondrii, Rickettsia helvetica and Spiroplasma spp. was measured by qPCR in unfed larvae, nymphs, and adults. Larvae and nymphs fed on calves performed significantly better compared to both in vitro groups. Adults fed on blood supplemented with gentamicin and B vitamins had a higher detachment proportion and weight compared to the group fed with B vitamins but without gentamicin. The detachment proportion and weights of females did not differ significantly between ticks fed on calves and in vitro with gentamicin, but the fecundity was significantly higher in ticks fed on calves. 16S rRNA sequencing showed a higher microbiome species richness in ticks fed on calves compared to ticks fed in vitro. A shift in microbiome composition, with Ca. Midichloria mitochondrii as dominant species in females fed as juveniles on calves and R. helvetica as the most abundant species in females previously fed in vitro was observed. Females fed in vitro without gentamicin showed significant lower loads of Ca. M. mitochondrii compared to females fed in vitro with gentamicin and ticks fed on calves. Spiroplasma spp. were exclusively detected in female ticks fed on cattle by qPCR, but 16S rRNA sequencing results also showed a low abundance in in vitro females exposed to gentamicin. In conclusion, the employed feeding method and gentamicin supplementation affected the ticks' microbiome composition and fecundity. Since these changes may have an impact on tick biology and vector competence, they should be taken into account in studies employing ATFS.

Identification and Characterization of Immunodominant Proteins from Tick Tissue Extracts Inducing a Protective Immune Response against Ixodes ricinus in Cattle.

Ixodes ricinus is the main vector of tick-borne diseases in Europe. An immunization trial of calves with soluble extracts of I. ricinus salivary glands (SGE) or midgut (ME) previously showed a strong response against subsequent tick challenge, resulting in diminished tick feeding success. Immune sera from these trials were used for the co-immunoprecipitation of tick tissue extracts, followed by LC-MS/MS analyses. This resulted in the identification of 46 immunodominant proteins that were differentially recognized by the serum of immunized calves. Some of these proteins had previously also drawn attention as potential anti-tick vaccine candidates using other approaches. Selected proteins were studied in more detail by measuring their relative expression in tick tissues and RNA interference (RNAi) studies. The strongest RNAi phenotypes were observed for MG6 (A0A147BXB7), a protein containing eight fibronectin type III domains predominantly expressed in tick midgut and ovaries of feeding females, and SG2 (A0A0K8RKT7), a glutathione-S-transferase that was found to be upregulated in all investigated tissues upon feeding. The results demonstrated that co-immunoprecipitation of tick proteins with host immune sera followed by protein identification using LC-MS/MS is a valid approach to identify antigen-antibody interactions, and could be integrated into anti-tick vaccine discovery pipelines.

Tick Importin-α Is Implicated in the Interactome and Regulome of the Cofactor Subolesin.

Ticks and tick-borne diseases (TBDs) represent a burden for human and animal health worldwide. Currently, vaccines constitute the safest and most effective approach to control ticks and TBDs. Subolesin (SUB) has been identified as a vaccine antigen for the control of tick infestations and pathogen infection and transmission. The characterization of the molecular function of SUB and the identification of tick proteins interacting with SUB may provide the basis for the discovery of novel antigens and for the rational design of novel anti-tick vaccines. In the present study, we used the yeast two-hybrid system (Y2H) as an unbiased approach to identify tick SUB-interacting proteins in an Ixodes ricinus cDNA library, and studied the possible role of SUB as a chromatin remodeler through direct interaction with histones. The Y2H screening identified Importin-α as a potential SUB-interacting protein, which was confirmed in vitro in a protein pull-down assay. The sub gene expression levels in tick midgut and fat body were significantly higher in unfed than fed female ticks, however, the importin-α expression levels did not vary between unfed and fed ticks but tended to be higher in the ovary when compared to those in other organs. The effect of importin-α RNAi was characterized in I. ricinus under artificial feeding conditions. Both sub and importin-α gene knockdown was observed in all tick tissues and, while tick weight was significantly lower in sub RNAi-treated ticks than in controls, importin-α RNAi did not affect tick feeding or oviposition, suggesting that SUB is able to exert its function in the absence of Importin-α. Furthermore, SUB was shown to physically interact with histone 4, which was corroborated by protein pull-down and western blot analysis. These results confirm that by interacting with numerous tick proteins, SUB is a key cofactor of the tick interactome and regulome. Further studies are needed to elucidate the nature of the SUB-Importin-α interaction and the biological processes and functional implications that this interaction may have.