Ticks are obligate hematophagous arthropods that transmit a wide range of pathogens to humans as well as wild and domestic animals. They also harbor a non-pathogenic microbiota, although our previous study has shown that the diverse bacterial microbiome in the midgut of Ixodes ricinus is quantitatively poor and lacks a core. In artificial infections by capillary feeding of ticks with two model bacteria (Gram-positive Micrococcus luteus and Gram-negative Pantoea sp.), rapid clearance of these microbes from the midgut was observed, indicating the presence of active immune mechanisms in this organ. In the current study, RNA-seq analysis was performed on the midgut of I. ricinus females inoculated with either M. luteus or Pantoea sp. or with sterile water as a control. While no immune-related transcripts were upregulated by microbial inoculation compared to that of the sterile control, capillary feeding itself triggered dramatic transcriptional changes in the tick midgut. Manual curation of the transcriptome from the midgut of unfed I. ricinus females, complemented by the proteomic analysis, revealed the presence of several constitutively expressed putative antimicrobial peptides (AMPs) that are independent of microbial stimulation and are referred to here as 'guard' AMPs. These included two types of midgut-specific defensins, two different domesticated amidase effector 2 (Dae2), microplusin/ricinusin-related molecules, two lysozymes, and two gamma interferon-inducible lysosomal thiol reductases (GILTs). The in vitro antimicrobial activity assays of two synthetic mature defensins, defensin 1 and defensin 8, confirmed their specificity against Gram-positive bacteria showing exceptional potency to inhibit the growth of M. luteus at nanomolar concentrations. The antimicrobial activity of midgut defensins is likely part of a multicomponent system responsible for the rapid clearance of bacteria in the tick midgut. Further studies are needed to evaluate the role of other identified 'guard' AMPs in controlling microorganisms entering the tick midgut.

• Klíčová slova
• Ixodes, Micrococcus luteus, antimicrobial peptide, defensin, immune system, midgut microbiome, tick,
• MeSH
• antimikrobiální peptidy metabolismus   MeSH
• gastrointestinální trakt mikrobiologie   imunologie   MeSH
• klíště * mikrobiologie   imunologie   MeSH
• Micrococcus luteus imunologie   MeSH
• proteomika MeSH
• stanovení celkové genové exprese MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antimikrobiální peptidy MeSH

BACKGROUND: Ticks are obligate hematophagous arthropods transmitting a wide range of pathogens to humans and animals. They also harbor a non-pathogenic microbiota, primarily in the ovaries and the midgut. In the previous study on Ixodes ricinus, we used a culture-independent approach and showed a diverse but quantitatively poor midgut bacterial microbiome. Our analysis also revealed the absence of a core microbiome, suggesting an environmental origin of the tick midgut microbiota. METHODS: A bacterial analysis of the midgut of adult females collected by flagging from two localities in the Czech Republic was performed. Using the culture-independent approach, we tested the hypothesis that the midgut microbiome is of the environmental origin. We also cultured indigenous bacteria from the tick midgut and used these to feed ticks artificially in an attempt to manipulate the midgut microbiome. RESULTS: The midgut showed a very low prevalence and abundance of culturable bacteria, with only 37% of ticks positive for bacteria. The culture-independent approach revealed the presence of Borrelia sp., Spiroplasma sp., Rickettsia sp., Midichloria sp. and various mainly environmental Gram-positive bacterial taxa. The comparison of ticks from two regions revealed that the habitat influenced the midgut bacterial diversity. In addition, the midgut of ticks capillary fed with the indigenous Micrococcus luteus (Gram-positive) and Pantoea sp. (Gram-negative) could not be colonized due to rapid and effective clearance of both bacterial taxa. CONCLUSIONS: The midgut microbiome of I. ricinus is diverse but low in abundance, with the exception of tick-borne pathogens and symbionts. The environment impacts the diversity of the tick midgut microbiome. Ingested extracellular environmental bacteria are rapidly eliminated and are not able to colonize the gut. We hypothesize that bacterial elimination triggered in the midgut of unfed adult females is critical to maintain low microbial levels during blood-feeding.

• Klíčová slova
• Capillary feeding, Culturing, High-throughput sequencing, Ixodes ricinus, Microbiome, Microbiome manipulation, Midgut,
• MeSH
• Borrelia * MeSH
• klíště * mikrobiologie   MeSH
• mikrobiota * MeSH
• Rickettsia * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH