Ixodes ricinus ticks act as vectors for numerous pathogens that present substantial health threats. Additionally, they harbor vertically transmitted symbionts, some of which have been linked to diseases. The difficulty of isolating and cultivating these symbionts has hampered our understanding of their biological role, their potential to cause disease, and their modes of transmission. To expand our understanding of the tick symbiont Midichloria mitochondrii and Rickettsia helvetica, which has been linked to disease in humans, we utilized deep sequencing on 16 individual adult female ticks collected from coastal dune and forested areas in the Netherlands. By employing a combination of second- and third-generation sequencing techniques, we successfully reconstructed the complete genomes of M. mitochondrii from 11 individuals, R. helvetica from eight individuals, and the mitochondrial genome from all ticks. Additionally, we visualized the location of R. helvetica in tick organs and constructed genome-scale metabolic models (GEMs) of both symbionts to study their environmental dependencies. Our analysis revealed a strong cophylogeny between M. mitochondrii and mitochondrial genomes, suggesting frequent maternal transmission. In contrast, the absence of cophylogeny between R. helvetica and the mitochondrial genomes, coupled with its presence in the receptaculum seminis of I. ricinus females, raises the possibility of paternal transmission of R. helvetica. Notably, the genetic diversity of R. helvetica was found to be very low, except for the rickA virulence gene, where the presence of up to 13 insertions of a 33 nt-long repeat led to significant variability. However, this variation could not account for the differences in infection prevalence observed across eight distinct locations in the Netherlands. By employing deep sequencing, it becomes feasible to extract complete genomes and genetic data of symbionts directly from their host organisms. This methodology serves as a robust means to gain fresh insights into their interactions. Our observations, which suggest paternal transmission of R. helvetica, a relatively unexplored mode of transmission in ticks, require validation through experimental investigations. The genetic variations identified in the rickA virulence gene of R. helvetica have the potential to influence the infectivity and transmission dynamics of R. helvetica.IMPORTANCETicks are vectors of numerous human pathogens; however, the microbial interactions within ticks and the mechanisms governing pathogen transmission remain poorly understood. This study uses deep sequencing of individual Ixodes ricinus to reconstruct high-quality genomes of endosymbionts and the mitochondrion of the tick, revealing previously undetected microbial dynamics. Notably, we recovered low-abundance Rickettsia and Midichloria genomes from single ticks and present evidence that suggests paternal transmission of R. helvetica. These findings offer novel insights into the ecology and evolution of tick-associated microbes and have implications for understanding the origins and spread of tick-borne diseases.

• Klíčová slova
• Ixodes ricinus, Midichloria mitochondrii, Rickettsia helvetica, cophylogenetic analysis, deep sequencing, genome reconstruction, genome-scale metabolic modeling, paternal transmission, symbionts,
• MeSH
• fylogeneze MeSH
• genom bakteriální MeSH
• genom mitochondriální MeSH
• klíště * mikrobiologie   genetika   MeSH
• Rickettsia * genetika   fyziologie   MeSH
• symbióza * genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Although the cat flea, Ctenocephalides felis, has been identified as the primary vector of Rickettsia felis, additional flea, tick, mite, and louse species have also been associated with this bacterium by molecular means; however, the role of these arthropods in the transmission of R. felis has not been clarified. Here, we succeeded in culture isolation of R. felis from a host-seeking castor bean tick, Ixodes ricinus, the most common tick in Slovakia. The bacterial isolation was performed on XTC-2 cells at 28 °C using the shell-vial technique. An evaluation of the growth properties was performed for both the XTC-2 and Vero cell lines. We observed R. felis in the infected host cells microscopically by Gimenez staining and immunofluorescence assay. The R. felis isolate was purified by gradient ultracentrifugation and visualized by electron microscopy. Fragments of the genes gltA, ompA, ompB, htrA, rpoB, sca4, rffE, and rrs were amplified and compared with the corresponding sequences of the type strain URRWXCal2 and other R. felis culture -isolated strains. We did not detect any nucleotide polymorphisms; however, plasmid pRFδ, characteristic of the standard strain, was absent in our isolate. Herein, we describe the first successful isolation and characterization of a tick-derived R. felis strain "Danube", obtained from an I. ricinus nymph.

• Klíčová slova
• Ixodes ricinus, Rickettsia felis, cell culture, shell-vial technique, vector-borne bacteria,
• MeSH
• buněčné linie MeSH
• členovci * MeSH
• klíště * mikrobiologie   MeSH
• Rickettsia felis * genetika   MeSH
• Rickettsia * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Tick-borne rickettsial diseases are caused by pathogens acquired from hard ticks. In particular, Rickettsia slovaca, a zoonotic infectious bacterium causing tick-borne lymphadenopathy (TIBOLA), is transmitted by the vectors Dermacentor spp. that can be found all over Europe. Although recent studies point out the extreme complexity of bacteria-induced effects in these blood-feeding vectors, the knowledge of individual molecules involved in the preservation and transmission of the pathogen is still limited. System biology tools, including proteomics, may contribute greatly to the understanding of pathogen-tick-host interactions. METHODS: Herein, we performed a comparative proteomics study of the tick vector Dermacentor reticulatus that was experimentally infected with the endosymbiotic bacterium R. slovaca. Rickettsia-free ticks, collected in the southern region of Slovakia, were infected with the bacterium by a capillary tube-feeding system, and the dynamics of infection was assessed by quantitative PCR method after 5, 10, 15 and 27 days. RESULTS: At the stage of controlled proliferation (at 27 dpi), 33 (from 481 profiled) differentially abundant protein spots were detected on a two-dimensional gel. From the aforementioned protein spots, 21 were successfully identified by tandem mass spectrometry. CONCLUSIONS: Although a few discovered proteins were described as having structural or housekeeping functions, the vast majority of the affected proteins were suggested to be essential for tick attachment and feeding on the host, host immune system evasion and defensive response modulation to ensure successful pathogen transmission.

• Klíčová slova
• Bacterial transmission, Blood-feeding, Comparative proteomics, Immune modulation, Protective antigens, TIBOLA, Tick vector,
• MeSH
• Dermacentor genetika   mikrobiologie   MeSH
• DNA bakterií MeSH
• infekce přenášené vektorem MeSH
• nemoci přenášené klíšťaty mikrobiologie   přenos   MeSH
• polymerázová řetězová reakce MeSH
• proteomika * MeSH
• Rickettsia genetika   patogenita   MeSH
• rickettsiové infekce přenos   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Geografické názvy
• Slovenská republika MeSH
• Názvy látek
• DNA bakterií MeSH