Although some studies have already been performed on Rhipicephalus sanguineus (Latreille) populations, there is still much disarray around its taxonomic classification. Rhipicephalus sanguineus, which is also known as " (brown dog tick) is a three-host tick that parasitizes many vertebrates, including occasional human hosts. It presents a worldwide distribution and is a vector of zoonosis with acknowledged public health importance. In Western Europe, R. turanicus (Pomerantsev) share many phenotypic features and is genetically closely related to R. sanguineus. Additionally, as these species are sympatric, they can be easily misidentified. However, by means of a thorough morphological traits analysis, it is possible to distinguish both species, especially resorting to scanning electron microscopy. This study was conducted using stereoscopic and SEM analyses, both associated with morphometric analysis, in order to help clarify the taxonomic status of this species complex. For that purpose, specimens resembling R. sanguineus were collected from dogs of the Midwestern Iberian Peninsula. Identification was performed based on taxonomical key characteristics. The obtained results allowed the authors to agree that a great morphological variability is found within R. sanguineus-like Iberian populations. Nevertheless, R. turanicus is easily identified by morphologic analysis, especially using reference specimens, and its presence in Portugal is suggested.

Rhipicephalus ticks are competent vectors of several pathogens, such as Spotted Fever Group Rickettsiae (SFGR) and many Babesia species. Within this genus, different R. sanguineus s.l. lineages show an unequal vector competence and resistance regarding some pathogenic strains. Current literature supports that tick endosymbionts may play an essential role in the transmission ability of a vector. Indeed, the microbial community of Rhipicephalus seems to be dominated by Coxiella-like endosymbionts (CLE). Still, their co-evolutionary associations with the complicated phylogeny of Rhipicephalus lineages and their transmissible pathogens remain unclear. We performed a phylogenetic congruence analysis to address whether divergent R. sanguineus s.l. lineages had a different symbiont composition. For that, we applied a PCR based approach to screen part of the microbial community present in 279 Rhipicephalus ticks from the Iberian Peninsula and Africa. Our analyses detected several qPCR-positive signals for both SFGR and Babesia species, of which we suggest R. sanguineus-tropical lineage as a natural vector of Babesia vogeli and R. sanguineus-temperate lineage of SFGR. The acquisition of 190 CLE sequences allowed to evaluate co-phylogenetic associations between the tick and the symbiont. With this data, we observed a strong but incomplete co-cladogenesis between CLE strains and their Rhipicephalus tick lineages hosts.

Rhipicephalus sanguineus Latreille (1806) (Ixodida: Ixodidae) is considered to be the most widely distributed tick and to have a vast range of habitats and hosts, including livestock, pets and wildlife. In addition to morphological differences, recent investigations using approaches based on molecular genetic markers have revealed the existence of different R. sanguineus lineages in different geographic regions. In this study, 475 ticks collected from dogs in the western Iberian peninsula were studied both morphologically and genetically, using 12S and 16S rDNA and COI gene markers in order to clarify the controversy over the systematic status of R. sanguineus sensu lato in Western Europe, and to compare the present data with those sourced from studies conducted in other regions of the world. Despite the high morphometric variability, particularly on spiracles in both genders and in female genitalia, data obtained with different genetic molecular markers show very low variability, suggesting the existence of a unique species. In addition, the phylogenetic analysis showed genetic uniformity, supporting the existence of a well-defined clade consisting of R. sanguineus s.l. specimens from Western Europe that are distinct from R. sanguineus s.l. from Africa. Furthermore, these data corroborate the existence of a polymorphic species in Western Europe, which requires to be consensually redescribed in view of its medical and veterinary importance in pathogen transmission.

Background Microbial communities can affect disease risk by interfering with the transmission or maintenance of pathogens in blood-feeding arthropods. Here, we investigated whether bacterial communities vary between Ixodes ricinus nymphs which were or were not infected with horizontally transmitted human pathogens. Methods Ticks from eight forest sites were tested for the presence of Borrelia burgdorferi sensu lato, Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis by quantitative polymerase chain reaction (qPCR), and their microbiomes were determined by 16S rRNA amplicon sequencing. Tick bacterial communities clustered poorly by pathogen infection status but better by geography. As a second approach, we analysed variation in tick microorganism community structure (in terms of species co-infection) across space using hierarchical modelling of species communities. For that, we analysed almost 14,000 nymphs, which were tested for the presence of horizontally transmitted pathogens B. burgdorferi s.l., A. phagocytophilum, and N. mikurensis, and the vertically transmitted tick symbionts Rickettsia helvetica, Rickettsiella spp., Spiroplasma ixodetis, and CandidatusMidichloria mitochondrii. Results With the exception of Rickettsiella spp., all microorganisms had either significant negative (R. helvetica and A. phagocytophilum) or positive (S. ixodetis, N. mikurensis, and B. burgdorferi s.l.) associations with M. mitochondrii. Two tick symbionts, R. helvetica and S. ixodetis, were negatively associated with each other. As expected, both B. burgdorferi s.l. and N. mikurensis had a significant positive association with each other and a negative association with A. phagocytophilum. Although these few specific associations do not appear to have a large effect on the entire microbiome composition, they can still be relevant for tick-borne pathogen dynamics. Conclusions Based on our results, we propose that M. mitochondrii alters the propensity of ticks to acquire or maintain horizontally acquired pathogens. The underlying mechanisms for some of these remarkable interactions are discussed herein and merit further investigation. Graphical Abstract

A multigene phylogeny including 24 Rhipicephalus species from the Afrotropical and Mediterranean regions, based on mitochondrial DNA genes (COI, 12S and 16S), was constructed based on Bayesian inference and maximum likelihood estimations. The phylogenetic reconstruction revealed 31 Rhipicephalus clades, which include the first molecular records of Rhipicephalus duttoni (Neumann), and Rhipicephalus senegalensis (Koch). Our results support the R. pulchellus, R. evertsi and R. pravus complexes as more phylogenetically close to Rhipicephalus (Boophilus) than to the remaining Rhipicephalus clades, suggesting two main monophyletic groups within the genus. Additionally, the phenotypic resembling R. sanguineus s.l. and Rhipicephalus turanicus (Pomerantsev) are here represented by nine clades, of which none of the R. turanicus assemblages appeared as distributed in the Iberian Peninsula. These results not only indicate that both species include more cryptic diversity than the already reported, but also suggest that R. turanicus distribution is less extended than previously anticipated. This analysis allowed to improve species identification by exposing cryptic species and reinforced mtDNA markers suitability for intra/inter-species clarification analyses. Incorporating new species molecular records to improve phylogenetic clarification can significantly improve ticks' identification methods which will have epidemiologic implications on public health.