Manuel de Rojas

Background: The tick microbiome is shaped by environmental filters and host biological traits. Microbial co-occurrence networks offer a powerful framework to map inter-microbial interactions, node topological roles, and community stability beyond standard diversity metrics; however, how tick sex modulates these structural networks remains poorly understood. Methods: We sequenced the V3–V4 variable region of the 16S rRNA gene from 86 adult Hyalomma lusitanicum ticks (37 males and 49 females) collected across six ecogeographical localities in Andalusia during 2023. A refined dataset of 5570 high-confidence Amplicon Sequence Variants (ASVs) was analyzed. Alpha and beta diversity patterns were evaluated using PERMANOVA models, and sex-stratified co-occurrence networks were constructed using Spearman’s rank correlations and the Zi-Pi topological framework to assess network complexity and keystone roles. Results: The primary obligate endosymbiont Francisella dominated the bacteriome (60–90% relative abundance), followed by Candidatus Midichloria. PERMANOVA models confirmed that geographical locality (R² = 0.111, p = 0.003) and host sex (R² = 0.095, p = 0.001) exerted strong, independent influences on microbial composition. Female ticks harbored a highly streamlined bacterial profile. Conversely, males exhibited significantly higher alpha diversity (p < 0.001), reflecting host-sex physiological and behavioral differences that create a broader ecological niche allowing diverse environmental and transient taxa (Acinetobacter, Sphingomonas, Stenotrophomonas, Brevundimonas) to colonize. Network analysis revealed a striking sexual dimorphism: female networks were highly centralized and anchored by a single global hub (Roseomonas), whereas male networks lacked global hubs and displayed higher topological complexity, achieving stability through a decentralized architecture sustained by 68 inter-module connectors (compared to 24 in females). Despite these macro-topological differences, a core set of seven connector ASVs was conserved across both sexes. Conclusions: These findings demonstrate that host sex shapes the ecological niche of the tick microbiome and drives distinct network topological strategies to maintain community stability, providing new insights into the structural ecology of tick–microbe interactions.

Ticks are obligate hematophagous arthropods and major vectors of diverse bacterial, parasitic, and viral pathogens. They host complex microbial communities that critically influence their biology, fitness, and interactions with pathogens. Using 16 S rRNA gene amplicon sequencing at the amplicon sequence variant (ASV) resolution combined with co-occurrence network analysis, we characterized the bacterial communities of questing adult ticks collected in the Cazorla, Segura y Las Villas Natural Park (Jaén, Spain). A total of 83 adult ticks (27 males and 56 females) representing six sympatric species, Dermacentor marginatus ( n = 39), Haemaphysalis punctata ( n = 9), H. sulcata ( n = 15), Hyalomma lusitanicum ( n = 7), Ixodes ricinus s. l. ( n = 2), and Rhipicephalus bursa ( n = 11), were analyzed. After stringent quality filtering, 3.77 million high-quality reads were recovered and resolved into 407 ASVs. Due to the inherent resolution limits of the V3–V4 region for species-level discrimination, taxonomic assignments were conservatively consolidated at the genus level (101 bacterial genera). Across all tick species, the bacteriome was heavily dominated by Pseudomonadota (98.6%), with species-specific differences primarily driven by variation in obligate symbionts. Coxiella -associated ASVs predominated in multiple tick species, whereas H. lusitanicum exhibited strong dominance by Francisella and secondary representation of Candidatus Midichloria . Alpha and beta diversity analyses revealed distinct compositional patterns shaped by host identity, with lower dispersion observed in D. marginatus and R. bursa , and greater variability among Hyalomma and Haemaphysalis individuals. Spearman-based co-occurrence network analysis indicated a highly cooperative and modular structure (> 98% positive correlations) across tick species. Notably, dominant endosymbionts ( Coxiella , Rickettsia , and Francisella ) occupied peripheral or weakly connected positions within the networks. In contrast, ecological connectivity was governed by a distinct set of low-abundance taxa, including Roseomonas , Friedmanniella , Methylobacterium , Sphingomonas , Aureimonas , Conexibacter , Marmoricola , Mycobacterium , and Nocardioides , which acted as central hubs bridging network modules, a topology robustly validated by an independent, composition-aware (SparCC) reanalysis within the D. marginatus cohort. These comparative findings demonstrate a functional decoupling between abundance and connectivity in tick microbiomes, highlighting how the “rare microbiome” can drive the topological organization and potential stability of microbial communities across sympatric host species.

The family Rhinonyssidae includes endoparasitic, blood-feeding mites that are parasitic on birds and that remain largely unstudied despite their potential role as vectors or reservoirs for various pathogens, like other Dermanyssoidea. Traditionally, the taxonomy of the group has been based on morphometric characteristics, which makes identification very difficult in many groups of closely related species. On the other hand, studies on the phylogenetic relationships within this group of mites have been neglected until the early years of the present century. In this study, twelve species belonging to five different species complexes were identified, and domains D1–D3 28S rRNA of each one were sequenced, for the first time, to investigate the sequence variation and its taxonomic implications for phylogenetic inference. Our data indicate that this molecular marker can effectively differentiate between species within the “motacillae”, “sairae”, “pari”, and “hirsti” complexes of the genus Ptilonyssus and the “melloi” and “columbae” complexes of the genus Tinaminyssus. Furthermore, the phylogenetic tree that can be derived from the domain D1–D3 28S rRNA sequences presented in this study is congruent with the current taxonomy of the Rhinonyssidae. This research calls for a reassessment of the taxonomic status of some group of species.

Members of the family Rhinonyssidae are tiny hematophagous endoparasitic mites that inhabit the nasal cavities of birds and can cause trauma to their hosts. Traditionally, identifying species in this group has relied on observing their morphometrical characteristics. Nevertheless, determining species within this particular group has become more challenging due to the rising number of newly discovered species. Moreover, the morphometrical traits vary depending on the specific genus or group of species being studied. In this study, the complete internal transcribed spacer ITS1, 5.8S rDNA, and ITS2 regions of the ribosomal DNA from eighteen species of rhinonyssid mites belonging to four genera were sequenced to assess the utility of this genomic region in resolving taxonomic questions in this group and to estimate the phylogenetic relationships among the species. Mites were collected by dissecting the nasal cavities of birds under a stereomicroscope. Specimens used for morphometrical analyses were cleared in 85% lactic acid for 1–48 h and mounted in Hoyer’s medium. Other specimens were preserved at −20 °C for molecular studies. From the data obtained in this study, it can be concluded that a thorough review and an accurate morphometrical identification and determination of the discriminatory traits are needed in this group of mites. Moreover, although the ITS1–5.8S–ITS2 fragment solves different taxonomic and phylogenetic problems at the species level, it would be necessary to test new molecular markers, or even a combination of nuclear and mitochondrial markers or different domains of the nuclear 28S rDNA, to discover a reliable taxonomic situation for rhinonyssids.

Rhinonyssidae (Mesostigmata) is a family of nasal mites only found in birds. All species are hematophagous endoparasites, which may damage the nasal cavities of birds, and also could be potential reservoirs or vectors of other infections. However, the role of members of Rhinonyssidae as disease vectors in wild bird populations remains uninvestigated, with studies of the microbiomes of Rhinonyssidae being almost non-existent. In the nasal mite (Tinaminyssus melloi) from rock doves (Columba livia), a previous study found evidence of a highly abundant putatively endosymbiotic bacteria from Class Alphaproteobacteria. Here, we expanded the sample size of this species (two different hosts- ten nasal mites from two independent samples per host), incorporated contamination controls, and increased sequencing depth in shotgun sequencing and genome-resolved metagenomic analyses. Our goal was to increase the information regarding this mite species and its putative endosymbiont. We obtained a metagenome assembled genome (MAG) that was estimated to be 98.1% complete and containing only 0.9% possible contamination. Moreover, the MAG has characteristics typical of endosymbionts (namely, small genome size an AT bias). Overall, our results support the presence of a potential endosymbiont, which is the first described for avian nasal mites to date, and improve the overall understanding of the microbiota inhabiting these mites.

Rhinonyssidae (Mesostigmata) is a family of nasal mites only found in birds. All species are hematophagous endoparasites, which may damage the nasal cavities of birds, and also could be potential reservoirs or vectors of other infections. However, the role of members of Rhinonyssidae as disease vectors in wild bird populations remains uninvestigated, with studies of the microbiomes of Rhinonyssidae being almost non-existent. In the nasal mite ( Tinaminyssus melloi ) from rock doves ( Columba livia ), a previous study found evidence of a highly abundant putatively endosymbiotic bacteria from Class Alphaproteobacteria. Here, we expanded the sample size of this species, incorporated contamination controls, and increased sequencing depth in shotgun sequencing and genome-resolved metagenomic analyses. Our goal was to increase the information regarding this mite species with its putative endosymbiont. Our results support the endosymbiotic nature of this bacterial taxon, which is the first described for bird’s nasal mites to date, and improve the overall understanding of the microbiota inhabiting these mites.

Rhynonyssidae is a family of endoparasitic hematophagous mites, which are still largely unknown even though they could act as vector or reservoir of different pathogens like dermanyssids. Sampling requirements have prevented deeper analysis. Rhinonyssids have been explored in a few host specimens per species, leading to undetailed morphological descriptions and inaccurate epidemiology. We explore the relationships established between these parasites in two Columbiformes urban birds (domestic pigeon (Columba livia domestica) and Eurasian collared dove (Streptopelia decaocto)), assesing 250 individuals of each type in Seville (Spain). As expected, Mesonyssus melloi (Castro, 1948) and Mesonyssus columbae (Crossley, 1950) were found in domestic pigeons, and Mesonyssus streptopeliae (Fain, 1962) in Eurasian collared doves. However, M. columbae was found for the first time in Eurasian collared doves. This relationship could be common in nature, but sampling methodology or host switching could also account for this result. An additional unknown specimen was found in a Eurasian collared dove, which could be a new species or an aberrant individual. We also provide an epidemiological survey of the three mite species, with M. melloi being the most common one followed by M. streptopeliae and M. columbae. High variation between previous epidemiological measurements and ours highlights the importance of developing deeper studies to uncover the factors regulating mite prevalence and intensities of infection.

Complete Mitochondrial Genomes and Bacterial Metagenomic Data From Two Species of Parasitic Avian Nasal-Mites (Rhinonyssidae: Mesostigmata)