Molecular detection of Rickettsia spp. in ticks (Ixodida: Argasidae and Ixodidae) and mites (Trombiculoidae) from a brazilian taxonomic collection

Molecular detection of Rickettsia spp. in ticks (Ixodida: Argasidae and Ixodidae) and mites (Trombiculoidae) from a brazilian taxonomic collection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular detection of Rickettsia spp. in ticks (Ixodida: Argasidae and Ixodidae) and mites (Trombiculoidae) from a brazilian taxonomic collection Almir Rogério Pepato, Nikolay V. Anisimov, Jefferson C. Carvalho Farias da Silva, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8190507/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Rickettsia species are arthropod-hosted endosymbionts with a wide range of ecologies, including induction of parthenogenesis, male-killing, and even plant pathogens with potential horizontal transmission through a plant host , although they attract greater attention for including many vertebrate pathogens. The genus is classified into phylogenetic and serological groups, heterogeneous concerning their ecologies. This study aims at screen ticks and mites deposited in Acarological Collection at Center of Taxonomical Collections at Universidade Federal de Minas Gerais, Brazil, for rickettsial occurrences, making available genetic data on hosts and bacteria. Endogenous mitochondrial markers for ticks and mitochondrial and nuclear genes for chiggers were sequenced for sample quality control and species identification. A total of 38 soft ticks belonging to the genus Ornithodoros (at least six putative species), 18 hard ticks belonging to the genus Amblyomma (7 spp) , and 52 chiggers belonging to genus Whartonia (2 spp) led to sequences. Polymerase Chain reactions for a Citrate S ynthase gene fragment led to 21 sequences grouped in the phylogroups Bellii, Spotted Fever, and Transitional, with one sequence of Rickettsia obtained from Amblyomma dubitatum (identified as R. bellii ), eight sequences from Ornithodoros ( classified in five or six strains associated to the phylogroups Belli, Spotted Fever and Transitional) and twelve associate to Whartonia pachywhartoni (three likely in the Spotted Fever and nine in the Transitional phylogroup). Considering the diversity of rickettsial occurrences in Ornithodoros , with four of the nine strains detected only in O. fonsecai , a species known to bite humans, the medical relevance of Rickettsia associated with caves and bats needs to be further investigated. Taxonomic collections One Health Ectoparasites Vertebrates Taxonomy Cave Bat gltA Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The Alphaproteobacteria genus Rickettsia belongs to the diversified order Rickettsiales, which includes many intracellular obligate symbionts (Dumler and Walker 2005), a condition apparently convergently acquired (Castelli et al. 2024). The evolution of the ability to invade and hijack eukaryotic cells along the trend toward losing pathways for aminoacid and nucleotide synthesis make them unable to survive extracellularly, turning a group difficult to laboratory manipulation and the use of traditional techniques for bacterial study (McGinn and Lamason 2021). On other hand, since some species of Rickettsia cause life-threatening diseases such as the Rocky Mountain spotted fever and typhus, the genus has attracted considerable attention, particularly in medicine (Raoult and Roux 1997; Parola et al. 2005), prompting molecular studies on their infection mechanisms (Uchiyama et al. 2006, Ge and Rikihisa 2011).The genus Rickettsia encompasses a broad diversity of hosts and infection mechanisms, extending beyond its potential threat to human and other vertebrates (Perlman et al. 2006; Weinert et al. 2009; McGinn and Lamason 2021), with the transmission to humans and animals, mainly through arthropod bites such as mites, ticks and insect hosts, with a predominance in ticks (Parola et al. 2013, El Karkouri et al. 2022). Moreover, their close relationship to mitochondria makes them key models in evolutionary biology (Fitzpatrick et al. 2006, El Karkouri et al. 2022). Overall, using multiloci and phylogenomic analysis, the genera could be divided in a few major phylogroups: Spotted Fever group (SFG), the Transitional group (TRG) the Typhus group (TG), the Canadensis group (CG), and the Bellii group (BG) as well some less diversified lineages such as Scapularis, Adalia, Helvetica, Meloidiae and Rhyzobius. These lineages were recovered in multiloci and genomic scale phylogenetic analyses in reasonably congruent topologies, which branching order will be used to order an overview on the genus diversity below (Murray et al. 2016, Perlman et al. 2006, Weinert et al. 2009, El Karkouri et al. 2022, Davison et al. 2022). Former groups Torix and Megaira are recently reputed as separate genera (Davison et al. 2022). They have the largest range of hosts, from microeukaryotes to arthropods. Remaining Rickettsia are arthropod symbionts, and comprise the arthropod borne pathogens. Infections of basal Rickettsia lead to a diversity of phenotypes including male-killing (Hurst and Jiggins 2000), parthenogenesis induction (Stouthamer et al. 2001), mutualists required for oogenesis (Perotti et al. 2006), facultative mutualism (Jaenike 2012), and other mechanisms of sex-ratio distortion (Takahashi et al. 1997, Weinert et al. 2007, Himler et al. 2011) The basal groups Rhyzobius and Meloidae are both named after beetles, even though each also comprise Dipteran as hosts. Despite fragmentary knowledge on both lineages, populations of Rhyzobius litura have female-biased sex-ratios, suggesting sex-ratio distortion (Weinert et al. 2007). The following branch from Rickettsia tree is the Bellii group, all associated with arthropods, comprising hexapoda (Diptera, Coleoptera, Hemiptera, Hymenoptera, Lepidoptera and Neuroptera), ticks (Ixodidae and Argasidae), and mites (Mesostigmata). The Bellii group is associated with a wide range of adaptive phenotypes, including parthenogenesis induction, male-killing, uncharacterised sex-ratio distortion, requirement for oogenesis and facultative mutualism, and possible vertebrate pathogenicity (Weinert 2015). This group includes the single Rickettsia that acts as a plant pathogen with potential horizontal transmission through a plant host (Davis et al. 1998). The Adalia group is named after ladybird beetles but also includes Dipteran and Collembola hosts (Weinert et al. 2009), the occurrence of male-killing species in the Adalia and Bellii groups is probably homoplastic (Weinert 2015). The Canadensis group, includes bacteria with hard tick hosts (Ixodidae), and the beetle Coccotrypes dactyliperda , which is sterile when cured of Rickettsia and Wolbachia (Zchori-Fein et al. 2006), but if the Rickettsia is responsible for this phenomena, it will be homoplastic with representatives of the Torix and Transitional groups (Weinert 2015). The following groups have their internal relationships with low support and incongruency among datasets, despite being included in a clade with high support (Murray et al. 2016, Perlman et al. 2006, Weinert et al. 2009, El Karkouri et al. 2022, Davison et al. 2022). Historically and in current clinical practice, the SFG and TG groups were recognized based on serological characteristics (Stewart & Stewart 2021). The SFG includes, among others, R. montanensis , R. massiliae , R. japonica , R. peacockii , R. rickettsii , R. conorii , R. sibirica , and R. parkeri (Weinert et al. 2009, Davison et al. 2022). All members are associated with ticks (Ixodida), and includes the first recognized and one of more lethal pathogens among Rickettsia , R. rickettsii (Parola et al. 2013). On other hand, the pathogenicity of R. parkeri was not recognized until 60 years after its description, being responsible for milder cases of Spotted fever (Parola et al. 2005). The group includes an even more interesting case, R. peacockii . The species is perpetuated transstadially and transovarially in ticks, and the presence of R. peacockii within ovaries interfering in the ability of R. rickettsii to infect the ovarian tissues and to be transovarially transmitted to progeny (Parola et al. 2005). However, R. peacockii does not invade the salivary glands, precluding its transmission during biting (Niebylski et al. 1997), and possesses an ompA gene that contains three premature stop codons, hence is unable to produce a functional ompA protein, crucial for pathogenicity (Baldridge et al. 2004). The Typhus group comprises two named species, Rickettsia typhi and Rickettsia prowazekii, primarily associated with fleas (Siphonaptera) and lice (Phthiraptera) (Parola et al. 2005). The Transitional group contains named species such as R. felis , R. australis , and R. akari , and lineages occurring associated to Ixodida, Diptera, Hemiptera, Hymenoptera, Psocoptera, and Siphonaptera (Weinert 2015). Rickettsia felis is vectored by cat fleas to cats and humans, to which it has been associated with fevers and spots, while a closely related strain is required for oogenesis in the booklouse and is thought to cause parthenogenesis (Thepparit et al. 2011). Rickettsia australis causes Queensland tick typhus, transmitted to humans through the bites of the hard ticks Ixodes holocyclus or Ixodes tasmani . It has mostly mild symptoms – fever, headache, and myalgia followed by the development of a maculopapular or vesicular rash, an inoculation eschar (65% of cases), and lymphadenopathy (71%) (McBride et al. 2007). Finally, the closely related R. akari , the causative agent of rickettsialpox, has as invertebrate host mites belonging to the order Mesostigmata, specifically Liponyssoides sanguineus , despite it is suspected of being transmitted by ticks too (e.g. Iweriebor et al. 2017). Mites belonging to the epifamily Trombiculoidae (=Trombiculidae sensu Kudryashova 1998) include species with larvae parasite of vertebrates, with a few exceptions (with invertebrate hosts) that are likely reversals (Costa et al. 2024, Felska et al. 2018, Vasconcelos et al. 2017). They differ from all other ectoparasites mites for not being hematophagous or tissue boring, but feeding upon host tissues through the stylostome, a straw-like structure by interaction of mite saliva and host tissue (Shatrov 2009). Furthermore harboring Orientia tsutsugamushi and other members of genus Orientia (Martínez-Valdebenito et al. 2024), bacteria belonging to the genus Rickettsia are frequent in Trombiculoidae mites, being detected in chiggers feeding on small mammals and birds in Brazil, USA, Slovakia, and in many places across Asia (Choi et al. 2007, Tsui et al. 2007, Huang et al. 2017, Miťková et al. 2015, Jacinavicius et al. 2019, Linsuwanon et al. 2021, Kuo et al. 2022, Ponnusamy et al. 2022, Bassini-Silva et al. 2018, 2023). Most of them were grouped in the Transitional group, implying that these bacteria in Trombiculoidae may be arthropod symbionts, vertebrate pathogens or both. Some molecularly detected lineages, such as ‘ Rickettsia sp. clone MB74-1’ and ‘ Rickettsia sp. TwKM02’ and related bacteria occurring in South Korea seems to be specific to chiggers and are seldom found in the rodent host or coinfesting ectoparasites (Choi et al. 2007, Kuo et al. 2022, Tsui et al. 2007). Acarological taxonomic collections in a One Health context According to Stärk and colleagues (2015): “One Health surveillance describes the systematic collection, validation, analysis, interpretation of data and dissemination of information collected on humans, animals and the environment to inform decisions for more effective, evidence- and system-based health interventions”. It is clear from its very definition that many aspects of this approach may interplay with the objectives of taxonomic collections, which harbors relevant samples from natural or synanthropic communities, including potential vectors and hosts of symbionts. The Acarological collection at “Centro de Coleções Taxonômicas da UFMG” – Acronym UFMG-AC (Zhang, 2018) – is a repository started in 2012, totaling, to October/2023, 16,361 specimens, mounted on slides or in alcohol (Pepato 2024). These mites were collected in all regions of Brazil and several countries in the Americas, Europe, Asia and Oceania. The collection stands out for its sampling of mites from caves and coastal habitats, comprising approximately 150 different families, providing a solid basis for taxonomic and ecological studies, and type material (Pepato 2024). The cave dwelling mites include a substantial sample of members of Epifamily Trombiculoidae and Order Ixodida, mainly from Minas Gerais State. The objective of this study is applying the One Health approach to these individuals, by the molecular detection of the Rickettsia diversity hosted by these groups, making the information on their occurrence available as far as possible. Material and methods Specimen sampling and collection Essayed individuals were deposited in the Acarological collection at “Centro de Coleções Taxonômicas da UFMG”, Acronym UFMG-AC (Zhang, 2018). Specimens were variably preserved in alcohol 70-100%. As the original sampling context of most mites was not that of employing molecular techniques, but an inventory of invertebrate communities, most mites were stored for days to months in 70% ethanol at room temperature before having the fixative replaced by absolute alcohol and kept at -20 o C upon arrival at UFMG-AC. Up to October/2023, the UFMG-AC collection had 580 Trombiculoidae occurrences, including larval and post larval individuals, most of the former found parasitizing bats, often caught inside caves or near to their entrance, and 144 Argasidae and 138 Ixodidae, mostly caught off host, on caves soil. DNA extraction and morphological identification Genomic DNA was extracted from single specimens using QIAamp® DNA Micro kit (Qiagen) for larvae and the Wizard® Genomic DNA Purification Kit (Promega) for nymph and adult stages, following the manufacturer’s protocol, except by using two steps of the final elution in the former, leading to a final volume of 50 μl and by removing and preserving the specimen’s exoskeleton after the protein digestion step. Most exoskeletons recovered after DNA extraction were mounted on permanent microscope slides using Hoyer’s medium, following the protocol described by Walter and Krantz (2009), and kept in an oven at 50–55 °C for approximately one week or until completely dry. Slides were examined under a Leica DM2500 light microscope equipped with an ICC50 W digital camera and phase contrast optics. In the case of post larval ticks, specimens we a re preserved in 70% ethanol and carefully examined under Leica M125 stereomicroscopes, as described by Muñoz-Leal et al. (2017). Ticks and mites were identified using morphological keys and original species descriptions, for post larval stages of hard ticks (Ixodidae) following Barros-Battesti et al. (2006) and Martins et al. (2010) and soft tick larvae and post larval (Argasidae) by Dantas-Torres et al. (2019). Since the generic classification of Argasidae remains under debate without consensus (Mans et al. 2021, Kneubehl et al. 2022), in the present study we adopted the classification of Guglielmone et al. (2003) sensu Hoogstraal (1985) for the Argasidae genera, until this taxonomic problem is solved. Gomes-Almeida et al. (2023) included most of the chiggers reported in this study and was consulted for the identification of two species of the genus Whartonia . PCR amplification of mitochondrial host genes and Rickettsia screening, sequencing and chromatogram checking Conventional PCR and nested PCR assays were initially performed to amplify two mitochondrial gene fragments: (i) For ticks, a ~410 bp fragment of the mitochondrial large ribosomal subunit (16S) gene was amplified using primers proposed by Mangold et al. (1998): 16S + 1 (5'-CTG CTC AAT GAT TTT TTA AAT TGC TGT GG-3') and 16S–1 (5' -CCG GTC TGA ACT CAG ATC AAG T-3'). The cycling conditions used were the same as in Gomes-Almeida and Pepato (2021), consisting of an initial denaturing step at 94 °C for 2 min, followed by 35 cycles of denaturing at 94 °C for 30 s, annealing for 35 s and extension at 72 °C for 45 s. The annealing temperature increased by 0.3 °C every cycle during the first seven cycles (from 47 °C to 48.8 °C), followed by 28 cycles using an annealing temperature of 50 °C. A final extension step at 72 °C for 5 min. (ii) For mites and ticks, a ~1200 bp fragment of mitochondrial cytochrome c oxidase subunit I (cox-1) gene was amplified using a nested PCR, following the primers proposed by Klimov et al. (2018). The first PCR step using primers COX1 16F (5'-TGA NTW TTT TCH ACW AAY CAY AA-3') and COX1 1324R (5'-CDG WRT AHC GDC GDG GTA T-3'), followed by a second PCR step with primers including a M13 tail: COX1 25Fshort (5'-CHA CWA AYC AYA ARR AYA-3') and COX1 1282R (5'-CCW VYT ARD CCT ARR AAR TGT TG-3'). The cycling conditions used followed Gomes-Almeida et al. (2023, 2024) that included an initial denaturation at 94 °C for 2 mins, followed by 35 cycles of 94 °C for 30 s, 40 °C for 30 s, and 72 °C for 2 min, with a final extension at 72 °C for 7 min. (iii) We used DNA samples that have been extracted and tested for the COI and 28S genes in previous studies, as described by Gomes-Almeida et al. (2023). Samples were screened for Rickettsia spp. using conventional PCR targeting a ~350 bp fragment of the citrate synthase ( gltA ) gene of all members of the genus Rickettsia , following the primers and protocols described by Labruna et al. (2004): CS 78 (5'-GCAAGTATCGGTGAGGATGTAAT-3') and CS 323 (5'-GCTTCCTTAAAATTCAATAAATCAGGAT-3'). The thermal cycling conditions included an initial denaturation step of 2 min at 95 °C, followed by 40 cycles comprising denaturation at 95 °C for 15 s, annealing at 48 °C for 30 s, and extension at 72 °C for 30 s. A final extension step was performed at 72 °C for 7 min. Extractions from which gltA sequences could be obtained were used in attempts to obtain additional loci, in order to further characterize the lineages detected. Following Weinert et al. (2009), we attempted to obtain sequences from genes the rickettsial 16SrDNA (Small Subunit Ribosomal DNA), atpA (ATP synthase subunit alpha), and coxA (cytochrome C oxidase subunit I) were performed by using the same primers pairs employed by Weinert et al. (2009). All PCR reactions were performed in 20 μl of final volume with Platinum Taq DNA Polymerase (Invitrogen) in a Mastercycler nexus (Eppendorf) thermocycler. The master mix for initial PCR contained 2.0 μl of PCR buffer (1X), 1.4 μl MgCl 2 (50 mM), 1.4 μL of dNTP (10 mM each) and 0.8 μl of each oligonucleotide primer (10uM), to which 1–3 μl of genomic DNA or alternatively 0.5 μl of parent PCR products for nested reactions was added. All reactions included a negative control (ultrapure water) and, for Rickettsi a spp. screening, a positive control ( Rickettsia rickettsii ). All PCR products found positive in 1% agarose gel electrophoresis were purified using the Ampure® (Agencourt) kit and sequenced using a 3730 DNA Analyzer and BigDyeTM Terminator v3.1 (Applied Biosystems) according to the manufacturer's protocol. For nested PCR (COI), sequencing was performed using the M13 forward (5'TGTAAAACGACGGCCAGT-3’) and reverse (5'-CAGGAAACAGCTATGACC-3') primers. For all other markers, the primers employed in sequencing were the same as for PCRs. Forward and reverse chromatograms were checked, edited and assembled into contigs using software ChromasPro 1.41 (Technelysium Pty Ltd). All sequences generated for this study were compared with available mites or bacteria sequences using the NCBI BLAST feature (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) (Altschul et al. 1997) and deposited in the GenBank database. Host endogenous genome and gltA sequences are summarized in the supplementary Tables S1-S2. Alignment and phylogenetic inference The mitochondrial 16S sequences were employed in an integrative approach to assist soft tick identification. They were aligned in MAFFT (Katoh et al. 2002) using the E-INS-i strategy (Katoh et al. 2005) implemented in the MAFFT server at https://mafft.cbrc.jp/alignment/server/. The Scoring matrix for nucleotide sequences employed was the 200 PAM/K=2. The sequences obtained here were combined to sequences from all Argasidae species recorded from Brazil according to Dantas-Torres et al. (2019), and an undescribed putative new species reported in Jorge et al. (2022), referred by the authors as Ornithodoros sp. Ubajara. Alignment features and distances were calculated using MEGA X (Kumar et al. 2018). The model for sequence evolution was chosen using the Bayesian Information Criterion, calculated in ModelFinder (Kalyaanamoorthy et al, 2017) implemented in IQ-TREE 3 (Wong et al. 2025). IQ-TREE 3 also was employed for inferring the maximum likelihood, and Ultrafast Bootstrap and the SHlike approximate likelihood ratio test (SH-aLRT), both calculated with 1000 replicates. The 16S tree was employed as input for PTP Maximum Likelihood species delimitation analyses (Zhang et al. 2013), at the bPTP webserver (https://species.h-its.org/ptp/). Sequences from Trombiculoidae are summarized in Supplementary Table S2. The sequences from gltA gene were combined in an alignment with sequences from GenBank in order to identify the affinities of the Rickettsia detected, as summarized in Supplementary Table S3: (A) representatives of the main lineages of Rickettsia , including basal groups Rhyzobius and Meloidae; (B) Sequences from Rickettsia detected in chiggers, as reviewed by Chaisiri et al 2023; (C) Matches with sequences deposited in GenBank using the online implemented BLATn, during the preliminary sequence processing described above. After preliminary automatic alignment with the aid of the BioEdit 7.2.1 software (Hall, 1999) sequences had their extremities trimmed and checked for the maintenance of the reading frame in the program MEGA X (Kumar et al. 2018). Multiloci analyses from rickettsial markers were performed for those terminals for which at least two markers could be obtained. It was built on Weinert et al. (2009) study. GenBank accessions for retrieved and newly obtained sequences are summarized in Supplementary Table S4. Bayesian phylogenetic inference was performed in MrBayes 3.2.7 (Ronquist et al. 2012) and maximum likelihood inference was performed in IQ-TREE 3 (Wong et al. 2025). Results Detection attempts were performed in a total of 363 specimens, comprising 158 ticks (Ixodida: Argasidae and Ixodidae) and 205 mites (Trombiculoidea), including larvae, nymphs and adults. These specimens were collected in localities in the Brazilian states of Minas Gerais, Pará, and São Paulo, most of them (327) from caves or cave associated fauna. Among them, only those that led to sequences were depicted in Fig. 1, and listed in Supplementary Table S1 and S2. A total of 38 soft ticks belonging to the genus Ornithodoros , 18 hard ticks belonging to the genus Amblyomma, and 52 chiggers belonging to genus Whartonia led to sequences. Arthropod hosts identification Sequences of mitochondrial 16S and COI were employed for molecular species delimitation in ticks (Fig. 2). Concerning Argasidade, all belonging to the genus Ornithodoros , a total of 25 specimens were sequenced for 16S, 20 for COI, and seven were sequenced for both markers. The aligned 16S new sequences are 424 – 418 bps long, counting 135 variable positions out 432 positions, among them 99 parsimony informative. The COI nucleotide sequences counted 330 variable positions out of 973 bps, among them 320 parsimony informative. Translated into aminoacids, it led to 43 out 324 positions, with 39 parsimony informative positions. Among Argasidae, the species Ornithodoros cavernicolous Dantas-Torres, Venzal & Labruna, 2012 and Ornithodoros fonsecai (Labruna & Venzal, 2009) could be identified both morphologically and molecularly beyond any reasonable doubt. The presence of cheeks and legs with micromammillate cuticle points these ticks as members of the mostly bat-associated soft ticks of the subgenus Alectorobiu s (Dantas-Torres et al. 2012; Labruna & Venzal, 2009). Both species were originally described from specimens obtained in caves or associated with bats, exactly as in our case. Similar to Oliveira et al (2024), 16S sequences from O. fonsecai grouped into two haplogroups, those obtained from Itambé do Mato Dentro, Lagoa Santa and Pedro Leopoldo Municipalities grouped with sequences from Mato Grosso do Sul state – Cerrado biome, type locality – and Rio de Janeiro – Atlantic Forest biome, whereas the specimens from the Caatinga – Pernambuco and Ceará states – and Cerrado biomes – Mato Grosso state – grouped with sequences of ticks from Luislândia municipality (Fig. 2A). Despite all O. fonsecai sequenced came from the same state, the later municipality is in a dry region, distant ~ 560 kms from the other localities, hence the division between an haplogroup inhabiting drier environments and other to more humidity seems to stand. The highest p-distance observed among terminals from the two haplogroups is 0.043, observed between the sequence from Mato Grosso do Sul (GQ120967) and sequences from Ceará state (KX781699, OK256967). Among sequences within both haplogroups was observed an average p-distance = 0.006. Molecular data also clearly grouped ticks collected in caves from Minas Gerais with the undescribed “ Ornithodoros sp. Ubajara”. Jorge et al. (2022), referred this way after the Ubajara municipality, Ceará State, Northeast Brazil, from caves in the semi arid Caatinga biome, their 16S sequences have an average distance p = 0.005, and PTP analyses clearly recovers them as co-specific. Six 16S sequences grouped with Ornithodoros saraivai Muñoz-Leal & Labruna, 2017 (Fig. 1A), but neither PTP nor the pairwise distances p= 0.079-0.050, support unambiguously assigning the specimens to such species. A least 16S sequence from an individual was recovered as sister to clade including Ornithodoros yumatensis Cooley & Kohls, 1941 (GenBank: KX668415) and Ornithodoros . brodyi Matheson, 1935 (GenBank: KY454706), but with even higher pairwise distances (pairwise p = 0.121 and p = 0.138) are suggestive of a new species. These ticks should be further investigated taxonomically. Most individuals sequenced for COI could be unambiguously assigned to O. cavernicolous , O. fonsecai , and Ornithodoros sp. Ubajara thanks terminals sequenced for both 16S and COI (Fig. 2C). In contrast to the 16S marker, there was no available sequence of the COI marker for the type specimens of O. saraivai . Even so, of the specimens morphologically identified as O. saraivai and confirmed by 16S sequences (Fig. 2A), at least three of them, all from Conceição do Mato Dentro, generated COI sequences that formed a distinct clade (Fig. 2C), in which one of the sequences showed a considerable p-distance (= 0.136) and without PTP support for being cospecific. Two ticks from Janaúba municipality, Minas Gerais state, morphologically identified as Ornithodoros sp., was recovered in a clade along Ornithodoros cerradoensis Muñoz-Leal, Martins & Labruna, 2020 (NC_067907, average p-distance = 0.102). All ixodid belonging to Amblyomma could be identified to the species level, both through molecules and morphology, comprising seven species: Amblyomma brasiliense Aragão, 1908 (N=3), Ambyomma cajennense (Fabricius, 1787) (N=1), Amblyomma dubitatum Neumann, 1899 (N=1), Amblyomma nodosum Neumann, 1899 (N=1), Amblyomma ovale Kock, 1844 (N=3), Amblyomma pacae Aragão, 1911 (N=1), and Amblyomma sculptum Berlese, 1888 (N= 8). The same for Whartonia , with most host sequences already reported in previous studies, with 44 individuals identified as W hartonia pachywhartoni Vercammen-Grandjean, 1966 and eight as W hartonia nudosetosa (Wharton, 1938). Rickettsia detection A total of 36 samples tested positive for Rickettsia spp. from ticks (Ixodida: Amblyomma dubitatum and Argasidae: Ornithodoros spp.) and mites (Trombiculoidea: Whartonia pachywhartoni ), all collected from cave soil or bats (Fig. 1). From these samples, 21 gltA gene partial sequences were successfully obtained (Supplementary Table S1-S2). The gltA alignment, after adding GenBank terminals (Supplementary Table S3) comprised 77 sequences, 379 columns, 165 variable, and 125 parsimony-informative sites. The best-fit model was the K3Pu+F+G4 (Kimura 3-parameter model with unequal base frequencies and a gamma distribution) chosen according to BIC. Despite being only 379 bp long, our more comprehensive dataset, the gltA gene tree, led to a topology that roughly corresponds to the trees obtained by whole genome or multi loci analyses, as follows: (Rhyzobius, (Meloidae, (Belli, ((Adalia, Canadensis), (Typhus, Transitional, Spotted Fever)))). The new sequences were recovered in the gene tree in three phylogroups, the transitional (TRG), spotted fever (SFG) andbellii groups (Fig. 3). Based on their DNA sequences, we group them in nine strains, as described below and shown in Fig. 3. Strain 1 : The single Rickettsia detected from a hard tick, a female identified as Amblyomma dubitatum (UFMG-AC 170244, GenBank AC1985), collected dwelling on a cave floor, belongs to the bellii group. The Blastn search querying gltA sequences revealed 70 hits with 100% identity with R. belli , detected from Ixodidae (genera Amblyomma , Dermacentor , Ixodes , and Haemaphysalis ) from USA, El Salvador, Costa Rica, Colombia, Argentina and widespread in Brazil, including localities scattered across the country. The family Argasidae harbored nine Rickettsia occurrences, including sequences recovered in the three phylogroups (TRG, SFG, andbellii groups). Strain 2 : In the belli Group, the sequence obtained from the Ornithodoros sp. 2 ( O. yumatensis + O. brodyi clade) female (UFMG AC 180276), collected near to the entrance of an iron ore cave in Minas Gerais state, a BLASTn search recovered a 98.94% identity with the sequence identified as Rickettsia sp. ALSK (GenBank KX254162), obtained from the flea Xenopsylla minax (Siphonaptera,Pulicidae) from the Alataw Pass, Xinjiang, China according to the GenBank register. Strain 3 : The sequence obtained from a female of O. fonsecai (UFMG AC 172234), from the twilight zone floor of a limestone cave in the southern portion of São Paulo state, was recovered in the Belli Group too. It has an identity of 99.47% with Rickettsia sp. RDa420 (GenBank AF497584) detected in Dermacentor auratus removed from a bear in Thailand (Parola et al. 2003). Strain 4 : A sequence from one female (UFMG-AC 210291, aphotic zone of a limestone cave) appear nested in the TRG (Fig. 3), with an identity of 99.47% with the sequence GenBank MF175748, from a Amblyomma sculptum individual collected in the southern Brazilian state of Paraná, and 98.94% identity with 13 sequences identified as Rickettsia asembonensis , eleven obtained from Ctenocephalides felis associated to dogs, nine in the Brazilian amazonian state of Rondônia and two from Costa Rica, a sequence obtained from C. canis in Kenia, and a sequence from a human patient from Malaysa. Strain 5 : The Rickettsia sequences from three individuals (two O. fonsecai and one O. aff. saraivai ) grouped in a basal clade among TRG. Two of them, obtained in Caves from Minas Gerais, composed strain 5 as follows: one female of the O. saraivai Clade (UFMG-AC 170467, cave soil) and a female identified as O. fonsecai (UFMG-AC 1608677), both from twilight zone of a limestone caves. These sequences are identical and had 60 Blastn hits with sequences identified as R. parkeri , R. sibirica , R. africae and other unidentified bacteria, with 98.67% identity, all Rickettsia belonging to the SFG. Strain 6 : The sequence from a O. fonsecai female (UFMG-AC 171498), from twilight zone of a limestone cave, had 52 hits with 99.20 % identity with Rickettsia identified as R. raoultii , R. conori i, and unidentified bacteria. Strain 7 : Other two sequences were obtained from an Ornithodoros fonsecai (UFMG-AC 171333) and a nymph of O. aff saravai (UFMG-AC 173021) both collected on the cave floor in the twilight zone. They were nested in the SFG and are identical to a sequence identified as ‘ Candidatus Rickettsia wissemanii’ (GenBank LT558852) obtained from O. hasei ticks found on the bat Noctilio albiventris from French Guiana (Tahir et al 2016). All rickettsial sequences obtained from Trombiculoidae were obtained from Whartonia pachywhartoni individuals. Strain 8 : The strain with more sequences (nine terminals) comprise larvae associated to the bats Carollia perspicillata (UFMG AC 210163, 210170), Diphylla ecaudata (UFMG AC 210180), Mimon bennetti (UFMG AC 210101), larvae found dwelling free on a cave floor (UFMG AC 210337, 210339-210341) and a male (UFMG AC 170297). The Blastn hit with maximum similarity was with Rickettsia australis str. Cutlack (CP003338), which belongs to TRG, with 97.61% identity. Strain 9 : The other clade included three sequences, all obtained from W. pachywhartoni males (UFMG AC 170228, 170255, 170291) dwelling on the floor of iron ore caves in the twilight and aphotic zones. Blastn searches recovered a hit with 98.14 % identity with Rhipicephalus sp. tick nymph from Nesokia indica (Rodentia, Muridae) in Pakistan. The following most similar hits (97.87%) are SFG members ( R. raultii , R. conorii , R. montanensis ). However, in the phylogenetic tree, strain 8 sequences were sister to the TG clade composed by R. prowazekii and R. typhi (Fig. 3). Multiloci analyses It was possible to obtain sequences from additional markers for bacteria belonging to all above delimited strain except by the Strain 9. The concatenated alignment, after adding GenBank terminals (Supplementary Table S4) comprised 57 sequences, 2966 positions, 892 variable, and 604 parsimony-informative sites. The best-fit partitioned model, chosen according to BIC, divided the sequences into two partitions, the first with ribosomal 16S sequences and the second merging all protein coding genes. The model TPM3u+R2 (AC=CG, AG=CT, AT=GT and unequal base frequencies) was assigned to the first partition, while de model GTR+F+I+G4 (General time reversible model with unequal rates, unequal base frequencies , a proportion of invariant sites, and a gamma distribution) for the second. The topology of rickettsial groups recovered by the analyses as follows: (Rhyzobius, (Meloidae, (Belli, (Adalia, (Canadensis, ( R. monasensis (Typhus, (Transitional, Spotted Fever)))))))). Note, however, that the Canadensis group was not supported, appearing as a paraphyletic group in the ML tree (Fig.4). Rickettsia belli (Strain 1), the only occurring in an Amblyomma species, could be sequenced for the four markers and grouped with the other two terminals belonging to this species. The Strains 2 and 3, as in the gltA analyses, were recovered in the Belli Group. Whereas the support to the group is absolute, however, their internal relationship received a lower support. The endosymbiont of the Ornithodoros sp. 2 (UFMG AC 180276) Rickettsia , the Strain 2, group along to lacewing and Brachys tessellatus endosymbionts received a support of UFBoot =78, SH-aLRT = 96.2, and PP = 0.65, and the placement of O. fonsecai (UFMG AC 172234) in a clade with Bombyiid bee fly, Acyrthosiphon and Rickettisia associated with Carica papaya and Empoasca papayae received a support of UFBoot = 91, SH-aLRT = 51, and PP = 0.76. The strain 4, from another O. fonsecai specimen (UFMG AC 210291), was recovered in a well-supported clade along R. felis (UFBoot = 96, SH-aLRT =83.9, and PP = 1.00). The Strain 8, associated with nine specimens of W. pachywhasrtoni , was recovered in a clade with R. akari with considerable support (UFBoot = 89, SH-aLRT =75.5, and PP = 0.93). The remaining strains were associated with the SFG, which recovered a support of UFBoot =94, SH-aLRT = 88.1, and PP = 0.99. The strain 5 is the second branch in the SFG tree, whereas strains 7 and 8 grouped in a clade that received a good support (UFBoot = 99, SH-aLRT =99.3, and PP = 1.00), both grouped with ‘ Candidatus R. wissemanii ’ . Discussion Retrieving molecular data from natural history collections Despite that many samples had their DNA degraded due the storage conditions prior its inclusion in the CCT-UFMG Acarological collection, hence precluding amplification and sequencing, this study shows that a great deal of bacterial diversity still may be retrieved from taxonomical collections, integrating geographical information, ectoparasites integrative taxonomy and, in the case of ticks and chiggers, vertebrate sources. The microbiome of insects include not just potential pathogens, but also can have profound effects on their hosts, from negative interactions in the form of pathogenesis and parasitism to highly beneficial associations in the form of nutrient provisioning and immune defense (Short et al. 2018). The same is true for mites and similar to entomological collections the effort in sequencing even historical samples may lead to relevant contributions to the understanding of their microbiomes. Integrative taxonom y of ticks Even though not being its main focus, this study could bring some new occurrences of Ornithodoros species and possible new species. In the case of O. fonsecai it is remarkable that both haplogroups detected in Oliveira et al (2024) could be found in this study, up to now congruent to one haplogroup associated to localities more arid than to the other. A possible direction for future investigation concerning this species is testing if the distribution of those haplogroups corresponds to ongoing local adaptation. The still unnamed species “ Ornithodoros sp Ubajara” recorded by.Jorge et al (2022), proved to be abundant in caves around southern portion of Espinhaço Range (Lagoa Santa and Pedro Leopoldo), including larvae parasitizing the nectivorous bat Glossophaga soricina (Phyllostomidae). Despite the specimens reported herewith came from caves in transitional areas between the Atlantic Forest and Cerrado, apart almost 1800 km from the original records, the p-distances and PTP analyses left no doubts about its identity. The sequences grouped with Ornithodoros saraivai warrants further investigation, since they exhibit considerable divergence and the ticks reported here may constitute a species complex. Finally, some specimens may belong to undescribed species, such as the nymph UFMG AC 180276, collected near the entrance of a cave in the Santa Maria de Itabira municipality, Minas Gerais state. All these novelties related to Argasidae are not unexpected, since many new species were recently described (Dantas-Torres et al. 2019). Rickettsia diversity The phylogenetic diversity found in the Rickettsia sequenced herewith likely imply a similar ecological diversity, and a possible explanation to this diversity lies on its association with bats and caves used as roosts by bats. Among detected Rickettsia spp., only Rickettsia bellii was found associated with an Ixodid species, Amblyomma dubitatum , primarily a capybara ( Hydrochoerus hydrochaeris ) ectoparasite, but already found parasitizing the bat Glossophaga soricina (Phyllostomidae) (Nava et al. 2010). Rickettsia bellii presents distinct lineages for North and South America (Krawczak et al 2018), and the gltA sequence obtained here groups with other South-American bacteria. It is regarded as nonpathogenic for vertebrates, despite experimental studies showing that inoculation of this rickettsia produces eschars in rabbits and guinea pigs (Ogata et al 2006). The other arthropod hosts for Rickettisia spp in this study, the genera Ornithodoros and Whartonia , are primarily or at least putative bat ectoparasites that spend some time dwelling on the cave floor. Hence, most of rickettsial diversity found in this study came from arthropods that have stages on and off bat hosts, being integrated during part of their lives in the ecology of the cave soil, including examples of hyperparasitism by other mites, such as Erythraeidae larvae (e.g. Bassini-Silva et al 2019). For instance, Rickettsia is recorded from blood and tissue samples of bats, including disparate world regions as in South Africa and Swaziland, where a lineage related R. conorii was detected (Dietrich et al. 2016), Romania, where a high prevalence of Rickettsia monacensis was detected in insectivorous bats (Matei et al. 2021), China, where it was detected the presence of several SFG agents(Zhao et al 2020). It is also often detected in bats ectoparasites such as ticks (especially Argasidae), mites, and flies (reviewed in Matei et al. 2021), where the SFG and TRG were detected, the later with sequences related to R. felis . In our study, along occurrences related to bacteria already found in bat ectoparasites, as ‘ Candidatus Rickettsia wissemanii’ found in Ornithodoros hasei from French Guiana, Brazil and Argentina (Tahir et al. 2016, Colombo et al. 2020; Luz et al. 2019), above referred as strain 7 (and also the related strain 6), to R. felis (strain 4) and included in the SFG (strain 5 and likely strain 9), we recorded some lineages that seems to be found for the first time harbored by bat ectoparasites. It is the case for strains 2 and 3, both nested in the Bellii group, and in the multiloci analyses with terminals with phenotypes so diverse as male-killing and plant pathogens (Hurst and Jiggins 2000, Davis et al. 1998). It is also the case for strain 8, found in chiggers. It nests in the TRG group along R. australis and R. akari, both of medical relevance, being the causative agents of the Queensland Tick Typhus and rickettsialpox (Stewart et al 2017, Denison et al 2014). Ornithodoros in caves as a pathogen vector for humans Soft ticks are mostly reported as human parasites by benefiting from the conditions provided by the human houses and synanthropic fauna, including bats (Bermúdez et al. 2021). However, the increasing use of caves as recreational and touristic spots, or its use in economical activities like mining, bring humans in contact to the subterranean fauna, and may make biting of soft ticks more common. Jorge et al (2022) reported biting by O. cavernicolous and O. fonsecai , the later already reported to occur in other studies (Labruna and Venzal 2009; Santiago et al. 2019). As in this article we could find four out nine strains of Rickettsia in this species, warranting the need of a close inspection of its potential role in the appearance of emergent rickettsiosis. Finally, it is noteworthy that research on Rickettsia spp. in ticks has been strongly focused on hard ticks of the Ixodidae family. For example, until 2021, there were 599 records of 31 species of Rickettsia reported from 50 species of Ixodidae ticks in the neotropical region (Estrada-Peña et al. 2021). On the other hand, until the present study, there were only 15 records of Rickettsia in ticks of the Argasidae family in the neotropics, as follows: Rickettsia lusitaniae in Ornithodoros yumatensis and Ornithodoros sp. from Mexico (Sánchez-Montes et al 2016, Hornok et al 2019), Rickettsia bellii in Ornithodoros talaje from Mexico (Guzmán-Cornejo et al. 2022), Rickettsia felis in Ornithodoros puertoricensis from Mexico (Ballados-González et al. 2023), ‘ Candidatus Rickettsia nicoyana’ in Ornithodoros knoxjonesi from Costa Rica (Moreira-Soto et al 2017), Rickettsia sp. in Ornithodoros rietcorreai from Brazil (Muñoz-Leal et al. 2019a), Rickettsia sp. in Ornithodoros cf. mimon from Brazil (Dantas-Torres et al. 2022), Rickettsia sp. strain Itinguçú in Ornithodoros faccinii from Brazil (Peixoto et al. 2021), ' Candidatus Rickettsia wissemanii' in O. hasei from Brazil, French Guiana and Argentina (Tahir et al. 2016, Colombo et al. 2020; Luz et al. 2019; Dornelas Júnior et al. 2025), Rickettsia sp. in Ornithodoros amblus from Peru and Ornithodoros peruvianus from Chile (Duron et al. 2017), and Rickettsia sp. in Ornithodoros sp. from Chile (Muñoz-Leal et al. 2019b). Undoubtedly, the present study shows a diversity of Rickettsia spp. in Argasidae never before reported in a single study, contributing significantly to a better understanding of this important group of tick-borne bacteria, which has been neglected in argasids. Statements and Declarations Acknowledgements This study was supported by funding from FAPEMIG (Call 01/2022- Demanda Universal, process RDP 01535-22) and by the Ministry of Science and Higher Education of the Russian Federation, University of Tyumen, that supported the research project and work plan for the exchange of the doctoral student Nikolay Anisimov during his stay in Brazil in 2023. The authors thank the Program for Technological Development in Tools for Health-PDTISFIOCRUZ, especially to Renata de B. R. Oliveira and Nathalia S. Carvalho, who assisted with sequencing and the company Carste Ciência e Meio Ambiente for depositing the specimens at UFMG AC. Finally, authors thank Danilo Gonçalves Saraiva ( in memoriam ) for donating the DNA extraction positive to Rickettsia rickettisii employed as positive control in our essays. Funding This study was supported by funding from FAPEMIG (Call 01/2022- Demanda Universal , process RDP 01535-22) and by the Ministry of Science and Higher Education of the Russian Federation, University of Tyumen, that supported the research project and work plan for the exchange of the doctoral student Nikolay Anisimov during his stay in Brazil in 2023. Competing Interests The authors have no relevant financial or non-financial interests to disclose Author Contributions Almir R. Pepato, Nikolay V. Anisimov, and Brenda K. Gomes-Almeida contributed to the study conception and design. Material preparation, data collection were performed by Almir R. Pepato, Nikolay V. Anisimov, and Brenda K. Gomes-Almeida, Teofania H. D. Amorim Vidigal, and Jefferson C. Carvalho Farias da Silva. Analyses were performed by Almir R. 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Syst Appl Acarol 23 (12): 2432–2446. https://doi.org/10.11158/saa.23.12.12 Zhao S, Yang M, Liu G, Hornok S, Zhao S, Sang C, Tan W, Wang Y ( 2020) Rickettsiae in the common pipistrelle Pipistrellus pipistrellus (Chiroptera: Vespertilionidae) and the bat soft tick Argas vespertilionis (Ixodida: Argasidae). Parasit Vectors 13(1):10 https://doi.org/10.1186/s13071-020-3885-x Additional Declarations No competing interests reported. Supplementary Files MoleculardetectionofRickettsiasupplementarytables.docx Supplementary material: GenBank accession numbers and collection data. Supplementary Table captions Table S1 Sample data of Ixodida (Argasidae and Ixodidae)tested for the gltA gene, including taxonomic identification, collection locality and host, and voucher numbers of the voucher material housed at UFMG Acarological Collection (UFMG AC). Stage/sex: L, larva; N, Nymph. Host or substrate: CS, Cave soil; A, aphotic; E, entrance; T, twilight; PESNT, Parque Estadual Serra Nova e Talhado; PES, Parque Estadual do Sumidouro. 16S rDNA, Large Subunit Mitochondrial Ribosomal DNA; COI, Mitochondrial Cytochrome C Oxidase subunit I; gltA , Citrate Synthase. Table S2 Sample data of chiggers belonging to the genus Whartonia Ewing, 1944 including taxonomic identification, collection locality and host, and voucher numbers of the voucher material housed at UFMG Acarological Collection (UFMG AC). Stage/sex: L, larva; DN, Deutonymph. Host or substrate: CS, Cave soil; A, aphotic; E, entrance; T, twilight. 28S, Large Subunit Nuclear Ribosomal DNA; COI, Mitochondrial Cytochrome C Oxidase subunit I; gltA , Citrate Synthase. Table S3 GenBank accession numbers for Rickettsia terminals included in phylogenetic analyses of gltA (citrate synthase) gene, along information on their host and the phenotype it causes. Table S4 GenBank accession numbers for Rickettsia sequences employed in the multiloci analyses, grouped according to their phylogenetic or serological groups. Abbreviations: 16SrDNA, Small Subunit Bacterial Ribosomal DNA); gltA, citrate synthase; atpA, ATP synthase subunit alpha; and CoxA, bacterial cytochrome C oxidase subunit I. SupplementaryFigS1.tif Supplementary material: Bayesian analyses Figure S1. Bayesian majority rule consensus tree from trees sampled during the stationary Markov Chain, based on nucleotide sequences of citrate synthase (gltA) of Rickettsia spp. Values associated with branches are Posterior Probabilities. SupplementaryFigS2.tif Supplementary material: Bayesian analyses Figure S2. Bayesian majority rule consensus tree from trees sampled during the stationary Markov Chain, based on nucleotide sequences of citrate synthase (gltA), 16SrDNA (Small Subunit Ribosomal DNA), atpA (ATP synthase subunit alpha), and coxA (cytochrome C oxidase subunit I) of Rickettsia spp. Values associated with branches are Posterior Probabilities. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8190507","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":552105911,"identity":"dc26a76d-04be-4633-85fb-b4ee15fcb818","order_by":0,"name":"Almir Rogério Pepato","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBADORBx4AHxGhIYjMFaEkjRktgAoYkA/LObjz34+cMmfX7Y4YdAW+zkdBsIaJG4cyzdsCchLXfj7TQDoJZkY7MDhKy5kWMmwZNwOHfj7ASQlgOJ2whpkb+R/03yT8LhdMPZ6R+I02JwI4dNGmhLgrx0DpG2GN45Zm4sk5ZmuEE6p+BAggERfpG73fzs4RsbG3n52embP3yosJMj7H0JBjaIC8EqDQgpR9Yi30CM6lEwCkbBKBiRAAA0n0d6axoZdwAAAABJRU5ErkJggg==","orcid":"","institution":"Universidade Federal de Minas Gerais","correspondingAuthor":true,"prefix":"","firstName":"Almir","middleName":"Rogério","lastName":"Pepato","suffix":""},{"id":552105912,"identity":"110aa33f-655d-4635-ab5a-0de87862943c","order_by":1,"name":"Nikolay V. 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13:04:29","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":220153,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8190507/v1/d7420d511b5f9317a0e387c4.html"},{"id":97248710,"identity":"82653dea-542b-4491-a14e-f2f1719285ed","added_by":"auto","created_at":"2025-12-02 13:06:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6977777,"visible":true,"origin":"","legend":"\u003cp\u003eMap depicting sampling localities for tested mites with negative results for \u003cem\u003eRickettsia\u003c/em\u003e(blue), positive in electrophoresis gel but that failed sequencing (yellow) and successfully sequenced individuals (red). A– All sampling sites across Brazil; B– Detailed views of Minas Gerais, highlighting overlapping sampling points; C– Detailed views of the southern Espinhaço Range (Quadrilátero Ferrífero region); D– Detailed views of the central Espinhaço Range. Squares: Argasidae; stars: Ixodidae; and circles: Leeuwenhoekiidae. Espinhaço Range is shaded in gray.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8190507/v1/5eb0a647cd345a48d5b4bb39.png"},{"id":97162862,"identity":"fffc8a95-3b88-460c-a1d2-34cf123ea9e6","added_by":"auto","created_at":"2025-12-01 13:03:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2340849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA-\u003c/strong\u003e Maximum likelihood phylogenetic tree including mitochondrial 16S rDNA sequences from Brazilian \u003cem\u003eOrnithodoros\u003c/em\u003e(Argasidae) species along newly obtained sequences; \u003cstrong\u003eB-\u003c/strong\u003e Maximum likelihood phylogenetic tree including mitochondrial 16S rDNA sequences from \u003cem\u003eAmblyomma\u003c/em\u003e (Ixodidae) newly obtained sequences along co-specific sequences available in GenBank; \u003cstrong\u003eC\u003c/strong\u003e- Maximum likelihood phylogenetic tree including mitochondrial COI sequences of all newly sequenced Argasidae and Ixodidae, plus the sequences available in GenBank with close hits in BLASTn. Values associated with branches are SHlike approximate likelihood ratio test and UltraFast Bootstrap (SH-aLRT), respectively, both calculated with 1000 replicates. The newly sequenced terminals are indicated in blue for those negative for rickettsial \u003cem\u003egltA\u003c/em\u003e, yellow for positive amplification but failed sequencing, and red for \u003cem\u003eRickettsia\u003c/em\u003esuccessfully sequenced. *, indicates terminals sequenced both to 16S and COI.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8190507/v1/c62ec9073e9fff795d7ac218.png"},{"id":97162864,"identity":"bd5a9c81-0f8b-437c-915a-8eaae8b1ae89","added_by":"auto","created_at":"2025-12-01 13:03:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1757163,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree based on nucleotide sequences of citrate synthase (\u003cem\u003egltA\u003c/em\u003e) of \u003cem\u003eRickettsia \u003c/em\u003espp. Main phylogenetic and serological groups are shaded. Values associated with branches are SHlike approximate likelihood ratio test and UltraFast Bootstrap (SH-aLRT), respectively, both calculated with 1000 replicates. Newly sequenced terminals are in red as well the strains as referred in the main text.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8190507/v1/acd6c57d01b10fff523869b2.png"},{"id":97162867,"identity":"26dc6834-1a93-4f1a-ae7a-56ff4a186ef6","added_by":"auto","created_at":"2025-12-01 13:03:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1918127,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood phylogenetic tree based on nucleotide sequences based on concatenated alignment of Rickettsial genes 16S, ATPase, Cytochrome c Oxidase and Citrate Synthase. Main phylogenetic and serological groups are shaded. Values associated with branches are SHlike approximate likelihood ratio test and UltraFast Bootstrap (SH-aLRT), respectively, both calculated with 1000 replicates, and Posterior Probabilities calculated in the Bayesian analysis. The posterior probability is not indicated when = 100%. Newly sequenced terminals are in red as well the strains as referred in the main text.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8190507/v1/6a8c44771d80d84803a56241.png"},{"id":97664492,"identity":"3f47227d-1eba-482a-97fa-97918585a3b8","added_by":"auto","created_at":"2025-12-08 09:05:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12891482,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8190507/v1/6ea6e85d-3f6d-499b-a6ef-bdbc9c0c0bda.pdf"},{"id":97162885,"identity":"d266cbc6-07b4-4d60-a9b5-a3e3e540de19","added_by":"auto","created_at":"2025-12-01 13:03:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5691039,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary material: \u003c/strong\u003eGenBank accession numbers and collection data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table captions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1 \u003c/strong\u003eSample data of Ixodida (Argasidae and Ixodidae)tested for the \u003cem\u003egltA\u003c/em\u003egene, including taxonomic identification, collection locality and host, and voucher numbers of the voucher material housed at UFMG Acarological Collection (UFMG AC). Stage/sex: L, larva; N, Nymph. Host or substrate: CS, Cave soil; A, aphotic; E, entrance; T, twilight;\u003cstrong\u003e \u003c/strong\u003ePESNT,\u003cstrong\u003e \u003c/strong\u003eParque Estadual Serra Nova e Talhado; PES, Parque Estadual do Sumidouro. 16S rDNA, Large Subunit Mitochondrial Ribosomal DNA; COI, Mitochondrial Cytochrome C Oxidase subunit I; \u003cem\u003egltA\u003c/em\u003e, Citrate Synthase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2\u003c/strong\u003e Sample data of chiggers belonging to the genus \u003cem\u003eWhartonia \u003c/em\u003eEwing, 1944 including taxonomic identification, collection locality and host, and voucher numbers of the voucher material housed at UFMG Acarological Collection (UFMG AC). Stage/sex: L, larva; DN, Deutonymph. Host or substrate: CS, Cave soil; A, aphotic; E, entrance; T, twilight. 28S, Large Subunit Nuclear Ribosomal DNA; COI, Mitochondrial Cytochrome C Oxidase subunit I; \u003cem\u003egltA\u003c/em\u003e, Citrate Synthase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S3 \u003c/strong\u003eGenBank accession numbers for \u003cem\u003eRickettsia\u003c/em\u003eterminals included in phylogenetic analyses of gltA (citrate synthase) gene, along information on their host and the phenotype it causes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S4 \u003c/strong\u003eGenBank accession numbers for \u003cem\u003eRickettsia\u003c/em\u003e sequences employed in the multiloci analyses, grouped according to their phylogenetic or serological groups. Abbreviations: 16SrDNA, Small Subunit Bacterial Ribosomal DNA); gltA, citrate synthase; atpA, ATP synthase subunit alpha; and CoxA, bacterial cytochrome C oxidase subunit I.\u003c/p\u003e","description":"","filename":"MoleculardetectionofRickettsiasupplementarytables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8190507/v1/2ec02f1a7d72eaf0f0874c0f.docx"},{"id":97249091,"identity":"37241860-b245-4a10-b749-2a6e326224de","added_by":"auto","created_at":"2025-12-02 13:10:20","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3876320,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary material: \u003c/strong\u003eBayesian analyses\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S1\u003c/strong\u003e. Bayesian majority rule consensus tree from trees sampled during the stationary Markov Chain, based on nucleotide sequences of citrate synthase (gltA) of \u003cem\u003eRickettsia\u003c/em\u003e spp. Values associated with branches are Posterior Probabilities.\u003c/p\u003e","description":"","filename":"SupplementaryFigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8190507/v1/d3e38b10ce5e2231d7b10339.tif"},{"id":97162868,"identity":"6f543b36-49c5-4d9e-8951-244b04a685d2","added_by":"auto","created_at":"2025-12-01 13:03:57","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3641852,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary material: \u003c/strong\u003eBayesian analyses\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S2\u003c/strong\u003e. Bayesian majority rule consensus tree from trees sampled during the stationary Markov Chain, based on nucleotide sequences of citrate synthase (gltA), 16SrDNA (Small Subunit Ribosomal DNA), atpA (ATP synthase subunit alpha), and coxA (cytochrome C oxidase subunit I) of \u003cem\u003eRickettsia\u003c/em\u003e spp. Values associated with branches are Posterior Probabilities.\u003c/p\u003e","description":"","filename":"SupplementaryFigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8190507/v1/180b76cb78488d1236ff669b.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular detection of Rickettsia spp. in ticks (Ixodida: Argasidae and Ixodidae) and mites (Trombiculoidae) from a brazilian taxonomic collection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Alphaproteobacteria genus \u003cem\u003eRickettsia\u003c/em\u003e belongs to the diversified order Rickettsiales, which includes many intracellular obligate symbionts (Dumler and Walker 2005), a condition apparently convergently acquired (Castelli et al. 2024). The evolution of the ability to invade and hijack eukaryotic cells along the trend toward losing pathways for aminoacid and nucleotide synthesis make them unable to survive extracellularly, turning a group difficult to laboratory manipulation and the use of traditional techniques for bacterial study (McGinn and Lamason 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn other hand, since some species\u0026nbsp;of \u003cem\u003eRickettsia\u003c/em\u003e cause life-threatening diseases such as the Rocky Mountain spotted fever and typhus, the genus has attracted considerable attention, particularly in medicine (Raoult and Roux 1997; Parola et al. 2005), prompting molecular studies on their infection mechanisms (Uchiyama et al. 2006, Ge and Rikihisa 2011).The genus \u003cem\u003eRickettsia\u003c/em\u003e encompasses a broad diversity of hosts and infection mechanisms, extending beyond its potential threat to human and other vertebrates (Perlman et al. 2006; Weinert et al. 2009; McGinn and Lamason 2021), with the transmission to humans and animals, mainly through arthropod bites such as mites, ticks and insect hosts, with a predominance in ticks (Parola et al. 2013, El Karkouri et al. 2022). Moreover, their close relationship to mitochondria makes them key models in evolutionary biology (Fitzpatrick et al. 2006, El Karkouri et al. 2022).\u003c/p\u003e\n\u003cp\u003eOverall, using multiloci and phylogenomic analysis, the genera could be divided in a few major phylogroups: Spotted Fever group (SFG), the Transitional group (TRG) the Typhus group (TG), the Canadensis group (CG), and the Bellii group (BG) as well some less diversified lineages such as Scapularis, Adalia, Helvetica, \u0026nbsp; Meloidiae and Rhyzobius. These lineages were recovered in multiloci and genomic scale phylogenetic analyses in reasonably congruent topologies, which branching order will be used to order an overview on the genus diversity below (Murray et al. 2016, Perlman et al. 2006, Weinert et al. 2009, El Karkouri et al. 2022, Davison et al. 2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFormer groups Torix and Megaira are recently reputed as separate genera (Davison et al. 2022). They have the largest range of hosts, from microeukaryotes to arthropods. Remaining \u003cem\u003eRickettsia\u003c/em\u003e are arthropod symbionts, and comprise the arthropod borne pathogens. Infections of basal \u003cem\u003eRickettsia\u003c/em\u003e lead to a diversity of phenotypes including male-killing (Hurst and Jiggins 2000), parthenogenesis induction (Stouthamer et al. 2001), mutualists required for oogenesis (Perotti et al. 2006), facultative mutualism (Jaenike 2012), and other mechanisms of sex-ratio distortion (Takahashi et al. 1997, Weinert et al. 2007, Himler et al. 2011)\u003c/p\u003e\n\u003cp\u003eThe basal groups Rhyzobius and Meloidae are both named after beetles, even though each also comprise Dipteran as hosts. Despite fragmentary knowledge on both lineages, populations of \u003cem\u003eRhyzobius litura\u003c/em\u003e have female-biased sex-ratios, suggesting sex-ratio distortion (Weinert et al. 2007).\u003c/p\u003e\n\u003cp\u003eThe following branch from \u003cem\u003eRickettsia\u003c/em\u003e tree is the Bellii group, all associated with arthropods, comprising hexapoda (Diptera, Coleoptera, Hemiptera, Hymenoptera, Lepidoptera and Neuroptera), ticks (Ixodidae and Argasidae), and mites (Mesostigmata). The Bellii group is associated with a wide range of adaptive phenotypes, including parthenogenesis induction, male-killing,\u0026nbsp;uncharacterised\u0026nbsp;sex-ratio distortion, requirement for oogenesis and facultative mutualism, and possible vertebrate pathogenicity \u0026nbsp;(Weinert 2015). This group includes the single \u003cem\u003eRickettsia\u003c/em\u003e that acts as a plant pathogen with potential horizontal transmission through a plant host (Davis et al. 1998).\u003c/p\u003e\n\u003cp\u003eThe Adalia group is named after ladybird beetles but also includes Dipteran and Collembola hosts (Weinert et al. 2009), the occurrence of male-killing species in the Adalia and Bellii groups is probably homoplastic (Weinert 2015). The Canadensis group, includes bacteria with hard tick hosts (Ixodidae), and the beetle \u003cem\u003eCoccotrypes dactyliperda\u003c/em\u003e, which is sterile when cured of\u003cem\u003e\u0026nbsp;Rickettsia\u0026nbsp;\u003c/em\u003eand \u003cem\u003eWolbachia\u003c/em\u003e (Zchori-Fein et al. 2006), but if the \u003cem\u003eRickettsia\u003c/em\u003e is responsible for this phenomena, it will be homoplastic with representatives of the Torix and Transitional groups (Weinert 2015).\u003c/p\u003e\n\u003cp\u003eThe following groups have their internal relationships with low support and incongruency among datasets, despite being included in a clade with high support (Murray et al. 2016, Perlman et al. 2006, Weinert et al. 2009, El Karkouri et al. 2022, Davison et al. 2022).\u003c/p\u003e\n\u003cp\u003eHistorically and in current clinical practice, the SFG and TG groups were recognized based on serological characteristics (Stewart \u0026amp; Stewart 2021). The SFG includes, among others, \u003cem\u003eR. montanensis\u003c/em\u003e, \u0026nbsp;\u003cem\u003eR. massiliae\u003c/em\u003e, \u003cem\u003eR. japonica\u003c/em\u003e, \u003cem\u003eR. peacockii\u003c/em\u003e, \u003cem\u003eR. rickettsii\u003c/em\u003e, \u003cem\u003eR. conorii\u003c/em\u003e, \u003cem\u003eR. sibirica\u003c/em\u003e, and \u003cem\u003eR. parkeri\u003c/em\u003e (Weinert et al. 2009, Davison et al. 2022). All members are associated with ticks (Ixodida), and includes the first recognized and one of more lethal pathogens among \u003cem\u003eRickettsia\u003c/em\u003e, \u003cem\u003eR. rickettsii\u0026nbsp;\u003c/em\u003e(Parola et al. 2013). On other hand, the pathogenicity of \u003cem\u003eR. parkeri\u0026nbsp;\u003c/em\u003ewas not recognized until 60 years after its description, being responsible for milder cases of Spotted fever (Parola et al. 2005). The group includes an even more interesting case, \u003cem\u003eR. peacockii\u003c/em\u003e. The species is\u0026nbsp;perpetuated\u0026nbsp;transstadially and transovarially in ticks,\u0026nbsp;and\u0026nbsp;the presence of \u003cem\u003eR. peacockii\u003c/em\u003e within ovaries interfering\u0026nbsp;in\u0026nbsp;the ability of \u003cem\u003eR. rickettsii\u003c/em\u003e to infect the ovarian tissues and to be transovarially transmitted to progeny (Parola et al. 2005). However,\u0026nbsp;\u003cem\u003eR. peacockii\u003c/em\u003e does not invade the salivary glands, precluding its transmission during biting (Niebylski et al. 1997), and \u0026nbsp;possesses an \u003cem\u003eompA\u003c/em\u003e gene that contains three premature stop codons, hence is unable to produce a functional \u003cem\u003eompA\u003c/em\u003e protein, crucial for pathogenicity (Baldridge et al. 2004).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;Typhus\u0026nbsp;group comprises two named species, \u003cem\u003eRickettsia\u003c/em\u003e \u003cem\u003etyphi\u003c/em\u003e and \u003cem\u003eRickettsia prowazekii,\u003c/em\u003e primarily\u0026nbsp;associated with fleas (Siphonaptera) and lice (Phthiraptera) (Parola et al. 2005). The Transitional group contains named species such as \u003cem\u003eR. felis\u003c/em\u003e, \u003cem\u003eR. australis\u003c/em\u003e, and \u003cem\u003eR. akari\u003c/em\u003e, and lineages occurring associated to Ixodida, Diptera, Hemiptera, Hymenoptera, Psocoptera, and Siphonaptera (Weinert 2015). \u003cem\u003eRickettsia felis\u003c/em\u003e is vectored by cat fleas to cats and humans, to which it\u0026nbsp;has been associated with\u0026nbsp;fevers and spots, while a closely related strain is required for oogenesis in the booklouse and is thought to cause parthenogenesis (Thepparit et al. 2011). \u003cem\u003eRickettsia australis\u003c/em\u003e causes Queensland tick typhus, transmitted to humans through the bites of the hard ticks \u0026nbsp;\u003cem\u003eIxodes holocyclus\u003c/em\u003e or \u003cem\u003eIxodes tasmani\u003c/em\u003e. It has mostly mild symptoms – fever, headache, and myalgia followed by the development of a maculopapular or vesicular rash, an inoculation eschar (65% of cases), and lymphadenopathy (71%) (McBride et al. 2007). Finally, the closely related \u003cem\u003eR. akari\u003c/em\u003e, the causative agent of rickettsialpox, has as invertebrate host mites belonging to the order Mesostigmata, specifically \u003cem\u003eLiponyssoides sanguineus\u003c/em\u003e, despite it is suspected of being transmitted by ticks too (e.g. Iweriebor et al. 2017).\u003c/p\u003e\n\u003cp\u003eMites belonging to the epifamily Trombiculoidae (=Trombiculidae \u003cem\u003esensu\u003c/em\u003e Kudryashova 1998) include species with larvae parasite of vertebrates, with a few exceptions (with invertebrate hosts) that are likely reversals (Costa et al. 2024, Felska et al. 2018, Vasconcelos et al. 2017). They differ from all other ectoparasites mites for not being hematophagous or tissue boring, but feeding upon host tissues through the stylostome, a straw-like structure by interaction of mite saliva and host tissue (Shatrov 2009). Furthermore harboring \u003cem\u003eOrientia\u003c/em\u003e \u003cem\u003etsutsugamushi\u0026nbsp;\u003c/em\u003eand other members of genus \u003cem\u003eOrientia\u0026nbsp;\u003c/em\u003e(Martínez-Valdebenito et al. 2024), bacteria belonging to the genus \u003cem\u003eRickettsia\u003c/em\u003e are frequent in Trombiculoidae mites, being detected in chiggers feeding on small mammals and birds in Brazil, USA, Slovakia, and in many places across Asia (Choi et al. 2007, Tsui et al. 2007, Huang et al. 2017, Miťková et al. 2015, Jacinavicius et al. 2019, Linsuwanon et al. 2021, Kuo et al. 2022, Ponnusamy et al. 2022, Bassini-Silva et al. 2018, 2023). Most of them were grouped in the Transitional group, implying that these bacteria in Trombiculoidae may be arthropod symbionts, vertebrate pathogens or both. Some molecularly detected lineages, such as ‘\u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003esp. clone MB74-1’ and ‘\u003cem\u003eRickettsia\u003c/em\u003e sp. TwKM02’ and related bacteria occurring in South Korea seems to be specific to chiggers and are seldom found in the rodent host or coinfesting ectoparasites (Choi et al. 2007, Kuo et al. 2022, \u0026nbsp;Tsui et al. 2007).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcarological taxonomic collections in a One Health context\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Stärk and colleagues (2015): “One Health surveillance describes the systematic collection, validation, analysis, interpretation of data and dissemination of information collected on humans, animals and the environment to inform decisions for more effective, evidence- and system-based health interventions”. It is clear from its very definition that many aspects of this approach may interplay with the objectives of taxonomic collections, which harbors relevant samples from natural or synanthropic communities, including potential vectors and hosts of symbionts.\u003c/p\u003e\n\u003cp\u003eThe Acarological collection at “Centro de Coleções Taxonômicas da UFMG” – \u0026nbsp; Acronym UFMG-AC (Zhang, 2018) – \u0026nbsp; is a repository started in 2012, totaling, to October/2023, 16,361 specimens, mounted on slides or in alcohol (Pepato 2024). These mites were collected in all regions of Brazil and several countries in the Americas, Europe, Asia and Oceania. The collection stands out for its sampling of mites from caves and coastal habitats, comprising approximately 150 different families, providing a solid basis for taxonomic and ecological studies, and type material (Pepato 2024). The cave dwelling mites include a substantial sample of members of Epifamily Trombiculoidae and Order Ixodida, mainly from Minas Gerais State. The objective of this study is applying the One Health approach to these individuals, by the molecular detection of the \u003cem\u003eRickettsia\u003c/em\u003e diversity hosted by these groups, making the information on their occurrence available as far as possible.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSpecimen sampling and collection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEssayed individuals were deposited in the Acarological collection at \u0026ldquo;Centro de Cole\u0026ccedil;\u0026otilde;es Taxon\u0026ocirc;micas da UFMG\u0026rdquo;, Acronym UFMG-AC (Zhang, 2018). Specimens were variably preserved in alcohol 70-100%. As the original sampling context of most mites was not that of employing molecular techniques, but an inventory of invertebrate communities, most mites were stored for days to months in 70% ethanol at room temperature before having the fixative replaced by absolute alcohol and kept at -20 \u003csup\u003eo\u003c/sup\u003eC upon arrival at UFMG-AC.\u003c/p\u003e\n\u003cp\u003eUp to October/2023, the UFMG-AC collection had 580 Trombiculoidae occurrences, including larval and post larval individuals, most of the former found parasitizing bats, often caught inside caves or near to their entrance, and 144 Argasidae and 138 Ixodidae, mostly caught off host, on caves soil.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDNA extraction and morphological identification\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA was extracted from single specimens using QIAamp\u0026reg; DNA Micro kit (Qiagen) for larvae and the Wizard\u0026reg; Genomic DNA Purification Kit (Promega) for nymph and adult stages, following the manufacturer\u0026rsquo;s protocol, except by using two steps of the final elution in the former, leading to a final volume of 50 \u0026mu;l and by removing and preserving the specimen\u0026rsquo;s exoskeleton after the protein digestion step.\u003c/p\u003e\n\u003cp\u003eMost exoskeletons recovered after DNA extraction\u0026nbsp;were\u0026nbsp;mounted on permanent microscope slides using Hoyer\u0026rsquo;s medium, following the protocol described by Walter and Krantz (2009), and kept in an oven at 50\u0026ndash;55 \u0026deg;C for approximately one week or until completely dry. Slides were examined under a Leica DM2500 light microscope equipped with an ICC50 W digital camera and phase contrast optics. In the case of post larval ticks, specimens\u0026nbsp;\u003cins cite=\"mailto:Almir%20Pepato\" datetime=\"2025-11-24T04:17\"\u003ewe\u003c/ins\u003e\u003cdel cite=\"mailto:Almir%20Pepato\" datetime=\"2025-11-24T04:17\"\u003ea\u003c/del\u003ere preserved in 70% ethanol and carefully examined under Leica M125 stereomicroscopes, as described by Mu\u0026ntilde;oz-Leal et al. (2017).\u003c/p\u003e\n\u003cp\u003eTicks and mites were identified using morphological keys and original species descriptions, for post larval stages of hard ticks (Ixodidae) following Barros-Battesti et al. (2006) and Martins et al. (2010) and soft tick larvae and post larval (Argasidae) by Dantas-Torres et al. (2019). Since the generic classification of Argasidae remains under debate without consensus (Mans et al. 2021, Kneubehl et al. 2022), in the present study we adopted the classification of Guglielmone et al. (2003) sensu Hoogstraal (1985) for the Argasidae genera, until this taxonomic problem is solved.\u003c/p\u003e\n\u003cp\u003eGomes-Almeida et al. (2023) included most of the chiggers reported in this study and was consulted for the identification of two species of the genus \u003cem\u003eWhartonia\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePCR amplification of mitochondrial host genes and\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eRickettsia \u003cem\u003escreening, sequencing and chromatogram checking\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConventional PCR and nested PCR assays were initially performed to amplify two mitochondrial gene fragments:\u003c/p\u003e\n\u003cp\u003e(i) For ticks, a ~410 bp fragment \u0026nbsp;of the mitochondrial large ribosomal subunit (16S) gene was amplified using primers proposed by Mangold et al. (1998): 16S + 1 (5\u0026apos;-CTG CTC AAT GAT TTT TTA AAT TGC TGT GG-3\u0026apos;) and 16S\u0026ndash;1 (5\u0026apos; -CCG GTC TGA ACT CAG ATC AAG T-3\u0026apos;). The cycling conditions used were the same as in Gomes-Almeida and Pepato (2021), consisting of an initial denaturing step at 94 \u0026deg;C for 2 min, followed by 35 cycles of denaturing at 94 \u0026deg;C for 30 s, annealing for 35 s and extension at 72 \u0026deg;C for 45 s. The annealing temperature increased by 0.3 \u0026deg;C every cycle during the first seven cycles (from 47 \u0026deg;C to 48.8 \u0026deg;C), followed by 28 cycles using an annealing temperature of 50 \u0026deg;C. A final extension step at 72 \u0026deg;C for 5 min.\u003c/p\u003e\n\u003cp\u003e(ii) For mites and ticks, a ~1200 bp fragment of mitochondrial cytochrome c oxidase subunit I (cox-1) gene \u0026nbsp;was amplified using a nested PCR, following the primers proposed by Klimov et al. (2018). \u0026nbsp;The first PCR step using primers COX1 16F (5\u0026apos;-TGA NTW TTT TCH ACW AAY CAY AA-3\u0026apos;) and COX1 1324R (5\u0026apos;-CDG WRT AHC GDC GDG GTA T-3\u0026apos;), followed by a second PCR step with primers including a M13\u0026nbsp;tail:\u0026nbsp;COX1 25Fshort (5\u0026apos;-CHA CWA AYC AYA ARR AYA-3\u0026apos;) and COX1 1282R (5\u0026apos;-CCW VYT ARD CCT ARR AAR TGT TG-3\u0026apos;). The cycling conditions used followed Gomes-Almeida et al. (2023, 2024) that included an initial denaturation at 94 \u0026deg;C for 2 mins, followed by 35 cycles of 94 \u0026deg;C for 30 s, 40 \u0026deg;C for 30 s, and 72 \u0026deg;C for 2 min, with a final extension at 72 \u0026deg;C for 7 min.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(iii) We used DNA samples that have been extracted and tested for the COI and 28S genes in previous studies, as described by Gomes-Almeida et al. (2023).\u003c/p\u003e\n\u003cp\u003eSamples were screened for\u003cem\u003e\u0026nbsp;Rickettsia\u003c/em\u003e spp. using conventional PCR targeting a ~350 bp fragment \u0026nbsp;of the citrate synthase (\u003cem\u003egltA\u003c/em\u003e)\u0026nbsp;gene of all members of the genus \u003cem\u003eRickettsia\u003c/em\u003e, following the primers and protocols described by Labruna et al. (2004): CS 78 (5\u0026apos;-GCAAGTATCGGTGAGGATGTAAT-3\u0026apos;) and CS 323 (5\u0026apos;-GCTTCCTTAAAATTCAATAAATCAGGAT-3\u0026apos;).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The thermal cycling conditions included an initial denaturation step of 2 min at 95 \u0026deg;C, followed by 40 cycles comprising denaturation at 95 \u0026deg;C for 15 s, annealing at 48 \u0026deg;C for 30 s, and extension at 72 \u0026deg;C for 30 s. A final extension step was performed at 72 \u0026deg;C for 7 min.\u003c/p\u003e\n\u003cp\u003eExtractions from which \u003cem\u003egltA\u003c/em\u003e sequences could be obtained were used in attempts to obtain additional loci, in order to further characterize the lineages detected. Following Weinert et al. (2009), we\u0026nbsp;attempted\u0026nbsp;to obtain sequences from genes the rickettsial\u0026nbsp;16SrDNA (Small Subunit Ribosomal DNA), \u003cem\u003eatpA\u003c/em\u003e (ATP synthase subunit alpha), and \u0026nbsp;\u003cem\u003ecoxA\u003c/em\u003e (cytochrome C oxidase subunit I)\u0026nbsp;were performed by using the same primers pairs employed by Weinert et al. (2009).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll PCR reactions were performed in 20 \u0026mu;l of final volume with Platinum Taq DNA Polymerase (Invitrogen) in a Mastercycler nexus (Eppendorf) thermocycler. The master mix for initial PCR contained 2.0 \u0026mu;l of PCR buffer (1X), 1.4 \u0026mu;l MgCl 2 (50 mM), 1.4 \u0026mu;L of dNTP (10 mM each) and 0.8 \u0026mu;l of each oligonucleotide primer (10uM), to which 1\u0026ndash;3 \u0026mu;l of genomic DNA or alternatively 0.5 \u0026mu;l of parent PCR products for nested reactions was added. All reactions included a negative control (ultrapure water) and, for \u003cem\u003eRickettsi\u003c/em\u003ea spp. screening, a positive control (\u003cem\u003eRickettsia rickettsii\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eAll PCR products found positive in 1% agarose gel electrophoresis were purified using the Ampure\u0026reg; (Agencourt) kit and sequenced using a 3730 DNA Analyzer and BigDyeTM Terminator v3.1 (Applied Biosystems) according to the manufacturer\u0026apos;s protocol. For nested PCR (COI), sequencing was performed using the M13 forward \u0026nbsp;(5\u0026apos;TGTAAAACGACGGCCAGT-3\u0026rsquo;) and reverse (5\u0026apos;-CAGGAAACAGCTATGACC-3\u0026apos;) primers. For all other markers, the primers employed in sequencing were the same as for PCRs.\u003c/p\u003e\n\u003cp\u003eForward and reverse chromatograms were checked, edited and assembled into contigs using software ChromasPro 1.41 (Technelysium Pty Ltd). All sequences generated for this study were compared with available mites or bacteria sequences using the NCBI BLAST feature (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) (Altschul et al. 1997) and deposited in the GenBank database. Host endogenous genome and gltA sequences are summarized in the supplementary Tables S1-S2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAlignment and phylogenetic inference\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mitochondrial 16S sequences were employed in an integrative approach to assist soft tick identification. They were aligned in MAFFT (Katoh et al. 2002) using the E-INS-i strategy (Katoh et al. 2005) implemented in the MAFFT server at https://mafft.cbrc.jp/alignment/server/. The Scoring matrix for nucleotide sequences employed was the 200 PAM/K=2. The sequences obtained here were combined to sequences from all Argasidae species recorded from Brazil according to Dantas-Torres et al. (2019), and an undescribed putative new species reported in Jorge et al. (2022), referred by the authors as \u003cem\u003eOrnithodoros\u003c/em\u003e sp. Ubajara. Alignment features and distances were calculated using MEGA X (Kumar et al. 2018). The model for sequence evolution was chosen using the Bayesian Information Criterion, calculated in ModelFinder (Kalyaanamoorthy et al, 2017) implemented in IQ-TREE 3 (Wong et al. 2025). \u0026nbsp;IQ-TREE 3 also was employed for inferring the maximum likelihood, and Ultrafast Bootstrap and the SHlike approximate likelihood ratio test (SH-aLRT), both calculated with 1000 replicates. The 16S tree was employed as input for PTP Maximum Likelihood species delimitation analyses (Zhang et al. 2013), at the bPTP webserver (https://species.h-its.org/ptp/). Sequences from Trombiculoidae are summarized in Supplementary Table S2.\u003c/p\u003e\n\u003cp\u003eThe sequences from \u003cem\u003egltA\u003c/em\u003e gene were combined in an alignment with sequences from GenBank in order to identify the affinities of the \u003cem\u003eRickettsia\u003c/em\u003e detected, as summarized in Supplementary Table S3: (A) representatives of the main lineages of \u003cem\u003eRickettsia\u003c/em\u003e, including basal groups Rhyzobius and Meloidae; (B) Sequences from \u0026nbsp;\u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003edetected in chiggers, as reviewed by Chaisiri et al 2023; (C) Matches with sequences deposited in GenBank using the online implemented BLATn, during the preliminary sequence processing described above. After preliminary automatic alignment with the aid of the BioEdit 7.2.1 software (Hall, 1999) sequences had their extremities trimmed and checked for the maintenance of the reading frame in the program MEGA X (Kumar et al. 2018).\u003c/p\u003e\n\u003cp\u003eMultiloci analyses from rickettsial markers \u0026nbsp;were \u0026nbsp;performed for those terminals for which at least two markers could be obtained. It was built on Weinert et al. (2009) study. GenBank accessions for retrieved and newly obtained sequences are summarized in Supplementary Table S4.\u003c/p\u003e\n\u003cp\u003eBayesian phylogenetic inference was performed in MrBayes 3.2.7 (Ronquist et al. 2012) and maximum likelihood inference was performed in IQ-TREE 3 (Wong et al. 2025).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDetection attempts were performed in a total of 363 specimens, comprising 158 ticks (Ixodida: Argasidae and Ixodidae) and 205 mites (Trombiculoidea), including larvae, nymphs and adults. These specimens were collected in localities in the Brazilian states of Minas Gerais, Par\u0026aacute;, and S\u0026atilde;o Paulo, most of them (327) from caves or cave associated fauna. Among them, only those that led to sequences were depicted in Fig. 1, and listed in \u0026nbsp;Supplementary Table S1 and S2. A total of 38 soft ticks belonging to the genus \u003cem\u003eOrnithodoros\u003c/em\u003e, 18 hard ticks belonging to the genus \u003cem\u003eAmblyomma,\u0026nbsp;\u003c/em\u003eand 52 chiggers belonging to genus \u003cem\u003eWhartonia\u003c/em\u003e\u0026nbsp; led to sequences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eArthropod hosts identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequences of mitochondrial 16S and COI were employed for molecular species delimitation in ticks (Fig. 2). Concerning Argasidade, all belonging to the genus\u0026nbsp;\u003cem\u003eOrnithodoros\u003c/em\u003e, a total of 25 specimens were sequenced for 16S, 20 for COI, and seven were sequenced for both markers. The aligned 16S new sequences are 424 \u0026ndash; 418 bps long, counting 135 variable positions out 432 positions, among them 99 parsimony informative. The COI nucleotide sequences counted 330 variable positions\u0026nbsp;out of 973\u0026nbsp;bps, among them 320 parsimony informative. Translated into aminoacids, it led to\u0026nbsp;43\u0026nbsp;out 324 positions, with\u0026nbsp;39\u0026nbsp;parsimony informative positions.\u003c/p\u003e\n\u003cp\u003eAmong Argasidae, the species\u0026nbsp;\u003cem\u003eOrnithodoros\u003c/em\u003e\u003cem\u003e\u0026nbsp;cavernicolous\u003c/em\u003e Dantas-Torres, Venzal \u0026amp; Labruna, 2012 and\u0026nbsp;\u003cem\u003eOrnithodoros\u003c/em\u003e\u003cem\u003e\u0026nbsp;fonsecai\u003c/em\u003e (Labruna \u0026amp; Venzal, 2009) could be identified both morphologically and molecularly beyond any reasonable doubt. The presence of cheeks and legs with micromammillate cuticle points these ticks as members of the mostly bat-associated soft ticks of the subgenus \u003cem\u003eAlectorobiu\u003c/em\u003es (Dantas-Torres et al. 2012; Labruna \u0026amp; Venzal, 2009). Both species were originally described from specimens obtained in caves or associated with bats, exactly as in our case. Similar to Oliveira et al (2024), 16S sequences from \u003cem\u003eO. fonsecai\u0026nbsp;\u003c/em\u003egrouped into two haplogroups, those obtained from Itamb\u0026eacute; do Mato Dentro, Lagoa Santa and Pedro Leopoldo Municipalities grouped with sequences from Mato Grosso do Sul state \u0026ndash; Cerrado biome, type locality \u0026ndash; and Rio de Janeiro \u0026ndash; Atlantic Forest biome, whereas the specimens from the Caatinga \u0026ndash; Pernambuco and Cear\u0026aacute; states \u0026ndash; and Cerrado biomes \u0026ndash; Mato Grosso state \u0026ndash; \u0026nbsp;grouped with sequences of ticks from Luisl\u0026acirc;ndia municipality (Fig. 2A). Despite all \u003cem\u003eO. fonsecai\u0026nbsp;\u003c/em\u003esequenced came from the same state, the later municipality is in a dry region, distant ~ 560 kms from the other localities, hence the division between an haplogroup inhabiting drier environments and other to more humidity seems to stand. The highest p-distance observed among terminals from the two haplogroups is 0.043, observed between the sequence from Mato Grosso do Sul (GQ120967) and sequences from Cear\u0026aacute; state (KX781699, OK256967). Among sequences within both haplogroups was observed an average p-distance = 0.006.\u003c/p\u003e\n\u003cp\u003eMolecular data also clearly grouped ticks collected in caves from Minas Gerais with the undescribed \u0026ldquo;\u003cem\u003eOrnithodoros\u003c/em\u003e sp. Ubajara\u0026rdquo;. Jorge et al. (2022), referred this way after the Ubajara municipality, Cear\u0026aacute; State, Northeast Brazil, from caves in the semi arid Caatinga biome, their 16S sequences have an average distance p = 0.005, and PTP analyses clearly recovers them as co-specific.\u003c/p\u003e\n\u003cp\u003eSix 16S sequences grouped with \u003cem\u003eOrnithodoros\u003c/em\u003e\u003cem\u003e\u0026nbsp;saraivai\u003c/em\u003e Mu\u0026ntilde;oz-Leal \u0026amp; Labruna, 2017 (Fig. 1A), but neither PTP nor the pairwise distances p= 0.079-0.050, support unambiguously assigning the specimens to such species. A least 16S sequence from an individual was recovered as sister to clade including \u003cem\u003eOrnithodoros\u003c/em\u003e\u003cem\u003e\u0026nbsp;yumatensis\u003c/em\u003e Cooley \u0026amp; Kohls, 1941 (GenBank: KX668415) and \u003cem\u003eOrnithodoros\u003c/em\u003e\u003cem\u003e. brodyi\u003c/em\u003e Matheson, 1935 (GenBank: KY454706), but with even higher pairwise distances (pairwise p = 0.121 and p = 0.138) are suggestive of a new species. These ticks should be further investigated taxonomically.\u003c/p\u003e\n\u003cp\u003eMost individuals sequenced for COI could be unambiguously assigned to \u003cem\u003eO. cavernicolous\u003c/em\u003e, \u003cem\u003eO. fonsecai\u003c/em\u003e, and \u003cem\u003eOrnithodoros\u0026nbsp;\u003c/em\u003esp. Ubajara thanks terminals sequenced for both 16S and COI (Fig. 2C). In contrast to the 16S marker, there was no available sequence of the COI marker for the type specimens of \u003cem\u003eO. saraivai\u003c/em\u003e. Even so, of the specimens morphologically identified as \u003cem\u003eO. saraivai\u003c/em\u003e and confirmed by 16S sequences (Fig. 2A), at least three of them, all from Concei\u0026ccedil;\u0026atilde;o do Mato Dentro, generated COI sequences that formed a distinct clade (Fig. 2C), in which one of the sequences showed a considerable p-distance (= 0.136) and without PTP support for being cospecific. Two ticks from Jana\u0026uacute;ba municipality, Minas Gerais state, morphologically identified as \u003cem\u003eOrnithodoros\u0026nbsp;\u003c/em\u003esp., was recovered in a clade along \u003cem\u003eOrnithodoros cerradoensis\u003c/em\u003e Mu\u0026ntilde;oz-Leal, Martins \u0026amp; Labruna, 2020 (NC_067907, average p-distance = 0.102).\u003c/p\u003e\n\u003cp\u003eAll ixodid belonging to \u003cem\u003eAmblyomma\u003c/em\u003e could be identified to the species level, both through molecules and morphology, comprising seven species:\u0026nbsp;\u003cem\u003eAmblyomma brasiliense\u003c/em\u003e Arag\u0026atilde;o, 1908 (N=3), \u003cem\u003eAmbyomma cajennense\u003c/em\u003e (Fabricius, 1787) (N=1), \u003cem\u003eAmblyomma dubitatum\u003c/em\u003e Neumann, 1899 (N=1), \u003cem\u003eAmblyomma nodosum\u003c/em\u003e Neumann, 1899 (N=1), \u003cem\u003eAmblyomma ovale\u0026nbsp;\u003c/em\u003eKock, 1844 (N=3), \u003cem\u003eAmblyomma pacae\u003c/em\u003e Arag\u0026atilde;o, 1911 (N=1), and \u003cem\u003eAmblyomma sculptum\u003c/em\u003e Berlese, 1888 (N= 8).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The same for \u003cem\u003eWhartonia\u003c/em\u003e, with most host sequences already reported in previous studies, with 44 individuals identified as \u003cem\u003eW\u003c/em\u003e\u003cem\u003ehartonia\u003c/em\u003e \u003cem\u003epachywhartoni\u0026nbsp;\u003c/em\u003eVercammen-Grandjean, 1966\u0026nbsp;and eight as \u003cem\u003eW\u003c/em\u003e\u003cem\u003ehartonia\u003c/em\u003e\u003cem\u003e\u0026nbsp;nudosetosa\u003c/em\u003e (Wharton, 1938).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRickettsia\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;detection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 36 samples tested positive for \u003cem\u003eRickettsia\u003c/em\u003e spp. from ticks (Ixodida: \u003cem\u003eAmblyomma dubitatum\u0026nbsp;\u003c/em\u003eand Argasidae: \u003cem\u003eOrnithodoros\u0026nbsp;\u003c/em\u003espp.) and mites (Trombiculoidea: \u003cem\u003eWhartonia pachywhartoni\u003c/em\u003e), all collected from cave soil or bats (Fig. 1). From these samples, 21\u003cem\u003e\u0026nbsp;gltA\u003c/em\u003e gene partial sequences were successfully obtained (Supplementary Table S1-S2).\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003egltA\u003c/em\u003e alignment, after adding GenBank terminals (Supplementary Table S3) comprised 77 sequences, 379 columns, 165 variable, and 125 parsimony-informative sites. The best-fit model was the K3Pu+F+G4 (Kimura 3-parameter model with unequal base frequencies \u0026nbsp;and a gamma distribution) chosen according to BIC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite being only 379 bp long, our more comprehensive dataset, the \u003cem\u003egltA\u003c/em\u003e gene tree, led to a topology that roughly corresponds to the trees obtained by whole genome or multi loci analyses, as follows: (Rhyzobius, \u0026nbsp; (Meloidae, (Belli, ((Adalia, Canadensis), (Typhus, Transitional, Spotted Fever)))). The new sequences were recovered in the \u0026nbsp;gene tree in three phylogroups, the transitional (TRG), spotted fever (SFG) andbellii groups (Fig. 3).\u0026nbsp;Based on their DNA sequences, we group them in nine strains, as described below and shown in Fig. 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain 1\u003c/strong\u003e: The single \u003cem\u003eRickettsia\u003c/em\u003e detected from a hard tick, a\u0026nbsp;female identified as \u003cem\u003eAmblyomma dubitatum\u003c/em\u003e (UFMG-AC 170244, GenBank AC1985), collected dwelling on a cave floor, belongs to the \u0026nbsp;bellii group. The Blastn search querying \u003cem\u003egltA\u003c/em\u003e sequences revealed 70 hits with 100% identity with \u003cem\u003eR. belli\u003c/em\u003e, detected from Ixodidae (genera \u003cem\u003eAmblyomma\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Dermacentor\u003c/em\u003e, \u003cem\u003eIxodes\u003c/em\u003e, and \u003cem\u003eHaemaphysalis\u003c/em\u003e) from USA, El Salvador, Costa Rica, Colombia, Argentina and widespread in Brazil, including localities scattered across the country.\u003c/p\u003e\n\u003cp\u003eThe family Argasidae harbored nine \u003cem\u003eRickettsia\u003c/em\u003e occurrences, including sequences recovered in the three phylogroups (TRG, SFG, andbellii groups).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain 2\u003c/strong\u003e: In the belli Group, the sequence obtained from the\u0026nbsp;\u003cem\u003eOrnithodoros\u003c/em\u003e sp. 2 (\u003cem\u003eO. yumatensis\u0026nbsp;\u003c/em\u003e+\u003cem\u003e\u0026nbsp;O. brodyi\u003c/em\u003e clade) female (UFMG AC 180276), collected near to the entrance of an iron ore cave in Minas Gerais state, a BLASTn search recovered a 98.94% identity with the sequence identified as \u003cem\u003eRickettsia\u003c/em\u003e sp. ALSK (GenBank KX254162), obtained from the flea \u003cem\u003eXenopsylla minax\u003c/em\u003e (Siphonaptera,Pulicidae) from the Alataw Pass, Xinjiang, China \u0026nbsp;according to the GenBank register.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain 3\u003c/strong\u003e:\u0026nbsp;The sequence obtained from a female of\u0026nbsp;\u003cem\u003eO.\u003c/em\u003e\u003cem\u003e\u0026nbsp;fonsecai\u003c/em\u003e (UFMG AC 172234), from the twilight zone floor of a limestone cave in the southern portion of S\u0026atilde;o Paulo state,\u0026nbsp;was recovered in the Belli Group too. It has an identity of 99.47% with \u003cem\u003eRickettsia\u003c/em\u003e sp. RDa420 (GenBank AF497584) detected in\u003cem\u003e\u0026nbsp;Dermacentor auratus\u003c/em\u003e removed from a bear in Thailand (Parola et al. 2003).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain 4\u003c/strong\u003e: A sequence from one female (UFMG-AC 210291, aphotic zone of a limestone cave) appear nested in the TRG (Fig. 3), with an identity of 99.47% with the sequence GenBank MF175748, from a \u003cem\u003eAmblyomma sculptum\u003c/em\u003e individual collected in the southern Brazilian state of Paran\u0026aacute;, and 98.94% identity with 13 sequences identified as\u003cem\u003e\u0026nbsp;Rickettsia asembonensis\u003c/em\u003e, eleven obtained from \u003cem\u003eCtenocephalides felis\u003c/em\u003e associated to dogs, nine in the Brazilian amazonian state of Rond\u0026ocirc;nia and two from Costa Rica, a sequence obtained from \u003cem\u003eC. canis\u003c/em\u003e in Kenia, and a sequence from a human patient from Malaysa.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain 5\u003c/strong\u003e:\u0026nbsp;The \u003cem\u003eRickettsia\u003c/em\u003e sequences from three individuals (two \u003cem\u003eO. fonsecai\u0026nbsp;\u003c/em\u003eand one \u003cem\u003eO.\u0026nbsp;\u003c/em\u003eaff.\u003cem\u003e\u0026nbsp;saraivai\u003c/em\u003e) grouped in a basal clade among TRG. Two of them, obtained in Caves from Minas Gerais, composed strain 5 as follows: one female of the \u003cem\u003eO. saraivai\u003c/em\u003e Clade\u0026nbsp;(UFMG-AC 170467, cave soil) and a female identified as \u003cem\u003eO. fonsecai\u0026nbsp;\u003c/em\u003e(UFMG-AC 1608677), both from twilight zone of a limestone caves. These sequences are identical and had 60 Blastn hits with sequences identified as \u003cem\u003eR. parkeri\u003c/em\u003e, \u003cem\u003eR. sibirica\u003c/em\u003e, \u003cem\u003eR. africae\u003c/em\u003e and other unidentified bacteria, with 98.67% identity, all\u0026nbsp;\u003cem\u003eRickettsia\u003c/em\u003e belonging to the SFG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain 6\u003c/strong\u003e: The sequence \u0026nbsp;from a \u003cem\u003eO. fonsecai\u0026nbsp;\u003c/em\u003efemale (UFMG-AC 171498), from twilight zone of a limestone cave, had 52 hits with 99.20 % identity with \u003cem\u003eRickettsia\u003c/em\u003e identified as \u003cem\u003eR. raoultii\u003c/em\u003e, \u003cem\u003eR. conori\u003c/em\u003ei, and unidentified bacteria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain 7\u003c/strong\u003e: Other two sequences were obtained from an \u003cem\u003eOrnithodoros\u003c/em\u003e \u003cem\u003efonsecai\u003c/em\u003e (UFMG-AC 171333) and a nymph of \u003cem\u003eO.\u003c/em\u003e aff \u003cem\u003esaravai\u003c/em\u003e (UFMG-AC 173021) both collected on the cave floor in the twilight zone. They were nested in the SFG and are identical to a sequence identified as\u0026nbsp;\u0026lsquo;\u003cem\u003eCandidatus\u003c/em\u003e Rickettsia wissemanii\u0026rsquo;\u0026nbsp;(GenBank LT558852) obtained from \u003cem\u003eO. hasei\u003c/em\u003e ticks found on the bat \u003cem\u003eNoctilio albiventris\u003c/em\u003e from French Guiana (Tahir et al 2016).\u003c/p\u003e\n\u003cp\u003eAll rickettsial sequences obtained from Trombiculoidae were obtained from \u003cem\u003eWhartonia pachywhartoni\u0026nbsp;\u003c/em\u003eindividuals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain 8\u003c/strong\u003e: The strain with more sequences (nine terminals) comprise larvae associated to the bats \u003cem\u003eCarollia perspicillata\u0026nbsp;\u003c/em\u003e(UFMG AC 210163, 210170), \u003cem\u003eDiphylla ecaudata\u0026nbsp;\u003c/em\u003e(UFMG AC 210180), \u003cem\u003eMimon bennetti\u0026nbsp;\u003c/em\u003e(UFMG AC 210101), larvae found dwelling free on a cave floor (UFMG AC 210337, 210339-210341) and a male (UFMG AC 170297). The Blastn hit with maximum similarity was with \u003cem\u003eRickettsia australis\u003c/em\u003e str. Cutlack (CP003338), which belongs to TRG, with 97.61% identity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain 9\u003c/strong\u003e: The other clade included three sequences, all obtained from \u003cem\u003eW. pachywhartoni\u0026nbsp;\u003c/em\u003emales (UFMG AC 170228, 170255, 170291) dwelling on the floor of iron ore caves in the twilight and aphotic zones. Blastn searches recovered a hit with 98.14 % identity with \u003cem\u003eRhipicephalus\u003c/em\u003e sp. tick nymph from \u003cem\u003eNesokia indica\u003c/em\u003e (Rodentia, Muridae) in Pakistan. The following most similar hits (97.87%) are SFG members (\u003cem\u003eR. raultii\u003c/em\u003e, \u003cem\u003eR. conorii\u003c/em\u003e, \u003cem\u003eR. montanensis\u003c/em\u003e).\u0026nbsp;However, in the phylogenetic tree, strain 8 sequences were sister to the TG clade composed by \u003cem\u003eR. prowazekii\u0026nbsp;\u003c/em\u003eand \u003cem\u003eR. typhi\u0026nbsp;\u003c/em\u003e(Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMultiloci analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was\u0026nbsp;possible to obtain\u0026nbsp;sequences from additional markers for bacteria belonging to all above delimited strain except by the Strain 9. The concatenated alignment, after adding GenBank terminals (Supplementary Table S4) comprised 57 sequences, 2966 positions, 892 variable, and 604 parsimony-informative sites. The best-fit partitioned model, chosen according to BIC, divided the sequences into two partitions, the first with ribosomal 16S sequences and the second merging all protein\u0026nbsp;coding\u0026nbsp;genes. The model TPM3u+R2 (AC=CG, AG=CT, AT=GT and unequal base\u0026nbsp;frequencies) was assigned to the first partition, while de model GTR+F+I+G4 (General time reversible model with unequal rates, unequal base frequencies , a proportion of invariant sites, and a gamma distribution) for the second.\u003c/p\u003e\n\u003cp\u003eThe topology of rickettsial groups recovered by the analyses as follows: (Rhyzobius, \u0026nbsp;(Meloidae, (Belli, (Adalia, (Canadensis, (\u003cem\u003eR. monasensis\u003c/em\u003e (Typhus, (Transitional, Spotted Fever)))))))). Note, however, that the \u0026nbsp;Canadensis group was not supported, appearing as a paraphyletic group in the ML tree (Fig.4).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRickettsia belli\u0026nbsp;\u003c/em\u003e(Strain 1), the only occurring in an \u003cem\u003eAmblyomma\u003c/em\u003e species, could be sequenced for the four markers and grouped with the other two terminals belonging to this species. The Strains 2 and 3, as in the gltA analyses, were recovered in the Belli Group. Whereas the support to the group is absolute, however, their internal relationship received a lower support. The endosymbiont of the\u0026nbsp;\u003cem\u003eOrnithodoros\u003c/em\u003e sp. 2 (UFMG AC 180276) \u003cem\u003eRickettsia\u003c/em\u003e, the Strain 2, group along to lacewing and \u003cem\u003eBrachys tessellatus\u003c/em\u003e endosymbionts received a support of UFBoot =78, SH-aLRT =\u0026nbsp;96.2, and PP = 0.65, and the placement of \u003cem\u003eO. fonsecai\u0026nbsp;\u003c/em\u003e(UFMG AC 172234) in a clade with Bombyiid bee fly,\u0026nbsp;\u003cem\u003eAcyrthosiphon\u003c/em\u003e and \u003cem\u003eRickettisia\u003c/em\u003e associated with \u003cem\u003eCarica papaya\u0026nbsp;\u003c/em\u003eand \u003cem\u003eEmpoasca papayae\u0026nbsp;\u003c/em\u003e received a support of UFBoot = 91, SH-aLRT =\u0026nbsp;51, and PP = 0.76.\u003c/p\u003e\n\u003cp\u003eThe strain 4, from another \u003cem\u003eO. fonsecai\u0026nbsp;\u003c/em\u003especimen (UFMG AC 210291), was recovered in a well-supported clade along \u003cem\u003eR. felis\u0026nbsp;\u003c/em\u003e(UFBoot = 96, SH-aLRT =83.9, and PP = 1.00). The Strain 8, associated with nine specimens of \u003cem\u003eW. pachywhasrtoni\u003c/em\u003e, was recovered in a clade with \u003cem\u003eR. akari\u003c/em\u003e with considerable support (UFBoot = 89, SH-aLRT =75.5, and PP = 0.93).\u003c/p\u003e\n\u003cp\u003eThe remaining strains were associated\u0026nbsp;with the\u0026nbsp;SFG, which recovered a support of UFBoot =94, SH-aLRT =\u0026nbsp;88.1, and PP = 0.99. The strain 5 is the second branch in the SFG tree, whereas strains 7 and 8 grouped in a clade that received a good support (UFBoot =\u0026nbsp;99, SH-aLRT =99.3, and PP = 1.00), both grouped with\u0026nbsp;\u0026lsquo;\u003cem\u003eCandidatus\u0026nbsp;\u003c/em\u003eR. wissemanii\u003cem\u003e\u0026rsquo;\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eRetrieving molecular data from natural history collections\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDespite that many samples had their DNA degraded due the storage conditions prior its inclusion in the CCT-UFMG Acarological collection, hence precluding amplification and sequencing, this study shows that a great deal of bacterial diversity still may be retrieved from taxonomical collections, integrating geographical information, \u0026nbsp;ectoparasites integrative taxonomy and, in the case of ticks and chiggers, vertebrate sources. The microbiome of insects include not just potential pathogens, but also can have profound effects on their hosts, from negative interactions in the form of pathogenesis and parasitism to highly beneficial associations in the form of nutrient provisioning and immune defense (Short et al. 2018). The same is true for mites and similar to entomological collections the effort in sequencing even historical samples may\u0026nbsp;lead\u0026nbsp;to relevant contributions to the understanding of their microbiomes.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIntegrative taxonom\u003c/em\u003ey\u003cem\u003e\u0026nbsp;of ticks\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEven though not being its main focus, this study could bring some new occurrences of\u0026nbsp;\u003cem\u003eOrnithodoros\u003c/em\u003e species and possible new species.\u003c/p\u003e\n\u003cp\u003eIn the case of \u003cem\u003eO. fonsecai\u003c/em\u003e it is remarkable that both haplogroups detected in Oliveira et al (2024) could be found in this study, up to now congruent to one haplogroup associated to localities more arid than to the other. A possible direction for future investigation concerning this species is testing if the distribution of those haplogroups corresponds to ongoing local\u0026nbsp;adaptation.\u003c/p\u003e\n\u003cp\u003eThe still unnamed species “\u003cem\u003eOrnithodoros\u003c/em\u003esp Ubajara” recorded by.Jorge et al (2022), proved to be abundant in caves around southern portion of Espinhaço Range (Lagoa Santa and Pedro Leopoldo), including larvae parasitizing the nectivorous bat \u003cem\u003eGlossophaga soricina\u0026nbsp;\u003c/em\u003e(Phyllostomidae). Despite the specimens reported herewith came from caves in transitional areas between the Atlantic Forest and Cerrado, apart almost 1800 km from the original records, the p-distances \u0026nbsp;and PTP analyses left no doubts about its identity.\u003c/p\u003e\n\u003cp\u003eThe sequences grouped with\u0026nbsp;\u003cem\u003eOrnithodoros\u003c/em\u003e\u003cem\u003e\u0026nbsp;saraivai\u0026nbsp;\u003c/em\u003ewarrants further investigation, since they exhibit considerable divergence and the ticks reported here may constitute a species complex. Finally, some specimens may belong to undescribed species,\u0026nbsp;such as the\u0026nbsp;nymph UFMG AC 180276, collected near the entrance of a cave in the Santa Maria de Itabira municipality, Minas Gerais state. All these novelties related to Argasidae\u0026nbsp;are\u0026nbsp;not unexpected, since many new species were recently described (Dantas-Torres et al. 2019).\u003c/p\u003e\n\u003cp\u003eRickettsia \u003cem\u003ediversity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe phylogenetic diversity found in the \u003cem\u003eRickettsia\u003c/em\u003e sequenced herewith likely imply a similar ecological diversity, and a possible explanation to this diversity lies on its association with bats and caves used as roosts by bats.\u003c/p\u003e\n\u003cp\u003eAmong detected \u003cem\u003eRickettsia\u003c/em\u003e spp., only \u003cem\u003eRickettsia bellii\u003c/em\u003e was found associated with an Ixodid species, \u003cem\u003eAmblyomma dubitatum\u003c/em\u003e, primarily a capybara (\u003cem\u003eHydrochoerus hydrochaeris\u003c/em\u003e) ectoparasite, but already found parasitizing the bat \u003cem\u003eGlossophaga soricina\u0026nbsp;\u003c/em\u003e(Phyllostomidae) (Nava et al. 2010). \u003cem\u003eRickettsia bellii\u0026nbsp;\u003c/em\u003epresents distinct lineages for North and South America (Krawczak et al 2018), and the \u003cem\u003egltA\u0026nbsp;\u003c/em\u003esequence obtained here groups with other\u0026nbsp;South-American bacteria. It is regarded as nonpathogenic for vertebrates, despite experimental studies\u0026nbsp;showing\u0026nbsp;that inoculation of this rickettsia produces eschars in rabbits and guinea pigs (Ogata et al 2006).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe other arthropod hosts for \u003cem\u003eRickettisia\u003c/em\u003e spp in this study, the genera\u0026nbsp;\u003cem\u003eOrnithodoros\u003c/em\u003e and \u003cem\u003eWhartonia\u003c/em\u003e, are primarily or at least putative bat ectoparasites that spend some time\u0026nbsp;dwelling\u0026nbsp;on the cave floor. Hence, most of rickettsial diversity found in this study came from arthropods that have stages on and off bat hosts, being integrated during part of their lives in the ecology of the cave soil, including examples of hyperparasitism by other mites, such as Erythraeidae larvae (e.g. Bassini-Silva et al 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor instance, \u003cem\u003eRickettsia\u003c/em\u003e is recorded from blood and tissue samples of bats, including disparate world regions as in South Africa and Swaziland,\u0026nbsp;where a\u0026nbsp;lineage related \u003cem\u003eR. conorii\u003c/em\u003e was detected (Dietrich et al. 2016), Romania, where a high prevalence of \u003cem\u003eRickettsia monacensis\u003c/em\u003e was detected in insectivorous bats (Matei et al. 2021), China, where it was detected the presence of\u0026nbsp;several SFG agents(Zhao et al 2020). It is also often detected in bats ectoparasites such as ticks (especially Argasidae), mites, and flies (reviewed in Matei et al. 2021), where the SFG and TRG were detected, the later with sequences related to \u003cem\u003eR. felis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn our study, along occurrences related to bacteria already found in bat ectoparasites, as\u0026nbsp;‘\u003cem\u003eCandidatus\u003c/em\u003e Rickettsia wissemanii’ found in \u003cem\u003eOrnithodoros hasei\u003c/em\u003e from French Guiana, Brazil and Argentina (Tahir et al. 2016, Colombo et al. 2020; Luz et al. 2019), above referred as strain 7 (and also the related strain 6), to \u003cem\u003eR. felis\u0026nbsp;\u003c/em\u003e(strain 4) and included in the SFG (strain 5 and likely strain 9), we recorded some lineages that seems to be found for the first time harbored by bat ectoparasites.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is the case for strains 2 and 3, both nested in the Bellii group, and in the multiloci analyses with terminals with phenotypes so diverse as male-killing and plant pathogens (Hurst and Jiggins 2000, Davis et al. 1998). It is also the case for strain 8, found in chiggers. It nests in the TRG group along \u003cem\u003eR. australis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eR. akari,\u003c/em\u003e both of medical relevance, being the causative agents of the Queensland Tick Typhus and rickettsialpox (Stewart et al 2017, Denison et al 2014).\u003c/p\u003e\n\u003cp\u003eOrnithodoros\u0026nbsp;\u003cem\u003ein caves as a pathogen vector for humans\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSoft ticks are mostly reported as human parasites by benefiting\u0026nbsp;from the\u0026nbsp;conditions provided by the human houses and synanthropic fauna, including bats (Bermúdez et al. 2021). However, the increasing use of caves as recreational and touristic spots, or\u0026nbsp;its use in\u0026nbsp;economical activities like mining, bring humans in contact to the subterranean fauna, and may make biting of soft ticks more common. Jorge et al (2022) reported biting by \u003cem\u003eO. cavernicolous\u003c/em\u003e and \u003cem\u003eO. fonsecai\u003c/em\u003e, the later already reported to occur in other studies (Labruna and Venzal 2009; Santiago et al. 2019). As in this article we could find four out nine strains of \u003cem\u003eRickettsia\u003c/em\u003e in this species,\u0026nbsp;warranting\u0026nbsp;the need of a close\u0026nbsp;inspection\u0026nbsp;of its\u0026nbsp;potential role in the appearance of emergent rickettsiosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, it is noteworthy that research on \u003cem\u003eRickettsia\u003c/em\u003e spp. in ticks has been strongly focused on hard ticks of the Ixodidae family. For example, until 2021, there were 599 records of 31 species of \u003cem\u003eRickettsia\u003c/em\u003e reported from 50 species of Ixodidae ticks in the neotropical region (Estrada-Peña et al. 2021). On the other hand, until the present study, there were only 15 records of \u003cem\u003eRickettsia\u003c/em\u003e in ticks of the Argasidae family in the neotropics, as follows: \u003cem\u003eRickettsia lusitaniae\u0026nbsp;\u003c/em\u003ein \u003cem\u003eOrnithodoros yumatensis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOrnithodoros\u003c/em\u003e sp. from Mexico (Sánchez-Montes et al 2016, Hornok et al 2019), \u003cem\u003eRickettsia bellii\u0026nbsp;\u003c/em\u003ein \u003cem\u003eOrnithodoros talaje\u0026nbsp;\u003c/em\u003efrom Mexico (Guzmán-Cornejo et al. 2022), \u003cem\u003eRickettsia felis\u003c/em\u003e in \u003cem\u003eOrnithodoros puertoricensis\u003c/em\u003e from Mexico (Ballados-González et al. 2023), ‘\u003cem\u003eCandidatus\u003c/em\u003e Rickettsia nicoyana’ in \u003cem\u003eOrnithodoros knoxjonesi\u003c/em\u003e from Costa Rica (Moreira-Soto et al 2017), \u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003esp. in \u003cem\u003eOrnithodoros rietcorreai\u0026nbsp;\u003c/em\u003efrom Brazil (Muñoz-Leal et al. 2019a), \u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003esp. in \u003cem\u003eOrnithodoros\u003c/em\u003e cf. \u003cem\u003emimon\u003c/em\u003e from Brazil (Dantas-Torres et al. 2022), \u003cem\u003eRickettsia\u003c/em\u003e sp. strain Itinguçú in \u003cem\u003eOrnithodoros faccinii\u003c/em\u003e from Brazil (Peixoto et al. 2021), '\u003cem\u003eCandidatus\u003c/em\u003e Rickettsia wissemanii' in \u003cem\u003eO. hasei\u0026nbsp;\u003c/em\u003efrom Brazil, French Guiana and Argentina (Tahir et al. 2016, Colombo et al. 2020; Luz et al. 2019; Dornelas Júnior et al. 2025), \u003cem\u003eRickettsia\u003c/em\u003e sp. in \u003cem\u003eOrnithodoros amblus\u0026nbsp;\u003c/em\u003efrom Peru and \u003cem\u003eOrnithodoros peruvianus\u0026nbsp;\u003c/em\u003efrom Chile (Duron et al. 2017), and \u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003esp. in \u003cem\u003eOrnithodoros\u0026nbsp;\u003c/em\u003esp. from Chile (Muñoz-Leal et al. 2019b). Undoubtedly, the present study shows a diversity of \u003cem\u003eRickettsia\u003c/em\u003e spp. in Argasidae never before reported in a single study, contributing significantly to a better understanding of this important group of tick-borne bacteria, which has been neglected in argasids.\u003c/p\u003e"},{"header":"Statements and Declarations ","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by funding from FAPEMIG (Call 01/2022-\u0026nbsp;Demanda Universal, process RDP 01535-22) and by the Ministry of Science and Higher Education of the Russian Federation, University of Tyumen, that supported the research project and work plan for the exchange of the doctoral student Nikolay Anisimov during his stay in Brazil in 2023. The authors thank the Program for Technological Development in Tools for Health-PDTISFIOCRUZ, especially to Renata de B. R. Oliveira and Nathalia S. Carvalho, who assisted with sequencing and the company Carste Ci\u0026ecirc;ncia e Meio Ambiente for depositing the specimens at UFMG AC. Finally, authors thank Danilo Gon\u0026ccedil;alves Saraiva (\u003cem\u003ein memoriam\u003c/em\u003e) for donating the DNA extraction positive to \u003cem\u003eRickettsia rickettisii\u0026nbsp;\u003c/em\u003eemployed as positive control in our essays.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by funding from FAPEMIG (Call 01/2022- \u003cstrong\u003eDemanda Universal\u003c/strong\u003e, process RDP 01535-22) and by the Ministry of Science and Higher Education of the Russian Federation, University of Tyumen, that supported the research project and work plan for the exchange of the doctoral student Nikolay Anisimov during his stay in Brazil in 2023.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlmir R. Pepato, Nikolay V. Anisimov, and Brenda K. Gomes-Almeida contributed to the study conception and design. Material preparation, data collection were performed by Almir R. Pepato, Nikolay V. Anisimov, and Brenda K. Gomes-Almeida, Teofania H. D. Amorim Vidigal, and Jefferson C. Carvalho Farias da Silva. Analyses were performed by Almir R. Pepato. The manuscript was written by Almir R. Pepato, Marcelo B. Labruna and Brenda K. Gomes-Almeida. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch4\u003eData Availability\u003c/h4\u003e\n\u003cp\u003eSequences were deposited in GenBank. Accession numbers are refereed in the supplementary tables [partially available, remaining accessions will be provided upon its release by GenBank]. Voucher material is deposited in the \u0026nbsp; AcarologIcal Collection at Centro de Cole\u0026ccedil;\u0026otilde;es Taxon\u0026ocirc;micas da UFMG (UFMG AC)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbarca K, L\u0026oacute;pez J, Acosta-Jamett G, Mart\u0026iacute;nez-Valdebenito C (2013) \u003cem\u003eRickettsia felis\u003c/em\u003e in \u003cem\u003eRhipicephalus sanguineus\u003c/em\u003e from two distant Chilean cities. Vector Borne Zoonotic Dis. 13(8):607-9 https://doi.org/10.1089/vbz.2012.1201\u003c/li\u003e\n \u003cli\u003eAltschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389\u0026ndash;3444\u003c/li\u003e\n \u003cli\u003eAnstead CA, Chilton NB. A novel \u003cem\u003eRickettsia\u003c/em\u003e species detected in Vole Ticks (\u003cem\u003eIxodes angustus\u003c/em\u003e) from Western Canada. 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Ticks Tick Borne Dis. 15(3):102331. https://doi.org/10.1016/j.ttbdis.2024.102331.\u003c/li\u003e\n \u003cli\u003eParola P, Cornet JP, Sanogo YO, Miller RS, Thien HV, Gonzalez JP, Raoult D, Telford III SR, Wongsrichanalai C (2003) Detection of \u003cem\u003eEhrlichia\u003c/em\u003e spp, \u003cem\u003eAnaplasma\u003c/em\u003e spp. \u003cem\u003eRickettsia\u003c/em\u003e spp, and other eubacteria in ticks from the Thai-Myanmar border and Vietnam. 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(1997) Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae. Int J Syst Bacteriol. 47(2):252-61. https://doi.org/10.1099/00207713-47-2-252\u003c/li\u003e\n \u003cli\u003eS\u0026aacute;nchez-Montes S, Guzm\u0026aacute;n-Cornejo C, Mart\u0026iacute;nez-N\u0026aacute;jera Y, Becker I, Venzal JM, Labruna MB (2016) \u003cem\u003eRickettsia lusitaniae\u0026nbsp;\u003c/em\u003eassociated with \u003cem\u003eOrnithodoros yumatensis\u003c/em\u003e (Acari: Argasidae) from two caves in Yucatan, Mexico. Ticks Tick Borne Dis 7(6):1097-1101. https://doi.org/10.1016/j.ttbdis.2016.09.003.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eShatrov AB (2009) Stylostome formation in trombiculid mites (Acariformes: Trombiculidae). Exp Appl Acarol 49(4): 261-280 https://doi.org/10.1007/s10493-009-9264-0.\u003c/li\u003e\n \u003cli\u003eShort AEZ, Dikow T, Moreau CS (2018) Entomological Collections in the Age of Big Data. 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Ticks Tick Borne Dis. 7(6):1089-1096 https://doi.org/10.1016/j.ttbdis.2016.09.004\u003c/li\u003e\n \u003cli\u003eTakahashi M, Urakami H, Yoshida Y, Furuya Y, Misumi H, Hori E, Kawamura A Jr, Tanaka H (1997) Occurrence of high ratio of males after introduction of minocycline in a colony of \u003cem\u003eLeptotrombidium fletcheri\u003c/em\u003e infected with \u003cem\u003eOrientia tsutsugamushi\u003c/em\u003e. Eur J Epidemiol 13(1): 79-86. https://doi.org/10.1023/a:1007341721795\u003c/li\u003e\n \u003cli\u003eThepparit C, Sunyakumthorn P, Guillotte ML, Popov VL, Foil LD, Macaluso KR (2011) Isolation of a rickettsial pathogen from a non-hematophagous arthropod. PLoS One 6(1): e16396. https://doi.org/10.1371/journal.pone.0016396\u003c/li\u003e\n \u003cli\u003eTsui PY, Tsai KH, Weng MH, Hung YW, Liu YT, Hu KY, Lien JC, Lin PR, Shaio MF, Wang HC, Ji DD (2007) Molecular detection and characterization of spotted fever group \u003cem\u003eRickettsiae\u003c/em\u003e in Taiwan. 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Pathog Dis 10;79(5):ftab024. https://doi.org/ 10.1093/femspd/ftab024\u003c/li\u003e\n \u003cli\u003eZchori-Fein E, Chandresh B, Harari AR (2006) Oogenesis in \u003cem\u003eCoccotrypes dactyliperda\u003c/em\u003e (Coleoptera: Curculionidae: Scolytinae) depends on symbiotic bacteria. Physiol Entomol 31: 164\u0026ndash;169\u003c/li\u003e\n \u003cli\u003eZhang J, Kapli P, Pavlidis P, Stamatakis, AA (2013) General species delimitation method with applications to phylogenetic placements. Bioinform. 29(22): 2869-2876 https://doi.org/10.1093/bioinformatics/btt499\u003c/li\u003e\n \u003cli\u003eZhang Z-Q (2018) Repositories for mite and tick specimens: acronyms and their nomenclature. Syst Appl Acarol 23 (12): 2432\u0026ndash;2446. https://doi.org/10.11158/saa.23.12.12\u003c/li\u003e\n \u003cli\u003eZhao S, Yang M, Liu G, Hornok S, Zhao S, Sang C, Tan W, Wang Y ( 2020) \u003cem\u003eRickettsiae\u003c/em\u003e in the common pipistrelle \u003cem\u003ePipistrellus pipistrellus\u003c/em\u003e (Chiroptera: Vespertilionidae) and the bat soft tick \u003cem\u003eArgas vespertilionis\u003c/em\u003e (Ixodida: Argasidae). Parasit Vectors 13(1):10 https://doi.org/10.1186/s13071-020-3885-x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Taxonomic collections, One Health, Ectoparasites, Vertebrates, Taxonomy, Cave, Bat gltA ","lastPublishedDoi":"10.21203/rs.3.rs-8190507/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8190507/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eRickettsia\u003c/span\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003especies are\u003c/span\u003e arthropod-hosted endosymbionts with a wide range of ecologies, including induction of parthenogenesis, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003emale-killing, and even plant pathogens with potential horizontal transmission through a plant host\u003c/span\u003e, although they attract greater attention for including many vertebrate pathogens. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe genus is classified into phylogenetic and serological groups, heterogeneous concerning their ecologies.\u003c/span\u003e This study aims at screen ticks and mites deposited in Acarological Collection at Center of Taxonomical Collections at Universidade Federal de Minas Gerais, Brazil, for rickettsial occurrences, making available genetic data on hosts and bacteria. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eEndogenous mitochondrial markers for ticks and mitochondrial and nuclear genes for chiggers were sequenced for sample quality control and species identification. A total of 38 soft ticks belonging to the genus\u003c/span\u003e \u003cem\u003eOrnithodoros\u003c/em\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e(at least six putative species), 18 hard ticks belonging to the genus\u003c/span\u003e \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eAmblyomma\u003c/span\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e(7 spp)\u003c/span\u003e, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand 52 chiggers belonging to genus\u003c/span\u003e \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eWhartonia\u003c/span\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e(2 spp) led to sequences.\u003c/span\u003e Polymerase \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eChain reactions for a Citrate\u003c/span\u003e S\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eynthase gene fragment led to 21 sequences grouped in the phylogroups Bellii, Spotted Fever, and Transitional, with one sequence of\u003c/span\u003e \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eRickettsia\u003c/span\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eobtained from\u003c/span\u003e \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eAmblyomma dubitatum\u003c/span\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e(identified as\u003c/span\u003e \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eR. bellii\u003c/span\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e), eight sequences from\u003c/span\u003e \u003cem\u003eOrnithodoros\u003c/em\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e(\u003c/span\u003eclassified \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ein five or six strains associated to the phylogroups Belli, Spotted Fever and Transitional) and twelve associate to\u003c/span\u003e \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eWhartonia pachywhartoni\u003c/span\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e(three likely in the Spotted Fever and nine in the Transitional phylogroup). Considering the diversity of rickettsial occurrences in\u003c/span\u003e \u003cem\u003eOrnithodoros\u003c/em\u003e, with four of the nine strains detected only in \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eO. fonsecai\u003c/span\u003e, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ea species known to bite humans, the medical\u003c/span\u003e relevance of Rickettsia associated with caves and bats needs to be further investigated.\u003c/p\u003e","manuscriptTitle":"Molecular detection of Rickettsia spp. in ticks (Ixodida: Argasidae and Ixodidae) and mites (Trombiculoidae) from a brazilian taxonomic collection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 13:03:52","doi":"10.21203/rs.3.rs-8190507/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5bbf0d9b-6cb1-4447-8543-578862cfea75","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-21T14:39:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 13:03:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8190507","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8190507","identity":"rs-8190507","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}