Microbiome and mitogenomics of the chigger mite Pentidionis agamae: Potential role as an Orientia vector and associations with divergent clades of Wolbachia and Borrelia

Microbiome and mitogenomics of the chigger mite Pentidionis agamae: Potential role as an Orientia vector and associations with divergent clades of Wolbachia and Borrelia | 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 Microbiome and mitogenomics of the chigger mite Pentidionis agamae: Potential role as an Orientia vector and associations with divergent clades of Wolbachia and Borrelia Hadil A. Alkathiry, Samia Q. Alghamdi, Amit Sinha, Gabriele Margos, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3837555/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background Trombiculid mites are globally distributed, highly diverse arachnids that largely lack molecular resources such as whole mitogenomes for the elucidation of taxonomic relationships. Trombiculid larvae (chiggers) parasitise vertebrates and can transmit bacteria ( Orientia spp.) responsible for scrub typhus, a zoonotic febrile illness. Orientia tsutsugamushi causes most cases of scrub typhus and is endemic to the Asia-Pacific Region, where it is transmitted by Leptotrombidium spp. chiggers. However, in Dubai, Candidatus Orientia chuto was isolated from a case of scrub typhus and is also known to circulate among rodents in Saudi Arabia and Kenya, although its vectors remain poorly defined. In addition to Orientia , chiggers are often infected with other potential pathogens or arthropod-specific endosymbionts, but their significance for trombiculid biology and public health is unclear. Results Pooled chiggers of 10 species were collected from rodents in southwestern Saudi Arabia and screened for Orientia DNA by PCR. Two species ( Microtrombicula muhaylensis and Pentidionis agamae ) produced positive results for the htrA gene, although Ca . Orientia chuto DNA was confirmed by Sanger sequencing only in P. agamae . Metagenomic sequencing of three pools of P. agamae provided evidence for two other bacterial associates: a spirochaete and a Wolbachia symbiont. Phylogenetic analysis of 16S rRNA and multi-locus sequence typing genes placed the spirochaete in a clade of micromammal-associated Borrelia spp. that are widely-distributed globally with no known vector. For the Wolbachia symbiont, a genome assembly was obtained that allowed phylogenetic localisation in a novel, divergent clade. Cytochrome c oxidase I ( coi ) gene barcodes for Saudi Arabian chiggers enabled comparisons with global chigger diversity, revealing several cases of discordance with classical taxonomy. Complete mitogenome assemblies were obtained for the three P. agamae pools and almost 50 SNPs were identified, despite a common geographic origin. Conclusions P. agamae was identified as a potential vector of Ca. Orientia chuto on the Arabian Peninsula. The detection of an unusual Borrelia sp. and a divergent Wolbachia symbiont in P. agamae indicated links with chigger microbiomes in other parts of the world, while coi barcoding and mitogenomic analyses greatly extended our understanding of inter- and intraspecific relationships in trombiculid mites. Scrub typhus chiggers metagenomics Orientia Wolbachia Borrelia mitochondrial genome Acomys dimidiatus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Chiggers, the larval stage of trombiculid mites, are miniscule ectoparasites that feed on a wide range of terrestrial vertebrates and humans are incidental hosts for some species. The two main potential clinical impacts of chigger infestations are trombiculiasis (or “scrub itch”), which is an allergic dermatitis caused by hypersensitivity reactions to mite saliva [ 1 ], and acquisition of scrub typhus, which is caused by Orientia spp. These are obligate intracellular bacteria in the family Rickettsiaceae (order Rickettsiales) maintained in chiggers as vertically transmitted symbionts [ 2 ]. Scrub typhus is the more serious chigger-related condition, since the median mortality of the untreated disease in the Asia-Pacific region (where is it is caused by Orientia tsutsugamushi ) is 6% [ 3 ]. Two other Orientia spp. are recognised although not yet formally described: Candidatus Orientia chiloensis [ 4 ], which has only been reported from Chile (~ 100 cases to date, none fatal), and Candidatus Orientia chuto, which is known from a single, nonfatal human case contracted in Dubai [ 5 ], but has also been detected in chiggers in Kenya [ 6 ] and wild rodents in Saudi Arabia [ 7 ]. Only two genera of chiggers are known to transmit Orientia spp. to humans; these are Leptotrombidium spp. across the Asia-Pacific (vectors of O. tsutsugamushi ) [ 2 ] and Herpetacarus spp. in Chile (vectors of Ca . O. chiloensis) [ 8 ]. However, Orientia spp. have been found in several other chigger genera that are thought to maintain infection in wild hosts, including Microtrombicula spp. from Kenya, in which Ca . O. chuto was detected [ 6 ]. The chigger vector(s) of Ca . O. chuto in the Arabian Peninsula remain unknown. In addition to Orientia spp., a number of other potentially pathogenic bacteria and viruses have been reported from chiggers in targeted surveys or 16S rRNA amplicon sequencing, including Bartonella spp., Rickettsia spp., Borrelia spp., Anaplasma spp., hantaviruses, and Dabie bandavirus (reviewed in [ 9 ]). Among the bacteria, the genera Rickettsia (family Rickettsiaceae) and Anaplasma (family Anaplasmataceae) are obligate intracellular organisms related to Orientia . They utilise a range of arthropod hosts (primarily ticks, mites, fleas, or lice for Rickettsia spp.; or predominantly ticks for Anaplasma spp.), and many species can be transmitted to humans, causing potentially severe disease. Major pathogens of medical significance include Rickettsia rickettsii (aetiological agent of Rocky Mountain spotted fever) and Anaplasma phagocytophilum (agent of human granulocytic anaplasmosis), both of which can be fatal if not treated promptly [ 10 ]. The spotted fever group of rickettsiae is transmitted mainly by ticks, while one member of the more recently defined transitional group of rickettsiae, Rickettsia akari (agent of rickettsialpox), is vectored by gamasid mites [ 11 ]. Detection of Rickettsia spp. DNA in chiggers has been reported from geographically diverse locations [ 12 – 15 ], but a role in transmission of rickettsiae to vertebrates has not been established, and at least some of these rickettsiae may be arthropod-specific symbionts. Interestingly, DNA of both Rickettsia spp. [ 16 ] and A. phagocytophilum [ 17 ] has been amplified from unfed chiggers, which is strong evidence for vertical transmission and long-term symbiotic relationships. The Gram-negative, facultatively intracellular genus Bartonella (order Hyphomicrobiales) comprises bacteria that infect vertebrate erythrocytes and are highly prevalent in micromammals, especially rodents. It is generally accepted that they are maintained in the mammalian populations by arthropod vectors (sandflies for Bartonella bacilliformis ; and fleas, lice, and perhaps ticks for other species) [ 18 ]. Of the many species within the genus, B. bacilliformis, Bartonella quintana , and Bartonella henselae are the most important human pathogens and only the latter (agent of cat-scratch fever) is zoonotic [ 19 ]. Other zoonotic, rodent-associated Bartonella spp. have been reported from various chigger species in Southeast Asia, but data supporting a vector role for them in human disease remain circumstantial [ 20 , 21 ]. The genus Borrelia includes the causative agents of Lyme borreliosis [also referred to as Lyme disease (LD) in the USA] and relapsing fever (RF) borreliosis [ 22 ]. These spirochaetal bacteria are commonly maintained in natural transmission cycles by tick vectors, and rodents are important reservoirs for many of the human-pathogenic species [ 23 ]. While evidence for Borrelia spp. in both trombiculid and gamasid mites has been reported, their vector status remains questionable [ 24 – 26 ]. One group of spirochaetal bacteria of uncertain taxonomic status has been found previously in the tissues of small mammals, but a vector for this micromammal-specific clade has yet to be identified [ 27 , 28 ]. Lastly, vertically-transmitted endobacteria that do not infect vertebrates (predominantly Wolbachia , Rickettsiella and Cardinium ) have been detected in several chigger microbiome studies (reviewed in [ 9 ]), all of which were performed on Asian species of trombiculids. However, any potential phenotypic effects of these symbionts (such as cytoplasmic incompatibility) [ 29 ], or inhibition or enhancement of pathogen transmission [ 30 ] by chiggers, remain unexplored. Compounding these knowledge gaps regarding the vector biology and microbiome of chiggers, the population genetics and molecular systematics of trombiculid mites remain in their infancy. Only two nuclear genomes (both from Leptotrombidium spp. [ 31 , 32 ]) and five mitogenomes (three from Leptotrombidium spp. [ 33 ]) for chiggers are publicly available. Most other genetic data for trombiculid mites consist of cytochrome c oxidase I ( coi ) DNA barcodes, but even these display poor geographic representation, with most being obtained from Southeast Asia [ 34 ], East Asia [ 35 ] and Europe [ 36 ], with none available for the Middle East. Here, we present evidence that the chigger Pentidionis agamae may be a vector of Ca . O. chuto in Saudi Arabia. Moreover, applying a metagenomic approach, we obtained complete mitogenomes from this species and place it in the phylogenetic context of other Saudi Arabian chigger species, as well as trombiculid diversity worldwide, through analysis of coi barcodes. Finally, sequences from two additional, non- Orientia bacterial associates of P. agamae are shown to represent a poorly described, micromammal-associated Borrelia sp. and a member of a novel, deep-branching clade of Wolbachia symbionts. Results Chigger sampling In total, 156 rodents were captured, belonging to six different species: Acomys dimidiatus , Meriones rex , Mus musculus , Ochromyscus yemeni and Rattus rattus (Additional File 1: Table S1 ). A total 7,329 chiggers were recovered from 27 and 55 rodents in ‘Asir and Al-Bahah provinces respectively. Of these, 4,226 chiggers belonging to 20 trombiculid species were identified (Table 1 ). The remaining chiggers were excluded from the study as they were damaged, or the important identifying features were absent. Table 1 Chigger species and numbers found at two sampling locations in Saudi Arabia. Chigger species Subfamily and tribe Province ‘Asir Al-Bahah Schoengastiella hypoderma Gahrliepiinae - 6 Walchia parvula Gahrliepiinae 1 - Odontacarus thesigeri Leeuwenhoekiinae - 3 Ascoschoengastia browni Trombiculinae: Schoengastiini 32 112 Helenicula lukshumiae Trombiculinae: Schoengastiini 171 29 Schoutedenichia asirensis Trombiculinae: Schoengastiini 5 1 Schoutedenichia originalis Trombiculinae: Schoengastiini 17 17 Schoutedenichia saudi Trombiculinae: Schoengastiini 346 153 Schoutedenichia zarudnyi Trombiculinae: Schoengastiini 227 878 Ericotrombidium caucasicum Trombiculinae: Trombiculini 411 5 Ericotrombidium kazeruni Trombiculinae: Trombiculini 735 - Microtrombicula abyssinica Trombiculinae: Trombiculini 2 - Microtrombicula felis Trombiculinae: Trombiculini 28 - Microtrombicula hoogstraali Trombiculinae: Trombiculini - 1 Microtrombicula hyracis Trombiculinae: Trombiculini 2 - Microtrombicula muhaylensis Trombiculinae: Trombiculini 305 157 Microtrombicula peltifera Trombiculinae: Trombiculini - 8 Microtrombicula saperoi Trombiculinae: Trombiculini 1 - Microtrombicula traubi Trombiculinae: Trombiculini - 15 Pentidionis agamae Trombiculinae: Trombiculini 87 471 Ca . O. chuto in P. agamae Orientia screening by qPCR ( traD ) and nested PCR ( htrA ) was performed on 165 pools of chiggers, consisting of 3,286 individuals (Additional File 1: Table S2). A single pool each of P. agamae (R9P) and M. muhaylensis (R19M) – both obtained from A. dimidiatus hosts in ‘Asir province - yielded positive amplification in the traD qPCR assay. However, Sanger sequencing of the htrA nested PCR product only produced a high-quality sequence from R9P for further analyses. The htrA sequence from R9P formed a single clade (bootstrap 100) with the htrA sequences from Ca O. chuto reported from the tissues of A. dimidiatus captured from ‘Asir Province (MR25, MR26Ki, MR26Li) in our previous study [ 7 ], and the sequences from Saudi Arabia remained in a single clade (bootstrap 86) distinct from Ca O. chuto from Dubai, United Arab Emirates (UAE) (Fig. 1 ). Genetic pairwise distance calculated between the Ca. O. chuto htrA sequences (based on 659 bp) from Saudi Arabia also showed that R9P htrA is more closely related to the sequences from ‘Asir Province (MR25, MR26Ki, MR26Li: pairwise distance = 0.003) than sequences from Al-Bahah Province (AR33 and AR43: pairwise distance = 0.017). Following this finding, the R9P pool of P. agamae was subjected to metagenomic sequencing using Illumina technology to obtain additional genes for comparison with the sequenced culture isolate of Ca . O. chuto str. Dubai [ 5 ]. Since P. agamae is a potential vector of Ca O. chuto based on the PCR screening, an additional two pools of P. agamae , Pa1 and Pa2, obtained from the ‘Asir region in a previous sampling effort [ 37 ], were also subjected to metagenomic sequencing. The Kraken2 assignment found contigs assigned to Orientia (Additional File 1: Table S3). A single contig from R9P overlapped 137 bp at the 3′ end of the htrA sequence from Sanger sequencing at 100% identity, indicating that the contig did not contain the full-length coding sequence of the gene. We did not find the htrA sequence from the contigs from Pa2; however, BLASTn analyses showed 99%-100% matches of the contigs to various O. tsutsugamushi strains (Additional File 1: Table S4). Diamond BLASTx revealed matches to a number of O. tsutsugamushi proteins, namely dihydrolipoyl dehydrogenase, toprim domain protein, conjugal transfer protein TraN, transposase and two different hypothetical proteins (Additional File 1: Table S4). None of the contigs from P. agamae pool Pa1 were verified as Orientia sequences from BLASTn analyses. Wolbachia and Rickettsia Contigs assigned to Wolbachia were also found in all three pools (Additional File 1: Table S3), with over 200 identified in Pa2. An improved Wolbachia assembly was obtained from this pool by mapping the short reads to metaSPAdes and Megahit-assembled contigs, and reassembling the mapped reads using metaSpades. This workflow resulted in a new draft assembly with BUSCO improvement from 60.4–78.8%. Maximum likelihood phylogeny placed this assembly, which we designate as w Paga, in its own clade (new supergroup X – bootstrap 100) and this was close to the more divergent clades, including supergroups W, M, L, E, and I (Fig. 2 ). As genome assemblies for other Wolbachia symbionts from acariform mites (the mould mite Tyrophagus putrescentiae [ 38 ] and the quill mite Syringophilopsis turdi [ 39 ]) were made available recently on NCBI, we included these in the phylogenomic analysis. We determined that they were both very distinct from w Paga, with w Tput from T . putrescentiae displaying closer affinities with supergroup M (Fig. 2 ), whereas w Stur from S. turdi is a member of a distinct, more distant supergroup (P) [ 39 ]. A number of contigs were also assigned as Rickettsia (Additional File 1: Table S3). However, further verification with BLASTn analyses revealed that most of these contigs either had no match to any existing sequences in GenBank, or matched with Rickettsia sequences with low percentage identity (< 95%, data not shown), suggesting the presence of more genetically distant Rickettsiales bacteria. Detection of a micromammal-associated Borrelia sp. in P. agamae All three P. agamae pools had contigs assigned as Borrelia (Additional File 1: Table S3). We recovered sequences for 16S rRNA and several genes from the multi-locus sequence typing (MLST) scheme [ 40 ] - clpX , recG and uvrA - from R9P contigs, which were used to construct phylogenetic trees with other published spirochaete sequences from GenBank. We were unable to recover these genes from Pa1 and Pa2. In the 16S rRNA phylogenetic tree (Fig. 3 ), the Borrelia sp. from R9P clustered with Borrelia spp. previously reported from micromammals (mainly rodents), namely Borrelia sp. isolates R57 [ 41 ], BRAUS (TIS 37), CA682, and ALEPB216 [ 27 , 28 ]. A discrepancy in the tree topology was observed between the current and previously published 16S rRNA trees [ 27 , 28 ]. The rodent-associated Borrelia spp., called “rodent group” here, were placed in a sister clade to the other Borrelia groups in the published studies, but clustered with the RF borreliae in our tree. This discrepancy could be due to the inclusion of different Borrelia strains available and also the impact of sequence length. With the shortest sequence [429 bp; namely the sequence from Borrelia sp. isolate BRAUS (TIS 37)] removed from the analyses, the tree topology reverted to one in which the rodent group formed a sister clade to the other Borrelia groups (Additional File 1: Fig. S1 ), similar to previous studies [ 27 , 28 ]. This observation indicates that accurate phylogenetic placement of the rodent group borreliae will require more genes from the MLST scheme [ 40 ]. Since MLST gene sequences for the rodent group Borrelia spp. are not currently available, Borrelia sp. R9P forms a sister clade to other known Borrelia spp. from the LD and RF groups in the phylogenies based on the concatenated matrix of clpX , recG and uvrA (Fig. 4 ), or each of the individual MLST genes (Additional file 1: Fig. S2). Mitochondrial assembly Circularised mitochondrial genomes were assembled separately from Pa1 and Pa2 (14,755 bp). The MitoZ pipeline produced a linear mitochondrial assembly for R9P, which was then circularised (14,753 bp) with an additional step based on overlapping sequences at the end of the linear assembly. Both assemblies from Pa2 and R9P appeared to be almost identical to Pa1 (Additional File 1: Fig. S3), with 99.92% and 99.76% identity, respectively. The base composition of the mitochondrial genomes was approximately 45% (A), 25% (T), 10% (C), and 20% (G). Maximum likelihood phylogeny based on a partial coi gene fragment, combining data from the mitogenomic assemblies and additional coi PCR products from archived specimens, placed P. agamae in a single clade with Schoutedenichia centralkwangtunga (KY971498.1) from Laos and Walchia hayashii (NC010595.1) from Japan, with a bootstrap value of 94 (Fig. 5 ). This is surprising, as Walchia belongs to a different subfamily (Gahrliepiinae) than Pentidionis and Schoutedenichia ( Trombiculinae ), and the two latter genera belong to different tribes (Trombiculini and Schoengastiini, respectively) [ 42 , 43 ]. Further inconsistencies of species placements were observed in this phylogeny. For instance, W. hayashii (represented by a complete mitogenomic assembly but lacking an accompanying publication) did not cluster within the clade containing the other Walchia spp. (all from Southeast Asia), and Schoutedenichia sp. D454 (OQ924405.1) from Albania seemed more closely related to Blankaartia acuscutellaris from Laos instead of S. centralkwangtunga. To provide the first molecular taxonomic data for chiggers from the Middle East, we generated coi barcodes for Ericotrombidium caucasicum , E. kazeruni , Ascoschoengastia browni , Microtrombicula felis , M. peltifera , M. traubi , M. muhaylensis , Schoutedenichia zarudnyi , S. saudi , and Helenicula lukshumiae , which were described from Saudi Arabia in our previous studies [ 7 , 44 ]. For Ericotrombidium spp., Microtrombicula spp., and Helenicula spp., these were the first barcodes available for each genus and comparisons with congeneric species were thus not possible, although in each case, the genus formed a monophyletic group (Fig. 5 ). However, while the two Schoutedenichia spp. from Saudi Arabia clustered with S. centralkwangtunga , A. browni displayed closer affinities with Hirsutiella zachvatkini from Poland than to Ascoschoengastia indica from Thailand/Laos. Interestingly, the H. lukshumiae specimens were placed on a deep branch despite the classification of Helenicula in the tribe Schoengastiini with Schoutedenichia and Ascoschoengastia (Fig. 5 . Additional File 1: Table S1 ). Nevertheless, the placements of Ericotrombidium and Hirsutiella appear to conform with the current classification system of chiggers based on larval morphology: (i) clustering of Ericotrombidium with Leptotrombidium (the former genus was described as a subgenus of the latter); (ii) clustering of H. zachvatkini with Neotrombicula ( Hirsutiella is considered as a subgenus of Neotrombicula by some authors [ 45 , 46 ]); and (iii) clustering of A. indica with Microtrombicula ( Ascoschoengastia and Microtrombicula , although they belong to different tribes, in fact differ from each other by a single trait – trichobothria that are expanded in the former genus and flagelliform in the latter [ 47 ]). The affinity between two chigger species, namely N. gallinarum (tribe Schoengastiini) and B. acuscutellaris (tribe Trombiculini), which prefer avian hosts despite belonging to strikingly different genera and different tribes, was also noteworthy. Annotation of the assembly from Pa1 yielded thirteen protein CDS, two rRNAs and sixteen tRNAs (Fig. 6 , Additional file 1: Fig. S4). We were able to identify the six missing tRNAs ( trnL1 , tnrL2 , trnA , tnrR , trnG and trnV ) by manually inspecting the conserved anti-codon regions in the alignments between the current assembly and the other five available mitochondrial assemblies from trombiculid mites (Additional file 1: Fig. S5). However, their predicted secondary structures appeared to have no T-arms and hence lack the typical clover leaf structure, or appeared to be extremely truncated (for trnA ). Relative to Pa1, 12 SNPs were observed in Pa2 and 36 SNPs (including two deletions) were detected in R9P. The SNPs in the Pa2 assembly were found in trnT , as well as in the coi , cob and nad5 genes, causing non-synonymous substitutions in these protein CDS (Additional File 1: Table S6). A single non-synonymous substitution was observed in coi , while two non-synonymous substitutions were found each for cob and nad5 . When gene arrangements were analysed, the mitochondrial genome from P. agamae displayed closest synteny to W. hayashii , with rearrangement of the positions of the control region and trnQ (Fig. 7 ). The control region for P. agamae lies upstream of rrnS , and trnQ lies downstream of rrnL . Unlike the mitochondrial genomes for Ascoschoengastia sp. TATW-1, Leptotrombidium deliense or Leptotrombidium pallidum , there was no duplication of any mitochondrial genes in P. agamae . Discussion The ecology of Ca O. chuto has remained enigmatic since its discovery over a decade ago. The endemic region of this pathogen is potentially vast, with evidence of circulation across the Arabian Peninsula [ 5 , 7 ], East Africa [ 6 ], and perhaps West Africa [ 48 ]. Despite this wide range, only one human case of scrub typhus caused by this species has been reported, which was contracted in Dubai [ 5 ]; however, no studies on Ca . O. chuto in chiggers or non-human vertebrate hosts in the UAE have been published to date. Following the publication of details of the clinical isolate of Ca . O. chuto from Dubai [ 5 ], pathogen DNA was detected in one pool of Microtrombicula spp. chiggers (of five pools from multiple host species screened), which was obtained from a Natal multimammate mouse ( Mastomys natalensis ) in Baringo county, Kenya [ 6 ]. More recently, Ca . O. chuto DNA was amplified from the tissues of 7.3% ( n = 82) rodents (Eastern spiny mice - Acomys dimidiatus , or Wagner’s gerbil - Dipodillus dasyurus ) trapped in ‘Asir and Al-Bahah provinces of Saudi Arabia [ 7 ]. Most of the positive rodents lacked chigger infestations, but chiggers of five species ( E. caucasicum, E. kazeruni, S. saudi, S. zarudnyi , and M. hoogstraali ) were obtained from two infected individuals and were shown to be negative for Orientia DNA. The current study, in which a much more extensive collection of Saudi chiggers was screened, represents the first report of Ca . O. chuto DNA from potential vector species from the Arabian Peninsula (albeit > 1,500 km distant from the clinical case reported from Dubai). Since the htrA PCR amplicon from the pool of M. muhaylensis failed to provide a high-quality sequence, the apparent positive result for this chigger species remains provisional. However, it is noteworthy that this species is in the same genus as the positive chigger pool reported from Kenya [ 6 ]. For P. agamae , molecular evidence for infection with Ca . O. chuto was obtained using Sanger sequencing of a htrA nested PCR product and metagenomic sequencing via Illumina short-read technology. Unfortunately, no additional Orientia genes could be assembled using the Illumina data from P. agamae pool R9P to obtain further phylogenetic information for comparison with the Dubai isolate. This means that current data for Ca . O. chuto in Saudi Arabia is comprised of only six sequences from a single gene [ 7 ], and the Kenyan data by two gene sequences (16S rRNA and htrA ) from a single sample [ 6 ]. Nevertheless, the htrA sequences exhibit differences that correspond to geographic distance, with the R9P sequence from P. agamae collected in ‘Asir clustering with sequences from rodents obtained from the same province, and genetic distances increasing stepwise compared with sequences from Al-Bahah province, UAE, or Kenya, respectively. The lack of other Orientia sequence data in pool R9P suggests a very low level of Ca . O. chuto DNA in this sample, perhaps representing only a single positive chigger; neither can we rule out traces of host-derived pathogen DNA from mite mouthparts or gut contents in the absence of systemic chigger infection. Future studies could attempt to obtain additional Orientia genome data by sequence capture with DNA extracts from human samples and chigger specimens, which has been performed successfully using specific probes for O. tsutsugamushi [ 49 ]. Interestingly, while an htrA sequence could not be recovered from the metagenomic dataset for another P. agamae pool, Pa2, several other Orientia genes were identified in this sample. These had closest matches to O. tsutsugamushi sequences, especially the Karp-like strain UT176, which is a clinical isolate from Thailand [ 50 ]. However, genes from the multi-locus sequence typing scheme for O. tsutsugamushi were not assembled and caution is needed in interpreting these data as evidence of O. tsutsugamushi in Saudi Arabia, as the only Ca . O. chuto genome assembly available (str. Dubai) is incomplete [ 5 ]. Notwithstanding this limitation, the detection of Orientia sequences in this second pooled DNA sample from P. agamae adds to the evidence that this chigger species may act as a vector, at least between wild hosts. Unfortunately, in common with other chigger species in the Middle East, P. agamae is poorly studied with limited host records. Prior to our rodent studies in Saudi Arabia, P. agamae was only known from agamid lizard hosts in the Persis region of Iran [ 51 ] and around Lake Tiberias (Galilee) [ 52 ], although it is widespread on A. dimidiatus in both ‘Asir [ 37 ] and Al-Bahah [ 44 ] provinces. Whether P. agamae could bite humans and act as a clinically-relevant scrub typhus vector is an important open question, especially as the mountainous regions of southwest Saudi Arabia are popular destinations for tourists seeking cooler temperatures in the summer months. With respect to the origin of the only confirmed case of scrub typhus in the Middle East, limited data are available on the chigger fauna of UAE, with four chigger species reported recently from a very small sample of A. dimidiatus ( n = 3) [ 53 ]. However, P. agamae was not present among these. In the past five years, interest in the trombiculid mite microbiome has blossomed on the back of technological advances that have enabled 16S rRNA amplicon sequencing studies on low-input DNA samples. The current study constitutes the first genuine metagenomic analysis of a trombiculid mite since the publication of the Leptotrombidium deliense genome [ 31 ], thus providing the potential to obtain multiple gene sequences or even genome assemblies for members of the chigger microbiome. Here, we found a Wolbachia symbiont of P. agamae ( w Paga) to be sufficiently represented to allow a genome assembly and phylogenomic analysis. Wolbachia has been detected previously from trombiculid mites in Southeast Asia and East Asia using 16S rRNA amplicon sequencing [ 16 , 54 ], but the use of a single conserved gene has precluded robust phylogenetic placement. Our data locate w Paga firmly among the early-branching clades of Wolbachia that have been poorly studied compared with the ubiquitous, so-called “pandemic” supergroups (A and B) [ 55 ], but it is sufficiently distinct to constitute the first member of a new supergroup. Unfortunately, we did not recover the 16S rRNA sequence from w Paga, but its position on a long branch is consistent with that of a previous reported symbiont from Leptotrombidium scutellare in Japan [ 16 ]. It has been hypothesized that Wolbachia evolved in the soil milieu [ 56 ] through associations with parasitic nematodes of plants (supergroup L [ 57 ]) or saprotrophic flies (W [ 58 ]), and may have been horizontally transmitted via plants, honeydew, and/or insect carcasses to other hosts of early-branching Wolbachia clades including the banana aphid (M), springtails (E), oribatid mites (E), and fleas (I) – the latter being detritivorous in the larval stage. In accordance with this model, the free-living lifecycle of trombiculid mites proceeds underground, where the nymphal and adult stages predate small edaphic arthropods or their eggs. We also assigned a phylogenetic placement to another Wolbachia symbiont of mite origin, w Tput from T. putrescentiae , which was close to supergroup M but may be a member of another novel clade. While renowned as a pest of stored foodstuffs, T . putrescentiae is also common in outdoor agricultural biomes [ 59 ] and is likely to share habitats with other hosts of the non-pandemic Wolbachia clades listed above. In addition to Wolbachia , Borrelia spp. have been reported from trombiculid mites from several locations worldwide. Spirochaetes of the LD clade have been detected molecularly in harvest mites ( Neotrombicula autumnalis ) in Europe [ 24 ] and this chigger has been shown to acquire borreliae experimentally from infected rodents [ 26 ]. There is also some evidence for vertical transmission of LD borreliae in harvest mites [ 24 , 26 ], while unassigned Borrelia spp. 16S rRNA sequences have been detected at high prevalence in chiggers collected from wild micromammals in Thailand [ 54 , 60 ]. In the current study, we were able to acquire multiple gene sequences for a chigger-associated Borrelia spp. for the first time, allowing robust phylogenetic classification. Surprisingly, the sequences associated with P. agamae were not of LD or RF Borrelia spp. origin but belonged to a clade associated with rodents and shrews previously reported from Spain [ 41 ], California [ 28 ], and New South Wales [ 27 ]. On the basis of 16S rRNA and groEL gene sequences, this clade (originally described from Spain as isolate R57) has been known to be distinct from the LD and RF groups for nearly two decades, but its biology has remained enigmatic. Importantly, it has never been detected in arthropods or mammalian blood, but only ear punch biopsies. Our data suggest that chiggers (many species of which have a predilection for the pinna and ear canal as feeding sites) [ 61 ] may be the vector for this micromammal clade of borreliae. While we cannot rule out that the Borrelia spp. DNA is an incidental finding due to ingestion of host tissue fluid by chiggers, the fact we could assemble several genes from the organism coupled with the absence of prior PCR detection in hard ticks that are often contaminated with host skin, renders this possibility less likely. In the past five years, molecular barcoding (primarily based on the mitochondrial coi gene) has been applied to chigger mites to determine whether low-throughput morphological identification can be supplanted, or at least complemented, by less laborious procedures. The first study to analyse coi barcodes from multiple chigger species, which was conducted in South-East Asia, demonstrated that the technique reliably binned individual specimens by morphotyped species and clustered subgenera in cognate groups [ 34 ]. However, certain species exhibited multiple haplotypes, sometimes even if recovered from the same individual host. Importantly, barcoding studies of European chiggers have revealed clear cases both of phenotypic plasticity within trombiculid species (which is linked to the host species used for larval development) [ 62 ] and cryptic diversity, where single chigger morphotypes show genetic distances similar to that between recognised species [ 63 ]. In the current study, while most species clustered by subgenus when compared with published barcodes, Ascoschoengastia spp. and Schoutedenichia spp. were striking exceptions. Moreover, the system of subfamilies and tribes within the family Trombiculidae that has existed for over half-a-century was not reflected in the coi -based phylogeny. A clear example of this was the apparent affinity of Pentidionis with Schoutedenichia , despite their classification in different tribes (Trombiculini and Schoengastiini, respectively). While these findings suggest that the classification of trombiculid mites based on larval morphology has significant limitations, phylogenetic relationships cannot be resolved using a single mitochondrial gene, and there is an urgent need to develop multi-locus-based approaches to trombiculid taxonomy. Here, we were able to successfully generate complete mitogenomic assemblies from three pools of P. agamae , which is the first time multiple mitogenomes from a single chigger species have been obtained for intraspecific comparisons. Notably, we found tRNA gene annotation to be dependent on manual comparisons with available trombiculid mitogenomes due to previously recognised non-canonical features of these genes in multiple acariform taxa [ 64 – 66 ]. Several SNPs were identified between pools of P. agamae , including non-synonymous substitutions, despite the mites being collected from the same province. Unfortunately, the paucity of whole mitogenome data from other trombiculid species severely limited interspecific comparisons. This is particularly problematic, as of the five other complete mitogenomes available from trombiculid mites, three are from a single genus ( Leptotrombidium spp.) [ 33 ], and one of the non- Leptotrombidium assemblies is from a mite identified to genus level only ( Ascoschoengastia sp. TATW-1). While mitochondrial gene order in P. agamae was most closely related that of W. hayashii , the phylogenetic position of the latter in the coi tree was unexpected, as it did not cluster with published sequences available for five Walchia spp. from South-East Asia. No information on how W. hayashii specimens were identified prior to sequencing is available, as the mitogenome record on NCBI is not linked to a publication and the depositors are no longer active in research. Thus, it is unclear if this is a case of misidentification or if the subgenus Walchia is paraphyletic. Since we have demonstrated that assembly of chigger mitogenomes is feasible using ethanol-preserved pools and Illumina technology, which is declining rapidly in cost per sample, we hope these results with spur routine sequencing of trombiculid mitogenomes. Indeed, this has happened already for ticks, revolutionising phylogenetics for the Ixodidae and Argasidae [ 67 ]. However, it is important that phylogenetically-informative nuclear markers such as ITS2 are also utilised due to differing evolutionary rates between nuclear and mitochondrial genomes [ 68 ], the potential for vertically-transmitted symbionts such as Wolbachia to cause cytonuclear discordance [ 69 ], and the possibility that trombiculid species may hybridise [ 70 ]. Conclusions Our PCR-based screening and sequencing of chigger mites from Saudi Arabia has revealed P. agamae as a potential vector of Ca . O. chuto, but further research is required to determine if this species may be anthropophilic and thus important in scrub typhus epidemiology in the Middle East. Moreover, this first metagenomic analysis of a trombiculid mite outside the genus Leptotrombidium has enabled deeper insights into chigger-associated Wolbachia and Borrelia bacteria that were only known previously from 16S rRNA gene data, as well as providing a reference mitogenome for the genus Pentidionis and initial evidence for intraspecific variation. Overall, the metagenomic approach we applied here has demonstrated its potential to generate complete mitogenomes for phylogenetic and population genetic studies of trombiculids with relative ease; furthermore, it can greatly improve our understanding of chigger microbiomes that so far have been studied predominantly by 16S rRNA amplicon-based methods. Methods Chigger collection and identification Rodents were trapped overnight in southwestern Saudi Arabia at sites in ‘Asir (October 2020) and Al-Bahah (August 2021) provinces (Fig. 8 ) as described previously [ 7 ]. Rodents were euthanized by inhalational anaesthetic isoflurane overdose or dislocated in the cervical region. The identification of rodents was based on morphological features and confirmed molecularly through the amplification of cytB gene fragment [ 7 ]. Each rodent was carefully inspected for chiggers including inside ears, removed chiggers were preserved in 70% ethanol. The fieldwork was approved by the Saudi Wildlife Authority (approval no. 288/33/A) and Animal Welfare and Ethics Review Board of the University of Liverpool. As representative specimens, 10% of chiggers were selected by purposive sampling and fixed permanently using Berlese fluid (TCS Bioscience Ltd, Buckingham, UK). The measurements and identification of chiggers were performed on a fluorescence microscope (ZEISS Axio Imager M2 microscope through GT Vision GXCapture-T software). The remaining chiggers were identified without the usage of mountant and pooled on the basis of species from each rodent (Additional File 1: Table S2). Molecular detection of Orientia sp. Genomic DNA from chigger pools were extracted using the Qiagen DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol. DNA concentration and quality were assessed by a Qubit High Sensitivity dsDNA Quantification Assay kit (Invitrogen) and NanoDrop One/One C Microvolume UV-Vis Spectrophotometer (Thermo Scientific). A quantitative PCR assay (qPCR) targeting the multicopy traD gene was used in the initial screening of chigger pools for detection of Orientia sp. [ 71 ]. Positive samples were subjected to a nested PCR assay for amplification of the htrA gene [ 6 ]. The PCR amplicons were purified and submitted to Eurofins Genomics ( https://www.eurofins.com ) for Sanger sequencing in both directions. Paired sequences were aligned to generate a corrected consensus and manually quality-trimmed using Bioedit 7.2.5 [ 72 ] to produce the final sequence for phylogenetic analyses. Illumina sequencing Illumina library preparation and sequencing from chigger pools were performed at the Centre for Genomic Research (CGR) at the University of Liverpool. NEBNext Ultra II FS kit paired- end libraries (2×150 bp) with a 350 bp insert were generated and sequenced on an Illumina NovaSeq 6000 using SP or S4 chemistry. The CGR performed the following read curation: the raw fastq files were trimmed for the presence of Illumina adapter sequences using Cutadapt v1.2.1 [ 73 ] with option -O 3; the reads were further trimmed using Sickle v1.200 with a minimum window quality score of 20 ( https://github.com/najoshi/sickle ); and reads shorter than 15 bp after trimming were removed. Metagenomic assembly and taxonomic classification. Trimmed paired-end Illumina reads were assembled using metaSPAdes [ 74 ] or Megahit [ 75 ] genome assemblers. When the memory requirements for metaSPAdes exceeded the memory available on our servers, we removed the reads mapped to A. dimidiatus (GCA_907164435.1) to reduce the proportion of animal host sequences and used the unmapped reads for assembly with metaSPAdes. Short-read mapping was performed using bowtie2 v2.5.1 [ 76 ]. We applied Kraken2 classification using the NCBI non-redundant nucleotide database (02/05/2023) for taxonomic classification. Contigs classified to taxon of interest were extracted from the Kraken2 output using the “extract_kraken_reads. py” script from KrakenTools [ 77 ]. Sequence annotation for further verification of the assigned contigs was performed by a DIAMOND BLASTX v2.0.14.152 [ 78 ] search against the NCBI non-redundant protein database and BLASTn search against the NCBI non-redundant nucleotide database. For Wolbachia assembly, paired-end read sequences were separately mapped to the contigs produced from metaSPAdes and Megahit using bowtie2 and re-paired and merged using FLASH v1.2.11 [ 79 ]. Mapped reads were reassembled with metaSPAdes, and Blobtoolkit v4.2.1 was used to remove eukaryotic sequences and sequences with no BLASTn hits in the assembly [ 80 ]. Prokka v1.14.6 was used for gene prediction and annotation [ 81 ]. Genome completeness was assessed by the Benchmarking Universal Single-Copy Orthologs (BUSCO) pipeline 5.0 and the rickettsiales_odb10 database [ 82 ]. Mitochondrial genome assembly The P. agamae mitochondrial genome was assembled using the MitoZ toolkit v3.3 [ 83 ] with additional annotations of protein coding sequences (CDS), ribosomal RNA (rRNA) and transfer RNA (tRNA) sequences using the MITOS2 web service [ 84 ], which produces circular and non-circular assemblies. The non-circular assembly was artificially circularised using the Simple-Circularise python script ( https://github.com/Kzra/Simple-Circularise ). Missing tRNAs were identified by aligning the assembly with existing mitochondrial genomes from trombiculid mites available in NCBI GenBank ( Leptotrombidium pallidum , AB180098.1; Leptotrombidium deliense , AB194044.1; Leptotrombidium akamushi , NC_007601.1; Walchia hayashii , NC_010595.1; and Ascoschoengastia sp. TATW-1, AB300501.1) and manually inspecting the presence of conserved regions for tRNAs. Alignments of putative tRNA sequences were performed with MAFFT v6.864b [ 85 ] as described above and visualised using Jalview v2 [ 86 ]. RNA secondary structures were predicted using mfold [ 87 ] in the UNAFold web service ( http://www.unafold.org ) and the predicted structures for the tRNAs from L. pallidum [ 88 ] as a reference. The mitochondrial assembly was visualised using the web version of OGDRAW [ 89 ] to produce the circular genome plot in Fig. 6 . Alignments of the mitochondrial assembly and the detection of single nucleotide polymorphisms (SNPs) were performed using nucmer and dnadiff from the MUMmer4 package [ 90 ]. Genome synteny was visualised using genoPlotR [ 91 ]. Existing chigger mitogenomes analysed for gene arrangements comprised Ascoschoengastia sp. TATW-1, W. hayashii , L. deliense and L. pallidum (accession nos. above). Sequences for the cytochrome oxidase I ( coi ) gene were generated from pooled archived chigger specimens described in our earlier studies [ 37 , 44 ] by PCR amplification using the HCO2198 and LCO1490 primers [ 92 ] followed by Sanger sequencing. Phylogenetic tree construction and genetic pairwise distance calculation Sequences for genes of interest were aligned using MAFFT v6.864b [ 85 ] along with existing sequences from NCBI GenBank. Concatenated alignments and partition files were generated using FASconCAT-G ( https://github.com/PatrickKueck/FASconCAT-G ). Maximum-likelihood phylogenies were produced from the single or concatenated nucleotide alignments using IQTREE v2.2.2.9[ 93 ] with 1,000 ultra-fast bootstraps [ 94 ] and best model selection from ModelFinder [ 95 ]. The Interactive Tree of Life online tool was used to visualize the consensus trees produced ( https://itol.embl.de ) and to generate the tree figures. Numbers at nodes represent ultra-fast bootstrap values and tree scales represent number of nucleotide substitutions. Genetic pairwise distances were calculated from alignments using the DistanceCalculator class from the Bio.Phylo.TreeConstruction module in Biopython v1.79 [ 96 ]. For the Wolbachia phylogenomic tree, Orthofinder v2.5.4 was used to produce a set of orthologous sequences [ 97 ]. Protein sequences for each single-copy orthogroup (OG) were aligned using MAFFT v7.149b [ 85 ]. Gblocks v0.91b was used to trim noisy or poorly aligned protein positions [ 98 ]. The trimmed alignments were concatenated into a supermatrix used to construct maximum likelihood trees in IQTREE v2.1.2. We used ModelFinder within IQTREE to determine the appropriate model for each protein. Branch support was calculated using the following options in IQTREE: (i) ultra- fast bootstrap, (ii) SH- aLRT support, (iii) local bootstrap support and (iv) aBayes Bayesian support, with all options set to 1,000, and all options produced highly similar values. Values from the ultra-fast bootstrap option [ 99 ] were displayed along with the consensus trees in the final figures. Declarations Ethics approval and consent to participate: The fieldwork was approved by the Saudi Wildlife Authority (approval no. 288/33/A) and Animal Welfare and Ethics Review Board of the University of Liverpool. Consent for publication: Not applicable Availability of data and materials: Sequencing reads and assembled sequences produced in this study have been deposited in NCBI GenBank with the BioProject accession number PRJNA1031942. The following sequences were also deposited in NCBI Genbank: The Orientia sp. htrA sequence was deposited with the accession number OR966881. Borrelia sp. sequences were deposited with accession numbers OR817655 and OR817732-OR817734. Chigger mitochondrial coi sequences were deposited with accession numbers OR820617-OR820651. The mitochondrial genome assembly for P. agamae Pa1 was deposited with the accession number OR817658. Competing interests: The authors declare no competing interests. Funding:  This work was funded by a doctoral scholarship from Imam Mohammad Ibn Saud Islamic University to H.A.A., and A.S. is funded by New England Biolabs. A.N.A is supported by the Researchers Supporting Project number (RSPD2023R602), King Saud University, Riyadh, Saudi Arabia. A.A.S. is supported by the Ministry of Science and Higher Education of the Russian Federation (cooperative agreement No. 122031100263-1). Authors' contributions: H.A.A., S.Q.A. and B.L.M. designed the study; H.A.A., S.Q.A. and A.N.A. selected field sites and trapped rodents; H.A.A., S.Q.A. and A.A.S. identified rodents and mites; H.A.A. and S.Q.A. performed DNA extractions, PCR assays and sequence analysis; H.A.A., A.S., J.J.K. and A.C.D. conducted metagenomic and phylogenetic/genomic analyses; J.J.K assembled and analysed mitochondrial sequences; B.L.M. supervised the study; H.A.A., B.L.M. and J.J.K. wrote the first manuscript draft. G.M., A.S, A.A.S, A.N.A., and A.C.D reviewed and edited the manuscript. All authors read and approved the manuscript. The authors declare no conflict of interest. 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Supplementary Files AlkathiryAdditionalFile1.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 29 Feb, 2024 Reviews received at journal 28 Feb, 2024 Reviews received at journal 11 Feb, 2024 Reviewers agreed at journal 29 Jan, 2024 Reviewers agreed at journal 27 Jan, 2024 Reviewers agreed at journal 27 Jan, 2024 Reviewers invited by journal 23 Jan, 2024 Editor assigned by journal 22 Jan, 2024 Editor invited by journal 16 Jan, 2024 Submission checks completed at journal 16 Jan, 2024 First submitted to journal 05 Jan, 2024 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-3837555","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267530539,"identity":"89f3e89e-48df-4976-b341-0ed35c4ee375","order_by":0,"name":"Hadil A. Alkathiry","email":"","orcid":"","institution":"Imam Mohammad Ibn Saud Islamic University","correspondingAuthor":false,"prefix":"","firstName":"Hadil","middleName":"A.","lastName":"Alkathiry","suffix":""},{"id":267530540,"identity":"ffeeaa34-1625-438d-8454-46acdb9ef1bf","order_by":1,"name":"Samia Q. Alghamdi","email":"","orcid":"","institution":"Al-Baha University","correspondingAuthor":false,"prefix":"","firstName":"Samia","middleName":"Q.","lastName":"Alghamdi","suffix":""},{"id":267530541,"identity":"278a94a0-63ed-42e0-95e3-2baa99154a57","order_by":2,"name":"Amit Sinha","email":"","orcid":"","institution":"New England Biolabs (United States)","correspondingAuthor":false,"prefix":"","firstName":"Amit","middleName":"","lastName":"Sinha","suffix":""},{"id":267530542,"identity":"9135328d-fb72-4d61-aca3-f15e384f5ab0","order_by":3,"name":"Gabriele Margos","email":"","orcid":"","institution":"Bavarian Health and Food Safety Authority","correspondingAuthor":false,"prefix":"","firstName":"Gabriele","middleName":"","lastName":"Margos","suffix":""},{"id":267530543,"identity":"243f303b-c2c9-407c-b0ab-c0f743958d04","order_by":4,"name":"Alexandr A. Stekolnikov","email":"","orcid":"","institution":"Zoological Institute of the Russian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Alexandr","middleName":"A.","lastName":"Stekolnikov","suffix":""},{"id":267530544,"identity":"cb299699-e5f1-46d8-a0fb-beaec7dd6e8b","order_by":5,"name":"Abdulaziz N. Alagaili","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Abdulaziz","middleName":"N.","lastName":"Alagaili","suffix":""},{"id":267530545,"identity":"65d8aad6-fd75-401d-b6a4-5d138491a933","order_by":6,"name":"Alistair C. Darby","email":"","orcid":"","institution":"University of Liverpool","correspondingAuthor":false,"prefix":"","firstName":"Alistair","middleName":"C.","lastName":"Darby","suffix":""},{"id":267530546,"identity":"8f797af1-fac6-4cd9-9f0e-ea024cd562f2","order_by":7,"name":"Benjamin L. Makepeace","email":"","orcid":"","institution":"University of Liverpool","correspondingAuthor":false,"prefix":"","firstName":"Benjamin","middleName":"L.","lastName":"Makepeace","suffix":""},{"id":267530547,"identity":"6a54d4b8-ace1-4351-83a4-152e274e3596","order_by":8,"name":"Jing Jing Khoo","email":"data:image/png;base64,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","orcid":"","institution":"University of Liverpool","correspondingAuthor":true,"prefix":"","firstName":"Jing","middleName":"Jing","lastName":"Khoo","suffix":""}],"badges":[],"createdAt":"2024-01-05 15:02:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3837555/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3837555/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49892363,"identity":"54b3359a-0b2f-4652-95c6-a849d46d9361","added_by":"auto","created_at":"2024-01-19 20:53:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":24119,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood tree of \u003cem\u003eOrientia htrA\u003c/em\u003esequence detected from \u003cem\u003eP. agamae\u003c/em\u003e R9P pool (in bold) from Saudi Arabia. Tree was constructed based on 1,500 nucleotide positions and the best-fit model according to BIC: K3Pu+F+I+R2. The tree was rooted mid-point. Ultra-fast bootstrap values above 80 are displayed on branches.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3837555/v1/969c177ce497500d80eb302c.png"},{"id":49892975,"identity":"7faa52ef-b1f1-4f0a-9586-2c1dcc3b7d5c","added_by":"auto","created_at":"2024-01-19 21:01:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":126210,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood phylogeny based on the concatenated alignments of 35 single copy orthologs (4,365 amino acid sites) from \u003cem\u003eWolbachia\u003c/em\u003e using a partitioned best-fit model for each ortholog. Letters represent the major clades or supergroups; \u003cem\u003ew\u003c/em\u003ePaga (in green type) constitutes new supergroup X and is distinct from the symbionts of \u003cem\u003eTyrophagus putrescentiae\u003c/em\u003e (\u003cem\u003ew\u003c/em\u003eTput) and \u003cem\u003eSyringophilopsis turdi\u003c/em\u003e (\u003cem\u003ew\u003c/em\u003eStur) (in orange type).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3837555/v1/81161ba5763984038cacd62f.jpeg"},{"id":49893276,"identity":"17bb158e-d22b-4aa5-a872-b1bf7f66545b","added_by":"auto","created_at":"2024-01-19 21:09:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63843,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood tree of spirochaete \u003cem\u003e16S rRNA\u003c/em\u003e sequence detected from \u003cem\u003eP. agamae\u003c/em\u003e R9P pool (in bold) from Saudi Arabia. Tree was constructed with 1,866 nucleotide sites and the best-fit model according to BIC was TIM3+F+R3. Ultra-fast bootstrap values above 80 are indicated with black circles on the branches. The tree was rooted mid-point.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3837555/v1/4ee8620260fbb45cf401e371.png"},{"id":49892974,"identity":"87098780-db91-400d-9bdc-783f20067e07","added_by":"auto","created_at":"2024-01-19 21:01:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6806,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum-likelihood tree of Spirochaetia including \u003cem\u003eP. agamae \u003c/em\u003e(in bold) based on 3,669 bp of concatenated \u003cem\u003eclpX\u003c/em\u003e, \u003cem\u003erecG\u003c/em\u003e and \u003cem\u003euvrA \u003c/em\u003esequences, with best-fit model determined for each gene separately. The tree was rooted mid-point. Accession numbers for MLST gene sequences used in analyses were given in Table S5 (Additional file 1)\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3837555/v1/6982720fe743ee37ad31c6f6.png"},{"id":49892371,"identity":"0d6b43eb-46ff-4d95-af31-2bcc1b61a8ad","added_by":"auto","created_at":"2024-01-19 20:53:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":145498,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood tree based on partial cytochrome oxidase I gene (\u003cem\u003ecoi\u003c/em\u003e) of Trombiculidae. Sequences generated in this study were highlighted in blue. Phylogeny was constructed based on 397 nucleotide positions and best-fit model according to BIC: TIM+F+I+G4. Ultrafast bootstrap values between 90 and 100 are indicated. \u003cem\u003eBdellidae \u003c/em\u003esp. was used as an outgroup.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3837555/v1/1617e0a995a338d6aae9e0bb.png"},{"id":49892364,"identity":"f2832d02-f037-4a79-8ad9-d63ee1c12249","added_by":"auto","created_at":"2024-01-19 20:53:38","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":614855,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondrial genome from \u003cem\u003eP. agamae\u003c/em\u003e. Outer ring shows CDS on the (+)-strand; inner ring shows CDS on the (-)-strand. The grey circle represents the GC content from low to high (outermost to innermost) and the circle inside the plot indicates the 50% threshold.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3837555/v1/e86c267f859d95004af527ac.jpeg"},{"id":49893764,"identity":"d4db340a-ca6d-41e9-a670-448e8a1066fc","added_by":"auto","created_at":"2024-01-19 21:17:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":73143,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondrial genomes of trombiculid mites. The assembly for \u003cem\u003eP. agamae\u003c/em\u003ewas generated from the current study. Annotations for tRNAs are not shown in the figure. Blocks above the black line indicate genes on the (+)-strand, while blocks under the black line indicate genes on the (-)-strand. Blue blocks indicate the positions for protein CDS. Yellow blocks indicate the positions for rRNA. Pink blocks indicate the positions of control regions. Genomes were linearised at the position of the \u003cem\u003ecoi \u003c/em\u003egene to facilitate comparisons.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3837555/v1/371ae519d77412f8536430a7.png"},{"id":49892367,"identity":"52942bea-b48d-47e2-a8b7-359148b07671","added_by":"auto","created_at":"2024-01-19 20:53:38","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":211965,"visible":true,"origin":"","legend":"\u003cp\u003eA map of Saudi Arabia highlighting ‘Asir and Al-Bahah provinces (outlined in red) where rodent sampling was conducted.\u003c/p\u003e","description":"","filename":"floatimage83.png","url":"https://assets-eu.researchsquare.com/files/rs-3837555/v1/02df1e5cbbbbbd99c81617e9.png"},{"id":49894006,"identity":"ffdb268d-2134-43ed-be1b-f174b95b43d0","added_by":"auto","created_at":"2024-01-19 21:25:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2477648,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3837555/v1/e7512c76-77c4-46e0-bc8c-3c7f611d9a7f.pdf"},{"id":49892366,"identity":"aed81b8c-e73e-416e-833e-52be672c4dd9","added_by":"auto","created_at":"2024-01-19 20:53:38","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1260041,"visible":true,"origin":"","legend":"","description":"","filename":"AlkathiryAdditionalFile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3837555/v1/b2e5992c6b8262a32404e4cf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Microbiome and mitogenomics of the chigger mite Pentidionis agamae: Potential role as an Orientia vector and associations with divergent clades of Wolbachia and Borrelia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChiggers, the larval stage of trombiculid mites, are miniscule ectoparasites that feed on a wide range of terrestrial vertebrates and humans are incidental hosts for some species. The two main potential clinical impacts of chigger infestations are trombiculiasis (or \u0026ldquo;scrub itch\u0026rdquo;), which is an allergic dermatitis caused by hypersensitivity reactions to mite saliva [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], and acquisition of scrub typhus, which is caused by \u003cem\u003eOrientia\u003c/em\u003e spp. These are obligate intracellular bacteria in the family Rickettsiaceae (order Rickettsiales) maintained in chiggers as vertically transmitted symbionts [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Scrub typhus is the more serious chigger-related condition, since the median mortality of the untreated disease in the Asia-Pacific region (where is it is caused by \u003cem\u003eOrientia tsutsugamushi\u003c/em\u003e) is 6% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Two other \u003cem\u003eOrientia\u003c/em\u003e spp. are recognised although not yet formally described: \u003cem\u003eCandidatus\u003c/em\u003e Orientia chiloensis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which has only been reported from Chile (~\u0026thinsp;100 cases to date, none fatal), and \u003cem\u003eCandidatus\u003c/em\u003e Orientia chuto, which is known from a single, nonfatal human case contracted in Dubai [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], but has also been detected in chiggers in Kenya [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and wild rodents in Saudi Arabia [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Only two genera of chiggers are known to transmit \u003cem\u003eOrientia\u003c/em\u003e spp. to humans; these are \u003cem\u003eLeptotrombidium\u003c/em\u003e spp. across the Asia-Pacific (vectors of \u003cem\u003eO. tsutsugamushi\u003c/em\u003e) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and \u003cem\u003eHerpetacarus\u003c/em\u003e spp. in Chile (vectors of \u003cem\u003eCa\u003c/em\u003e. O. chiloensis) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, \u003cem\u003eOrientia\u003c/em\u003e spp. have been found in several other chigger genera that are thought to maintain infection in wild hosts, including \u003cem\u003eMicrotrombicula\u003c/em\u003e spp. from Kenya, in which \u003cem\u003eCa\u003c/em\u003e. O. chuto was detected [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The chigger vector(s) of \u003cem\u003eCa\u003c/em\u003e. O. chuto in the Arabian Peninsula remain unknown.\u003c/p\u003e \u003cp\u003eIn addition to \u003cem\u003eOrientia\u003c/em\u003e spp., a number of other potentially pathogenic bacteria and viruses have been reported from chiggers in targeted surveys or 16S rRNA amplicon sequencing, including \u003cem\u003eBartonella\u003c/em\u003e spp., \u003cem\u003eRickettsia\u003c/em\u003e spp., \u003cem\u003eBorrelia\u003c/em\u003e spp., \u003cem\u003eAnaplasma\u003c/em\u003e spp., hantaviruses, and Dabie bandavirus (reviewed in [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]). Among the bacteria, the genera \u003cem\u003eRickettsia\u003c/em\u003e (family Rickettsiaceae) and \u003cem\u003eAnaplasma\u003c/em\u003e (family Anaplasmataceae) are obligate intracellular organisms related to \u003cem\u003eOrientia\u003c/em\u003e. They utilise a range of arthropod hosts (primarily ticks, mites, fleas, or lice for \u003cem\u003eRickettsia\u003c/em\u003e spp.; or predominantly ticks for \u003cem\u003eAnaplasma\u003c/em\u003e spp.), and many species can be transmitted to humans, causing potentially severe disease. Major pathogens of medical significance include \u003cem\u003eRickettsia rickettsii\u003c/em\u003e (aetiological agent of Rocky Mountain spotted fever) and \u003cem\u003eAnaplasma phagocytophilum\u003c/em\u003e (agent of human granulocytic anaplasmosis), both of which can be fatal if not treated promptly [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The spotted fever group of rickettsiae is transmitted mainly by ticks, while one member of the more recently defined transitional group of rickettsiae, \u003cem\u003eRickettsia akari\u003c/em\u003e (agent of rickettsialpox), is vectored by gamasid mites [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Detection of \u003cem\u003eRickettsia\u003c/em\u003e spp. DNA in chiggers has been reported from geographically diverse locations [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], but a role in transmission of rickettsiae to vertebrates has not been established, and at least some of these rickettsiae may be arthropod-specific symbionts. Interestingly, DNA of both \u003cem\u003eRickettsia\u003c/em\u003e spp. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and \u003cem\u003eA. phagocytophilum\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] has been amplified from unfed chiggers, which is strong evidence for vertical transmission and long-term symbiotic relationships.\u003c/p\u003e \u003cp\u003eThe Gram-negative, facultatively intracellular genus \u003cem\u003eBartonella\u003c/em\u003e (order Hyphomicrobiales) comprises bacteria that infect vertebrate erythrocytes and are highly prevalent in micromammals, especially rodents. It is generally accepted that they are maintained in the mammalian populations by arthropod vectors (sandflies for \u003cem\u003eBartonella bacilliformis\u003c/em\u003e; and fleas, lice, and perhaps ticks for other species) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Of the many species within the genus, \u003cem\u003eB. bacilliformis, Bartonella quintana\u003c/em\u003e, and \u003cem\u003eBartonella henselae\u003c/em\u003e are the most important human pathogens and only the latter (agent of cat-scratch fever) is zoonotic [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Other zoonotic, rodent-associated \u003cem\u003eBartonella\u003c/em\u003e spp. have been reported from various chigger species in Southeast Asia, but data supporting a vector role for them in human disease remain circumstantial [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eBorrelia\u003c/em\u003e includes the causative agents of Lyme borreliosis [also referred to as Lyme disease (LD) in the USA] and relapsing fever (RF) borreliosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These spirochaetal bacteria are commonly maintained in natural transmission cycles by tick vectors, and rodents are important reservoirs for many of the human-pathogenic species [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. While evidence for \u003cem\u003eBorrelia\u003c/em\u003e spp. in both trombiculid and gamasid mites has been reported, their vector status remains questionable [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. One group of spirochaetal bacteria of uncertain taxonomic status has been found previously in the tissues of small mammals, but a vector for this micromammal-specific clade has yet to be identified [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLastly, vertically-transmitted endobacteria that do not infect vertebrates (predominantly \u003cem\u003eWolbachia\u003c/em\u003e, \u003cem\u003eRickettsiella\u003c/em\u003e and \u003cem\u003eCardinium\u003c/em\u003e) have been detected in several chigger microbiome studies (reviewed in [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]), all of which were performed on Asian species of trombiculids. However, any potential phenotypic effects of these symbionts (such as cytoplasmic incompatibility) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], or inhibition or enhancement of pathogen transmission [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] by chiggers, remain unexplored.\u003c/p\u003e \u003cp\u003eCompounding these knowledge gaps regarding the vector biology and microbiome of chiggers, the population genetics and molecular systematics of trombiculid mites remain in their infancy. Only two nuclear genomes (both from \u003cem\u003eLeptotrombidium\u003c/em\u003e spp. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]) and five mitogenomes (three from \u003cem\u003eLeptotrombidium\u003c/em\u003e spp. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]) for chiggers are publicly available. Most other genetic data for trombiculid mites consist of cytochrome \u003cem\u003ec\u003c/em\u003e oxidase I (\u003cem\u003ecoi\u003c/em\u003e) DNA barcodes, but even these display poor geographic representation, with most being obtained from Southeast Asia [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], East Asia [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and Europe [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], with none available for the Middle East.\u003c/p\u003e \u003cp\u003eHere, we present evidence that the chigger \u003cem\u003ePentidionis agamae\u003c/em\u003e may be a vector of \u003cem\u003eCa\u003c/em\u003e. O. chuto in Saudi Arabia. Moreover, applying a metagenomic approach, we obtained complete mitogenomes from this species and place it in the phylogenetic context of other Saudi Arabian chigger species, as well as trombiculid diversity worldwide, through analysis of \u003cem\u003ecoi\u003c/em\u003e barcodes. Finally, sequences from two additional, non-\u003cem\u003eOrientia\u003c/em\u003e bacterial associates of \u003cem\u003eP. agamae\u003c/em\u003e are shown to represent a poorly described, micromammal-associated \u003cem\u003eBorrelia\u003c/em\u003e sp. and a member of a novel, deep-branching clade of \u003cem\u003eWolbachia\u003c/em\u003e symbionts.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChigger sampling\u003c/h2\u003e \u003cp\u003eIn total, 156 rodents were captured, belonging to six different species: \u003cem\u003eAcomys dimidiatus\u003c/em\u003e, \u003cem\u003eMeriones rex\u003c/em\u003e, \u003cem\u003eMus musculus\u003c/em\u003e, \u003cem\u003eOchromyscus yemeni\u003c/em\u003e and \u003cem\u003eRattus rattus\u003c/em\u003e (Additional File 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A total 7,329 chiggers were recovered from 27 and 55 rodents in \u0026lsquo;Asir and Al-Bahah provinces respectively. Of these, 4,226 chiggers belonging to 20 trombiculid species were identified (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The remaining chiggers were excluded from the study as they were damaged, or the important identifying features were absent.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChigger species and numbers found at two sampling locations in Saudi Arabia.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eChigger species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSubfamily and tribe\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eProvince\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lsquo;Asir\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAl-Bahah\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSchoengastiella hypoderma\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGahrliepiinae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eWalchia parvula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGahrliepiinae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eOdontacarus thesigeri\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeeuwenhoekiinae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAscoschoengastia browni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Schoengastiini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e112\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHelenicula lukshumiae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Schoengastiini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSchoutedenichia asirensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Schoengastiini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSchoutedenichia originalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Schoengastiini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSchoutedenichia saudi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Schoengastiini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSchoutedenichia zarudnyi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Schoengastiini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e878\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricotrombidium caucasicum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Trombiculini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e411\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEricotrombidium kazeruni\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Trombiculini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e735\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMicrotrombicula abyssinica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Trombiculini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMicrotrombicula felis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Trombiculini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMicrotrombicula hoogstraali\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Trombiculini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMicrotrombicula hyracis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Trombiculini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMicrotrombicula muhaylensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Trombiculini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e305\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e157\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMicrotrombicula peltifera\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Trombiculini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMicrotrombicula saperoi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Trombiculini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMicrotrombicula traubi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Trombiculini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePentidionis agamae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrombiculinae: Trombiculini\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e471\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCa\u003c/b\u003e. \u003cb\u003eO. chuto in\u003c/b\u003e \u003cb\u003eP. agamae\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eOrientia\u003c/em\u003e screening by qPCR (\u003cem\u003etraD\u003c/em\u003e) and nested PCR (\u003cem\u003ehtrA\u003c/em\u003e) was performed on 165 pools of chiggers, consisting of 3,286 individuals (Additional File 1: Table S2). A single pool each of \u003cem\u003eP. agamae\u003c/em\u003e (R9P) and \u003cem\u003eM. muhaylensis\u003c/em\u003e (R19M) \u0026ndash; both obtained from \u003cem\u003eA. dimidiatus\u003c/em\u003e hosts in \u0026lsquo;Asir province - yielded positive amplification in the \u003cem\u003etraD\u003c/em\u003e qPCR assay. However, Sanger sequencing of the \u003cem\u003ehtrA\u003c/em\u003e nested PCR product only produced a high-quality sequence from R9P for further analyses. The \u003cem\u003ehtrA\u003c/em\u003e sequence from R9P formed a single clade (bootstrap 100) with the \u003cem\u003ehtrA\u003c/em\u003e sequences from \u003cem\u003eCa\u003c/em\u003e O. chuto reported from the tissues of \u003cem\u003eA. dimidiatus\u003c/em\u003e captured from \u0026lsquo;Asir Province (MR25, MR26Ki, MR26Li) in our previous study [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and the sequences from Saudi Arabia remained in a single clade (bootstrap 86) distinct from \u003cem\u003eCa\u003c/em\u003e O. chuto from Dubai, United Arab Emirates (UAE) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Genetic pairwise distance calculated between the \u003cem\u003eCa.\u003c/em\u003e O. chuto \u003cem\u003ehtrA\u003c/em\u003e sequences (based on 659 bp) from Saudi Arabia also showed that R9P \u003cem\u003ehtrA\u003c/em\u003e is more closely related to the sequences from \u0026lsquo;Asir Province (MR25, MR26Ki, MR26Li: pairwise distance\u0026thinsp;=\u0026thinsp;0.003) than sequences from Al-Bahah Province (AR33 and AR43: pairwise distance\u0026thinsp;=\u0026thinsp;0.017).\u003c/p\u003e \u003cp\u003eFollowing this finding, the R9P pool of \u003cem\u003eP. agamae\u003c/em\u003e was subjected to metagenomic sequencing using Illumina technology to obtain additional genes for comparison with the sequenced culture isolate of \u003cem\u003eCa\u003c/em\u003e. O. chuto str. Dubai [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Since \u003cem\u003eP. agamae\u003c/em\u003e is a potential vector of \u003cem\u003eCa\u003c/em\u003e O. chuto based on the PCR screening, an additional two pools of \u003cem\u003eP. agamae\u003c/em\u003e, Pa1 and Pa2, obtained from the \u0026lsquo;Asir region in a previous sampling effort [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], were also subjected to metagenomic sequencing. The Kraken2 assignment found contigs assigned to \u003cem\u003eOrientia\u003c/em\u003e (Additional File 1: Table S3). A single contig from R9P overlapped 137 bp at the 3\u0026prime; end of the \u003cem\u003ehtrA\u003c/em\u003e sequence from Sanger sequencing at 100% identity, indicating that the contig did not contain the full-length coding sequence of the gene. We did not find the \u003cem\u003ehtrA\u003c/em\u003e sequence from the contigs from Pa2; however, BLASTn analyses showed 99%-100% matches of the contigs to various \u003cem\u003eO. tsutsugamushi\u003c/em\u003e strains (Additional File 1: Table S4). Diamond BLASTx revealed matches to a number of \u003cem\u003eO. tsutsugamushi\u003c/em\u003e proteins, namely dihydrolipoyl dehydrogenase, toprim domain protein, conjugal transfer protein TraN, transposase and two different hypothetical proteins (Additional File 1: Table S4). None of the contigs from \u003cem\u003eP. agamae\u003c/em\u003e pool Pa1 were verified as \u003cem\u003eOrientia\u003c/em\u003e sequences from BLASTn analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWolbachia\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eRickettsia\u003c/b\u003e\u003c/p\u003e \u003cp\u003eContigs assigned to \u003cem\u003eWolbachia\u003c/em\u003e were also found in all three pools (Additional File 1: Table S3), with over 200 identified in Pa2. An improved \u003cem\u003eWolbachia\u003c/em\u003e assembly was obtained from this pool by mapping the short reads to metaSPAdes and Megahit-assembled contigs, and reassembling the mapped reads using metaSpades. This workflow resulted in a new draft assembly with BUSCO improvement from 60.4\u0026ndash;78.8%. Maximum likelihood phylogeny placed this assembly, which we designate as \u003cem\u003ew\u003c/em\u003ePaga, in its own clade (new supergroup X \u0026ndash; bootstrap 100) and this was close to the more divergent clades, including supergroups W, M, L, E, and I (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As genome assemblies for other \u003cem\u003eWolbachia\u003c/em\u003e symbionts from acariform mites (the mould mite \u003cem\u003eTyrophagus putrescentiae\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and the quill mite \u003cem\u003eSyringophilopsis turdi\u003c/em\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]) were made available recently on NCBI, we included these in the phylogenomic analysis. We determined that they were both very distinct from \u003cem\u003ew\u003c/em\u003ePaga, with \u003cem\u003ew\u003c/em\u003eTput from \u003cem\u003eT\u003c/em\u003e. \u003cem\u003eputrescentiae\u003c/em\u003e displaying closer affinities with supergroup M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), whereas \u003cem\u003ew\u003c/em\u003eStur from \u003cem\u003eS. turdi\u003c/em\u003e is a member of a distinct, more distant supergroup (P) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA number of contigs were also assigned as \u003cem\u003eRickettsia\u003c/em\u003e (Additional File 1: Table S3). However, further verification with BLASTn analyses revealed that most of these contigs either had no match to any existing sequences in GenBank, or matched with \u003cem\u003eRickettsia\u003c/em\u003e sequences with low percentage identity (\u0026lt;\u0026thinsp;95%, data not shown), suggesting the presence of more genetically distant \u003cem\u003eRickettsiales\u003c/em\u003e bacteria.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of a micromammal-associated\u003c/b\u003e \u003cb\u003eBorrelia\u003c/b\u003e \u003cb\u003esp. in\u003c/b\u003e \u003cb\u003eP. agamae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAll three \u003cem\u003eP. agamae\u003c/em\u003e pools had contigs assigned as \u003cem\u003eBorrelia\u003c/em\u003e (Additional File 1: Table S3). We recovered sequences for 16S rRNA and several genes from the multi-locus sequence typing (MLST) scheme [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] - \u003cem\u003eclpX\u003c/em\u003e, \u003cem\u003erecG\u003c/em\u003e and \u003cem\u003euvrA\u003c/em\u003e - from R9P contigs, which were used to construct phylogenetic trees with other published spirochaete sequences from GenBank. We were unable to recover these genes from Pa1 and Pa2. In the 16S rRNA phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), the \u003cem\u003eBorrelia\u003c/em\u003e sp. from R9P clustered with \u003cem\u003eBorrelia\u003c/em\u003e spp. previously reported from micromammals (mainly rodents), namely \u003cem\u003eBorrelia\u003c/em\u003e sp. isolates R57 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], BRAUS (TIS 37), CA682, and ALEPB216 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A discrepancy in the tree topology was observed between the current and previously published 16S rRNA trees [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The rodent-associated \u003cem\u003eBorrelia\u003c/em\u003e spp., called \u0026ldquo;rodent group\u0026rdquo; here, were placed in a sister clade to the other \u003cem\u003eBorrelia\u003c/em\u003e groups in the published studies, but clustered with the RF borreliae in our tree. This discrepancy could be due to the inclusion of different \u003cem\u003eBorrelia\u003c/em\u003e strains available and also the impact of sequence length. With the shortest sequence [429 bp; namely the sequence from \u003cem\u003eBorrelia\u003c/em\u003e sp. isolate BRAUS (TIS 37)] removed from the analyses, the tree topology reverted to one in which the rodent group formed a sister clade to the other \u003cem\u003eBorrelia\u003c/em\u003e groups (Additional File 1: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), similar to previous studies [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This observation indicates that accurate phylogenetic placement of the rodent group borreliae will require more genes from the MLST scheme [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Since MLST gene sequences for the rodent group \u003cem\u003eBorrelia\u003c/em\u003e spp. are not currently available, \u003cem\u003eBorrelia\u003c/em\u003e sp. R9P forms a sister clade to other known \u003cem\u003eBorrelia\u003c/em\u003e spp. from the LD and RF groups in the phylogenies based on the concatenated matrix of \u003cem\u003eclpX\u003c/em\u003e, \u003cem\u003erecG\u003c/em\u003e and \u003cem\u003euvrA\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), or each of the individual MLST genes (Additional file 1: Fig. S2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial assembly\u003c/h2\u003e \u003cp\u003eCircularised mitochondrial genomes were assembled separately from Pa1 and Pa2 (14,755 bp). The MitoZ pipeline produced a linear mitochondrial assembly for R9P, which was then circularised (14,753 bp) with an additional step based on overlapping sequences at the end of the linear assembly. Both assemblies from Pa2 and R9P appeared to be almost identical to Pa1 (Additional File 1: Fig. S3), with 99.92% and 99.76% identity, respectively. The base composition of the mitochondrial genomes was approximately 45% (A), 25% (T), 10% (C), and 20% (G).\u003c/p\u003e \u003cp\u003eMaximum likelihood phylogeny based on a partial \u003cem\u003ecoi\u003c/em\u003e gene fragment, combining data from the mitogenomic assemblies and additional \u003cem\u003ecoi\u003c/em\u003e PCR products from archived specimens, placed \u003cem\u003eP. agamae\u003c/em\u003e in a single clade with \u003cem\u003eSchoutedenichia centralkwangtunga\u003c/em\u003e (KY971498.1) from Laos and \u003cem\u003eWalchia hayashii\u003c/em\u003e (NC010595.1) from Japan, with a bootstrap value of 94 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This is surprising, as \u003cem\u003eWalchia\u003c/em\u003e belongs to a different subfamily \u003cem\u003e(Gahrliepiinae)\u003c/em\u003e than \u003cem\u003ePentidionis\u003c/em\u003e and \u003cem\u003eSchoutedenichia\u003c/em\u003e (\u003cem\u003eTrombiculinae\u003c/em\u003e), and the two latter genera belong to different tribes (Trombiculini and Schoengastiini, respectively) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurther inconsistencies of species placements were observed in this phylogeny. For instance, \u003cem\u003eW. hayashii\u003c/em\u003e (represented by a complete mitogenomic assembly but lacking an accompanying publication) did not cluster within the clade containing the other \u003cem\u003eWalchia\u003c/em\u003e spp. (all from Southeast Asia), and \u003cem\u003eSchoutedenichia\u003c/em\u003e sp. D454 (OQ924405.1) from Albania seemed more closely related to \u003cem\u003eBlankaartia acuscutellaris\u003c/em\u003e from Laos instead of \u003cem\u003eS. centralkwangtunga.\u003c/em\u003e To provide the first molecular taxonomic data for chiggers from the Middle East, we generated \u003cem\u003ecoi\u003c/em\u003e barcodes for \u003cem\u003eEricotrombidium caucasicum\u003c/em\u003e, \u003cem\u003eE. kazeruni\u003c/em\u003e, \u003cem\u003eAscoschoengastia browni\u003c/em\u003e, \u003cem\u003eMicrotrombicula felis\u003c/em\u003e, \u003cem\u003eM. peltifera\u003c/em\u003e, \u003cem\u003eM. traubi\u003c/em\u003e, \u003cem\u003eM. muhaylensis\u003c/em\u003e, \u003cem\u003eSchoutedenichia zarudnyi\u003c/em\u003e, \u003cem\u003eS. saudi\u003c/em\u003e, and \u003cem\u003eHelenicula lukshumiae\u003c/em\u003e, which were described from Saudi Arabia in our previous studies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. For \u003cem\u003eEricotrombidium\u003c/em\u003e spp., \u003cem\u003eMicrotrombicula\u003c/em\u003e spp., and \u003cem\u003eHelenicula\u003c/em\u003e spp., these were the first barcodes available for each genus and comparisons with congeneric species were thus not possible, although in each case, the genus formed a monophyletic group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, while the two \u003cem\u003eSchoutedenichia\u003c/em\u003e spp. from Saudi Arabia clustered with \u003cem\u003eS. centralkwangtunga\u003c/em\u003e, \u003cem\u003eA. browni\u003c/em\u003e displayed closer affinities with \u003cem\u003eHirsutiella zachvatkini\u003c/em\u003e from Poland than to \u003cem\u003eAscoschoengastia indica\u003c/em\u003e from Thailand/Laos. Interestingly, the \u003cem\u003eH. lukshumiae\u003c/em\u003e specimens were placed on a deep branch despite the classification of \u003cem\u003eHelenicula\u003c/em\u003e in the tribe Schoengastiini with \u003cem\u003eSchoutedenichia\u003c/em\u003e and \u003cem\u003eAscoschoengastia\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Additional File 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Nevertheless, the placements of \u003cem\u003eEricotrombidium\u003c/em\u003e and \u003cem\u003eHirsutiella\u003c/em\u003e appear to conform with the current classification system of chiggers based on larval morphology: (i) clustering of \u003cem\u003eEricotrombidium\u003c/em\u003e with \u003cem\u003eLeptotrombidium\u003c/em\u003e (the former genus was described as a subgenus of the latter); (ii) clustering of \u003cem\u003eH. zachvatkini\u003c/em\u003e with \u003cem\u003eNeotrombicula\u003c/em\u003e (\u003cem\u003eHirsutiella\u003c/em\u003e is considered as a subgenus of \u003cem\u003eNeotrombicula\u003c/em\u003e by some authors [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]); and (iii) clustering of \u003cem\u003eA. indica\u003c/em\u003e with \u003cem\u003eMicrotrombicula\u003c/em\u003e (\u003cem\u003eAscoschoengastia\u003c/em\u003e and \u003cem\u003eMicrotrombicula\u003c/em\u003e, although they belong to different tribes, in fact differ from each other by a single trait \u0026ndash; trichobothria that are expanded in the former genus and flagelliform in the latter [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]). The affinity between two chigger species, namely \u003cem\u003eN. gallinarum\u003c/em\u003e (tribe Schoengastiini) and \u003cem\u003eB. acuscutellaris\u003c/em\u003e (tribe Trombiculini), which prefer avian hosts despite belonging to strikingly different genera and different tribes, was also noteworthy.\u003c/p\u003e \u003cp\u003eAnnotation of the assembly from Pa1 yielded thirteen protein CDS, two rRNAs and sixteen tRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Additional file 1: Fig. S4). We were able to identify the six missing tRNAs (\u003cem\u003etrnL1\u003c/em\u003e, \u003cem\u003etnrL2\u003c/em\u003e, \u003cem\u003etrnA\u003c/em\u003e, \u003cem\u003etnrR\u003c/em\u003e, \u003cem\u003etrnG\u003c/em\u003e and \u003cem\u003etrnV\u003c/em\u003e) by manually inspecting the conserved anti-codon regions in the alignments between the current assembly and the other five available mitochondrial assemblies from trombiculid mites (Additional file 1: Fig. S5). However, their predicted secondary structures appeared to have no T-arms and hence lack the typical clover leaf structure, or appeared to be extremely truncated (for \u003cem\u003etrnA\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eRelative to Pa1, 12 SNPs were observed in Pa2 and 36 SNPs (including two deletions) were detected in R9P. The SNPs in the Pa2 assembly were found in \u003cem\u003etrnT\u003c/em\u003e, as well as in the \u003cem\u003ecoi\u003c/em\u003e, \u003cem\u003ecob\u003c/em\u003e and \u003cem\u003enad5\u003c/em\u003e genes, causing non-synonymous substitutions in these protein CDS (Additional File 1: Table S6). A single non-synonymous substitution was observed in \u003cem\u003ecoi\u003c/em\u003e, while two non-synonymous substitutions were found each for \u003cem\u003ecob\u003c/em\u003e and \u003cem\u003enad5\u003c/em\u003e. When gene arrangements were analysed, the mitochondrial genome from \u003cem\u003eP. agamae\u003c/em\u003e displayed closest synteny to \u003cem\u003eW. hayashii\u003c/em\u003e, with rearrangement of the positions of the control region and \u003cem\u003etrnQ\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The control region for \u003cem\u003eP. agamae\u003c/em\u003e lies upstream of \u003cem\u003errnS\u003c/em\u003e, and \u003cem\u003etrnQ\u003c/em\u003e lies downstream of \u003cem\u003errnL\u003c/em\u003e. Unlike the mitochondrial genomes for \u003cem\u003eAscoschoengastia\u003c/em\u003e sp. TATW-1, \u003cem\u003eLeptotrombidium deliense\u003c/em\u003e or \u003cem\u003eLeptotrombidium pallidum\u003c/em\u003e, there was no duplication of any mitochondrial genes in \u003cem\u003eP. agamae\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe ecology of \u003cem\u003eCa\u003c/em\u003e O. chuto has remained enigmatic since its discovery over a decade ago. The endemic region of this pathogen is potentially vast, with evidence of circulation across the Arabian Peninsula [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], East Africa [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and perhaps West Africa [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Despite this wide range, only one human case of scrub typhus caused by this species has been reported, which was contracted in Dubai [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]; however, no studies on \u003cem\u003eCa\u003c/em\u003e. O. chuto in chiggers or non-human vertebrate hosts in the UAE have been published to date. Following the publication of details of the clinical isolate of \u003cem\u003eCa\u003c/em\u003e. O. chuto from Dubai [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], pathogen DNA was detected in one pool of \u003cem\u003eMicrotrombicula\u003c/em\u003e spp. chiggers (of five pools from multiple host species screened), which was obtained from a Natal multimammate mouse (\u003cem\u003eMastomys natalensis\u003c/em\u003e) in Baringo county, Kenya [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. More recently, \u003cem\u003eCa\u003c/em\u003e. O. chuto DNA was amplified from the tissues of 7.3% (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;82) rodents (Eastern spiny mice - \u003cem\u003eAcomys dimidiatus\u003c/em\u003e, or Wagner\u0026rsquo;s gerbil - \u003cem\u003eDipodillus dasyurus\u003c/em\u003e) trapped in \u0026lsquo;Asir and Al-Bahah provinces of Saudi Arabia [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Most of the positive rodents lacked chigger infestations, but chiggers of five species (\u003cem\u003eE. caucasicum, E. kazeruni, S. saudi, S. zarudnyi\u003c/em\u003e, and \u003cem\u003eM. hoogstraali\u003c/em\u003e) were obtained from two infected individuals and were shown to be negative for \u003cem\u003eOrientia\u003c/em\u003e DNA.\u003c/p\u003e \u003cp\u003eThe current study, in which a much more extensive collection of Saudi chiggers was screened, represents the first report of \u003cem\u003eCa\u003c/em\u003e. O. chuto DNA from potential vector species from the Arabian Peninsula (albeit\u0026thinsp;\u0026gt;\u0026thinsp;1,500 km distant from the clinical case reported from Dubai). Since the \u003cem\u003ehtrA\u003c/em\u003e PCR amplicon from the pool of \u003cem\u003eM. muhaylensis\u003c/em\u003e failed to provide a high-quality sequence, the apparent positive result for this chigger species remains provisional. However, it is noteworthy that this species is in the same genus as the positive chigger pool reported from Kenya [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For \u003cem\u003eP. agamae\u003c/em\u003e, molecular evidence for infection with \u003cem\u003eCa\u003c/em\u003e. O. chuto was obtained using Sanger sequencing of a \u003cem\u003ehtrA\u003c/em\u003e nested PCR product and metagenomic sequencing via Illumina short-read technology. Unfortunately, no additional \u003cem\u003eOrientia\u003c/em\u003e genes could be assembled using the Illumina data from \u003cem\u003eP. agamae\u003c/em\u003e pool R9P to obtain further phylogenetic information for comparison with the Dubai isolate. This means that current data for \u003cem\u003eCa\u003c/em\u003e. O. chuto in Saudi Arabia is comprised of only six sequences from a single gene [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and the Kenyan data by two gene sequences (16S rRNA and \u003cem\u003ehtrA\u003c/em\u003e) from a single sample [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Nevertheless, the \u003cem\u003ehtrA\u003c/em\u003e sequences exhibit differences that correspond to geographic distance, with the R9P sequence from \u003cem\u003eP. agamae\u003c/em\u003e collected in \u0026lsquo;Asir clustering with sequences from rodents obtained from the same province, and genetic distances increasing stepwise compared with sequences from Al-Bahah province, UAE, or Kenya, respectively. The lack of other \u003cem\u003eOrientia\u003c/em\u003e sequence data in pool R9P suggests a very low level of \u003cem\u003eCa\u003c/em\u003e. O. chuto DNA in this sample, perhaps representing only a single positive chigger; neither can we rule out traces of host-derived pathogen DNA from mite mouthparts or gut contents in the absence of systemic chigger infection. Future studies could attempt to obtain additional \u003cem\u003eOrientia\u003c/em\u003e genome data by sequence capture with DNA extracts from human samples and chigger specimens, which has been performed successfully using specific probes for \u003cem\u003eO. tsutsugamushi\u003c/em\u003e [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, while an \u003cem\u003ehtrA\u003c/em\u003e sequence could not be recovered from the metagenomic dataset for another \u003cem\u003eP. agamae\u003c/em\u003e pool, Pa2, several other \u003cem\u003eOrientia\u003c/em\u003e genes were identified in this sample. These had closest matches to \u003cem\u003eO. tsutsugamushi\u003c/em\u003e sequences, especially the Karp-like strain UT176, which is a clinical isolate from Thailand [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, genes from the multi-locus sequence typing scheme for \u003cem\u003eO. tsutsugamushi\u003c/em\u003e were not assembled and caution is needed in interpreting these data as evidence of \u003cem\u003eO. tsutsugamushi\u003c/em\u003e in Saudi Arabia, as the only \u003cem\u003eCa\u003c/em\u003e. O. chuto genome assembly available (str. Dubai) is incomplete [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Notwithstanding this limitation, the detection of \u003cem\u003eOrientia\u003c/em\u003e sequences in this second pooled DNA sample from \u003cem\u003eP. agamae\u003c/em\u003e adds to the evidence that this chigger species may act as a vector, at least between wild hosts. Unfortunately, in common with other chigger species in the Middle East, \u003cem\u003eP. agamae\u003c/em\u003e is poorly studied with limited host records. Prior to our rodent studies in Saudi Arabia, \u003cem\u003eP. agamae\u003c/em\u003e was only known from agamid lizard hosts in the Persis region of Iran [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and around Lake Tiberias (Galilee) [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], although it is widespread on \u003cem\u003eA. dimidiatus\u003c/em\u003e in both \u0026lsquo;Asir [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and Al-Bahah [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] provinces. Whether \u003cem\u003eP. agamae\u003c/em\u003e could bite humans and act as a clinically-relevant scrub typhus vector is an important open question, especially as the mountainous regions of southwest Saudi Arabia are popular destinations for tourists seeking cooler temperatures in the summer months. With respect to the origin of the only confirmed case of scrub typhus in the Middle East, limited data are available on the chigger fauna of UAE, with four chigger species reported recently from a very small sample of \u003cem\u003eA. dimidiatus\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. However, \u003cem\u003eP. agamae\u003c/em\u003e was not present among these.\u003c/p\u003e \u003cp\u003eIn the past five years, interest in the trombiculid mite microbiome has blossomed on the back of technological advances that have enabled 16S rRNA amplicon sequencing studies on low-input DNA samples. The current study constitutes the first genuine metagenomic analysis of a trombiculid mite since the publication of the \u003cem\u003eLeptotrombidium deliense\u003c/em\u003e genome [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], thus providing the potential to obtain multiple gene sequences or even genome assemblies for members of the chigger microbiome. Here, we found a \u003cem\u003eWolbachia\u003c/em\u003e symbiont of \u003cem\u003eP. agamae\u003c/em\u003e (\u003cem\u003ew\u003c/em\u003ePaga) to be sufficiently represented to allow a genome assembly and phylogenomic analysis. \u003cem\u003eWolbachia\u003c/em\u003e has been detected previously from trombiculid mites in Southeast Asia and East Asia using 16S rRNA amplicon sequencing [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], but the use of a single conserved gene has precluded robust phylogenetic placement. Our data locate \u003cem\u003ew\u003c/em\u003ePaga firmly among the early-branching clades of \u003cem\u003eWolbachia\u003c/em\u003e that have been poorly studied compared with the ubiquitous, so-called \u0026ldquo;pandemic\u0026rdquo; supergroups (A and B) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], but it is sufficiently distinct to constitute the first member of a new supergroup. Unfortunately, we did not recover the 16S rRNA sequence from \u003cem\u003ew\u003c/em\u003ePaga, but its position on a long branch is consistent with that of a previous reported symbiont from \u003cem\u003eLeptotrombidium scutellare\u003c/em\u003e in Japan [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It has been hypothesized that \u003cem\u003eWolbachia\u003c/em\u003e evolved in the soil milieu [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] through associations with parasitic nematodes of plants (supergroup L [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]) or saprotrophic flies (W [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]), and may have been horizontally transmitted via plants, honeydew, and/or insect carcasses to other hosts of early-branching \u003cem\u003eWolbachia\u003c/em\u003e clades including the banana aphid (M), springtails (E), oribatid mites (E), and fleas (I) \u0026ndash; the latter being detritivorous in the larval stage. In accordance with this model, the free-living lifecycle of trombiculid mites proceeds underground, where the nymphal and adult stages predate small edaphic arthropods or their eggs. We also assigned a phylogenetic placement to another \u003cem\u003eWolbachia\u003c/em\u003e symbiont of mite origin, \u003cem\u003ew\u003c/em\u003eTput from \u003cem\u003eT. putrescentiae\u003c/em\u003e, which was close to supergroup M but may be a member of another novel clade. While renowned as a pest of stored foodstuffs, \u003cem\u003eT\u003c/em\u003e. \u003cem\u003eputrescentiae\u003c/em\u003e is also common in outdoor agricultural biomes [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] and is likely to share habitats with other hosts of the non-pandemic \u003cem\u003eWolbachia\u003c/em\u003e clades listed above.\u003c/p\u003e \u003cp\u003eIn addition to \u003cem\u003eWolbachia\u003c/em\u003e, \u003cem\u003eBorrelia\u003c/em\u003e spp. have been reported from trombiculid mites from several locations worldwide. Spirochaetes of the LD clade have been detected molecularly in harvest mites (\u003cem\u003eNeotrombicula autumnalis\u003c/em\u003e) in Europe [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and this chigger has been shown to acquire borreliae experimentally from infected rodents [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. There is also some evidence for vertical transmission of LD borreliae in harvest mites [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], while unassigned \u003cem\u003eBorrelia\u003c/em\u003e spp. 16S rRNA sequences have been detected at high prevalence in chiggers collected from wild micromammals in Thailand [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. In the current study, we were able to acquire multiple gene sequences for a chigger-associated \u003cem\u003eBorrelia\u003c/em\u003e spp. for the first time, allowing robust phylogenetic classification. Surprisingly, the sequences associated with \u003cem\u003eP. agamae\u003c/em\u003e were not of LD or RF \u003cem\u003eBorrelia\u003c/em\u003e spp. origin but belonged to a clade associated with rodents and shrews previously reported from Spain [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], California [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and New South Wales [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. On the basis of 16S rRNA and \u003cem\u003egroEL\u003c/em\u003e gene sequences, this clade (originally described from Spain as isolate R57) has been known to be distinct from the LD and RF groups for nearly two decades, but its biology has remained enigmatic. Importantly, it has never been detected in arthropods or mammalian blood, but only ear punch biopsies. Our data suggest that chiggers (many species of which have a predilection for the pinna and ear canal as feeding sites) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] may be the vector for this micromammal clade of borreliae. While we cannot rule out that the \u003cem\u003eBorrelia\u003c/em\u003e spp. DNA is an incidental finding due to ingestion of host tissue fluid by chiggers, the fact we could assemble several genes from the organism coupled with the absence of prior PCR detection in hard ticks that are often contaminated with host skin, renders this possibility less likely.\u003c/p\u003e \u003cp\u003eIn the past five years, molecular barcoding (primarily based on the mitochondrial \u003cem\u003ecoi\u003c/em\u003e gene) has been applied to chigger mites to determine whether low-throughput morphological identification can be supplanted, or at least complemented, by less laborious procedures. The first study to analyse \u003cem\u003ecoi\u003c/em\u003e barcodes from multiple chigger species, which was conducted in South-East Asia, demonstrated that the technique reliably binned individual specimens by morphotyped species and clustered subgenera in cognate groups [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, certain species exhibited multiple haplotypes, sometimes even if recovered from the same individual host. Importantly, barcoding studies of European chiggers have revealed clear cases both of phenotypic plasticity within trombiculid species (which is linked to the host species used for larval development) [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] and cryptic diversity, where single chigger morphotypes show genetic distances similar to that between recognised species [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In the current study, while most species clustered by subgenus when compared with published barcodes, \u003cem\u003eAscoschoengastia\u003c/em\u003e spp. and \u003cem\u003eSchoutedenichia\u003c/em\u003e spp. were striking exceptions. Moreover, the system of subfamilies and tribes within the family Trombiculidae that has existed for over half-a-century was not reflected in the \u003cem\u003ecoi\u003c/em\u003e-based phylogeny. A clear example of this was the apparent affinity of \u003cem\u003ePentidionis\u003c/em\u003e with \u003cem\u003eSchoutedenichia\u003c/em\u003e, despite their classification in different tribes (Trombiculini and Schoengastiini, respectively). While these findings suggest that the classification of trombiculid mites based on larval morphology has significant limitations, phylogenetic relationships cannot be resolved using a single mitochondrial gene, and there is an urgent need to develop multi-locus-based approaches to trombiculid taxonomy.\u003c/p\u003e \u003cp\u003eHere, we were able to successfully generate complete mitogenomic assemblies from three pools of \u003cem\u003eP. agamae\u003c/em\u003e, which is the first time multiple mitogenomes from a single chigger species have been obtained for intraspecific comparisons. Notably, we found tRNA gene annotation to be dependent on manual comparisons with available trombiculid mitogenomes due to previously recognised non-canonical features of these genes in multiple acariform taxa [\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Several SNPs were identified between pools of \u003cem\u003eP. agamae\u003c/em\u003e, including non-synonymous substitutions, despite the mites being collected from the same province. Unfortunately, the paucity of whole mitogenome data from other trombiculid species severely limited interspecific comparisons. This is particularly problematic, as of the five other complete mitogenomes available from trombiculid mites, three are from a single genus (\u003cem\u003eLeptotrombidium\u003c/em\u003e spp.) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and one of the non-\u003cem\u003eLeptotrombidium\u003c/em\u003e assemblies is from a mite identified to genus level only (\u003cem\u003eAscoschoengastia\u003c/em\u003e sp. TATW-1). While mitochondrial gene order in \u003cem\u003eP. agamae\u003c/em\u003e was most closely related that of \u003cem\u003eW. hayashii\u003c/em\u003e, the phylogenetic position of the latter in the \u003cem\u003ecoi\u003c/em\u003e tree was unexpected, as it did not cluster with published sequences available for five \u003cem\u003eWalchia\u003c/em\u003e spp. from South-East Asia. No information on how \u003cem\u003eW. hayashii\u003c/em\u003e specimens were identified prior to sequencing is available, as the mitogenome record on NCBI is not linked to a publication and the depositors are no longer active in research. Thus, it is unclear if this is a case of misidentification or if the subgenus \u003cem\u003eWalchia\u003c/em\u003e is paraphyletic.\u003c/p\u003e \u003cp\u003eSince we have demonstrated that assembly of chigger mitogenomes is feasible using ethanol-preserved pools and Illumina technology, which is declining rapidly in cost per sample, we hope these results with spur routine sequencing of trombiculid mitogenomes. Indeed, this has happened already for ticks, revolutionising phylogenetics for the Ixodidae and Argasidae [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. However, it is important that phylogenetically-informative nuclear markers such as ITS2 are also utilised due to differing evolutionary rates between nuclear and mitochondrial genomes [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e], the potential for vertically-transmitted symbionts such as \u003cem\u003eWolbachia\u003c/em\u003e to cause cytonuclear discordance [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], and the possibility that trombiculid species may hybridise [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur PCR-based screening and sequencing of chigger mites from Saudi Arabia has revealed \u003cem\u003eP. agamae\u003c/em\u003e as a potential vector of \u003cem\u003eCa\u003c/em\u003e. O. chuto, but further research is required to determine if this species may be anthropophilic and thus important in scrub typhus epidemiology in the Middle East. Moreover, this first metagenomic analysis of a trombiculid mite outside the genus \u003cem\u003eLeptotrombidium\u003c/em\u003e has enabled deeper insights into chigger-associated \u003cem\u003eWolbachia\u003c/em\u003e and \u003cem\u003eBorrelia\u003c/em\u003e bacteria that were only known previously from 16S rRNA gene data, as well as providing a reference mitogenome for the genus \u003cem\u003ePentidionis\u003c/em\u003e and initial evidence for intraspecific variation. Overall, the metagenomic approach we applied here has demonstrated its potential to generate complete mitogenomes for phylogenetic and population genetic studies of trombiculids with relative ease; furthermore, it can greatly improve our understanding of chigger microbiomes that so far have been studied predominantly by 16S rRNA amplicon-based methods.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eChigger collection and identification\u003c/h2\u003e \u003cp\u003eRodents were trapped overnight in southwestern Saudi Arabia at sites in \u0026lsquo;Asir (October 2020) and Al-Bahah (August 2021) provinces (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) as described previously [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Rodents were euthanized by inhalational anaesthetic isoflurane overdose or dislocated in the cervical region. The identification of rodents was based on morphological features and confirmed molecularly through the amplification of \u003cem\u003ecytB\u003c/em\u003e gene fragment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Each rodent was carefully inspected for chiggers including inside ears, removed chiggers were preserved in 70% ethanol. The fieldwork was approved by the Saudi Wildlife Authority (approval no. 288/33/A) and Animal Welfare and Ethics Review Board of the University of Liverpool. As representative specimens, 10% of chiggers were selected by purposive sampling and fixed permanently using Berlese fluid (TCS Bioscience Ltd, Buckingham, UK). The measurements and identification of chiggers were performed on a fluorescence microscope (ZEISS Axio Imager M2 microscope through GT Vision GXCapture-T software). The remaining chiggers were identified without the usage of mountant and pooled on the basis of species from each rodent (Additional File 1: Table S2).\u003c/p\u003e\u003cp\u003e \u003cb\u003eMolecular detection of\u003c/b\u003e \u003cb\u003eOrientia\u003c/b\u003e \u003cb\u003esp.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenomic DNA from chigger pools were extracted using the Qiagen DNeasy Blood \u0026amp; Tissue Kit (Qiagen) according to the manufacturer\u0026rsquo;s protocol. DNA concentration and quality were assessed by a Qubit High Sensitivity dsDNA Quantification Assay kit (Invitrogen) and NanoDrop One/One\u003csup\u003eC\u003c/sup\u003e Microvolume UV-Vis Spectrophotometer (Thermo Scientific). A quantitative PCR assay (qPCR) targeting the multicopy \u003cem\u003etraD\u003c/em\u003e gene was used in the initial screening of chigger pools for detection of \u003cem\u003eOrientia\u003c/em\u003e sp. [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Positive samples were subjected to a nested PCR assay for amplification of the \u003cem\u003ehtrA\u003c/em\u003e gene [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The PCR amplicons were purified and submitted to Eurofins Genomics (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.eurofins.com\u003c/span\u003e\u003cspan address=\"https://www.eurofins.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for Sanger sequencing in both directions. Paired sequences were aligned to generate a corrected consensus and manually quality-trimmed using Bioedit 7.2.5 [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] to produce the final sequence for phylogenetic analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eIllumina sequencing\u003c/h2\u003e \u003cp\u003eIllumina library preparation and sequencing from chigger pools were performed at the Centre for Genomic Research (CGR) at the University of Liverpool. NEBNext Ultra II FS kit paired- end libraries (2\u0026times;150 bp) with a 350 bp insert were generated and sequenced on an Illumina NovaSeq 6000 using SP or S4 chemistry. The CGR performed the following read curation: the raw fastq files were trimmed for the presence of Illumina adapter sequences using Cutadapt v1.2.1 [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e] with option -O 3; the reads were further trimmed using Sickle v1.200 with a minimum window quality score of 20 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/najoshi/sickle\u003c/span\u003e\u003cspan address=\"https://github.com/najoshi/sickle\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e); and reads shorter than 15 bp after trimming were removed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetagenomic assembly and taxonomic classification.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTrimmed paired-end Illumina reads were assembled using metaSPAdes [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] or Megahit [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e] genome assemblers. When the memory requirements for metaSPAdes exceeded the memory available on our servers, we removed the reads mapped to \u003cem\u003eA. dimidiatus\u003c/em\u003e (GCA_907164435.1) to reduce the proportion of animal host sequences and used the unmapped reads for assembly with metaSPAdes. Short-read mapping was performed using bowtie2 v2.5.1 [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. We applied Kraken2 classification using the NCBI non-redundant nucleotide database (02/05/2023) for taxonomic classification. Contigs classified to taxon of interest were extracted from the Kraken2 output using the \u0026ldquo;extract_kraken_reads. py\u0026rdquo; script from KrakenTools [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Sequence annotation for further verification of the assigned contigs was performed by a DIAMOND BLASTX v2.0.14.152 [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] search against the NCBI non-redundant protein database and BLASTn search against the NCBI non-redundant nucleotide database.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eWolbachia\u003c/em\u003e assembly, paired-end read sequences were separately mapped to the contigs produced from metaSPAdes and Megahit using bowtie2 and re-paired and merged using FLASH v1.2.11 [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Mapped reads were reassembled with metaSPAdes, and Blobtoolkit v4.2.1 was used to remove eukaryotic sequences and sequences with no BLASTn hits in the assembly [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Prokka v1.14.6 was used for gene prediction and annotation [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Genome completeness was assessed by the Benchmarking Universal Single-Copy Orthologs (BUSCO) pipeline 5.0 and the rickettsiales_odb10 database [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial genome assembly\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eP. agamae\u003c/em\u003e mitochondrial genome was assembled using the MitoZ toolkit v3.3 [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e] with additional annotations of protein coding sequences (CDS), ribosomal RNA (rRNA) and transfer RNA (tRNA) sequences using the MITOS2 web service [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e], which produces circular and non-circular assemblies. The non-circular assembly was artificially circularised using the Simple-Circularise python script (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/Kzra/Simple-Circularise\u003c/span\u003e\u003cspan address=\"https://github.com/Kzra/Simple-Circularise\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Missing tRNAs were identified by aligning the assembly with existing mitochondrial genomes from trombiculid mites available in NCBI GenBank (\u003cem\u003eLeptotrombidium pallidum\u003c/em\u003e, AB180098.1; \u003cem\u003eLeptotrombidium deliense\u003c/em\u003e, AB194044.1; \u003cem\u003eLeptotrombidium akamushi\u003c/em\u003e, NC_007601.1; \u003cem\u003eWalchia hayashii\u003c/em\u003e, NC_010595.1; and \u003cem\u003eAscoschoengastia\u003c/em\u003e sp. TATW-1, AB300501.1) and manually inspecting the presence of conserved regions for tRNAs. Alignments of putative tRNA sequences were performed with MAFFT v6.864b [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e] as described above and visualised using Jalview v2 [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. RNA secondary structures were predicted using mfold [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e] in the UNAFold web service (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.unafold.org\u003c/span\u003e\u003cspan address=\"http://www.unafold.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the predicted structures for the tRNAs from \u003cem\u003eL. pallidum\u003c/em\u003e [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e] as a reference. The mitochondrial assembly was visualised using the web version of OGDRAW [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e] to produce the circular genome plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Alignments of the mitochondrial assembly and the detection of single nucleotide polymorphisms (SNPs) were performed using nucmer and dnadiff from the MUMmer4 package [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. Genome synteny was visualised using genoPlotR [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. Existing chigger mitogenomes analysed for gene arrangements comprised \u003cem\u003eAscoschoengastia\u003c/em\u003e sp. TATW-1, \u003cem\u003eW. hayashii\u003c/em\u003e, \u003cem\u003eL. deliense\u003c/em\u003e and \u003cem\u003eL. pallidum\u003c/em\u003e (accession nos. above). Sequences for the cytochrome oxidase I (\u003cem\u003ecoi\u003c/em\u003e) gene were generated from pooled archived chigger specimens described in our earlier studies [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] by PCR amplification using the HCO2198 and LCO1490 primers [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e] followed by Sanger sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic tree construction and genetic pairwise distance calculation\u003c/h2\u003e \u003cp\u003eSequences for genes of interest were aligned using MAFFT v6.864b [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e] along with existing sequences from NCBI GenBank. Concatenated alignments and partition files were generated using FASconCAT-G (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/PatrickKueck/FASconCAT-G\u003c/span\u003e\u003cspan address=\"https://github.com/PatrickKueck/FASconCAT-G\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Maximum-likelihood phylogenies were produced from the single or concatenated nucleotide alignments using IQTREE v2.2.2.9[\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e] with 1,000 ultra-fast bootstraps [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e] and best model selection from ModelFinder [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. The Interactive Tree of Life online tool was used to visualize the consensus trees produced (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de\u003c/span\u003e\u003cspan address=\"https://itol.embl.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and to generate the tree figures. Numbers at nodes represent ultra-fast bootstrap values and tree scales represent number of nucleotide substitutions. Genetic pairwise distances were calculated from alignments using the DistanceCalculator class from the Bio.Phylo.TreeConstruction module in Biopython v1.79 [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the \u003cem\u003eWolbachia\u003c/em\u003e phylogenomic tree, Orthofinder v2.5.4 was used to produce a set of orthologous sequences [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]. Protein sequences for each single-copy orthogroup (OG) were aligned using MAFFT v7.149b [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Gblocks v0.91b was used to trim noisy or poorly aligned protein positions [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. The trimmed alignments were concatenated into a supermatrix used to construct maximum likelihood trees in IQTREE v2.1.2. We used ModelFinder within IQTREE to determine the appropriate model for each protein. Branch support was calculated using the following options in IQTREE: (i) ultra- fast bootstrap, (ii) SH- aLRT support, (iii) local bootstrap support and (iv) aBayes Bayesian support, with all options set to 1,000, and all options produced highly similar values. Values from the ultra-fast bootstrap option [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e] were displayed along with the consensus trees in the final figures.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eThe fieldwork was approved by the Saudi Wildlife Authority (approval no. 288/33/A) and Animal Welfare and Ethics Review Board of the University of Liverpool.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eSequencing reads and assembled sequences produced in this study have been deposited in NCBI GenBank with the BioProject accession number PRJNA1031942. The following sequences were also deposited in NCBI Genbank: The \u003cem\u003eOrientia\u003c/em\u003e sp. \u003cem\u003ehtrA\u003c/em\u003e sequence was deposited with the accession number OR966881. \u003cem\u003eBorrelia\u003c/em\u003e sp. sequences were deposited with accession numbers OR817655 and OR817732-OR817734. Chigger mitochondrial \u003cem\u003ecoi\u0026nbsp;\u003c/em\u003esequences were deposited with accession numbers OR820617-OR820651. The mitochondrial genome assembly for \u003cem\u003eP. agamae\u003c/em\u003e Pa1 was deposited with the accession number OR817658.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003c/strong\u003eThis work was funded by a doctoral scholarship from Imam Mohammad Ibn Saud Islamic University to H.A.A., and A.S. is funded by New England Biolabs. A.N.A is supported by the Researchers Supporting Project number (RSPD2023R602), King Saud University, Riyadh, Saudi Arabia. A.A.S. is supported by the Ministry of Science and Higher Education of the Russian Federation (cooperative agreement No. 122031100263-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u0026nbsp;\u003c/strong\u003eH.A.A., S.Q.A. and B.L.M. designed the study; H.A.A., S.Q.A. and A.N.A. selected field sites and trapped rodents; H.A.A., S.Q.A. and A.A.S. identified rodents and mites; H.A.A. and S.Q.A. performed DNA extractions, PCR assays and sequence analysis; H.A.A., A.S., J.J.K. and A.C.D. conducted metagenomic and phylogenetic/genomic analyses; J.J.K assembled and analysed mitochondrial sequences; B.L.M. supervised the study; H.A.A., B.L.M. and J.J.K. wrote the first manuscript draft. G.M., A.S, A.A.S, A.N.A., and A.C.D reviewed and edited the manuscript. All authors read and approved the manuscript. The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e We acknowledge the important contribution of Camille Glazer to the chigger barcoding work, who sadly passed away in 2023. We are grateful to Al-Bahah University for access to research facilities during fieldwork.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information (optional):\u003c/strong\u003e Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWomersley H. The scrub-typhus and scrub-itch mites (Trombiculidae, Acarina) of the Asiatic-Pacific region. Rec S Aust Mus. 1952;10:1\u0026ndash;680.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElliott I, Pearson I, Dahal P, Thomas NV, Roberts T, Newton PN. Scrub typhus ecology: a systematic review of \u003cem\u003eOrientia\u003c/em\u003e in vectors and hosts. 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Syst Biol. 2008;57:758\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Scrub typhus, chiggers, metagenomics, Orientia, Wolbachia, Borrelia, mitochondrial genome, Acomys dimidiatus","lastPublishedDoi":"10.21203/rs.3.rs-3837555/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3837555/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTrombiculid mites are globally distributed, highly diverse arachnids that largely lack molecular resources such as whole mitogenomes for the elucidation of taxonomic relationships. Trombiculid larvae (chiggers) parasitise vertebrates and can transmit bacteria (\u003cem\u003eOrientia\u003c/em\u003e spp.) responsible for scrub typhus, a zoonotic febrile illness. \u003cem\u003eOrientia tsutsugamushi\u003c/em\u003e causes most cases of scrub typhus and is endemic to the Asia-Pacific Region, where it is transmitted by \u003cem\u003eLeptotrombidium\u003c/em\u003e spp. chiggers. However, in Dubai, \u003cem\u003eCandidatus\u003c/em\u003e Orientia chuto was isolated from a case of scrub typhus and is also known to circulate among rodents in Saudi Arabia and Kenya, although its vectors remain poorly defined. In addition to \u003cem\u003eOrientia\u003c/em\u003e, chiggers are often infected with other potential pathogens or arthropod-specific endosymbionts, but their significance for trombiculid biology and public health is unclear.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePooled chiggers of 10 species were collected from rodents in southwestern Saudi Arabia and screened for \u003cem\u003eOrientia\u003c/em\u003e DNA by PCR. Two species (\u003cem\u003eMicrotrombicula muhaylensis\u003c/em\u003e and \u003cem\u003ePentidionis agamae\u003c/em\u003e) produced positive results for the \u003cem\u003ehtrA\u003c/em\u003e gene, although \u003cem\u003eCa\u003c/em\u003e. Orientia chuto DNA was confirmed by Sanger sequencing only in \u003cem\u003eP. agamae\u003c/em\u003e. Metagenomic sequencing of three pools of \u003cem\u003eP. agamae\u003c/em\u003e provided evidence for two other bacterial associates: a spirochaete and a \u003cem\u003eWolbachia\u003c/em\u003e symbiont. Phylogenetic analysis of 16S rRNA and multi-locus sequence typing genes placed the spirochaete in a clade of micromammal-associated \u003cem\u003eBorrelia\u003c/em\u003e spp. that are widely-distributed globally with no known vector. For the \u003cem\u003eWolbachia\u003c/em\u003e symbiont, a genome assembly was obtained that allowed phylogenetic localisation in a novel, divergent clade. Cytochrome c oxidase I (\u003cem\u003ecoi\u003c/em\u003e) gene barcodes for Saudi Arabian chiggers enabled comparisons with global chigger diversity, revealing several cases of discordance with classical taxonomy. Complete mitogenome assemblies were obtained for the three \u003cem\u003eP. agamae\u003c/em\u003e pools and almost 50 SNPs were identified, despite a common geographic origin.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eP. agamae\u003c/em\u003e was identified as a potential vector of \u003cem\u003eCa.\u003c/em\u003e Orientia chuto on the Arabian Peninsula. The detection of an unusual \u003cem\u003eBorrelia\u003c/em\u003e sp. and a divergent \u003cem\u003eWolbachia\u003c/em\u003e symbiont in \u003cem\u003eP. agamae\u003c/em\u003e indicated links with chigger microbiomes in other parts of the world, while \u003cem\u003ecoi\u003c/em\u003e barcoding and mitogenomic analyses greatly extended our understanding of inter- and intraspecific relationships in trombiculid mites.\u003c/p\u003e","manuscriptTitle":"Microbiome and mitogenomics of the chigger mite Pentidionis agamae: Potential role as an Orientia vector and associations with divergent clades of Wolbachia and Borrelia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-19 20:53:34","doi":"10.21203/rs.3.rs-3837555/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-01T04:15:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-28T15:38:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-11T16:59:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3a19fc57-f807-4068-8707-c51bf0533643","date":"2024-01-29T12:01:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4ef85d1f-c6f4-41d4-b21f-53db38125861","date":"2024-01-27T14:15:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"503da7d9-0813-4cd0-b66e-b02e23eefa1a","date":"2024-01-27T13:01:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-23T11:32:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-22T09:19:31+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-01-17T02:38:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-17T02:36:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2024-01-05T14:56:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"04468ac6-b128-465f-b1fe-35c04ea98ffa","owner":[],"postedDate":"January 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-04-11T03:47:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-19 20:53:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3837555","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3837555","identity":"rs-3837555","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}