General Microbiota of the Soft Tick Ornithodoros turicata Parasitizing the Bolson Tortoise (Gopherus flavomarginatus) in the Mapimi Biosphere Reserve, Mexico

The general bacterial microbiota of the soft tick Ornithodoros turicata found on Bolson tortoises (Gopherus flavomarginatus) were analyzed using next generation sequencing. The main aims of the study were to establish the relative abundance of bacterial taxa in the tick, and to document the presence of potentially pathogenic species for this tortoise, other animals, and humans. The study was carried-out in the Mapimi Biosphere Reserve in the northern-arid part of Mexico. Bolson tortoises (n = 45) were inspected for the presence of soft ticks, from which 11 tortoises (24.4%) had ticks in low loads (1–3 ticks per individual). Tick pools (five adult ticks each) were analyzed through 16S rRNA V3–V4 region amplification in a MiSeq Illumina, using EzBioCloud as a taxonomical reference. The operational taxonomic units (OTUs) revealed 28 phyla, 84 classes, 165 orders, 342 families, 1013 genera, and 1326 species. The high number of taxa registered for O. turicata may be the result of the variety of hosts that this tick parasitizes as they live inside G. flavomarginatus burrows. While the most abundant phyla were Proteobacteria, Actinobacteria, and Firmicutes, the most abundant species were two endosymbionts of ticks (Midichloria-like and Coxiella-like). Two bacteria documented as pathogenic to Gopherus spp. were registered (Mycoplasma spp. and Pasteurella testudinis). The bovine and ovine tick-borne pathogens A. marginale and A. ovis, respectively, were recorded, as well as the zoonotic bacteria A. phagocytophilum, Coxiella burnetii, and Neoehrlichia sp. Tortoises parasitized with O. turicata did not show evident signs of disease, which could indicate a possible ecological role as a reservoir that has yet to be demonstrated. In fact, the defense mechanisms of this tortoise against the microorganisms transmitted by ticks during their feeding process are still unknown. Future studies on soft ticks should expand our knowledge about what components of the microbiota are notable across multiple host–microbe dynamics. Likewise, studies are required to better understand the host competence of this tortoise, considered the largest terrestrial reptile in North America distributed throughout the Chihuahuan Desert since the late Pleistocene.


Location and Environmental Conditions
The study was carried-out in the Mapimi Biosphere Reserve in Mexico, which includes part of the states of Chihuahua, Coahuila, and Durango (26 • 00 and 26 • 10 N, 104 • 10 and 103 • 20 W; Figure 1). The Bolson de Mapimi has been defined as an endoreic basin, which includes diverse small sub-basins intermixed along valleys with a mean altitude of 1150 m. This area has very arid climate [20], with an average annual temperature of 25.5 • C, and an average annual precipitation of 264 mm [21]. The predominant vegetation in the reserve is rosette and microphile scrub, as well as halophyte, and gypsophila plants [22]. Interestingly, the microregion concentrates the richest herpetofauna across the whole Chihuahuan desert, having diverse endemic species, including the Bolson tortoise [23].
All the methods and activities of this study were in strict accordance with accepted guidelines for ethical use, care, and welfare of animals in research at the international level [19]. The federal approval reference number is SEMARNAT-SGPA/DGVS/08406/16. This research has been approved by the Facultad de Ciencias Biológicas UJED ethic committee on 5 February 2017 (ethic code: 0023). The files used in this study (Supplementary files: P1.fasta, P2.fasta and P3.fasta) were deposited into the NCBI Sequence Read Archive (SRA) database (SRA Accession Number: PRJNA649587).

Location and Environmental Conditions
The study was carried-out in the Mapimi Biosphere Reserve in Mexico, which includes part of the states of Chihuahua, Coahuila, and Durango (26°00′ and 26°10′ N, 104°10′ and 103°20′ W; Figure 1). The Bolson de Mapimi has been defined as an endoreic basin, which includes diverse small subbasins intermixed along valleys with a mean altitude of 1150 m. This area has very arid climate [20], with an average annual temperature of 25.5 °C, and an average annual precipitation of 264 mm [21]. The predominant vegetation in the reserve is rosette and microphile scrub, as well as halophyte, and gypsophila plants [22]. Interestingly, the microregion concentrates the richest herpetofauna across the whole Chihuahuan desert, having diverse endemic species, including the Bolson tortoise [23].

Field Work
From May to July 2017, Bolson tortoises were captured by hand from 900 to 1300 h and from 1700 to 2100 h. The search for ticks was carefully carried out on the carapace, neck and skin folds of all four limbs. A total of 45 individuals of G. flavomarginatus were captured and revised, but only 11 tortoises (24.4%) carried soft ticks on the carapace or over the skin (Figures 2 and 3). Tortoises carried from one to three ticks per individual. A total of 17 adult ticks were collected and taxonomic keys were used to determine the species [24]. Each tick was placed in an individual 1.5 mL tube containing 500 uL ethanol (70%), 500 uL hydrogen peroxide (H2O2), and 200 uL of ultrapurified H2O; each tube was vortexed for 15 s to remove tick surface contaminants [25]. Later, three pools were formed (five ticks each) and were deposited in BashingBead™ Zymo Research™ cell lysis tubes, containing 750 μL of lysing/stabilizing solution. The tubes were processed in a cellular disruptor (TerraLyzer™) for 20 s. The two extra ticks were deposited in the Entomological collection of the Facultad de Ciencias Biológicas, Universidad Juárez del Estado de Durango, México, for reference purposes.

Field Work
From May to July 2017, Bolson tortoises were captured by hand from 900 to 1300 h and from 1700 to 2100 h. The search for ticks was carefully carried out on the carapace, neck and skin folds of all four limbs. A total of 45 individuals of G. flavomarginatus were captured and revised, but only 11 tortoises (24.4%) carried soft ticks on the carapace or over the skin (Figures 2 and 3). Tortoises carried from one to three ticks per individual. A total of 17 adult ticks were collected and taxonomic keys were used to determine the species [24]. Each tick was placed in an individual 1.5 mL tube containing 500 uL ethanol (70%), 500 uL hydrogen peroxide (H 2 O 2 ), and 200 uL of ultrapurified H 2 O; each tube was vortexed for 15 s to remove tick surface contaminants [25]. Later, three pools were formed (five ticks each) and were deposited in BashingBead™ Zymo Research™ cell lysis tubes, containing 750 µL of lysing/stabilizing solution. The tubes were processed in a cellular disruptor (TerraLyzer™) for 20 s. The two extra ticks were deposited in the Entomological collection of the Facultad de Ciencias Biológicas, Universidad Juárez del Estado de Durango, México, for reference purposes.

DNA Extraction, Visualization, and Quantification
DNA was extracted from the pools using the Xpedition™ Tissue/insect DNA MiniPrep kit (Zymo Research Corp., Irvine, CA, USA) in a laminar UV flow hood in sterile conditions. Then, the DNA was placed on a 1.2% agarose gel at 80 V for 45 min in a BIORAD electrophoresis chamber (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The DNA visualization was carried out in a GelMax™ photo documenter (UVP LLC, Upland, CA, USA). The amount of DNA obtained was measured in a Qubit™ fluorometer (Invitrogen, Carlsbad, CA, USA). Then, the V3-V4 region of the 16S rRNA gene was amplified using the following primers [26]: S-D-Bact-0341-b-S-17, 5 -CCTACGGGNGGCWGCAG-3 and S-D-Bact-0785-a-A-21, 5 -GACTACHVGGGTATCTAATCC-3 .

Bioinformatics Analyses
The DNA sequences were analyzed on MGLinux, in a VM Oracle VirtualBox using Quantitative Insights into Microbial Ecology bioinformatics software (QIIME, Boulder, CO, USA) [29]. Both forward and reverse sequences were assembled using the PEAR program [30] with an overlap of 50 bp, a minimum reading length of 430 bp, and a maximum of 470 bp, a quality criterion Q30 (one false base for every 1000 bases) with p < 0.0001. Then the files were converted to the FASTA format, and chimeric sequences were discarded with USEARCH [31]. Thereafter, the operational taxonomic units (OTUs) were selected with the UCLUST method [31] at 97% similarity; a representative sequence for each OTU was obtained, and the taxonomy was assigned and taken as reference to the EzBioCloud database [32]. Next, the OTUs table was built in the Biom format (biological observation matrix) [33] separating domains. A simple random rarefaction process was performed [34] in order to obtain a standardized Biom file for all pools. The Shannon and Simpson alpha diversity indexes were calculated using the standardized Biom file; the mean ± standard deviation for each index was obtained. The relative abundance for the phylum level was represented as a bar chart using Excel, and family and genus levels were visualized as heatmaps using Morpheus software (Broad Institute, Cambridge, MA, USA) [35]; hierarchical clustering (average linkage method with Euclidean distance) was used to visualize pools dendrograms. Each genus and/or species of bacteria registered for O. turicata was consulted in the available literature to indicate its possible pathogenic potential for tortoises or zoonotic potential for humans.

Results
The average number of sequences assembled was 196,564. After taxonomic designation an average of 190,769 bacterial sequences was obtained. The average number of OTUs was 44,369 (Table 1). Simple random rarefaction was made at 100,000 sequences, since at this point the number of taxa of the three pools reached asymptotes. From the standardized Biom file, the OTUs resulted in 28 phyla, 84 classes, 165 orders, 342 families, 1013 genera, and 1326 species. Abbreviations are ChS = chimeric sequences eliminated, QS = quality sequences after chimeras elimination, BS = bacteria sequences after taxonomy designation, and OTUs = operational taxonomic units.

Discussion
Our working hypothesis stated that the microbiota of O. turicata would contain potentially pathogenic bacteria for the Bolson tortoise Gopherus flavomarginatus, other animals, and humans. Therefore, and based on the obtained results, this study demonstrated the presence of some potentially pathogenic and zoonotic bacteria in the microbiota of O. turicata.
Alphaproteobacteria and Gammaproteobacteria classes were the most abundant in O. turicata microbiota, as has been observed in other tick species [41]. These classes are relevant in the microbiota of all tick species because they group the main intracellular endosymbionts (Arsenophonus-like, Coxiella-like, Fransicella-like, Midichloria-like, Rickettsia-like, and Wolbachia-like) that inhabit the organs of these arthropods [6,42]. These endosymbionts have generally been reported to predominate in the arthropod microbiota and may interfere with the transmission dynamics of pathogenic bacteria [43,44]. In the present study, O. turicata showed only two genera of intracellular endosymbionts, Midichloria and Coxiella. An unidentified species Midichloria-like endosymbiont was the dominant taxon in the O. turicata microbiota, although Candidatus Midichloria mitochondrii (CMM) species was also abundant in this tick. CMM has been reported in a large number of tick genera (Amblyomma spp., Dermacentor spp., Haemaphysalis spp., Hyalomma spp., Ixodes spp., and Rhipicephalus spp.) in different countries of Europe, Asia, and North America [41,45,46]. It is considered a facultative non-pathogenic mutualistic bacterium that lodges in the reproductive tissues of female ticks, specifically in the ovarian mitochondria, where they sometimes invade and destroy this organ [47,48]. By remaining in this organ, vertical transmission to the next generation is carried out. However, its presence has also been recorded in salivary glands from which it is transmitted to vertebrate hosts during tick feeding [49]. Since all the body of O. turicata was analyzed in the present study, it is not possible at this time to determine in which organs occur both unknown Midichloria-like and CMM. However, it is feasible that they occur in the reproductive organs since some studies have analyzed the possible evolutionary process of intracellular endosymbionts of arthropods, and apparently transovarian transmission is the common factor for all species [50]. Bacterial diversity studies in each organ of O. turicata are required to clarify this question.
The second most abundant bacterial species in O. turicata microbiota in our study was a Coxiella-like endosymbiont. This type of endosymbiont does not appear to be pathogenic and are relatively common in the microbiota of various tick species around the world [51,52]. These microorganisms are known to infect tick ovaries and then transmission occurs vertically through the egg cytoplasm, but they have also been found in Malpighi tubules where they may provide essential nutrients to their host [7]. It should be noted the presence of C. burnetii in the present study. This is a zoonotic bacterium that causes Q-fever in both animals and humans. In particular, for the Ornithodoros genus there are reports of C. burnetii in O. tartakovskyi, O. papillipes and O. alactagalis in the former Soviet Union [53], O. moubata in Japan [54], and O. sonrai in Senegal [55]. According to Balashov and Daiter (1973) [53] and Eldin et al. (2017) [56], transmission of C. burnetii is transovarian in most ticks, except for Ixodes holocyclus, O. hermsi, and O. turicata in which it is transestadial. Direct transmission of C. burnetii from infected ticks to humans is not well documented and may occur only rarely in nature [57]. However, the main transmission route is via inhalation of contaminated fecal material from ticks [58]. Since the personnel that monitor G. flavomarginatus populations each year in the Mapimi Biosphere Reserve should manipulate tortoises, it is advisable to take precautions to avoid this airborne infection.
Mycoplasma spp. and Pasteurella spp. are bacterial genera that have been reported as part of the microbiota of hard ticks, as in Ixodes simplex and I. ventalloi, respectively [59,60]. In the present study with O. turicata, the presence of Mycoplasma spp. and Pasteurella testudinis in low abundances was recorded. An interesting fact is that these bacteria along with some viruses have been reported as some of the possible causes of upper respiratory disease (URTD) in tortoises, causing a decrease in the populations of G. agassizii and G. polyphemus in the USA [61,62]. It is still unknown if these bacteria can be transmitted directly to the host, and in the case of G. flavomarginatus if they could cause disease. For now, these bacteria should remain as potential pathogens for this tortoise, until further studies of vector competence discard this possibility.
Other relevant species recorded in O. turicata microbiota were the obligate intracellular bacteria Anaplasma phagocytophilum, A. marginale, A. ovis, and Neoehrlichia sp. These species are important for animal and human health because they typically infect hematopoietic or endothelial cells [63]. Anaplasma phagocytophilum is a tick-borne pathogen that causes human granulocytic anaplasmosis mainly in Asia, Europe, and the USA; A. marginale and A. ovis are worldwide tick-borne bacteria causing bovine and ovine anaplasmosis respectively [64,65]. The genus Neoehrlichia was discovered in 1999 as an Ehrlichia-like bacterium [66], which was later described as Candidatus Neoehrlichia mikurensis [67], an emerging tick-borne pathogen detected in ticks and rodents, causing human systemic inflammatory syndrome in Asia and Europe [68,69]. In America, Candidatus Neoehrlichia lotoris was registered in a free-living raccoon associated with tick-infested populations [70], which suggests that it is transmitted to mammals by ticks. Undoubtedly, specific molecular studies are needed to determine which Neoehrlichia species is the one registered in O. turicata, nevertheless, its zoonotic potential is latent.
According to Gofton et al. (2015b) [71], a limitation of 16S bacterial community profiling in ticks is that a high proportion of sequences will belong to bacterial endosymbionts, as CMM as observed in O. turicata in the present study. This high abundance may mask the presence of less abundant bacteria with zoonotic potential, as for example the genus Borrelia, which was not detected in our study. Species such as B. mazzottii recorded in O. talaje in Mexico [72], as well as B. parkeri in O. parkeri, B. hermsii in O. hermsi, and B. turicatae in O. turicata in the USA [73], are important indicators of the presence of this bacterial genus in the Ornithodoros ticks of America. Perhaps, by carrying out detailed molecular studies in O. turicata in the Mapimi Biosphere Reserve, Borrelia and other zoonotic bacteria such as Rickettsia could be identified. In the same way, since this massive sequencing technique does not reach the species level in many genera documented here for O. turicata, it is necessary to carry out specific PCR studies for the most relevant bacteria involved in the biology of this tick and those bacteria important for the health of animals and humans.
It is important to comment that the present study was carried out at a single time of the year, without distinguishing sexes of O. turicata to provide an initial overview of the microbiota of this tick species. However, various studies indicate that the microbiota of ticks is dynamic and can change among seasons of the year due to the effect of temperature and humidity, between males and females, between different feeding statuses, among life stages, etc. [3,4]. Therefore, it is guaranteed that detailed studies on these issues will further be carried out.
Finally, the high number of bacterial taxa recorded for O. turicata in the present study could be due to the variety of hosts that this tick parasitizes within the burrows of G. flavomarginatus, performing a rapid feeding process that usually lasts an hour on average [14,74]. In this way the general feeding habits of O. turicata may be keeping pathogenic microbes in circulation, thereby ensuring their survival in the ecosystem. In our study, all G. flavomarginatus tortoises found carrying O. turicata ticks were apparently healthy, having the possible ecological role of reservoirs of pathogenic bacteria [75,76]. The fact that tortoises have long lives can favor the maintenance of pathogen cycles under normal conditions [77,78]. However, after thousands of years of coevolution, a microbial balance between O. turicata and G. flavomarginatus must have been reached, although it is not yet known whether this tortoise's defense mechanism is resistance (i.e., capacity to limit pathogen loads) or tolerance (i.e., capacity to survive damage caused by a given pathogen load) [79][80][81]. To clarify this enquiry, it will be necessary to determine the bacteria that G. flavomarginatus carries in the blood and whether they were transmitted by O. turicata. Studies of resistance mechanisms and vector competence in this tortoise will also be relevant from the point of view of immunity and the eco-epidemiology of zoonotic diseases, respectively.

Conclusions
Our study established the general relative abundance of bacterial taxa in the soft tick O. turicata, documenting the presence of potentially pathogenic species for the Bolson tortoise G. flavomarginatus, other animals and humans in the Mapimi Biosphere Reserve, Durango, Mexico. The most abundant phyla were Proteobacteria, Actinobacteria, and Firmicutes. Additionally, Alphaproteobacteria and Gammaproteobacteria classes were the most abundant in O. turicata microbiota. The most abundant species were Midichloria-like and Coxiella-like (endosymbionts of ticks). Mycoplasma spp. and Pasteurella testudinis, both potentially pathogenic to the Bolson tortoise, were registered. Additionally, the bovine and ovine tick-borne pathogens A. marginale and A. ovis, respectively, as well as the zoonotic bacteria A. phagocytophilum, Coxiella burnetii, and Neoehrlichia sp. were founded in O. turicata. Future studies on soft ticks should expand our knowledge about what components of the microbiota are notable across multiple host-microbe dynamics. Likewise, studies are required to better understand the reservoir competence of this tortoise, considered the largest terrestrial reptile in North America distributed throughout the Chihuahuan Desert since the late Pleistocene.