Introduction

Ticks are strict hematophagous ectoparasites of vertebrate animals and humans. Distributed all over the world, they are implicated in the transmission of several pathogens (viruses, bacteria, protozoa, and helminths) of medical and veterinary importance. Accordingly, ticks are considered to be the second world wild vectors of human diseases behind mosquitos, but they are the first vectors of pathogen diseases in domestic and wild animals [1, 2].

Livestock farming is one of the main agricultural sectors in North African countries and Tunisia [3]. Ticks are widespread in Tunisia over different geographical regions, Hyalomma spp are the most prevalent ticks infesting cattle [4, 5].

Tick infestation represents a major threat in cattle breeding due to their capacity to transmit three major tick-borne pathogens (TBPs) and to induce different diseases specifically anaplasmosis, babesiosis and theileriosis [4, 6, 7]. Tropical theileriosis, which is caused by the protozoan Theileria annulata and transmitted by Hyalomma scupense, is the major tick-borne disease affecting North African and Tunisian cattle [8–10].

Besides pathogens, ticks also harbor a large variety of symbiotic and commensal microorganisms [11]. Symbiotic bacteria play important roles in tick survival particularly via the synthesis of some essential vitamins and cofactors, especially vitamins of group B, which are lacking in the highly specialized hematophagous tick diet [12–14]. These bacteria are implicated in tick adaptation to environmental stress, they are also essential for tick’s reproductive fitness [11, 12]. The symbiotic bacteria can also influence the colonization, maintenance and transmission of pathogens [15, 16]. Ticks carry at least 10 different genera of maternally inherited endosymbiotic bacteria [12, 13]. Three of these endosymbionts namely Coxiella, Francisella and M. mitochondrii are specific to ticks. Coxiella is the most frequent endosymbiont which presents in two-thirds of tick species [12, 17]. M. mitochondrii which infect the mitochondria has been reported in some tick species, while Francisella has only been detected in a few tick species [18]. Indeed, Francisella, Coxiella and Rickettsia endosymbionts are obligate nutritional symbiotic bacteria that have conserved all the major genes encoding for vitamins B synthesis despite their highly small genome [19–21]. For instance, Coxiella endosymbiont carries the genes encoding for vitamins B7 (biotin), B2 (riboflavin), B9 (folic acid), and their cofactors [22, 23]. In the Rickettsia endosymbionts genome, Hunter’s team confirmed the presence of all genes encoding for the biosynthesis of folic acid [21]. Also, Francisella endosymbiont genome includes all the genetic pathways for the synthesis of folic acid, biotin, and riboflavin [16, 24].

The importance of B vitamins in tick survival and development has been established by Duron et al. in 2018 with Francisella in the soft tick Ornithodoros moubata [13]. An experimental elimination of the symbiont using antibiotic treatment was followed by a reduced tick survival and the appearance of some physical abnormalities, interestingly, these effects were fully restored with a supplement of vitamin B. Similar experiments with Coxiella in Amblyomma americanum, Haemaphysalis longicornis, and Rhipicephalus microplus resulted in lower reproductive fitness and prevented even the development into adults in R. microplus. While, being one of the maternally transmitted endosymbiotic, the role of M. mitochondrii in their host biology has not been well established. However, some studies have suggested their role in molting and tick blood meal metabolism following the increase of this symbiont after feeding [25, 26].

Taken altogether the above general data are highlighting the important role of obligate symbionts in tick biology and reproduction, opening a potential window of opportunities for developing prototypes of innovative and environmentally friendly control measures targeting these symbionts.

In North Africa and Tunisia, the majority of studies on livestock and zoonotic tick-borne pathogens have focused essentially on pathogens and diseases identification, and their epidemiology and control using conventional approaches. However, almost there is no data on microbiota and endosymbionts of the most dominant and important Tunisian tick species of the Hyalomma genus. In this context, our work intends to provide the first comprehensive study carried out on major Hyalomma species in Tunisia and more generally in North Africa, for describing, firstly, the microbial diversity and richness of tick microbiota in Hyalomma ticks infecting livestock in different bioclimatic and geographic regions in Tunisia, and establishing secondly, the prevalence of their endosymbiotic bacteria. We aim in the present work to focus more, and for the first time to our knowledge, on the symbionts associated with the different life stages of the monotropic H. scupense cattle tick, the vector of tropical theileriosis, and to carry out a comparative study with the adult stages of other dominant cattle Hyalomma tick species in Tunisia.

Material and methods

Study area and tick sampling

A total of 776 adult ticks were sampled during June, July and August 2020. Adult ticks were collected from cattle in 6 different bioclimatic stages and 9 Tunisian administrative governorates to explore the whole microbial diversity of Tunisian ticks and to investigate their endosymbionts. For this purpose, cattle barns were selected according to previous tropical theileriosis cases, sampling information’s and geographical coordinates were summarized in Table 1. Furthermore, engorged H. scupense nymphs were collected from walls of some cattle breeding barns between October and November 2020 from El Hissiene site (upper semi-arid stage, Ariana, Tunisia: N37°0’14,50469" E10°10’18,06892).

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Table 1. Bioclimatic stage, governorate, location, delegation, farms visited, surveyed cattle and ticks collected.

https://doi.org/10.1371/journal.pone.0268172.t001

Prevalence and transmission of symbionts

Independent Francisella and Rickettsia endosymbionts identification was performed using PCR by amplifying the rRNA 16S and glta gene with specific primers (Table 2). PCR conditions set with denaturation step at 94°C for 7 min followed by 40 cycles at 94°C for 45s, 62°C for 30s and 72°C for 45s with a final extension at 72°C for 7 min. The negative DNA template for symbiont was verified systemically by the amplification of ticks’ 18S rRNA gene using universal primers of eucaryotic. PCR products of glta and 16S were purified and sequenced from both directions. Sequences were verified using BLAST.

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Table 2. Targeted genes, primers, and nucleotide sequences per tick species.

https://doi.org/10.1371/journal.pone.0268172.t002

To compare the prevalence of symbionts between sex and life stage of H. scupense ticks, the test chi 2 was performed. Results were considered significant at 5% threshold.

Results

Microbial diversity in ticks

We have essentially focused on the characterization of the microbial composition and microbiota diversity, richness, and their variation between male and female as well as between different life stages of H. scupense. We have also carried out a comparative study with H. marginatum and H. excavatum which adult stage commonly infest Tunisian cattle in Tunisia. In this regard, 16S rRNA (V3-V4 region) sequencing and metagenomic analysis were established. The relevant accession numbers used to access to our data are available on the following links https://www.ncbi.nlm.nih.gov/sra/PRJNA791601 (for all data).

Denoising reads with deblurring were generated 97081 features where the greatest number of reads was observed in female H. scupense and the lowest number of reads was detected in male H. scupense. The denoising allowed the selection of 124 representative sequences Operational Taxonomic Unit (OTU) with a mean length of 148 bp. The taxonomic assignment of OTUs was carried out using the Silva 16S database with a cutoff of 99%, the classifier was trained using the qiime feature-classifier algorithm for V3-V4 primers.

Bacteria profiling from different identified livestock species showed that proteobacteria were the dominant phylum with more than 97% of relative percent abundance (RPA) followed by Actinobacteria, Firmicutes, and Deinococcota. Overall, 16 bacterial families were detected among those Rickettsiacea, Francisellacea, Enterobacteriaceae, Bacillaceae, and Brevibacillacea were the most predominant families in the examined tick species (Fig 1). In the male of H. scupense, Enterobacteriaceae was the most prevalent bacterial family with 69.5% RPA followed by Rickettsiaceae 22.9% and Francisellacea 2.56%. However, Francisellacea was the dominant family in the female of H. scupense with 78.8% of RPA followed by Rickettsiaceae with 19.9% of RPA. In the two other Hyalomma species, Rickettsiaceae were the most abundant followed by Francisellacea.

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Fig 1. Family level taxonomic composition of Hyalomma excavatum, H. marginatum and H. scupense adult ticks.

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Three symbiotic bacterial genera were detected in the analyzed ticks, these were Francisella, Rickettsia and M. mitochondrii. Francisella and Rickettsia genera were detected in all three species of adult Hyalomma, they were also present in all the stages of H. scupense (Fig 2), however at different RPA. The highest RPA of Francisella was recorded in the females of H. scupense with an RPA of 78.8%, followed by H. marginatum sample with an RPA of 37.9%. However, the lowest rate was observed in the male of H. scupense. The Rickettsia genera make up the vast majority of H. excavatum with 70.8% RPA, it was also detected at a high abundance of 34.5% in H. marginatum. M. mitochondrii was highly present in H. marginatum and H. excavatum with an RPA of 34.5 and 17.3% respectively.

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Fig 2. Relative abundance (%) of bacterial genera according to tick species and life stage of Hyalomma scupense.

Organisms listed in the legend appear from top to bottom in order of highest relative abundance.

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We have also detected some genera of pathogen bacteria for instance; Ehrlichia, Erwinia, Dietzia, Escherichia, Shigella, Pseudomonas, as well as the two other symbiotic/pathogenic bacteria: Francisella and Rickettsia. To examine the possible difference in bacterial population structure between species, sexes, and life stages, alpha diversity parameters were calculated (Table 3 and Fig 3).

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Fig 3. Alpha diversity and richness parameters of Hyalomma scupense microbiota.

a: Shannon alpha diversity between species; c: Chao alpha diversity between species; b: Chao alpha diversity between life stages.

https://doi.org/10.1371/journal.pone.0268172.g003

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Table 3. Alpha diversity indexes.

https://doi.org/10.1371/journal.pone.0268172.t003

Notably, there was a significant difference between adult species, H. excavatum and H. marginatum samples have a significantly more diverse microbiome than H. scupense as indicated by the Chao and Simpson indexes (Table 3 and Fig 3). H. scupense, female ticks have less microbial diversity than males, furthermore, there was a significant difference between the relative abundance of the bacterial genus and species detected in female versus male ticks (p<0.05), indeed, 40 species were detected in males while 35 species were recorded in females (Fig 4). Indeed, Francisella endosymbionts constituted by far a dominant percentage of the microbiome of female ticks comparatively to male ticks, 78.8% and 2.51% respectively. Although a significant difference between different stages was detected since adult ticks exhibit a greater extent of microbial diversity. We then examined the microbial richness using the Shannon diversity index (Table 3) and similar trends were observed with the Chao index because H. scupense samples have less rich diversity than the other two species. However, in the case of H. scupense, nymphs were by far the richest in diversity relative to the two other life stages.

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Fig 4. Genus’s level taxonomic composition of male and female Hyalomma scupense ticks.

https://doi.org/10.1371/journal.pone.0268172.g004

Discussion

Previous surveys on ticks microbiota have shown carriage of multiple symbionts which are very important for their physiology, fecundity, and vector competence [32–34]. Currently, several studies were carried out on microbial populations in ticks such as Ixodes, Rhipiciphalus, Haemaphysalis, Amblyomma, Dermacentor [35–41]. However, little is known about the diverse microbiome of the tick genus Hyalomma associated with cattle. Moreover, there is no information regarding the microbial population structure of the most dominant Tunisian cattle tick H. scupense despite its veterinary and economic importance. It is of great interest to know crucial details on the microbial composition and their symbionts to improve our knowledge about tick bacterial communities. Therefore, this investigation may shed light on the vectorial competency of these vectors to transmit pathogens to vertebrate hosts. We present here the first characterization of the whole microbial communities of the monotropic cattle tick H. scupense according to the stages and sex and its comparison with the microbial diversity of adults H. scupense tick of two other dominant Hyalomma species which adult stages are infesting cattle, namely H. excavatum and H. marginatum.

Next generation sequencing of the hypervariable V3-V4 region of bacterial 16S rRNA gene was performed using Hyalomma specimens with Illumina Miseq. Similar to other studies, we found that the most dominant phylum was Proteobacteria. Additionally, the bacterial microbiota of Hyalomma was dominated by three principal genera; namely the two symbiotic/pathogenic bacteria Francisella and Rickettsia, and the endosymbiotic M. mitochondrii. Previous studies on other genera like Rhipicephalus have shown that they were mainly infected with Coxiella and Rickettsia with an association of Coxiella-like endosymbionts with females [37, 42]. A more recent study on the microbiome of Hyalomma species infesting livestock in Pakistan showed that Francisella, Rickettsia, and Coxiella were the most dominant tick bacterial identified species [43, 44]. Nevertheless, our results correlate with another survey on ticks in Turkey, where Coxiella was not detected in Hyalomma samples, while Francisella and Rickettsia predominate in the microbiome of H. excavatum and H. marginatum [28]. In our study, it was not entirely surprising that we detected the genus Francisella at high relative abundance associated with the absence of Coxiella endosymbiont, despite the higher rate of transovarial transmission and the abundance of Coxiella endosymbiont in several other tick genera. This endosymbiont was detected using NGS in a large variety of hard ticks, including Amblyomma americanum [45], Haemaphysalis longicornis [35], Ornithodoros amblus [46], Rhipicephalus microplus [47], and Rhipicephalus sanguineus [37]. Our findings are similar to those of Duron et al. (2015) who detected at low frequency Coxiella endosymbiont in approximately one-third of their targeted species [47]. Furthermore, Gehrart and his team suggested that Francisella has replaced Coxiella in the Gulf Coast tick, Amblyomma maculatum [48].

We suggest that the lack of detection of Coxiella infection in our samples and the abundance of another maternally inherited endosymbiont may be due to the replacement and competition of this ancient endosymbiont with other maternally inherited endosymbionts. In our case, we have revealed the presence of three other genera of maternally inherited bacteria: Francisella, M. mitochondrii and Rickettsia endosymbiont in all tick specimens. The high distribution of Francisella among all our tick species suggests that they may have independently replaced Coxiella endosymbiont as an alternative obligate symbiont. The importance of Francisella in Tunisian Hyalomma ticks is probably the result of the interaction and coevolution between the microbiome community and the tick’s ecological modification and restricted diet. Although it is important to emphasize that physiological and nutritional investigation on the symbiotic functions of Francisella are required to validate their role as alternative obligate symbionts. Moreover, Genome sequencing of these bacteria demonstrates that they evolved by developing adaptative mechanisms implicated in tick survival however, they have highly conserved the major genes responsible for the synthesis of vitamins B, cofactors, and some amino acids like Coxiella endosymbiont despite their restraint genome [19–21, 24, 48].

Interestingly, we report here, using high throughput sequencing, the first detection of the obligate intracellular mitochondrial tick endosymbiont M. mitochondrii in eggs of Hyalomma tick with a high abundance of more than 65%, however this density was considerably decreased in the other stages of H. scupense (nymphs and adults), along with the increase of other two endosymbionts. The distribution and the transovarial transmission of this intracellular endosymbiont in H. scupense suggested that this association might be obligate, and have a potential role in the physiological fitness of H. scupense tick. Furthermore, this bacterium is significantly abundant in H. marginatum and to a lesser extent in H. excavatum with RPA of 34.5 and 17.8% respectively. Recently, Selmi et al. reported that M. mitochondrii was prevalent at a rate of 8% in partially fed H. dromedarii and H. impeltatum ticks collected from dromedaries in Tunisia [49]. A recent study on the detection of pathogens and M. mitochodrii in Egyptian cattle ticks using the Reverse Line Blot hybridization (RLB) showed that 11.6% of pools of H. excavatum are infected with M. mitochondrii while only 2.9% of R. annulatus pools are infected by this bacteria [50]. Moreover, the detection of this endosymbiotic bacterium in other hard ticks is very variable [26, 51]. M. mitochondrii is well-described in I. ricinus [25] where it was detected in the mitochondria of ovaries. These bacteria which are included in the α-Proteobacteria entered the mitochondria of the oocyst cells. About 94–100% of adult females of I. ricinus are infected with this bacterium with a 100% transovarial transmission rate [25].

Additionally, we have studied various aspects of microbial diversity including species, sex, and life stage. The investigation of the microbial diversity with Haylomma species indicates that the adults of H. marginatum and H. excavatum exhibits more diverse and richer microbiota than H. scupense. Previous microbial diversity studies in three Hyalomma species collected in Turkey also indicated that H. excavatum and H. marginatum harbor more diverse microbiota than H. aegyptium [28]. This diversity is influenced by several determinants, mainly, environmental factors like habitat [52], geographical dispersion [53], bioclimatic conditions [54], sample location [55], soil and plant associated bacteria [56], and tick’s host [38, 57].

H. marginatum and H. excavatum are probably exposed to more complex environmental conditions as they pass through different hosts during their development particularly at juvenile stages [58]. Moreover, it is well known that blood meals have a strong impact on tick microbial diversity, composition, and species richness as mentioned by Swei and Kwan [38]. Indeed, ticks acquire more microorganisms, including pathogens, from hosts during their blood meals. More diverse hosts and larger numbers of vertebrate hosts hypothetically drive to more diverse microbiota [59]. This is probably the case for the outdoor H. marginatum and H. excavatum ticks, indeed, these tick juveniles feed, according to their tropism, on a variety of small terrestrial mammals and birds, whilst their adults can engorge on a variety of large herbivores such as goats, sheep, cattle, horses, camels [60, 61]. In contrast, H. scupense, exhibits a domestic behavior, and cattle are by far the almost exclusive host for adults and juveniles in Tunisia [5, 62]. Other studies found that the use of different tick species feeding on the same host don’t even have the same microbiomes, suggesting, therefore, that the tick microbiome is, to a certain degree, governed by species specific determinants [63]. Microbial composition was also affected by sex. Females H. scupense microbiota was less diverse and rich as compared to males, but inherited a high relative abundance of endosymbionts than males especially their OTUs number. We also found a higher prevalence of Francisella in females, especially in engorged females (100%). Similar results were reported in other studies that typically [42, 64, 65] In the same way, Van Treuren and his team found that females show less diverse microbiota than males [53]. The increased RPA of Rickettsia and Francisella endosymbiont that we recorded in females relative to males could be an adaptation to reproductive requirements and transovarial transmission. In addition, we demonstrated efficient maternal transmission of these two endosymbionts.

According to our metagenomic analysis, we found a significant loss of bacterial species richness in adults when compared to nymphs. Francisella and Rickettsia become dominant at older life stages correlated with the loss of microbiome richness. Our results are similar to another study on the microbiome of Ixodes pacificus [38]. This change is obviously due to the variation on tick activity and their metabolic function between their different life stages and sex. Indeed, tick’s obligatory endosymbiont have a crucial role in its development, nutrition, reproductive fitness, and tick adaptation to new environmental conditions in the tick life cycle [11, 59]. Furthermore, it is also important to consider the potential role of the environmental conditions and the difference of the ecological niches between species during their life cycle in the acquisition and the expression of microbiota like for instance the case of the domestic H. scupense with the outdoor ticks H. marginatum and H. excavatum.

Our results on symbionts of Hyalomma ticks may contribute to improve our knowledge on the interaction between tick bacterial communities and their hosts opening the way to develop subsequently applied research targeting the development of new control options against ticks and tick-borne pathogens. Bacterial endosymbionts may affect tick’s physiology and their reproductive fitness, they may also influence tick’s vectorial capacity for transmitted pathogens, and finally, they may interact with the tick hosts with potential veterinary and zoonotic outcomes in particular for Francisella and Rickettsia bacteria.

Conclusion

Overall, the analysis of the bacterial diversity within three Hyalomma species reveals that H. marginatum and H. excavatum have greater diversity than H. scupense. Furthermore, microbial diversity and composition vary according to the tick’s life stage and sex in the specific case of H. scupense. The endosymbionts Francisella, M. mitochondrii and Rickettsia were shown to be the most prevalent in Hyalomma. According to our findings, all studied Hyalomma tick species possess zoonotic bacteria genera and could potentially operate as vectors for various zoonoses. Rickettsia and Francisella are the most common zoonotic bacteria found in Hyalomma ticks.

This study provides general information about microbial communities in Hyalomma ticks and their endosymbionts. This information provides critical clues for future studies aimed at the prevention and control of neglected tick and tick-borne diseases in the area using innovative approaches.

Acknowledgments

The authors thank Mr. Limam Sassi and Mr. Taoufik Lahmar for their technical support. The authors acknowledge also all the veterinarians for their valuable help and all the farmers that participated to this study.