Ticks evolved to hematophagy through the acquisition of key genomic adaptations (Gulia-Nuss, 2016; Jia et al., 2020) and the evolution of mutualistic interactions with microbial symbionts (Buysse and Duron, 2021; Duron and Gottlieb, 2020). Ticks rely on vertebrate blood as their sole food source throughout their development, although it is nutritionally unbalanced. Blood contains high levels of proteins, iron, and salts, but low levels of carbohydrates, lipids, and vitamins. To overcome these dietary challenges, tick genomes present large repertoires of genes related to vitamin and lipid shortages, iron management, and osmotic homeostasis (Gulia-Nuss, 2016; Jia et al., 2020). Yet, tick genomes do not contain genes for synthesis of some essential vitamins, thus, vitamin-provisioning pathways have indirectly arisen from symbiotic associations with transovarially transmitted intracellular bacteria (Ben-Yosef et al., 2020; Duron et al., 2018; Duron and Gottlieb, 2020; Gerhart et al., 2016; Gottlieb et al., 2015; Guizzo et al., 2017; Smith et al., 2015). This partnership allowed the long-term acquisition of heritable biochemical functions by ticks and favored their radiation into the nearly 900 tick species that currently exist (Duron and Gottlieb, 2020). This co-evolutionary process is reflected by microbial communities dominated by tick nutritional symbionts (Bonnet and Pollet, 2021; Narasimhan and Fikrig, 2015).

Most tick species typically harbor a single lineage of nutritional symbiont, but multiple symbiont lineages exist in different tick species (Duron and Gottlieb, 2020). The most common lineages belong to the Coxiella-like endosymbiont group and to the Francisella-like endosymbiont group (CLE and FLE, hereafter) (Azagi et al., 2017; Binetruy et al., 2020; Duron et al., 2017; Machado-Ferreira et al., 2016), but a few tick species may rely on other symbionts (Duron et al., 2017), such as the Rickettsia-like endosymbionts (Narasimhan et al., 2021) and the intramitochondrial bacterium Midichloria (Sassera et al., 2006). Once deprived of their nutritional symbionts, ticks show a complete stop of growth and moulting, as well as lethal physical abnormalities (Ben-Yosef et al., 2020; Duron et al., 2018; Guizzo et al., 2017; Zhong et al., 2007), which can be fully restored with an artificial supplement of B vitamins (Duron et al., 2018). Nutritional symbionts of ticks exhibit a high degree of lifestyle specialization toward mutualism. They have been found primarily in two tick organs, the ovaries and Malpighian tubules, while other organs have only been found sporadically infected (Buysse et al., 2019; Duron and Gottlieb, 2020). Ovary infection is consistent with vertical transmission of nutritional symbionts in developing oocytes. A high density of nutritional symbionts in Malpighian tubules supports a nutritional role for these symbionts, as Malpighian tubules are involved in excretion and osmoregulation. A likely hypothesis is that nutritional symbionts use tick hemolymph compounds to synthesize de novo B vitamins (Duron and Gottlieb, 2020).

Genomes of CLE and FLE consistently contain complete biosynthesis pathways of the same three B vitamins: biotin (vitamin B₇), riboflavin (B₂), and folate (B₉) (Duron et al., 2018; Gerhart et al., 2018; Gerhart et al., 2016; Gottlieb et al., 2015; Guizzo et al., 2017; Nardi et al., 2021; Smith et al., 2015). These three pathways form a set of core symbiotic genes essential for ticks (Duron and Gottlieb, 2020) while other functions are disposable. As such, the genomes of CLE, FLE, and Midichloria underwent massive reduction as observed in other nutritional endosymbionts of arthropods with a specialized diet (Duron et al., 2018; Gerhart et al., 2018; Gerhart et al., 2016; Gottlieb et al., 2015; Nardi et al., 2021; Sassera et al., 2011; Smith et al., 2015). This reduction process is primarily initiated as a consequence of relaxed selection on multiple genes which are not essential within the host stable and nutrient-rich cellular environment (Bennett and Moran, 2015; McCutcheon et al., 2019). Over time, genes encoding for functions such as motility, regulation, secondary-metabolite biosynthesis, and defense are pseudogenized and then definitively jettisoned (Bennett and Moran, 2015; McCutcheon et al., 2019).

Coevolution of hosts and their nutritional symbionts over millions of years is expected to result in stable co-cladogenesis (Bennett and Moran, 2015). However, in sap-feeding insects where symbionts compensate for nutritional deficiencies of the sap diet, cases of unstable interactions have been observed (Bennett and Moran, 2015; McCutcheon et al., 2019). Indeed, as the symbiont genome decays, repair mechanisms can be lost, leading to faster pseudogenization, which can affect genes in all functional categories including those involved in nutritional supplementation (Bennett and Moran, 2015; McCutcheon et al., 2019). This may ultimately lead to overly reduced and maladapted genomes, limiting beneficial contributions to hosts and opening the road to wholesale replacement by new symbiont(s) (Bennett and Moran, 2015; McCutcheon et al., 2019; Sudakaran et al., 2017). This degeneration–replacement model has been proposed in sap-feeding insects (Campbell et al., 2015; Lukasik et al., 2018; Manzano-Marın et al., 2020; Matsuura et al., 2018), but is difficult to document as such replacements are expected to be transient (McCutcheon et al., 2019). In parallel, other forms of multi-partite interactions have been reported, where a novel bacterium with similar function to a pre-existing symbiont enters a bipartite system (Husnik and McCutcheon, 2016; Santos-Garcia et al., 2018; Takeshita et al., 2019). This can lead to relaxation of conservative selection pressure on the original symbionts, which can lose genes that were previously fundamental, ultimately leading to a tripartite stable symbiosis (Husnik and McCutcheon, 2016; Takeshita et al., 2019).

In this study, we document a complex nutritional endosymbiosis in the invasive bont-legged tick Hyalomma marginatum characterized by a maladapted ancestral symbiont genome compensated by gene functions of a more recently acquired symbiont. This large tick species usually feeds on humans and a diversity of mammals and birds in the Mediterranean climatic zone but has recently expanded its range to the north, invading Western Europe (Vial et al., 2016). Hyalomma marginatum can transmit Crimean-Congo hemorrhagic fever virus, an emerging disease affecting humans in the northern Mediterranean Basin, as well as protozoan parasites and intracellular bacterial pathogens such as Rickettsia aeschlimannii (Vial et al., 2016). Previous studies detected the presence of diverse highly prevalent bacterial symbionts across H. marginatum populations (Di Lecce et al., 2018), but their contribution to tick nutrition was not studied. A previous study showed that high densities of FLE can be observed in Malpighian tubules and ovaries of H. marginatum, while infections in other organs are scattered (Azagi et al., 2017). Here, we sequenced three microbial metagenomes of H. marginatum in three distinct locations, and determined the nutritional interactions in this unique symbiosis system.

In this study, we show that the tick H. marginatum is a complex symbiotic system, combining genes of diverse phylogenetic origins to generate nutritional adaptations to obligate hematophagy. This tick harbors a microbiome functionally analogous to those observed in other tick species, dominated by intracellular bacterial symbionts producing the core set of B vitamins (biotin, riboflavin, and folate) essential for tick growth and survival. However, while in other tick species, as in other obligate blood feeders, production of all three core B vitamins is performed by a single nutritional symbiont (Binetruy et al., 2020; Duron et al., 2017; Duron and Gottlieb, 2020), H. marginatum harbors two bacterial partners whose core B vitamins biosynthetic capacities depend on a combination of genes, with indigenous and exogenous phylogenetic origins. This multi-partner nutritional symbiosis is stably maintained at least at the tick species level, as supported by our founding of genetically similar FLE and Midichloria present in H. marginatum populations from geographically distant regions.

Genome reconstructions confirmed the key role of symbionts in H. marginatum nutrition, but contrary to other tick species, no single symbiont does compensate for all nutritional needs. The FLE-Hmar genomes contain the genes to synthesize folate and riboflavin but its biotin synthesis pathway is degraded, and thus non-functional. Genome reduction is a conserved trait in all FLEs, but this process has extended in the FLE of H. marginatum, with an additional loss of genes in most COG functional categories. This process could have been triggered by the pseudogenization of the mismatch repair system (mutSL), leaving the FLE of H. marginatum with only one DNA repair system, nucleotide excision repair (uvrABCD). Absence of the mutSL system, coupled with strict clonality and a small population size during transovarial transmission, have probably led the FLE- Hmar genomes to accumulate deleterious mutations, that resulted in maladaptation, and ultimately limited their beneficial contributions to ticks.

In the context of FLE genome degradation, co-infection with Midichloria prevents the breakdown and collapse of nutritional symbiosis in H. marginatum; that is, the Midichloria genome contains an intact biotin biosynthesis operon, and thus compensates for genome decay of FLE. Inversely, Midichloria has only partial pathways for riboflavin and folate and hence cannot meet the nutritional needs of H. marginatum for these B vitamins. Consequently, the co-infection of FLE with Midichloria forms a cooperative system essential for the nutritional symbiosis in H. marginatum. In addition, complementary heme synthesis genes are present in the FLE and Midichloria genomes, and these symbionts may be an additional source of heme, perhaps conferring a fitness advantage to H. marginatum during periods of starvation. In animals, heme is essential for the function of all aerobic cells, playing an important role in controlling protein synthesis and cell differentiation. Complete biosynthetic heme gene pathways exist in most animals, but not in ticks that need an exogenous source of heme (Gulia-Nuss, 2016; Jia et al., 2020). Vertebrate blood is usually considered to be their unique source for this essential cofactor (Perner et al., 2016), however ticks spend most of their life off host, and thus the presence of heme genes in FLE and Midichloria genomes may also indicate symbiont provisioning of heme in H. marginatum.

The co-localization of FLE and Midichloria in tick ovaries suggests that vertical transmission has led to their stable co-inheritance through tick generations, linking their evolutionary fate, as observed in other symbiotic systems of arthropods (McCutcheon et al., 2019; Vautrin and Vavre, 2009). This co-transmission fidelity creates the conditions under which cooperative interactions can emerge. The stable coexistence of FLE and Midichloria genomes makes various genes redundant, as we observed for B vitamin and heme synthesis pathways. Furthermore, the covariance of FLE and Midichloria densities in ticks is positive, suggesting that these two symbionts are not in direct competition but rather cooperative. However, FLE is about 100-times more abundant than Midichloria, which can be suggestive of competition. Indeed, negative interactions among these endosymbionts can occur because their tick host provides a limited amount of resources and space. Under this hypothesis, the density of Midichloria can be depressed by FLE, which could be more competitive. Regardless of the issue of this interaction, FLE and Midichloria depend on each other since their host tick would not survive without their combined production of B vitamins. The asymmetrical abundance of FLE and Midichloria may have further implications in nutritional endosymbiosis. First, it suggests that, at lower abundance, Midichloria can fully bridge the biotin requirements for H. marginatum. Second, it could imply that biotin (produced by Midichloria) is required at much lower abundance than riboflavin (FLE) and folate (FLE and Midichloria), or that Midichloria could over-express its biotin genes, possibly thanks to the streamlined structure of the biotin operon, and finally produce biotin at the same abundance than riboflavin and folate.

While the nutritional association of FLE with Hyalomma species has persisted for millions of years (Azagi et al., 2017), the nutritional role of Midichloria seems more recent in this tick genus. Co-infection with Midichloria was reported in at least nine Hyalomma species (Supplementary file 1d), but in most species, such as H. excavatum and H. impeltatum (Azagi et al., 2017; Selmi et al., 2019), Midichloria is present at much lower frequencies than would be expected for an obligate nutritional mutualist, suggesting that it is instead a facultative (i.e., not essential) association. Interestingly, the Hyalomma species apparently devoid of Midichloria, H. asiaticum, harbors an FLE with intact biotin, folate, and riboflavin synthesis pathways (Buysse and Duron, 2021). In H. marginatum, the decay of the FLE genome may have been accelerated by the presence of Midichloria and the redundancy of biotin biosynthesis, a process also reported in sap-feeding insects (Husnik and McCutcheon, 2016; Manzano-Marın et al., 2020). While it is clear that two events have led to the current status (loss of biotin genes and acquisition of Midichloria), the order by which these happened is unclear. Midichloria could have rescued a decaying symbiosis, or could have led, with its entrance in the system, to relaxation of selective constraints on FLE. Based on the common presence of Midichloria at lower frequencies in other Hyalomma species, a scenario of gradual parallel occurrence of these two events cannot be ruled out.

Our analyses also indicated that the biotin operon detected in the Midichloria of H. marginatum has an exogenous origin, being acquired following an ancient lateral gene transfer pre-dating the divergence between the Midichloria of H. marginatum and the one of I. ricinus. Accumulating genomic sequences indicate that this streamlined biotin operon (denoted in some studies as the Biotin synthesis Operon of Obligate intracellular Microbes—BOOM Driscoll et al., 2020) has experienced transfers between distantly related endosymbionts inhabiting arthropod cells including Wolbachia (Driscoll et al., 2020; Gerth and Bleidorn, 2016; Ju et al., 2020; Nikoh et al., 2014), Rickettsia (Gillespie et al., 2012), Cardinium (Penz et al., 2012), and Legionella (Ríhová et al., 2017). Remarkably, the invasive nature of this streamlined biotin operon has contributed not only to the emergence of novel nutritional symbioses in H. marginatum with Midichloria, but also in other blood feeders, such as bedbugs with Wolbachia (Nikoh et al., 2014) and rodent lice with Legionella (Ríhová et al., 2017), or in sap feeders as white flies with Cardinium (Santos-Garcia et al., 2014) and planthoppers with Wolbachia (Ju et al., 2020). Until recently, lateral gene transfer was thought to be rare in intracellular symbionts, as they reside in confined and isolated environments. However, co-infections of unrelated endosymbionts within the same host cell have created freely recombining intracellular bacterial communities, a unique environment previously defined as the ‘intracellular arena’ (Kent and Bordenstein, 2010). The detection of the streamlined biotin operon in Wolbachia, Rickettsia, and Cardinium—three of the most common intracellular symbionts of arthropods (Duron et al., 2008)—corroborates the role of such ‘intracellular arenas’ in the evolutionary dynamics of nutritional symbioses, especially as the biotin operon can be found on plasmids (Gillespie et al., 2012).

The macroevolutionary and ecological consequences of acquiring nutritional symbionts are immense, ticks would simply not exist as strict blood feeders without such mutualistic interactions (Duron and Gottlieb, 2020). Our investigations corroborate that nutritional symbionts of ticks have all converged towards an analogous process of B vitamin provisioning. While past investigations showed that this evolutionary convergence consists in severe degeneration of symbiont genomes accompanied by preservation of biotin, folate, and riboflavin biosynthesis pathways (Buysse and Duron, 2021; Duron and Gottlieb, 2020), nutritional endosymbiosis in H. marginatum is influenced by a more complex web of interactions resulting in a dynamic mosaic of symbiont combinations and offering novel evolutionary possibilities.

The bacterial genomes obtained in this study were deposited in European Nulceotide Archive (ENA) under the project PRJEB44779 and accession numbers GCA_910592745 (FLE-Hmar-ES), GCA_910592815 (FLE-Hmar-IL), GCA_910592705 (FLE-Hmar-IT), GCA_910592695 (Midi-Hmar-ES), GCA_910592725 (Midi-Hmar-IL), GCA_910592865 (Midi-Hmar-IT) and GCA_910592765 (RAES-Hmar-IT). Metagenome reads were deposited in ENA under accession numbers ERR7255744 (Hmar-ES), ERR7255745 (Hmar-IL) and ERR7255743 (Hmar-IT). Command lines used for the different genomic analyses are available on GitHub (https://github.com/mariebuysse/Hmar-2021; copy archived at swh:1:rev:a459aeecb87d3fae17cf6ab8317654feadb62dba).

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Author details

1. Marie Buysse

   1. MIVEGEC (Maladies Infectieuses et Vecteurs : Ecologie, Génétique, Evolution et Contrôle), Univ. Montpellier (UM) - Centre National de la Recherche Scientifique (CNRS) - Institut pour la Recherche et le Développement (IRD), Montpellier, France
   2. Centre of Research in Ecology and Evolution of Diseases (CREES), Montpellier, France, Montpellier, France

   Contribution

   Formal analysis, Investigation, Writing – original draft, Writing – review and editing

   Contributed equally with

   Anna Maria Floriano

   For correspondence

   marie.buysse@ird.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8160-2470

2. Anna Maria Floriano

   1. Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Pavia, Italy
   2. Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic

   Contribution

   Formal analysis, Investigation, Writing – original draft, Writing – review and editing

   Contributed equally with

   Marie Buysse

   Competing interests

   No competing interests declared

3. Yuval Gottlieb

   Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

   Contribution

   Conceptualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

4. Tiago Nardi

   Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Pavia, Italy

   Contribution

   Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1248-9873

5. Francesco Comandatore

   Department of Biomedical and Clinical Sciences L. Sacco and Pediatric Clinical Research Center, University of Milan, Milan, Italy

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

6. Emanuela Olivieri

   Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Pavia, Italy

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

7. Alessia Giannetto

   Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

8. Ana M Palomar

   Center of Rickettsiosis and Arthropod-Borne Diseases (CRETAV), San Pedro University Hospital- Center of Biomedical Research from La Rioja (CIBIR), Logroño, Spain

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5461-5874

9. Benjamin L Makepeace

   Institute of Infection, Veterinary & Ecological Sciences, University of Liverpool, Liverpool, United Kingdom

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

10. Chiara Bazzocchi

    Department of Veterinary Medicine, University of Milan, Lodi, Italy

    Contribution

    Data curation, Investigation

    Competing interests

    No competing interests declared

11. Alessandra Cafiso

    Department of Veterinary Medicine, University of Milan, Lodi, Italy

    Contribution

    Data curation, Investigation

    Competing interests

    No competing interests declared

12. Davide Sassera

    Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Pavia, Italy

    Contribution

    Conceptualization, Writing – original draft, Writing – review and editing

    Contributed equally with

    Olivier Duron

    Competing interests

    No competing interests declared

13. Olivier Duron

    1. MIVEGEC (Maladies Infectieuses et Vecteurs : Ecologie, Génétique, Evolution et Contrôle), Univ. Montpellier (UM) - Centre National de la Recherche Scientifique (CNRS) - Institut pour la Recherche et le Développement (IRD), Montpellier, France
    2. Centre of Research in Ecology and Evolution of Diseases (CREES), Montpellier, France, Montpellier, France

    Contribution

    Conceptualization, Writing – original draft, Writing – review and editing

    Contributed equally with

    Davide Sassera

    Competing interests

    No competing interests declared

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