Intracellular pathogens, reproductive parasites and endosymbionts in ticks

The advent of high-throughput sequencing has revealed a vast array of intracellular bacteria that associate with ticks (Andreotti et al., Reference Andreotti, Pérez de León, Dowd, Guerrero, Bendele and Scoles2011; Carpi et al., Reference Carpi, Cagnacci, Wittekindt, Zhao, Qi, Tomsho, Drautz, Rizzoli and Schuster2011; Qiu et al., Reference Qiu, Nakao, Ohnuma, Kawamori and Sugimoto2014; Narasimhan and Fikrig, Reference Narasimhan and Fikrig2015). In addition to pathogens such as Anaplasma spp., Ehrlichia spp., Rickettsia spp., Francisella tularensis and Coxiella burnetii (Potgieter and Stoltsz, Reference Potgieter, Stoltsz, Coetzer and Tustin1994; Parola and Raoult, Reference Parola and Raoult2001; Bown et al., Reference Bown, Begon, Bennett, Woldehiwet and Ogden2003; Jongejan and Uilenberg, Reference Jongejan and Uilenberg2004; Parola et al., Reference Parola, Paddock and Raoult2005; de la Fuente et al., Reference de la Fuente, Estrada-Pena, Venzal, Kocan and Sonenshine2008; Dantas-Torres et al., Reference Dantas-Torres, Chomel and Otranto2012; Kamani et al., Reference Kamani, Morick, Mumcuoglu and Harrus2013; Latrofa et al., Reference Latrofa, Dantas-Torres, Giannelli and Otranto2014; Ereqat et al., Reference Ereqat, Nasereddin, Vayssier-Taussat, Abdelkader, Al-Jawabreh, Zaid, Azmi and Abdeen2016; Regier et al., Reference Regier, Ballhorn and Kempf2017), ticks occasionally contain bacteria that are assumed to be reproductive parasites (Ahantarig et al., Reference Ahantarig, Trinachartvanit, Baimai and Grubhoffer2013; Narasimhan and Fikrig, Reference Narasimhan and Fikrig2015; Bonnet et al., Reference Bonnet, Binetruy, Hernández-Jarguín and Duron2017; Duron et al., Reference Duron, Binetruy, Noël, Cremaschi, McCoy, Arnathau, Plantard, Goolsby, Pérez de León and Heylen2017). For instance, Wolbachia spp. that are closely related to those that manipulate insect reproduction have been detected in Ixodes and Rhipicephalus (Benson et al., Reference Benson, Gawronski, Eveleigh and Benson2004; Zhang et al., Reference Zhang, Norris and Rasgon2011; Hirunkanokpun et al., Reference Hirunkanokpun, Ahantarig, Baimai and Trinachartvanit2018; Chao et al., Reference Chao, Castillo and Shih2021). However, the impact Wolbachia have on tick reproduction is unknown and requires further investigation. In fact, detection of Wolbachia in I. ricinus has been linked to the presence of an endoparasitoid wasp, suggesting that ticks may not be natural hosts of Wolbachia (Plantard et al., Reference Plantard, Bouju-Albert, Malard, Hermouet, Capron and Verheyden2012; Lejal et al., Reference Lejal, Chiquet, Aubert, Robin, Estrada-Peña, Rue, Midoux, Mariadassou, Bailly, Cougoul, Gasqui, Cosson, Chalvet-Monfray, Vayssier-Taussat and Pollet2021).

Another suspected reproductive parasite present in ticks is Rickettsiella spp., which is prevalent in Ixodes and Ornithodoros ticks and is thought to cause sex ratio distortions in parthenogenetic Ixodes woodi (Kurtti et al., Reference Kurtti, Palmer and Oliver2002; Carpi et al., Reference Carpi, Cagnacci, Wittekindt, Zhao, Qi, Tomsho, Drautz, Rizzoli and Schuster2011; Leclerque and Kleespies, Reference Leclerque and Kleespies2012; Anstead and Chilton, Reference Anstead and Chilton2014; Duron et al., Reference Duron, Noël, Mccoy, Bonazzi, Sidi-Boumedine, Morel, Vavre, Zenner, Jourdain and Durand2015, Reference Duron, Binetruy, Noël, Cremaschi, McCoy, Arnathau, Plantard, Goolsby, Pérez de León and Heylen2017; Bonnet et al., Reference Bonnet, Binetruy, Hernández-Jarguín and Duron2017). Other tick-associated intracellular bacteria include Arsenophonus sp. that may decrease the questing success of Dermacentor variabilis and Amblyomma americanum ticks, and Spiroplasma ixodetis, Cardinium spp. and Lariskella spp. with unknown functions (Kurtti et al., Reference Kurtti, Munderloh, Andreadis, Magnarelli and Mather1996; Grindle et al., Reference Grindle, Tyner, Clay and Fuqua2003; Benson et al., Reference Benson, Gawronski, Eveleigh and Benson2004; Henning et al., Reference Henning, Greiner-Fischer, Hotzel, Ebsen and Theegarten2006; Clay et al., Reference Clay, Klyachko, Grindle, Civitello, Oleske and Fuqua2008; Dergousoff and Chilton, Reference Dergousoff and Chilton2010; Mediannikov et al., Reference Mediannikov, Subramanian, Sekeyova, Bell-Sakyi and Raoult2012; Kagemann and Clay, Reference Kagemann and Clay2013; Qiu et al., Reference Qiu, Nakao, Ohnuma, Kawamori and Sugimoto2014; Bell-Sakyi et al., Reference Bell-Sakyi, Palomar and Kazimirova2015; Duron et al., Reference Duron, Binetruy, Noël, Cremaschi, McCoy, Arnathau, Plantard, Goolsby, Pérez de León and Heylen2017; Aivelo et al., Reference Aivelo, Norberg and Tschirren2019).

Ticks also harbour intracellular bacteria that are thought to improve tick fitness. These ‘endosymbionts’ include Coxiella endosymbionts (CEs), Francisella endosymbionts (FEs), Rickettsia endosymbionts (REs) and Candidatus Midichloria mitochondrii (CMM) (Noda et al., Reference Noda, Munderloh and Kurtti1997; Sun et al., Reference Sun, Scoles, Fish and O'Neill2000; Sassera et al., Reference Sassera, Beninati, Bandi, Bouman, Sacchi, Fabbi and Lo2006; Clay et al., Reference Clay, Klyachko, Grindle, Civitello, Oleske and Fuqua2008). CEs are found in a variety of hard and soft ticks and are the most common endosymbionts identified in ticks worldwide (Andreotti et al., Reference Andreotti, Pérez de León, Dowd, Guerrero, Bendele and Scoles2011; Lalzar et al., Reference Lalzar, Harrus, Mumcuoglu and Gottlieb2012; Qiu et al., Reference Qiu, Nakao, Ohnuma, Kawamori and Sugimoto2014; Duron et al., Reference Duron, Binetruy, Noël, Cremaschi, McCoy, Arnathau, Plantard, Goolsby, Pérez de León and Heylen2017), FEs are present in soft ticks (e.g. Ornithodoros moubata, O. porcinus porcinus) and hard ticks (e.g. Dermacentor sp., Amblyomma sp.) (Noda et al., Reference Noda, Munderloh and Kurtti1997; Sun et al., Reference Sun, Scoles, Fish and O'Neill2000; Gerhart et al., Reference Gerhart, Moses and Raghavan2016, Reference Gerhart, Auguste Dutcher, Brenner, Moses, Grubhoffer and Raghavan2018; Duron et al., Reference Duron, Binetruy, Noël, Cremaschi, McCoy, Arnathau, Plantard, Goolsby, Pérez de León and Heylen2017) and REs are present in hard ticks of the genera Ixodes, Dermacentor, Amblyomma, Haemaphysalis and Rhipicephalus (Clay et al., Reference Clay, Klyachko, Grindle, Civitello, Oleske and Fuqua2008; Ahantarig et al., Reference Ahantarig, Malaisri, Hirunkanokpun, Sumrandee, Trinachartvanit and Baimai2011; Carpi et al., Reference Carpi, Cagnacci, Wittekindt, Zhao, Qi, Tomsho, Drautz, Rizzoli and Schuster2011; Lalzar et al., Reference Lalzar, Harrus, Mumcuoglu and Gottlieb2012; Duron et al., Reference Duron, Binetruy, Noël, Cremaschi, McCoy, Arnathau, Plantard, Goolsby, Pérez de León and Heylen2017; Gall et al., Reference Gall, Scoles, Magori, Mason and Brayton2017). CMM, which replicates within host mitochondria, was first detected in I. ricinus, but recent reports indicate its presence in other tick species as well (Lewis, Reference Lewis1979; Zhu et al., Reference Zhu, Aeschlimann and Gern1992; Sacchi et al., Reference Sacchi, Bigliardi, Corona, Beninati, Lo and Franceschi2004; Epis et al., Reference Epis, Sassera, Beninati, Lo, Beati, Piesman, Rinaldi, McCoy, Torina, Sacchi, Clementi, Genchi, Magnino and Bandi2008; Duron et al., Reference Duron, Binetruy, Noël, Cremaschi, McCoy, Arnathau, Plantard, Goolsby, Pérez de León and Heylen2017). All ticks studied to date contain 1 or more of CE, FE, RE or CMM, suggestive of their importance to tick biology. Data currently available from genomic and experimental studies that support endosymbiont function are discussed below.

Genomic evidence for endosymbiont function

Genomes of bacteria that form long-term associations with eukaryotic hosts tend to lose genes that are not under selection while retaining genes that benefit the bacterium or host (McCutcheon and Moran, Reference McCutcheon and Moran2012; Bennett and Moran, Reference Bennett and Moran2015; McCutcheon et al., Reference McCutcheon, Boyd and Dale2019). Befitting this pattern, tick endosymbionts have degraded genomes with intact pathways for the production of B vitamins, suggesting a role for endosymbionts in provisioning these essential nutrients to ticks (Hunter et al., Reference Hunter, Torkelson, Bodnar, Mortazavi, Laurent, Deason, Thephavongsa and Zhong2015; Smith et al., Reference Smith, Driscoll, Gillespie and Raghavan2015; Gerhart et al., Reference Gerhart, Moses and Raghavan2016, Reference Gerhart, Auguste Dutcher, Brenner, Moses, Grubhoffer and Raghavan2018; Guizzo et al., Reference Guizzo, Parizi, Nunes, Schama, Albano, Tirloni, Oldiges, Vieira, Oliveira, Leite, Gonzales, Farber, Martins, Vaz and Oliveira2017; Tsementzi et al., Reference Tsementzi, Castro Gordillo, Mahagna, Gottlieb and Konstantinidis2018; Olivieri et al., Reference Olivieri, Epis, Castelli, Varotto Boccazzi, Romeo, Desirò, Bazzocchi, Bandi and Sassera2019; Buysse et al., Reference Buysse, Floriano, Gottlieb, Nardi, Comandatore, Olivieri, Giannetto, Palomar, Makepeace, Bazzocchi, Cafiso, Sassera and Duron2021b) (Fig. 1). Reconstruction of the vitamin biosynthesis pathway of CEs in the ixodid tick A. americanum (CLEAA) and the argasid tick O. amblus (CLEOA) revealed that they have complete pathways to produce thiamine (vitamin B₁), riboflavin (vitamin B₂), niacin (vitamin B₃), biotin (vitamin B₇) and folate (vitamin B₉) (Smith et al., Reference Smith, Driscoll, Gillespie and Raghavan2015; Duron and Gottlieb, Reference Duron and Gottlieb2020). CLEAA also has partial pathways for the synthesis of pantothenic acid (vitamin B₅) and pyridoxine (vitamin B₆) (Smith et al., Reference Smith, Driscoll, Gillespie and Raghavan2015). The genomes of the CEs in R. turanicus (CRt) and A. sculptum (CeAS-UFV) have complete pathways to synthesize riboflavin, biotin and folate and partial pathways for niacin, pantothenic acid and pyridoxine (Gottlieb et al., Reference Gottlieb, Lalzar and Klasson2015; Duron and Gottlieb, Reference Duron and Gottlieb2020). The genome of CeAS-UFV also possesses a partial pathway for the synthesis of thiamine (Duron and Gottlieb, Reference Duron and Gottlieb2020), and the CE in R. microplus (CERM) has complete pathways for the synthesis of riboflavin, pyridoxine, biotin and folate and a partial pathway for the synthesis of thiamine (Gottlieb et al., Reference Gottlieb, Lalzar and Klasson2015; Smith et al., Reference Smith, Driscoll, Gillespie and Raghavan2015; Guizzo et al., Reference Guizzo, Parizi, Nunes, Schama, Albano, Tirloni, Oldiges, Vieira, Oliveira, Leite, Gonzales, Farber, Martins, Vaz and Oliveira2017).

Figure 1. B vitamin biosynthesis pathways in tick endosymbionts. Pathway for the synthesis of cobalamin (vitamin B₁₂) was not detected in any tick endosymbiont genome. CE, Coxiella endosymbiont; FE, Francisella endosymbiont; CMM, Candidatus Midichloria mitochondrii.

Similar to CEs, analysis of the genomes of the FEs in O. moubata (FLE-Om), A. maculatum (FLE-Am) and F. persica, the endosymbiont of the soft tick Argas arboreus, showed that they possess complete pathways for the synthesis of riboflavin, biotin and folate (Gerhart et al., Reference Gerhart, Auguste Dutcher, Brenner, Moses, Grubhoffer and Raghavan2018; Duron and Gottlieb, Reference Duron and Gottlieb2020) (Fig. 1). Furthermore, FLE-Am and FLE-Om may be able to utilize aspartate to synthesize pantothenic acid (vitamin B₅) but F. persica seems to only possess a partial pathway for this process (Gerhart et al., Reference Gerhart, Auguste Dutcher, Brenner, Moses, Grubhoffer and Raghavan2018). In the case of Rickettsia symbionts, metabolic reconstruction suggests that R. buchneri in I. scapularis and Rickettsia sp. phylotype G021 in I. pacificus are likely able to synthesize folate but no other B vitamins (Hunter et al., Reference Hunter, Torkelson, Bodnar, Mortazavi, Laurent, Deason, Thephavongsa and Zhong2015; Bodnar et al., Reference Bodnar, Fitch, Rosati and Zhong2018). For CMM, metabolic reconstructions showed that genomes encode complete pathways for the biosynthesis of riboflavin, biotin and folate suggesting that the intra-mitochondrial symbiont could also provide B vitamins to its tick partner (Sassera et al., Reference Sassera, Lo, Epis, D'Auria, Montagna, Comandatore, Horner, Peretó, Luciano, Franciosi, Ferri, Crotti, Bazzocchi, Daffonchio, Sacchi, Moya, Latorre and Bandi2011; Olivieri et al., Reference Olivieri, Epis, Castelli, Varotto Boccazzi, Romeo, Desirò, Bazzocchi, Bandi and Sassera2019).

Many cofactors and coenzymes that are critical to the functioning of essential enzymes are derived from B vitamins (Douglas, Reference Douglas2017). Several CEs and FEs contain pathways for the production of cofactors and coenzymes from B vitamins (Guizzo et al., Reference Guizzo, Parizi, Nunes, Schama, Albano, Tirloni, Oldiges, Vieira, Oliveira, Leite, Gonzales, Farber, Martins, Vaz and Oliveira2017; Duron et al., Reference Duron, Morel, Noël, Buysse, Binetruy, Lancelot, Loire, Ménard, Bouchez, Vavre and Vial2018; Gerhart et al., Reference Gerhart, Auguste Dutcher, Brenner, Moses, Grubhoffer and Raghavan2018; Nardi et al., Reference Nardi, Olivieri, Kariuki, Sassera and Castelli2020; Brenner et al., Reference Brenner, Muñoz-Leal, Sachan, Labruna and Raghavan2021). For example, CERM, CLEAA and CRt could synthesize flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) from riboflavin and coenzyme A from pantothenate (Guizzo et al., Reference Guizzo, Parizi, Nunes, Schama, Albano, Tirloni, Oldiges, Vieira, Oliveira, Leite, Gonzales, Farber, Martins, Vaz and Oliveira2017). In addition, CLEAA encodes genes for the synthesis of nicotinamide adenine dinucleotide phosphate (NADP⁺⁾ and CLEAA, CLEOA and CERM possess complete pathways to produce lipoic acid (Gottlieb et al., Reference Gottlieb, Lalzar and Klasson2015; Smith et al., Reference Smith, Driscoll, Gillespie and Raghavan2015; Guizzo et al., Reference Guizzo, Parizi, Nunes, Schama, Albano, Tirloni, Oldiges, Vieira, Oliveira, Leite, Gonzales, Farber, Martins, Vaz and Oliveira2017; Duron and Gottlieb, Reference Duron and Gottlieb2020). Analysis also found that the genome of FLE-Om possessed complete pathways for the biosynthesis of lipoic acid, FAD and coenzyme A (Duron et al., Reference Duron, Morel, Noël, Buysse, Binetruy, Lancelot, Loire, Ménard, Bouchez, Vavre and Vial2018; Gerhart et al., Reference Gerhart, Auguste Dutcher, Brenner, Moses, Grubhoffer and Raghavan2018). Additional analysis of FLE-Om and F. persica genomes using tools available in the microbial genome portal BioCyc (Karp et al., Reference Karp, Billington, Caspi, Fulcher, Latendresse, Kothari, Keseler, Krummenacker, Midford, Ong, Ong, Paley and Subhraveti2017) revealed that both symbionts encode complete pathways for the biosynthesis of several other cofactors that could be useful to their tick hosts: iron-sulphur cluster [2Fe-2S], dipyrromethane, FMN, heme, molybdopterin, nicotinamide adenine dinucleotide (NAD⁺), NADP⁺, pyridoxal 5′-phosphate, S-adenosyl-L-methionine and thiamine diphosphate (A. Kolo, unpublished).

Apart from provisioning vitamins and cofactors, endosymbionts may also supply amino acids and other metabolites that boost tick fitness (Fig. 2). For instance, in silico flux balance analyses of CRt and CE of R. sanguineus (CRs) identified excessive production of the amino acid proline, which is thought to play a significant role in arthropods due to its involvement in energy production and nitrogen metabolism (O'Donnell and Donini, Reference O'Donnell, Donini, Weihrauch and O'Donnell2017; Tsementzi et al., Reference Tsementzi, Castro Gordillo, Mahagna, Gottlieb and Konstantinidis2018). Similarly, CERM encodes genes for the production of essential amino acids, and FLE-Am appears to have the metabolic capacity to produce the amino acids cysteine, threonine, tyrosine, tryptophan, phenylalanine and serine from pyruvate (Gerhart et al., Reference Gerhart, Moses and Raghavan2016). Additionally, FEs encode enzymes that recycle nitrogen by incorporating ammonia, a metabolic waste product, into the synthesis of glutamine, as done by several insect endosymbionts (Sabree et al., Reference Sabree, Kambhampati and Moran2009; Hansen and Moran, Reference Hansen and Moran2011; Gerhart et al., Reference Gerhart, Auguste Dutcher, Brenner, Moses, Grubhoffer and Raghavan2018).

Figure 2. Putative functions of tick endosymbionts. ^G represents information based on genome sequences and ^E indicates data derived from experimental studies. CE, Coxiella endosymbiont; FE, Francisella endosymbiont; RE, Rickettsia endosymbiont; CMM, Candidatus Midichloria mitochondrii.

CMM genome encodes proteins that could assist I. ricinus in its response to oxidative stress and in energy metabolism (Olivieri et al., Reference Olivieri, Epis, Castelli, Varotto Boccazzi, Romeo, Desirò, Bazzocchi, Bandi and Sassera2019) (Fig. 2). These proteins include the cytochrome cbb₃ oxidases that may support ATP production with reduced levels of oxygen concentration during oogenesis, heme exporter proteins, a protoheme ferro-lyase, superoxide dismutase, ferrochelatase, nucleotide tlc translocases and a pathway for the synthesis of lipoic acid, a cofactor that forms part of diverse enzyme complexes of electron transport chains in mitochondria (Packer et al., Reference Packer, Witt and Tritschler1995; Sassera et al., Reference Sassera, Lo, Epis, D'Auria, Montagna, Comandatore, Horner, Peretó, Luciano, Franciosi, Ferri, Crotti, Bazzocchi, Daffonchio, Sacchi, Moya, Latorre and Bandi2011; Olivieri et al., Reference Olivieri, Epis, Castelli, Varotto Boccazzi, Romeo, Desirò, Bazzocchi, Bandi and Sassera2019). Proteins such as constituents of the major facilitator superfamily and the drug/metabolite transporter superfamily potentially responsible for the transportation of fluids and ions such as sodium, protons and potassium and the uptake of organic compounds like amino acids were also annotated in the genome of CMM suggesting that the bacterium may play a role in the maintenance of osmoregulation and water balance in I. ricinus during blood feeding (Olivieri et al., Reference Olivieri, Epis, Castelli, Varotto Boccazzi, Romeo, Desirò, Bazzocchi, Bandi and Sassera2019). Lastly, a recently acquired CMM in Hyalomma marginatum seems to compensate for the loss of biotin and heme biosynthesis genes in the co-infecting FE (Buysse et al., Reference Buysse, Floriano, Gottlieb, Nardi, Comandatore, Olivieri, Giannetto, Palomar, Makepeace, Bazzocchi, Cafiso, Sassera and Duron2021b), probably because both endosymbionts together provide critical metabolites to ticks as observed in several insects (Husnik and McCutcheon, Reference Husnik and McCutcheon2016; Santos-Garcia et al., Reference Santos-Garcia, Juravel, Freilich, Zchori-Fein, Latorre, Moya, Morin and Silva2018; Takeshita et al., Reference Takeshita, Yamada, Kawahara, Narihiro, Ito, Kamagata and Shinzato2019).

Experimental evidence for endosymbiont function

Administration of antibiotics that diminish endosymbiont populations leads to reduced reproductive success in ticks, suggesting a fitness-boosting role for endosymbionts (Fig. 2). For instance, exposure of A. americanum to tetracycline and rifampicin led to a decrease in CE load and concomitant reduction in reproductive fitness, as evidenced by a setback in the rate of oviposition and significantly lower number of ticks that emerged from eggs (Zhong et al., Reference Zhong, Jasinskas and Barbour2007). Similarly, injection of tetracycline into R. microplus and its cattle host led to reduced levels of CERM and a delay in the development of the tick at the metanymph phase (Guizzo et al., Reference Guizzo, Parizi, Nunes, Schama, Albano, Tirloni, Oldiges, Vieira, Oliveira, Leite, Gonzales, Farber, Martins, Vaz and Oliveira2017), and administration of ofloxacin to R. sanguineus led to reduced burdens of CRs accompanied by an increase in time for adult females to feed to repletion, a reduced engorgement weight and a decreased capacity to lay eggs (Ben-Yosef et al., Reference Ben-Yosef, Rot, Mahagna, Kapri, Behar and Gottlieb2020; An et al., Reference An, Bhowmick, Liang, Suo, Liao, Zhao and Han2022). Likewise, tetracycline administration to female H. longicornis led to reduced densities of the Coxiella symbiont in the ovaries and malpighian tubules, which led to significant changes in tick engorgement weight, feeding time, number of eggs laid and the length of the oviposition period (Zhang et al., Reference Zhang, Li, Zhang, Qiu, Li, Li and Liu2017).

The reduction in tick fitness associated with endosymbiont loss could be due to the diminished availability of B vitamins that are vital to tick development. Antibiotic treatment of O. moubata reduced the tick's FE burden and interrupted with normal nymph feeding and moulting, which forestalled the development of viable adult female ticks and significantly lowered the emergence of male ticks. Providing a B vitamin mixture (thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folate and cobalamin) to O. moubata restored its reproductive fitness, indicating a role for FLE-Om in provisioning B vitamins required for normal tick development and reproduction (Duron et al., Reference Duron, Morel, Noël, Buysse, Binetruy, Lancelot, Loire, Ménard, Bouchez, Vavre and Vial2018).

Experiments also suggest that endosymbionts may contribute to the blood-feeding capacity of ticks (Fig. 2). Treatment of H. longicornis nymphs with tetracycline reduced the levels of the Coxiella symbiont (CHI), which in turn reduced the blood intake of the tick (Zhong et al., Reference Zhong, Zhong, Peng, Zhou, Wang, Tang and Wang2021). Intriguingly, it is the metabolite chorismate produced by CHI rather than B vitamins or cofactors that likely influences tick blood intake. The study showed that chorismate promotes the production of 5-hydroxytryptamine (serotonin), a bioamine whose stability in the midgut and synganglion of the tick regulates the amount of blood ingested by the tick (Zhong et al., Reference Zhong, Zhong, Peng, Zhou, Wang, Tang and Wang2021). Similar to H. longicornis, administration of antibiotics to R. sanguineus and R. microplus also reduced the density of CEs and tick blood intake, and transcriptomic analysis of CERM-free R. microplus metanymphs revealed that genes associated with tick blood-feeding capacity such as DAP-36, lipocalin, trypsin inhibitor-like family, Kunitz-type inhibitors, cystatin and evasins were significantly under-expressed (Guizzo et al., Reference Guizzo, Parizi, Nunes, Schama, Albano, Tirloni, Oldiges, Vieira, Oliveira, Leite, Gonzales, Farber, Martins, Vaz and Oliveira2017; Zhang et al., Reference Zhang, Li, Zhang, Qiu, Li, Li and Liu2017; Ben-Yosef et al., Reference Ben-Yosef, Rot, Mahagna, Kapri, Behar and Gottlieb2020; An et al., Reference An, Bhowmick, Liang, Suo, Liao, Zhao and Han2022). Collectively, these data indicate that CEs improve blood feeding across tick species, but further studies are required to confirm whether endosymbiont-produced chorismate is the key metabolite that drives this process in all ticks.

CMM is the most common endosymbiont associated with I. ricinus and feeding females with tetracycline-containing bovine blood produced CMM-free ticks within 2 generations. Larvae that hatched from eggs laid by CMM-free females consistently performed poorly during blood feeding, suggesting that CMM is required for the emergence of larvae with intact blood-feeding ability (Guizzo et al., Reference Guizzo, Hatalová, Frantová, Zurek, Kopáček and Perner2023). Similar to the above findings, I. ricinus nymphs fed with gentamicin-treated blood had significantly lower engorgement weights, lower moulting proportions and lower weights of moulted unfed adult females in comparison to nymphs fed on antibiotic-free blood (Militzer et al., Reference Militzer, Pinecki Socias and Nijhof2023). These studies show that CMM, in addition to CEs, enhances blood intake by ticks. Interestingly, increased blood feeding by ticks seems to benefit the endosymbionts as well. For example, Francisella symbionts in H. doenitzi significantly increased in number during blood feeding, and Rickettsia sp. phylotype G021 and CMM multiplied massively in I. pacificus and I. ricinus, respectively, following blood meals (Sassera et al., Reference Sassera, Lo, Bouman, Epis, Mortarino and Bandi2008; Cheng et al., Reference Cheng, Lane, Moore and Zhong2013; Liu et al., Reference Liu, Yu, Liu, Li, Wang, Zhang and Liu2016). Thus, improved intake of blood, which nourishes both tick and endosymbiont, seems to be one of the major benefits of long-term symbiosis between ticks and intracellular bacteria.

Finally, D. variabilis and A. americanum larvae infected with Rickettsia symbionts displayed increased motility than uninfected larvae [42]. The locomotive ability of newly hatched larvae was determined on flat and inclined surfaces and Rickettsia-containing larvae displayed increased locomotive speed relative to uninfected larvae. Tick motility plays a role in host-questing success; thus, infection with Rickettsia symbionts may impact the disease risk posed by tick-borne pathogens.