Grappling with the Tick Microbiome

Abstract

Ixodes scapularis and Ixodes pacificus, are the predominant vectors of multiple human pathogens including Borrelia burgdorferi, one of the causative agents of Lyme disease, in North America. Differences in the habitats and host preferences of these closely-related tick species presents an opportunity to examine key aspects of the tick microbiome. While, advances in sequencing technologies, have accelerated a descriptive understanding of the tick microbiome, molecular and mechanistic insights into the tick microbiome is only beginning to emerge. Progress is stymied by technical difficulties to manipulate the microbiome and by biological variables related to the life cycle of Ixodid ticks. This review will highlight these challenges and examine avenues to understand the significance of the tick microbiome in tick biology.

Hematophagous arthropod microbiome

There is an increasing interest in determining the role of the microbiomes of hematophagous arthropod vectors of disease in pathogen acquisition and transmission, as well as in the life cycle of the vector itself. Of particular interest has been the arthropod gut microbiome, since acquiring and utilizing a bloodmeal is central to the life cycle of the vector and hinges on the physical and functional integrity of the gut. Further, pathogens acquired by the vector most often have to transit through the gut and this brings the vector, the microbiome, and the pathogen in close proximity, a crucible of interactions that may impact vectorial capacity. Hematophagous (see Glossary) arthropods have a restricted diet, feeding predominantly on vertebrate blood. Comparison of the microbiome composition of different hematophagous arthropods demonstrates that dietary bloodmeal is not the only determinant of microbiome composition [1] and that the microbiome composition is broadly related to specific arthropod genera. The microbiome compositions of hematophagous arthropods are also determined based on whether they are obligate or facultative blood-feeders [2]. Obligate blood feeders such as ticks, bed bugs and tsetse flies have evolved to rely on microbial endosymbionts to supplement several B-vitamins such as biotin, folate, and riboflavin that are deficient in blood [2, 3]. Thus, in obligate feeders, we observe a convergent evolution or co-cladogenesis [2, 3] that favors associations with microbial endosymbionts such as Wigglesworthia in Tsetse flies, Wolbachia in bed bugs, and Rickettsia, Francisella, Midichloria or Coxiella-like symbionts in ticks that encode analogous functions critical to circumvent nutritional deficiencies in the blood meal [2, 3]. These endosymbionts reside in bacteriocytes associated with the gut, or with reproductive organs [2, 3]. Facultative blood feeders like mosquitoes obtain their nutrients from additional food sources and do not appear to demonstrate nutritional dependence on specific bacteria [4].

Eubacterial organisms of hematophagous arthropods have been shown to have significant effects on arthropod evolution and ecology [1]. It is important to note that the microbiome also includes viral and eukaryotic microbes [2, 3]. Although, examination of the virome and eukaryotic microbiome has been hampered by cumbersome analysis and bioinformatic pipelines, technological advancements are providing the momentum to describe the viromes and eukaryotic microbiomes of arthropod vectors to enhance our understanding of the arthropod microbiome in further detail [5-9]. Functional understanding of arthropod microbiota has largely come from studies on hematophagous dipteran and hemipteran insect vectors of disease. Important functions associated with arthropod microbiome include the production of B-vitamins [2, 3], manipulation of reproductive behaviors including cytoplasmic incompatibility [10] parthenogenesis, male killing, and protection of the host against infection by pathogens, and parasites [11-13], possibly via modulation of innate immune responses [14-17].

Ticks are obligate blood feeding members of phylum Arthropoda, class Arachnida sub class Acari and are evolutionarily distant from the class Insecta; they transmit human and livestock pathogens worldwide [18]. The two primary families of tick species are Ixodidae (hard ticks) and Argasidae (soft ticks) and a monospecific family, Nuttalliellidae [19]. The feeding habits of hard and soft ticks vary significantly, with hard ticks feeding once in each developmental stage on a limited number of hosts, and soft ticks feeding on several different hosts at more frequent intervals [20]. While hard and soft ticks harbor microbiomes of varying complexities [21], a detailed understanding of their composition, role in tick biology and vectorial capacity is only beginning to emerge. This review will focus on I. pacificus and I. scapularis, two Ixodes species that are endemic to North America [22]. These ticks transmit multiple human pathogens [18] and indeed these pathogens represent frequent microbial residents of the tick microbiome. This review will dwell only on the non-pathogenic components of the microbiome and summarize our current understanding of the bacterial microbiome of these tick species, and highlight knowledge gaps that remain to be bridged to achieve a functional understanding of the tick microbiome.

Ixodes scapularis and Ixodes pacificus

I. scapularis and I. pacificus are the principal vectors of Borrelia burgdorferi, one of the causative agents of Lyme disease in the United States of America [22]. I. scapularis is endemic to the Northeast, upper Midwest of the USA, and southeastern parts of Canada. I. pacificus is endemic predominantly to the west coast of the USA. In addition to B. burgdorferi, both species have also been known to carry Anaplasma phagocytophilum, that causes human anaplasmosis, Babesia microti, the agent of babesiosis [18], and Borrelia miyamotoi, that causes a relapsing fever-like disease [23]. I. scapularis also serves as a vector of Powassan virus that causes encephalitis [24], Borrelia mayonii that causes Lyme disease, and Ehrlichia eauclairensis, a minor causative agent of ehrlichiosis [25]. I. pacificus has been shown to carry spotted fever group Rickettsia [26], although there is no reported transmission of Rickettsia from I. pacificus to vertebrate hosts. Both I. scapularis and I. pacificus larval and nymphal stages tend to feed on small-to-medium sized mammals, birds, mice, squirrels, deer, and humans; notably, I. pacificus also feed on lizards [27]. Differences in host preferences and geographic distributions between these two species significantly influence their microbiome compositions (Figure 1) and offer novel opportunities to gain mechanistic insights into tick-microbiome interactions.

Figure 1. Microbiome composition and distribution map of Ixodes pacificus and Ixodes scapularis.

I. pacificus and I scapularis, the two vectors of Lyme disease in the United States, with the most commonly reported blood meal hosts for juvenile and adult stages of the tick. Summaries of major eubacterial components of the adult tick microbiomes are displayed in the pie charts for each tick species. Illustration created by Ms Mona Luo.

Opportunities for acquisition of microbiota

Despite their comparatively long life-spans, I. scapularis and I. pacificus have limited and discrete opportunities for microbial acquisition or loss over their life span. The first opportunity for establishing the microbiome is seeded from the adult female tick to her offspring through transovarial transmission. After that, ticks can also acquire microbes from their environment and from blood feeding on vertebrate hosts. Before and after feeding to repletion on a vertebrate host, I. scapularis and I. pacificus ticks come in direct contact with soil microbes. Bacterial entry into the tick is predominantly through trans-ovarial, oral or cuticular routes. The majority of the soil microbiome is comprised of Acidobacteria, with bacteria from the phyla Verrucomicrobia, Bacteroidetes, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Betaproteobacteria, Plantomycetes, and Actinobacteria seen at lower relative abundances [28] and some of these have been found to be associated with the tick microbiome [29]. There has been ongoing debate as to whether these shared soil bacteria are environmental contaminants or whether they constitute an important part of the intrinsic tick microbiome [30]. The microbiome of the mammalian skin plays a vital role in shaping mammalian immunobiology [31]. Therefore, we must be careful not to dismiss the role of microbiota on the surface of ticks.

In a field experiment, Couper et al. [32] directly tested the influence of environmental exposure time on the I. pacificus microbiome by placing larval I. pacificus in field enclosures buried in the soil for different amounts of time ranging from 0 to 6 weeks and found that field exposed larvae acquire microbial richness. This was also supported by another study on I. scapularis that coupled microbiome analysis with midgut immuno-staining and found that some taxa isolated from external wash samples were also detected internally such as Bacillus, Enterobacteriaceae, and Pseudomonas [33]. Further, lab-reared I. scapularis and I. pacificus display distinct microbiomes compared to field-collected ticks and are generally characterized by lower microbial diversity presumably due to a more sterile environment compared to field-collected ticks [15, 21, 34, 35].

Host blood meal and tick microbiome

The intimate relationship between Ixodes spp. and their blood meal hosts prompted investigations on whether the host influences the tick microbiome. Several studies found that there is little correlation between host skin or blood and the microbiomes of hard ticks such as I. scapularis and D. variabilis [35-37]. Instead, those studies found that the most important factor in structuring tick microbiomes was tick species [36]. When the microbiomes of D. variabilis and I. scapularis that fed on two rodent blood meal hosts, prairie voles (Microtus ochrogaster) and white-footed mice (Peromyscus leucopus), were analyzed, the overwhelmingly dominant factor influencing microbiome composition was the tick species [37]. Landesman et al’s study [38]. indicates that host species can also affect overall microbiome composition as well as the relative proportion of the endosymbiont, Rickettsia buchneri. The life history of I. pacificus may be well suited to address this question because its dominant blood meal host is not a rodent but a reptile [39-41], the western fence lizard (Sceloporus occidentalis). This species is Borrelia refractory [42], meaning infected I. pacificus that feed on S. occidentalis are cleared of their B. burgdorferi infections [43]. It is perhaps because of this unusual property of S. occidentalis that Swei and Kwan [44] observed that nymphal I. pacificus that fed on lizards as larvae had significantly lower microbiome species richness and higher relative abundance of the Rickettisial endosymbiont compared to ticks that fed on Peromyscus mice as larvae. A recent study of several hard tick species found that generalist ticks that feed on diverse hosts have more diverse microbiomes than nest dwelling or one-host tick species [45]. These findings suggest that the host bloodmeal does have an impact on the tick microbiome composition, due perhaps to as-yet undefined factors in the bloodmeal that may modulate microbial compositions directly or indirectly by regulating tick innate immune responses.

Inter-stadial changes in microbiome

How the tick’s microbiome changes throughout its life is not well characterized. The transmission of obligate endosymbionts, a core component of the tick microbiome, from one generation to the next and between tick life stages is a well-documented phenomenon in hard ticks [46, 47]. In Ixodes scapularis, the most commonly reported endosymbiont to date is Rickettsia buchneri [48-51]. Meanwhile, I. pacificus is frequently, and potentially ubiquitously, associated with Rickettsia genomospecies G021 which clusters closely with R. buchneri based on several folate synthesis loci [52]. Rickettsia genomospecies G021 is distinct from the spotted fever group Rickettsia genomospecies G022 observed by Phan et al in I. pacificus [26]. The importance of rickettsial endosymbionts is believed to lie in the nutritional benefit they provide to the tick, particularly of B-vitamins such as folate that ticks lack from having a strictly hematophagous diet [53]. Gillespie et al [54] have shown that the genome of R. buchneri encodes 2 functional biotin operons, not observed in other rickettsial species. R. buchneri, the predominant endosymbiont of I. scapularis [55], may therefore be unique in having the potential to provide biotin in addition to folate to the tick host. Many microbiome studies have documented a pattern of increasing relative abundance of endosymbionts through development of Ixodes spp. from larva, to nymph, to adult stages [35, 48, 56-58]. Rickettsia accumulation by adult female ticks is thought to be an adaptation to facilitate transovarial transmission of the endosymbiont to eggs, underlining the importance of endosymbionts to tick survival and development [47, 52, 59]. It is worth noting that I. pacificus and I. scapularis are associated with Rickettsial endosymbionts but not Coxiella or Francisella-like endosymbionts observed in other tick species [60] (Table 1). The biological significance of this preferential association of Rickettsial endosymbionts with I. scapularis and I. pacificus remains to be understood.

Table 1.

Bacterial endosymbionts of selected hard tick vectors of human diseases in the USA.

Tick vector	Predominant endosymbionts	Localization	Reference
Ixodes scapularis	Rickettsia buchneri	Ovaries, salivary glands, guts	[99, 100]
Ixodes pacificus	Rickettsia buchneri-like (GO21)	Ovary, midgut	[34, 101]
Ixodes ricinus	Candidatus midichloria, Francisella, Spiroplasma, Rickettsia	Ovaries, trachea, salivary glands, malphigian tubules	[74, 102]
Dermacentor andersoni	Rickettsia peacockii, Rickettsia belli, Francisella spp, Arsenophonous spp,	Salivary glands, guts	[103]
Amblyomma americanum	Coxiella-like, Rickettsia spp*	Ovaries, malphigian tubules, salivary glands, muscles, gut	[104, 105]
Amblyomma maculatum	Francisella-like endosymbiont and Candidatus Midichloria mitochondrii	Salivary glands, gut, ovaries	[106, 107]
Haemaphysalis longicornis	Coxiella-like, Francisella-like	Malphigian tubules and ovaries	[108, 109]

^*Amblyomma americanum also harbors Rickettsia amblyommatis, a potential pathogen [110].

In multiple studies of I. scapularis that examined the relative abundance within sequenced 16S rRNA amplicons, adult female ticks were observed to exhibit a higher proportion of Rickettsia endosymbionts than males and have lower microbiome diversity as measured by Shannon diversity or overall richness [35, 48, 56, 57]. However, Tokarz et al performed detailed qPCR analysis of male and female ticks separately and did not observe significant differences in bacterial abundance between male and female I. scapularis [48]. In contrast to I. scapularis, I. pacificus males and females have more similar microbiome profiles based on species diversity and the proportions of Rickettsia endosymbiont [34]. Looking to other Ixodes species, Ixodes ricinus resembles I. scapularis in exhibiting a higher relative abundance of Rickettsia in female ticks relative to males [61] but other species, such as Ixodes ovatus and Ixodes persulcatus exhibited higher alpha diversity and lower relative abundance of endosymbionts in female ticks [62]. Thus, there is evidence that the patterns of overall microbial diversity and the relative abundance of a key endosymbiont at the adult stage can vary depending on the species of tick under examination and the methods used for quantification. When drawing conclusions on general patterns across life stages, it is also important to distinguish between field and lab-collected ticks and to identify the stage of blood feeding (i.e. questing or engorged ticks)[63]. When ticks are sampled from their host at various stages of engorgement, the Rickettsia endosymbiont dominates the tick microbiome because it replicates prolifically during blood feeding [36, 46].

In another study that examined several hard tick species, Chicana et al. [45] reported similar patterns of reduced microbiome species richness and diversity through life stages of both I. pacificus and two Dermacentor species (D. occidentalis and D. variabilis). At the same time, the relative abundance of the dominant endosymbiont abundance increased along life stage development in several of the species examined [45]. On average, adult stages had 50% of larval richness, while nymphs had intermediate levels of richness. This pattern may be due to competitive interactions between the components of the tick microbiome or could reflect the gradual loss of transient, environmentally acquired microbes typically associated with the larval stage [32]. There was no evidence of competition between microbial species based on checkboard score (C-score) analysis which compares co-occurrence of OTUs with random simulations of microbiome community assembly [32]. The loss of species through time within a single life stage suggests that environmental microbes can assimilate into the tick microbiome but that host filtration through immune or physiological processes may remove the vast majority of microbes [32, 33]. Gene function analysis of microbiomes through the duration of this experiment did not find functionally different microbiomes here or in another study [63], suggesting that the tick microbiome is functionally stable and potentially redundant [32, 64].

Geographic changes and regional patterns in microbiome composition

Thus far, there has been limited evidence of microbiome structuring in I. scapularis or I. pacificus based on habitat or region [37, 45], but investigation of microbiomes across a broader spatial scale has not been systematically attempted (Box 1). A comparative genomic study of six Ixodid tick species has suggested the importance of ecogeographical fauna on the distribution of pathogenic bacteria in tick [65]. Consideration of the spatial scale in designing microbiome experiments will be important in future studies to draw more general conclusions [66]. Rickettsia species, the most commonly reported endosymbionts in I. scapularis and I. pacificus microbiomes is not observed in equal abundance in ticks from all geographic regions [36, 37, 45, 57, 67]. In the southeastern USA, Rickettsia spp. was not common in I. scapularis and instead, an uncharacterized Enterobacteriaceae was the most common element of the microbiome and while some specimens had low abundance, some samples had no measurable reads corresponding to Rickettsia [57]. Meanwhile, in Canada, Rickettsia were more common in the Atlantic region (New Brunswick and Nova Scotia) but further west in Ontario, Pseudomonadaceae and Enterobacteriaceae appear to be the most abundant element of the I. scapularis microbiome [67]. Some of this geographic variation may be traced to genetic history or life cycle differences in the tick population. Although, I. scapularis is considered one species from the southeast and north through the upper Midwest and eastern Canada, there are considerable life cycle differences between populations of I. scapularis in the northeast versus the southern Atlantic coast including different questing modality [68], host associations, and life cycle [69]. Ixodes scapularis from the northeastern US tend to quest above the leaf litter, feed on small mammals and shrews, and are more likely to attach to people. Genetic analyses suggest that there is some genetic structuring that limits gene flow from the northern and southern populations of I. scapularis in the eastern US [70, 71] which may influence reported microbiome differences. A detailed comparative genomic study [65] also highlighted the multifactorial impact of ecogeographic fauna on the bacterial distribution in ticks. Therefore, studies that examine the alignment of tick population genetics with microbiome profiles could help shed new insights into microbial diversity in ticks from different regions (Box 1).

Box 1. Addressing disparities in tick microbiome compositions

The disparate compositions of the Ixodes microbiome observed by different studies could be explained in-part by technical and sample processing differences. Even with a unified protocol differences may be observed due to the differential impact of biotic and abiotic factors on the tick. Careful assessment of the microbiome composition under different conditions may help resolve the inconsistencies.

1. A systematic assessment of seasonal variations in microbiome compositions may serve as a prologue to how climate change may impact tick microbiome.

2. A systematic analysis of the tick microbiome composition in the context of geographic variations. ecology and host preference.

3. A detailed examination of genetic changes in Ixodes ticks and correlation with microbiome composition

Box 2. Tools to manipulate the tick microbiome

In order to obtain a mechanistic understanding of the impact of specific bacteria on tick biology it will be critical to improve strategies to robustly manipulate the tick microbiota.

1. As artificial /membrane feeding systems for feeding ticks become more amenable for routine use in many laboratories, strategies to generate ticks with little or no environmentally acquired microbiota may be feasible simply by adding combinations of antibiotics in the medium. Given the long-life cycle of ticks, long-term maintenance will require germ-free isolators in conjunction with artificial membrane feeding systems.

2. Optimizing robust strategies to generate gnotobiotic ticks, the ability to add and remove specific microbiota, and co-relate the cause and effect of specific microbiota on tick biology will be another important milestone. This will spur biocontrol strategies to prevent tick-borne diseases.

Disparities in the assessment of microbiome compositions

An important decision in amplicon-based microbiome library preparation is the selection of the gene target. Most microbiome analyses use amplicon-based methods targeting the 16S rRNA region because it is highly conserved in prokaryotes but also has several regions of hypervariability, termed V1-V9. Of the 9 hypervariable regions, V1, V3 and V4 were the most informative [57, 67] while V9 gave the least reliable estimates of diversity [72, 73]. Most tick microbiome studies focus on the V3 and V4 regions, making cross study comparisons more feasible. Meta genomic sequence analysis [65], metatranscriptomic RNA-seq approaches in combination with metaproteomic approaches are also being utilized to obtain a comprehensive description of the microbiota [74]

There is considerable debate on the complexity of the microbiome composition of Ixodes species with some reporting tens of different genera [61, 75, 76] and some reporting significantly less diversity [33, 77]. It is critical to understand that the tick microbiome is influenced at the macro (population) and micro (organ/tissue) level in the context of biotic and abiotic factors [57, 66, 78]. The external surface of ticks can be contaminated by environmental microbes and cloud our assessment of bona fide members of the microbiome [33] and stringent surface sterilization protocols including bleach, hydrogen peroxide, and 70 % ethanol may be required [17, 79]. Despite stringent surface sterilization Couper et al. [32] identified likely contaminants such as Propionibacterium, a common resident on human skin. Whether these bacteria should be discarded as contaminants or regarded as transient, yet relevant passengers, remains a conundrum. Instead of using whole ticks, dissection of specific tissues such as guts, or salivary glands in conjunction with robust visualization strategies, pooling multiple low input samples [33], adding internal positive controls [80], and incorporating negative controls [81-83] may also help clarify the true composition of the tick microbiome. Analysis of core taxa (as defined as occurring at more than 5% of sequence reads in a majority of the samples) in the tick microbiome can be employed to assess important taxa that constitute a consistent and important presence [84].

Microbiome -tick Immunome Interactions

There is general consensus that the Ixodes microbiome is composed predominantly of Rickettsial endosymbionts [33, 55, 79, 85] and other bacterial genera such as Enterococcus, Pseudomonas, Staphylococcus, Lysinibacillus, and Bacillus occur at much lower abundance [32, 33, 77]. Bacterial members that associate with the tick, even if transiently and in much lower abundance, may impact the tick and the pathogens it harbors directly, or indirectly. A study in 2014 demonstrated that the microbiome plays an important role in facilitating B. burgdorferi colonization of the tick gut [15]. I. scapularis larvae raised in sterile containers associated with significantly decreased relative abundance of Acinetobacter spp, Brevibacterium spp, Lysinibacillus spp and Staphylococcus spp compared to that in normal lab-reared ticks. Dysbiosed larvae were also less effectively colonized by B. burgdorferi compared to larvae raised in normal containers. The microbiome was suggested to impact the Janus kinase (JAK)/ Signal transducer and activator of transcription (STAT) pathway of the tick and modulate the peritrophic matrix (PM), a key component of gut barrier integrity [15]. The PM was shown to provide an effective barricade against luminal contents as spirochetes colonized the gut epithelium [15]. Ross et al [33] showed that B. burgdorferi lacks interbacterial effector immunity genes that would be critical for it to survive in a polymicrobial milieu indicating the need for B. burgdorferi to escape the gut lumen and take cover under the PM. Consistent with this rationale, abundance of Pseudomonas, Bacillus or Enterobacteriacea was negatively correlated with B. burgdorferi abundance [86].

The JAK/STAT pathway is an evolutionarily conserved and key signaling pathway invoked in repair and remodeling of the gut epithelial cells and in activating immune responses in arthropods [87, 88]. The tick genome encodes all the key components of this pathway [89] except Upd (unpaired), a cytokine-like molecule released upon damage to the epithelial cells, that is essential for activation of the JAK/STAT pathway [90]. The observation that tick gut microbiota modulate the JAK/STAT pathway [15] raises the possibility that bacterial components may also activate this pathway either by serving as Upd surrogates or by other mechanisms that remains to be understood. Chou et al’s study has shown that I. scapularis-microbiota associations are ancient [91] and that during the course of evolution I. scapularis likely co-opted and domesticated a type VI secretion amidase effector gene (Dae2) from one of its gut-associated bacteria to protect itself from invading bacteria, including B. burgdorferi. A recent study by Hayes et al [92] has shown that tick salivary Dae2 has a broad spectrum antibacterial activity and is delivered into the bite site during feeding. Dae2 acts on skin commensal bacteria such as Staphylococci spp to preempt their entry into the tick gut. RNAi-mediated silencing of Dae2 resulted in increased abundance of skin-resident Staphylococci in the gut and impaired tick feeding. Effector molecules such as these antimicrobial peptides may control the abundance of environmental bacteria that enter the tick and tick gut from the soil, bloodmeal, or even the host skin (Figure 2). This may explain the relatively simple microbiome of the tick relative to the complex microbiomes of the environment including soil and host skin that the tick intimately associates with during it’s off-host and on-host phases respectively.

Figure 2. Environmental encounters that facilitate microbiota acquisition.

I. scapularis and I. pacificus come in contact with environmental bacteria including soil microbiota during their off-host phase and with vertebrate skin microbiota during feeding. Defense responses of haemocytes (H) in the haemolymph such as phagocytosis and antimicrobial peptides may thwart some of these microbiota from infecting the tick. Salivary defense responses including antimicrobial peptides secreted by the salivary glands (SG) may control skin microbiota from infecting the tick. Immune responses of the gut may also control microbiota that enter the gut. The stand-off between microbiota and the tick helps modulate the immune milieu, barrier integrity, nutrient status, tick biology and consequently the vectorial capacity. Dashed arrow indicates transovarial transmission of specific microbiota that inoculates eggs laid by mated females. Illustration created by Biorender.com.

I. scapularis has also been shown to express proteins such as IAFGP (Ixodes Antifreeze Glycoprotein), an antifreeze glycoprotein. IAFGP was originally identified as an antifreeze protein that was increased in A. phagocytophilum-infected ticks [93] and suggested to provide a survival advantage during overwintering of nymphal ticks. Careful analysis revealed that IAFGP was also an antibacterial protein that effectively impaired bacterial biofilm formation [94]. Abraham et al [95] showed that increased IAFGP expression during A.phagocytophilum acquisition from the mammalian host altered the composition of tick gut microbiota. This, in turn, resulted in a compromised barrier integrity facilitating A. phagocytophilum exit from the gut to the salivary glands [95]. While the integrity of the peritrophic matrix was critical for successful B. burgdorferi colonization [15], the converse was invoked in A. phagocytophilum infection [95]. This suggests that microbiota compositions may also determine the frequency and success of coinfections with these bacterial pathogens in endemic areas. Demonstrating a thematic pattern in controlling microbiota composition in the tick gut, it was observed that tick feeding induced the expression of a protein of I. scapularis with a Reeler domain, known as PIXR, that inhibits Gram-positive bacterial biofilm formation [77]. Abrogation of PIXR function by antibodies or by RNAi-mediated silencing resulted in increased biofilm formation, altered microbiome composition and impaired B. burgdorferi colonization of the tick. PIXR abrogation-mediated changes in the gut microbiome composition had no impact on the JAK/STAT pathway [77] nor on the PM integrity, invoking other interactions between microbiota and tick that influence B. burgdorferi colonization and remain to be deciphered.

Immunodeficiency pathway or IMD pathway is an evolutionarily conserved innate immune signaling pathway that activates the transcription factor NF-kB leading to the expression of antimicrobial peptides that predominantly control Gram-negative bacteria in arthropods [96]. Key components of the canonical IMD pathway are not represented in the I. scapularis genome [89]. This may seem an evolutionary adaptation to maintain critical Gram-negative bacteria such as Rickettsia [55] in the tick, and to avoid inadvertent activation of the IMD pathway. However, Shaw et al [97] showed that despite lacking several components of the IMD pathway including PGRP-LC -critical to sense the Diaminopimelic acid (DAP)-type peptidoglycans that decorate Gram-negative bacterial cell walls, the IMD pathway in Ixodes is functional, and is activated by infection-derived lipid components of bacteria including B. burgdorferi and A. phagocytophilum. As a better understanding of the unique IMD pathway of Ixodes tick unfolds, we may gain new insights into microbiota that may or may not have adapted to this unusual IMD pathway.

Tokarz et al [8], Sakimoto et al [7] have used unbiased deep sequencing to characterize the virome of several tick species including I. scapularis and show that viruses of Bunyaviridae, Rhabdoviridae and Chuviridae families are predominant members of the tick microbiome. Detailed and targeted studies will be required to understand interactions between these viruses, the bacteria in the microbiome and tick–immune pathway/s (Box 2). Importantly, to make progress in our understanding of the tick microbiome, and its unique interactions with its microbial partners we must develop robust tools to generate germ-free ticks and gnotobiotic ticks -this remains a major challenge in this field (Box 2). Kurlovs et al [98] utilized various antibiotics including ciproflaxin to reduce Rickettsial endosymbiont levels in I. pacificus and observed no impact on fecundity or egg hatching. In recent studies, Oliver et al [99] similarly used microinjection and artificial feeding strategies to introduce the antibiotic ciproflaxin into female I. scapularis to effectively eliminate R. buchneri from the tick microbiome. Interestingly, their initial studies also suggest that R. buchneri elimination has no impact on fecundity and tick development raising questions regarding the role of this endosymbiont in tick reproduction and development and pave the way for determining the vectorial competence of R. buchneri-deficient ticks.

Concluding remarks

The long-life cycle of the tick, variations in developmental stage, age, and gender of ticks, and spatial distribution of ticks present disparate microbiome compositions and confound conclusive determination of the bona-fide members of the tick microbiome. The core tick microbiome is simple, and the reported diversity of bacterial genera in the tick microbiome likely represents transient associations with bacteria in the environment including from soil, leaf litter, host skin, or host blood meal. These transient microbial associations are under surveillance by tick innate immune responses and are cleared by effector molecules such as antibacterial peptides or potentially excreted due to the absence of cognate adhesins to engage with the tick gut. Nevertheless, it is in the context of this constant friction with environment-associated microbes that ticks also encounter tick-transmitted pathogens of human disease. Increased immune surveillance may clear the environmental microbes, but, this is likely to be energetically costly for the tick and must warrant careful fine-tuning of the immune responses. Presumably, tick-borne pathogens have evolved ways to suppress, circumvent or even co-opt these immune responses. Indeed, this highlights a fundamental difference between these transient microbial passengers and the stable pathogenic microbial inhabitants that survive through the tick’s developmental stages and raises important questions about tick-microbe interactions (see Outstanding Questions). The time is ripe to direct scientific efforts to unravel a functional and mechanistic understanding of the tick microbiome, albeit transient, and its interactions with the tick, and implicit in this is also the understanding of how tick-borne pathogens are sustained and transmitted.

Outstanding Questions.

• Pathogens such as B. burgdorferi are stably maintained unlike environmental/transient bacteria that appear to be cleared. What are the mechanisms that drive this differential infection of ticks?

• Does the microbiome composition change when specific tick innate immune pathways are impaired or abrogated?

• Does the microbiome composition change in the context of specific tick-borne pathogens under similar environmental exposures?

Highlights.

• Ixodes scapularis and Ixodes pacificus harbor a simple primary microbiome composed predominantly of the endosymbiont Rickettsia buchneri.

• The tick microbiome also includes microbiota acquired from the environment and represents a transient microbiome.

• The tick microbiome composition is in a state of flux and is likely influenced by multiple biotic and abiotic factors.

• The microbiome composition is regulated by immune responses of the tick at the vector-host and environment-vector interface.

• Understanding the interactions between the tick microbiota -tick-borne pathogens and the tick immune responses will reveal new insights into tick biology.

Glossary

Bacteriocyte

Specialized giant cells observed in certain arthropods that harbor endosymbionts. Endosymbionts provide nutrients such as vitamins, and amino acids to the host.

B-vitamins

Water soluble vitamins synthesized by microorganisms. Folate and Biotin are one of this class of 8 vitamins.

Endosymbiont

Microorganisms that live within the body cells of another organism. These may or may not live in specialized cells of the host organism and may or may not always be a mutualistic relationship. Endosymbionts may be transferred vertically (from parent to offspring) or horizontally (free living symbionts are acquired by the host organism from the environment).

Hematophagous

Feeding on vertebrate hosts to obtain blood as the predominant source of nutrition

IMD pathway

This is a conserved immune signaling pathway in insects that is involved in the activation antibacterial responses predominantly towards gram-negative bacteria.

JAK/STAT pathway

This is a conserved signaling pathway found in arthropods and mammals and is involved in key events including immunity, cell division, repair and remodeling. The main components of this pathway include a transmembrane receptor that engages with signals in the external milieu , an intracellular Janus kinase (JAK) that is associated with the transmembrane receptor and signal transducer and activator of transcription proteins (STAT).

Microbiome

Community of microorganisms that inhabit a specific niche such as within an animal host and include commensals, mutualistic, and parasitic organisms.

Transovarial transmission

Transmission of microorganisms through oocytes from mother to offspring.

Upd

Upd is a secreted protein encoded by the upd gene in Drosophila that activates the JAK/STAT signaling pathway by binding to the transmembrane receptor of the JAK/STAT pathway.

16S rRNA

Ribosomal RNA of the 16S subunit of bacterial ribosome.