Abstract
Hard ticks of the order Ixodidae serve as vectors for numerous human pathogens, including the causative agent of Lyme Disease Borrelia burgdorferi. Tick-associated microbes can influence pathogen colonization, offering the potential to inhibit disease transmission through engineering of the tick microbiota. Here, we investigate whether B. burgdorferi encounters abundant bacteria within the midgut of wild adult Ixodes scapularis, its primary vector. Through the use of controlled sequencing methods and confocal microscopy, we find that the majority of field-collected adult I. scapularis harbor limited internal microbial communities that are dominated by endosymbionts. A minority of I. scapularis ticks harbor abundant midgut bacteria and lack B. burgdorferi. We find that the lack of a stable resident midgut microbiota is not restricted to I. scapularis since extension of our studies to I. pacificus, Amblyomma maculatum, and Dermacentor spp showed similar patterns. Finally, bioinformatic examination of the B. burgdorferi genome revealed the absence of genes encoding known interbacterial interaction pathways, a feature unique to the Borrelia genus within the phylum Spirochaetes. Our results suggest that reduced selective pressure from limited microbial populations within ticks may have facilitated the evolutionary loss of genes encoding interbacterial competition pathways from Borrelia.
Introduction
Vector-borne pathogens infect over one billion people annually and have expanded at an alarming rate in recent years (Harvell et al., 2002; Jones et al., 2013; Keesing et al., 2010; Vora, 2008). Lyme disease, which is caused by the tick-borne bacterial pathogen Borrelia burgdorferi, is the fifth-most reported infectious disease in the United States, corresponding to over 90% of vector-borne infections in North America (Kugeler et al., 2015; Radolf et al., 2012). B. burgdorferi transits an enzootic cycle between small mammal reservoir hosts and other mammals, vectored by ticks of the genus Ixodes (Radolf et al., 2012). Transmission of B. burgdorferi to humans via tick bite results in a constellation of inflammatory symptoms requiring an antibiotic treatment regimen for resolution within 2-3 weeks in most cases (Berende et al., 2016; Wormser et al., 2006). While a vaccine targeting B. burgdorferi had been approved by the FDA, it was subsequently withdrawn and currently there is an urgent need for new strategies to control tick-borne disease transmission (Nigrovic and Thompson, 2007).
Colonization resistance against pathogens mediated by commensal microorganisms is one such proposed strategy (Buffie and Pamer, 2013; Finney et al., 2015). Studies in mosquitos and tsetse flies, among others, have motivated the characterization of the endogenous microbiota associated with vectors in hopes of identifying means by which pathogen transmission can be interrupted through direct or indirect interactions (Cirimotich et al., 2011a; Cirimotich et al., 2011b; Wang et al., 2017). One mechanism that may govern interactions between pathogens and the microbiota involves direct competition. Many bacteria possess elaborate mechanisms that can mediate interbacterial competition in polymicrobial environments, including specialized pathways such as the type VI secretion system, which delivers toxic effector proteins to target cells (Hibbing et al., 2010; Russell et al., 2014). It is thought that the presence and repertoire of interbacterial systems in a given bacterial genome reflects to a certain degree the selective pressures that organism faces in its natural niche (Zhang et al., 2012). Indeed, these antagonistic systems contribute to bacterial fitness in complex bacterial communities such as the mammalian gut (Kommineni et al., 2015; Verster et al., 2017; Wexler et al., 2016).
The genus Borrelia comprises a group of pathogenic bacteria that rely upon hematophagous arthropods for infectious transmission to humans, including ticks of the order Ixodida (Radolf et al., 2012). In hard ticks (Ixodidae), surveys of the internal microbiota have to-date identified bacteria that could potentially restrict pathogen transmission (Andreotti et al., 2011; Budachetri et al., 2014; Budachetri et al., 2016; Clay et al., 2008; Clayton et al., 2015; Hawlena et al., 2012; Khoo et al., 2016; Nakao et al., 2013; Narasimhan et al., 2014; Rynkiewicz et al., 2015; Swei and Kwan, 2016; Trout Fryxell and DeBruyn, 2016; van Treuren et al., 2015; Williams-Newkirk et al., 2014; Zolnik et al., 2016). However, significant variation in the diversity and identity of tick-associated bacteria was observed in these studies – rendering the potential for bacterial interference within ticks uncertain. In this study, we aimed to determine whether I. scapularis possesses a diverse midgut microbiota that might be encountered by B. burgdorferi. Most previous studies of the microbiome of hard ticks have utilized high-throughput sequencing technologies. We sought to augment sequencing with direct measurements of bacterial load and visualization of bacteria within adult and nymphal I. scapularis ticks by confocal microscopy. With these complementary approaches, we provide evidence that hard ticks lack a stable midgut microbiota.
Results
Borrelia lacks interbacterial effector–immunity genes
Members of the phylum Spirochaetes inhabit diverse environments, from the dense and competitive oral cavity and gastrointestinal tract of mammals to the midguts of arthropods like termites and ticks. How members of the phylum Spirochaetes engage in interactions with other bacteria is not understood. We therefore sought to characterize the distribution of interbacterial effector and immunity genes in the genomes of Spirochaetes. We first compiled a database of Spirochaetes genomes, encompassing a total of 63 genomes representing all major genera. These genomes were queried for the presence of homologs of 220 interbacterial effector and immunity genes (Zhang et al., 2012), including those with characterized domains found associated with contact-dependent inhibition (Hayes et al., 2010), type VI secretion system (Russell et al., 2014), and ESX/T7SS antagonistic pathways (Cao et al., 2016; Whitney et al., 2017). This analysis revealed the presence of genes encoding interbacterial effector and immunity domains throughout the Spirochaetes, particularly in species known to inhabit polymicrobial environments such as the mammalian gut microbiome (Treponema succinifaciens and Brachyspira spp) and the oral microbiome (Treponema denticola) (Figure 1A). We failed to detect effector gene homologs and identified only a limited group of immunity gene homologs encoded by any species within the genus Borrelia, including representatives from the sensu stricto and sensu lato genospecies (Becker et al., 2016). Since the phylum Spirochaetes is considered to be monophyletic, with extant genera descending from a single common ancestor (Paster et al., 1991), parsimony supports the conclusion that Borrelia lost interbacterial effector genes early in the evolution of the genus. Parallel investigation of the genomes of tick-associated endosymbionts and tick-transmitted intracellular pathogens including Rickettsia, Coxiella, Anaplasma, and Ehrlichia revealed that these genomes also largely lack interbacterial effector– immunity genes (Figure 1B).
Assessment of bacterial abundance and diversity in I. scapularis
The evolutionary loss of effector–immunity genes from the genus Borrelia led us to hypothesize that interbacterial interactions might be limited within I. scapularis. We therefore sought to quantify the microbial communities associated with wild I. scapularis. We first isolated DNA from the dissected viscera (a combination of internal tissues that included midgut, reproductive tissues, and salivary glands) of 61 adult ticks collected from 5 distinct geographic sites in the Midwestern US. We then performed quantitative polymerase chain reaction (qPCR) targeting conserved regions of the 16S rRNA gene. Our analysis demonstrated that the mean internal bacterial load of unfed adult I. scapularis ticks varies over several orders of magnitude (Figure 2A). Male ticks analyzed harbored less bacterial load than did females (4.5x105 vs 5.3x107, t-test p-value < 0.001, N = 61). qPCR with primers specific to the B. burgdorferi flaB gene revealed infection frequencies similar to that previously reported from the Midwestern geographic regions (Figure S1A) (Hamer et al., 2014). To investigate the differences in bacterial community underlying the sex-specific differences that we observed, we performed 16S rRNA gene sequencing on the same samples used for qPCR analysis. In addition, we sequenced the external washes from a subset of ticks, and a water-only control for each group. 178 OTUs were detected in the water-only controls, including taxa commonly implicated in reagent contamination and some previously reported to be associated with ticks, such as Sphingomonas and Comamonas (Nakao et al., 2013; Narasimhan et al., 2014; van Treuren et al., 2015). These OTUs were subsequently eliminated from all other samples in downstream analyses. To describe the taxa 6 that comprise I. scapularis-associated bacterial communities following removal of water-borne contamination, we calculated the taxonomic relative abundance averaged across all internal and external samples. This revealed that 258 (95%) of 270 OTUs fell below 1% relative abundance when averaged. In contrast, other taxa exceeded 1% average relative abundance in both internal and external samples, including Bacillus, Pseudomonas, and Enterobacteriaceae (Figure S2). Importantly, the taxon that was most abundant in internal samples but entirely absent in external samples was the genus Rickettsia (average relative abundance of 52%), which includes the dominant Rickettsia endosymbiont of I. scapularis (Gillespie et al., 2012). The relative abundance of Rickettsia exhibited a positive correlation with total bacterial load (Figure 2B), indicating that Rickettsia is the primary driver of bacterial abundance in most I. scapularis ticks. The family Spirochaetaceae also was often abundant in viscera samples but absent in washes (Figure S2).
The number of taxa with low relative abundance could indicate that the internal environment of I. scapularis is particularly well suited to fostering diverse microbial communities. However, an alternate possibility is that low abundance OTUs represent signal from contamination. Indeed, several studies have examined the effect of low biomass input on diversity metrics in 16S rRNA gene sequencing surveys, with the notable result that alpha diversity (within-sample diversity) correlated negatively with the abundance of input DNA (Jervis-Bardy et al., 2015; Lauder et al., 2016; Salter et al., 2014). We therefore examined the relationship between alpha diversity and bacterial load as quantified by qPCR in our I. scapularis samples. When examined across all 61 I. scapularis samples, we found a strong negative correlation between total bacterial load and alpha diversity that was independent of geographic origin (Spearman’s rho = −0.74, p < 0.001) (Figure 2C and S3). These data imply that the low bacterial biomass associated with adult I. scapularis ticks can impact the interpretation of 16S rRNA gene sequencing surveys by artificially inflating alpha diversity. We further observed a strong negative correlation between bacterial load and the relative abundance of the group of taxa contributing to 1% or less of the total across all I. scapularis samples (Figure 2D). We found only six taxa to be present in all I. scapularis viscera samples, represented by eight OTUs, including the genera Rickettsia, Borrelia, Pseudomonas, Francisella, and Escherichia, and the family Enterobacteriaceae (Figure S2). Linear discriminant analysis effect size (LEfSe) revealed the taxa most likely to explain differences between external wash samples and internal viscera samples. External samples are characterized by taxa from the phyla Proteobacteria and Actinobacteria, while viscera samples are distinguished by the orders Spirochaetales and Rickettsiales (Figure S4) (Segata et al., 2011).
While the internal bacteria associated with most wild adult I. scapularis are dominated by Rickettsia and B. burgdorferi, a minority of samples exhibit high relative abundances of three putatively environmental taxa, including the genera Bacillus and Pseudomonas, and the family Enterobacteriaceae (Figure 3A) all of which have been previously described as associated with I. scapularis (Moreno et al., 2006; Steinhaus, 1946; van Treuren et al., 2015). Colonization of ticks appeared to be independent for each taxon since, although co-occurrence was detected in a few samples, most ticks harbored only one dominant environmental taxon. While total bacterial load was not correlated with colonization by environmental bacteria, samples with high abundance of these bacteria – deriving from each of the five geographically-isolated collection sites – were less frequently infected by B. burgdorferi than expected (Figure 3B, C). This observation suggests that colonization by Bacillus, Pseudomonas, and Enterobacteriaceae may limit B. burgdorferi infection.
Immuno-staining and confocal microscopy reveals I. scapularis midgut biogeography
To orthogonally and directly validate our findings of a limited internal microbiome in ticks, we turned to confocal microscopy in order to visualize bacteria distribution and localization within I. scapularis. Our sequencing results suggest that Rickettsia comprise the dominant internal microbial inhabitants of most wild adult I. scapularis ticks. However, we questioned i) if our analyses failed to detect low-abundance yet highly diverse communities of midgut-associated bacteria masked by the high abundance of Rickettsia and ii) if the limited midgut colonization by environmental bacteria that we observed could be visualized and therefore validated. Ticks present a number of challenges that hinder standard microscopy techniques. These challenges include the physical barrier of the thick outer cuticle, a high degree of internal auto-fluorescence derived from the cuticle, and remnants of previous blood-meals including heme crystals (Sojka et al., 2013). In order to preserve tissue integrity and organization, we utilized formalin-fixation and paraffin embedding of dissected ticks, followed by thin sectioning and staining for visualization. Use of biotin-labeled ISH probes allowed us to perform tyramide-signal amplification (TSA), dramatically increasing the signal to noise ratio within tick sections.
With these methods, we characterized the distribution of bacteria within tissues of individual adult I. scapularis ticks (N=41). Abundant and DAPI-intense cocci that co-stained with probes targeting universally conserved 16S rRNA sequences (EU338) were observed in the cytoplasm of ovarian cells of all female I. scapularis ticks (Figure S5). These are presumed to be vertically transmitted Rickettsia (Noda et al., 1997). In striking contrast to ovaries, the midgut of most unfed adult I. scapularis lacked bacilli or cocci as visualized by DAPI signal or ISH signal localized to the lumen or luminal epithelium (Figure 5A, 90%, N=41 total ticks). This differed for B. burgdorferi-infected I. scapularis ticks, in which spirochete cells constituted the only detected midgut bacteria (Figure 4A, S5). However, a minority of I. scapularis ticks from a single site (CA) exhibited midgut colonization by bacterial cells with distinct cocci and bacilli cellular morphologies (10% of total I. scapularis examined, N=41) (Figure 4B). Since I. scapularis nymphs have been reported to possess a diverse midgut biofilm-forming microbiome, with very low relative abundance of Rickettsia (Abraham et al., 2017; Narasimhan et al., 2014), we considered the possibility that while adult I. scapularis ticks may lack a diverse internal microbiome, earlier life stages could be colonized to a greater extent. To test this possibility, we imaged sections prepared from whole-mounted I. scapularis nymphs, using similar formalin fixation and staining methodology as before. We found that I. scapularis nymphs also exhibit low DAPI and ISH probe staining within midgut tissues (Figure S6). Since nymphs are considerably smaller than adults, they may be more sensitive to artefactual noise associated with low-biomass high-throughput sequencing. In support of this notion, the alpha diversity of the Ixodes pacificus microbiome negatively correlates with life stage progression from larvae to adults as body size increases (Swei and Kwan, 2016).
16S rRNA gene sequencing of diverse wild adult ticks
We next sought to test whether our findings of a limited internal bacterial load and restricted diversity in I. scapularis were generalizable across Ixodidae. Vector competence for Borrelia spp. varies across genera within the Ixodidae. Ixodid ticks also vector pathogenic Anaplasma and Ehrlichia spp which must transit the midgut during their enzootic cycles (Ismail et al., 2010). We collected six species of hard tick from multiple geographic locations, including species from the genera Amblyomma, Dermacentor, and Ixodes. qPCR-based assessment of internal bacterial load revealed a variability similar to that of I. scapularis, with Amblyomma maculatum exhibiting the highest load and broadest variation across individual ticks (Figure 5A). As observed for I. scapularis, the dominant bacterial taxa associated with each tick species were species-specific endosymbionts (Figure 5B). These include Francisella and Rickettsia in Amblyomma maculatum, Francisella in Dermacentor spp, and Rickettsia in Ixodes spp, as has been previously reported (Budachetri et al., 2014; Rynkiewicz et al., 2015). Although alpha diversity varies broadly across species (Figure 5C), negative correlations with total bacterial load across hard tick samples (an exception being I. pacificus) supported a similar pattern as seen for I. scapularis (Figure 5D).
Beta diversity (between-samples) analysis using the weighted UniFrac metric (which accounts for differences in taxon abundance between samples) revealed significant clustering of samples by tick genus (ADONIS, p=0.001) (Figure S7A), as expected for stable microbiomes or those largely composed of endosymbionts. In contrast, external wash samples cluster together regardless of which tick they were associated with. This suggests that external microbiomes share common features (eg. the presence of diverse low-abundance taxa and lack of highly abundant endosymbionts) that differ from internal microbiomes of ticks. This interpretation is supported by beta diversity analyses using the unweighted UniFrac metric (in which each OTU is given equal weight regardless of relative abundance in the sample) which revealed no such internal-external (Figure S7B) or genus-level clustering, suggesting that the low-abundance taxa in samples do not sufficiently drive differences between tick genera.
Finally, we sought to extend our microscopy findings across hard tick species. Similar to I. scapularis, we found that A. maculatum, D. variabilis, and I. pacificus ticks lack abundant midgut-associated bacteria, while females belonging to these species contained abundant DAPI-intense cocci within ovaries (Figure S8). We also observed that midgut sections near ovaries sometimes contained localized micro-colonies, similar to those shown to be Rickettsia in I. scapularis. These may be species-specific endosymbionts, as previously reported (Clay et al., 2008; Klyachko et al., 2007).
Discussion
Here we have provided multiple lines of evidence showing that unfed wild hard ticks possess a limited internal microbiome. This is in contrast to reports that possession of a diverse microbiome is a characteristic feature of hard ticks across species and life stages (Abraham et al., 2017; Andreotti et al., 2011; Budachetri et al., 2014; Budachetri et al., 2016; Nakao et al., 2013; Narasimhan et al., 2014; Swei and Kwan, 2016; Trout Fryxell and DeBruyn, 2016; van Treuren et al., 2015; Zolnik et al., 2016). Our report adds to the growing literature suggesting that not all animals are intimately associated with a complex and abundant internal microbiome (Hammer et al., 2017; Jing et al., 2014). We find that measurements of high diversity derived from high-throughput sequencing of tick samples is largely a function of low bacterial biomass. This is a widespread problem inherent in high-throughput sequencing studies that rely upon PCR-amplification during the sample preparation process (Glassing et al., 2016; Kim et al., 2017). We propose that pooling multiple samples to increase input biomass is one method that might lower noise from reagent-based or external contamination (Gall et al., 2016).
There is great interest in exploiting natural microbial communities to benefit human health and combat disease. One such avenue of active research involves leveraging the native microbial communities associated with medically-relevant arthropod vectors (like mosquitos and ticks) to prevent pathogen transmission (Cirimotich et al., 2011b; Narasimhan and Fikrig, 2015).
We note that B. burgdorferi may be particularly susceptible to inhibition by competitor bacteria if encountered due to a paucity of genetic mechanisms for direct interbacterial interactions. This is supported by our findings that the increased abundance of Bacillus, Enterobacteriaceae, and Pseudomonas within the midgut is associated with decreased B. burgdorferi infection. Notably, these bacteria were also detected in external wash samples. This intriguing pattern suggests that some tick-associated external bacteria can colonize the I. scapularis midgut and, in doing so, might competitively exclude B. burgdorferi, either by displacement or by inhibition of infection during a subsequent blood meal. Members of these taxa encode an arsenal of mechanisms with which to compete with other bacterial cells, including type IV, VI, and VII secretion systems, contact-dependent inhibition (CDI) mechanisms, and the ability to produce bacteriocins (Cao et al., 2016; Hayes et al., 2010; Riley and Wertz, 2002; Russell et al., 2014; Souza et al., 2015). It is unknown if environmental bacteria can persist within the midgut during transstadial molts, but it is worth noting that detection of Bacillus and other bacteria within ticks long preceded the 16S rRNA gene sequencing era (Steinhaus, 1946). Direct inhibition of B. burgdorferi by tick-associated bacteria remains an active area of investigation by our laboratories.
Despite the detection of environmental bacteria within ticks, our findings suggest that the midgut of hard ticks may largely be an environment that is ill-suited for bacterial growth, perhaps due in part to the exceedingly low levels of the vital nutrient thiamin in I. scapularis (Zhang et al., 2016). While B. burgdorferi has evolved unique metabolic strategies in order to persist in this thiamin-limited environment, other less-specialized bacteria may not be able to do so. Additional factors that might limit the overall bacterial load of hard ticks are the possession of conserved or unique innate immunity factors that could target bacteria (Chou et al., 2014; Palmer and Jiggins, 2015), heme toxicity (Anzaldi and Skaar, 2010), and the effects of nutrient limitation and desiccation during the extended period between blood meals (Radolf et al., 2012; Sonenshine and Roe, 2014).
Borrelia burgdorferi may encounter a limited diversity of bacteria within the I. scapularis midgut, yet co-infections with other pathogens such as Anaplasma are common (Hamer et al., 2014). The mechanisms by which B. burgdorferi co-exists with other microbial pathogens within the tick remain unexplored. While Anaplasma, Ehrlichia, and Rickettsia spp are intracellular and may not engage in direct physical interactions with Borrelia spp, these bacteria may influence colonization through other means (Kocan et al., 2010; Simhadri et al., 2017). B. burgdorferi may not frequently encounter diverse bacteria within the midgut of the tick, however, multi-strain infections may be common within single ticks (Durand et al., 2017; Durand et al., 2015; Herrmann et al., 2013; Strandh and Råberg, 2015; Voordouw, 2015). Furthermore, it is as yet unclear the extent to which different B. burgdorferi strains engage in interactions or how multi-strain infections might influence transmission to humans and pathogenicity.
Our bioinformatic analyses suggests that the genus Borrelia lost genes for mediating interbacterial interactions during the course of its evolution. While Ixodes are the only vectors for B. burgdorferi in North America, ticks of the genus Ornithodoros (soft ticks) vector the relapsing fever spirochete B. hermsii (Dworkin et al., 2002). We did not detect interbacterial effector–immunity gene homologues encoded by B. hermsii. Although soft ticks were not included in our 16S rRNA gene sequencing or microscopy, we speculate that Ornithodoros, like Ixodidae, harbors a limited internal microbiome. Our results further imply that lice of the order Phthiraptera (class Insecta), which vectors B. duttonii and B. recurrentis, may also possess a limited internal microbiome (Lescot et al., 2008). Borrelia spp may have therefore evolved to exploit evolutionarily divergent hematophagous arthropods that share the common lack of a stable midgut microbiota.
Methods
Tick collection
Ticks were collected from the following sites: oak woodland habitat in Klickitat River Canyon, Washington (Ixodes pacificus and Dermacentor andersonii); oak-hickory forest in Wolf Creek State Park, Illinois (Ixodes scapularis and Dermacentor variabilis); oak-dominated forest in Gordie Mikkelson Wildlife Management Area and Carlos Avery Wildlife Management Area, Minnesota (abbreviated GM and CA, Ixodes scapularis and Dermacentor variabilis); red pine forest in Kettle Moraine State Forest Southern Unit (KM), mixed hardwood forest in Big Eau Pleine County Park (BEP), and oak-hickory forest in Sandberg Woods Conservancy (SC), Wisconsin (Ixodes scapularis); oak forest near McPherson Preserve, Oklahoma (Amblyomma maculatum). Ticks were collected by the flagging and dragging methods and shipped in 50mL Falcon tubes with damp paper towels on wet ice to retain moisture. Species, developmental stage, and sex of individual ticks were determined by visual inspection.
DNA isolation
Live ticks were washed three times with sterile water then dried before immobilization on a glass slide using double-sided tape. 21-gauge needles were used to remove the cuticle, and sterile forceps used to dissect and remove the viscera into 500uL sterile deionized water for subsequent DNA purification. Fresh needles were exchanged, and forceps sterilized by washing three times in sterile water and 70% ethanol between each dissection. The first external wash sample was saved, and pooled across individuals of the same species, sex, and geographical collection location. Samples were frozen at −80°C prior to DNA isolation. Frozen tissue samples were thawed and subsequently homogenized via glass bead-beating on a MiniBeadBeater (Biospec Products) for two cycles of one minute duration each. Subsequently, phenol-chloroform-isoamyl alcohol extraction of total nucleic acids was performed with a RNase treatment to remove RNA. Following extraction, DNA pellets were resuspended in 25uL sterile deionized water.
Quantitative PCR
The abundance of bacteria in single tick samples was determined by quantitative PCR using SsoAdvanced Universal SYBR Green Supermix (Biorad) on a BioRad CFX96 Real Time System C1000. Primers used targeted the 16S rRNA gene (331f and 797r(Nadkarni et al., 2002)) or the B. burgdorferi flaB gene (Jewett et al., 2007). Standard curves were generated from serial dilutions of purified genomic DNA prepared from monocultures of Escherichia coli DH5a or B. burgdorferi B31A3. Samples were run in technical triplicates with the mean of each triplicate used for later analysis.
16S rRNA gene sequencing and analysis
Genomic DNA from tick samples, external washes, and water controls was submitted for sequencing and individually barcoded for high-throughput sequencing of V3-V4 16S rRNA amplicons on an Illumina MiSeq, in three separate runs (performed by MrDNA). Sequencing reads were subsequently demultiplexed and merged into a single fastq file. The UPARSE pipeline was used to process the samples using default settings (Edgar, 2010, 2013). Following taxonomy prediction and OTU assignment, the OTU table were filtered to only retain OTUs that appeared at greater than 1% relative abundance in at least one sample. Alpha-diversity and beta-diversity metrics were calculated in QIIME (MacQIIME v1.9.0) (Caporaso et al., 2010). For determination of B. burgdorferi infection status, tick samples in which the sum of the relative abundances of the family Spirochaetaceae exceeded 1% were considered to be infected, while those less than 1% were considered to be uninfected. The sum of the relative abundance of the genera Bacillus and Pseudomonas was calculated for samples for comparison of B. burgdorferi infection status. For LEfSe analysis, the OTU table was converted to relative abundances and, along with associated metadata, was uploaded to the Huttenhower Lab Galaxy web application. Statistical tests and generation of plots were performed in R version 3.3.2 (R Development Core Team, 2013).
Histology
Preparing tick sections
Ticks were washed three times in sterile water and immobilized with double-sided tape attached to glass slides. Cuticles were removed with sterile 21-gauge needles. Following removal of the cuticle, dissected ticks were fixed in formalin for at least 5 days at room temperature. Fixed ticks were then embedded in paraffin and four 4-micron sections were prepared from each tick. Slides were deparaffinized in a series of washes in xylenes, followed by rehydration in washes of decreasing percent ethanol solutions.
Antibody-based microscopy
Deparaffinized slides were first subjected to a 40 minute incubation in sodium citrate buffer at 95oC in a vegetable steamer (Black and Decker). Slides were then washed in 2X SSC + 0.3% Tween 20, blocked in PBS + 5% BSA, and incubated with BacTrace fluorescein isothiocyanate goat anti-Borrelia whole cell antibodies (Kirkegard and Perry Laboratories) for 1 hour at room temperature or overnight at 4oC. Slides were then washed, dried, and mounted in ProLong Diamond plus DAPI (Thermo Fisher Scientific). Coverslips were sealed with nail polish.
In situ hybridization analysis
Deparaffinized slides were washed in 2X SSC + 0.3% Tween20 then were lightly digested with Proteinase K to allow probe access into tissues, washed and dried. Probes used were EU338 (Amann et al., 1995) or specific to Rickettsia. For Rickettsia-specific probes, the sequence of the Rick_1442 16S rRNA probe (Vannini et al., 2005) was aligned to the publically available REIS genome (Gillespie et al., 2012) and confirmed to be identical. ISH-probes (5’ biotin labeled) were diluted 1:100 to 50uM in ethylene carbonate hybridization buffer (15% ethylene carbonate, 20% dextran sulfate, 600mM NaCl, 10mM sodium citrate (Matthiesen and Hansen, 2012). Slides were covered with parafilm, placed in humidified chambers, and allowed to incubate for 1 hour at 42°C. Slides were subsequently washed in 2X SSC at 37°C then allowed to air dry. Alexa Fluor 488-coupled tyramide signal amplification (Thermo Fisher Scientific T20912) was performed to increase probe signal above background autofluorescence from tick midgut digestive products (Biegala et al., 2002; Sojka et al., 2013). Slides were mounted in Diamond Prolong mounting medium plus DAPI and coverslips sealed with nail polish.
Imaging
Confocal microscopy was performed at the University of Washington Keck Microscopy Center, on a Leica SP8X confocal microscope. 2-micron stacks were imaged at 63x magnification, using 6X averaging. Images were extracted from raw.lif files, maximum projected, channels merged, cropped, and pseudocolored using Fiji (Schindelin et al., 2012).
Identification of interbacterial effector–immunity gene homologues in genomes
Multi-alignments for 220 putative and validated effector and immunity proteins were acquired from the supplemental materials of Zhang et al. (Zhang et al., 2012). Amino acid seed sequences of interbacterial effector and immunity proteins were queried via tBLASTn against a custom database of 63 Spirochetes genomes acquired from RefSeq, or a database of 22 genomes for tick-associated endosymbionts and pathogens from the phylum Proteobacteria. All hits with e-values less than or equal to 10−3 across the full length of the seed sequence were considered to be homologues of interbacterial effector or immunity proteins. The distribution of effector and immunity genes per genome within each genus was calculated by normalizing the total number of BLAST hit by the number of genomes searched.
Author contributions
BDR, SC, and JDM conceived the study. BDR, SC, and JDM designed the study. BDR, SC, BH, and MCR conducted experimental work. XL, TJ, DN, and JB collected ticks. BDR, SC, and JDM wrote the paper. All authors read and approved the paper.
Data accessibility
Sequencing data was deposited at the NCBI SRA, BioProject accession # pending.
Author information
The authors declare that no competing financial information exists. Correspondence should be directed to bdross@uw.edu, seemay.chou@ucsf.edu, and mougous@uw.edu. 20
Acknowledgements
We thank the Fred Hutch Experimental Histopathology Core facility and Dan Long at Rocky Mountain Labs for tick sectioning and slide preparation. We thank Barbara Simon, Jim Ruppa, David Simon, and June Reznikoff for their indefatigable efforts in collection of ticks from the Klickitat River “Ant Ranch”, and Susan Little for collection of Amblyomma ticks. We thank the UW Keck Imaging Center for providing equipment and assistance in confocal microscopy and acknowledge its support from the NIH (S10 OD016240). We thank Mr. DNA for sequencing support. We are grateful to colleagues for careful review of the manuscript and members of the Mougous lab for helpful discussions. This work was supported by National Institutes of Health grant R21AI114923 (JDM), and the Burroughs Wellcome Fund (JDM). BDR was supported by a Simons Foundation-sponsored Life Sciences Research Foundation postdoctoral fellowship.