Summary
Ticks in the family Ixodidae are important vectors of zoonoses, including Lyme disease (LD), which is caused by spirochete bacteria from the Borreliella (Borrelia) burgdorferi sensu lato complex. The blacklegged tick (Ixodes scapularis) continues to expand across Canada, creating hot spots of elevated LD risk at the leading edge of its expanding range. Current efforts to understand the risk of pathogen transmission associated with I. scapularis in Canada focus primarily on targeted screens, while natural variation in the tick microbiome remains poorly understood. Using multiomics consisting of 16S metabarcoding and ribosome-depleted, whole-shotgun RNA transcriptome sequencing, we examined the microbial communities associated with adult I. scapularis (n = 32), sampled from four tissue types (whole tick, salivary glands, midgut, and viscera) and three geographical locations within a LD hot spot near Kingston, Ontario, Canada. The communities consisted of both endosymbiotic and known or potentially pathogenic microbes, including RNA viruses, bacteria, and a Babesia sp. intracellular parasite. We show that β-diversity is significantly higher between the bacterial communities of individual tick salivary glands and midguts than that of whole ticks. Linear discriminant analysis effect size (LEfSe) determined that the three potentially pathogenic bacteria detected by V4 16S rRNA sequencing also differed among dissected tissues only, including a Borrelia strain from the B. burgdorferi sensu lato complex, Borrelia miyamotoi, and Anaplasma phagocytophilum. Importantly, we find coinfection of I. scapularis by multiple microbes, in contrast to diagnostic protocols for LD, which typically focus on infection from a single pathogen of interest (B. burgdorferi sensu stricto).
IMPORTANCE As a vector of human health concern, blacklegged ticks (Ixodes scapularis) transmit pathogens that cause tick-borne diseases (TBDs), including Lyme disease (LD). Several hot spots of elevated LD risk have emerged across Canada as I. scapularis expands its range. Focusing on a hot spot in southeastern Ontario, we used high-throughput sequencing to characterize the microbiome of whole ticks and dissected salivary glands and midguts. Compared with whole ticks, salivary glands and midguts were more diverse and associated with distinct bacterial communities that are less dominated by Rickettsia endosymbiont bacteria and are enriched for pathogenic bacteria, including a B. burgdorferi sensu lato-associated Borrelia sp., Borrelia miyamotoi, and Anaplasma phagocytophilum. We also found evidence of coinfection of I. scapularis by multiple pathogens. Overall, our study highlights the challenges and opportunities associated with the surveillance of the microbiome of I. scapularis for pathogen detection using metabarcoding and metatranscriptome approaches.
Methodology
Tick collection and nucleic acid extraction.
Adult I. scapularis were collected at the Lemoine Point (LP) Conservation Area (44°13′, 56.228″N; 76°36′, 45.795″W) within the municipality of Kingston Ontario, Murphy’s Point (MB) Provincial Park (44°46′, 54.7098′N; 76°14′, 30.1122′W), and Queen’s University Biological Station (QB) (44°34′, 4.6524′N; 76°19′, 57.0966′W). Samples were collected from the QB site in 2016 and from all three of the sites in 2017. Questing adults were captured by flagging with 1-m2 white flannel fabric attached to an aluminum bar along a transect of 25 m, checking for attached ticks at 5-m intervals. Ticks were removed by hand or with forceps, placed in 2-mL screwcap tubes containing 70% ethanol, and later stored at −20 or −80°C. Prior to extraction, ticks were submerged in a 1% bleach solution for 30 s and rinsed with lab-grade water. Different extraction methods were used for the DNA of internal tissue, DNA of whole ticks, and RNA of whole ticks, as follows.
We dissected 12 female ticks to remove internal tissue from the exoskeleton and to separate the salivary glands and midgut from the remaining internal viscera for separate extraction and sequencing. The midgut and salivary glands were isolated successfully from 10 ticks. Internal tissues were macerated separately in 50 μL of extraction buffer containing 1 mM EDTA, 25 mM NaCl, 10 mM Tris-HCl (pH 8.0), and 200 μg mL−1 proteinase K (VWR). The DNA was purified using solid-phase reversible immobilization (SPRI) beads at 2.5× volume and resuspended in 20 μL laboratory-grade water.
In addition to the dissected ticks, we extracted DNA from 17 whole female ticks. Following sterilization as above, these ticks were dried at room temperature, frozen to −80°C, and pulverized in a Next Advance Bullet Blender Storm 24 device at 100 Hz in 2-mL tubes containing equal volumes of 0.2- and 0.5-mm low-binding ZrO beads (SPEX) for 3 to 4 min, refreezing every 1 min until no large visible fragments remained. Next, each sample was incubated with 500 μL of preheated cetyltrimethylammonium bromide (CTAB) buffer (100 mM Tris-HCl [pH 8.0], 25 mM EDTA, 1.5 M NaCl, 3% CTAB, 1% polyvinylpyrrolidone, and 1% β-mercaptoethanol) at 62°C for 30 min. To isolate the DNA, 500 μL of chloroform was added, the samples were centrifuged at 1,300 × g for 15 min, and then the supernatant was transferred to a new microcentrifuge tube. The DNA was precipitated in 1 mL prechilled 100% ethanol, mixed by inversion, and then incubated at −20°C for 30 min. The DNA was pelleted by centrifugation at ~21,000 × g for 15 min, washed twice with 1 mL 75% ethanol, and resuspended in 15 μL laboratory-grade water.
We isolated RNA from one male and three female ticks using the same sterilizing and pulverizing steps as those for the whole-tick DNA protocol above but with 500 μL TRIzol reagent (Invitrogen) following the manufacturer’s protocol and resuspending purified RNA in 15 μL lab-grade water.