Microbiome Composition and Borrelia Detection in Ixodes scapularis Ticks at the Northwestern Edge of Their Range.

Lyme disease-causing Borrelia burgdorferi has been reported in 10-19% of Ixodes ticks from Alberta, Canada, where the tick vector Ixodes scapularis is at the northwestern edge of its range. However, the presence of Borrelia has not been verified independently, and the bacterial microbiome of these ticks has not been described. We performed 16S rRNA bacterial surveys on female I. scapularis from Alberta that were previously qPCR-tested in a Lyme disease surveillance program. Both 16S and qPCR methods were concordant for the presence of Borrelia. The 16S studies also provided a profile of associated bacteria that showed the microbiome of I. scapularis in Alberta was similar to other areas of North America. Ticks that were qPCR-positive for Borrelia had significantly greater bacterial diversity than Borrelia-negative ticks, on the basis of generalized linear model testing. This study adds value to ongoing tick surveillance and is a foundation for deeper understanding of tick microbial ecology and disease transmission in a region where I. scapularis range expansion, induced by climate and land use changes, is likely to have increasing public health implications.

Within-population diversity of bacterial microbiomes in winter ticks (Dermacentor albipictus).

The bacterial microbiome of ticks is notoriously diverse, but the factors leading to this diversity are poorly understood. We sequenced bacterial 16S rRNA amplicons from individual winter ticks, Dermacentor albipictus, to assess whether their one-host life cycle is associated with reduced bacterial diversity. On average, about 100 bacterial genera were found for individual ticks. Francisella-like endosymbiont (FLE) dominated bacterial communities, particularly in female ticks and in ticks that had fed. The remainder of the winter tick microbiome was highly variable. In addition to FLE, the main bacterial genera associated with winter ticks on elk were Pseudomonas, Ehrlichia, Asinibacterium, Acinetobacter and Streptococcus, although sequences associated with hundreds of other minor bacterial genera were detected. A complex interaction between richness and evenness was revealed in comparisons among tick life stages, using the Hill number series to show trends in diversity with decreasing emphasis on rare members of the assemblage. Male ticks had a significantly greater number of bacterial genera than females or nymphs, while males had greater evenness than females and similar evenness to nymphs. We intentionally sampled ticks from a single host species, North American elk, from a single location in Alberta, Canada, to constrain the ecological and blood meal variation that individuals experience through their life cycle. In spite of this, we found that the number of bacterial genera detected in this one-host tick system was remarkably diverse. The high taxonomic variability of the minor components of the winter tick microbiome suggests that this part of their microbiome diversity should be examined for functional significance.

Comparison of bacterial 16S rRNA variable regions for microbiome surveys of ticks.

Ticks vector diverse pathogenic bacteria that are important to identify in public health and veterinary contexts. Technological advances in high throughput sequencing have given an unprecedented opportunity to comprehensively characterize bacterial associates of ticks, but recent studies have used different 16S rRNA variable regions and sequence read lengths with little consideration of whether they reveal the same bacterial diversity. We compare the effectiveness of bacterial surveys using three library preparations across nine 16S variable regions and a set of 12 tick specimens (Acari: Ixodidae). We identify the bacterial assemblages present in extractions from wild-collected Ixodes scapularis from two regions of Canada, and provide the first microbiome survey for Ixodes angustus. Four bacterial families accounted for most diversity, with Rickettsiaceae being replaced as most common by Enterobacteriaceae or Pseudomonadaceae in some I. scapularis, and Francisellaceae being most abundant in I. angustus. The commercially available Ion 16S kit, based on 6 amplicons representing 16S regions V2, V3, V4, V67, V8 and V9, gave the most comprehensive estimates of bacterial families, with the Ion V4 amplicon generally giving the highest estimated diversity. Sequencing of the V4 amplicon by the MR DNA commercial service also provided cost effective assays of tick microbiomes that were within the range of results from the Ion 16S kit. Subtraction of the number of reads found in an extraction control sample lowered estimates of the number of bacterial families by approximately half. Our study shows that diversity patterns obtained from 16S microbiome surveys depend on the amplicon and protocol used, demonstrating that more than one marker region is needed to provide reliable inferences.