Influence of tick sex and geographic region on the microbiome of Dermacentor variabilis collected from dogs and cats across the United States

As tick-borne diseases continue to increase across North America, current research strives to understand how the tick microbiome may affect pathogen acquisition, maintenance, and transmission. Prior high throughput amplicon-based microbial diversity surveys of the widespread tick Dermacentor variabilis have suggested that life stage, sex, and geographic region may influence the composition of the tick microbiome. Here, adult D. variabilis ticks (n = 145) were collected from dogs and cats from 32 states with specimens originating from all four regions of the United States (West, Midwest, South, and Northeast), and the tick microbiome was examined via V4 -16S rRNA gene amplification and Illumina sequencing. A total of 481,246 bacterial sequences were obtained (median 2924 per sample, range 399-11,990). Fifty genera represented the majority (> 80%) of the sequences detected, with the genera Allofrancisella and Francisella being the most abundant. Further, 97%, 23%, and 5.5% of the ticks contained sequences belonging to Francisella spp., Rickettsia spp., and Coxiella spp., respectively. No Ehrlichia spp. or Anaplasma spp. were identified. Co-occurrence analysis, by way of correlation coefficients, between the top 50 most abundant genera demonstrated five strong positive and no strong negative correlation relationships. Geographic region had a consistent effect on species richness with ticks from the Northeast having a significantly greater level of richness. Alpha diversity patterns were dependent on tick sex, with males exhibiting higher levels of diversity, and geographical region, with higher level of diversity observed in ticks obtained from the Northeast, but not on tick host. Community structure, or beta diversity, of tick microbiome was impacted by tick sex and geographic location, with microbiomes of ticks from the western US exhibiting a distinct community structure when compared to those from the other three regions (Northeast, South, and Midwest). In total, LEfSe (Linear discriminant analysis Effect Size) identified 18 specific genera driving these observed patterns of di-versity and community structure. Collectively, these findings highlight the differences in bacterial diversity of D. variabilis across the US and supports the interpretation that tick sex and geographic region affects microbiome composition across a broad sampling distribution.