12/6/2023 0 Comments Coluber constrictor foxii mich2019), and as yet has not been as widely tested or used as qPCR. Digital PCR is relatively new to the eDNA toolkit, especially for snake eDNA detection ( Hunter et al. Over the last 10 years, quantitative PCR (qPCR) species-specific assays have increasingly been developed to assess occupancy of aquatic and terrestrial snake species, such as the invasive Burmese python ( Python bivittatus) or the threatened Louisiana pinesnake ( Pituophis ruthveni) ( Hunter et al. 2020), and detection of critical habitat for species at risk (e.g., Feng et al. 2021), occupancy modelling (e.g., Neto et al. 2022), comparing regional biodiversity (e.g., van der Heyde et al. 2021), assessing diet (e.g., Wanniarachchi et al. Opportunistic wild turkey predation has been suggested as a factor negatively affecting persistence of various species at risk on the island, including blue racers ( MacKinnon 2005) and other taxa of concern (e.g., small-mouthed salamanders, Ambystoma texanum) ( Environment and Climate Change Canada 2020).Įnvironmental DNA tools (e.g., quantitative PCR and metabarcoding) are increasingly used for a range of applications in conservation and ecology, including diagnosing the presence of pathogens (e.g., Boukerb et al. Wild turkeys were re-introduced to Pelee Island in 2002 ( Environment and Climate Change Canada 2020) and have since flourished despite being hunted. These factors are exacerbated by the possibility of snake fungal disease ( Ophidiomyces ophiodiicola) and wild turkey ( Meleagris gallopavo) predation ( COSEWIC 2012 Environment and Climate Change Canada 2017). Conservation efforts are challenging because of competing demands for land (e.g., cottages and homes), climate change, small population size, and isolation from their mainland counterparts in the USA, diminishing the probability of replenishment from southern populations. On Pelee Island, the species prefers habitats that include grasslands, alvars, and open woodlands ( Willson and Rouse 2002). Threats to the blue racer include habitat loss and degradation (e.g., of overwintering and nesting sites) and road mortality ( Willson and Rouse 2002). The blue racer is also listed as endangered under Ontario’s Endangered Species Act, 2007, and under Schedule 1 of the federal Species at Risk Act (SARA), 2002. The Committee on the Status of Endangered Wildlife in Canada (COSEWIC) designated the blue racer as endangered in 1991, and this status was retained in re-assessments in both 20. It occurs in northern USA (Ohio, Indiana, Illinois, Michigan, Wisconsin, and Iowa) but in Canada is restricted to the eastern two-thirds of Pelee Island (western Lake Erie, Ontario). The blue racer ( Coluber constrictor foxii (Baird and Girard, 1853)) is a large, non-venomous subspecies of the eastern racer, with a limited distribution in North America. Our assay should be effective for other environmental samples and can be used for investigating other factors negatively affecting blue racers, for example, helping to quantify blue racer habitat suitability and site occupancy. More faecal samples collected at strategic locations during snake peak activity on Pelee Island would enable a more thorough assessment of the possibility of turkey predation. We detected no racer DNA in any wild turkey faecal sample. Our assay is specific, can detect the target species at very low levels of concentration (0.002 copies/µL), and can accurately quantify copy numbers ≥ 0.26 copies/µL. As wild turkey predation has been suggested to negatively affect racers, we tested the assay on eight wild turkey faecal samples. We tested the assay in silico and in vitro using DNA of blue racers and co-occurring snake species and estimated the LOD and LOQ using synthetic DNA. We designed and evaluated the performance of an environmental DNA droplet digital PCR assay that can be used for multiple facets of conservation of this species. The species is threatened by multiple factors, including habitat degradation and loss, road mortality, persecution, and potentially predation. The only population of the endangered blue racer ( Coluber constrictor foxii) in Canada occurs on Pelee Island, Ontario.
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