Using environmental DNA for detection of Batrachochytrium salamandrivorans in natural water

Rapid, early, and reliable detection of invasive pathogenic microorganisms is essential in order to either predict or delineate an outbreak, and monitor appropriate mitigation measures. The chytrid fungus Batrachochytrium salamandrivorans is expanding in Europe, and infection with this fungus may cause massive mortality in urodelans (salamanders and newts). In this study, we designed and validated species‐specific primers and a probe for detection of B. salamandrivorans in water. In a garden pond in close proximity to the B. salamandrivorans index site in the Netherlands, B. salamandrivorans‐infected newts had been detected in 2015 and have been monitored since. In 2016 and 2017, no B. salamandrivorans was detected at this site, but in 2018 B. salamandrivorans flared up in this isolated pond which allowed validation of the technique in situ. We here present the development of an environmental DNA technique that successfully detects B. salamandrivorans DNA in natural waterbodies even at low concentrations. This technique may be further validated to play a role in B. salamandrivorans range delineation and surveillance in both natural waterbodies and in captive collections

Environmental DNA for conservation

Biodiversity must be documented before it can be conserved. However, it may be difficult to document species with few individuals (Thompson, 2013; Goldberg et al., 2016), thus it requires a multitude of tools to detect species that occur in low numbers or are elusive (see the various chapters in this volume). One tool that has become useful for conservation efforts utilizes environmental DNA, which is DNA shed into the environment by organisms (eDNA; Taberlet et al., 2018). Typically this involves taking environmental samples such as soil, water, air, or using biological surrogates for sampling biodiversity (e.g. leeches, sponges, carrion flies, etc.; Schnell et al., 2012; Calvignac-Spencer et al., 2013; Lynggaard et al., 2019; Mariani et al., 2019) and using laboratory approaches to concentrate, isolate, and test for target DNA through polymerase chain reaction (PCR) amplification (Taberlet et al., 2018). The utilization of eDNA for species detection is part of a larger field of non-invasive DNA sampling, which more broadly includes collecting DNA passively from wildlife, through collection of faeces, saliva, feathers, hair, or other methods of sampling shed DNA. Environmental DNA has been used to document presence/absence of a target species (Ficetola et al., 2008a, 2008b; Himter et al., 2017) or to quantify relative abundance for biodiversity from varied environments such as the arctic (e.g. Leduc et al., 2019; Von Duyke et al., 2019), marine (e.g. Port et al., 2016; Jo et al, 2017; Stoeckle et al., 2018), freshwater (e.g. Lacoursi^re-Roussel et al., 2016; Doi et al., 2017), and tropical (e.g. Schnell et al., 2012; Gogarten et al, 2020) ecosystems. The application of this technology includes the detection of invasive species, pathogens (including DNA and RNA), species of conservation concern, and biodiversity (Acevedo-Whitehouse et al., 2010; Rees et al., 2014; Sakai et al, 2019). In this world -of \u27fast-paced technological advances, not all new methods prove useful in an applied context. Although eDNA has not been used regularly in biodiversity conservation for more than a decade, it has proven to be an extremely practical and informative tool. The utility of eDNA is supported by ongoing advancements and development of novel applications. There is no easy way to standardize the application or methods of eDNA as the conservation question, and the target system must drive the selection of a range of options at every step. However, guidelines now exist for the best practices of optimizing a sampling scheme and sample processing for eDNA applications (Goldberg et al., 2016; Jeunen et al., 2019; Klymus et al., 2020; The eDNA Society, 2019; Shu et al, 2020). Further, the ranks of experienced eDNA practitioners have expanded globally; thus, it is fairly easy to find expert consultation. Therefore, it is now practical and prudent to adopt eDNA in the service of biodiversity conservation efforts

Environmental DNA Detection of Invasive Species

Invasive species cause catastrophic changes to environments they are introduced into. Early detection offers the best chance at controlling the spread and mitigating any potential damages caused by the invaders. Environmental DNA (eDNA) has emerged as a more sensitive and cost effective alternative to traditional survey approaches to detection. In this study, I designed primer-probe sets for use in quantitative PCR detection of three invasive Centrarchid species, Largemouth Bass (Micropterus salmoides), Smallmouth Bass (Micropterus dolomieu) and Black Crappie (Pomoxis nigromaculatus). I surveyed 21 water bodies in Maine during two seasons (winter and spring). I designed and validated a sampling device and protocol for through-ice water sampling for eDNA. I detected target species in all lakes where they were known to be present as well as five previously unconfirmed lakes. Through hierarchical occupancy modeling I estimated the cumulative probabilities of presence, collection and detection of eDNA at three levels of the surveys (sites, samples, qPCR replicates). Although my toolsets were effective during both seasons, spring samples contained much higher concentrations of eDNA and hierarchical occupancy models showed this season to have much higher average power to detect target species than winter. Winter is still a viable season for sampling, providing fewer contamination concerns, and with a more robust sampling protocol, would be able to provide a high level of confidence of detection. Based on my dataset, and in order to have \u3e95% confidence of detection at each level of the survey for simple detection of presence/absence, I recommend sampling from a minimum of four sites per lake, taking three samples per site and conducting five qPCR replicates in spring and sampling from a minimum of seven sites per lake, taking five samples per site and conducting seven qPCR replicates in winter

Testing environmental DNA sampling and predictive modeling as means to investigate wood frog (Rana sylvatica) distribution in Alaska and Northern Canada

Thesis (M.S.) University of Alaska Fairbanks, 2017Global amphibian declines over the past 30+ years have led to a greater awareness of amphibian conservation issues. Few amphibian species occur in northern landscapes, however, and the species that do occur are widely dispersed and at the northern extent of their range. Accordingly, amphibian research is not prioritized in northern landscapes. Deficient monitoring practices have resulted in incomplete distribution knowledge that impedes the management of wood frogs (Rana sylvatica) in Alaska and northern Canada. I developed an environmental DNA detection assay to complement monitoring practices at the northern extent of the wood frog's range. This assay was tested to be species-specific, allowing it to be implemented in areas where wood frogs may co-occur with other amphibian species. It can detect wood frog DNA in environmental samples to a concentration of 1.83 x 10⁻³ pg/μL. I further demonstrate that environmental DNA occurrence data can be used to predict wood frog distribution in the Fairbanks North Star Borough. I combined environmental DNA occurrence data with environmental GIS data and analyzed the resulting dataset with machine learning algorithms to define an ecological niche for the wood frog. This niche, when extrapolated to the landscape, results in a species distribution model that attains 74% predictive accuracy. Lastly, I conducted an environmental DNA mega-transect survey along the Elliot/Dalton Highway corridor in Alaska. I combined the results of this survey with citizen science occurrence data from past and current monitoring projects to create a set of alternative occurrence data. This alternative data was combined with environmental GIS data and analyzed with machine learning algorithms to create a species distribution model that achieves 92% predictive accuracy across Alaska and the Yukon Territory, Canada. These results improve upon prior species distribution models developed for wood frogs in Alaska. They provide deeper insights into potential wood frog distribution at high latitudes and elevations in Alaska, where anecdotal observations have previously been recorded. Adoption and widespread use of an environmental DNA monitoring protocol in under-sampled regions of Alaska and northern Canada will generate larger datasets with wider geographic coverage, leading to models with even higher predictive accuracy. Alternative data, including that obtained from environmental DNA and citizen science monitoring, can boost efforts to further develop baseline knowledge of wood frog occurrence in these areas. Species distribution models generated in this research can help guide these efforts. Increasing knowledge of wood frog distribution may assist conservation managers to designate critical habitat, study climate impacts, and make more informed decisions regarding amphibians in northern landscapes.Chapter 1 Development, validation, and evaluation of an assay for the detection of wood frogs (Rana sylvatica) in environmental DNA -- Chapter 2 Application of environmental DNA-based occurrence data in modeling wood frog (Rana sylvatica) distribution in Interior Alaska -- Chapter 3 A reassessment of wood frog (Rana sylvatica) distribution in Alaska and northern Canada based on environmental DNA and citizen science -- Conclusion -- References -- Appendices

ranacapa: An R package and Shiny web app to explore environmental DNA data with exploratory statistics and interactive visualizations.

Environmental DNA (eDNA) metabarcoding is becoming a core tool in ecology and conservation biology, and is being used in a growing number of education, biodiversity monitoring, and public outreach programs in which professional research scientists engage community partners in primary research. Results from eDNA analyses can engage and educate natural resource managers, students, community scientists, and naturalists, but without significant training in bioinformatics, it can be difficult for this diverse audience to interact with eDNA results. Here we present the R package ranacapa, at the core of which is a Shiny web app that helps perform exploratory biodiversity analyses and visualizations of eDNA results. The app requires a taxonomy-by-sample matrix and a simple metadata file with descriptive information about each sample. The app enables users to explore the data with interactive figures and presents results from simple community ecology analyses. We demonstrate the value of ranacapa to two groups of community partners engaging with eDNA metabarcoding results

Phylogenetic classification of short environmental DNA fragments

Metagenomics is providing striking insights into the ecology of microbial communities. The recently developed massively parallel 454 pyrosequencing technique gives the opportunity to rapidly obtain metagenomic sequences at a low cost and without cloning bias. However, the phylogenetic analysis of the short reads produced represents a significant computational challenge. The phylogenetic algorithm CARMA for predicting the source organisms of environmental 454 reads is described. The algorithm searches for conserved Pfam domain and protein families in the unassembled reads of a sample. These gene fragments (environmental gene tags, EGTs), are classified into a higher-order taxonomy based on the reconstruction of a phylogenetic tree of each matching Pfam family. The method exhibits high accuracy for a wide range of taxonomic groups, and EGTs as short as 27 amino acids can be phylogenetically classified up to the rank of genus. The algorithm was applied in a comparative study of three aquatic microbial samples obtained by 454 pyrosequencing. Profound differences in the taxonomic composition of these samples could be clearly revealed