Abstract
Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.
Highlights
Environmental DNA is DNA extracted from environmental samples without first isolating the target organisms or their parts [1, 2]
In vitro, and in situ testing of the new bigheaded carp quantitative PCR (qPCR) assay showed no evidence of amplification from DNA of non-target species, and Sanger sequencing of in situ testing samples confirmed amplification of the targeted 100 bp D-loop mitochondrial DNA (mtDNA) amplicon from bigheaded carp (Table S1)
The standard operating procedure for Environmental DNA (eDNA)-based monitoring of bigheaded carp regularly detects H. molitrix eDNA, and occasionally H. nobilis eDNA, in surveillance of invasion pathways to the Great Lakes [27, 28], but in this study it failed to detect one H. molitrix and five H. nobilis in a 0.08 hectare pond (Table 2). This unexpected result suggests that bigheaded carp eDNA concentration is often higher in these invasion pathways than it was in the pond
Summary
Environmental DNA (eDNA) is DNA extracted from environmental samples (e.g., soil, water, air) without first isolating the target organisms or their parts [1, 2]. Macrobial eDNA is the DNA of large organisms such as animals or plants that occurs in environmental samples. Macrobial eDNA has been studied since 1991 in fields such as human forensics [4], agricultural transgenics [5], paleogenetics [6], and fecal pollution source tracking [7], it was only in 2008 that it was first used for aquatic macrofauna [8]. Aqueous macrobial eDNA has garnered particular interest [9, 10] as a simple and sensitive way to detect rare aquatic macrofauna such as invasive or endangered vertebrates and invertebrates [11,12,13,14,15,16,17,18,19,20]. Direct observation of rare organisms often has low detection probability [21], limited seasons [22], high costs [23], and increased risk of harming sensitive species [24]
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