Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA

Abstract

Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi-aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water-borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P. bivittatus-specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles.

History, applications, methodological issues and perspectives for the use environmental DNA (eDNA) in marine and freshwater environments

“…Methodological papers trying to make approximations between fish biomass, abundance and eDNA detection probability were also published during the same time period by Dejean et al (2011) and Takahara, Minamoto, Yamanaka, Hideyuki & Kawabata (2012). Recently, studies have been focused on modeling persistence and detection using multiple biotic and abiotic factors such as vectors, system volume, sample volume, eDNA dynamics, stream flow, discharge and particle size (Piaggio, Engeman, Hopken, Humphrey, Keacher, Bruce, & Michael, 2013;Schmidt, Kery, Ursenbasher, Hyman, & Collins, 2013;Barnes, Turner, Jerde, Renshaw, Chadderton, & Lodge, 2014;Pilliod, Goldberg, Arkle, & Waits, 2014;Turner, Barnes, Charles, Jones, Xu, Jerde, & Lodge, 2014).…”

“…Distributions of organisms and biomass in freshwater systems have been correlated with eDNA concentration (Takahara et al, , 2013. Thus eDNA concentration has been used as a proxy for population distribution in amphibians (Ficetola et al, 2008;Goldberg et al, 2011), fishes (Mahon, Jerde, Galaska, Bergner, Chadderton, Lodge, Hunter, & Nico, 2012;Minamoto, Yamanaka, Takahara, Honjo, & Kawabata, 2012) and reptiles species (Piaggio et al, 2013). In marine species no studies have been conducted to determine the relationship between eDNA concentration and species distribution, abundance and biomass.…”

“…Persistence estimates have been determined for different taxonomic groups: from 15 to 30 days for fresh water fishes (Dejean et al, 2011& Takahara et al, 2012a, 15 to 30 days for amphibians (Ficetola et al, 2008;Goldberg et al, 2011), 0.9 to 7 days for marine mammals (Foote et al, 2012), 21 days for mudsnails (Goldberg, Sepulveda, Ray, Baumgardt, & Waits, 2013), and 14 days for reptiles (Piaggio et al, 2013). In addition to density, size and life history features of the target taxa, eDNA persistence can be influenced by other biotic factors such as bacterial and fungal concentrations (Dejean et al, 2011).…”

“…Leading causes of PCR inhibition include high salinity in marine systems (Foote et al, 2012) and humic/fluvic acids, tannins and polyphenols in freshwater environments (Matheson, Gurney, Esau, & Lehto, 2010). Thus, a DNA extraction kit that removes potential inhibitors is recommended along with appropriate PCR controls (Piaggio et al 2013). Specifically for marine environments eDNA detection is considered to be local due to the reduced persistence period (Foote et al, 2012;Thomsen et al 2012b).…”

Characterization, optimization, and validation of environmental DNA (eDNA) markers to detect an endangered aquatic mammal

“…The amount and geographical range of eDNA can thereby be used to infer species distributions and/or the conservation status of a population, and has been widely applied for ecological surveillance of rare, cryptic, or otherwise hard-to-study taxa, including endangered (Jerde et al 2011;Thomsen et al 2012b;Laramie et al 2015), endemic (Fukumoto et al 2015), and recently colonized invasive species Piaggio et al 2014;Dejean et al 2012;Hunter et al 2015). Research consensus to date illustrates eDNA as a sensitive, efficient, effective and noninvasive method (Rees et al 2014) well-suited for work in conservation management.…”