EnvironmentalDNAsurveillance for invertebrate species: advantages and technical limitations to detect invasive crayfishProcambarus clarkiiin freshwater ponds

Abstract

Summary1. The introduction of non-native species is a major threat to biodiversity. While eradication programs of well-established invaders are costly and hazardous for non-target species, the early detection of a non-native species at low density is critical for preventing biological invasions in recipient ecosystems. Recent studies reveal that environmental DNA (eDNA) is a powerful tool for detecting target species in aquatic ecosystems, but these studies focus mostly on fish and amphibians. 2. We examine the reliability of using eDNA to detect the presence of an invasive freshwater crustacean species, the red swamp crayfish Procambarus clarkii. Species-specific primers and probes were designed; their specificity was tested using in silico PCR simulations and against tissues of other crayfish species. Limits of detection and quantification were specified for the target DNA sequence by means of quantitative PCR amplifications on dilution series of known amount of P. clarkii DNA. 3. The method was applied to water samples collected in 158 ponds in a French Nature Park, and results were compared to a traditional method using food-baited funnel traps. Environmental DNA had a better detection efficiency but predominantly led to divergent results compared with the trapping method. While habitat features partly explained the failure of crayfish detection by trapping, detection by eDNA was problematic at low crayfish abundances. When P. clarkii was detected, the estimated concentrations of crayfish DNA in water samples were always below the limit of quantification for the target DNA sequence. 4. Synthesis and applications. The combination of environmental DNA (eDNA) and conventional trapping methods is recommended to monitor the invasion by P. clarkii in small waterbodies such as ponds. However, the risk of mortality for non-target species, notably amphibians, has to be carefully evaluated before large-scale deployment of traps. Contrary to fish and amphibians, a low amount of extracellular DNA in water is suspected to be the major limitation for crayfish detection by molecular approaches. Current advancements in PCR technology, together with optimization of the water sampling method, promise upcoming developments of eDNA detection for aquatic invertebrate species.

The ecology of environmental DNA and implications for conservation genetics

“…The high-profile invasion of bigheaded carps Hypophthalmichthys molitrix and H. nobilis into waters of the midwestern United States (USA) has also represented a frequent target of eDNA surveillance efforts (Jerde et al 2011;Mahon et al 2013;Turner et al 2014b). Other eDNA surveillance targets in natural waters have included Bluegill Sunfish (Lepomis macrochirus) invading ponds in Japan (Takahara and Minamoto 2013), New Zealand mudsnails (Potamopyrgus antipodarum) in streams of Idaho, USA , PontoCaspian zebra mussels (Dreissena polymorpha) in the midwestern USA (Egan et al 2013), Louisiana crayfish (Procambarus clarkii) in France (Tréguier et al 2014), and African Jewelfish (Hemichromis letourneuxi) and Burmese pythons (Python bivittatus) in Florida, USA's ponds ) and wetlands (Piaggio et al 2014), respectively.…”

Improving herpetological surveys in eastern North America using the environmental DNA method

“…Yet, a number of eDNA studies have shown positive correlations between the eDNA concentrations obtained from quantitative (qPCR) analyses and population abundance indices (Lacoursière-Roussel et al 2016a, 2016bPilliod et al 2013;Takahara et al 2012;Thomsen et al 2012b;Wilcox et al 2013). However, the ability to quantify population abundance using eDNA is still debated (Iversen et al 2015;Roussel et al 2015;Tréguier et al 2014) and the relative sensitivity of both methods is largely unknown.…”

Ground-truthing Phylotype Assignments for Antarctic Invertebrates

“…With this study, we have complemented research that has compared metabarcoding markers (e.g. : [59,60]), focused on invertebrates [61][62][63] and provided 'ground-truthing' for replicated metabarcoding data by means of morphological data [62][63][64]. We also expand the range of studies investigating the effect of analysis parameters on metabarcoding data sets [35,60,65,66] including the quality of reference data [67,68].…”

“…Generally, retrieval of a completely overlapping species inventory between morphological and sequence-based approaches is difficult to achieve due to inherent biases of each approach [61][62][63]. This limitation somewhat constrains comparisons between sequence-based and morphology based taxonomy assignments as performed here.…”

“…Apart from Araeolaimida (Nematoda) and Adinetida (Rotifera), Antarctic phylotype data did not contain taxa detected also by visual inspection (morphology). These absences may be caused by (a) absences of target organisms in the sample, (b) incomplete/ imperfect DNA extraction, (c) poor PCR performance of markers, (d) inappropriate removal of reads during sequence processing or (e) incorrect taxonomy assignment due to lacking reference data [62,63,72]. Sub-samples of Antarctic soils used for sequence generation may have lacked taxa identified visually (a, above), but extraction of large soil quantities (400 g) performed here makes biased DNA extract composition (b, above) unlikely [73].…”