Andrew R. Mahon

From molecules to management: Adopting DNA-based methods for monitoring biological invasions in aquatic environments ☆

Recent technological advances have driven rapid development of DNA-based methods designed to facilitate detection and monitoring of invasive species in aquatic environments. These tools promise to improve on traditional monitoring approaches by enhancing detection sensitivity, reducing analytical turnaround times and monitoring costs, and increasing specificity of target identifications. However, despite the promise of DNA-based monitoring methods, the adoption of these tools in decision-making frameworks remains challenging. Here, rather than explore technical aspects of method development, we examine impediments to effective translation of those methods into management contexts. In addition to surveying current use of DNA-based tools for aquatic invasive species monitoring, we explore potential sources of uncertainty associated with molecular technologies and possibilities for limiting that uncertainty and effectively communicating its implications for decision-making. We pay particular attention to the recent adoption of DNA-based methods for detection of invasive Asian carp species in the United States Great Lakes region, as this example illustrates many of the challenges associated with applying molecular tools to achieve desired management outcomes. Our goal is to provide a useful assessment of the obstacles associated with integrating DNA-based methods into aquatic invasive species management, and to offer recommendations for future efforts aimed at overcoming those obstacles.

Conservation in a cup of water: estimating biodiversity and population abundance from environmental DNA

Three mantras often guide species and ecosystem management: (i) for preventing invasions by harmful species, ‘early detection and rapid response’; (ii) for conserving imperilled native species, ‘protection of biodiversity hotspots’; and (iii) for assessing biosecurity risk, ‘an ounce of prevention equals a pound of cure.’ However, these and other management goals are elusive when traditional sampling tools (e.g. netting, traps, electrofishing, visual surveys) have poor detection limits, are too slow or are not feasible. One visionary solution is to use an organism’s DNA in the environment (eDNA), rather than the organism itself, as the target of detection. In this issue of Molecular Ecology, Thomsen et al. (2012) provide new evidence demonstrating the feasibility of this approach, showing that eDNA is an accurate indicator of the presence of an impressively diverse set of six aquatic or amphibious taxa including invertebrates, amphibians, a fish and a mammal in a wide range of freshwater habitats. They are also the first to demonstrate that the abundance of eDNA, as measured by qPCR, correlates positively with population abundance estimated with traditional tools. Finally, Thomsen et al. (2012) demonstrate that next‐generation sequencing of eDNA can quantify species richness. Overall, Thomsen et al. (2012) provide a revolutionary roadmap for using eDNA for detection of species, estimates of relative abundance and quantification of biodiversity.

Open‐ocean barriers to dispersal: a test case with the Antarctic Polar Front and the ribbon worm Parborlasia corrugatus (Nemertea: Lineidae)

Open‐ocean environments provide few obvious barriers to the dispersal of marine organisms. Major currents and/or environmental gradients potentially impede gene flow. One system hypothesized to form an open‐ocean dispersal barrier is the Antarctic Polar Front, an area characterized by marked temperature change, deep water, and the high‐flow Antarctic Circumpolar current. Despite these potential isolating factors, several invertebrate species occur in both regions, including the broadcast‐spawning nemertean worm Parborlasia corrugatus. To empirically test for the presence of an open‐ocean dispersal barrier, we sampled P. corrugatus and other nemerteans from southern South America, Antarctica, and the sub‐Antarctic islands. Diversity was assessed by analyzing mitochondrial 16S rRNA and cytochrome c oxidase subunit I sequence data with Bayesian inference and tcs haplotype network analysis. Appropriate neutrality tests were also employed. Although our results indicate a single well‐mixed lineage in Antarctica and the sub‐Antarctic, no evidence for recent gene flow was detected between this population and South American P. corrugatus. Thus, even though P. corrugatus can disperse over large geographical distances, physical oceanographic barriers (i.e. Antarctic Polar Front and Antarctic Circumpolar Current) between continents have likely restricted dispersal over evolutionary time. Genetic distances and haplotype network analysis between South American and Antarctic/sub‐Antarctic P. corrugatus suggest that these two populations are possibly two cryptic species.

Critical considerations for the application of environmental DNA methods to detect aquatic species

Summary Species detection using environmental DNA (eDNA) has tremendous potential for contributing to the understanding of the ecology and conservation of aquatic species. Detecting species using eDNA methods, rather than directly sampling the organisms, can reduce impacts on sensitive species and increase the power of field surveys for rare and elusive species. The sensitivity of eDNA methods, however, requires a heightened awareness and attention to quality assurance and quality control protocols. Additionally, the interpretation of eDNA data demands careful consideration of multiple factors. As eDNA methods have grown in application, diverse approaches have been implemented to address these issues. With interest in eDNA continuing to expand, supportive guidelines for undertaking eDNA studies are greatly needed. Environmental DNA researchers from around the world have collaborated to produce this set of guidelines and considerations for implementing eDNA methods to detect aquatic macroorganisms. Critical considerations for study design include preventing contamination in the field and the laboratory, choosing appropriate sample analysis methods, validating assays, testing for sample inhibition and following minimum reporting guidelines. Critical considerations for inference include temporal and spatial processes, limits of correlation of eDNA with abundance, uncertainty of positive and negative results, and potential sources of allochthonous DNA. We present a synthesis of knowledge at this stage for application of this new and powerful detection method.

Validation of eDNA surveillance sensitivity for detection of Asian carps in controlled and field experiments.

In many North American rivers, populations of multiple species of non-native cyprinid fishes are present, including black carp (Mylpharyngodon piceus), grass carp (Ctenopharyngodon idella), bighead carp (Hypophthalmichthys nobilis), silver carp (Hypophthalmichthys molitrix), common carp (Cyprinus carpio), and goldfish (Carassius auratus). All six of these species are found in the Mississippi River basin and tracking their invasion has proven difficult, particularly where abundance is low. Knowledge of the location of the invasion front is valuable to natural resource managers because future ecological and economic damages can be most effectively prevented when populations are low. To test the accuracy of environmental DNA (eDNA) as an early indicator of species occurrence and relative abundance, we applied eDNA technology to the six non-native cyprinid species putatively present in a 2.6 river mile stretch of the Chicago (IL, USA) canal system that was subsequently treated with piscicide. The proportion of water samples yielding positive detections increased with relative abundance of the six species, as indicated by the number of carcasses recovered after poisoning. New markers for black carp, grass carp, and a common carp/goldfish are reported and details of the marker testing to ensure specificity are provided.

Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding.

Freshwater fauna are particularly sensitive to environmental change and disturbance. Management agencies frequently use fish and amphibian biodiversity as indicators of ecosystem health and a way to prioritize and assess management strategies. Traditional aquatic bioassessment that relies on capture of organisms via nets, traps and electrofishing gear typically has low detection probabilities for rare species and can injure individuals of protected species. Our objective was to determine whether environmental DNA (eDNA) sampling and metabarcoding analysis can be used to accurately measure species diversity in aquatic assemblages with differing structures. We manipulated the density and relative abundance of eight fish and one amphibian species in replicated 206‐L mesocosms. Environmental DNA was filtered from water samples, and six mitochondrial gene fragments were Illumina‐sequenced to measure species diversity in each mesocosm. Metabarcoding detected all nine species in all treatment replicates. Additionally, we found a modest, but positive relationship between species abundance and sequencing read abundance. Our results illustrate the potential for eDNA sampling and metabarcoding approaches to improve quantification of aquatic species diversity in natural environments and point the way towards using eDNA metabarcoding as an index of macrofaunal species abundance.

Quantifying Environmental DNA Signals for Aquatic Invasive Species Across Multiple Detection Platforms

The use of molecular surveillance techniques has become popular among aquatic researchers and managers due to the improved sensitivity and efficiency compared to traditional sampling methods. Rapid expansion in the use of environmental DNA (eDNA), paired with the advancement of molecular technologies, has resulted in new detection platforms and techniques. In this study we present a comparison of three eDNA surveillance platforms: traditional polymerase chain reaction (PCR), quantitative PCR (qPCR), and digital droplet PCR (ddPCR) in which water samples were collected over a 24 h time period from mesocosm experiments containing a population gradient of invasive species densities. All platforms reliably detected the presence of DNA, even at low target organism densities within the first hour. The two quantitative platforms (qPCR and ddPCR) produced similar estimates of DNA concentrations. The analyses completed with ddPCR was faster from sample collection through analyses and cost approximately half the expenditure of qPCR. Although a new platform for eDNA surveillance of aquatic species, ddPCR was consistent with more commonly used qPCR and a cost-effective means of estimating DNA concentrations. Use of ddPCR by researchers and managers should be considered in future eDNA surveillance applications.

Shear and AC Field Enhanced Carbon Nanotube Impedance Assay for Rapid, Sensitive, and Mismatch-Discriminating DNA Hybridization.

Other than concentrating the target molecules at the sensor location, we demonstrate two distinct new advantages of an open-flow impedance-sensing platform for DNA hybridization on carbon nanotube (CNT) surface in the presence of a high-frequency AC electric field. The shear-enhanced DNA and ion transport rate to the CNT surface decouples the parasitic double-layer AC impedance signal from the charge-transfer signal due to DNA hybridization. The flow field at high AC frequency also amplifies the charge-transfer rate across the hybridized CNT and provides shear-enhanced discrimination between DNA from targeted species and a closely related congeneric species with three nucleotide mismatches out of 26 bases in a targeted attachment region. This allows sensitive detection of hybridization events in less than 20 min with picomolar target DNA concentrations in a label-free CNT-based microfluidic detection platform.

Evolutionary history of Southern Ocean Odontaster sea star species (Odontasteridae; Asteroidea)

We investigated the recent evolutionary history of demersal sea stars in the genus Odontaster throughout the Western Antarctic waters and on the South American shelf. The mitochondrial 16S ribosomal and cytochrome c oxidase subunit I (COI) genes were sequenced from adult and larval specimens. TCS parsimony network analysis and Bayesian inference were used to examine evolutionary history. Hierarchical AMOVA and mitochondrial DNA diversity statistics were also computed. Additionally, morphological characters were used. In assessing O. validus, we discovered morphological and range descriptions of Odontaster species to be inaccurate and include other Odontaster species in the Southern Ocean. We found O. meridionalis on both sides of the Antarctic circumpolar current (ACC) and Antarctic polar front (APF), whereas O. validus and O. penicillatus do not appear to have permeated these oceanographic features. Additionally, we discovered previously unrecognized species of Odontaster. Subsequent examination revealed diagnostic morphological differences in the number of spinelets on the abactinal and actinal plates. Mitochondrial characterization of Odontaster species suggests their recent history has been influenced by the APF and ACC in different ways. With the exception of O. meridionalis, Odontaster species are restricted to either side of the Drake Passage. O. validus shows genetic connectivity throughout sampled Antarctic waters.

Rapid on-chip genetic detection microfluidic platform for real world applications

The development of genetic detection protocols for field applications is an important aspect of modern medical diagnostic technology and environmental monitoring. In this paper, we report a rapid, portable, and inexpensive DNA hybridization technique using a bead-based microfluidic platform that functions by passing fluorescently labeled target DNA through a chamber packed with functionalized beads within a microfluidic channel. DNA hybridization is then assessed using a digital camera attached to a Clare Chemical DR-45M dark reader non-UV transilluminator that uses visible light as an excitation source and a blue and amber filter to reveal fluorescence. This microfluidic approach significantly enhances hybridization by reducing the diffusion time between target DNA and the silica surface. The use of probe-functionalized beads as solid support also enhances the sensitivity and limit of detection due to a larger surface area per unit volume. This platform could be adapted for use in medical applications and environmental monitoring, including the detection of harmful organisms in the ballast water of ships.