eDNA Detection Research Articles

Estimating eDNA shedding and decay rates for muskellunge in early stages of development

AbstractEstimating population size and species distribution is essential for fisheries management and conservation. Traditionally, estimates rely on live‐capture and visual surveys; however, these approaches are challenging for low density or elusive species and sensitive habitats. Environmental DNA (eDNA) has shown potential to improve fisheries management, offering a sensitive tool for species detection while reducing some of the unintended harm, uncertainties, and cost of traditional approaches. For eDNA to be incorporated into quantitative population estimates, variability in factors such as DNA shedding and decay must be understood. We assess shedding and decay rates for three developmental stages of muskellunge (Esox masquinongy), a recreational species of social and economic importance and an apex predator of conservation priority in the St. Lawrence River. By housing fish at different biomass levels, we attempt to assess how crowding, and underlying behavioral and/or metabolic responses, affects shedding and decay. Additionally, we collected water from spawning bays and compared eDNA detections to live‐capture data. Total eDNA shedding rates for muskellunge were similar to values reported in previous studies of freshwater fishes and ranged from 9.92 × 103 copies/h/fish for larvae to 1.32 × 106 copies/h/fish for juveniles. Adjusting shedding rates for fish mass revealed no significant difference between larvae and juveniles. eDNA decay rates varied between life stages and experimental aquaria, with coefficients ranging from 0.064 to 0.259. We were unable to detect eDNA from hardened embryos even at high density. Muskellunge DNA was quantified in over 27% of samples from spawning bays, including bays where muskellunge were not captured with traditional approaches. The recovery of muskellunge eDNA in quantifiable levels, often in the absence of live capture, highlights the potential for eDNA approaches to supplement muskellunge monitoring and management. eDNA shedding and decay rates estimated here may also aid in interpretation of future data.

A Review of Next-Generation Wildlife Monitoring using Environmental DNA (eDNA) Detection and Next-Generation Sequencing in Malaysia

The use of environmental DNA (eDNA) as the genetic tool to monitor biodiversity has been increasing across the world, including Malaysia. Over a decade, the technique has become widely used in wildlife conservation with the technologies called next-generation sequencing (NGS). Unfortunately, as one of the top megadiverse countries, Malaysia is still behind in this field since eDNA methods outperform traditional surveys. Hence, in this study, we analyzed the paper related to eDNA studies in Malaysia, which focused on wildlife monitoring. We conducted a systematic bibliographic search and quantitative review of studies published before June 2021 from Google Scholar and Scopus database. Forty published eDNA studies were found, and each paper was classified based on five themes (species identification, diet assessment, health monitoring, resolve taxonomic, whole-genome sequencing) according to the study analysis. This study aims to identify gaps in eDNA in Malaysia, which can later be useful for future conservation actions and development by applying technology in wildlife monitoring.

Two for the price of one: eDNA metabarcoding reveals temporal and spatial variability of mussel and fish co‐distributions in Michigan riverine systems

AbstractFreshwater mussels (family Unionidae) are among the world's most endangered taxa, with almost 75% of North American taxa classified as a species of concern, threatened, or endangered. Despite the critical importance of comprehensive distributional data for the conservation of unionids and fishes, these data are often lacking because of the labor and resources associated with traditional survey methods. During their larval stage, unionid mussels use various fish species as obligate hosts, making native fish species vital to unionid persistence and an understanding of host distribution similarly important. Here, we utilized an eDNA metabarcoding approach to evaluate patterns of co‐distribution of unionid mussels and fishes along ~362 km of the densely sampled Grand River network as well as the outlets of 19 tributaries along the eastern shore of Lake Michigan, USA. We detected a total of 21 mussel and 40 fish taxa, with distinctive composition of both mussel and fish assemblages across tributaries and differences in fish taxa between sampling periods. Notably, we detected more mussel taxa within the Grand River watershed than at the outlets of all 20 rivers combined. Within the Grand River network, two fish taxa (Pylodictus olivaris and Cyprinella) were found more frequently in areas of high mussel diversity, and three fish taxa more frequently in areas of low mussel diversity (Umbra, Leuciscidae, and Etheostoma). There was little difference between eDNA detections of mussels from samples collected in June versus August, but we detected significantly more fish taxa in August compared to June. Taken together, our findings demonstrate the value of eDNA metabarcoding for evaluating co‐distribution of ecologically connected taxa. The use of eDNA as a tool for determining distributions of mussels and their obligate hosts may facilitate conservation efforts for these imperiled taxa.

Assessing the breeding phenology of a threatened frog species using eDNA and automatic acoustic monitoring.

Climate change has driven shifts in breeding phenology of many amphibians, causing phenological mismatches (e.g., predator-prey interactions), and potentially population declines. Collecting data with high spatiotemporal sensitivity on hibernation emergence and breeding times can inform conservation best practices. However, monitoring the phenology of amphibians can be challenging because of their cryptic nature over much of their life cycle. Moreover, most salamanders and caecilians do not produce conspicuous breeding calls like frogs and toads do, presenting additional monitoring challenges. In this study, we designed and evaluated the performance of an environmental DNA (eDNA) droplet digital PCR (ddPCR) assay as a non-invasive tool to assess the breeding phenology of a Western Chorus Frog population (Pseudacris maculata mitotype) in Eastern Ontario and compared eDNA detection patterns to hourly automatic acoustic monitoring. For two eDNA samples with strong PCR inhibition, we tested three methods to diminish the effect of inhibitors: diluting eDNA samples, adding bovine serum albumin to PCR reactions, and purifying eDNA using a commercial clean-up kit. We recorded the first male calling when the focal marsh was still largely frozen. Chorus frog eDNA was detected on April 6th, 6 days after acoustic monitoring revealed this first calling male, but only 2 days after males attained higher chorus activity. eDNA signals were detected at more sampling locales within the marsh and eDNA concentrations increased as more males participated in the chorus, suggesting that eDNA may be a reasonable proxy for calling assemblage size. Internal positive control revealed strong inhibition in some samples, limiting detection probability and quantification accuracy in ddPCR. We found diluting samples was the most effective in reducing inhibition and improving eDNA quantification. Altogether, our results showed that eDNA ddPCR signals lagged behind male chorusing by a few days; thus, acoustic monitoring is preferable if the desire is to document the onset of male chorusing. However, eDNA may be an effective, non-invasive monitoring tool for amphibians that do not call and may provide a useful complement to automated acoustic recording. We found inhibition patterns were heterogeneous across time and space and we demonstrate that an internal positive control should always be included to assess inhibition for eDNA ddPCR signal interpretations.

Environmental DNA survey to detect an endemic cryptic fish, Anablepsoides cryptocallus, in tropical freshwater streams

Abstract Martinique island is a biodiversity hotspot in the Lesser Antilles that harbours many endemic species. One of these, Anablepsoides cryptocallus, also known as ‘Poisson gale’, is the only endemic freshwater fish of Martinique and a species with a poorly understood distribution range. In this study, an environmental DNA (eDNA) detection protocol was developed, validated, and optimized, targeting a short fragment (125 bp) of the A. cryptocallus COI mitochondrial gene, to investigate the presence of this species in Martinique. Fifty‐seven sites spread over 43 permanent rivers and two wetlands were sampled using both eDNA and conventional fishing (dip net capture). Presence was confirmed in 27 sites using eDNA detection, and in nine sites by dip net fishing. eDNA‐based detection of A. cryptocallus was effective and less time‐consuming than conventional fishing, making it a relevant tool for future studies throughout the island. Even though A. cryptocallus was found to be present in a significant number of sites, many sites previously known for this species were found to be negative, highlighting the need for continuous monitoring. There is now an urgent need to propose protection status for this endemic species to preserve its preferential habitats, as these are being increasingly threatened by human activities that are leading to habitat loss and fragmentation.

DNA Barcoding of Trichobilharzia (Trematoda: Schistosomatidae) Species and Their Detection in eDNA Water Samples

We designed and tested species-specific PCR primers to detect Trichobilharzia species via environmental DNA (eDNA) barcoding in selected Austrian water bodies. Tests were performed with eDNA samples from the field as well as with artificial samples from the lab, where snails releasing cercariae were kept in aquariums. From two localities, Trichobilharzia was documented based on the release of cercariae from snails, enabling morphological species identification. In both cases, the corresponding species were detected via eDNA: Trichobilharzia szidati and Trichobilharzia physellae. Nonetheless, the stochasticity was high in the replicates. PCR tests with aquarium water into which the cercariae had been released allowed eDNA detection even after 44 days. As in the PCRs with eDNA samples from the field, positive results of these experiments were not obtained for all samples and replicates. PCR sensitivity tests with dilution series of T. szidati genomic DNA as well as of PCR amplification products yielded successful amplification down to concentrations of 0.83 pg/µL and 0.008 pg/µL, respectively. Our results indicate that the presumed species specificity of PCR primers may not be guaranteed, even if primers were designed for specific species. This entails misidentification risks, particularly in areas with incomplete species inventories.

Comparison of droplet digital PCR and real-time quantitative PCR for quantitative detection of the parasitic ciliate Ichthyophthirius multifiliis in the water environment.

Ichthyophthiriasis, caused by the parasitic ciliate Ichthyophthirius multifiliis (Ich), is considered one of the most harmful diseases affecting freshwater fish globally. It can cause mass mortalities of fish in intensive farming systems. In such systems, it is thus necessary to detect and quantify the number of Ich in the water so that control measures can be implemented before Ichthyophthiriasis breaks out. In recent years, molecular diagnostic methods have become increasingly important in aquaculture. Real-time quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) have become robust assays for detecting pathogens. In this study, a set of specific primers and a TaqMan-minor groove binder probe targeting the small-subunit rDNA (SSU rDNA) of Ich were developed. They were used in qPCR and ddPCR assays to compare the performance of these two different methods in quantitatively detecting Ich. After optimizing the reaction conditions, both qPCR and ddPCR assays were found to have high linearity and quantitative correlations for standard plasmid DNA. When used for the detection of Ich eDNA in water samples, the qPCR assay had a wider detection range, making it a suitable method to screen for the prevalence of Ichthyophthiriasis. However, the ddPCR approach had higher sensitivity, which would help provide advance notice of the disease in complex water environmental samples.

Environmental DNA as a detection tool for small-bodied, cryptic, threatened fish in a highly turbid freshwater lake system

Context Where conservation efforts are undertaken to decrease downward trends in fish populations, comparatively few resources are directed to small-bodied cryptic species. The true extent of the decline of many of these species is therefore often unknown. Where surveys have occurred, they are frequently limited by budget and hence effort. Consequently, there is a risk that rare species may not be physically captured despite their presence at a site. Such an outcome has dire consequences for the conservation of remnant populations of threatened fish. To counter possible false negative detections, environmental DNA is increasingly being used in conjunction with, or as a precursor to, physical surveys. The Southern Purple-spotted Gudgeon (Mogurnda adspersa) is a small, threatened freshwater fish native to Australia. Recent surveys captured M. adspersa in two highly turbid waterbodies in north-central Victoria. This capture represented the first detection of the species in the state in more than 20 years. Because these waterbodies are part of a network of hydrologically connected systems, it was suspected that the species likely had a broader distribution in the region. Aims To develop a probe-based eDNA assay for M. adspersa and compare its sensitivity against a physical sampling program. Methods Detection (presence/absence) between eDNA and traditional surveys was compared across multiple sites. Key results eDNA presents an effective tool for determining the presence of M. adspersa. The eDNA survey demonstrated significant clustering of eDNA detections towards the outlets of lakes sampled, suggesting concentrated eDNA at this point, or that, due to the channels being shallower, the eDNA of resident individuals may be less diluted. Conclusions Based on these results, future survey of rare, cryptic species in highly turbid lake systems should in the first instance include a broad scale eDNA survey, with sampling concentrated at outlet channels. Implications The likely most cost-effective approach to determining the presence/absence of rare species in lake systems is the collection of eDNA samples at outlet channels.

Monitoring threatened species with environmental DNA and open ecological data: Local distribution and habitat preferences of scalloped hammerhead sharks (Sphyrna lewini)

Evidence-based environmental management is urgently required to enable progress towards global conservation commitments. To fill large-scale biodiversity knowledge gaps, governments, organisations and communities must adopt innovative ecological monitoring techniques. The emergence of environmental DNA (eDNA) survey methods, as well as the increased availability of in situ ocean observation and satellite remote sensing data, offers opportunities to overcome significant logistic and financial challenges previously associated with traditional biodiversity monitoring. Here, a 1.5 year-long eDNA survey reveals the spatial and temporal presence of ‘critically endangered’ scalloped hammerhead sharks (Sphyrna lewini) in Apra Harbor, Guam. The survey confirms the presence of S. lewini in the region, and shows eDNA detections are most frequent at the beginning of the dry season, in January. Combined analysis of eDNA results with open (freely available) ecological data identifies that S. lewini presence is associated with increases in water velocity, turbidity and wind direction, as well as decreases in temperature and wind speed. Additionally, the data indicate S. lewini are most frequent at sampling sites characterised by high turbidity, shallow depths, and large distances from the harbor entrance. Information on local distribution and habitat preferences generated here is fundamental to the establishment of management protocols that aim to minimise negative anthropogenic impacts on S. lewini in Apra Harbor. The work provides an example of contemporary marine monitoring for the efficient generation of biodiversity data; an approach that will enable informed management decisions and help to reduce the risk and rate of species extinctions throughout the next decade.

Environmental DNA: State-of-the-art of its application for fisheries assessment in marine environments

Fisheries management involves a broad and complex set of tasks that are necessary to prevent overfishing and to help the recovery of overfished stock. Monitoring fishing activities based on two main sources, landings data and scientific surveys, is a challenging task. Fisheries collection data is often limited, which compromises the accuracy of the results obtained. Therefore, several emerging applications of molecular methods have the potential to provide unique understanding of ecological processes in marine environments and to build stronger empirical underpinnings for the Ecosystem-Based Fisheries Management. Environmental DNA (eDNA) is a complex mixture of genetic material shed by those organisms that inhabit a given environment, whereby DNA is extracted from an environmental sample without accessing the target organism. eDNA studies can be categorized into two main approaches, i) eDNA metabarcoding or semi-targeted (community) approaches and ii) species-specific or targeted approaches (single). Although both categories are often discussed, they differ drastically in their methodology, interpretations and accuracy. Both approaches involve a series of steps that include eDNA capture, preservation, extraction and amplification. This detection will depend on the affinity to the targeted taxa sequences and completeness and accuracy of DNA reference collection databases. The eDNA method applied in marine environments are probably the most challenging aquatic environments for applying this technique. This is because of the extreme relationship between water-volume to biomass, dynamics and the physical and chemical properties of seawater that affect dispersion, dilution and preservation. Here, we review the present application of this novel method in fishery assessment in marine environments. To date, many studies suggest that this method offers the potential to revolutionize fisheries monitoring, which will contribute to improving the range of tasks involved in fisheries management. The compelling conclusion is that the methodological steps including in eDNA surveys should be standardized and that research efforts should focus on developing appropriately validated tests to address environmental and sampling factors that may affect eDNA detection in marine environments in order to draw reliable conclusions. This bioassessment tool can assist fisheries professionals in achieve their research, management, and conservation objectives, but not as a replacement for time-proven assessment methods.

Validation of a Portable eDNA Detection Kit for Invasive Carps

Loop-mediated isothermal amplification (LAMP) is a rapid molecular detection technique that has been used as a diagnostic tool for detecting human and animal pathogens for over 20 years and is promising for detecting environmental DNA shed by invasive species. We designed a LAMP assay to detect the invasive carps, silver carp (Hypophthalmichthys molitrix), bighead carp (Hypophthalmichthys nobilis), black carp (Mylopharyngodon piceus), and grass carp (Ctenopharyngodon idella). To determine the sensitivity of the LAMP assay, we determined limit of detection (LOD) for each invasive carp species and compared with the performance of a grass carp quantitative PCR (qPCR) assay in LOD and in a mesocosm study. We used two grass carp densities, 3 juvenile grass carp in one mesocosm and 33 juvenile grass carp in the other. Prior to adding grass carp to the mesocosms, we added 68 kg of fathead minnows (Pimephales promelas) to each mesocosm to simulate farm ponds used for raising bait fish. We filtered 500 mL of water per sample to compare LAMP and qPCR analysis, and we collected 50 mL grab samples that were only analyzed using qPCR to gain additional data using a higher-throughput method to monitor environmental DNA (eDNA) levels throughout the study period. No eDNA for any of the four invasive carp species was detected in water collected from the mesocosms during the three days prior to adding grass carp. Forty-eight hours after grass carp addition to mesocosms, we detected grass carp eDNA in the mesocosm containing 33 grass carp using the LAMP assay. However, we failed to detect any grass carp DNA in the mesocosm containing 3 grass carp with the LAMP assay throughout the study. We analyzed the data using an occupancy model and found that the 500 mL filter samples yielded a higher eDNA capture probability than 50 mL grab samples in the mesocosm containing three grass carp but had similar eDNA capture probability in the mesocosm containing 33 grass carp. Both LAMP and qPCR reliably detected grass carp eDNA 2 days after grass carp addition, but detections were more consistent with qPCR. The LAMP assay may have utility for certain niche uses because it can be used to rapidly analyze eDNA samples and is robust to inhibition, despite having some limitations.

Detection of brook trout in spatiotemporally separate locations using validated eDNA technology

Brook trout are a species of conservation concern in Southwestern Ontario, Canada, and effective monitoring of their populations is crucial for making informed management decisions. Electrofishing is a traditional, yet invasive, method that allows for fish abundance estimation. Environmental DNA (eDNA) is an emerging molecular tool that presents a non-invasive alternative to electrofishing. This study was a collaborative effort between researchers in academia, industry, and an NGO, with the following objectives: 1) compare eDNA detections with electrofishing when monitoring brook trout populations in a site of known occupancy, 2) compare existing eDNA collection methods, and 3) extend eDNA surveys to regions of unknown occupancy that could be of conservation concern (Hanlon Creek and Twelve Mile Creek, Ontario). First, eDNA sampling methods were validated with standard electrofishing. Water samples were filtered in tandem at each site using two commercially available eDNA samplers. The results suggest a significant difference in total eDNA capture and incidence of PCR inhibitors between the two autosamplers. Brook trout eDNA was detected at all locations in Hanlon Creek in September and November, as well as 5/6 sampling locations in Twelve Mile Creek. Brook trout signal in Hanlon Creek was stronger in November compared to September 2019, suggesting possible spawning activity. Brook trout eDNA was also detected in Twelve Mile Creek where brook trout were previously unreported. This study provides a technical validation for the use of eDNA in brook trout monitoring and illustrates the opportunity to use eDNA surveys in regulated settings to complement and improve conventional biomonitoring methods for the management of elusive species.

Net overboard: Comparing marine eDNA sampling methodologies at sea to unravel marine biodiversity.

Environmental DNA (eDNA) analyses are powerful for describing marine biodiversity but must be optimized for their effective use in routine monitoring. To maximize eDNA detection probabilities of sparsely distributed populations, water samples are usually concentrated from larger volumes and filtered using fine-pore membranes, often a significant cost-time bottleneck in the workflow. This study aimed to streamline eDNA sampling by investigating plankton net versus bucket sampling, direct versus sequential filtration including self-preserving filters. Biodiversity was assessed using metabarcoding of the small ribosomal subunit (18S rRNA) and mitochondrial cytochrome c oxidase I (COI) genes. Multispecies detection probabilities were estimated for each workflow using a probabilistic occupancy modelling approach. Significant workflow-related differences in biodiversity metrics were reported. Highest amplicon sequence variant (ASV) richness was attained by the bucket sampling combined with self-preserving filters, comprising a large portion of microplankton. Less diversity but more metazoan taxa were captured in the net samples combined with 5 μm pore size filters. Prefiltered 1.2 μm samples yielded few or no unique ASVs. The highest average (~32%) metazoan detection probabilities in the 5 μm pore size net samples confirmed the effectiveness of preconcentration plankton for biodiversity screening. These results contribute to streamlining eDNA sampling protocols for uptake and implementation in marine biodiversity research and surveillance.

Experimental assessment of downstream environmental DNA patterns under variable fish biomass and river discharge rates

AbstractThe development of environmental DNA (eDNA) methods toward implementation as a cost‐effective, nonlethal tool for fish biomonitoring in lotic environments requires insights on the temporal and spatial distribution of eDNA in river systems. Yet, little is known on how downstream eDNA dispersal is affected by the combination of river discharge and source biomass effects. In this study, we aimed at unraveling the effect of source‐ and system‐specific processes on the stream reach of eDNA. We used a longitudinal cage study in two river sections characterized by a significantly different discharge rate, where two invasive and two native fish species were introduced under two contrasting biomass treatments. Using droplet digital PCR (ddPCR) analyses, we found that eDNA concentrations become strongly reduced 2 km downstream from the source, an effect that is strengthened with increasing river discharge and coinciding dilution effects. Higher discharge rates resulted in equal or even higher detection probabilities at increasing distance from the source. The introduction of high fish stock biomass resulted in an increase of eDNA concentrations, as well as detection probabilities and in parallel reduced the stochasticity of the measurements. A peak in eDNA concentrations at a downstream distance ranging between 300 m and 2 km confirms the complexity of plume‐shaped downstream eDNA patterns. Our results showed interspecific variation in eDNA emission and suggest species‐specific differences in eDNA persistence. This study underlines the impact of both river discharge rate and source biomass on downstream eDNA detection and dispersal patterns.

Environmental DNA detects Spawning Habitat of an ephemeral migrant fish (Anadromous Rainbow Smelt: Osmerus mordax)

BackgroundAnadromous rainbow smelt (Osmerus mordax) have experienced a large range reduction in recent decades and the status of remnant spawning populations is poorly known in Maine, where these fish have significant ecological, cultural, and commercial relevance. Defining the remnant range of anadromous smelt is more difficult than for many declining fish species because adults are only ephemerally present while spawning in small coastal streams at night during spring runoff periods when traditional assessments can be unreliable or even hazardous. We hypothesized that eDNA might facilitate improved survey efforts to define smelt spawning habitat, but that detection could also face challenges from adult eDNA quickly flushing out of these small stream systems. We combined daytime eDNA sampling with nighttime fyke netting to ascertain a potential window of eDNA detection before conducting eDNA surveys in four streams of varying abundance. Hierarchical occupancy modeling was in turn employed to estimate eDNA encounter probabilities relative to numbers of sampling events (date), samples within events, and qPCR replicates within samples.ResultsResults from the combined eDNA and fyke net study indicated eDNA was detectable over an extended period, culminating approximately 8–13 days following peak spawning, suggesting developing smelt larvae might be the primary source of eDNA. Subsequently, smelt eDNA was readily detected in eDNA surveys of four streams, particularly following remediation of PCR inhibitors. Hierarchical occupancy modeling confirmed our surveys had high empirical detection for most sites, and that future surveys employing at least three sampling events, three samples per event, and six qPCR replicates can afford greater than 90% combined detection capability in low abundance systems.ConclusionsThese results demonstrate that relatively modest eDNA sampling effort has high capacity to detect this ephemerally present species of concern at low to moderate abundances. As such, smelt eDNA detection could improve range mapping by providing longer survey windows, safer sampling conditions, and lower field effort in low density systems, than afforded by existing visual and netting approaches.

Identification of factors associated with Fasciola hepatica infection risk areas on pastures via an environmental DNA survey of Galba truncatula distribution using droplet digital and quantitative real‐time PCR assays

AbstractEnvironmental DNA (eDNA) is a powerful tool for identifying the spatial and temporal presence and density of species in a range of aquatic habitats. The analysis of eDNA has a wide range of application, one of which may be to inform of Fasciola hepatica infection risk on pastures based on the detection of its eDNA as well as that of its intermediate snail host, Galba truncatula eDNA. Here, droplet digital PCR (ddPCR) and quantitative real‐time PCR (qPCR) assays were developed to detect the eDNA of F. hepatica, and its intermediate snail host, G. truncatula in water samples collected from pastures grazed by cattle and/or sheep. Environmental factors associated with species presence, as detected via an eDNA survey, were identified using zero‐inflated linear mixed models. Sixty‐four habitats were sampled across six farms in Ceredigion, Wales, UK, with ddPCR and qPCR identifying 42 and 33 habitats to be positive for G. truncatula eDNA, respectively. G. truncatula eDNA was significantly less likely to be detected in habitats fully shaded by trees, those that contained black or dark brown soils and habitats that contained deep water pools (p < 0.05). Significantly higher G. truncatula eDNA concentrations were observed in habitats that tend to dry up during Summer (i.e., temporary habitats) (p < 0.05). ddPCR also identified five habitats to be positive for F. hepatica eDNA; however, questions remain regarding the utility of F. hepatica eDNA detection due to a lack of specificity toward infective F. hepatica larval stages. The results of this study inform of factors which influences G. truncatula distribution and ecology on pastures and also provided practical information for farmers to aid F. hepatica control in their flocks and herds.

Monochloramine Induces Release of DNA and RNA from Bacterial Cells: Quantification, Sequencing Analyses, and Implications.

Monochloramine (MCA) is a widely used secondary disinfectant to suppress microbial growth in drinking water distribution systems. In monochloraminated drinking water, a significant amount of extracellular DNA (eDNA) has been reported, which has many implications ranging from obscuring DNA-based drinking water microbiome analyses to posing potential health concerns. To address this, it is imperative for us to know the origin of the eDNA in drinking water. Using Pseudomonas aeruginosa as a model organism, we report for the first time that MCA induces the release of nucleic acids from both biofilms and planktonic cells. Upon exposure to 2 mg/L MCA, massive release of DNA from suspended cells in both MilliQ water and 0.9% NaCl was directly visualized using live cell imaging in a CellASIC ONIX2 microfluidic system. Exposing established biofilms to MCA also resulted in DNA release from the biofilms, which was confirmed by increased detection of eDNA in the effluent. Intriguingly, massive release of RNA was also observed, and the extracellular RNA (eRNA) was also found to persist in water for days. Sequencing analyses of the eDNA revealed that it could be used to assemble the whole genome of the model organism, while in the water, certain fragments of the genome were more persistent than others. RNA sequencing showed that the eRNA contains non-coding RNA and mRNA, implying its role as a possible signaling molecule in environmental systems and a snapshot of the past metabolic state of the bacterial cells.

Environmental DNA reflects common haplotypic variation

AbstractAnalysis of environmental DNA (eDNA) has gained widespread usage for taxonomically based biodiversity assessment. While interest in applying noninvasive eDNA monitoring for population genetic assessments has grown, its usage in this sphere remains limited. One barrier to uptake is that the effectiveness of eDNA detection below the species level remains to be determined for multiple species and environments. Here, we test the utility of this emergent technology to obtain within‐species haplotypic variation of New Zealand (NZ) blackfoot pāua (Haliotis iris). We compare mitochondrial haplotype diversity recovered from marine eDNA samples against traditional tissue samples of blackfoot pāua collected at the same NZ coastal site. Targeting the ATP8‐ATP6 region, we recovered four mitochondrial haplotypes from eDNA versus six haplotypes from tissue samples. Three common haplotypes were recovered with both eDNA and tissue samples, while only one out of three rare haplotypes – represented in tissue samples by one individual each – was recovered with our eDNA methods. We demonstrate that eDNA monitoring is an effective tool for recovering common genetic diversity from pāua, although rare (<5%) haplotypes are seldom recovered. Our results show the potential of eDNA to identify population‐level haplotypic diversity for gastropods in the marine environment below the species level. This work demonstrates that eDNA methods can be an effective, noninvasive tool for genetic monitoring. Noninvasive eDNA sampling could minimize target organism stress and human interaction enabling population genetic research for hard‐to‐sample, delicate, or sensitive species.

Detecting endangered pinto abalone (Haliotis kamtschatkana) using environmental DNA: Comparison of ddPCR, qPCR, and conventional diver surveys

AbstractAbalone populations along the Pacific Coast of North America are threatened. In the Salish Sea (Washington, USA), pinto abalone (Haliotis kamtschatkana) have failed to recover from intensive harvest after over 25 years of fishery closure, prompting a growing restoration effort. As these efforts expand, a persistent challenge is simply locating this rare and highly cryptic species in the field, limiting the ability to identify critical habitat and locate wild adults to serve as restoration broodstock. Here, we tested the use of environmental DNA (eDNA) to detect pinto abalone. Using a quantitative PCR (qPCR) assay previously developed for larval pinto abalone, we first evaluated its sensitivity to abalone eDNA in aquaria settings, finding a positive relationship between abalone biomass and the concentration of abalone DNA. We then tested abalone eDNA detection in the field by collecting replicate water samples from abalone restoration sites, using an occupancy model to estimate detection probability in relation to abalone biomass estimated via diver surveys. Both eDNA concentration and detection probability were positively associated with diver‐estimated abalone biomass. By modifying the assay for droplet digital PCR (ddPCR), detection probability increased by 32%–89% over qPCR. eDNA surveys using ddPCR had higher error (CV = 96.9%) than diver surveys (CV = 29.4%) but were more efficient, taking approximately 1/10th of the person‐hours per site of a diver survey. For the final phase of the study, we collected water samples at 80 sites throughout the region, obtaining positive abalone eDNA detections at 11 sites with qPCR and 19 additional sites with ddPCR. Our results provide novel survey data on abalone populations within the Salish Sea and show that eDNA is a viable tool for cost‐effective, efficient, and non‐invasive abalone detection.

Developing an effective marine eDNA monitoring: eDNA detection at pre-outbreak densities of corallivorous seastar (Acanthaster cf. solaris)

Outbreaks of the corallivorous Crown-of-Thorns Seastar (CoTS) Acanthaster cf. solaris contribute significantly to coral reef loss. Control of outbreaks is hampered because standard monitoring techniques do not detect outbreaks at early (low density) stages, thus preventing early intervention. We previously demonstrated that eDNA monitoring can detect CoTS at intermediate densities. Here, we test whether detection probability can be improved by (i) targeted site selection or collection at specific times and (ii) moving from an average eDNA copy number approach (based on the limit of quantification) to a presence/absence approach (based on the limit of detection). Using a dataset collected over three years and multiple reef sites, we demonstrated that adding water residence age, sea surface level and temperature into generalized linear models explained low amounts of variance of eDNA copy numbers. Site specific CoTS density, by contrast, was a significant predictor for eDNA copy numbers. Bayesian multi-scale occupancy modelling of the presence/absence data demonstrated that the probability of sample capture (θ) on most reefs with intermediate or high CoTS densities was >0.8. Thus, confirming CoTS presence on these reefs would only require 2–3 samples. Sample capture decreased with decreasing CoTS density. Collecting ten filters was sufficient to reliably (based on the lower 95 % Credibility Interval) detect CoTS below nominal outbreak levels (3 Ind. ha−1). Copy number-based estimates may be more relevant to quantify CoTS at higher densities. Although water residence age did contribute little to our models, sites with higher residence times may serve as sentinel sites accumulating eDNA. The approach based on presence or absence of eDNA facilitates eDNA monitoring to detect CoTS densities below outbreak thresholds and we continue to further develop this method for quantification.