A Spatial Modeling Approach to Predicting the Secondary Spread of Invasive Species Due to Ballast Water Discharge

Ballast water in ships is an important contributor to the secondary spread of invasive species in the Laurentian Great Lakes. Here, we use a model previously created to determine the role ballast water management has played in the secondary spread of viral hemorrhagic septicemia virus (VHSV) to identify the future spread of one current and two potential invasive species in the Great Lakes, the Eurasian Ruffe (Gymnocephalus cernuus), killer shrimp (Dikerogammarus villosus), and golden mussel (Limnoperna fortunei), respectively. Model predictions for Eurasian Ruffe have been used to direct surveillance efforts within the Great Lakes and DNA evidence of ruffe presence was recently reported from one of three high risk port localities identified by our model. Predictions made for killer shrimp and golden mussel suggest that these two species have the potential to become rapidly widespread if introduced to the Great Lakes, reinforcing the need for proactive ballast water management. The model used here is flexible enough to be applied to any species capable of being spread by ballast water in marine or freshwater ecosystems.     

Mitochondrial Genome Sequencing and Development of Genetic Markers for the Detection of DNA of Invasive Bighead and Silver Carp (Hypophthalmichthys nobilis and H. molitrix) in Environmental Water Samples from the United States

Invasive Asian bighead and silver carp (Hypophthalmichthys nobilis and H. molitrix) pose a substantial threat to North American aquatic ecosystems. Recently, environmental DNA (eDNA), genetic material shed by organisms into their environment that can be detected by non-invasive sampling strategies and genetic assays, has gained recognition as a tool for tracking the invasion front of these species toward the Great Lakes. The goal of this study was to develop new species-specific conventional PCR (cPCR) and quantitative (qPCR) markers for detection of these species in North American surface waters. We first generated complete mitochondrial genome sequences from 33 bighead and 29 silver carp individuals collected throughout their introduced range. These sequences were aligned with those from other common and closely related fish species from the Illinois River watershed to identify and design new species-specific markers for the detection of bighead and silver carp DNA in environmental water samples. We then tested these genetic markers in the laboratory for species-specificity and sensitivity. Newly developed markers performed well in field trials, did not have any false positive detections, and many markers had much higher detection rates and sensitivity compared to the markers currently used in eDNA surveillance programs. We also explored the use of multiple genetic markers to determine whether it would improve detection rates, results of which showed that using multiple highly sensitive markers should maximize detection rates in environmental samples. The new markers developed in this study greatly expand the number of species-specific genetic markers available to track the invasion front of bighead and silver carp and will improve the resolution of these assays. Additionally, the use of the qPCR markers developed in this study may reduce sample processing time and cost of eDNA monitoring for these species.     

Environmental DNA detection of aquatic invasive plants in lab mesocosm and natural field conditions

Aquatic invasive plant species cause negative impacts to economies and ecosystems worldwide. Traditional survey methods, while necessary, often do not result in timely detections of aquatic invaders, which can be cryptic, difficult to identify, and exhibit very rapid growth and reproduction rates. Environmental DNA (eDNA) is a relatively new method that has been used to detect multiple types of animals in freshwater and marine ecosystems through tissues naturally shed from the organism into the water column or sediment. While eDNA detection has proven highly effective in the detection of aquatic animals, we know less about the efficacy of eDNA as an effective surveillance tool for aquatic plants. To address this disparity, we designed mesocosm experiments with Elodea species to determine the ability to detect accumulation and degradation of the DNA signal for aquatic plants, followed by field surveillance of the highly invasive Hydrilla verticillata in freshwaters across several U.S. geographic regions. In both lab and field experiments, we designed a high sensitivity quantitative PCR assay to detect the aquatic plant species. In both experiments, plant eDNA detection was successful; we saw accumulation of DNA when plants were introduced to tanks and a decrease in DNA over time after plants were removed. We detected eDNA in the field in areas of known Hydrilla distribution. Employing eDNA detection for aquatic plants will strengthen efforts for early detection and rapid response of invaders in global freshwater ecosystems.     

Environmental DNA Marker Development with Sparse Biological Information: A Case Study on Opossum Shrimp (Mysis diluviana)

The spread of Mysis diluviana, a small glacial relict crustacean, outside its native range has led to unintended shifts in the composition of native fish communities throughout western North America. As a result, biologists seek accurate methods of determining the presence of M. diluviana, especially at low densities or during the initial stages of an invasion. Environmental DNA (eDNA) provides one solution for detecting M. diluviana, but building eDNA markers that are both sensitive and species-specific is challenging when the distribution and taxonomy of closely related non-target taxa are poorly understood, published genetic data are sparse, and tissue samples are difficult to obtain. To address these issues, we developed a pair of independent eDNA markers to increase the likelihood of a positive detection of M. diluviana when present and reduce the probability of false positive detections from closely related non-target species. Because tissue samples of closely-related and possibly sympatric, non-target taxa could not be obtained, we used synthetic DNA sequences of closely related non-target species to test the specificity of eDNA markers. Both eDNA markers yielded positive detections from five waterbodies where M. diluviana was known to be present, and no detections in five others where this species was thought to be absent. Daytime samples from varying depths in one waterbody occupied by M. diluviana demonstrated that samples near the lake bottom produced 5 to more than 300 times as many eDNA copies as samples taken at other depths, but all samples tested positive regardless of depth.     

Something in the water: biosecurity monitoring of ornamental fish imports using environmental DNA

The international trade in ornamental aquatic organisms represents an important vector in the spread of invasive species worldwide, but the accurate identification of imported organisms as part of a biosecurity surveillance program offers an opportunity to mitigate potential problems. Species level identification is historically conducted visually, and more recently, with the use of DNA barcoding. However, new diagnostic methods targeting extracellular environmental DNA (eDNA) can offer advantages over these approaches, being non-destructive and potentially more sensitive at low population densities of target organisms (e.g. in mixed consignments). Despite their recent introduction, techniques utilising eDNA are quickly becoming recognised as an important tool for invasion biologists and ecosystem managers. Here, we present a model for the development of a biosecurity protocol for ornamental fish identification using degraded eDNA molecules in water. We demonstrate how a DNA barcode reference library can be mined for informative short-length markers, and report repeatable and accurate detection at low densities of the target species. This study represents a framework for biosecurity agencies to develop eDNA procedures as an innovative management technique for routine surveillance of high risk imports. Future up-scaling of the method will open up prospects for long term monitoring of entire quarantine facilities for a variety of harmful species.     

Improved Methods for Capture,Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)

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.     

Development and application of environmental DNA surveillance for the threatened crucian carp (Carassius carassius)

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Development of environmental DNA (eDNA) methods for detecting high-risk freshwater fishes in live trade in Canada

Preventing the arrival, establishment, and spread of aquatic invasive species is an important step in protecting our aquatic environments. The use of detection tools, like DNA barcoding technologies, high-throughput sequencing and environmental DNA (eDNA) monitoring, is becoming increasingly important in preventing the introduction of potential invasive species. The combination of eDNA with realtime PCR (qPCR) provide the opportunity to have a rapid and specific detection. In this study, we developed a DNA sequence library that has sufficient depth and species coverage such that high-risk species can be confidently discriminated from legitimately imported and native species. A total of 12 species-specific qPCR assays were developed for the detection of 13 potential invasive species (pAIS) in bulk water samples. Detection of these species was also compared with a HTS approach. We have demonstrated the high sensitivity of qPCR assays using eDNA at very low densities, suggesting we could detect a low number of individuals mixed with non-target species in a simulated live shipment. For the detection of a targeted list of species, qPCR is advantageous. The mini-barcodes developed in this project offered a good sensitivity of detection, and HTS is a discovery tool that can be desirable when unlisted or numerous species need to be identified.     

Investigating the Potential Use of Environmental DNA (eDNA) for Genetic Monitoring of Marine Mammals

The exploitation of non-invasive samples has been widely used in genetic monitoring of terrestrial species. In aquatic ecosystems, non-invasive samples such as feces, shed hair or skin, are less accessible. However, the use of environmental DNA (eDNA) has recently been shown to be an effective tool for genetic monitoring of species presence in freshwater ecosystems. Detecting species in the marine environment using eDNA potentially offers a greater challenge due to the greater dilution, amount of mixing and salinity compared with most freshwater ecosystems. To determine the potential use of eDNA for genetic monitoring we used specific primers that amplify short mitochondrial DNA sequences to detect the presence of a marine mammal, the harbor porpoise, Phocoena phocoena, in a controlled environment and in natural marine locations. The reliability of the genetic detections was investigated by comparing with detections of harbor porpoise echolocation clicks by static acoustic monitoring devices. While we were able to consistently genetically detect the target species under controlled conditions, the results from natural locations were less consistent and detection by eDNA was less successful than acoustic detections. However, at one site we detected long-finned pilot whale, Globicephala melas, a species rarely sighted in the Baltic. Therefore, with optimization aimed towards processing larger volumes of seawater this method has the potential to compliment current visual and acoustic methods of species detection of marine mammals.     

Environmental DNA (eDNA) Sampling Improves Occurrence and Detection Estimates of Invasive Burmese Pythons

Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors. Generic sampling design and terminology are proposed to standardize and clarify interpretations of eDNA-based occupancy models.     

Early detection of a highly invasive bivalve based on environmental DNA (eDNA)

Management of non-indigenous invasive species (NIS) is challenging owing in part to limitations of early detection and identification. The advent of environmental DNA (eDNA) techniques provides an efficient way to detect NIS when their abundance is extremely low. However, eDNA-based methods often suffer from uncertain detection sensitivity, which requires detailed testing before applying these methods in the field. Here we developed an eDNA tool for early detection of the highly invasive golden mussel, Limnoperna fortunei, based on the mitochondrial cytochrome c oxidase subunit I gene (COI). Further, we tested technical issues, including sampling strategy and detection sensitivity, based on a laboratory experiment. We then applied the method to field samples collected from water bodies in China where this mussel has or is expected to colonize. Results showed that the detection limit varied extensively among our newly developed primer pairs, ranging from 4 × 10^?2 to 4 × 10^?6 ng of total genomic DNA. Laboratory detection was affected by the availability of eDNA (i.e., both mussel abundance and incubation time). Detection capacity was higher in laboratory samples containing re-suspended matter from the bottom layer versus that collected from the surface. Among 25 field sites, detection was 100% at sites with high mussel abundance and as low as 40% at sites with low abundance when tested using our most sensitive primer pair. Early detection of NIS present at low abundance in nature requires not only sensitive primers, but also an optimized sampling strategy to reduce the occurrence of false negatives. Careful selection and detailed testing of primer pairs ensures effective eDNA-based species detection in surveillance and management programs.     

Detecting the movement and spawning activity of bigheaded carps with environmental DNA

Bigheaded carps are invasive fishes threatening to invade the Great Lakes basin and establish spawning populations, and have been monitored using environmental DNA (eDNA). Not only does eDNA hold potential for detecting the presence of species, but may also allow for quantitative comparisons like relative abundance of species across time or space. We examined the relationships among bigheaded carp movement, hydrography, spawning and eDNA on the Wabash River, IN, USA. We found positive relationships between eDNA and movement and eDNA and hydrography. We did not find a relationship between eDNA and spawning activity in the form of drifting eggs. Our first finding demonstrates how eDNA may be used to monitor species abundance, whereas our second finding illustrates the need for additional research into eDNA methodologies. Current applications of eDNA are widespread, but the relatively new technology requires further refinement.     

Environmental DNA detection of rare and invasive fish species in two Great Lakes tributaries

The extraction and characterization of DNA from aquatic environmental samples offers an alternative, noninvasive approach for the detection of rare species. Environmental DNA, coupled with PCR and next‐generation sequencing (“metabarcoding”), has proven to be very sensitive for the detection of rare aquatic species. Our study used a custom‐designed group‐specific primer set and next‐generation sequencing for the detection of three species at risk (Eastern Sand Darter, Ammocrypta pellucida; Northern Madtom, Noturus stigmosus; and Silver Shiner, Notropis photogenis), one invasive species (Round Goby, Neogobius melanostomus) and an additional 78 native species from two large Great Lakes tributary rivers in southern Ontario, Canada: the Grand River and the Sydenham River. Of 82 fish species detected in both rivers using capture‐based and eDNA methods, our eDNA method detected 86.2% and 72.0% of the fish species in the Grand River and the Sydenham River, respectively, which included our four target species. Our analyses also identified significant positive and negative species co‐occurrence patterns between our target species and other identified species. Our results demonstrate that eDNA metabarcoding that targets the fish community as well as individual species of interest provides a better understanding of factors affecting the target species spatial distribution in an ecosystem than possible with only target species data. Additionally, eDNA is easily implemented as an initial survey tool, or alongside capture‐based methods, for improved mapping of species distribution patterns.     

Modeling the secondary spread of viral hemorrhagic septicemia virus (VHSV) by commercial shipping in the Laurentian Great Lakes

Researchers have only begun to study the role of shipping in the spread of invasive species in the Laurentian Great Lakes despite a well-documented history of introductions in these lakes due to ballast water release. Here, we determine whether ballast water discharge was a likely vector of spread of the fish disease, viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb), throughout the Great Lakes and St. Lawrence Seaway. Three models were developed to assess whether the spread of VHSV was due to (1) chance (random model), or (2) ballast water discharge (location model), and whether (3) increased propagule pressure, as measured by the number of visitations by ships carrying ballast water from VHSV infected areas, increased the likelihood of a discharge location becoming infected with VHSV (propagule pressure model). The third model was also used to assess the probable point of initial introduction of VHSV. Presence and absence accuracies and weighted Cohen’s kappa were calculated to determine which models best predicted observed presences and absences of VHSV. Location models explain the patterns of VHSV detections better than random models, and inclusion of “propagule pressure” often improved model fit; however, the relationship is weak likely because of a long lag time between introduction and detection, a high rate of false negatives in reporting, and the possible contribution of other vectors of spread. Montreal was also identified as the more likely introduction site of VHSV, rather than Lake St. Clair, the site where the virus was first detected.     

Quantifying site-level usage and certainty of absence for an invasive species through occupancy analysis of camera-trap data

Efficient implementation of management programs for invasive species depends on accurate surveillance for guiding prioritization of surveillance and control resources in space and time. Occupancy probabilities can be used to determine where surveillance should occur. Conversely, knowledge of the certainty of site-level absence is of special interest in situations where the objective is to completely remove populations despite substantial risk of re-invasion. Indeed, the decision to shift from emphasizing control activities over the full range to emphasizing reinvasion prevention, surveillance, and response near the borders, depends on accurate knowledge of absence across space. We used a dynamic occupancy model to monitor changes in the distribution of an invasive species, feral swine (Sus scrofa), based on camera-trap data collected as part of a management program from June 2014 to January 2016 in San Diego County, California. Site usage of feral swine declined overall. The most informative predictors of site usage were spatial (latitude and longitude). Site-level non-usage rates increased over time and in response to management removal efforts; and site-level usage rates were heavily impacted by having neighboring sites that were used. Combining the detection probability estimated from the occupancy model and Bayes Theorem, we demonstrated how certainty of local (site-level) absence can be estimated iteratively in time in areas with negative surveillance (no detections) data. Our framework provides a means for using management-based surveillance data to quantify certainty of site-level absence of an invasive species, allowing for adaptive prioritization of surveillance and control resources. Our approach is flexible for application to other species and types of surveillance (e.g., track-plates, eDNA).     

Occupancy in dynamic systems: accounting for multiple scales and false positives using environmental DNA to inform monitoring

Occupancy is an important metric to understand current and future trends in populations that have declined globally. In addition, occupancy can be an efficient tool for conducting landscape-scale and long-term monitoring. A challenge for occupancy monitoring programs is to determine the appropriate spatial scale of analysis and to obtain precise occupancy estimates for elusive species. We used a multi-scale occupancy model to assess occupancy of Columbia spotted frogs in the Great Basin, USA, based on environmental DNA (eDNA) detections. We collected three replicate eDNA samples at 220 sites across the Great Basin. We estimated and modeled ecological factors that described watershed and site occupancy at multiple spatial scales simultaneously while accounting for imperfect detection. Additionally, we conducted visual and dipnet surveys at all sites and used our paired detections to estimate the probability of a false positive detection for our eDNA sampling. We applied the estimated false positive rate to our multi-scale occupancy dataset and assessed changes in model selection. We had higher naïve occupancy estimates for eDNA (0.37) than for traditional survey methods (0.20). We estimated our false positive detection rate per qPCR replicate at 0.023 (95% CI: 0.016–0.033). When the false positive rate was applied to the multi-scale dataset, we did not observe substantial changes in model selection or parameter estimates. Conservation and resource managers have an increasing need to understand species occupancy in highly variable landscapes where the spatial distribution of habitat changes significantly over time due to climate change and human impact. A multi-scale occupancy approach can be used to obtain regional occupancy estimates that can account for spatially dynamic differences in availability over time, especially when assessing potential declines. Additionally, this study demonstrates how eDNA can be used as an effective tool for improved occupancy estimates across broad geographic scales for long-term monitoring.     

Real‐time PCR detection of Didemnum perlucidum (Monniot, 1983) and Didemnum vexillum (Kott, 2002) in an applied routine marine biosecurity context

Prevention and early detection are well recognized as the best strategies for minimizing the risks posed by nonindigenous species (NIS) that have the potential to become marine pests. Central to this is the ability to rapidly and accurately identify the presence of NIS, often from complex environmental samples like biofouling and ballast water. Molecular tools have been increasingly applied to assist with the identification of NIS and can prove particularly useful for taxonomically difficult groups like ascidians. In this study, we have developed real‐time PCR assays suited to the specific identification of the ascidians Didemnum perlucidum and Didemnum vexillum. Despite being recognized as important global pests, this is the first time specific molecular detection methods have been developed that can support the early identification and detection of these species from a broad range of environmental sample types. These fast, robust and high‐throughput assays represent powerful tools for routine marine biosecurity surveillance, as detection and confirmation of the early presence of species could assist in the timely establishment of emergency responses and control strategies. This study applied the developed assays to confirm the ability to detect Didemnid eDNA in water samples. While previous work has focused on detection of marine larvae from water samples, the development of real‐time PCR assays specifically aimed at detecting eDNA of sessile invertebrate species in the marine environment represents a world first and a significant step forwards in applied marine biosecurity surveillance. Demonstrated success in the detection of D. perlucidum eDNA from water samples at sites where it could not be visually identified suggests value in incorporating such assays into biosecurity survey designs targeting Didemnid species.     

Reliable eDNA detection and quantification of the European weather loach (Misgurnus fossilis)

The European weather loach (Misgurnus fossilis) is a cryptic and poorly known fish species of high conservation concern. The species is experiencing dramatic population collapses across its native range to the point of regional extinction. Although environmental DNA (eDNA)-based approaches offer clear advantages over conventional field methods for monitoring rare and endangered species, accurate detection and quantification remain difficult and quality assessment is often poorly incorporated. In this study, we developed and validated a novel digital droplet PCR (ddPCR) eDNA-based method for reliable detection and quantification, which allows accurate monitoring of M. fossilis across a number of habitat types. A dilution experiment under laboratory conditions allowed the definition of the limit of detection (LOD) and the limit of quantification (LOQ), which were set at concentrations of 0.07 and 0.14 copies μl^–1, respectively. A series of aquarium experiments revealed a significant and positive relationship between the number of individuals and the eDNA concentration measured. During a 3 year survey (2017–2019), we assessed 96 locations for the presence of M. fossilis in Flanders (Belgium). eDNA analyses on these samples highlighted 45% positive detections of the species. On the basis of the eDNA concentration per litre of water, only 12 sites appeared to harbour relatively dense populations. The other 31 sites gave a relatively weak positive signal that was typically situated below the LOQ. Combining sample-specific estimates of effective DNA quantity (Q_e) and conventional field sampling, we concluded that each of these weak positive sites still likely harboured the species and therefore they do not represent false positives. Further, only seven of the classified negative samples warrant additional sampling as our analyses identified a substantial risk of false-negative detections (i.e., type II errors) at these locations. Finally, we illustrated that ddPCR outcompetes conventional qPCR analyses, especially when target DNA concentrations are critically low, which could be attributed to a reduced sensitivity of ddPCR to inhibition effects, higher sample concentrations being accommodated and higher sensitivity obtained.     

A review of Ruffe (<Emphasis Type="Italic">Gymnocephalus cernua</Emphasis>) life history in its native versus non-native range

Invasive Ruffe (Gymnocephalus cernua) has caused substantial ecological damage in North America, parts of Western Europe, Scandinavian countries, and the United Kingdom. The objectives of this review are to define Ruffe’s native and non-native range, examine life history requirements, explore the life cycle, and differentiate between life stages. We compare data from its native and non-native ranges to determine if there are any differences in habitat, size, age, genotype, or seasonal migration. Literature from both the native and non-native ranges of Ruffe, with some rare, translated literature, is used. In each life stage, Ruffe exhibit plasticity with regard to chemical, physical, biological, and habitat requirements. Adult Ruffe has characteristics that allow them to adapt to a range of environments, including rapid maturation, relatively long life and large size (allowing them to reproduce many times in large batches), batch spawning, genotype and phenotype (having plasticity in their genetic expression), tolerance to a wide range of water quality, broad diet, and multiple dispersal periods. There is, however, variability among these characteristics between the native, non-native North American, and European non-native populations, which presents a challenge to managing populations based on life history characteristics. Monitoring and preventative strategies are important because, based on Ruffe’s variable life history strategies and its recent range expansion, all of the Laurentian Great Lakes and many other water bodies in the UK, Europe, and Norway are vulnerable to Ruffe establishment.