Environmental DNA Analysis Research Articles

Field Trials of an Autonomous eDNA Sampler in Lotic Waters.

Environmental DNA (eDNA) analysis has become a transformative technology, but sample collection methods lack standardization and sampling at effective frequencies requires considerable field effort. Autonomous eDNA samplers that can sample water at high frequencies offer potential solutions to these problems. We present results from four case studies using a prototype autonomous eDNA sampler as part of the U.S. Geological Survey's Rapid Environmental eDNA Assessment and Deployment Initiative & Network (READI-Net) project. These case studies involved short-term deployments of an eDNA autosampler (Smith-Root) across a range of riverine habitats with the objectives of (a) identifying what insights could be gained from high-frequency autosampling and (b) benchmarking these autosamples against manually collected samples. The high frequency autosampling revealed high temporal variability of eDNA concentrations and provided valuable insights about eDNA associations with environmental covariates, such as discharge and turbidity. Benchmarking assessments indicated autosamples had similar detection rates to manual samples and obtained similar or greater eDNA quantities. We did find minimal carryover contamination in autosampler field controls. We conclude that eDNA autosamplers have potential to improve freshwater biosurveillance by reducing logistical sampling barriers, standardizing collection methods, and clarifying the influence of environmental covariates on eDNA results.

Towards the integration of environmental DNA analysis to profile the upper mesopelagic fish layer in the Northeast Atlantic Ocean

Towards the integration of environmental DNA analysis to profile the upper mesopelagic fish layer in the Northeast Atlantic Ocean

The Core of the Matter-Importance of Identification Method and Biological Replication for Benthic Marine Monitoring.

Benthic macrofauna are important and widely used biological indicators of marine ecosystems as they have limited mobility and therefore integrate the effects of local environmental stressors over time. Recently, environmental DNA (eDNA) analysis has provided a potentially more resource-efficient approach for benthic biomonitoring than traditional morphology-based methods. Several studies have compared eDNA with morphology-based monitoring, but few have compared the two approaches using the exact same sediment cores. In addition, the meiofauna and pelagic organisms obtained as 'bycatch' using eDNA have largely been disregarded from comparisons. Here, we address these shortcomings through comparative invertebrate analyses of six sediment sample replicates from each of four stations in Denmark, using eDNA metabarcoding and morphological identification. Our results revealed large variation between the six replicates for both methods and little overlap in taxon compositions between methods. While the morphological dataset was dominated by molluscs and annelids, the eDNA dataset was dominated by arthropods and annelids. Using community composition data, we found that sampling stations could be distinguished both with eDNA and morphology. Finally, we inferred expected total richness from extrapolated accumulation curves of detected taxa from each method. This indicated that eDNA metabarcoding requires less replication than morphology for maximum coverage of diversity to be reached. However, both methods required high levels of replication, and our results on taxonomic composition add to the evidence that morphological and eDNA-based methods should preferably be used as complimentary tools for marine bioassessment.

Unveiling Aquatic Organism Health Through eProteins: A Contemporary Perspective

Environmental DNA (eDNA) analysis has transformed our understanding of aquatic ecosystems, but traditional methods often lack insights into organismal health. The emerging field of environmental protein (eProtein) analysis offers a novel approach to monitoring the physiological status of aquatic organisms. Recent advancements in proteomic technologies have enabled the detection and characterization of stress-responsive proteins in water samples, providing valuable insights into organismal health and environmental stressors. This perspective paper explores the potential of eProtein analysis for monitoring aquatic organism health, disease dynamics and reproductive cycles. Methodological advancements in protein extraction and mass spectrometry have enhanced the sensitivity and specificity of eProtein analysis, facilitating comprehensive molecular profiling and biomarker identification. By integrating eProtein analysis into aquaculture management and environmental monitoring, researchers can proactively manage fish health, mitigate disease outbreaks and safeguard aquatic ecosystems. Future research directions include refining extraction methods, establishing standardized protocols and leveraging interdisciplinary collaborations to maximize the potential of eProtein analysis for aquatic research and conservation.

Fish diversity assessment through conventional morphological identification and recent advances in Saudi Arabia: A review

Fish identification in the Red Sea, particularly in Saudi Arabia, has a long history. Because of the vast fish diversity in Saudi Arabia, proper species identification is required. Indeed, identifying fish species is critical for biodiversity conservation, food and drug safety, and sustainable fishery management. Numerous approaches have been used to identify fish species, including conventional morphological identification, next-generation sequencing (NGS), nanopore sequencing, DNA barcoding, and environmental DNA analysis. In this review, we collected as much scientific information as possible on species identification in Saudi Arabia. Our findings suggest that the identification process has advanced and spread rapidly and broadly, as evidenced by the discovery of new fish species in Saudi Arabia. The advantages and disadvantages of each method were discussed as part of a comprehensive comparison. This study aimed to provide further scientific knowledge to promote the growth of fish diversity worldwide. Keywords: fish diversity, identification process, Saudi Arabia.

The Situation of Counterfeited and Mislabeled Commercialized Edible Mushrooms in China and the Development of Possible Controls

Edible mushroom products, encompassing both cultivated and wild varieties, are highly favored by consumers due to their rich nutritional profiles, including significant levels of proteins and amino acids. These mushrooms have extensive applications across the food, pharmaceutical, and cosmetic industries, making the edible mushroom industry a vital component of global poverty alleviation efforts. Taking China as an example, the country produces over 45 million tons of edible mushrooms annually, accounting for 94.01% of the world’s total production, thereby establishing itself as the leading global producer of edible mushrooms. However, alongside the rapid expansion of this industry, concerns have emerged regarding counterfeit products and incidents of poisoning resulting from the consumption of toxic wild mushrooms. As follows, to advance the development and integrity of the mushroom production and processing industry: (1) This study presents the situation of counterfeit edible mushrooms and elucidates the factors contributing to the production of fraudulent products from both subjective and non-subjective perspectives. (2) We provide a detailed introduction to 22 varieties of freshly cultivated edible mushrooms and commonly encountered wild edible mushrooms in the Chinese consumer market, proposing the application of DNA barcoding, environmental DNA analysis, and other technologies for the future authentication of counterfeit mushroom products. (3) Concurrently, we present an overview of mushroom poisoning incidents in China from 2010 to 2023, emphasizing the challenges in mitigating the risks associated with wild mushroom consumption and preventing food poisoning, thereby necessitating heightened consumer caution. (4) Finally, we offer four recommendations aimed at ensuring the healthy, stable, and sustainable growth of the edible mushroom industry.

Dammed coastal waterways are less diverse, more homogenous, and dominated by non-native species: Comprehensive insights from quantitative analysis of environmental DNA

Small dams are commonplace worldwide and impact local and regional aquatic diversity by altering habitats and disrupting dispersal networks. Quantifying the local and regional impacts of dams requires nearly comprehensive species occurrence data. We used environmental DNA (eDNA) metabarcoding to test theoretical predictions about the impacts of dams on local and regional bony fish diversity within the Chesapeake Bay Watershed, USA. We analyzed eDNA from 465 sampling points within 34 waterbodies documenting the distributions of 61 species. On average, dammed waterbodies had approximately half (48 %) as many species per site as undammed (lower alpha diversity) and more homogenous species composition (lower beta diversity). Native migratory species were less than one tenth (0.08) as likely to be detected at dammed sites than undammed sites, native resident (non-migratory) species were one third (0.34) as likely, whereas introduced species were 2.6 times more likely to be detected. Our sampling and bioinformatics methods were validated by a diverse mock community control. Our results suggest that dams in coastal waterways homogenize fish metacommunities, reduce local biodiversity through dispersal limitation and habitat alteration, and favor the dominance of lentic-adapted introduced species while potentially restricting the spread of introduced catfish. Decisions to construct or decommission dams should consider local and regional impacts on biodiversity.

Complete mitochondrial genomes and phylogenetic analysis of native and non-native fishes in a national key wetland of China

Fish are considered objective indicators of environmental health and ecosystem stability. Establishing regional reference databases of mitochondrial genome sequences from local fish communities can significantly enhance fish monitoring using environmental DNA (eDNA) analysis. For non-native species, the eDNA technique provides early detection and rapid monitoring. It is also crucial to include fundamental genetic information for both native and non-native species in genetic databases. This study presents the complete mitochondrial genomes of 17 fish species inhabiting the Baiyangdian Basin, a national key wetland in China. The mitochondrial DNA of these fish was analyzed to investigate their characteristics, and their phylogeny was determined using maximum likelihood (ML) methods. Various analyses were performed, including the examination of nucleotide composition, evaluation of AT-skew and GC-skew, analysis of codon frequency, and determination of relative synonymous codon usage (RSCU) values, and assessment of selection pressure on protein-coding genes (PCGs). The analysis showed that all PCGs in all fish underwent purifying selection. Using Xenocyprididae as a representative, this study investigated the genetic selection tendencies of native and non-native fish species in the Baiyangdian Basin. Significant differences were found in five of the 13 PCGs: COI, COII, COIII, Cytb, and ATP8. Except for ATP8, the findings indicated that the genes of non-native species underwent stronger purifying selection during evolution compared to native species. Additionally, comparing the population ω values of non-native species to those of native species showed that the Cytb and COIII genes exhibited greater differential purification selection than COI and COII. These differences may be the result of the evolution of non-native species to migrate and adapt to the Baiyangdian Basin, thereby affecting the evolution of related genes.

Evaluating the quantitative performance of environmental DNA metabarcoding for freshwater zooplankton community: a case study in Lake Biwa, Japan.

Zooplankton monitoring is important for understanding their population dynamics and life history, ecosystem health, and environmental changes. Compared with traditional morphological identification, environmental DNA (eDNA) analysis allows for more sensitive and efficient monitoring of zooplankton diversity. Previous eDNA studies have primarily used metabarcoding approaches to reveal their richness and composition, whereas its performance in predicting zooplankton abundance remains understudied. We conducted water and bulk sampling in Lake Biwa, Japan, showing that the number of sequence reads by metabarcoding moderately correlated with eDNA concentrations estimated by quantitative real-time PCR (qPCR). In addition, the eDNA read number was significantly related to cladoceran and copepod abundance estimated by microscopy sorting, although there remained too much uncertainty in the read-abundance relationship. Moreover, there was a significant difference in species composition between eDNA metabarcoding and sorting. Although our results indicated the potential applicability of eDNA metabarcoding for quantifying multiple zooplankton abundance, several methodological validations in eDNA metabarcoding would also be required to optimize its performance in the future.

Methodological assessment for efficient collection of Schistosoma mansoni environmental DNA and improved schistosomiasis surveillance in tropical wetlands

Schistosomiasis, caused by trematodes of genus Schistosoma, is among the most seriously neglected tropical diseases. Although rapid surveillance of risk areas for Schistosoma transmission is vital to control schistosomiasis, the habitat and infection status of this parasite are difficult to assess. Environmental DNA (eDNA) analysis, involving the detection of extra-organismal DNA in water samples, facilitates cost-efficient and sensitive biomonitoring of aquatic environments and is a promising tool to identify Schistosoma habitat and infection risk areas. However, in tropical wetlands, highly turbid water causes filter clogging, thereby decreasing the filtration volume and increasing the risk of false negatives. Therefore, in this study, we aimed to conduct laboratory experiments and field surveys in Lake Victoria, Mbita, to determine the appropriate filter pore size for S. mansoni eDNA collection in terms of particle size and filtration volume. In the laboratory experiment, aquarium water was sequentially filtered using different pore size filters. Targeting >3 µm size fraction was found to be sufficient to capture S. mansoni eDNA particles, regardless of their life cycle stage (egg, miracidia, and cercaria). In the field surveys, GF/D (2.7 µm nominal pore size) filter yielded 2.5-times the filtration volume obtained with a smaller pore size filter and pre-filtration methods under the same time constraints. Moreover, a site-occupancy model was applied to the field detection results to estimate S. mansoni eDNA occurrence and detection probabilities and assess the number of water samples and PCR replicates necessary for efficient eDNA detection. Overall, this study reveals an effective method for S. mansoni eDNA detection in turbid water, facilitating the rapid and sensitive monitoring of its distribution and cost-effective identification of schistosomiasis transmission risk areas.

Development of eDNA Protocols for Detection of Endangered White Sturgeon (Acipenser transmontanus) in the Wild

ABSTRACTUnderstanding the distribution and habitat use of endangered species is essential for conservation efforts. Environmental DNA (eDNA) analysis has become a more common approach to defining species habitat occupancy through identification of residual DNA in water samples and has potential to detect populations that are in low abundance or use habitats over a large geographical range. Here, we optimized an eDNA protocol to detect the presence of the endangered white sturgeon (Acipenser transmontanus). We implemented lab‐based experiments to understand the sensitivity and persistence of white sturgeon eDNA and then applied these methods to habitats with known white sturgeon abundances categorized as high, low, or not present. Using quantitative PCR (qPCR) and a modified StrAci1N‐flap primer set, white sturgeon eDNA was detected in water collected from tanks holding white sturgeon down to a dilution of 10,000× (estimated eDNA concentration of 0.00035 μg/L—0.00176 μg/L). Following the removal of white sturgeon from the tanks, the eDNA signal decreased with time but could be detected for up to 7 days. In the field, all sites with high abundances of white sturgeon returned positive eDNA detections. We did not detect white sturgeon eDNA at sites with low abundance or in areas where they were not expected to be present. Results from this work further advance our interpretation of eDNA from wild populations and provide a noninvasive method to advance recovery efforts by identifying species presence in areas of suspected use or to guide additional inventory efforts.

Environmental DNA as Early Warning for Alien Species in Mediterranean Coastal Lagoons: Implications for Conservation and Management

Non-indigenous species (NIS) introduction notoriously threatens the Mediterranean Sea. In addition, Mediterranean coastal lagoons play a crucial role as nurseries for marine species, which new NIS arrivals can threaten. Therefore, monitoring and early warning of NIS presence is essential in preserving biodiversity. An innovative technique for rapid and accurate species identification and biodiversity screening is the application of environmental DNA (eDNA) metabarcoding. In this research, different Penaeidae (Arthropoda, Crustacea, Decapoda) NIS specimens were collected from a Mediterranean coastal lagoon after an early warning about a potentially invasive NIS arising from next-generation sequencing data. DNA barcoding of the DNA extracted from tissue samples and amplified with specifically designed primer pairs led to the recognition of Penaeus aztecus in this NATURA 2000 protected ecosystem for the first time. DNA barcoding from DNA isolated from the water where the living specimens were stored further validated the possibility of identifying P. aztecus starting from eDNA. This approach demonstrated the validity of environmental DNA analysis in the early screening of potentially invasive NIS presence in Mediterranean protected areas and ecosystems. This work describes an applicative example of the efficacy in improving the biomonitoring of lagoon ecosystems using molecular tools and it represents a guideline for the validation of eDNA metabarcoding data for the presence of potentially invasive species.

Optimising eDNA analysis for urban otter monitoring: seasonal patterns, detection strategies and prey availability

Significant advancements in environmental DNA (eDNA) analysis technology have led to its widespread adoption for species monitoring. However, the low DNA concentration in eDNA often raises concerns about PCR bias, which is a primary issue in enhancing the reliability of eDNA survey results. This study aims to identify the suitable eDNA analysis method and detection strategy for obtaining the presence data of Eurasian otters in an urban stream and to investigate the seasonal distribution patterns of Eurasian otters and their potential prey species. The eDNA survey revealed the presence of six out of the 13 mammal species recorded in fieldwork and literature reviews, including Canis lupus, Felis silvestris and Mustela sibirica. Notably, Eurasian otters were not detected in the eDNA metabarcoding conducted in April and October, despite traditional surveys confirming their presence. In contrast, qPCR assays successfully amplified Eurasian otters from the same samples that were analysed using metabarcoding. When evaluating detection criteria, based on the number of positive samples in repetitions, Eurasian otters were detected at 42.9% to 85.7% of the sampling sites in April and at all sampling sites in October. This suggests a higher detection probability of Eurasian otters in October, indicating a potential expansion of their home range during that season compared to April. Metabarcoding results revealed similar findings regarding fish species as traditional surveys, with Cyprinidae accounting for the largest proportion of fish species at the family level (April, 54.57%; October, 43.58%), followed by Gobiidae (April, 16.90%; October, 22.88%). At the species level, P. parva was the dominant fish species in Saetgang, constituting 5.68% and 6.35% of relative abundance in April and October, respectively. This implies that Eurasian otters as opportunistic predators, may increasingly take advantage of the availability of species within the family Cyprinidae, notably Pseudorasbora parva, as a food source within the study area during the months of April and October. This study highlights that qPCR is the more effective approach in urban areas to offer insights into otters’ distribution patterns, while metabarcoding is useful to provide the properties of the biological environment. Furthermore, this study indicated that it is necessary to determine the suitable eDNA analysis methods depending on the research purpose to obtain detection results effectively.

Off the conservation radar: the hidden story of Europe's tiny pea clams (Bivalvia: Sphaeriidae)

AbstractThis study highlights the conservation problems faced by the tiny freshwater bivalves of the family Sphaeriidae, also known as pea, pill, or fingernail clams (or mussels) in Europe. Despite their global distribution, assumed ecological importance, and potential uses, basic knowledge about their taxonomy, biology, and ecology is very limited and much lower than for the larger freshwater bivalve taxa. Immediate scientific priorities are required to fill knowledge gaps regarding their taxonomy, genetic diversity, distribution, reproductive cycle, ecosystem functions, and population trends. Such fundamental knowledge is necessary to identify specific threats and develop appropriate conservation actions. Deploying environmental DNA analysis at a large scale could be a valuable way to fill gaps in distribution and strengthen monitoring in areas where local taxonomic knowledge is lacking. Until taxon-specific management plans can be developed, we recommend that efforts concentrate on the general protection and restoration of wetland habitats, implementing pollution control measures, and managing invasive species. These actions should be complemented by community engagement through citizen science initiatives. Additionally, prioritising data collection to fill existing knowledge gaps and updating conservation statuses (Red Lists) based on comprehensive assessments will be crucial. Implementing these actions will provide a starting point for the broader protection of freshwater ecosystems, thus benefiting pea clams and other interconnected species within these habitats.

Macrobial airborne environmental DNA analysis: A review of progress, challenges, and recommendations for an emerging application.

In the context of looming global biodiversity loss, effective species detection represents a critical concern for ecological research and management. Environmental DNA (eDNA) analysis, which refers to the collection and taxonomic identification of genetic fragments that are shed from an organism into its surroundings, emerged approximately 15 years ago as a sensitive tool for species detection. Today, one of the frontiers of eDNA research concerns the collection and analysis of genetic material in dust and other airborne materials, termed airborne eDNA analysis. As the study of airborne eDNA matures, it is an appropriate time to review the foundational and emerging studies that make up the current literature, and use the reviewed literature to summarize, synthesize, and forecast the major challenges and opportunities for this advancing research front. Specifically, we use the "ecology of eDNA" framework to organize our findings across the origin, state, transport, and fate of airborne genetic materials in the environment, and summarize what is so far known of their interactions with surrounding abiotic and biotic factors, including population and community ecologies and ecosystem processes. Within this work we identify key challenges, opportunities, and future directions associated with the application of airborne eDNA development. Lastly, we discuss the development of applications, partnerships, and messaging that promote development and growth of the field. Together, the broad potential of eDNA analysis and the rate at which research is accelerating in this field suggest that the sky's the limit for airborne eDNA science.

Is it worthy to use environmental DNA instead of scientific trawling or video survey to monitor taxa in soft-bottom habitats?

Non-extractive techniques such as video analysis are increasingly used by scientists to study marine communities instead of extractive methods such as trawling. Currently, environmental DNA (eDNA) analysis is seen as a revolutionary tool to study taxonomic diversity. We aimed to determine which method is the most appropriate to describe fish and commercial invertebrate diversity comparing bottom trawl hauls, video transects and seawater eDNA. Our results reveal that video detected the lowest number of taxa and trawling the highest. eDNA analysis is powerful to describe marine bony fish communities, but some taxa of importance for the ecosystem such as elasmobranchs, crustaceans or molluscs are poorly detected. This may be due to several factors such as marker specificity, incomplete reference gene databases or low DNA release in the environment. For now, the various methods provide different information and none is exhaustive enough to be used alone for biodiversity characterisation.

Assessing benthic macroinvertebrate communities’ spatial heterogeneity in Mediterranean transitional waters through eDNA metabarcoding

Transitional waters are important habitats both for biodiversity and ecological functions, providing valuable natural resources and relevant ecosystem services. However, they are highly susceptible to climate changes and anthropogenic pressures responsible for biodiversity losses and require specific biomonitoring programs. Benthic macroinvertebrates are suitable as ecological indicators of transitional waters, being affected by biological, chemical, and physical conditions of the ecosystems about their life cycles and space-use behaviour. The advent of high-throughput sequencing technologies has allowed biodiversity investigations, at the molecular level, in multiple ecosystems and for different ecological guilds. Benthic macroinvertebrate communities’ composition has been investigated, at the molecular level, mainly through DNA extracted from sediments in marine and riverine ecosystems. In this work, benthic macroinvertebrate communities are explored through eDNA metabarcoding from water samples in a Mediterranean transitional water ecosystem. This research highlighted the validity of eDNA metabarcoding as an efficient tool for the assessment of benthic macroinvertebrate community structure in transitional waters, unveiling the spatial heterogeneity of benthic macroinvertebrate communities correlated to the measured environmental gradients. The results suggest that peculiar features of transitional water ecosystems, such as shallow waters and limited currents, facilitate the assessment of benthic macroinvertebrate communities through environmental DNA analysis from surface water samples, opening for more rapid and accurate monitoring programs for these valuable ecosystems.

A new flow path: eDNA connecting hydrology and biology

AbstractEnvironmental DNA (eDNA) has revolutionized ecological research, particularly for biodiversity assessment in various environments, most notably aquatic media. Environmental DNA analysis allows for non‐invasive and rapid species detection across multiple taxonomic groups within a single sample, making it especially useful for identifying rare or invasive species. Due to dynamic hydrological processes, eDNA samples from running waters may represent biodiversity from broad contributing areas, which is convenient from a biomonitoring perspective but also challenging, as hydrological knowledge is required for meaningful biological interpretation. Hydrologists could also benefit from eDNA to address unsolved questions, particularly concerning water movement through catchments. While naturally occurring abiotic tracers have advanced our understanding of water age distribution in catchments, for example, current geochemical tracers cannot fully elucidate the timing and flow paths of water through landscapes. Conversely, biological tracers, owing to their immense diversity and interactions with the environment, could offer more detailed information on the sources and flow paths of water to the stream. The informational capacity of eDNA as a tracer, however, is determined by the ability to interpret the complex biological heterogeneity at a study site, which arguably requires both biological and hydrological expertise. As eDNA data has become increasingly available as part of biomonitoring campaigns, we argue that accompanying eDNA surveys with hydrological observations could enhance our understanding of both biological and hydrological processes; we identify opportunities, challenges, and needs for further interdisciplinary collaboration; and we highlight eDNA's potential as a bridge between hydrology and biology, which could foster both domains.This article is categorized under: Science of Water > Hydrological Processes Science of Water > Methods Water and Life > Nature of Freshwater Ecosystems

Fine-scale spatial distribution of a fish community in artificial reefs investigated using an underwater drone and environmental DNA analysis

Although artificial reef (AR) effect evaluation is useful for planning the installation of high-rise ARs and their management, few studies have investigated them quantitatively. The fine-scale 2-dimensional fish distribution in ARs was estimated regarding current fields and vertical structures of 2 high-rise ARs (20 and 30 m high at 62 and 72 m depths, respectively) in Tateyama Bay, central Japan, using underwater drone recordings with vertical line transects and environmental DNA (eDNA) metabarcoding. The species detected by video surveys (21 taxa were identified to species, and 1 to genus) were fewer than by eDNA analysis (103 species and 6 genera), especially for pelagic, small-sized, and cryptic fish. Video surveys revealed that demersal fish increased with decreasing horizontal distance from the AR surface within 20 m, and the richness and total fish density were significantly higher upstream of the ARs. Conversely, the fish eDNA concentration showed different patterns, with significantly higher concentrations downstream of the ARs. The richness peaked at horizontal AR surfaces (e.g. reef top), but density of the dominant species peaked near the bottom by video survey. In comparison, eDNA analysis indicated lower richness and higher eDNA concentration of the dominant species at the reef top. Such discrepancies may be explained by the influence of eDNA transport or its specific behavior or buoyancy. Video surveys revealed the growth stage and sex information of 4 species from their morphology, which is not possible using eDNA analysis. This study shows that the advantages of each evaluation method can complement each other.

Research horizons for invasive marine species detection with eDNA/eRNA

The global marine ecosystem is changing rapidly as the result of biogeochemical cycles and ecosystem structure being altered by industrial civilization. Invasive marine species (IMS) are one of the most damaging regional consequences of human activity, and one of the most easily attributable to specific processes. This makes IMS introduction one of most tractable threats for management by appropriate policies. Once established, a different set of policies are required either to restrict IMS spread, or to attempt local eradication. The key ecosystem management tool for IMS damage mitigation is rapid, widely deployable IMS detection. Environmental Nucleic Acids (eNA), combining environmental DNA (eDNA) and environmental RNA (eRNA) analyses, have emerged as valuable tools for sensitive, cost-effective and readily deployable detection of IMS. Methods for IMS detection by eNA are still being developed through a widespread and active research community, so identifying the limitations of current processes will help prioritise eNA-based IMS detection research. We analysed and synthesised the opinions of expert marine ecosystem managers and researchers in Australia and New Zealand about the knowledge gaps and research needs for eNA-based IMS detection. This synthesis was placed in context with current research literature on what eNA technologies are currently providing as an IMS management tool; what problems exist with the current technology; and what could be done to improve this general approach. Our analyses produced a list of priorities that chart a path towards the best possible systems for IMS detection by eNA.