Use Of Environmental DNA Research Articles

Use of Environmental DNA to Evaluate the Spatial Distribution of False Kelpfish (Sebastiscus marmoratus) in Nearshore Areas of Gouqi Island

This study aims to explore the spatial distribution of false kelpfish (Sebastiscus marmoratus) in the mussel farming area, artificial reef areas of Gouqi Island (Shengsi, China), and natural areas using eDNA detection methods. Surface and bottom water samples were collected at 12 stations in November 2022 and April 2023, totaling 52 samples. We used species-specific primers and probes for quantitative PCR (qPCR) detection of Sebastiscus marmoratus eDNA. The eDNA concentrations differed seasonally (p < 0.05) and did not differ (p > 0.05) among the three sampling areas and two water layers. The greatest eDNA concentrations occurred in the surface layer during the spring. Higher concentrations of Sebastiscus marmoratus eDNA were also found in the mussel aquaculture area. Temperature exhibited a significant positive correlation with Sebastiscus marmoratus eDNA concentration (p < 0.05). Additionally, we developed linear equations predicting the relationship between environmental factors and environmental factors, providing a reference for future fishery resource surveys.

Environmental DNA as a complementary tool for biodiversity monitoring: A multi-technique and multi-trophic approach to investigate cetacean distribution and feeding ecology.

The use of environmental DNA (eDNA) to assess the presence of biological communities has emerged as a promising monitoring tool in the marine conservation landscape. Moreover, advances in Next-Generation Sequencing techniques, such as DNA metabarcoding, enable multi-species detection in mixed samples, allowing the study of complex ecosystems such as oceanic ones. We aimed at using these molecular-based techniques to characterize cetacean communities, as well as potential prey on the northern coast of Mainland Portugal. During four seasonal campaigns (summer 2021 to winter 2022/2023), seawater samples were collected along with visual records of cetacean occurrence. The eDNA isolated from 64 environmental samples was sequenced in an Illumina platform, with universal primers targeting marine vertebrates. Five cetacean species were identified by molecular detection: common dolphin (Delphinus delphis), bottlenose dolphin (Tursiops truncatus), Risso's dolphin (Grampus griseus), harbor porpoise (Phocoena phocoena) and fin whale (Balaenoptera physalus). Overall, except for the latter (not sighted during the campaigns), this cetacean community composition was similar to that obtained through visual monitoring, and the complementary results suggest their presence in the region all year round. In addition, the positive molecular detections of Balaenoptera physalus are of special relevance since there are no records of this species reported on scientific bibliography in the area. The detection of multiple known prey of the identified dolphins indicates an overlap between predator and prey in the study area, which suggests that these animals may use this coastal area for feeding purposes. While this methodological approach remains in a development stage, the present work highlights the benefits of using eDNA to study marine communities, with specific applications for research on cetacean distribution and feeding ecology.

Using grass inflorescence as source material for biomonitoring through environmental DNA metabarcoding

BackgroundOver the last decade, increasing attention has been directed to using different substrates as sources of environmental DNA (eDNA) in ecological research. Reports on the use of environmental DNA located on the surface of plant leaves and flowers have highlighted the utility of this DNA source in studies including, but not limited to, biodiversity, invasive species, and pollination ecology. The current study assesses grass inflorescence as a source of eDNA for detecting invertebrate taxa.Methods and resultsInflorescences from four common grass species in a central South African grassland were collected for high-throughput sequencing analysis. Universal COI primers were utilised to detect Metazoan diversity. The sequencing results allowed for the detection of three Arthropoda orders, with most OTUs assigned to fungal taxa (Ascomycota and Basidiomycota). Some biases were detected while observing the relative read abundance (RRA) results.DiscussionThe observed biases could be explained by the accidental inclusion of invertebrate specimens during sample collection and DNA extraction. Primer biases towards the amplified taxa could be another reason for the observed RRA results. This study provided insight into the invertebrate community associated with the four sampled grass species. It should be noted that with the lack of negative field controls, it is impossible to rule out the influence of airborne eDNA on the observed diversity associated with each grass species. The lack of the inclusion of PCR and extraction blanks in the sequencing step, as well as the inclusion of negative field controls, including other areas for refinement were highlighted, and suggestions were provided to improve the outcomes of future studies.

Environmental DNA Particle Size Distribution and Quantity Differ Across Taxa and Organelles

ABSTRACTThe use of environmental DNA to detect species is now widespread in freshwater ecology. However, the detectability of species depends on many factors, such as the quantity of eDNA particles available in the environment and their state (e.g., free DNA fragments, organellar, or aggregated DNA particles). To date, the most advanced knowledge of the production and state of DNA particles concerns teleosts. Most often, these studies target mitochondrial genes, since they are present in multiple copies in a cell. However, it is likely that the characteristics of eDNA molecules vary greatly among taxa and genetic compartments, with direct consequences for species detection. Using an indoor mesocosm experiment, we compared the rate of mitochondrial and nuclear eDNA production and particle size distribution (PSD) of four distinct and common aquatic taxa (zebrafish, tadpole, isopod and mollusk). The tank water was filtered through a series of filters with decreasing porosity and mitochondrial and nuclear eDNA at each size fraction were quantified by qPCR. We found that the production and the size of eDNA particles varied greatly among taxa and genetic compartments. For most taxa, the number of nuclear eDNA particles released in water was higher than that of mitochondrial origin. The PSD of mt‐eDNA showed a pattern common to all taxa: the relative number of particles increased from the smallest size fractions (0.2 μm and less) to the largest (over 1.2 μm), while the distribution of nu‐eDNA was very different from one taxon to another. We also observed a high temporal variability in the quantity of eDNA particles and in PSD, although the latter was more complex to model. These results call for caution in how to sample and analyze eDNA in aquatic environments, particularly for organisms that emit small particles in small quantities such as isopods.

A Guide to Environmental DNA Extractions for Non‐Molecular Trained Biologists, Ecologists, and Conservation Scientists

ABSTRACTEcologists, biologists, and conservation scientists are increasingly interested in the use of environmental DNA (eDNA) data for research and potentially decision‐making. While commercial DNA extraction kits are typically user‐friendly and accessible, they may fail to deliver the desired results with inherently complex eDNA samples, necessitating protocol optimization or educated selection of alternative approaches. To this end, knowledge of the basic steps and principles of DNA extractions is essential, but traditional education tracks in ecology, conservation, and environmental management typically do not include in‐depth training in molecular methods. The primary objective of this paper is to enable scientists with an ecological background and limited molecular training to understand the four key steps of eDNA isolations, and to use this expertise to their advantage. We describe the purpose of commonly used reagents and chemicals, point out alternatives for each key step, explain the impact of certain choices regarding isolation approaches on DNA integrity and purity, and highlight the possibility of a tailor‐made “mix and match” approach. We anticipate that this paper will enable field ecologists to develop a deeper understanding of the mechanisms and chemistry underlying eDNA extractions, thus allowing them to make informed decisions regarding the best eDNA extraction method for their research goals. Our intention is not to provide comprehensive, step‐by‐step protocols, but to offer guiding principles while highlighting alternative solutions. Finally, we hope that this paper will act as a useful resource to support knowledge transfer and teaching.

Development and Application of a Portable Environmental DNA Test for the Detection of Rafetus swinhoei in Viet Nam

ABSTRACTSwinhoe's (or Yangtze) giant softshell turtle (Rafetus swinhoei) is a large, critically endangered freshwater turtle and considered among the rarest species in the world. As of 2024, only two individuals have been confirmed to remain alive, one at the Suzhou Zoo in China and one in Xuan Khanh Lake, Viet Nam. The only hope for the long‐term survival of R. swinhoei is finding additional, as yet undiscovered, animals that have thus far eluded detection by traditional survey methods. In recent years, numerous studies have been published on the use of environmental DNA (eDNA) for species detection and monitoring. This method takes advantage of the persistence of DNA in the environment, such as in water, soil, and air. An organism's DNA is shed into the environment through urine, feces, and the sloughing of skin. Species‐specific quantitative polymerase chain reaction (qPCR) testing can be used to detect eDNA in samples collected from the environment. eDNA trials to detect R. swinhoei were initiated by the Asian Turtle Program and Washington State University in 2013. To expand the use of eDNA for species detection by conservationists, we developed and validated a first‐of‐its‐kind innovative point‐of‐detection (POD) qPCR platform for the rapid, onsite detection of R. swinhoei from water samples. Here we show that the portable eDNA test kit can be used for the successful detection of R. swinhoei in a large body of water and that pooling filters may be a useful strategy to reduce test costs and improve detection efficiency. Use of this test can expand the search for R. swinhoei in unexplored and understudied lakes, reservoirs, and other bodies of water where this species may be present and could inform field surveys utilizing eDNA for other threatened species that are rare in nature.

The use of environmental DNA for biodiversity monitoring in lentic and lotic ecosystems

Abstract Global biodiversity is facing an extinction crisis leading to increasing pressure on industries to monitor their potential environmental impact. Relatedly, there is demand for more efficient biodiversity monitoring methods, resulting in growing interest in the use of environmental DNA (eDNA). Many questions, however, regarding the reliability of this relatively novel method remain, particularly for non‐specialist end‐users of the technology. Here, the use of commercially available (in the UK) eDNA assays for monitoring freshwater fish and invertebrate biodiversity was compared to conventional surveillance techniques. Samples were collected from different habitats, on varying spatial scales and using multiple sampling regimes to assess how eDNA results were affected. For aquatic macroinvertebrates and fish, more taxa were detected by eDNA than conventional surveys conducted in parallel, and for fish, all taxa detected by conventional monitoring were confirmed by eDNA. For aquatic macroinvertebrates, several species were only detected through conventional methods, and the number of families detected by eDNA was lower than for conventional monitoring at all sites. eDNA results varied significantly between sampling locations within lentic sites and, for lotic sites, with the number of subsamples collected. Practical implication. This study demonstrates the need for bespoke sampling protocols when collecting eDNA samples. It also improves understanding of using eDNA for detecting aquatic taxa that could inform species surveillance protocols. These are essential if eDNA is to be used by practitioners as a regulatory monitoring tool.

Use of environmental DNA for early detection of invasive Elodea species in interior Alaska

Abstract Invasive Canadian waterweed Elodea canadensis and western waterweed E. nuttallii are a threat to native salmon Onchorhynchus spp. in Alaska. Early detection is important to stop or mitigate spread. We evaluated detection of environmental DNA (eDNA) as a tool for early detection of these invasive species in Alaska. First, we evaluated four quantitative real-time PCR assays, one for each species, and two targeting either species, using samples taken at known infestations in Chena Lakes and Potter Marsh, Alaska. We also deployed E. nuttallii in screened containers at the Small Arms Complex Pond, Fort Wainwright, Alaska (Test Pond) in 2018 and 2019 to evaluate the detection of eDNA as a function of distance from the containers. At the known infestations, we detected the eDNA of both species in water samples. However, in our Test Pond, we only detected Elodea eDNA in 2 of 126 samples collected in 2019. Both detections were from samples collected within 10 cm of the containers. There were no detections in 60 samples collected in 2018 at the Test Pond. While there are potential uses for the eDNA markers we developed (e.g., species identification), we found no evidence to support their use as an early detection tool for Elodea in Alaska.

Environmental DNA metabarcoding reflects spatiotemporal fish community shifts in the Scheldt estuary

Estuarine ecosystems face increasing anthropogenic pressures, necessitating effective monitoring methods to mitigate their impacts on the biodiversity they harbour. The use of environmental DNA (eDNA) based detection methods is increasingly recognized as a promising tool to complement other, potentially invasive monitoring techniques. Integrating such eDNA analyses into monitoring frameworks for large ecosystems is still challenging and requires a deeper understanding of the scale and resolution at which eDNA patterns may offer insights in species presence and community composition space and time. The Scheldt estuary, characterized by its diverse habitats and complex currents, is one of the largest Western European tidal river systems. Until now, it remains challenging to obtain accurate information on fish communities living in and migrating through this ecosystem, consequently confining our knowledge to specific locations. To explore the potential of eDNA based monitoring, we simultaneously combine stow net fishing with eDNA metabarcoding, to assess spatiotemporal shifts in the Scheldt estuary's fish communities. In total, we detected 71 fish species in the estuary using eDNA metabarcoding, partly overlapping with historic fish community data gathered at the different study locations and in contrast to only 42 species using stow net fishing during the same survey period. Community compositions found by both detection methods varied among sampling locations, driven by a clear correlation to the salinity gradient. Limited effects of sampling depth and tide were observed on the eDNA metabarcoding data, allowing a significant reduction of the eDNA sampling effort for future eDNA fish monitoring campaigns in this study system. Our results further demonstrate that seasonal shifts in fish species occurrence can be detected using eDNA metabarcoding. Combining eDNA metabarcoding and stow net fishing further enhances our understanding of this vital waterway's diverse fish populations, allowing a higher resolution and more efficient monitoring strategy.

Monitoring and predicting the presence and abundance of juvenile Atlantic salmon in tributaries according to habitat characteristics using environmental DNA

AbstractConservation of the Atlantic salmon Salmo salar requires to monitor the spatial distribution and abundance of juveniles at a local scale in tributaries. However, tributaries are rarely accounted for in monitoring programs despite their importance for juvenile life stages. This is mainly because inventories of young salmon populations in tributaries can be technically challenging with traditional methods, as the number of tributaries in a watershed can be important and their access limited compared to the main stem. In this study, we tested the use of environmental DNA (eDNA) to quantify the abundance of juvenile Atlantic salmon in tributaries. We successfully detected eDNA of juvenile Atlantic salmon in 19 tributaries of three main rivers of the Gaspé Peninsula (Québec, Canada) using quantitative real‐time PCR analyses. By comparing the eDNA approach with electrofishing surveys conducted in parallel to water sampling, we found that eDNA concentrations positively correlated with juvenile abundance, total biomass, and body surface area. The use of the allometrically scaled mass (ASM) instead of abundance improved the correlation. Furthermore, we demonstrated that the levels of eDNA molecules detected for juvenile Atlantic salmon were also correlated with water temperature and canopy cover measured in each tributary. Finally, we tested if eDNA concentrations measured in a tributary could be used as a reliable indicator of juvenile abundance or biomass in that tributary. We found that our models slightly better predicted juvenile biomass than juvenile abundance. The use of ASM did not improve model prediction, suggesting that further refinement would be required in the future. Our method will facilitate the implementation of conservation practices appropriate to the ecology of juvenile Atlantic salmon in tributaries.

A comparative study on eDNA-based detection of Siamese bat catfish (Oreoglanissiamensis) in wet and dry conditions

The use of environmental DNA (eDNA) analysis has demonstrated notable efficacy in detecting the existence of freshwater species, including those that are endangered or uncommon. This application holds significant potential for enhancing environmental monitoring and management efforts. However, the efficacy of eDNA-based detection relies on several factors. In this study, we assessed the impact of rainfall on the detection of eDNA for the Siamese bat catfish (Oreoglanis siamensis). Quantitative polymerase chain reaction (qPCR) analysis indicated that samples from days with average rainfall exceeding 35 mm (classified as heavy and very heavy rain) yielded negative results. While eDNA detection remains feasible on light or moderate rainy days, a noteworthy reduction in eDNA concentration and qPCR-positive likelihood was observed. Analysis across 12 sampling sites established a statistically significant negative relationship (p < 0.001) between eDNA detection and rainfall. Specifically, for each 1 mm increase in rainfall, there was an observed drop in eDNA concentration of 0.19 copies/mL (±0.14). The findings of this study provide definitive evidence that precipitation has a significant impact on the detection of eDNA in Siamese bat catfish. However, in the case of adverse weather conditions occurring on the day of sampling, our research indicates that it is acceptable to continue with the task, as long as the rainfall is not heavy or very heavy. To enhance the effectiveness of an eDNA survey, it is crucial to consider many factors related to climatic conditions. The aforementioned factor holds significant importance not only for the specific species under scrutiny but also for the broader dynamics of the climate.

Existing evidence on the use of environmental DNA as an operational method for studying rivers: a systematic map and thematic synthesis

BackgroundEnvironmental DNA (eDNA) is the DNA that can be extracted from an environmental sample, enabling the monitoring of whole biological communities across a large number of samples, at a potentially lower cost, which can significantly benefit river conservation. A systematic mapping protocol was designed to investigate the use of eDNA in rivers, specifically in terms of research topics, geographic and taxonomic biases, as well as information gaps. Furthermore, the potential research opportunities of eDNA in rivers and possible paths to find this kind of information on available platforms are identified.MethodsA published systematic map protocol was applied, consisting of a search for published articles and gray literature in two bibliographic databases and one search engine. All search results were submitted to a 2-stage screening for relevance and pertinence in accordance with pre-defined eligibility criteria. Data extraction and codification regarding country of study, year, taxonomic group, sequencing platform, and type of technique employed resulted in a publicly available database.ResultsFrom 7372 studies initially obtained by the search, 545 met the inclusion criteria spanning a period from 2003 to 2022. The five countries with most studies are: USA (134), Japan (61), China (54), Brazil (29) and the UK (25). The most used fragments to analyze DNA are 16S and COI, whilst 26S and 23S are the least used. Only 84 (15%) of the studies reported hypervariable regions, among which the most used are V4 and V5. Regarding taxonomic groups, fishes are most often studied (176), followed by bacteria (138) and virus (52), while fungi is the least studied group (3). Concerning data availability, 229 (42%) studies provided access to sequencing data.ConclusionsThis study presents a comprehensive analysis of the available evidence regarding the implementation of the eDNA methods in rivers. The findings indicate that since the year 2003, this approach has been applied to aquatic lotic systems, and their recent increase can be attributed to the development of Next-Generation-Sequencing technologies and their reduced costs. However, there is a bias towards high-income countries, particularly USA and Europe. Widespread use and applications of this approach at a global level would allow for the generation of a large amount of information that can be compared between countries to understand if responses of aquatic systems follow similar patterns worldwide.

Barcoding a glass eel migration: a snapshot of taxa migrating upstream in the Palu River, Central Sulawesi, Indonesia

Eels of the genus Anguilla are high value food fish with a catadromous life cycle. As tropical anguillid glass eels migrate to freshwater in multi-species schools, effective management of these and other diadromous resources requires reliable data. Previous research using two morphological methods concluded that between three and five anguillid eels recruit to the Palu River in Central Sulawesi, Indonesia with interannual and seasonal variations in species composition, size structure, and co-migrating taxa. Some glass eels and other taxa could not be identified based on external morphology. To clarify the taxonomic uncertainty regarding species recruiting to the Palu River this study applied DNA barcoding using the Cytochrome Oxidase I (COI) mitochondrial DNA (mtDNA) molecular marker. Taxa identified from the first sampling of glass eels and other taxa recruiting in these mixed schools included three anguillid eels, five other fishes, two crustacea and one mollusc, presenting a snapshot of taxa migrating upstream in the Palu River. Current GenBank accessions did not enable species level identification of two fish larvae (families Eleotridae and Tetraroginae). The sequences will add to Indonesian accessions in the GenBank repository and inform resource management, including the use of environmental DNA (eDNA) methods for monitoring Indonesian anguillid eels.

Environmental DNA (eDNA) removal rates in streams differ by particle size under varying substrate and light conditions

The use of environmental DNA (eDNA) as a sampling tool offers insights into the detection of invasive and/or rare aquatic species and enables biodiversity assessment without traditional sampling approaches, which are often labor-intensive. However, our understanding of the environmental factors that impact eDNA removal (i.e., how rapidly eDNA is removed from the water column by the combination of decay and physical removal) in flowing waters is limited. This limitation constrains predictions about the location and density of target organisms after positive detection. To address this question, we spiked Common Carp (Cyprinus carpio) eDNA into recirculating mesocosms (n = 24) under varying light (shaded versus open) and benthic substrate conditions (no substrate, bare substrate, and biofilm-colonized substrate). We then collected water samples from each mesocosm at four time points (40 min, 6 h, 18 h, and 48 h), and sequentially filtered the samples through 10, 1.0, and 0.2 μm filters to quantify removal rates for different eDNA particle sizes under varying light and substrate conditions. Combining all size classes, total eDNA removal rates were higher for mesocosms with biofilm-colonized substrate compared to those with no substrate or bare (i.e., no biofilm) substrate, which is consistent with previous findings linking biofilm colonization with increased eDNA removal and degradation. Additionally, when biofilm was present, light availability increased eDNA removal; eDNA levels fell below detection after 6–18 h for open mesocosms versus 18–48 h for shaded mesocosms. Among size classes, larger particles (>10 μm) were removed faster than small particles (1.0–0.2 μm). These results suggest that changes in the distribution of eDNA size classes over time (e.g., with downstream transport) and with differing environmental conditions could be used to predict the location of target organisms in flowing waters, which will advance the use of eDNA as a tool for species monitoring and management.

The Ecological Quality Status Assessment of Marine and Transitional Ecosystems: New Methods and Perspectives for the Future

Worldwide legislation emphasizes the need to monitor the health of aquatic ecosystems based on the response of biological quality elements to environmental conditions. A plethora of methodologies have been suggested in this sense. Lately, substantial efforts have led to the exploration of new biological quality elements from the meiobenthic compartment and the implementation of new methodologies based on environmental DNA. Due to their short life-cycles, meiofaunal organisms respond quickly to environmental variability. Changes in population dynamics and species composition are indicative of changes in environmental conditions. Recent pioneer studies have shown that biotic indices based on benthic foraminifera and nematodes can efficiently assess the health of transitional and marine ecosystems. The use of environmental DNA, as well as other fingerprinting techniques, is increasing in biomonitoring studies, and further calibrations are still needed to implement this method. The published papers in this Special Issue represent well the wide applicability of meiobenthic groups, i.e., benthic foraminifera and nematodes, allowing us to address a key ecological knowledge gap in order to convince decision makers and stakeholders about the advantage of introducing new biological quality elements in environmental biomonitoring.

Modelling environmental DNA transport in rivers reveals highly resolved spatio-temporal biodiversity patterns

The ever-increasing threats to riverine ecosystems call for novel approaches for highly resolved biodiversity assessments across taxonomic groups and spatio-temporal scales. Recent advances in the joint use of environmental DNA (eDNA) data and eDNA transport models in rivers (e.g., eDITH) allow uncovering the full structure of riverine biodiversity, hence elucidating ecosystem processes and supporting conservation measures. We applied eDITH to a metabarcoding dataset covering three taxonomic groups (fish, invertebrates, bacteria) and three seasons for a catchment sampled for eDNA at 73 sites. We upscaled eDNA-based biodiversity predictions to approximately 1900 reaches, and assessed α- and β-diversity patterns across seasons and taxonomic groups over the whole network. Genus richness predicted by eDITH was generally higher than values from direct eDNA analysis. Both predicted α- and β-diversity varied depending on season and taxonomic group. Predicted fish α-diversity increased downstream in all seasons, while invertebrate and bacteria α-diversity either decreased downstream or were unrelated to network position. Spatial β-diversity mostly decreased downstream, especially for bacteria. The eDITH model yielded a more refined assessment of freshwater biodiversity as compared to raw eDNA data, both in terms of spatial coverage, diversity patterns and effect of covariates, thus providing a more complete picture of freshwater biodiversity.

Molecular identity crisis: environmental DNA metabarcoding meets traditional taxonomy-assessing biodiversity and freshwater mussel populations (Unionidae) in Alabama.

The use of environmental DNA (eDNA) to assess aquatic biodiversity is a growing field with great potential for monitoring and managing threatened species, like freshwater mussel (Unionidae) populations. Freshwater mussels are globally imperiled and serve essential roles in aquatic systems as a food source and as a natural water filter making their management essential for ecosystem health. Unfortunately, mussel populations are often understudied, and challenges exist to accurately and efficiently describe the full suite of species present. Multispecies eDNA approaches may also be more challenging where freshwater mussel populations are most diverse due to ongoing and significant taxonomic restructuring that has been further complicated by molecular phylogenies using mitochondrial genes. For this study, we developed a microfluidic metabarcoding array that targets a wide range of species, from invertebrates to fishes, with an emphasis on detecting unionid mussels known to be present in the Sipsey River, Alabama. We compared mussel species diversity across six sites with well-studied mussel assemblages using eDNA surveys and traditional quadrat surveys in 2016. We examined how factors such as mussel population density, biomass and location in the river substrate impacted our ability to detect certain species; and investigated unexpected eDNA detections through phylogenetic analysis. Our eDNA results for fish and mussel species were broadly consistent with the data from traditional electrofishing and quadrat-based field surveys, although both community eDNA and conventional sampling detected species unique to that method. Our phylogenetic analysis agreed with other studies that treat Pleurobema decisum and P. chattanoogaense as synonymous species; however, they are still listed as unique species in molecular databases which complicates their identity in a metabarcoding assay. We also found that Fusconaia flava and F. cerina are indistinguishable from one another using a portion of the NADH dehydrogenase Subunit 1 (ND1) marker, which may warrant further investigation into whether or not they are synonymous. Our results show that many factors impacted our ability to detect and correctly identify Unionidae mussel species. Here we describe the obstacles we faced, including the murky phylogeny of Unionidae mussels and turbid river conditions, and our development of a potentially impactful freshwater mussel monitoring eDNA assay.

Assessing the potential use of environmental DNA for multifaceted genetic monitoring of cetaceans: Example of a wandering whale in a highly disturbed bay area

Environmental DNA (eDNA) sampling of water is a powerful method for comprehensive and noninvasive monitoring of aquatic animal species. However, there have been few reports of its application to cetacean species. On June 29, 2021, a whale (nicknamed Xiaobu) appeared in Dapeng Bay off Guangdong Province, China. We used eDNA technology to obtain information related to this whale (e.g., species identification and food resources) and to trace its possible origin. Fragments of four whale mitochondrial sequences (12S rDNA, 16S rDNA, cytochrome c oxidase subunit 1, and control region) were obtained from amplicons of eDNA collected in Dapeng Bay; sequence barcoding showed that this was an Eden’s whale (Balaenoptera edeni edeni Anderson 1879). Analysis of potential prey species (PPS) suggested that this whale might enter Dapeng Bay while tracking prey, mainly sardines (Sardinella lemuru, Sardinella gibbosa and Sardinella jussieui) and anchovies (Thryssa dussumieri, Thryssa vitrirostris and Thryssa kammalensis). Retrieval of eDNA metabarcoding data from samples collected in waters adjacent to Dapeng Bay (i.e. Lingding Bay and Daya Bay) revealed that Eden’s whale had appeared outside Lingding Bay up to 2 months prior to the appearance of this whale in Dapeng Bay (early April 2021). Overall, this study showed that eDNA is a highly effective noninvasive survey method for the accurate identification of target cetacean species and prey composition; it can be used to monitor megafauna that are under strict legal protection or to monitor megafauna with unknown conditions.

A validated protocol for eDNA-based monitoring of within-species genetic diversity in a pond-breeding amphibian

In light of the dramatic decline in amphibian biodiversity, new cost-efficient tools to rapidly monitor species abundance and population genetic diversity in space and time are urgently needed. It has been amply demonstrated that the use of environmental DNA (eDNA) for single-species detection and characterization of community composition can increase the precision of amphibian monitoring compared to traditional (observational) approaches. However, it has been suggested that the efficiency and accuracy of the eDNA approach could be further improved by more timely sampling; in addition, the quality of genetic diversity data derived from the same DNA has been confirmed in other vertebrate taxa, but not amphibians. Given the availability of previous tissue-based genetic data, here we use the common frog Rana temporaria Linnaeus, 1758 as our target species and an improved eDNA protocol to: (i) investigate differences in species detection between three developmental stages in various freshwater environments; and (ii) study the diversity of mitochondrial DNA (mtDNA) haplotypes detected in eDNA (water) samples, by amplifying a specific fragment of the COI gene (331 base pairs, bp) commonly used as a barcode. Our protocol proved to be a reliable tool for monitoring population genetic diversity of this species, and could be a valuable addition to amphibian conservation and wetland management.

Towards environmental detection, quantification, and molecular characterization of Anopheles stephensi and Aedes aegypti from experimental larval breeding sites

The invasion and establishment of An. stephensi mosquitoes in the Horn of Africa represents a significant regional threat, which may jeopardise malaria control, particularly in urban areas which were formally free from disease transmission. Novel vector surveillance methods are urgently needed, both agnostic to mosquito larval morphology, and simple to implement at the sampling stage. Using new multiplex TaqMan assays, specifically targeting An. stephensi and Ae. aegypti, we validated the use of environmental DNA (eDNA) for simultaneous vector detection in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as one second instar larva in 1L of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-sequencing panels for both vector species, was possible from eDNA shed by as few as 16–32 s instar larvae in 50 ml of water. An. stephensi eDNA, derived from emergent pupae for 24 h, was remarkably stable, and still detectable ~ 2 weeks later. eDNA surveillance has the potential to be implemented in local endemic communities and at points of country entry, to monitor the spread of invasive vector species. Further studies are required to validate the feasibility of this technique under field conditions.