The European green toad (Bufotes viridis) is currently in decline and considered endangered across the northern extent of its native range, with large investments in ongoing conservation and translocation efforts. To assist conservation efforts, survey methods must be established that are cost-effective, non-invasive, and rapidly deployable. Here we evaluated the effectiveness of eDNA metabarcoding for amphibian conservation across three objectives: (1) Test B. viridis probability of detection before and after translocation efforts in 3 ponds in & Ouml;land, Sweden. (2) Assess pond biodiversity and biotic interactions across & Ouml;land and Kalmar using eDNA metabarcoding. (3) Determine which surveyed sites are suitable for future translocation efforts. We found that the detection probability of B. viridis increased 100% 24 h after the translocation was initiated, whereby they were undetected prior to release. Additionally, we detected 11 fish species, 14 bird species, 9 mammal species, and 4 amphibian species across the translocated sites. The results from the 37 pond eDNA surveys resulted in the detection of 15 fish species, 38 bird species, 8 amphibian species, and 17 mammal species. Species richness of the surveyed ponds ranged from 1 to 24, with an average richness of 8. Co-occurrence analysis found significant associations between several species, including a significant negative association between amphibian occurrence and cattle and gray heron and positive associations with duck and common crane. Multi-Criteria Decision Analysis (MCDA) suggests 6 sites had consistent lower site rankings, indicating them as more favorable locations for future amphibian translocation efforts. Overall, these findings showcase eDNA high-throughput sequencing as a viable means to non-invasively assess European green toads and simultaneously assess wider community dynamics that may help evaluate the sustainability of reintroduced and endemic populations.

AbstractThe field of environmental DNA (eDNA) has rapidly advanced in recent years, providing a non‐invasive and time‐saving method for assessing biodiversity. The First International Environmental DNA (eDNA) Workshop in Hong Kong was held from 16 to 27 October 2023 and provided early career professionals with hands‐on training and collaboration opportunities in eDNA research. With support from The Croucher Foundation Limited (Hong Kong), the workshop covered all stages of an eDNA‐based research project, including study design, field sampling, eDNA extraction, library preparation, bioinformatics, statistical data analysis, and ethics in scientific research. Participants gained insights into the principles and practical applications of eDNA technology, emphasizing the importance of careful experimental design and data interpretation. The workshop also highlighted the need for standardized protocols, comprehensive and local DNA reference databases, and careful selection of primer sets to overcome current issues and limitations. Workshop participants expressed enthusiasm for the potential of eDNA metabarcoding as a valuable tool for ecological monitoring, biodiversity assessment, and conservation decision‐making. The future of eDNA research looks promising overall, with continued advancements in technology, collaboration among researchers, and the integration of eDNA into large‐scale ecological monitoring. Future iterations of the Hong Kong International eDNA workshop will continue to provide hands‐on training and collaboration opportunities for early career professionals interested in eDNA research, focusing on addressing current limitations and challenges in the field.

Polar bears (Ursus maritimus), Eurasian lynx (Lynx lynx) and snow leopards (Panthera uncia) are elusive large carnivores inhabiting snow-covered and remote areas. Their effective conservation and management are challenged by inadequate population information, necessitating development of novel data collection methods. Environmental DNA (eDNA) from snow tracks (footprints in snow) has identified species based on mitochondrial DNA, yet its utility for individual-based analyses remains unsolved due to challenges accessing the nuclear genome. We present a protocol for capturing nuclear eDNA from polar bear, Eurasian lynx and snow leopard snow tracks and verify it through genotyping at a selection of microsatellite markers. We successfully retrieved nuclear eDNA from 87.5% (21/24) of wild polar bear snow tracks, 59.1% (26/44) of wild Eurasian lynx snow tracks, and the single snow leopard sampled. We genotyped over half of all wild polar bear samples (54.2%, 13/24) at five loci, and 11% (9/44) of wild lynx samples and the snow leopard at three loci. Genotyping success from Eurasian lynx snow tracks increased to 24% when tracks were collected by trained rather than untrained personnel. Thirteen wild polar bear samples comprised 11 unique genotypes and two identical genotypes; likely representing 12 individual bears, one of which was sampled twice. Snow tracks show promise for use alongside other non-invasive and conventional methods as a reliable source of nuclear DNA for genetic mark-recapture of elusive and threatened mammals. The detailed protocol we present has utility for broadening end user groups and engaging Indigenous and local communities in species monitoring.

Wetlands are biodiversity hotspots that provide several essential ecosystem services. On a global scale, wetlands have greatly declined due to human activities. To counteract wetland loss, created wetlands are used as a conservation tool to facilitate biodiversity and provide habitats mainly for birds and amphibians. Fishes are likely to colonise the created wetlands and potentially affect the diversity and occurrence of amphibians. Still, species occurrence data for fish in created wetlands are largely lacking. Using eDNA metabarcoding, we investigated occurrence and co-occurrence patterns of fish and amphibian communities in 52 wetlands (some of which are ponds) created to benefit mainly bird and amphibian communities in south-central Sweden. Altogether, 17 fish and five amphibian species were detected in the created wetlands out of the 32 fish and six amphibian species found in the regional species pool. Amphibians were less common in wetlands physically connected to other wetlands. Connected wetlands were more fish-rich than isolated ones, suggesting potential fish avoidance. Additionally, the amphibian community occupied a narrower environmental niche compared to the fish community. Nevertheless, we observed only five statistically significant negative fish-amphibian species co-occurrences (out of 14 species considered). Even though our results suggest amphibian avoidance/exclusion from the created biodiversity wetlands, they also show that fish and amphibians frequently co-exist. Increased habitat heterogeneity in terms of water vegetation, size, shape, and structure of the wetland could be possible factors enabling the co-existence of these two taxa. With this study, we contribute to the general knowledge of fish occurrence patterns in created biodiversity wetlands. By comparing the frequencies of fish occurrence in natural and created wetlands, we have shown that there was some mismatch in what is common in natural compared to created wetlands. This mismatch probably comes from species-specific habitat requirements, stocking, and differences in detectability when using eDNA metabarcoding (small species detected) versus conventional multi-mesh gill-net methods (small species missed). Therefore, our results obtained using eDNA metabarcoding can complement the pre-existing knowledge of amphibian and fish associations and increase our understanding of how to create wetlands to facilitate biodiversity of several taxa.

DNA-based methods for biodiversity assessment This book represents a synthesis of knowledge and best practice in the field of DNA-based biomonitoring at the time of writing. It has been written with end-users of molecular tools in mind, as well as those who are new to the field in research settings and are looking to gain an overall grounding in the subject area. For each of the main types of sample (water, soil / sediment, bulk invertebrates and diatoms), and for each stage of the field and laboratory processes, we outline key considerations, decisions that need to be made, factors that might influence those decisions, and trade-offs inherent in the choices made. We hope that this will help users, practitioners, and those commissioning DNA-based monitoring programmes to navigate this large field and critically evaluate the strengths and weaknesses of different analysis workflows based on context, project aims and available resources.

Sound environmental management decisions - in accordance with the EU WFD for aquatic ecosystems – mainly depend on reliable species presence- and distribution- data. Here we present a workflow from sampling strategies to results and decision making using eDNA metabarcoding analyses for fish, amphibians, and mussels from habitat to landscape scales with focus on sampling strategies for "big data" in marine and freshwater ecosystems in Sweden. The project LifeDNAquatic highlights a solid eDNA pipeline and comparison of methods, which cover field planning and the entire pipeline generating data for Species Distribution Models (SDMs). Intense sampling over a large river catchment highlights previoulsy unanswered questionsand and provides insights to a priori settings for sampling strategies to retrieve "big data". The results provide novel insights to DNA distribution in the environment, seasonal and spatial changes in eDNA composition, and validation of data.

Coral reefs worldwide are rapidly declining due to increasing anthropogenic stressors and environmental changes, with large-scale mortalities of coral reefs observed in many locations across the globe. It has become clear that the microbiome of corals is important in understanding the causes of coral infections, although its exact role is yet to be fully understood. Here, we characterize the bacteria and fungi associated with the non-lesional and lesional (identified by discoloration and tissue loss) tissues of coral species from Vietnam. Metabarcoding of the bacterial 16S rRNA gene and the fungal ITS rRNA gene region were performed. We sampled across two Porites species with potentially multiple causes of stresses, yet the bacterial compositions of lesional regions were consistently different from non-lesional areas of the same coral. These differences were driven by a considerable and significant increase in OTU richness within the lesional region compared to the non-lesional region. While no single OTU was consistently associated with lesional tissue, indicator analysis revealed that nine OTUs were significantly more persistent in the lesional regions that could represent useful bioindicators of stress. Meanwhile, there were no indicator OTUs in the non-lesional region. Further investigations are needed to determine whether changing bacterial communities play a mechanistic role in inducing lesioning, or are opportunistically colonizing stressed corals.

Schistosomiasis is a waterborne, infectious disease with high morbidity and significant economic burdens affecting more than 250 million people globally. Disease control has, with notable success, for decades focused on drug treatment of infected human populations, but a recent paradigm shift now entails moving from control to elimination. To achieve this ambitious goal more sensitive diagnostic tools are needed to monitor progress towards transmission interruption in the environment, especially in low-intensity infection areas. We report on the development of an environmental DNA (eDNA) based tool to efficiently detect DNA traces of the parasite Schistosoma mansoni directly in the aquatic environment, where the non-human part of the parasite life cycle occurs. To our knowledge, this is the first report of the successful detection of S. mansoni in freshwater samples using aquatic eDNA. True eDNA was detected in as few as 10 cercariae/L water in laboratory experiments. The field applicability of the method was tested at known transmission sites in Kenya, where comparison of schistosome detection by conventional snail surveys (snail collection and cercariae shedding) with eDNA (water samples) showed 71% agreement between the methods. The eDNA method furthermore detected schistosome presence at two additional sites where snail shedding failed, demonstrating a higher sensitivity of eDNA sampling. We conclude that eDNA provides a promising new tool to significantly improve the environmental surveillance of S. mansoni . Given the proper method and guideline development, eDNA could become an essential future component of the schistosomiasis control tool box needed to achieve the goal of elimination.Significance Accurate detection and delineation of schistosomiasis transmission sites will be vital in on-going efforts to control and ultimately eliminate one of the most neglected tropical parasitic diseases affecting more than 250 million people worldwide. Conventional methods to detect parasites in the environment are cumbersome and have low sensitivity. We therefore developed an environmental DNA (eDNA) based method for schistosome detection in aquatic environments. Aquatic eDNA showed higher sensitivity than conventional snail surveys. We conclude that eDNA is a promising non-invasive and sensitive tool for environmental surveillance of schistosomiasis transmission. As the efforts and aims to control the disease are transitioning towards complete transmission interruption, this could be the robust and cost-effective surveillance tool needed in the “end game” of schistosomiasis.

Schistosomiasis is a water-based, infectious disease with high morbidity and significant economic burdens affecting >250 million people globally. Disease control has, with notable success, for decades focused on drug treatment of infected human populations, but a recent paradigm shift now entails moving from control to elimination. To achieve this ambitious goal, more sensitive diagnostic tools are needed to monitor progress toward transmission interruption in the environment, especially in low-intensity infection areas. We report on the development of an environmental DNA (eDNA)-based tool to efficiently detect DNA traces of the parasite Schistosoma mansoni directly in the aquatic environment, where the nonhuman part of the parasite life cycle occurs. This is a report of the successful detection of S. mansoni in freshwater samples by using aquatic eDNA. True eDNA was detected in as few as 10 cercariae per liter of water in laboratory experiments. The field applicability of the method was tested at known transmission sites in Kenya, where comparison of schistosome detection by conventional snail surveys (snail collection and cercariae shedding) with eDNA (water samples) showed 71% agreement between the methods. The eDNA method furthermore detected schistosome presence at two additional sites where snail shedding failed, demonstrating a higher sensitivity of eDNA sampling. We conclude that eDNA provides a promising tool to substantially improve the environmental surveillance of S. mansoni Given the proper method and guideline development, eDNA could become an essential future component of the schistosomiasis control tool box needed to achieve the goal of elimination.

Aqueous environmental DNA (eDNA) is an emerging efficient non-invasive tool for species inventory studies. To maximize performance of downstream quantitative PCR (qPCR) and next-generation sequencing (NGS) applications, quality and quantity of the starting material is crucial, calling for optimized capture, storage and extraction techniques of eDNA. Previous comparative studies for eDNA capture/storage have tested precipitation and open' filters. However, practical enclosed' filters which reduce unnecessary handling have not been included. Here, we fill this gap by comparing a filter capsule (Sterivex-GP polyethersulfone, pore size 022m, hereafter called SX) with commonly used methods. Our experimental set-up, covering altogether 41 treatments combining capture by precipitation or filtration with different preservation techniques and storage times, sampled one single lake (and a fish-free control pond). We selected documented capture methods that have successfully targeted a wide range of fauna. The eDNA was extracted using an optimized protocol modified from the DNeasy((R)) Blood & Tissue kit (Qiagen). We measured total eDNA concentrations and Cq-values (cycles used for DNA quantification by qPCR) to target specific mtDNA cytochrome b (cyt b) sequences in two local keystone fish species. SX yielded higher amounts of total eDNA along with lower Cq-values than polycarbonate track-etched filters (PCTE), glass fibre filters (GF) or ethanol precipitation (EP). SX also generated lower Cq-values than cellulose nitrate filters (CN) for one of the target species. DNA integrity of SX samples did not decrease significantly after 2weeks of storage in contrast to GF and PCTE. Adding preservative before storage improved SX results. In conclusion, we recommend SX filters (originally designed for filtering micro-organisms) as an efficient capture method for sampling macrobial eDNA. Ethanol or Longmire's buffer preservation of SX immediately after filtration is recommended. Preserved SX capsules may be stored at room temperature for at least 2weeks without significant degradation. Reduced handling and less exposure to outside stress compared with other filters may contribute to better eDNA results. SX capsules are easily transported and enable eDNA sampling in remote and harsh field conditions as samples can be filtered/preserved on site.

A majority of the sea cage farms in South East Asia are located close to coral reefs. This causes a conflict between conservation and food production since sea cage aquaculture has a number of negative impacts on coral reefs. The aim of this investigation was to assess the drivers causing the sea cage farmers to place their farms close to reefs and to examine some potential farming effects in detail i.e. usage of coral reef fish for grow out farming and feed. For some 3500 Vietnamese fish and lobster farms, we measured; the distance to the closest coastal city (proxy for infrastructure access), satellite derived Chl a (proxy for water quality), wind fetch, and the adjacent coastal slope and elevation. We also performed 159 semi-structured interviews with fish and lobster cage farmers from three regions in Vietnam. The interviews revealed that the choice of farming site is mainly determined by access to infrastructure, wind and wave shelter, and water quality. Although the farmers used coral reef services, e.g. coral reef derived seedlings, they were in general not aware of coral reef presence or did not find it important for selection of site. Both coral reefs and sea cage farms were found close to steep rocky coasts, which are favorable for corals, and provide sufficient depth for sea cages. Sea cages were always found on the leeward side of the coast where the wind fetch is low enough for the floating farms and their inhabitants. Most of the farms were located within 20 km from a coastal city confirming the importance of access to infrastructure. With few exceptions, sea cage farms were located in areas with good water quality, where also coral reefs are present. The study showed that several of the coral associated species groups farmed were dependent on wild caught seedlings and that 22% of the feed used at farms was "trashfish" of coral reef associated species. We consider the spatial correlation between sea cage farms and coral reefs as circumstantial and suggest that shared environmental preferences explain the farm distribution pattern, rather than access to ecosystem services provided by the nearby reef itself. We found no evidence that it is necessary for sea cage farms to be located near coral reefs and strongly recommend that sea cages are moved further away from coral reefs, but to areas still providing clear water, shelter and access to infrastructure.

The combined effects of the herbicide glyphosate and elevated temperature were studied on the tropical staghorn coral Acropora formosa, in Nha Trang bay, Vietnam. The corals were collected from two different reefs, one close to a polluted fish farm and one in a marine-protected area (MPA). In the laboratory, branches of the corals were exposed to the herbicide glyphosate at ambient (28 °C) and at 3 °C elevated water temperatures (31 °C). Effects of herbicide and elevated temperature were studied on coral bleaching using photography and digital image analysis (new colorimetric method developed here based on grayscale), chlorophyll a analysis, and symbiotic dinoflagellate (Symbiodinium, referred to as zooxanthellae) counts. All corals from the MPA started to bleach in the laboratory before they were exposed to the treatments, indicating that they were very sensitive, as opposed to the corals collected from the more polluted site, which were more tolerant and showed no bleaching response to temperature increase or herbicide alone. However, the combined exposure to the stressors resulted in significant loss of color, proportional to loss in chlorophyll a and zooxanthellae. The difference in sensitivity of the corals collected from the polluted site versus the MPA site could be explained by different symbiont types: the resilient type C3u and the stress-sensitive types C21 and C23, respectively. The additive effect of elevated temperatures and herbicides adds further weight to the notion that the bleaching of coral reefs is accelerated in the presence of multiple stressors. These results suggest that the corals in Nha Trang bay have adapted to the ongoing pollution to become more tolerant to anthropogenic stressors, and that multiple stressors hamper this resilience. The loss of color and decrease of chlorophyll a suggest that bleaching is related to concentration of chloro-pigments. The colorimetric method could be further fine-tuned and used as a precise, non-intrusive tool for monitoring coral bleaching in situ.

The protection, preservation and restoration of aquatic ecosystems and their functions are of global importance. For European states it became legally binding mainly through the EU-Water Framework Directive (WFD). In order to assess the ecological status of a given water body, aquatic biodiversity data are obtained and compared to a reference water body. The quantified mismatch obtained determines the extent of potential management actions. The current approach to biodiversity assessment is based on morpho-taxonomy. This approach has many drawbacks such as being time consuming, limited in temporal and spatial resolution, and error-prone due to the varying individual taxonomic expertise of the analysts. Novel genomic tools can overcome many of the aforementioned problems and could complement or even replace traditional bioassessment. Yet, a plethora of approaches are independently developed in different institutions, thereby hampering any concerted routine application. The goal of this Action is to nucleate a group of researchers across disciplines with the task to identify gold-standard genomic tools and novel eco-genomic indices for routine application in biodiversity assessments of European fresh- and marine water bodies. Furthermore, DNAqua-Net will provide a platform for training of the next generation of European researchers preparing them for the new technologies. Jointly with water managers, politicians, and other stakeholders, the group will develop a conceptual framework for the standard application of eco-genomic tools as part of legally binding assessments.

DNA obtained from environmental samples such as sediments, ice or water (environmental DNA, eDNA), represents an important source of information on past and present biodiversity. It has revealed an ancient forest in Greenland, extended by several thousand years the survival dates for mainland woolly mammoth in Alaska, and pushed back the dates for spruce survival in Scandinavian ice-free refugia during the last glaciation. More recently, eDNA was used to uncover the past 50 000 years of vegetation history in the Arctic, revealing massive vegetation turnover at the Pleistocene/Holocene transition, with implications for the extinction of megafauna. Furthermore, eDNA can reflect the biodiversity of extant flora and fauna, both qualitatively and quantitatively, allowing detection of rare species. As such, trace studies of plant and vertebrate DNA in the environment have revolutionized our knowledge of biogeography. However, the approach remains marred by biases related to DNA behaviour in environmental settings, incomplete reference databases and false positive results due to contamination. We provide a review of the field.

In the South China and Java Seas, cage farming is a recent regional activity, which since the year 2000 has experienced an annual growth of 29%. The region holds the highest diversity of marine life, which is partly or completely dependent on coral reefs. The increasingly growing coastal human population in the area relies on ecosystem goods and services provided by the reefs that are threatened by anthropogenic activities. Sea cage farming is one of the stressors negatively impacting coral reefs by being point sources of nutrients and other effluents. To date no systematic information is available on the physical location of marine farms in relation to the coral reefs. Little is known about the distance where impact from the farms can be detected on nearby coral reefs. The present survey aimed to fill this gap by assessing to what extent marine cage farms in South East Asia are placed in the vicinity of the reefs and at which distance stress indicators from the farms are observed. We used Google Earth satellite images to investigate the extension and spatial distribution of sea cage aquaculture in relation to the presence of coral reefs. The stress indicators were locally assessed in Central Vietnam by recording turf algal overgrowth, coral mortality, live coral and branching coral cover at increasing distances from the farms. We found that 90% of sea cage farms throughout the region clustered closer than 5 km from coral reefs and 50% of them closer than 1 km from reefs. In Taiwan, 71% of the cages were located within 100m from a reef. This pattern is nonrandom and could not be explained by the natural distribution of coral reefs; only 5% of the Vietnamese coast harbors coral reefs, and sea cage farms are present in these areas only. This indicates that the farms require similar conditions as the reefs including clear and shallow waters and protection against storms and wave action. We found that turf algal overgrowth decreased at 287 m +/- 54 m, dead coral at 1446m +/- 154 m, live coral cover increased at 566 +/- 221 m and branching corals increased at 867 m +/- 140 m from the cage farms. We conclude that proximity to coral reefs should be considered when planning future developments of sea cage aquaculture, and recommend that distances of at least 1.5 km should be kept.Statement of relevance: Consider coral reefs when planning sea cage aquaculture site. (C) 2015 Published by Elsevier B.V.

We aim to understand ontogenetic shifts in habitat use and feeding patterns by 2 fish species, Lutjanus fulviflamma and L. ehrenbergii, within a tropical seascape in East Africa. Stomach contents and stable isotope signatures of muscle tissues (delta C-13 and delta N-15) were compared between and within species. Fish of all life stages and potential food items were sampled from mangrove creeks, seagrass beds, and coral reefs around Mafia Island, Tanzania. Due to similarities in morphology between species, correct species identity was confirmed using genetic barcoding (mtDNA, partial sequence of cytochrome oxidase subunit I [COI]). Stable isotope analysis in R (based on mixing models) confirmed that delta C-13 and delta N-15 values in L. fulviflamma and L. ehrenbergii reflected those of prey items caught in different habitats. Diets and mean delta C-13 and delta N-15 values of muscle tissue differed between life stages of fish, indicating ontogenetic changes in habitat and diet. L. fulviflamma and L. ehrenbergii differed in diet and delta C-13 and delta N-15 values of muscle tissue, although they overlapped in habitat use, suggesting food resource partitioning between the 2 species. Furthermore, diet overlap indexes were low between subadult species in mangrove and seagrass or coral habitats. L. fulviflamma displayed a diet shift with decreasing importance of small crustaceans in juveniles and an increasing importance of prey fishes in subadults and adults. L. ehrenbergii showed the opposite pattern. The study verifies feeding interlinkage within the mangrove-seagrass-coral reef continuum in Mafia Island by providing strong evidence of ontogenetic migration. Understanding these connections will enhance our ability to manage tropical seascapes, and highlights the need to include multiple habitats in marine protected areas.