eDNA Surveys Research Articles

Assembly processes inferred from eDNA surveys of a pond metacommunity are consistent with known species ecologies

Technological advances are enabling ecologists to conduct large‐scale and structured community surveys. However, it is unclear how best to extract information from these novel community data. We metabarcoded 48 vertebrate species from their eDNA in 320 ponds across England and applied the ‘internal structure' approach, which uses joint species distribution models (JSDMs) to explain compositions as the result of four metacommunity processes: environmental filtering, dispersal, species interactions, and stochasticity. We confirm that environmental filtering plays an important role in community assembly, and find that species' estimated environmental preferences are consistent with known ecologies. We also detect negative biotic covariances between fish and amphibians after controlling for divergent environmental preferences, consistent with predator–prey interactions (likely mediated by predator avoidance behaviour), and we detect high spatial autocorrelation for the palmate newt, consistent with its hypothesised relict distribution. Promisingly, ecologically and spatially distinctive sites are better explained by their environmental covariates and geographic locations, respectively, revealing sites where environmental filtering and dispersal limitation act more strongly. These results are consistent with the recent proposal that applying JSDMs to species distribution patterns can help reveal the relative importance of environmental filtering, dispersal limitation, and biotic interaction processes for individual sites and species. Our results also highlight the value of the modern interpretation of metacommunity ecology, which embraces the fact that assembly processes differ among species and sites. We discuss how novel community data allow for several study design improvements that will strengthen the inference of metacommunity assembly processes from observational data.

A novel ddPCR™ assay for eDNA detection and quantification of Greater Amberjack Seriola dumerilli and three congeners in US waters: challenges and application to fisheries independent surveys.

Four Seriola species support recreational and commercial fisheries along the U.S. Atlantic Ocean and the Gulf of Mexico, with the S. dumerili Gulf of Mexico stock being overfished for over three decades. The study presented here is part of a fisheries-independent project initiated to determine an absolute abundance of S. dumerili, to expand biological knowledge of the species and to develop novel tools for fisheries management. Environmental DNA (eDNA) tools aimed at the detection and quantification of target species are starting to emerge in support of marine fisheries surveys. Key to progressing the field is Droplet Digital™ PCR (ddPCR™), a highly sensitive technique with advanced multiplexing and direct quantification capabilities that can provide fisheries scientists with improved interpretation of eDNA data. We developed and validated a novel tetraplex ddPCR™ assay able to detect and distinguish between S. dumerili, S. fasciata, S. rivoliana, and S. zonata from seawater eDNA samples. In order to groundtruth ddPCR™ data, and explore its capacity to provide abundance estimates, we compared ddPCR™ detections and quantifications to abundance data inferred from multiple camera (ROV, S-BRUV, chevron trap) and acoustic (VPS array) gears deployed during a fisheries research gear-calibration cruise. We demonstrated that with eDNA contamination controls and best practice protocols, it is viable to conduct eDNA research as part of a fisheries survey cruise. eDNA sampling was completed in less time than camera gears (15 min vs 2 h). Both eDNA and camera gears detected the presence of S. dumerili and S. rivoliana at both sites and all sampling days, but not S. fasciata and S. zonata. eDNA concentration data was higher for S. dumerili than S. rivoliana at both sites for all sampling days, in line with abundance patterns obtained from camera gears. The highest correlation (r = 0.97) was obtained between the measures of eDNA between gear deployments and ROV. Incorporating eDNA in fisheries surveys would not require additional days at sea and could improve precision in fish detection and abundance. eDNA can be a valuable complement to camera gears deployed in geographic areas or seasons with poor visibility conditions, where fish may be present but cannot be confidently identified to the species level. The high correlation obtained between ROV and eDNA data collected between gear deployments adds to a growing number of studies demonstrating the potential of eDNA as an indicator of abundance for fisheries stock assessments. Time-series data from a carefully designed eDNA survey, that estimates relative abundance, could be used as an index of relative abundance for the S. dumerili stock assessment. To achieve this, investment into follow-up studies with increased sample sizes and spatial and temporal replication would be necessary to allow for year-to-year comparisons and validate the robustness of the correlation observed.

Environmental DNA (eDNA) as a tool to detect Arctic grayling and their habitat preferences in the Northwest Territories, Canada

Environmental deoxyribonucleic acid (eDNA) assays represent a non-invasive approach for biomonitoring. To assess Arctic grayling ( Thymallus arcticus, Pallas, 1776) populations in the Little Nahanni watershed of Northwest Territories, Canada, an eDNA assay that has been previously validated for use in fish of the Beringia lineage was used in conjunction with traditional survey methods (i.e., electrofishing). Forty-six 100 m reaches of streams were assessed in August 2015. The assay successfully detected Arctic grayling of the Nahanni lineage, with an eDNA signal recorded at each of the 17 sites in which Arctic grayling were observed by traditional fish surveys, but also at 3 of the 29 sites where Arctic grayling were not observed. The presence of eDNA was related to habitat metrics via Random Forest and correlation analyses. Riffles and water temperature were identified as being predictive of Arctic grayling eDNA abundance; however, no significant relationship between eDNA abundance and biomass proxies (fish abundance and fork length metrics) could be established. The high congruence between traditional approaches and eDNA surveys suggests adoption of the latter method will enhance the temporal and spatial acuity of biomonitoring, thereby improving field assessment of Arctic grayling populations and contributing towards more effective conservation management of this species.

Quantifying the Detection Sensitivity and Precision of qPCR and ddPCR Mechanisms for eDNA Samples.

Environmental DNA (eDNA) detection employing quantitative PCR (qPCR) and droplet digital PCR (ddPCR) offers a non-invasive and efficient approach for monitoring aquatic organisms. Accurate and sensitive quantification of eDNA is crucial for tracking rare and invasive species and understanding the biodiversity abundance and distribution of aquatic organisms. This study compares the sensitivity and quantification precision of qPCR and ddPCR for eDNA surveys through Bayesian inference using latent parameters from both known concentration (standards) and environmental samples across three teleost fish species assays. The results show that ddPCR offers higher sensitivity and quantification precision, particularly at low DNA concentrations (< 1 copy/μL reaction), than qPCR. These findings highlight the superior performance of ddPCR for eDNA detection at low concentrations, guiding researchers towards more reliable methods for effective species monitoring. Additionally, this study indicates that a two-step (detection and concentration) model increased the precision of qPCR results, useful for enhancing the robustness of eDNA quantification. Furthermore, we investigated the lower limit of quantification for ddPCR, providing insights on how such limit can be extended, which could also be applied to qPCR.

Investigation on the Distribution of Bangana Tungting in Yuanshui Unique Fish Species National Aquatic Germplasm Resources Reserve Using Environmental DNA Technology.

Freshwater ecosystems face unprecedented challenges as the cumulative impact of human activities intensifies. While protected areas and species-specific conservation policies are widely implemented, their effectiveness remains difficult to gauge using traditional catch-based surveys. This research employed environmental DNA (eDNA) technology to assess the distribution of the endangered fish, Bangana tungting, within the Yuanshui Unique Fish Species National Aquatic Germplasm Resources Reserve (YUFRR) in Hunan, China. Over a 2-year period, we conducted comprehensive eDNA survey multiple sites within the YUFRR, confirming the species' continued existence in the area. In September 2022, B. tungting eDNA was detected at 8 of 60 sampling locations, while a follow-up survey in May 2023 identified its presence at 4 of 44 sites. Further analysis revealed critical environmental factors influencing B. tungting distribution, primarily dissolved oxygen concentration and the presence of physical barriers such as hydroelectric stations. Our data suggest a minimum dissolved oxygen tolerance threshold of 4 mg/L for this species. Moreover, we observed an inverse relationship between B. tungting detection rates and both the number of hydroelectric stations and their distance to sampling sites. This case study demonstrates the effectiveness of eDNA technology in mapping the distribution of endangered fish species like B. tungting and guiding conservation strategies. Our findings emphasize the crucial need to enhance environmental conditions, particularly water quality and habitat suitability, to ensure the effective conservation of B. tungting within the YUFRR.

Fish Diversity in a Little-Known Border River Between China, North Korea, and Russia, According to Traditional and eDNA Surveys

The Tumen River, which delineates the border between China, North Korea, and Russia, is a vital river in Northeast Asia. Understanding its current fish diversity holds significant scientific value for aquatic ecological protection. Therefore, traditional and eDNA sampling were conducted from 2022 to 2023. Integrating the historical literature with our field collection results reveals that the Tumen River has a total of 64 native fish species, of which 51 species have been surveyed in the wild. The fish composition is mainly dominated by cold-water species, with a high proportion of these being rare, endangered, and nationally protected. eDNA surveys are crucial complements in boundary rivers where traditional methods are not feasible. The results indicated that α diversity values were highest in the Hunchunhe River, attributed to the favorable natural geographical conditions and effective conservation efforts. Recently, the fish diversity in the Tumen River has been disrupted, primarily reflected in the absence of migratory species such as Oncorhynchus gorbuscha and Pseudaspius hakonensis. The study suggests that establishing nature reserves, preventing and controlling alien species, and strengthening international cooperation are key to protect fish diversity.

Taxonomic Blind Spots: A Limitation of Environmental DNA Metabarcoding‐Based Detection for Canadian Freshwater Fishes

ABSTRACTWith increasing utilization of eDNA metabarcoding for fish community assessment, it is critical to identify, address, and communicate its capabilities and limitations. One limitation of great concern is the reliability of taxonomic coverage. Taxonomic blind spots, defined as consistent false negatives for specific taxa despite known presence, reduce corroboration with conventional surveys and can limit the uptake of eDNA metabarcoding for biomonitoring. These blind spots result from gaps in reference sequence libraries, issues with taxonomic resolution, inefficient binding of universal primers to the DNA of certain species, and ineffective collection during the sampling of eDNA. To explore this, a multiproject empirical dataset was compiled and analyzed to evaluate the taxonomic coverage of eDNA metabarcoding for a subset of Canadian freshwater fishes using a standardized workflow for two genetic markers: 12S MiFish‐U and Vertebrate COI. The compiled dataset consists of species lists generated by eDNA surveys, paired conventional surveys, and historical records. In total, 59 fish species across 15 families were evaluated of which approximately 40% were unable to be consistently detected by either marker because of a blind spot. The 12S and COI markers also differed in which kinds of blind spots were most frequently observed, with 12S markers exhibiting more reference and resolution blind spots and the COI marker exhibiting more unclassified blind spots. Additionally, in silico primer testing exhibited inconsistent predictions for amplification when using multiple software packages, suggesting the need for further in vitro analysis to troubleshoot primer‐related blind spots. This study highlights the impact of these blind spots in taxonomic coverage on eDNA metabarcoding studies of Canadian freshwater fishes. The limitations imposed by taxonomic blind spots should be addressed in future optimization efforts as eDNA metabarcoding sees broader acceptance as an applied method for fish biomonitoring.

Hydrodynamics and Aquatic Vegetation Drive Spatial Patterns of Environmental DNA in Ponds

ABSTRACTEnvironmental DNA (eDNA) sampling is a powerful method for detecting aquatic species at low densities. However, eDNA may remain close to the source in lentic systems, decreasing the effectiveness of eDNA surveys. We conducted cage experiments with salamanders and simultaneous detailed hydrologic and wind measurements to investigate the influence of the physical environment on detection patterns of eDNA in ponds. We found much higher detection rates in the surface layer than at depth, and that aquatic vegetation reduced detection of eDNA produced in open water by 80%–94%. Within the surface mixed layer, detection rates were highest close to the source in the direction of water flow in the bottom half of the layer, and detections farthest from the source occurred when velocities in this sublayer were high. Detections were near zero even close to the source when this sublayer was flowing fast and away from the sampling point. The direction of water flow in this lower half of the surface mixed layer was negatively correlated with wind direction for most of the study. These spatial and temporal dynamics indicate that eDNA transport processes in ponds are highly complex. Sampling away from aquatic vegetation, in the surface mixed layer, and upwind of potential sources, in addition to sampling at many locations within a pond and considering temporal patterns, may improve detection of rare pond species. This work contributes to a growing body of literature characterizing the variability of eDNA detection in lentic systems.

Using eDNA Sampling to Identify Correlates of Species Occupancy Across Broad Spatial Scales

ABSTRACTAimSpecies presence–absence data can be time‐consuming and logistically difficult to obtain across large spatial extents. Yet these data are important for ensuring changes in species distributions are accurately monitored and are vital for ensuring appropriate conservation actions are undertaken. Here, we demonstrate how environmental DNA (eDNA) sampling can be used to systematically collect species occupancy data rapidly and efficiently across vast spatial domains to improve understanding of factors influencing species distributions.LocationSouth‐eastern Australia.MethodsWe use a widely distributed, but near‐threatened species, the platypus (Ornithorhynchus anatinus), as a test case and undertake an environmentally stratified systematic survey to assess the presence–absence of platypus eDNA at 504 sites across 584,292 km2 of south‐eastern Australia, representing ~37% of the species' extensive distribution. Site occupancy‐detection models were used to analyse how landscape‐ and site‐level factors affect platypus occupancy, enabling us to incorporate uncertainty at the different levels inherent in eDNA sampling (site, water sample replicate and qPCR replicate).ResultsPlatypus eDNA was detected at 272 sites (~54%) with platypuses more likely to occupy sites in catchments with increased runoff and less zero‐flow days, and sites with access to banks suitable for burrowing. Platypuses were less likely to occupy sites in catchments with a high proportion of shrubs and grasslands, or agricultural land use.Main ConclusionsThese data provide an important large‐scale validation of the landscape‐ and site‐level factors influencing platypus occupancy that can be used to inform future conservation efforts. Our case study shows that systematically designed, stratified eDNA surveys provide an efficient means to understand how environmental characteristics affect species occupancy across broad environmental gradients. The methods employed here can be applied to aquatic and semi‐aquatic species globally, providing unprecedented opportunities to understand biodiversity status and change and provide insights for current and future conservation actions.

A spatial matrix factorization method to characterize ecological assemblages as a mixture of unobserved sources: An application to fish eDNA surveys

Abstract Understanding how ecological assemblages vary in space and time is essential for advancing our knowledge of biodiversity dynamics and ecosystem functioning. Metabarcoding of environmental DNA (eDNA) is an efficient method for documenting biodiversity changes in both marine and terrestrial ecosystems. However, current methods fail to detect and display the biodiversity structure within and between eDNA samples limiting ecological and biogeographical interpretations. We present a spatial matrix factorization method that identifies optimal eDNA sample assemblages—called pools—assuming that taxonomic unit composition is based on a fixed number of unknown sources. These sources, in turn, represent taxonomic units sharing similar habitat properties or characteristics. The method aims to reduce the multi‐taxa composition structure into a low number of dimensions defined by these sources. This method is inspired by admixture analysis in population genetics. Using a marine fish eDNA survey on 263 sampling stations detecting 2888 molecular operational taxonomic units (MOTUs), we apply this method to analyse the biogeography and mixing patterns of fish assemblages at regional and large scales. At large scale, our analysis reveals six primary pools of fish samples characterized by distinct biogeographic patterns, with some mixtures between these pools. We identify pools composed of unique sources, corresponding to distinct and more isolated regions such as the Mediterranean and Scotia Seas. We also identify pools composed of a greater mix of sources, corresponding to geographically connected areas, such as tropical regions. Additionally, we identify the taxa underpinning the formation of each pool. In the regional analysis of Mediterranean eDNA samples, our method successfully identifies different pools, allowing the detection of not only geographic gradients but also human‐induced gradients corresponding to protection levels. Spatial matrix factorization adds a new method in community ecology, where each sample is considered as a mixture of K unobserved sources, to assess the dissimilarity of ecological assemblages revealing environmental and human‐induced gradients. Beyond the study of fish eDNA samples, this method has the potential to shed new light on any biodiversity survey and provide new bioindicators of global change.

Using Environmental DNA to Detect Radio-Tagged King Rails: Initial Validation Results and Needed Advancements

Abstract Secretive avian species can be difficult to detect. For such species, environmental DNA (eDNA) monitoring could improve our ability to estimate occupancy and population trends. There are often challenges associated with efforts to establish eDNA as an effective monitoring tool for species of conservation concern because it is difficult to hold nonmodel organisms in captivity to complete validation tests. To address this issue, we tracked four king rails Rallus elegans in Ohio, USA, in 2020 with radio telemetry to determine whether our eDNA monitoring approach could indicate king rail presence where these individuals occurred. Also, we collected eDNA near locations surveyed by human observers within potential habitat where king rail status was unknown. The eDNA surveys indicated king rail presence at one of five locations where we knew that an individual king rail had occurred within 30 h and at one of four locations where king rail presence was unknown but feasible. At these latter locations, human observers detected no king rails, although in 2022 human observers using traditional survey methods detected a king rail near the same location where our eDNA detection occurred in 2020. This study provides a preliminary, although informative, king rail eDNA monitoring methodology that we validated with radio telemetry data. We suggest ways to improve our approach to increase king rail detection rates and to add confidence in results.

Rapid and Cost‐Effective Platypus eDNA Detection in Waterways Using Loop‐Mediated Isothermal Amplification Assay: Advancing Conservation Efforts

ABSTRACTFreshwater ecosystems, home to a remarkable diversity of species, are facing severe threats from human activities such as climate change, habitat degradation, over‐extraction of water for irrigation, and pollution. The platypus, an iconic species in freshwater ecosystems around Australia, is threatened by all these activities, both singly and in combination. The scale and complexity of these intersecting and reinforcing threats makes cost‐effective monitoring tools essential to better understand how platypus populations are responding. In this study, we optimized a loop‐mediated isothermal amplification (LAMP) assay for the rapid and cost‐effective detection of platypus DNA in environmental water samples, offering an attractive alternative to quantitative polymerase chain reaction (qPCR). We improved a water filtration protocol for in‐field use, employing suitable filter membranes for processing large volumes of water, thereby maximizing DNA recovery from dilute samples. The limit of detection for the platypus LAMP (Plat‐LAMP) assay was determined to be 12.4 copies/μL using a standard plasmid positive reference and 7 × 10−6 ng/μL when applied to DNA extracted from platypus tissue, with improved sensitivity achieved through the incorporation of locked nucleic acid primers. In comparative testing against qPCR, the Plat‐LAMP assay exhibited greater sensitivity in detecting platypus DNA in known positive water samples collected from platypus habitat at Healesville Sanctuary. Furthermore, the Plat‐LAMP assay demonstrated 100% specificity when performed on water samples collected from non‐platypus habitats. In field testing across waterways in Victoria and New South Wales, the Plat‐LAMP assay detected platypus in 36.96% of samples, compared to 54.35% of samples using qPCR. These findings underscore the Plat‐LAMP assay's potential as a faster and more cost‐effective complementary method to qPCR, rendering it suitable for point‐of‐application water testing. The ability to conduct eDNA surveys without the need for cold‐chain logistics would significantly assist conservation organizations and water managers map platypus distributions and facilitate conservation efforts around Australia.

Comparing diversity and structure of freshwater fish assemblages using environmental DNA and gillnetting methods: A case study of a large deep reservoir in East China

Comparing diversity and structure of freshwater fish assemblages using environmental DNA and gillnetting methods: A case study of a large deep reservoir in East China

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.

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 &gt; Hydrological Processes Science of Water &gt; Methods Water and Life &gt; Nature of Freshwater Ecosystems

Optimising species detection probability and sampling effort in lake fish eDNA surveys

Environmental DNA (eDNA) metabarcoding is transforming biodiversity monitoring in aquatic environments. Such an approach has been developed and deployed for monitoring lake fish communities in Great Britain, where the method has repeatedly shown a comparable or better performance than conventional approaches. Previous analyses indicated that 20 water samples per lake are sufficient to reliably estimate fish species richness, but it is unclear how reduced eDNA sampling effort affects richness, or other biodiversity estimates and metrics. As the number of samples strongly influences the cost of monitoring programmes, it is essential that sampling effort is optimised for a specific monitoring objective. The aim of this project was to explore the effect of reduced eDNA sampling effort on biodiversity metrics (namely species richness and community composition) using algorithmic and statistical resampling techniques of a data set from 101 lakes, covering a wide spectrum of lake types and ecological quality. The results showed that reliable estimation of lake fish species richness could, in fact, usually be achieved with a much lower number of samples. For example, in almost 90% of lakes, 95% of complete fish richness could be detected with only 10 water samples, regardless of lake area. Similarly, other measures of alpha and beta-diversity were not greatly affected by a reduction in sample size from 20 to 10 samples. We also found that there is no significant difference in detected species richness between shoreline and offshore sampling transects, allowing for simplified field logistics. This could potentially allow the effective sampling of a larger number of lakes within a given monitoring budget. However, rare species were more often missed with fewer samples, with potential implications for monitoring of invasive or endangered species. These results should inform the design of eDNA sampling strategies, so that these can be optimised to achieve specific monitoring goals.

Can environmental DNA be used within pest insect agricultural biosecurity? Detecting khapra beetle within stored rice

AbstractEnvironmental DNA (eDNA) has recently emerged as an effective tool for invasive species biosecurity. We explored the use of eDNA for the detection of khapra beetle (Trogoderma granarium, Everts 1898), an invasive insect of cereal grains and other food products that has a high global economic impact. We developed a novel method for aggregating khapra beetle eDNA deposited in stored grain that entails washing a sample of rice, filtering the sample, and detecting trace beetle DNA using a standard qPCR workflow. To explore the performance of this method, we raised 500 khapra beetle larvae within 500 g of rice over a 14‐day period and then removed them. We then used this “spiked” rice to create a range of simulated densities of khapra beetle larvae. This lab approach mimics conditions that are comparable to field densities of ~1.4 to 180 beetles per 50 kg of rice (1/8 to 16 spiked rice grains per 100 g sample of clean rice), assuming DNA is uniformly distributed within the rice. We detected khapra beetle eDNA from all density levels tested. Logistic models revealed that eDNA amounts equivalent to what is left by ~1 khapra beetle larva in a 50 kg container of rice can be detected with 85% to &gt;97% certainty, depending on the number of qPCR technical replicates run per sample. Based on this model, we estimated that for one 50 kg container of rice where beetle DNA is uniformly distributed, a single sample of 100 g with six technical replicates would be sufficient to be &gt;99% certain that the container was free of khapra beetle eDNA (95% credible intervals: 97.7%–100%). Our results suggest that eDNA surveys may be useful as a cost‐effective, first‐step detection of khapra beetle in stored grain and provide a means to map the relative magnitude of khapra beetle transport pathways, informing allocation of conventional biosecurity inspection efforts.

Comparison of soil eDNA to camera traps for assessing mammal and bird community composition and site use.

Species detections often vary depending on the survey methods employed. Some species may go undetected when using only one approach in community-level inventory and monitoring programs, which has management and conservation implications. We conducted a comparative study of terrestrial mammal and bird detections in the spring and summer of 2021 by placing camera traps at 30 locations across a large military installation in northern Michigan, USA and testing replicate soil samples from these sites for environmental DNA (eDNA) using an established vertebrate metabarcoding assay. We detected a total of 48 taxa from both survey methods: 26 mammalian taxa (excluding humans, 24 to species and two to genus) and 22 avian taxa (21 to species and one to genus). We detected a relatively even distribution of mammalian taxa on cameras (17) and via eDNA analysis (15), with seven taxa detected from both methods. Most medium-to-large carnivores were detected only on cameras, whereas semi-fossorial small mammals were detected only via eDNA analysis. We detected higher bird diversity with camera traps (18 taxa) compared to eDNA analysis (eight taxa; four taxa were detected with both methods), but cameras alone were most effective at detecting smaller birds that frequently occupy arboreal environments. We also used Bayesian spatial occupancy models for two widely distributed game species (white-tailed deer, Odocoileus virginianus, and ruffed grouse, Bonasa umbellus) that were moderately detected with both survey methods and found species-specific site use (occupancy) estimates were similar between cameras and eDNA analysis. Concordant with similar studies, our findings suggest that a combination of camera trap and eDNA surveys could be most useful for assessing the composition of terrestrial mammal communities. Camera traps may be most efficient for assessing bird diversity but can be complemented with eDNA analysis, particularly for species that spend considerable time on the ground.

Rapid eDNA survey reveals a unique biodiversity hotspot: The Corubal River, West Africa

Abstract The Corubal (Guinea-Bissau) is a wild but underexplored river in West Africa. This study underscores the potential of environmental DNA (eDNA) surveys to fill biodiversity knowledge gaps in the region. We filtered large water volumes at 11 sites along the watershed, amplified multiple molecular markers, and performed high PCR (polymerase chain reaction) replication and in-depth sequencing. We recorded 2589 amplicon sequence variants, with accumulation curves indicating the need for additional sampling to achieve a thorough survey. The taxonomic assignments were constrained by the scarcity of genomic resources. We recorded 125 species of aquatic and terrestrial vertebrates, including 21 new to the country, predominantly fish (61.9%). Surprisingly, crocodiles were not detected, despite their known presence. There were 11 imperiled species, two of which are Critically Endangered (Western chimpanzee and the mussel Pleiodon ovatus). Our findings support the conservation importance of the Corubal, provide a baseline for future monitoring, and highlight the challenges and opportunities of eDNA surveys in remote tropical rivers.

Detection of environmental DNA of finless porpoise (Neophocaena asiaeorientalis) in Osaka Bay, Japan

Finless porpoises (Neophocaena asiaeorientalis) currently face population decline caused by significant human activities and are categorized as endangered on the International Union for Conservation of Nature (IUCN) Red List; however, information on their habitats is currently insufficient. Although conducting visual surveys to determine the distribution of cetaceans is common, visual observation of finless porpoises is challenging owing to their specific morphological and ecological characteristics. In this study, we developed an environmental DNA (eDNA) assay for species-specific detection of finless porpoises. To test the utility of the assay, we conducted a visual survey in parallel with an eDNA survey by collecting water samples from 50 sites throughout Osaka Bay. We visually found a finless porpoise at one location and detected eDNA at nine sites, including sites near the visual observation site and those with rare sightings of finless porpoises. Therefore, in this study, we suggest that the use of eDNA analysis for distribution surveys of finless porpoises will enable more efficient surveys. The proposed eDNA technique can not only be applied to the distribution surveys finless porpoises but also to those of other cetacean species.