Journal of AOAC International

ABSTRACTMetabarcoding of environmental DNA (eDNA) is becoming practically applied to fish monitoring and conservation surveys in estuaries. However, estuarine bays may be an unsuitable zone to use eDNA metabarcoding because they are affected by eDNA originating from upstream rivers. In this study, the transition of eDNA composition from river to bay was examined to investigate the influence of freshwater sources on the eDNA composition of the downstream bay. Samples were collected in a bay spanning around 1 km and an upstream river under high and low tide within 1 day in November and in January. The samples were analyzed by using eDNA metabarcoding for fish species and species‐specific quantitative analysis for the freshwater fish Cyprinus carpio. Our findings reveal that the eDNA of freshwater fishes was drastically diluted in the model estuarine bay. As a result, the relative‐read‐based composition clearly changed from riverine to marine environments, and the freshwater inflow had little effect on the relative‐read‐based composition at those sites. However, eDNA from freshwater fishes was widely detected in the bay by species‐specific and metabarcoding analysis, suggesting that fresh water may have a more significant impact when focusing on presence/absence‐based composition. Our study also found that the transition zone for the concentration of freshwater eDNA fluctuated spatiotemporally with tides, indicating that the degree of influence from the river varies with tide. Therefore, prior measurement of the distribution of freshwater fish eDNA at low tide would help to conservatively determine better sampling sites and design more reliable sampling in estuaries.

ABSTRACTHuman activities severely impact biodiversity, particularly in freshwater lakes. These habitats provide critical ecosystem services and, at the same time, suffer from river inflow, agricultural runoff, and urban discharge. DNA‐based techniques are preferred for monitoring biodiversity due to their effectiveness. However, pinpointing the causes of biodiversity decline across landscapes poses challenges due to the complex interactions between biodiversity and environmental drivers. In this study, we used an explainable multimodal machine learning approach that can integrate different types of data, such as biological, chemical, and physical data, to discover potential causes of biodiversity dynamics. This is done by identifying relationships between environmental drivers—plant protection products, physico‐chemical parameters and typology‐ and community biodiversity changes in 52 lake ecosystems. By analyzing benthic and pelagic lake communities, we found significant correlations between biodiversity and environmental drivers, such as plant protection products. Furthermore, our analysis allowed us to identify factors within these drivers responsible for biodiversity dynamics. Specifically, insecticides and fungicides were identified as the most important factors, followed by 43 physico‐chemical factors, including many heavy metals. Our holistic, data‐driven approach provides insights into large‐scale biodiversity changes and could inform conservation efforts and regulatory interventions to protect biodiversity from pollution.

ABSTRACTBeaked redfishes (Sebastes fasciatus and Sebastes mentella) of the northwest Atlantic have recently reached record abundance levels in the estuary and northern Gulf of St. Lawrence, dominated by Sebastes mentella. Knowledge of their diet composition is essential to understand the trophic role that these groundfish play in the ecosystem. The objective of the present study was to compare the performance of visual examination and DNA metabarcoding of stomach contents of the same individual redfish caught in the estuary and northern Gulf of St. Lawrence. Using a universal metazoan mitochondrial cytochrome c oxidase subunit I (COI) marker, we identified a total of 24 taxonomic groups, composed of 22 species and two genera in the content of 185 stomachs with DNA metabarcoding. We compared these results to the 25 prey types, eight identified at the genus and nine at the species level, obtained with visual stomach content analysis (SCA). While both techniques revealed a similar diet composition, our results showed that the SCA and DNA metabarcoding perform differently for particular prey categories, both in terms of detectability and taxonomic resolution, as well as in the estimated relative importance of weight and occurrence in the diet. The use of DNA metabarcoding along with SCA validates and improves the taxonomic resolution of visually determined prey, which supports the concept that both techniques provide useful complementary information on the diet of redfish and likely other fish species.

ABSTRACTTerrestrial vertebrates are experiencing worldwide population declines and species extinctions. To effectively conserve remaining populations and species, rapid, cost‐effective, and scalable methods are needed to complement longstanding monitoring methods. Increasingly, environmental DNA (eDNA)‐based approaches are being used for terrestrial vertebrate biomonitoring within a range of environments. However, as we move eDNA biomonitoring onto land, we are presented with a new set of challenges. This necessitates the development of “best‐practice” eDNA sample collection guidelines for terrestrial systems with the purpose of detecting terrestrial vertebrates. To address these needs, we conducted a systematic literature review of 143 peer‐reviewed papers applying eDNA to terrestrial vertebrate monitoring (excluding Lissamphibia) that were published between 2012 and 2023. We summarize the use of eDNA for terrestrial vertebrate biomonitoring, focusing on study design and field techniques. Over the decade we observe a steady growth in the annual number of publications, with 3 in 2012 and 33 in 2023. The majority of the reviewed studies targeted terrestrial mammals within temperate forest regions. While an equal number of studies focused on a metabarcoding approach to assess community taxon composition and/or species‐specific eDNA detection methods, novel uses are increasingly published. These include studies of animal behavior and population genetics. We record three types of sampling strategies, eight different substrate types, and seven different preservation methods, suggesting that there is no “one size fits all” eDNA‐based sampling methodology when detecting terrestrial vertebrates. With a multitude of study aims, across different environments, and target organisms with different ecologies, the standardization of eDNA sampling approaches in terrestrial systems is extremely challenging. We summarize in a table known factors influencing eDNA detection within terrestrial environments. Furthermore, we identify five key considerations to be addressed when sampling for eDNA studies targeting terrestrial vertebrate species, with the aim of guiding decision making.

ABSTRACTEnvironmental DNA (eDNA) is an increasingly popular, sensitive, and cost‐efficient method for studying biodiversity and detecting species. This noninvasive approach involves collecting environmental samples that contain genetic material shed by organisms into their surroundings. Due to the method's sensitivity, robust decontamination strategies are crucial, with sodium hypochlorite, commonly known as bleach, frequently employed. Despite its widespread use, there is no consensus on the most effective bleach concentration, leading to inconsistencies in how the chemical is used in research. This study aimed to determine the minimum concentration of bleach needed for effective decontamination. Genomic DNA of signal crayfish was treated with various concentrations of bleach, ranging from 0.01% to 5% (w/w). Results were observed using Qubit High Sensitivity reagents, quantitative PCR, agarose gel electrophoresis, and the Agilent TapeStation. Our results indicate that a minimum concentration of 0.5% (w/w) bleach is sufficient to prevent the detection of genomic DNA by the techniques tested. These results provide important insights into the use of bleach for decontamination in eDNA research. Establishing a standard bleach concentration for decontamination protocols will help to reduce inconsistencies and enhance the reliability of eDNA studies.

ABSTRACTMany species of reptiles and amphibians (herpetofauna) rely on wetlands that are being degraded and lost at a high rate. Characterization of herpetofauna diversity in different wetland types may help guide conservation strategies. However, traditional survey methods often involve sampling within small temporal windows, and the gear deployed may be taxonomically biased, thus, they may fail to accurately characterize species presence/absence and diversity. In contrast, environmental (e)DNA metabarcoding has been shown to effectively survey entire aquatic communities and can provide a useful complement to traditional surveys. The objective of this study was to design and optimize eDNA sampling and laboratory protocols for wetland herpetofauna. Protocols evaluated included different water sampling approaches (point versus transect sampling), seasonality of sampling, and choice of metabarcoding marker (mitochondrial 12S versus 16S rDNA). Samples collected from 10 sites across southern Michigan detected 17 amphibian and five reptile species, including four species of conservation concern (Ambystoma texanum, Clemmys guttata, Rana palustris, and Sternotherus odoratus). We observed no difference in the number of species detected between point and transect samples (p = 0.70), but point sampling required less time (p = 0.03) and allowed significantly larger volumes of water to be filtered (p = 1.13e‐5). No difference in species richness was observed between the 12S and 16S mitochondrial DNA markers (p = 0.96). However, a greater number of taxa were identifiable at the species level when using the 16S locus. There was also a significant difference in the number of species detected between early and late summer sampling periods (more species detected in the earlier period; p = 6.31e‐6), and some species were only found in the early or late sampling period. Sampling during multiple periods to fully characterize species composition, the use of point sampling, and the 16S mtDNA marker for herpetofauna eDNA metabarcoding studies may increase efficiency and reliability of results.

ABSTRACTBiological invasions represent a significant threat to global biodiversity. Population genetics plays a crucial role in addressing the invasion history of invasive species, as information on the genetic structure of local populations of invasive species is useful in estimating their source and dispersal. This study aimed to demonstrate the potential of an environmental DNA (eDNA)‐based approach for estimating the dispersal patterns of invasive species, using bluegill sunfish (Lepomis macrochirus), a freshwater fish introduced to Japan in 1960. We developed an eDNA haplotyping assay based on high‐throughput sequencing and validated its ability to reproduce the haplotype distribution of bluegill sunfish in Japan, which had previously been determined through DNA analysis of individual fish. We also detected a negative relationship between the number of detected haplotypes and the geographic distance from Lake Biwa, one of the initial introduction sites, to each study site. This genetic pattern can occur in introduced species as a result of serial founder events during their range expansion. Our results suggested that Lake Biwa is the source of bluegill sunfish distribution in Japan, which is consistent with the invasion records of this species. We demonstrated the potential of the eDNA haplotyping assay for estimating the dispersal patterns of invasive species, which would aid the preparation of countermeasures against emerging biological invasions by simultaneously enabling the early detection and tracking of invasive species.

ABSTRACTThe biodiversity of freshwater ecosystems globally is facing severe threats due to various anthropogenic stressors, such as habitat degradation, introduction of invasive species, and pollution. Assessing the effects of human‐induced environmental stressors on population and community persistence requires accurate biodiversity estimates. While environmental DNA (eDNA) metabarcoding has emerged as a promising tool, its effectiveness in capturing rapid biodiversity responses to acute stressors across levels of biological organization (community, population, and intra‐specific levels) remains to be investigated. In this study, we tested the efficacy of eDNA metabarcoding in assessing rapid changes in aquatic zooplankton and insect communities by conducting a two‐month mesocosm experiment with pulses of glyphosate‐based herbicide under contrasting nutrient levels (mesotrophic and eutrophic). We examined the effects of treatments on community assemblages, family richness, and intraspecific diversity, and compared our findings with those obtained through a microscopy approach. Metabarcoding revealed partially congruent ecological findings with microscopy, indicating its potential in assessing rapid community changes. The herbicide induced shifts in community composition and differentially impacted zooplankton and insect family richness (increase in insects, and decrease in crustaceans and rotifers), suggesting a gradient of tolerance to the herbicide among taxa and potential top‐down regulation by insect larvae that may counteract the advantage gained by herbicide‐tolerant zooplankton. Finally, we showed that nutrient enrichment exacerbated the negative effects of the herbicide on intraspecific diversity, highlighting concerns about genetic erosion. Our findings underscore the complexity of responses to herbicide and nutrient enrichment in freshwater ecosystems. We conclude that eDNA metabarcoding can not only be used to estimate rapid changes in invertebrate communities but also provides additional value by offering a broader perspective on diversity dynamics and potential cascading effects at different scales of biological organization.

ABSTRACTEnvironmental DNA (eDNA) analysis of terrestrial substrates, such as soil and sand, is a rapid and potentially cost‐effective way to monitor rare wildlife species. A promising use‐case in the southeastern United States is provided by the eastern indigo snake (Drymarchon couperi), for which accurate monitoring has been challenging due to large home ranges and low‐density populations. However, knowledge gaps regarding eDNA deposition and persistence in this system currently limit our ability to apply eDNA sampling effectively at the landscape scale. To overcome some of these gaps, we used an optimized soil and sand eDNA extraction protocol and species‐specific qPCR assay to conduct a full factorial experiment of eastern indigo snake DNA detection in sand as a function of the duration of snake presence and time since snake removal. We then used these data and a generalized linear mixed model to predict detection probability. Of the 224 total experimental samples, 68 (30.4%) tested positive for eastern indigo snake eDNA. Our model predicted that, with long periods in the enclosure and sampling soon after snake removal, eastern indigo snake eDNA is detectable 68.7% of the time. Eastern indigo snake DNA was detectable in as little as 100 s of snake presence in the enclosure (Pr = 21.1%) and for as long as 10 days after snake presence (Pr = 27.7%). These results suggest that DNA sampling in terrestrial systems may be an effective tool for increasing the temporal window of rare snake detection and a useful complement to existing sampling methods for eastern indigo snakes.

ABSTRACTEnvironmental DNA (eDNA) analysis is a powerful tool for quantifying and assessing the diversity of organisms in the environment. Unfortunately, isolating eDNA from aquatic environments is challenging due to the difficulties associated with water collection, preservation of samples during transportation, and onsite filtration. These processes are expensive and time‐consuming and can lead to eDNA degradation. These difficulties can be addressed by preserving eDNA in the collected water. In this study, we assessed the effect of short‐ and long‐term water storage using three different cationic surfactants on the half‐life of zebrafish (Danio rerio) mitochondrial DNA (mtDNA) in mesocosm water. The surfactants used were benzalkonium chloride (BAC), cetylpyridinium chloride (CPC), and cetyltrimethylammonium bromide (CTAB). We observed that CPC and CTAB treatment extended the half‐life of mtDNA by 3–5 times. Analysis by quantitative polymerase chain reaction (qPCR) demonstrated a mtDNA retention rate of 17.6%, 26.3%, and 2.2% for CPC, CTAB, and BAC, respectively, compared to 0.1% in untreated water after 30 days. The preservation of mtDNA by cationic surfactants was attributed to their bactericidal and cytotoxic properties as well as their electrostatic interaction with DNA molecules, as observed by spectrofluorometric analysis and subsequent precipitation. Our results demonstrated an inexpensive and convenient method to protect eDNA in water and improve its extraction.

ABSTRACTMarine metazoan biodiversity is accretively being explored through environmental DNA (eDNA) metabarcoding of seawater. However, knowledge gaps in the use of eDNA to study changes in diversity resulting from changing abiotic conditions still do exist. In order to address these gaps, we analyzed patterns of marine invertebrate biodiversity based on eDNA from water and sediment samples along a decreasing salinity gradient from the North Sea toward the Baltic Sea. eDNA was collected from surface (SW) and bottom (BW) water, and from the uppermost sediment layer (SE). To supplement the eDNA approach, we conducted parallel zooplankton (ZP) metabarcoding and morphological identification. DNA was extracted from eDNA and ZP samples, amplified using two universal primers that target of the mitochondrial cytochrome c oxidase subunit 1 (COI) and the nuclear ribosomal 18S rRNA genes, and paired‐end sequenced on Illumina Miseq. Metabarcoding detected 279 metazoan species (from 16 phyla) of which > 87% are known from the study area or adjacent regions. Communities identified in SW eDNA were a subset of communities identified in ZP metabarcoding. BW eDNA had additional benthic (mainly bivalve) species. Communities identified in SE eDNA were distinct from those in water eDNA and ZP metabarcoding, and mainly represented by in‐ and meiofauna. Out of all approaches, only ZP metabarcoding uncovered the expected decrease in species richness toward brackish conditions. Neither salinity nor spatial distance had a significant effect on species composition. All approaches revealed regional differences of which SE eDNA was least informative. The detection of holoplanktonic species from SE eDNA provided evidence for sinking of eDNA particles, dead organisms or the presence of resting eggs. Our study confirms the value of metabarcoding to identify the North Sea and Baltic Sea invertebrates and underscores the importance of combining multiple approaches to understand invertebrate biodiversity and its change in the marine realm.

ABSTRACTBats are a species‐rich mammalian order that provide a host of ecosystem services, but presently face threats from habitat loss, disease, climate change, and insect declines. Bat species often co‐occur with other ecologically similar bats, making them a suitable group in which to study niche overlap and partitioning. This study aimed to compare different non‐invasive sources of data on wildlife populations, while examining dietary, temporal, and spatial partitioning patterns among sympatric bat species. We used two different methods to assess niche partitioning among insectivorous bats at a site in the San Francisco Bay Area, California: (1) eDNA sequencing of bat feces that were collected weekly from a bat roost, and (2) nightly acoustic recordings of ultrasonic bat calls from recorders at multiple sites. Both the eDNA and acoustic data were collected over the course of an entire roosting season in 2020. We hypothesized that the insectivorous bats at this site would rely on one or more niche partitioning mechanisms to promote interspecific coexistence and limit competition. We found evidence of fine‐scale spatial partitioning of the broad community of bat species in our study area based on acoustic data, as well as temporal differences in activity of different species. The two species using the roosting site, Tadarida brasiliensis and Eptesicus fuscus, displayed some differences in the identities and relative abundances of prey species consumed, but both ultimately exhibited a strong reliance on dipterans and aquatic‐dependent insects. We demonstrate differences between the acoustic data and eDNA data, which has implications for how such datasets may be interpreted in future research. The study finds evidence of some types of niche partitioning in this community and characterizes baseline interactions between species, providing a foundation for future efforts to non‐invasively monitor for unexpected biological change in local ecosystems.

ABSTRACTEcosystems in coastal waters of Gulf of Maine (GOM) are undergoing environmental challenges in response to climate change and anthropogenic stressors. eDNA metabarcoding, a powerful tool for assessing the fish community structure, was used to identify fish communities in three types of GOM aquatic environments (sand, macroalgae, and eelgrass) in Maine and New Hampshire, USA. The available 12S rRNA fish universal primer analysis system (MiFish and 12S‐V5) was modified using nested polymerase chain reaction (PCR) to improve targeting of fish products and reduce non‐target products. The nested PCR strategy allowed successful amplification of 12S rRNA genes in fishes without production of non‐target products and identified 28 fish groups at the genus level. Presence/Absence data and Relative Abundance showed significant differences among locales but not among habitats. Myoxocephalus sp. were found at all sampling sites. Relative Abundance data revealed that Menidia menidia and Brevoortia sp. were statistical indicator species in Goosefare, Maine, and New castle, New Hampshire, respectively. Although beta diversity indicated that fish communities were not different across habitats, statistical analysis found that Pholis sp. and Ammodytes sp. were dominant species in macroalgae and sand, respectively. To our knowledge, this is the first metabarcoding study to assess fish communities in the Western Atlantic region using the MiFish primer set, and the study suggests that metabarcoding is useful for mapping geographic and temporal marine fish diversity.

ABSTRACTTraditional approaches for studying potential interactions in marine ecosystems often struggle to fully capture all taxa in a community, especially rare species. This issue is particularly challenging in coastal waters with high biodiversity and spatiotemporal dynamics. In this study, we employed environmental DNA (eDNA) metabarcoding, utilizing multiple marker genes, to comprehensively investigate interspecific interactions across various domains in the subtropical coastal waters of Hong Kong. The southern and eastern regions of Hong Kong waters exhibit distinct environmental seasonality, and our investigation focused on comparing the potential interaction networks and the keystone taxa between these two regions. The putative species interaction networks across various groups (i.e., bacteria, protists, and metazoans) were revealed by using weighted correlation network analysis (WGCNA). Our results showed that primary consumers, mainly dinoflagellates and ciliates, were the dominant actors within the interaction networks, although their distributions varied between the two regions. Bacterial taxa from the Pseudomonadota groups primarily constituted saprobes in the southern region, while exhibiting an even distribution in the eastern region. The interaction network in the southern region was larger but less stable compared to the eastern region. This could be attributed to the stronger responses of keystone taxa to environmental variations and the relatively higher number of connectors (e.g., Akashiwo and Protoperidinium within Dinophyceae) in the eastern region. Our findings highlight the versatility of eDNA metabarcoding for studying potential species interactions, providing critical insights into ecosystem structure and stability, and offering suggestions for marine biodiversity conservation.

ABSTRACTMarine biodiversity worldwide is rapidly declining, and nowhere is this more evident than in coastal ecosystems where the impacts of climate change and anthropogenic activities concentrate. The ongoing biodiversity crisis affects all components of the marine food web, but data required to monitor biodiversity shifts at continental scales are scarce and taxonomically and spatially heterogeneous. The application of environmental DNA metabarcoding can complement traditional approaches to monitoring marine biodiversity, but its efficiency in detecting large‐scale biogeographic breaks remains to be tested. Using 86 coastal surface water samples collected during the Canada C3 expedition in the summer of 2017, we investigated metazoan biodiversity across Canada's three oceans—North Pacific, Arctic and North Atlantic—using multi‐marker eDNA metabarcoding. The resulting dataset, combining information from seven separate amplicons, identified 1477 unique species ranging from zooplankton to marine mammals. We found that marine coastal biodiversity around Canada separated into four clusters that overlapped with known marine ecoregions, indicating a higher connectivity between the Arctic and Atlantic than between the Arctic and Pacific clusters. However, the detection of Pacific salmon eDNA in the Canadian Arctic suggests that these species may be extending their Pacific distribution range poleward. By comparing the distribution of eDNA with species occurrence recorded in the Ocean Biodiversity Information System (OBIS) for Canada and Alaska coastal waters, we identified 324 “unexpected” species. These results demonstrate the importance of primer selection for species‐specific applications of eDNA metabarcoding and provide a benchmark for further work aimed at validating species identification and map species distribution at large spatial scale. Our results showed that eDNA metabarcoding is a powerful method for monitoring biodiversity shifts at an interoceanic scale. Integrating eDNA into monitoring programs can provide valuable insights into biodiversity changes associated with climate change and contribute to filling gaps in the distribution of species‐at‐risk.