Mosquitoes on a chip-environmental DNA-based detection of invasive mosquito species using high-throughput real-time PCR.

The monitoring of mosquitoes is of great importance due to their vector competence for a variety of pathogens, which have the potential to imperil human and animal health. Until now mosquito occurrence data is mainly obtained with conventional monitoring methods including active and passive approaches, which can be time- and cost-consuming. New monitoring methods based on environmental DNA (eDNA) could serve as a fast and robust complementary detection system for mosquitoes. In this pilot study already existing marker systems targeting the three invasive mosquito species Aedes (Ae.) albopictus, Ae. japonicus and Ae. koreicus were used to detect these species from water samples via microfluidic array technology. We compared the performance of the high-throughput real-time PCR (HT-qPCR) system Biomark HD with real-time PCR (qPCR) and also tested the effect of different filter media (Sterivex® 0.45 µm, Nylon 0.22 µm, PES 1.2 µm) on eDNA detectability. By using a universal qPCR protocol and only 6-FAM-MGB probes we successfully transferred these marker systems on the HT-qPCR platform. All tested marker systems detected the target species at most sites, where their presence was previously confirmed. Filter media properties, the final filtration volume and observed qPCR inhibition did not affect measured Ct values via qPCR or HT-qPCR. The Ct values obtained from HT-qPCR were significantly lower as Ct values measured by qPCR due to the previous preamplification step, still these values were highly correlated. Observed incongruities in eDNA detection probability, as manifested by non-reproducible results and false positive detections, could be the result of methodological aspects, such as sensitivity and specificity issues of the used assays, or ecological factors such as varying eDNA release patterns. In this study, we show the suitability of eDNA-based detection of mosquito species from water samples using a microfluidic HT-qPCR platform. HT-qPCR platforms such as Biomark HD allow for massive upscaling of tested species-specific assays and sampling sites with low time- and cost-effort, thus this methodology could serve as basis for large-scale mosquito monitoring attempts. The main goal in the future is to develop a robust (semi)-quantitative microfluidic-based eDNA mosquito chip targeting all haematophagous culicid species occurring in Western Europe. This chip would enable large-scale eDNA-based screenings to assess mosquito diversity, to monitor species with confirmed or suspected vector competence, to assess the invasion progress of invasive mosquito species and could be used in pathogen surveillance, when disease agents are incorporated.

Effect of environmental DNA sampling resolution in detecting nearshore fish biodiversity compared to capture surveys.

Sampling and sequencing marine environmental DNA (eDNA) provides a tool that can increase our ability to monitor biodiversity, but movement and mixing of eDNA after release from organisms before collection could affect our inference of species distributions. To assess how conditions at differing spatial scales influence the inferred species richness and compositional turnover, we conducted a paired eDNA metabarcoding and capture (beach seining) survey of fishes on the coast of British Columbia. We found more taxa were typically detected using eDNA compared to beach seining. eDNA identified more taxa with alternative habitat preferences, and this richness difference was greater in areas of high seawater movement, suggesting eDNA has a larger spatial grain influenced by water motion. By contrast, we found that eDNA consistently missed low biomass species present in seining surveys. Spatial turnover of communities surveyed using beach seining differed from that of the eDNA and was better explained by factors that vary at small (10-1000s meters) spatial scales. Specifically, vegetation cover and shoreline exposure explained most species turnover from seining, while eDNA turnover was not explained by those factors and showed a distance decay pattern (a change from 10% to 25% similarity from 2 km to 10 km of distance), suggesting unmeasured environmental variation at larger scales drives its turnover. Our findings indicate that the eDNA sample grain is larger than that of capture surveys. Whereas seining can detect differences in fish distributions at scales of 10s-100s of meters, eDNA can best summarize fish biodiversity at larger scales possibly more relevant to regional biodiversity assessments.

Is it worth the extra mile? Comparing environmental DNA and RNA metabarcoding for vertebrate and invertebrate biodiversity surveys in a lowland stream.

Environmental DNA (eDNA) metabarcoding has emerged as a promising approach to assess biodiversity and derive ecological status classes from water samples. However, a limitation of eDNA surveys is that detected DNA molecules may originate from other places or even dead organisms, distorting local biodiversity assessments. Environmental RNA (eRNA) metabarcoding has recently been proposed as a complementary tool for more localized assessments of the biological community. In this study, we evaluated the effectiveness of eDNA and eRNA metabarcoding for inferring the richness and species distribution patterns of vertebrates and invertebrates in a Central European lowland river. We collected water samples and analyzed them using a 12S marker for vertebrates and a COI marker for invertebrates. We detected 31 fish, 16 mammal, 10 bird and one lamprey species in the vertebrate dataset. While results were largely consistent, we detected a higher number of species when analysing eRNA (mean = 30.89) than eDNA (mean = 26.16). Also, eRNA detections had a stronger local signature than eDNA detections when compared against species distribution patterns from traditional fish monitoring data. For invertebrates, we detected 109 arthropod, 22 annelid, 12 rotiferan, eight molluscan and four cnidarian species. In contrast to the pattern of vertebrate richness, we detected a higher richness using eDNA (mean = 41.37) compared to eRNA (mean = 22.42). Our findings primarily show that eDNA and eRNA-based detections are comparable for vertebrate and invertebrate taxa. Biological replication was important for both template molecules studied. Signal detections for vertebrates were more localized for eRNA compared to eDNA. Overall, the advantages of the extra steps needed for eRNA analyses depend on the study question but both methods provide important data for biodiversity monitoring and research.

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

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

Detection and transport of environmental DNA from two federally endangered mussels.

Environmental DNA (eDNA) offers a novel approach to supplement traditional surveys and provide increased spatial and temporal information on species detection, and it can be especially beneficial for detecting at risk or threatened species with minimal impact on the target species. The transport of eDNA in lotic environments is an important component in providing more informed descriptions of where and when a species is present, but eDNA transport phenomena are not well understood. In this study, we used species-specific assays to detect eDNA from two federally endangered mussels in two geographically distinct rivers. Using the eDNA concentrations measured from field samples, we developed a one-dimensional (1D) hydrodynamic transport model to predict the downstream fate and transport of eDNA. We detected eDNA from both federally endangered mussels across several seasons and flow rates and up to 3.5 km downstream from the source populations, but the detection rates and eDNA concentrations were highly variable across and within rivers and study reaches. Our 1D transport models successfully integrated the variability of the eDNA field samples into the model predictions and overall model results were generally within ±1 standard error of the eDNA field concentration values. Overall, the results of this study demonstrate the importance of optimizing the spatial locations from where eDNA is collected downstream from a source population, and it highlights the need to improve understanding on the shedding mechanisms and magnitude of eDNA from source populations and biogeomorphic processes that influence eDNA transport.

soibean: High-Resolution Taxonomic Identification of Ancient Environmental DNA Using Mitochondrial Pangenome Graphs.

Ancient environmental DNA (aeDNA) is becoming a powerful tool to gain insights about past ecosystems, overcoming the limitations of conventional fossil records. However, several methodological challenges remain, particularly for classifying the DNA to species level and conducting phylogenetic analysis. Current methods, primarily tailored for modern datasets, fail to capture several idiosyncrasies of aeDNA, including species mixtures from closely related species and ancestral divergence. We introduce soibean, a novel tool that utilizes mitochondrial pangenomic graphs for identifying species from aeDNA reads. It outperforms existing methods in accurately identifying species from multiple closely related sources within a sample, enhancing phylogenetic analysis for aeDNA. soibean employs a damage-aware likelihood model for precise identification at low coverage with a high damage rate. Additionally, we reconstructed ancestral sequences for soibean's database to handle aeDNA that is highly diverged from modern references. soibean demonstrates effectiveness through simulated data tests and empirical validation. Notably, our method uncovered new empirical results in published datasets, including using porpoise whales as food in a Mesolithic community in Sweden, demonstrating its potential to reveal previously unrecognized findings in aeDNA studies.

Important fish diversity maintenance status of the tributaries in a hotspot fish conservation area in the upper Yangtze River revealed by eDNA metabarcoding.

This study employed Environmental DNA (eDNA) barcoding technology to delve into the influence of the tributaries and mainstem on fish diversity and spatiotemporal distribution in a hotspot fish conservation area in the upper Yangtze River. A total of 123 fish species were detected, belonging to 7 orders, 19 families, and 77 genera. The composition of fish species in tributaries is similar to that in mainstem, with higher fish community diversity in tributaries during the spring and summer. Exploration of fish ecotypes revealed significant differences between mainstem and tributaries. The fish community is mainly influenced by key environmental factors such as water temperature, dissolved oxygen, electrical conductivity, and ammonia nitrogen, with a higher impact of these factors on tributaries than on mainstem. In conclusion, while tributaries and mainstem in the Jiangjin section exhibit similarities in fish community composition, there are notable differences in community structure and diversity. Therefore, the protection of not only mainstem but also tributaries and their associated fish habitats is crucial for promoting the overall health and sustainability.

Eukaryotic plankton species diversity and community structure in the Xiao Jiang River (the primary tributary of Upper Yangtze River), Yunnan.

The Xiao Jiang River, as a crucial element of ecological restoration in the upper reaches of the Yangtze River, plays an indispensable role in agricultural water utilization and water ecology within its watersheds. The water quality status of the Xiao Jiang River not only impacts local water-ecological equilibrium and economic benefits but also holds paramount importance for sustaining ecosystem health in the Yangtze River basin. Plankton surveys and environmental physicochemical detection were conducted in the major channel region of the Xiao Jiang River in dry and wet periods in 2022 to better understand the diversity of eukaryotic plankton and its community structure characteristics. Environmental DNA is an emerging method that combines traditional ecology with second-generation sequencing technology. It can detect species from a single sample that are difficult to find by traditional microscopy, making the results of plankton diversity studies more comprehensive. For the first time, environmental DNA was used to investigate eukaryotic plankton in the Xiao Jiang River . The results showed that a total of 881 species of plankton from 592 genera in 17 phyla were observed. During the dry period, 480 species belonging to 384 genera within17 phyla were detected, while, during the wet period, a total of 805 species belonging to 463 genera within 17 phyla were recorded. The phylum Ciliophora dominated the zooplankton, while the phylum Chlorophyta and Bacillariophyta dominated the phytoplankton. The presence of these dominant species indicate that the water quality conditions in the study area are oligotrophic and mesotrophic. Principal coordinate analysis and difference test showed that the number of plankton ASVs, abundance, species richness, dominating species, and diversity indices differed between the dry and wet periods. Spearman correlation analysis and redundancy analysis (RDA) of relative abundance data with environmental physicochemical factors revealed that water temperature (WT), dissolved oxygen (DO), potential of hydrogenacidity (pH), ammonia nitrogen (NH3-N), total nitrogen (TN), electrical conductivity (EC) and the determination of redox potential (ORP) were the main environmental physicochemical factors impacting the plankton community structure. The results of this study can serve as a provide data reference at the plankton level for water pollution management in the Xiao Jiang River, and they are extremely important for river ecological restoration and biodiversity recovery in the Yangtze River basin.

Multi-method survey rediscovers critically endangered species and strengthens Madagascar's freshwater fish conservation.

Freshwater ecosystems are crucial for global biodiversity through supporting plant and animal species and providing essential resources. These ecosystems are under significant threat, particularly in island environments such as Madagascar. Our study focuses on the Amboaboa River basin, home to the rare and endemic fish species Rheocles derhami, last recorded in 2013. To assess the status of this and other threatened fish species including Ptychochromis insolitus and Paretroplus gymnopreopercularis, and to understand freshwater fish population dynamics in this biodiversity hotspot, we conducted a comprehensive survey using both environmental DNA (eDNA) and traditional fishing methods. While traditional methods effectively captured a diverse range of species, including several invasive aliens and the critically endangered endemic species that were the focus of this study, the eDNA approach detected only a fraction of these introduced species and struggled to identify some critically endangered endemics at the species level. This highlights the value of combining methods to enhance species detection. We also investigated the trade-offs associated with multi-primer assessments in eDNA analysis, focusing on three different primer combinations targeting the 12S mitochondrial gene: MiFish, Tele02, and Riaz. Additionally, we provided 12S reference barcodes for 10 species across 9 genera of fishes from the region to increase the coverage of the public reference databases. Overall, our study elucidates the current state of freshwater biodiversity in the Amboaboa River basin and underscores the value of employing multiple methods for effective conservation strategies.

GoEnrich: creating high quality genomic DNA resources from limited voucher specimen tissues or museum specimens of at-risk species for conservation-friendly use in the validation of environmental DNA assays.

OBJECTIVE: Environmental DNA (eDNA) methods are crucial for monitoring populations, particularly rare and cryptic species. For confident eDNA application, rigorous assay validation is required including specificity testing with genomic DNA (gDNA). However, this critical step is often difficult to achieve as obtaining fresh tissue samples from at-risk species can be difficult, highly limited, or impossible. Natural history museum collections could serve as a valuable and ethical voucher specimen resource for eDNA assay validation. The present study demonstrates the effectiveness of whole genome amplification (WGA) in providing enough gDNA to assemble high quality mitogenomes from which robust targeted eDNA assays can be designed. RESULTS: Using fresh and historical museum tissue samples from six species spanning fish, birds, and mammals, we successfully developed a WGA method with an average yield of 380 to 1,268 ng gDNA per 20 µL reaction. This gDNA was used for whole genome shotgun sequencing and subsequent assembly of high quality mitogenomes using mtGrasp. These mitogenomes were then used to develop six new robust, targeted quantitative real time polymerase chain reaction-based eDNA assays and 200 ng WGA-enriched yielded satisfactory Cq values and near 100% detection frequencies for all assays tested. This approach offers a cost-effective and non-invasive alternative, streamlining eDNA research processes and aiding in conservation efforts.

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

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

Comparing the effectiveness of environmental DNA and camera traps for surveying American mink (Neogale vison) in northeastern Indiana.

While camera traps can effectively detect semi-aquatic mammal species, they are also often temporally and monetarily inefficient and have a difficult time detecting smaller bodied, elusive mammals. Recent studies have shown that extracting DNA from environmental samples can be a non-invasive, alternative method of detecting elusive species. Environmental DNA (eDNA) has not yet been used to survey American mink (Neogale vison), a cryptic and understudied North American mustelid. To help determine best survey practices for the species, we compared the effectiveness and efficiency of eDNA and camera traps in surveys for American mink. We used both methods to monitor the shoreline of seven bodies of water in northeastern Indiana from March to May 2021. We extracted DNA from filtered environmental water samples and used quantitative real-time PCR to determine the presence of mink at each site. We used Akaike's Information Criterion to rank probability of detection models with and without survey method as a covariate. We detected mink at four of the seven sites and seven of the 21 total survey weeks using camera traps (probability of detection (ρ) = 0.36). We detected mink at five sites and during five survey weeks using eDNA (ρ = 0.25). However, the highest probability of detection was obtained when both methods were combined, and data were pooled (ρ = 0.47). Survey method did not influence model fit, suggesting no difference in detectability between camera traps and eDNA. Environmental DNA was twice as expensive, but only required a little over half (58%) of the time when compared to camera trapping. We recommend ways in which an improved eDNA methodology may be more cost effective for future studies. For this study, a combination of both methods yielded the highest probability for detecting mink presence.

Recovering plant-associated arthropod communities by eDNA metabarcoding historical herbarium specimens.

Natural history collections are a priceless resource for understanding patterns and processes of biodiversity change in the Anthropocene.1 Herbaria, which house millions of historical plant records from all over the globe, are particularly valuable to study population genetics of the plants themselves and to understand the assembly of plant-associated microbial communities.2 Here we test if herbaria can serve yet another essential purpose, namely to provide information on the historical assembly of plant-arthropod interactions. The specificity and temporal stability of these associations are poorly known.3 Considering their pivotal role in the assembly of terrestrial food webs,4 this knowledge is paramount to understanding the consequences of global change. We use environmental DNA (eDNA) metabarcoding to characterize communities of plant-associated arthropods from archived herbarium specimens of different ages and origins. The herbarium specimens yield arthropod DNA across various ecological guilds and trophic levels over multiple decades. In an experiment, we also show that the typical dry storage of plants in herbaria does not alter the recovered arthropod diversity and community composition. By analyzing a time series of leaf samples from a forest monitoring project, we then characterize changes in arthropod biodiversity over two decades, showing that archived plants can also provide the time series data that are urgently needed to understand arthropod declines.5 This use of herbaria and plant archives promises unprecedented insights into plant-arthropod interactions and revolutionizes our ability to monitor spatiotemporal changes in interaction diversity.

A rapid phylogeny-based method for accurate community profiling of large-scale metabarcoding datasets.

Environmental DNA (eDNA) is becoming an increasingly important tool in diverse scientific fields from ecological biomonitoring to wastewater surveillance of viruses. The fundamental challenge in eDNA analyses has been the bioinformatical assignment of reads to taxonomic groups. It has long been known that full probabilistic methods for phylogenetic assignment are preferable, but unfortunately, such methods are computationally intensive and are typically inapplicable to modern next-generation sequencing data. We present a fast approximate likelihood method for phylogenetic assignment of DNA sequences. Applying the new method to several mock communities and simulated datasets, we show that it identifies more reads at both high and low taxonomic levels more accurately than other leading methods. The advantage of the method is particularly apparent in the presence of polymorphisms and/or sequencing errors and when the true species is not represented in the reference database.

Scanning amplicons with CRISPR-Dx detects endangered amphibians in environmental DNA.

More efficient methods for extensive biodiversity monitoring are required to support rapid measures to address the biodiversity crisis. While environmental DNA (eDNA) metabarcoding and quantitative PCR (qPCR) methods offer advantages over traditional monitoring approaches, their large-scale application is limited by the time and labour required for developing assays and/or for analysis. CRISPR (clustered regularly interspaced short palindromic repeats) diagnostic technologies (Dx) may overcome some of these limitations, but they have been used solely with species-specific primers, restricting their versatility for biodiversity monitoring. Here, we demonstrate the feasibility of designing species-specific CRISPR-Dx assays in silico within a short metabarcoding fragment using a general primer set, a methodology we term 'ampliscanning', for 18 of the 22 amphibian species in Switzerland. We sub-selected nine species, including three classified as regionally endangered, to test the methodology using eDNA sampled from ponds at nine sites. We compared the ampliscanning detections to data from traditional monitoring at these sites. Ampliscanning was successful at detecting target species with different prevalences across the landscape. With only one visit, we detected more species per site than three traditional monitoring visits (visual and acoustic detections by trained experts), in particular more elusive species and previously undocumented but expected populations. Ampliscanning detected 25 species/site combinations compared to 12 with traditional monitoring. Sensitivity analyses showed that larger numbers of field visits and PCR replicates are more important for reliable detection than many technical replicates at the CRISPR-Dx assay level. Given the reduced sampling and analysis effort, our results highlight the benefits of eDNA and CRISPR-Dx combined with universal primers for large-scale monitoring of multiple endangered species across landscapes to inform conservation measures.

Stability of environmental DNA methylation and its utility in tracing spawning in fish.

The use of environmental DNA (eDNA) is becoming prevalent as a novel method of ecological monitoring. Although eDNA can provide critical information on the distribution and biomass of particular taxa, the DNA sequences of an organism remain unaltered throughout its existence, which complicates the accurate identification of crucial events, including spawning. Therefore, we examined DNA methylation as a novel source of information from eDNA, considering that the methylation patterns in eggs and sperm released during spawning differ from those of somatic tissues. Despite its potential applications, little is known about eDNA methylation, including its stability and methods for detection and quantification. Therefore, we conducted tank experiments and performed methylation analysis targeting 18S rDNA through bisulphite amplicon sequencing. In the target region, eDNA methylation was not affected by degradation and was equivalent to the methylation rate of genomic DNA from somatic tissues. Unmethylated DNA, abundant in the ovaries, was detected in the eDNA released during fish spawning. These results indicate that eDNA methylation is a stable signal reflecting targeted gene methylation and further demonstrate that germ cell-specific methylation patterns can be used as markers for detecting fish spawning.

Taxonomic and abundance biases affect the record of marine eukaryotic plankton communities in sediment DNA archives.

Environmental DNA (eDNA) preserved in marine sediments is increasingly being used to study past ecosystems. However, little is known about how accurately marine biodiversity is recorded in sediment eDNA archives, especially planktonic taxa. Here, we address this question by comparing eukaryotic diversity in 273 eDNA samples from three water depths and the surface sediments of 24 stations in the Nordic Seas. Analysis of 18S-V9 metabarcoding data reveals distinct eukaryotic assemblages between water and sediment eDNA. Only 40% of Amplicon Sequence Variants (ASVs) detected in water were also found in sediment eDNA. Remarkably, the ASVs shared between water and sediment accounted for 80% of total sequence reads suggesting that a large amount of plankton DNA is transported to the seafloor, predominantly from abundant phytoplankton taxa. However, not all plankton taxa were equally archived on the seafloor. The plankton DNA deposited in the sediments was dominated by diatoms and showed an underrepresentation of certain nano- and picoplankton taxa (Picozoa or Prymnesiophyceae). Our study offers the first insights into the patterns of plankton diversity recorded in sediment in relation to seasonality and spatial variability of environmental conditions in the Nordic Seas. Our results suggest that the genetic composition and structure of the plankton community vary considerably throughout the water column and differ from what accumulates in the sediment. Hence, the interpretation of sedimentary eDNA archives should take into account potential taxonomic and abundance biases when reconstructing past changes in marine biodiversity.

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

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

Environmental DNA Reveals Geographic Distributions of Two eel Species, Anguilla japonica and A. marmorata, in Western Kyushu, Japan.

Some anguillid eels migrate thousands of kilometers from their spawning grounds, dispersing across vast geographic areas to fresh and brackish water habitats, where they settle and grow. Japanese eels (Anguilla japonica) and giant mottled eels (A. marmorata) are both found in Japan, although their distributions differ, and their exact distributions are poorly known. We assumed that topographic distribution patterns of Japanese and giant mottled eels must differ among and within rivers along the northwest coast of Kyushu, Japan. Environmental DNA (eDNA) analysis was conducted at 87 sites in 23 rivers. Japanese eel eDNA was detected in 19 rivers (82.6%) and that of giant mottled eels was detected in eight (34.8%). We detected giant mottled eel eDNA in five rivers where they were previously unknown. eDNA for Japanese eels was detected at six of nine sites in the north (66.7%), 13 of 23 sites in Omura (56.5%), and 37 of 55 sites in the south (67.3%). In contrast, giant mottled eel eDNA was detected at one of nine sites in the north (11.1%), no sites in Omura, and 15 of 55 sites in the south (27.3%). There was no correlation between eDNA concentrations of the two species at 10 sites in the five rivers where eDNA of both species was detected. These findings suggest differences in the distribution of the two eel species and the northern distributional limit of giant mottled eels in the area facing the East China Sea.

Measuring air metagenomic diversity in an agricultural ecosystem.

All species shed DNA during life or in death, providing an opportunity to monitor biodiversity via environmental DNA (eDNA). In recent years, combining eDNA, high-throughput sequencing technologies, bioinformatics, and increasingly complete sequence databases has promised a non-invasive and non-destructive environmental monitoring tool. Modern agricultural systems are often large monocultures and so are highly vulnerable to disease outbreaks. Pest and pathogen monitoring in agricultural ecosystems is key for efficient and early disease prevention, lower pesticide use, and better food security. Although the air is rich in biodiversity, it has the lowest DNA concentration of all environmental media and yet is the route for windborne spread of many damaging crop pathogens. Our work suggests that ecosystems can be monitored efficiently using airborne nucleic acid information. Here, we show that the airborne DNA of microbes can be recovered, shotgun sequenced, and taxonomically classified, including down to the species level. We show that by monitoring a field growing key crops we can identify the presence of agriculturally significant pathogens and quantify their changing abundance over a period of 1.5 months, often correlating with weather variables. We add to the evidence that aerial eDNA can be used as a source for biomonitoring in terrestrial ecosystems, specifically highlighting agriculturally relevant species and how pathogen levels correlate with weather conditions. Our ability to detect dynamically changing levels of species and strains highlights the value of airborne eDNA in agriculture, monitoring biodiversity changes, and tracking taxa of interest.