Environmental RNA (eRNA) analysis is conventionally expected to infer physiological information about organisms within their ecosystems, whereas environmental DNA (eDNA) analysis only infers their presence and abundance. Despite the promise of eRNA application, basic research on eRNA characteristics and dynamics is limited. The present study conducted aquarium experiments using zebrafish (Danio rerio) to estimate the particle size distribution (PSD) of eRNA in order to better understand the persistence state of eRNA particles. Rearing water samples were sequentially filtered using different pore-size filters, and the resulting size-fractioned mitochondrial cytochrome b (CytB) eDNA and eRNA data were modeled with the Weibull complementary cumulative distribution function (CCDF) to estimate the parameters characterizing the PSDs. It was revealed that the scale parameter (α) was significantly higher (i.e., the mean particle size was larger) for eRNA than eDNA, while the shape parameter (β) was not significantly different between them. This result supports the hypothesis that most eRNA particles are likely in a protected, intra-cellular state, which mitigates eRNA degradation in water. Moreover, these findings also imply the heterogeneous dispersion of eRNA relative to eDNA and suggest an efficient method of eRNA collection using a larger pore-size filter. Further studies on the characteristics and dynamics of eRNA particles should be pursued in the future.

Stream ecosystems are under pressure due to multiple stressors. Restoration measures can halt further degradation and improve their ecological status. However, assessment of the effectiveness of the implemented measures is often insufficient because of logistic and financial constraints. DNA-metabarcoding has been proposed to scale up sample processing, although its application as a diagnostic tool has received less attention. The aim of our study was to evaluate if DNA-metabarcoding of stream macroinvertebrates can be used to compute a stressor-specific index to assess the effectiveness of a stream restoration project. For this purpose, we sampled the upstream, restored, and downstream section of a recently restored lowland stream in the Netherlands. At each site, we applied three different methods of macroinvertebrate identification: morphological identification of bulk samples (morphology), DNA-metabarcoding of the same bulk samples (DNA) and metabarcoding of eDNA extracted from the water (eDNA). First, we compared the community composition identified by each method. The communities identified by morphology and DNA were highly similar, whereas the communities generated by the eDNA differed. Second, we analysed whether the identification methods could be used to assess the effectiveness of the restoration project, focussing on a stressor-specific index for flow as the restoration measures aimed at improving flow conditions. Both the morphology and bulk DNA samples indicated improved flow conditions in the restored section of the stream (i.e., less stress from the reduction or absence of flow than in the unrestored sections). Contrary, the eDNA-water samples did not differentiate the amount of stress throughout the catchment, although applying recent developments in eDNA sampling could lead to more robust results. In conclusion, this study forms proof of concept that DNA from bulk samples can be utilized to assess the effectiveness of restoration measures, showing the added value of this approach for water managers.

Fungi play a vital role in ecosystem functioning, yet significant knowledge gaps persist in understanding their diversity and distribution leading to uncertainties about their threat status and extinction risk. This is partly owed to the difficulty of monitoring fungi using traditional fruiting body surveys. The present study evaluates airborne environmental DNA (eDNA) sampling as a monitoring tool with a focus on grassland macrofungi. We applied active and passive air sampling methods, complemented by extensive field surveys of waxcap and clavarioid fungi-species groups of high relevance for conservation. Twenty-nine species were recorded during the field surveys, 19 of which were also detectable by ITS2 metabarcoding of the collected samples. An additional 12 species from the studied genera were identified exclusively in air eDNA. We found that the patterns of species detection and read abundance in air samples reflected the abundance and occurrence of fruiting bodies on the field. Dispersal kernels fitted for the three dominant species predicted rapidly decreasing spore concentrations with increasing distance from fruitbodies. Airborne assemblages were dominated by a high diversity of common species, while rare and threatened red-listed species were under-represented, which underscores the difficulty in detecting rare species, not only in conventional surveys. Considering the benefits and drawbacks of air sampling and fruitbody surveys, we conclude that air sampling serves as a cost- and time-efficient tool to characterize local macrofungal communities, providing the potential to facilitate and improve future fungal monitoring efforts.

Environmental DNA metabarcoding is increasingly implemented in biodiversity monitoring, including phytoplankton studies. Using 21 mock communities composed of seven unicellular diatom and dinoflagellate algae, assembled with different composition and abundance by controlling the number of cells, we tested the accuracy of an eDNA metabarcoding protocol in reconstructing patterns of alpha and beta diversity. This approach allowed us to directly evaluate both qualitative and quantitative metabarcoding estimates. Our results showed non-negligible rates (17-25%) of false negatives (i.e., failure to detect a taxon in a community where it was included), for three taxa. This led to a statistically significant underestimation of metabarcoding-derived alpha diversity (Wilcoxon p = 0.02), with the detected species richness being lower than expected (based on cell numbers) in 8/21 mock communities. Considering beta diversity, the correlation between metabarcoding-derived and expected community dissimilarities was significant but not strong (R2 = 0.41), indicating suboptimal accuracy of metabarcoding results. Average biovolume and rDNA gene copy number were estimated for the seven taxa, highlighting a potential, though not exhaustive, role of the latter in explaining the recorded biases. Our findings highlight the importance of mock communities for assessing the reliability of phytoplankton eDNA metabarcoding studies and identifying their limitations.

The world\'s largest rivers are home to diverse, endemic, and threatened fish species. However, their sheer sizes make large-scale biomonitoring challenging. While environmental DNA (eDNA) metabarcoding has become an established monitoring approach in smaller freshwater ecosystems, its suitability for large rivers may be challenged by the sheer extent of their cross sections (>1 km wide and tens of meters deep). Here, we sampled fish eDNA from multiple vertical layers and horizontal locations from two cross sections of the lower reach of the Yangtze River in China. Over half of the ASVs (amplicon sequence variants) were detected in only a single combination of the vertical layers and horizontal locations, with ∼7% across all combinations. We estimated the need to sample >100 L of water across the cross-sectional profiles to achieve ASV richness saturation, which translates to ∼60 L of water at the species level. No consistent pattern emerged for prioritizing certain depth and horizontal samples, yet we underline the importance of sampling and integrating different layers and locations simultaneously. Our study highlights the significance of spatially stratified sampling and sampling volumes when using eDNA approaches. Specifically, we developed and tested a scalable and broadly applicable strategy that advances the monitoring and conservation of large rivers.

The present study was designed to assess the effectiveness of the eDNA metabarcoding approach to determine the phytoplankton composition in the marine environment with a special focus on mucilage episodes in the Sea of Marmara. For this purpose, the samples were collected from 5 different sites located in the Sea of Marmara and the northern Aegean Sea during the mucilage episode in June 2021. The phytoplankton diversity was analyzed morphologically and by 18S rRNA gene amplicon sequencing, and the dataset of both methods was compared, accordingly. The results showed significant differences between methods in terms of composition and the abundance of the phytoplankton groups. While Miozoa was the most abundant group by metabarcoding, light microscopy (LM) indicated a dominance of Bacillariophyta. Katablepharidophyta was found at lower abundances by the metabarcoding (representing < 1% of the community); the members of this phylum were not observed by a microscope. At the lower taxonomic levels, Chaetoceros was the only genus detected in all samples by both methods. Additionally, while mucilage-forming Gonyaulax fragilis, Cylindrotheca closterium, and Thalassiosira rotula were detected to species-level by LM, metabarcoding was able to determine these organisms at the genus level. On the other hand, the genus Arcocellulus was found in all metabarcoding datasets and not detected by microscopy. The results indicated that metabarcoding can detect a greater number of genera and reveal taxa that were overlooked by light microscopy but to develop a complete picture of phytoplankton diversity in the sample, microscopical observations still are in need.

Loss of biodiversity is a serious concern, and amphibians are particularly threatened. Most small salamanders in Japan are endangered. Distributional information is fundamental to the conservation of these rare species; however, small salamanders are generally difficult to locate or catch. Environmental DNA analysis is an effective survey method for monitoring such rare species. The conventional polymerase chain reaction (PCR) method, which combines PCR amplification with subsequent electrophoresis, and the real-time PCR method, which uses fluorescent material, are commonly used for this purpose. In this study, a comparison of these two detection methods was conducted using a rare salamander species, Hynobius boulengeri, as a model case. We compared three points: (i) detection sensitivity, (ii) influence of environmental factors related to detection, and (iii) time and financial costs of the two methods. To perform this comparison, we developed a real-time PCR detection assay, conducted field surveys, and compared the time and financial costs of conventional and real-time PCR methods. The comparison showed no statistical difference in the detection sensitivity from field samples, and the effects of environmental factors tended to be similar. In addition, the financial cost was lower for the conventional PCR method while the time cost was lower for the real-time PCR method. Therefore, selecting eDNA detection methods based on objectives, time, and financial costs will promote efficient monitoring and contribute to the conservation of rare species.

ddPCR is becoming one of the most widely used tool in the field of eDNA-based aquatic monitoring. Although emulsion PCR used in ddPCR confers a partial mitigation to inhibition due to the high number of reactions for a single sample (between 10K and 20K), it is not impervious to it. Our results showed that inhibition impacts the amplitude of fluorescence in positive droplets with a different intensity among rivers. This signal fluctuation could jeopardize the use of a shared threshold among samples from different origin, and thus the accurate assignment of the positive droplets which is particularly important for low concentration samples such as eDNA ones: amplification events are scarce, thus their objective discrimination as positive is crucial. Another issue, related to target low concentration, is the artifactual generation of high fluorescence droplets (\'stars\'). Indeed, these could be counted as positive with a single threshold solution, which in turn could produce false positive and incorrect target concentration assessments. Approximating the positive and negative droplets distribution as normal, we proposed here a double threshold method accounting for both high fluorescence droplets (\'stars\') and PCR inhibition impact in delineating positive droplets clouds. In the context of low concentration template recovered from environmental samples, the application of this method of double threshold establishment could allow for a consistent sorting of the positive and negative droplets throughout ddPCR data generated from samples with varying levels of inhibitor contents. Due to low concentrations template and inhibition effects, Quantasoft software produced an important number of false negatives and positive comparatively to the double threshold method developed here. This case study allowed the detection of the invasive crayfish P. leniusculus in 32 out of 34 sampled sites from two main rivers (Alzette and Sûre) and five of their tributaries (Eisch, Attert, Mamer, Wiltz and Clerve).

The development of a standardized eDNA detection process is the primary step in improving the accuracy and efficiency of eDNA detection. In this study, primers and probes with high specificity were selected to identify the COI gene of Acanthopagrus latus. Through experiments on the influence of different water quantities, methods of water sample preservation and water bathing times on the result of eDNA detection, the accuracy of this method for extracted water samples was improved. Specifically, a water bathing time of 6 h provided an optimal eDNA concentration from the water sample. After 6 h, the concentration began to decrease, so 6 h was determined to be the best water bathing time for A. latus. Five water extraction volumes (250 mL, 500 mL, 1 L, 2 L, and 3 L) were tested, and there was a positive correlation between water extraction volume and the DNA concentration in the water sample. Different water sample preservation methods were also compared, and it was found that at ≤7 d, the concentration obtained with the cryopreservation method for different water extraction volumes was higher than that obtained with the ethanol preservation method. In this study, we established and optimized a technical procedure for eDNA-based detection of A. latus in aquatic environments. We hope to apply this method in field investigations and provide a reference for the study of eDNA in other fishes.

Environmental DNA (eDNA) integrated with metabarcoding is a promising and powerful tool for species composition and biodiversity assessment in aquatic ecosystems and is increasingly applied to evaluate fish diversity. To date, however, no standardized eDNA-based protocol has been established to monitor fish diversity. In this study, we investigated and compared two filtration methods and three DNA extraction methods using three filtration water volumes to determine a suitable approach for eDNA-based fish diversity monitoring in the Pearl River Estuary (PRE), a highly anthropogenically disturbed estuarine ecosystem. Compared to filtration-based precipitation, direct filtration was a more suitable method for eDNA metabarcoding in the PRE. The combined use of DNeasy Blood and Tissue Kit (BT) and traditional phenol/chloroform (PC) extraction produced higher DNA yields, amplicon sequence variants (ASVs), and Shannon diversity indices, and generated more homogeneous and consistent community composition among replicates. Compared to the other combined protocols, the PC and BT methods obtained better species detection, higher fish diversity, and greater consistency for the filtration water volumes of 1 000 and 2 000 mL, respectively. All eDNA metabarcoding protocols were more sensitive than bottom trawling in the PRE fish surveys and combining two techniques yielded greater taxonomic diversity. Furthermore, combining traditional methods with eDNA analysis enhanced accuracy. These results indicate that methodological decisions related to eDNA metabarcoding should be made with caution for fish community monitoring in estuarine ecosystems.
环境DNA（eDNA）宏条形码技术是评估水生生态系统中物种组成和生物多样性的强有力工具，并已广泛应用于鱼类多样性的监测。然而，目前，用于监测鱼类多样性的eDNA技术尚未标准化。该研究分别比较了两种过滤方法、三种过滤水样体积以及三种DNA提取方法的DNA产出效果，以期为珠江口等人为干扰频繁的河口生态系统提供一种合适的基于eDNA的鱼类多样性监测方法。结果表明，与基于过滤的沉淀法相比，直接过滤法更适用于珠江口的eDNA宏条形码技术研究；DNeasy Blood and Tissue Kit（BT）和苯酚/氯仿（PC）提取方法具有较高的DNA产量、扩增子序列变体（ASV）丰度和香农多样性指数，并可以获得更均匀和一致的鱼类群落组成；其中，PC和BT分别在提取过滤水样体积为1000 mL和2000 mL时，获得更高的物种检测率、鱼类多样性和种类组成一致性。与底拖网相比，eDNA宏条形码技术在珠江口鱼类多样性调查中具有更高的灵敏度，监测到的种类数量更多，但两种方法在种类组成上具有一定的差异，二者的结合有利于提高鱼类多样性监测的准确性。这些结果表明，在河口生态系统的鱼类群落监测中，eDNA宏条形码技术流程中相关步骤的方法选择需要慎重考虑。.