Using digital PCR to predict ciliate abundance from ribosomal RNA gene copy numbers.

Ciliates play a key role in most ecosystems. Their abundance in natural samples is crucial for answering many ecological questions. Traditional methods of quantifying individual species, which rely on microscopy, are often labour-intensive, time-consuming and can be highly biassed. As a result, we investigated the potential of digital polymerase chain reaction (dPCR) for quantifying ciliates. A significant challenge in this process is the high variation in the copy number of the taxonomic marker gene (ribosomal RNA [rRNA]). We first quantified the rRNA gene copy numbers (GCN) of the model ciliate, Paramecium tetraurelia, during different stages of the cell cycle and growth phases. The per-cell rRNA GCN varied between approximately 11,000 and 130,000, averaging around 50,000 copies per cell. Despite these variations in per-cell rRNA GCN, we found a highly significant correlation between GCN and cell numbers. This is likely due to the coexistence of different cellular stages in an uncontrolled (environmental) ciliate population. Thanks to the high sensitivity of dPCR, we were able to detect the target gene in a sample that contained only a single cell. The dPCR approach presented here is a valuable addition to the molecular toolbox in protistan ecology. It may guide future studies in quantifying and monitoring the abundance of targeted (even rare) ciliates in natural samples.

The Multiple States of Environmental DNA and What Is Known about Their Persistence in Aquatic Environments.

Increased use of environmental DNA (eDNA) analysis for indirect species detection has spurred the need to understand eDNA persistence in the environment. Understanding the persistence of eDNA is complex because it exists in a mixture of different states (e.g., dissolved, particle adsorbed, intracellular, and intraorganellar), and each state is expected to have a specific decay rate that depends on environmental parameters. Thus, improving knowledge about eDNA conversion rates between states and the reactions that degrade eDNA in different states is needed. Here, we focus on eukaryotic extraorganismal eDNA, outline how water chemistry and suspended mineral particles likely affect conversion among each eDNA state, and indicate how environmental parameters affect persistence of states in the water column. On the basis of deducing these controlling parameters, we synthesized the eDNA literature to assess whether we could already derive a general understanding of eDNA states persisting in the environment. However, we found that these parameters are often not being measured or reported when measured, and in many cases very few experimental data exist from which to draw conclusions. Therefore, further study of how environmental parameters affect eDNA state conversion and eDNA decay in aquatic environments is needed. We recommend analytic controls that can be used during the processing of water to assess potential losses of different eDNA states if all were present in a water sample, and we outline future experimental work that would help determine the dominant eDNA states in water.

Trade-offs between reducing complex terminology and producing accurate interpretations from environmental DNA: Comment on "Environmental DNA: What's behind the term?" by Pawlowski et al., (2020).

In a recent paper, "Environmental DNA: What's behind the term? Clarifying the terminology and recommendations for its future use in biomonitoring," Pawlowski et al. argue that the term eDNA should be used to refer to the pool of DNA isolated from environmental samples, as opposed to only extra-organismal DNA from macro-organisms. We agree with this view. However, we are concerned that their proposed two-level terminology specifying sampling environment and targeted taxa is overly simplistic and might hinder rather than improve clear communication about environmental DNA and its use in biomonitoring. This terminology is based on categories that are often difficult to assign and uninformative, and it overlooks a fundamental distinction within eDNA: the type of DNA (organismal or extra-organismal) from which ecological interpretations are derived.

Reliable eDNA detection and quantification of the European weather loach (Misgurnus fossilis).

The European weather loach (Misgurnus fossilis) is a cryptic and poorly known fish species of high conservation concern. The species is experiencing dramatic population collapses across its native range to the point of regional extinction. Although environmental DNA (eDNA)-based approaches offer clear advantages over conventional field methods for monitoring rare and endangered species, accurate detection and quantification remain difficult and quality assessment is often poorly incorporated. In this study, we developed and validated a novel digital droplet PCR (ddPCR) eDNA-based method for reliable detection and quantification, which allows accurate monitoring of M. fossilis across a number of habitat types. A dilution experiment under laboratory conditions allowed the definition of the limit of detection (LOD) and the limit of quantification (LOQ), which were set at concentrations of 0.07 and 0.14 copies µl-1 , respectively. A series of aquarium experiments revealed a significant and positive relationship between the number of individuals and the eDNA concentration measured. During a 3 year survey (2017-2019), we assessed 96 locations for the presence of M. fossilis in Flanders (Belgium). eDNA analyses on these samples highlighted 45% positive detections of the species. On the basis of the eDNA concentration per litre of water, only 12 sites appeared to harbour relatively dense populations. The other 31 sites gave a relatively weak positive signal that was typically situated below the LOQ. Combining sample-specific estimates of effective DNA quantity (Qe ) and conventional field sampling, we concluded that each of these weak positive sites still likely harboured the species and therefore they do not represent false positives. Further, only seven of the classified negative samples warrant additional sampling as our analyses identified a substantial risk of false-negative detections (i.e., type II errors) at these locations. Finally, we illustrated that ddPCR outcompetes conventional qPCR analyses, especially when target DNA concentrations are critically low, which could be attributed to a reduced sensitivity of ddPCR to inhibition effects, higher sample concentrations being accommodated and higher sensitivity obtained.

Three-tiered authentication of herbal traditional Chinese medicine ingredients used in women's health provides progressive qualitative and quantitative insight.

Traditional Chinese Medicine (TCM) herbal products are increasingly used in Europe, but prevalent authentication methods have significant gaps in detection. In this study, three authentication methods were tested in a tiered approach to improve accuracy on a collection of 51 TCM plant ingredients obtained on the European market. We show the relative performance of conventional barcoding, metabarcoding and standardized chromatographic profiling for TCM ingredients used in one of the most diagnosed disease patterns in women, endometriosis. DNA barcoding using marker ITS2 and chromatographic profiling are methods of choice reported by regulatory authorities and relevant national pharmacopeias. HPTLC was shown to be a valuable authentication tool, combined with metabarcoding, which gives an increased resolution on species diversity, despite dealing with highly processed herbal ingredients. Conventional DNA barcoding as a recommended method was shown to be an insufficient tool for authentication of these samples, while DNA metabarcoding yields an insight into biological contaminants. We conclude that a tiered identification strategy can provide progressive qualitative and quantitative insight in an integrative approach for quality control of processed herbal ingredients.

Harnessing eDNA metabarcoding to investigate fish community composition and its seasonal changes in the Oslo fjord.

In the face of global ecosystem changes driven by anthropogenic activities, effective biomonitoring strategies are crucial for mitigating impacts on vulnerable aquatic habitats. Time series analysis underscores a great significance in understanding the dynamic nature of marine ecosystems, especially amidst climate change disrupting established seasonal patterns. Focusing on Norway's Oslo fjord, our research utilises eDNA-based monitoring for temporal analysis of aquatic biodiversity during a one year period, with bi-monthly sampling along a transect. To increase the robustness of the study, a taxonomic assignment comparing BLAST+ and SINTAX approaches was done. Utilising MiFish and Elas02 primer sets, our study detected 63 unique fish species, including several commercially important species. Our findings reveal a substantial increase in read abundance during specific migratory cycles, highlighting the efficacy of eDNA metabarcoding for fish composition characterization. Seasonal dynamics for certain species exhibit clear patterns, emphasising the method's utility in unravelling ecological complexities. eDNA metabarcoding emerges as a cost-effective tool with considerable potential for fish community monitoring for conservation purposes in dynamic marine environments like the Oslo fjord, contributing valuable insights for informed management strategies.

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.

Molecular analyses of carangid fish diets reveal inter-predation, dietary overlap, and the importance of early life stages in trophic ecology.

Carangid fishes are commercially important in fisheries and aquaculture. They are distributed worldwide in both tropical and subtropical marine ecosystems. Their role in food webs is often unclear since their diet cannot be easily identified by traditional gut content analysis. They are suspected to prey on pelagic and benthic species, with clupeiform fishes being important dietary items for some species, though it is unknown whether carangids share food resources or show trophic segregation. Here, we used metabarcoding to overcome traditional challenges of taxonomic approaches to analyze the diet of seven carangid species caught as bycatch in the Brazilian southwest Atlantic sardine fishery. Stomach contents were processed from the following species: Caranx crysos, Caranx latus, Chloroscombrus chrysurus, Hemicaranx amblyrhynchus, Oligoplites saliens, Selene setapinnis, and Trachinotus carolinus. Identified diets were dominated by teleost fishes. The C. latus diet was the most distinct among the seven species, preferentially consuming Engraulis anchoita, but H. amblyrhynchus, O. saliens, and S. setapinnis also showed a trend of predominantly consuming small pelagic fishes. Finally, we found evidence of inter-predation in carangids, especially strong between S. setapinnis and C. crysos, suggesting that consumption of early life stages may result in indirect competition through reduced recruitment in these fishes. These findings provide unprecedented insights into the biodiversity in marine ecosystems, especially the poorly known diet of carangid fishes.

Horizon scan of DNA-based methods for quality control and monitoring of herbal preparations.

Herbal medicines and preparations are widely used in healthcare systems globally, but concerns remain about their quality and safety. New herbal products are constantly being introduced to the market under varying regulatory frameworks, with no global consensus on their definition or characterization. These biologically active mixtures are sold through complex globalized value chains, which create concerns around contamination and profit-driven adulteration. Industry, academia, and regulatory bodies must collaborate to develop innovative strategies for the identification and authentication of botanicals and their preparations to ensure quality control. High-throughput sequencing (HTS) has significantly improved our understanding of the total species diversity within DNA mixtures. The standard concept of DNA barcoding has evolved over the last two decades to encompass genomic data more broadly. Recent research in DNA metabarcoding has focused on developing methods for quantifying herbal product ingredients, yielding meaningful results in a regulatory framework. Techniques, such as loop-mediated isothermal amplification (LAMP), DNA barcode-based Recombinase Polymerase Amplification (BAR-RPA), DNA barcoding coupled with High-Resolution Melting (Bar-HRM), and microfluidics-based methods, offer more affordable tests for the detection of target species. While target capture sequencing and genome skimming are considerably increasing the species identification resolution in challenging plant clades, ddPCR enables the quantification of DNA in samples and could be used to detect intended and unwanted ingredients in herbal medicines. Here, we explore the latest advances in emerging DNA-based technologies and the opportunities they provide as taxa detection tools for evaluating the safety and quality of dietary supplements and herbal medicines.

Authentication of milk thistle commercial products using UHPLC-QTOF-ESI + MS metabolomics and DNA metabarcoding.

BACKGROUND: Milk thistle is one of the most popular hepatoprotectants, and is often sold in combination with other ingredients. Botanical supplements are known to be vulnerable to contamination and adulteration, and emerging technologies show promise to improve their quality control. METHODS: Untargeted and semi-targeted metabolomics based on UHPLC-QTOF-ESI+MS techniques, UV spectrometry, and DNA metabarcoding using Illumina MiSeq were used to authenticate eighteen milk thistle botanical formulations (teas, capsules, tablets, emulsion). RESULTS: Untargeted metabolomics separated 217 molecules and by multivariate analysis the discrimination between the different preparations was established. The semi-targeted metabolomics focused on 63 phytochemicals, mainly silymarin flavonolignans and flavonoids, that may be considered as putative biomarkers of authenticity. All formulations contained molecules from silymarin complexes at different levels. The quantitative evaluation of silybins was done using in parallel UV spectrometry and UHPLC-QTOF-ESI+MS and their correlations were compared. DNA metabarcoding detected milk thistle in eleven out of sixteen retained preparations, whereas two others had incomplete evidence of milk thistle despite metabolomics validating specific metabolites, e.g., silymarin complex, identified and quantified in all samples. Meanwhile, the DNA metabarcoding provided insights into the total species composition allowing the interpretation of the results in a broad context. CONCLUSION: Our study emphasizes that combining spectroscopic, chromatographic, and genetic techniques bring complementary information to guarantee the quality of the botanical formulations.

Complementary authentication of Chinese herbal products to treat endometriosis using DNA metabarcoding and HPTLC shows a high level of variability.

Traditional Chinese Medicine (TCM) is popular for the treatment of endometriosis, a complex gynecological disease that affects 10% of women globally. The growing market for TCMs has yielded a significant incentive for product adulteration, and although emerging technologies show promise to improve their quality control, many challenges remain. We tested the authenticity of two traditional Chinese herbal formulae used in women's healthcare for the treatment of endometriosis, known as Gui Zhi Fu Ling Wan (FL) and Ge Xia Zhu Yu Tang (GX). Dual-locus DNA metabarcoding analysis coupled with high-performance thin-layer chromatography (HPTLC) were used to authenticate 19 FL and six GX commercial herbal products, as well as three ad hoc prepared artificial mixtures. HPTLC was able to detect most of the expected ingredients via comparative component analysis. DNA metabarcoding was able to detect an unexpected species diversity in the products, including 38 unexpected taxa. Chromatography has a resolution for all species indirectly through the identification of marker compounds for the different species ingredients. Metabarcoding on the other hand yields an overview of species diversity in each sample, but interpretation of the results can be challenging. Detected species might not be present in quantities that matter, and without validated quantification, some detected species can be hard to interpret. Comparative analysis of the two analytical approaches also reveals that DNA for species might be absent or too fragmented to amplify as the relevant chemical marker compounds can be detected but no amplicons are assigned to the same species. Our study emphasizes that integrating DNA metabarcoding with phytochemical analysis brings valuable data for the comprehensive authentication of Traditional Chinese Medicines ensuring their quality and safe use.

Merging two eDNA metabarcoding approaches and citizen-science-based sampling to facilitate fish community monitoring along vast Sub-Saharan coastlines.

The coastline of Sub-Saharan Africa hosts highly diverse fish communities of great conservation value, which are also key resources for local livelihoods. However, many costal ecosystems are threatened by overexploitation and their conservation state is frequently unknown due to their vast spatial extent and limited monitoring budgets. Here, we evaluated the potential of citizen science-based eDNA surveys to alleviate such chronic data deficiencies and assessed fish communities in Mozambique using two 12S metabarcoding primer sets. Samples were either collected by scientific personnel or trained community members and results from the two metabarcoding primers were combined using a new data merging approach. Irrespective of the background of sampling personnel, a high average fish species richness was recorded (38 ± 20 OTUs per sample). Individual sections of the coastline largely differed in the occurrence of threatened and commercially important species, highlighting the need for regionally differentiated management strategies. A detailed comparison of the two applied primer sets revealed an important trade-off in primer choice with MiFish primers amplifying a higher number of species but Riaz primers performing better in the detection of threatened fish species. This trade-off could be partly resolved by applying our new data-merging approach, which was especially designed to increase the robustness of multiprimer assessments in regions with poor reference libraries. Overall, our study provides encouraging results but also highlights that eDNA-based monitoring will require further improvements of, for example, reference databases and local analytical infrastructure to facilitate routine applications in Sub-Saharan Africa.

Predation increases multiple components of microbial diversity in activated sludge communities.

Protozoan predators form an essential component of activated sludge communities that is tightly linked to wastewater treatment efficiency. Nonetheless, very little is known how protozoan predation is channelled via bacterial communities to affect ecosystem functioning. Therefore, we experimentally manipulated protozoan predation pressure in activated-sludge communities to determine its impacts on microbial diversity, composition and putative functionality. Different components of bacterial diversity such as taxa richness, evenness, genetic diversity and beta diversity all responded strongly and positively to high protozoan predation pressure. These responses were non-linear and levelled off at higher levels of predation pressure, supporting predictions of hump-shaped relationships between predation pressure and prey diversity. In contrast to predation intensity, the impact of predator diversity had both positive (taxa richness) and negative (evenness and phylogenetic distinctiveness) effects on bacterial diversity. Furthermore, predation shaped the structure of bacterial communities. Reduction in top-down control negatively affected the majority of taxa that are generally associated with increased treatment efficiency, compromising particularly the potential for nitrogen removal. Consequently, our findings highlight responses of bacterial diversity and community composition as two distinct mechanisms linking protozoan predation with ecosystem functioning in activated sludge communities.

Improving the reliability of eDNA data interpretation.

Global declines in biodiversity highlight the need to effectively monitor the density and distribution of threatened species. In recent years, molecular survey methods detecting DNA released by target-species into their environment (eDNA) have been rapidly on the rise. Despite providing new, cost-effective tools for conservation, eDNA-based methods are prone to errors. Best field and laboratory practices can mitigate some, but the risks of errors cannot be eliminated and need to be accounted for. Here, we synthesize recent advances in data processing tools that increase the reliability of interpretations drawn from eDNA data. We review advances in occupancy models to consider spatial data-structures and simultaneously assess rates of false positive and negative results. Further, we introduce process-based models and the integration of metabarcoding data as complementing approaches to increase the reliability of target-species assessments. These tools will be most effective when capitalizing on multi-source data sets collating eDNA with classical survey and citizen-science approaches, paving the way for more robust decision-making processes in conservation planning.

Development and application of eDNA-based tools for the conservation of white-clawed crayfish.

eDNA-based methods represent non-invasive and cost-effective approaches for species monitoring and their application as a conservation tool has rapidly increased within the last decade. Currently, they are primarily used to determine the presence/absence of invasive, endangered or commercially important species, but they also hold potential to contribute to an improved understanding of the ecological interactions that drive species distributions. However, this next step of eDNA-based applications requires a thorough method development. We developed an eDNA assay for the white-clawed crayfish (Austropotamobius pallipes), a flagship species of conservation in the UK and Western Europe. Multiple subsequent in-situ and ex-situ validation tests aimed at improving method performance allowed us to apply eDNA-based surveys to evaluate interactions between white-clawed crayfish, crayfish plague and invasive signal crayfish. The assay performed well in terms of specificity (no detection of non-target DNA) and sensitivity, which was higher compared to traditional methods (in this case torching). The eDNA-based quantification of species biomass was, however, less reliable. Comparison of eDNA sampling methods (precipitation vs. various filtration approaches) revealed that optimal sampling method differed across environments and might depend on inhibitor concentrations. Finally, we applied our methodology together with established assays for crayfish plague and the invasive signal crayfish, demonstrating their significant interactions in a UK river system. Our analysis highlights the importance of thorough methodological development of eDNA-based assays. Only a critical evaluation of methodological strengths and weaknesses will allow us to capitalise on the full potential of eDNA-based methods and use them as decision support tools in environmental monitoring and conservation practice.

Influence of accuracy, repeatability and detection probability in the reliability of species-specific eDNA based approaches.

Environmental DNA (eDNA) barcoding has a high potential to increase the cost-efficiency of species detection and monitoring in aquatic habitats. However, despite vast developments in the field, many published assays often lack detailed validation and there is little to no commonly (agreed upon) standardization of protocols. In this study, we evaluated the reliability of eDNA detection and quantification using published primers and assays targeting the Freshwater Pearl Mussel as a model organism. We first assessed limits of detection for two different target genes (COI and 16S) following the MIQE guidelines, and then tested the reliability of quantification in a double-blind mesocosm experiment. Our results reveal that different methodological indicators, namely accuracy, repeatability and detection probability affected the reliability of eDNA measurement at the different levels tested. The selection of the optimal analytical method was mainly determined by detection probability. Both the COI and 16S assays were highly specific for the targeted organism and showed similar accuracy and repeatability, whilst the limit of detection was clearly lower for the COI based approach. In contrast, the reliability of eDNA quantification hinged on repeatability, reflected by the scattering (r2 = 0.87) around the relationship between eDNA and mussel density in mesocosms. A bootstrapping approach, which allowed for the assignment of measures associated with repeatability of samples, revealed that variability between natural replicates (i.e. accuracy) strongly influenced the number of replicates required for a reliable species detection and quantification in the field.

Combining ddPCR and environmental DNA to improve detection capabilities of a critically endangered freshwater invertebrate.

Isogenus nubecula is a critically endangered Plecoptera species. Considered extinct in the UK, I. nubecula was recently rediscovered (in one location of the River Dee, Wales), after 22 years of absence. In a similar way to many other species of Perlodidae, I. nubecula could be utilised as a bio-indicator, for assessing water quality and health status of a given freshwater system. However, conventional monitoring of invertebrates via kick-sampling, is invasive and expensive (time consuming). Further, such methods require a high level of taxonomic expertise. Here, we compared the traditional kick-sampling method with the use of eDNA detection using qPCR and ddPCR-analyses. In spring 2018, we sampled eDNA from twelve locations on the River Dee. I. nubecula was detected using kick-sampling in five of these locations, three locations using both eDNA detection and kick-sampling and one location using eDNA detection alone - resulting in a total of six known and distinct populations of this critically endangered species. Interestingly, despite the eDNA assay being validated in vitro and in silico, and results indicating high sensitivity, qPCR analysis of the eDNA samples proved to be ineffective. In contrast, ddPCR analyses resulted in a clear detection of I. nubecula at four locations suggesting that inhibition most likely explains the large discrepancy between the obtained qPCR and ddPCR results. It is therefore important to explore inhibition effects on any new eDNA assay. We also highlight that ddPCR may well be the best option for the detection of aquatic organisms which are either rare or likely to shed low levels of eDNA into their environment.

Rediscovery of the critically endangered 'scarce yellow sally stonefly' Isogenus nubecula in United Kingdom after a 22 year period of absence.

The critically endangered 'scarce yellow sally stonefly' Isogenus nubecula (Newman, 1833) (Plecoptera: Perlodidae) was rediscovered in the United Kingdom (UK) in 2017. This rediscovery comes after a 22-year period of absence despite numerous surveys since its last record in 1995. This species is one of the rarest stoneflies in the UK and Europe and its rediscovery is of international significance, being the westernmost point in Europe where the species is found, with the next nearest populations occurring in Austria and western Hungary, Slovakia, and central Sweden. The species is classed as pRDB2 (vulnerable), however is not listed in the British Red Data Book despite only being present (as far as records detail) in one river, the River Dee in North Wales, UK. Only fourteen individuals were caught and the need for conservation of this rare stonefly is therefore of paramount importance. We have made recommendations for the need to increase survey effort using environmental DNA (eDNA) techniques in order to fully understand the species range in this river and those in the surrounding area. The DNA sequence of I. nubecula has been uploaded on GenBank for further genetic studies. Captive rearing could also be explored with possible reintroductions to sites within its former UK range.