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ISSN (Online) 2603-3925
Published by Pensoft Homepage  [45 journals]
  • Food products identified as source of a foodborne disease outbreak by a
           fast and robust likelihood estimation

    • Abstract: ARPHA Conference s 4: e68945
      DOI : 10.3897/aca.4.e68945
      Authors : Jakub Fusiak, Kyrre Kausrud, Marion Gottschald, Dominic Tölle, Marco Rügen, Birgit Lewicki, Isaak Gerber, Michele Kayser, Alexander Falenski, Armin Weiser, Solveig Jore, Madelaine Norström, Katja Alt : Identifying a specific product causing a foodborne disease outbreak can be difficult, especially when dealing with a large amounts of suspicious food items and weak epidemiological evidence. A previously described likelihood model (Norström et al. 2015), improved within the OHEJP NOVA project, helps to prioritize food products that should be sampled for laboratory analysis. It is the aim of our study to integrate this approach into state of the art tracing software FoodChain-Lab (FCL; https://foodrisklabs.bfr.bund.de/foodchain-lab) developed at BfR to facilitate outbreak investigations.The model improved by Kausrud et al. in R (Ihaka and Gentleman 1996) uses wholesale data, the distribution of disease cases and census data to sort food items by their estimated likelihood to be the source of an outbreak. We developed a fast and secure intuitive software module using the Web Assembly technology (Haas et al. 2017) allowing professionals to embed the module easily into other applications. We integrated the module into the FCL web application for tracing (FCL Web; https://fcl-portal.bfr.berlin) to provide an intuitive and user-friendly solution. This solution combines a simple data input with extended data wrangling to make the calculation of the NOVA model as easy as possible. Since the model can be executed directly inside the web browser and therefore does not rely on any server environment, the possibility of data leakage can be highly reduced. The implementation of the advanced likelihood model into FCL Web increase the availability of this model and provides investigators easy, fast and reliable usage to improve outbreak investigation workflows.  HTML XML PDF
      PubDate: Fri, 28 May 2021 10:00:00 +030
  • One Health EJP - RaDAR model inventory: a user-friendly tool for
           annotating and exchanging models

    • Abstract: ARPHA Conference s 4: e68936
      DOI : 10.3897/aca.4.e68936
      Authors : Jakub Fusiak, Annemarie Käsbohrer : The lack of a harmonized model exchange formats among modelling tools impedes communication between researchers, since the exchange and usage of existing models in various software environments can be very difficult. The RaDAR model inventory aims to provide a platform to exchange models among professionals utilizing the Food Safety Knowledge Exchange (FSKX) Format (de Alba Aparicio et al. 2018) as a harmonized model exchange format. FSKX defines a framework that encodes all relevant data, metadata, and model scripts in an exchangeable file format. However, the creation of such a file can be a time-consuming and difficult process. To increase the usage of the FSK standard, we developed the RaDAR model inventory web application that targets the process of creating an FSKX file for the end user. Our inventory aims to be a user-friendly tool that allows users to create, read, edit, write, execute and compile FSKX files within the web browser. The possibility of sharing models with the public or a specific group of people facilitates collaboration and the exchange of information. Since the RaDAR model inventory is based on the open-source technology of Project Jupyter (Granger and Perez 2021), it can support nearly all relevant programming languages executed within a reproducible cloud-computing environment. The intuitive nature of the RaDAR model along with its wide range of features reduce the threshold for contribution to a harmonized model exchange format and eases collaboration. The RaDAR model inventory can be accessed at http://ejp-radar.eu. HTML XML PDF
      PubDate: Fri, 28 May 2021 10:00:00 +030
  • How much time have we got'

    • Abstract: ARPHA Conference s 4: e68934
      DOI : 10.3897/aca.4.e68934
      Authors : Kyrre Kausrud, Karin Lagesen, Ryan Easterday, Jason Whittington, Wendy Turner, Cecilia Wolff, Marie Feiring, Nils Stenseth : Here we present a developing probabilistic simulation model and tool to assess likely lead times from emergence to detection and arrival for new emerging infectious diseases (EIDs). Key aspects include combining real-world data available on multiple scales with a flexible underlying disease model.As demonstrated by the SARS-CoV-2 pandemic and other emerging infectious diseases, there is a need for scenario exploration for mitigation, surveillance and preparedness strategies. Existing simulation engines have been assessed but found to offer an insufficient set of features with regards to flexibility and control over processes, disease model structure and data sets incorporated for a wider enough range of diseases, circumstances, cofactors and scenarios (Heslop et al. 2017) to suit our aims.We are therefore developing the first version of a simulation model designed to be able to incorporate a diverse range of disease models and data sources including multiple transmission and infectivity stages, multiple host species, varying and evolving virulence, socioeconomic differences, climate events and public health countermeasures. It is designed to be flexible with respect to implementing both improvements in the model structure and data as they become available. It is based on a discrete-time (daily) structure where spatial movement and transition between categories and detection are stochastic rates dependent on spatial data and past states in the model, while being informed by the most suitable data available (Fig. 1).The probability of detection is in itself treated as a probabilistic process and treated as a variable dependent on socioeconomic factors and parameterized by past performance, yet open for manipulation in scenario exploration regarding surveillance and reporting effectiveness.Pathogen hotspot data are sourced from literature and included as a probabilistic assessment of emergence as well as a source of cofactor data (Allen et al. 2017), population data are adressed (Leyk et al. 2019) for utility and combined with data on local connectivity (Nelson et al. 2019) and transnational movement patterns (Recchi et al. 2019Fig. 1), as well as an increasing set of ecological and socioeconomic candidate variables.Model parameterization relies on a machine learning framework with matching to the often partial data available for known relevant disease cases as the training data, and assessing them for plausible ranges of input for new, hypothetical EIDs.As parameterizations improve, the range of scenarios to explore will incorporate effects of climate change and multiple stressors. When a suitable version becomes available it will be shared under a MIT license. HTML XML PDF
      PubDate: Fri, 28 May 2021 10:00:00 +030
  • Practical aspects of implementing the IRIDA system as a solution for One
           Health bioinformatics analyses

    • Abstract: ARPHA Conference s 4: e68913
      DOI : 10.3897/aca.4.e68913
      Authors : Jeevan Karloss Antony-Samy, Georgios Marselis, Eve Fiskebeck, Taran Skjerdal, Camilla Sekse, Karin Lagesen : Managing sequence data, associated metadata, bioinformatics analyses and results can be challenging. In a One Health context, the challenge is even larger as there are many actors involved, many diverse types of results need to be produced, and the ensuing process data, such as software versions and options have to be tracked for auditing purposes. In addition, results must often be produced rapidly to be actionable, and non-bioinformaticians should be able to perform the the analyses. Therefore,  a graphical user interface (preferably web system) with pipelines and visualization tools are needed to do these analyses. The Public Health Agency of Canada has together with other actors developed the web based system IRIDA (https://www.irida.ca) which uses Galaxy for analyses. IRIDA comes with a set of pipelines, visualization tools and a project based data management system that allows for fine grained data access control, which satisfies many of the requirements that a One Health bioinformatics platform dictates.  However, as is often the case with a system meant to satisfy high demands, the platform is not trivial to set up and adapt for local use. In our setup, we are using two web servers, two database servers and one file server. The IRIDA web server provides the user interface. The Galaxy web server receives commands from IRIDA, executes the commands and returns results. Each web server has a database that keeps their respective metadata: user information, file locations and results. The actual files are stored on the fileserver. This spoke-and-wheel infrastructure was implemented to ensure minimum disruption of service if a component should go down.  To get the necessary compute resources for this system, we are contracting with the Norwegian Research and Education Cloud (NREC), which offers Infrastructure as a Service (IaaS) services for Norwegian institutions and universities. NREC utilizes template VM images which can be instantiated according to need. The automated configuration and orchestration of images ensure that we can have dynamic access to resources according to need. This dynamic scaling is accomplished through collaboration with Elixir Norway. They have implemented the Pulse software which can check usage and instantiate and take down virtual machines as needed. At the Institute, we have spent close to two years on exploring and setting up this system. We have learned that it is important to not underestimate the amount compute resources needed to get a solid setup. However, having enough compute is irrelevant without knowledgeable staff. IRIDA comes with many features, which require considerable prior knowledge to adapt and set up in a local infrastructure. This includes knowledge on webservers, database systems, linux administration and Galaxy systems administration. The complexity dictates that these systems need to be set up and managed by in-house IT trained staff that will be able to tend the system along the way. It is also very important to maintain interactions with the users of the system, to ensure that the setup produces results that are useful to the users. To accomplish this, bioinformaticians are needed to develop pipelines and visualizations that give results that will on their own be easy for users to interpret in a biologically correct manner. Last but not least - such systems require a significant investment from the institution, thus it is important to showcase the benefits that the system will provide.    HTML XML PDF
      PubDate: Fri, 28 May 2021 10:00:00 +030
  • Sykdomspulsen One Health - A real time surveillance system in an
           infrastructure coping with half a million analysis a day

    • Abstract: ARPHA Conference s 4: e68891
      DOI : 10.3897/aca.4.e68891
      Authors : Clemence Koren, David Swanson, Gry Grøneng, Gunnar Rø, Petter Hopp, Malin Jonsson, Richard White : Sykdomspulsen is a real time surveillance system developed by the Norwegian Institute of Public Health (NIPH) for One Health surveillance and the surveillance of other infectious diseases in humans like respiratory diseases and lately covid-19.The One Health surveillance comprise of Campylobacter data from humans and chicken farms and also includes diagnosis codes from doctor appointments and weather data with analysis forecasting outbreaks in Norway. It is a joint project between the Norwegian Institute of Public Health (NIPH) and the Norwegian Veterinary Institute (NVI), under the framework of the OHEJP NOVA (Novel approaches for design and evaluation of cost-effective surveillance across the food chain) and MATRIX (Connecting dimensions in One-Health surveillance) projects.The system relies on two pillars, the first being an analytics infrastructure which in real time retrieves data from tens of sources, cleans and harmonizes it, then runs over half a million analyses each day and produces over 20 000 000 rows of results to be used every day. The analytics infrastructure is based on R. Results are notably being used by NIPH for the monitoring of covid-19 development and the surveillance of other transmittable diseases such as influenza and gastro-intestinal illness. The analytics framework also generates hundreds of reports every day, directed at dissemination to municipal health authorities. This framework is not currently publicly available, but an open-source release is expected by the end of 2021.The second pilar is an interactive R Shiny dashboard platform, which is used for communicating the data and the model results to partner organisations. It allows for the easy creation of a website where public and animal health researchers and food safety experts can view real time analyses. This dashboard combines the powerful data visualisation and analysis strength of R with the accessibility, flexibility, structure and interactivity of web-based platforms.The result is a real time interactive surveillance system, that is supported by a solid infrastructure and streamlined data flow, and shared with actors through a beautiful and user-friendly website, based entirely on R. HTML XML PDF
      PubDate: Fri, 28 May 2021 10:00:00 +030
  • Making Linked Data accessible for One Health Surveillance with the "One
           Health Linked Data Toolbox"

    • Abstract: ARPHA Conference s 4: e68821
      DOI : 10.3897/aca.4.e68821
      Authors : Taras Günther, Matthias Filter, Fernanda Dórea : In times of emerging diseases, data sharing and data integration are of particular relevance for One Health Surveillance (OHS) and decision support. Furthermore, there is an increasing demand to provide governmental data in compliance to the FAIR (Findable, Accessible, Interoperable, Reusable) data principles. Semantic web technologies are key facilitators for providing data interoperability, as they allow explicit annotation of data with their meaning, enabling reuse without loss of the data collection context. Among these, we highlight ontologies as a tool for modeling knowledge in a field, which simplify the interpretation and mapping of datasets in a computer readable medium; and the Resource Description Format (RDF), which allows data to be shared among human and computer agents following this knowledge model. Despite their potential for enabling cross-sectoral interoperability and data linkage, the use and application of these technologies is often hindered by their complexity and the lack of easy-to-use software applications.To overcome these challenges the OHEJP Project ORION developed the Health Surveillance Ontology (HSO). This knowledge model forms a foundation for semantic interoperability in the domain of One Health Surveillance. It provides a solution to add data from the target sectors (public health, animal health and food safety) in compliance with the FAIR principles of findability, accessibility, interoperability, and reusability, supporting interdisciplinary data exchange and usage. To provide use cases and facilitate the accessibility to HSO, we developed the One Health Linked Data Toolbox (OHLDT), which consists of three new and custom-developed web applications with specific functionalities. The first web application allows users to convert surveillance data available in Excel files online into HSO-RDF and vice versa. The web application demonstrates that data provided in well-established data formats can be automatically translated in the linked data format HSO-RDF. The second application is a demonstrator of the usage of HSO-RDF in a HSO triplestore database. In the user interface of this application, the user can select HSO concepts based on which to search and filter among surveillance datasets stored in a HSO triplestore database. The service then provides automatically generated dashboards based on the context of the data. The third web application demonstrates the use of data interoperability  in the OHS context by using HSO-RDF to annotate meta-data, and in this way link datasets across sectors. The web application provides a dashboard to compare public data on zoonosis surveillance provided by EFSA and ECDC.The first solution enables linked data production, while the second and third provide examples of linked data consumption, and their value in enabling data interoperability across sectors. All described solutions are based on the open-source software KNIME and are deployed as web service via a KNIME Server hosted at the German Federal Institute for Risk Assessment. The semantic web extension of KNIME, which is based on the Apache Jena Framework, allowed a rapid an easy development within the project. The underlying open source KNIME workflows are freely available and can be easily customized by interested end users.With our applications, we demonstrate that the use of linked data has a great potential strengthening the use of FAIR data in OHS and interdisciplinary data exchange. HTML XML PDF
      PubDate: Fri, 28 May 2021 10:00:00 +030
  • INSaFLU-TELE-Vir: an open web-based bioinformatics suite for influenza and
           SARS-CoV-2 genome-based surveillance

    • Abstract: ARPHA Conference s 4: e68845
      DOI : 10.3897/aca.4.e68845
      Authors : Miguel Pinheiro, Ricardo Pais, Joana Isidro, Miguel Pinto, Carlijn Bogaardt, Joaquin Prada, Daniel Horton, João Gomes, Vítor Borges : A new era of virus surveillance is emerging based on the real-time monitoring of virus evolution at whole-genome scale (World Health Organization 2021). Although national and international health authorities have strongly recommended this technological transition, especially for influenza and SARS-CoV-2 (World Health Organization 2021, Revez et al. 2017), the implementation of genomic surveillance can be particularly challenging due to the lack of bioinformatics infrastructures and/or expertise to process and interpret next-generation sequencing (NGS) data (Oakeson et al. 2017).We developed and implemented INSaFLU-TELE-Vir platform (https://insaflu.insa.pt/) (Borges et al. 2018), which is an influenza-  and SARS-CoV-2-oriented bioinformatics free web-based suite that handles primary NGS data (reads) towards the automatic generation of the main “genetic requests'' for effective and timely laboratory surveillance. By handling NGS data collected from any amplicon-based schema (making it applicable for other pathogens), INSaFLU-TELE-Vir enables any laboratory to perform multi-step and intensive bioinformatics analyses in a user-oriented manner without requiring advanced training.INSaFLU-TELE-Vir handles NGS data collected from distinct sequencing technologies (Illumina, Ion Torrent and Oxford Nanopore Technologies), with the possibility of constructing comparative analyses using different technologies. It gives access to user-restricted sample databases and project management, being a transparent and flexible tool specifically designed to automatically update project outputs as more samples are uploaded. Data integration is thus cumulative and scalable, fitting the need for both routine surveillance and outbreak investigation activities.The bioinformatics pipeline consists of six core steps:read quality analysis and improvement,human betacoronaviruses (including SARS-CoV-2 Pango lineages) and influenza type/subtype classification,mutation detection and consensus generation,coverage analysis,alignment/phylogeny,intra-host minor variant detection (and automatic detection of putative mixed infections).The multiple outputs are provided in nomenclature-stable and standardized formats that can be visualized and explored in situ or through multiple compatible downstream applications for fine-tuned data analysis.Novel features are being implemented into the INSaFLU-TELE-Vir bioinformatics toolkit as part of the OHEJP TELE-Vir (https://onehealthejp.eu/jrp-tele-vir/) project, including rapid detection of selected genotype-phenotype associations, and enhanced geotemporal data visualization.All the code is available in github (https://github.com/INSaFLU) with the possibility of a local docker installation (https://github.com/INSaFLU/docker). A detailed documentation and tutorial is also available (https://insaflu.readthedocs.io/en/latest/).In summary, INSaFLU supplies public health laboratories and researchers with an open and user-friendly framework, potentiating a strengthened and timely multi-country genome-based virus surveillance. HTML XML PDF
      PubDate: Fri, 28 May 2021 10:00:00 +030
  • The Glossaryfication Web Service – an automated glossary creation tool
           to support One Health communication

    • Abstract: ARPHA Conference s 4: e68843
      DOI : 10.3897/aca.4.e68843
      Authors : Estibaliz Lopez de Abechuco, Nazareno Scaccia, Taras Günther, Matthias Filter : Efficient communication and collaboration across sectors is an important precondition for true One Health Surveillance (OHS) activities. Despite the overall willingness to embrace the One Health paradigm, it is still challenging to accomplish this in day-to-day practice due to the differences in terminology and interpretation of sector-specific terms. In this sense, simple interventions like the inclusion of integrative glossaries in OHS documents (e.g. reports, research papers and guidelines) would help to reduce misunderstandings and could significantly improve the written communication in OHS. Here, we present the Glossaryfication Web Service that generates a document-specific glossary for any text file provided by the user. The web service automatically adds the available definitions with their corresponding references for the words in the document that match with terms in the user-selected glossaries.The Glossaryfication Web Service was developed to provide added value to the OHEJP Glossary that was developed within the OHEJP project ORION. The OHEJP Glossary improves the communication and collaboration among OH sectors by providing an online resource that lists relevant OH terms and sector-specific definitions. The Glossaryfication Web Service supports the practical use of the curated OHEJP Glossary and can also source information from other glossaries relevant for OH professionals (currently supporting the online CDC, WHO and EFSA glossaries).The Glossaryfication Web Service was created using the open-source software KNIME and the KNIME Text Processing extension (https://www.knime.com/knime-text-processing). The Glossaryfication KNIME workflow is deployed on BfR’s KNIME Server infrastructure providing an easy-to-use web interface where the users can upload their documents (any text-type file e.g. PDF, Word, Excel) and select the desired glossary to compare with. The Glossaryfication KNIME workflow reads in the document provided via the web interface and applies natural language processing (e.g. text cleaning, stemming), transforming (bag-of-words generation) and information retrieval methods to identify the matching terms in the selected glossaries.The Glossaryfication Web Service generates as an output a table containing all the terms that match with the selected glossaries. It also provides the available definitions, corresponding references and additional meta-information, e.g. the term frequency, i.e., how often each term appears in the given text, and the sectoral classification (only for the OHEJP Glossary terms). Furthermore, the workflow generates a tag cloud where the terms are categorized as: (i) exact match when the term in the text matches exactly with the entry of this term in the glossary; (ii) inexact match when the term appears in the text slightly modified (e.g. plural forms or suffixes) and (iii) non-matching that corresponds to all the other words appearing in the text that do not match with any glossary term. Through the user interface, the users can then choose if they want to download the whole list of terms, select only the exact/inexact matching terms, or just choose those terms and definitions that match with the meaning intended for this term in the user-provided document. The resulting table of terms can be downloaded as an Excel file and added to the user’s document as a document-specific glossary.The Glossaryfication Web Service provides an easy-to-adopt solution to enrich documents and reports with more comprehensive and unambiguous glossaries. Furthermore, it improves the referentiality of terms and definitions from different OH sectors. An additional feature provided by the Glossaryfication Web Service is the possibility of extending its use to other glossaries from other national or international institutions allowing the user to customize this glossary creation service.  HTML XML PDF
      PubDate: Fri, 28 May 2021 10:00:00 +030
  • FoodChain-Lab Web: An integrative modular software to visualise and
           analyse complex global food supply chain networks during foodborne

    • Abstract: ARPHA Conference s 4: e68835
      DOI : 10.3897/aca.4.e68835
      Authors : Marion Gottschald, Birgit Lewicki, Alexander Falenski, Marco Rügen, Isaak Gerber, Jakub Fusiak, Dominic Tölle, Annemarie Käsbohrer, Armin Weiser : In times of globalised food and feed trade, powerful integrative software tools are essential to solve foodborne crises quickly and reliably. The FoodChain-Lab web application (FCL Web; https://fcl-portal.bfr.berlin/) is such a tool. FCL Web is free and open-source software which helps to trace back and forward food along complex global supply chains during foodborne disease outbreaks or other food-related events. In the framework of One Health EJP COHESIVE, the efforts of several national and international tracing-related software projects are integrated within FCL Web to provide a modular tracing platform following the One Health approach.FCL Web unifies interactive tracing data visualisation, analysis as well as reporting - and in the future data collection - in one modular tracing platform (Fig. 1). The interactive analysis module was developed in a project with EFSA and offers automated visualisation of supply chains based on the needs of the user. A data table displays key information on involved food business operators and food items and includes comprehensive filter functions to analyse the information given in the table. The analysis module also helps to run simulations on hypothetical cross contamination or geographic clustering events during outbreaks via a scoring algorithm for deliveries and food business operators. A pilot version of a reporting module was integrated in FCL Web as well to display tracing, sample and case information in a format suitable for publishing tracing results in outbreak reports. A web-based tracing data collection mask offering a guided and structured data assessment with access to curated data was developed in a national project and will be integrated in FCL Web soon. Its multi-language design allows for potential European-wide use. In the future, more modules, e.g. to analyse genome sequencing data in the context of tracing are planned for FCL Web.With its features and its integrative approach, FCL Web blends seamlessly into a list of crucial tracing tool projects in Europe. In the future, these tools will be strongly interconnected to serve several tracing purposes on the local, national or European level. Hence, there is a need to improve interoperability of the tools e.g. via a universal data exchange format. HTML XML PDF
      PubDate: Fri, 28 May 2021 10:00:00 +030
  • An interactive online IT tool to aim the environmental surveillance of
           veterinary antibiotics in agriculture and pasture lands

    • Abstract: ARPHA Conference s 4: e68820
      DOI : 10.3897/aca.4.e68820
      Authors : Antonio Rodríguez, Ana de la Torre : The undermining of the therapeutic effectiveness of antibiotics by their widespread use is causing the emergence of antimicrobial resistance, which is a major threat for both animal and human health. Since most veterinary antibiotics employed in livestock production are excreted essentially unaltered, they have been identified as major contributors of environmental contamination. However, the efforts of monitoring antimicrobial effects are focused on humans and livestock, neglecting the environment. The European Union institutions recognized this gap in the appreciation of the issue, and adopted an approach that includes to prioritize environmental tracking and to build the tools to make it economically accessible. This abstract has three main targets. Firstly, to fill the gap applying the IT methodological approach (the soil vulnerability map to antibiotic contamination) developed by De La Torre et al. (2012). Secondly, to identify the main livestock species and scenarios (agriculture and pasture) to be prioritized in surveillance efforts. Finally, to implement the code of agriculture practices and the stocking rates of grazing animals based on high vulnerability areas for antibiotic contamination. To facilitate the implementation of this risk evaluation procedure, we developed an interactive tool that allows to obtain downloadable maps of soil vulnerability to contamination for several land use (agriculture and pasture) and livestock (cattle, pig and chicken) scenarios for any veterinary antibiotics. Additionally, the tool allows to obtain a plot of the mean vulnerability of each considered administrative unit. We implemented the European Union countries as an example, but the tool could be applied to individual countries or even regional or sub-national scales. HTML XML PDF
      PubDate: Fri, 28 May 2021 10:00:00 +030
  • Performance and cost-efficiency of eDNA and eRNA capture methodologies:
           experimental assessment using cultured microalgae

    • Abstract: ARPHA Conference s 4: e65098
      DOI : 10.3897/aca.4.e65098
      Authors : Anastasija Zaiko, Ulla von Ammon, Jacqui Stuart, Kirsty Smith, Richard Yao, Melissa Welsh, Xavier Pochon, Holly Bowers : Following the recent leap in biotechnologies and particularly in high-throughput sequencing techniques, environmental DNA and RNA (eDNA and eRNA) are increasingly being used for biodiversity assessments and monitoring of complex ecosystems – lakes, streams and coastal waters. Growing interest and affordability of eDNA/eRNA based tools have led to the emergence of manifold protocols for capturing genetic material from variable biological matrices, e.g. soil, water, feces, biofilms, etc. The variability in eDNA and eRNA material (ranging from free-floating molecules to cellular complexes to intact organisms) and its patchy distribution in the environment may substantially affect effective capture from the environment. Therefore, it is extremely challenging for stakeholders to standardize or choose optimal protocols, impeding incorporation of eDNA/eRNA methods into routine monitoring and surveillance programs.Although there is still no consensus on the standardized workflow for processing eDNA/eRNA samples, the common practice for water samples is to concentrate nucleic acids (NA) via filtration, ranging from 0.22 to 20-micron pore size. Although using the smallest pore is assumed efficient for NA capture from a wide range of material (including sub-cellular particles), a trade-off between detection of the meaningful molecular signal and time(cost)-efficiency is needed. Using finer pore membranes increases the likelihood of clogging and prohibits processing larger volumes of water, thus reducing the chances of detecting rare biodiversity. Moreover, large sample volumes may be accompanied by increased concentrations of inhibitory substances (e.g. humic compounds), suppressing target molecular signal.Comparative studies involving formal cost-efficiency assessments are lacking, restricting informed decision-making around the optimized sampling approach for addressing a particular research or surveillance question. Identifying the optimal combination of time effort and signal detection efficiency is particularly crucial for targeted surveillance, e.g. detection and monitoring of nuisance organisms, endangered and indicator taxa or other species of particular economic or cultural importance. Although it has been previously shown that larger pore filters can be as efficient for species detection from waterborne eDNA, more data are needed on the amount and type of NA material capture and loss.Here, we present a comparison study using an easily cultured microalgal species (Alexandrium pacificum) as a proxy to test the effectiveness of different filter membranes (cellulose acetate membranes of 5 μm, 1.2 μm or 0.45 μm pore size, and positively charged nylon membrane with 1.2 μm pore size) in the context of targeted species detection. We performed an efficiency analysis to identify the method that delivered the optimal use of resources. A tiered experimental design was applied to: i) assess the impact of membranes on capturing various fractions of target eDNA/eRNA (intact cells, partially lysed cells, naked NAs) spiked into pre-filtered and ambient environmental seawater and ii) establish efficiency and utility of different membranes in terms of optimizing the performance – maximized output (capture of target eDNA and eRNA) balanced against minimized time and cost input.The results showed no statistically significant difference between membranes for capturing DNA signal from intact and partially lysed cell treatments. However, positively charged nylon membranes were more efficient in capturing naked NAs, as well as RNA from partially lysed cells. In terms of time effort and volume processed, higher efficacy was reported for the larger pore size cellulose membranes. However positively charged nylon consistently performed better for capturing RNA signal across treatments. HTML XML PDF
      PubDate: Mon, 15 Mar 2021 09:30:00 +020
  • Environmental DNA methods for the analysis of macroorganismal populations

    • Abstract: ARPHA Conference s 4: e65549
      DOI : 10.3897/aca.4.e65549
      Authors : Hideyuki Doi : Environmental DNA (eDNA) methods have been widely used to investigate the distribution and abundance/biomass of macroorganisms. eDNA methods analyze DNA collected directly from the environment, such as from water, soil, and air. The techniques have been applied to many taxa inhabiting various aquatic and terrestrial ecosystems. The recent development of eDNA methods has revolutionized the way we assess macroorganisms in natural environments.In this talk, I will present current developments of eDNA methodology, especially with regard to population analysis using various DNA measurement methods. For example, 1) eDNA was used to assess fish species distributions and abundance/biomass (Takahara et al. 2012, Doi et al. 2017a), 2) quantitative PCR of sedimentary DNA was applied to sediment core samples to detect the DNA of three dominant fish species spanning the last 300 years (Kuwae et al. 2020), and 3) new methods for sampling eDNA from water (Doi et al. 2017b) and on-site measurement (Doi et al. 2020) will be presented. I end by addressing the need for standardized protocols for eDNA monitoring to enable broader uptake of eDNA technology (Minamoto et al. 2021). HTML XML PDF
      PubDate: Mon, 8 Mar 2021 16:47:58 +0200
  • The pathway of molecular methods from research to routine use

    • Abstract: ARPHA Conference s 4: e65556
      DOI : 10.3897/aca.4.e65556
      Authors : Meissner Kristian : In this talk I explore how to advance molecular methods from tools in the research domain to routine use in national biomonitoring. I outline the necessity of common guidance, networks, international pilot studies and cooperation with officials to achieve the goal of method uptake into routine use. Lastly, I will explain the role that international method standardization plays in speeding up the uptake process of molecular methods into routine biomonitoring. HTML XML PDF
      PubDate: Mon, 8 Mar 2021 16:47:52 +0200
  • Degradation factors of environmental DNA evaluated by experiments and

    • Abstract: ARPHA Conference s 4: e65552
      DOI : 10.3897/aca.4.e65552
      Authors : Tatsuya Saito, Hideyuki Doi : Environmental DNA (eDNA) methods have been developed to detect organisms' distributions and abundance/biomass in various environments. eDNA degradation is critical for eDNA evaluation, but, the dynamics and mechanisms of eDNA degradation are largely unknown, especially when considering different eDNA sources, e.g., cell-derived and fragmental DNA. In this study, we conducted the degradation experiments (Saito and Doi 2020a) and a meta-analysis (Saito and Doi 2020b). Firstly, we experimentally evaluated the degradation rates of eDNA derived from multiple sources, including fragmental DNA (the DNA of internal positive control, IPC), free cells from Oncorhynchus kisutch, and the resident species (Saito and Doi 2020a). We conducted the experiments with pond and seawater to evaluate the differences between freshwater and marine habitats. Our results showed that eDNA derived from the both cells and fragmental DNA decreased exponentially in the both sea and pond samples. The degradation of eDNA from the resident species showed similar behavior to the cell-derived eDNA.As a meta-analysis, we complied the degradation rates of eDNA in laboratory experiment and field studies from 28 studies (Saito and Doi 2020b). We also collected the related factors, including water sources, water temperature, DNA regions, and PCR amplicon lengths of the measured DNA. Our results suggested that water temperature and amplicon length were significantly related to the degradation rate of eDNA. From the simulation based on the 95% quantile model, we predicted the maximum degradation rate of eDNA in various combinations of water temperature and PCR amplicon length. HTML XML PDF
      PubDate: Mon, 8 Mar 2021 16:47:47 +0200
  • Accurate long-read eDNA metabarcoding of North Sea fish using Oxford
           Nanopore sequencing

    • Abstract: ARPHA Conference s 4: e65550
      DOI : 10.3897/aca.4.e65550
      Authors : Karlijn Doorenspleet, Lara Jansen, Saskia Oosterbroek, Reindert Nijland : To halt North Sea ecosystem degradation, accurate and intensive monitoring of the North Sea ecosystem and its fish is vital to correctly inform management decisions. DNA based techniques and especially the use of environmental (e)DNA from seawater can become a powerful monitoring tool. However, current eDNA based metabarcoding approaches are based on genetic target regions of
      PubDate: Mon, 8 Mar 2021 16:47:46 +0200
  • Can eDNA metabarcoding offer a catchment-based approach for biodiversity

    • Abstract: ARPHA Conference s 4: e65651
      DOI : 10.3897/aca.4.e65651
      Authors : Laura Allen, Martin Wilkes, Marco Van De Wiel, Mike Morris, Alex Dumbrell, Alessia Bani : In previous studies eDNA metabarcoding has been demonstrated as a viable tool for catchment-level biodiversity sampling in rivers (Deiner et al. 2016). However, questions still remain over the appropriate sampling protocol for large spatial scale sampling. River reaches are composed of multiple habitats with species composition varying from one to the next (Costa and Melo 2007). Therefore, how many spatial replicates are needed to reliably represent the river network' Is the previously used approach to sample at every river confluence (Deiner et al. 2016) sufficient or is more needed' These questions were addressed using a case study in the headwaters of the Cound Brook, a tributary to the River Severn in Shropshire, UK.Two sub-catchments of the Cound Brook were used. One sub-catchment had a sample taken at the most downstream point before the confluence. Additionally, a sample at the upstream extent of the same sub-catchment was taken to estimate any correlation between the species found at the beginning of the river reach and at the end. Another sub-catchment also had the same up- and downstream sample design. However, in between was a systemic sampling regime every 500 m. This is to test if increasing the spatial resolution gave significantly different results to the sparser sampling regime.At each sample location, a 1 L water sample was sequentially filtered through membranes of three different mesh sizes: 5µm, 0.45µm and 0.2µm. Sequential filtering was performed because DNA resides in two forms in the environment (Civade et al. 2017), within whole cells (cellular DNA) and outside of cells (extracellular). The theory is that the coarser filters predominantly collect cellular DNA and the finer filters collect predominantly extracellular DNA of increasingly smaller fragment lengths. Consequently, sequential filtering could represent DNA degradation in the environment (Fig. 1). Also, Turner et al. 2014 suggested that the larger particles could determine very recent or local organisms. Therefore, we hypothesised that the DNA collected by the coarser filters would represent local diversity and the DNA collected by the finer filters would reflect biodiversity further upstream.Initial results suggest sequential filtering through the 5µm and 0.45µm filters caught detectable levels of eDNA where the 0.2µm did not catch enough to show up through gel electrophoresis. The relevance of the initial finding suggests that if we only used a 5µm filter the data collected at 0.45µm could have been discarded. Further investigations of any differences in species compositions between filters and the relationships to other sampling locations is still to be determined. This ongoing research is intended to determine the appropriate sampling protocol for a large-scale biodiversity assessment regime combining eDNA metabarcoding and species distribution modelling. HTML XML PDF
      PubDate: Mon, 8 Mar 2021 16:47:29 +0200
  • Welcome to DNAQUA2021 International Conference

    • Abstract: ARPHA Conference s 4: e65590
      DOI : 10.3897/aca.4.e65590
      Authors : Florian Leese, Agnès Bouchez, Charlotte Frie, Alexander Weigand : Dear participants of DNAQUA2021 International Conference,Undoubtedly, DNAQUA2021 is a major highlight of the EU COST Action DNAqua-Net (CA15219). Even though we cannot claim that the organisation of DNAQUA2021 was a piece of cake, it is simply wonderful to see the great interest in this event. With 1,498 registered participants from 79 nations, 204 contributed talks and posters for only two and a half days, the conference shows how timely and relevant research on DNA-based aquatic bioassessment and monitoring is. As the managing team of DNAqua-Net, we could have hardly imagined the impact of DNAqua-Net back in 2015, when we wrote the proposal (Leese et al. 2016). Yet, the more we are now delighted and thankful to see the success. Together with many experts from many different countries, taxonomists, ecologists, geneticists and bioinformaticians, we have made significant methodological progress. Above all, we have succeeded in connecting biomonitoring experts all across Europe and beyond. With more than 100 scientific publications from DNAqua-Net's five working groups, the research impact of the network is obvious. Furthermore, with "Metabarcoding and Metagenomics" (MBMG), we have established an international journal for basic and applied aspects of genetic bioassessment and monitoring. However, in many ways, the impact of DNAqua-Net goes far beyond the mere scientific progress. Capacity building e.g. via barcoding projects have been initiated in many countries, validation studies were co-designed by researchers and stakeholders from the applied sector and launched - even across several countries as for example the SCANDNAnet project shows. DNAqua-Net has supported over 50 research exchanges that fostered close cooperation among the institutions and countries. Also, DNAqua-Net accompanied the fourth Joint Danube Survey (JDS4) and conducted the (e)DNA-based surveys for fish, benthic invertebrates, phytobenthos and the sediment community. Last but not least, we have developed many essential pieces of an applied concept for future implementation of DNA-based methods together with various stakeholders at national and international level. Here, of particular importance was the establishment of a working group within the European Standardisation Organisation CEN on DNA and eDNA-based methods (CEN/TC230/WG28). We are particularly grateful also to our colleagues from 'beyond Europe' that have supported us, participated in workshops, discussions and training schools, invited us to their national meetings on DNA and eDNA-based biomonitoring on five continents. The implementation of (e)DNA-based methods into bioassessment and monitoring programs of our rivers, lakes, oceans and the groundwater, will be particularly successful if we sustainably stay connected across countries, generations, cultures and disciplines  (Fig. 1).Many of the findings from basic to applied research will be presented at DNAQUA2021. We are particularly pleased that so many early career researchers present their findings. Please take the chance and discuss with them (but not only with them) about their findings. With "Spatial.Chat" we offer you a nice and intuitive environment that allows for some 'real' conference spirit even in these COVID-19 virtual meeting times.Now enjoy two and a half days packed with fascinating insights from (e)DNA-based aquatic biomonitoring. Take the chance, foster and extend your collaborations. We hope to see and discuss with you over the next days at DNAQUA2021 and beyond.THANK YOU!Florian, Agnès, Charly & Alex (Fig. 2) HTML XML PDF
      PubDate: Mon, 8 Mar 2021 16:47:27 +0200
  • Using meta-barcoding tools to monitor primate meat consumption at
           dedicated establishments in Guinea-Bissau, West Africa

    • Abstract: ARPHA Conference s 4: e65575
      DOI : 10.3897/aca.4.e65575
      Authors : Maria Ferreira da Silva, Mariato Camará, Bastian Egeter, Tania Minhós, Michael Bruford, Raquel Godinho : Guinea-Bissau (GB) is a regional stronghold for primate conservation. Ten primates occur in the country, including the Western chimpanzee (Pan troglodytes verus) and two colobus monkeys (Colobus polykomos and Piliocolobus badius temminckii). Primate meat is consumed at households and bushmeat-dedicated establishments, locally named "Abafatório". Such establishments are mentioned to be common in urban areas since the 1980s and to be specialized in serving primate meat while drinking alcoholic beverages. The meat is typically cooked in a stew and eaten with bread. However, as the trade and consumption of primate meat are illegal activities, the location of Abafatório establishments and details of the trade, namely species being consumed, are usually hidden from outsiders. Here, we characterize illicit bushmeat commerce and consumption at six Abafatórios of a small town. Our team visited the establishments every week for 15 months (2015-2017) and collected data on the type and prices of meals and gathered tissue samples taken from carcasses by establishment owners. A meta-barcoding approach (cytb and 12S mitochondrial DNA regions and Illumina MiSeq next-generation sequencing technology) was used to identify tissue samples to the species level. Two types of establishments can be distinguished – “restaurants” and “snack-bars”. Restaurants are similar to the ones found by previous works in the capital city where primate meat is sold as a dish containing few pieces of stewed meat. Snack-bars are smaller and the meat is sold inexpensively and by the piece. In the present study, 249 tissue samples were identified to be from four primates (Cercopithecus campbelli, Chlorocebus sabaeus, Papio papio, and Erythrocebus patas) and four Artiodactyla (Philantomba maxwellii, Tragelaphus scriptus, Potamochoerus porcus and Phacochoerus africanus). Primates represented approximately 92% of all species consumed across establishments, and C. campbelli was the most traded species. Our work suggests that primate meat is monetarily accessible for locals in rural areas and that the trade at Abafatórios may have extensive negative consequences to primate conservation, in particular, the reduction of primates' populations in the southern part of GB. Our work quantifies and identifies the species consumed in Abafatório establishments for the first time and highlights the need to improve regulation and law enforcement in Guinea-Bissau. HTML XML PDF
      PubDate: Mon, 8 Mar 2021 16:47:05 +0200
  • Exploring the use of new water quality indicators based on microbial

    • Abstract: ARPHA Conference s 4: e65420
      DOI : 10.3897/aca.4.e65420
      Authors : Luciana Griffero, Emiliano Pereira-Flores, Belén González, Andrés Pérez, Cecilia Alonso : The growing concern for the quality of water in aquatic ecosystems makes it essential to develop new indicators that allow evaluating and predicting their state against anthropogenic impact. Microorganisms are able to reflect quickly changes in their habitat, through both its taxonomic and functional characteristics, and that is why they are in consideration as indicators of environmental quality (*1). The objective of this work was to identify attributes of the composition and functionality of microbial communities, to be evaluated as an indicator of water quality, focusing on emerging pollutants (ECs). For that, ECs and bacterial communities were analyzed along the basins of two coastal lagoons encompassing an anthropogenic gradient, looking for taxonomic and functional indicators. Taxonomic indicators were looked using Illumina sequencing of 16S RNAr gene V4 region followed by identification of amplicon secuence variants (ASVs) and taxonomic annotation. In the case of functional indicators, shotgun sequencing was used added to identification and annotation of open reading frames (ORFs). Clustering techniques were implemented to define groups of sites based on the concentration of different categories of ECs. Then, the indicator value analysis (IndVal) (*2) was performed to identify taxonomic and functional traits that could be used as indicators of those groups. Finally, each sample was assigned to the corresponding group based on the indicators.A first analysis involved the search of taxonomic indicators for all the set of samples including three groups of sites of low, medium and high impact of emerging contamination. It was possible to find indicators with a very high IndVal value for the three groups of samples. All indicators were based on the co-occurrence of three ASVs belonging to several of the most abundant bacteria phyla (Actinobacteria, Bacteroidetes, Cyanobacteria, Planctomycetes, Proteobacteria). The bacterial indicators correctly assigned 100% and 93% of the samples to their corresponding group for streams and lagoons respectively.Then, a comparison between taxonomic and functional indicators using a subset of 41 samples was made, including two groups of samples: high and low-medium impact. Both the taxonomic and functional indicators showed high IndVal values for the high and low impact groups, being the highest in the case of functional genes Table 1. The high impact group was perfectly predicted for both taxonomic and functional indicators. The low-medium impact group was perfectly predicted by the functional indicators and 85% of the samples were correctly assigned by the taxonomic indicators.In conclusion, widespread availability of NGS technology allows for deep characterization of microbial diversity, enabling the use of robust ecological tools. Taking into account the high indval and prediction values, taxonomic and functional bacterial indicators appear as promissory candidates to evaluate for aquatic systems monitoring and conservation strategies. HTML XML PDF
      PubDate: Fri, 5 Mar 2021 16:00:00 +0200
  • The challenges and opportunities for implementation of eDNA biomonitoring
           in riverine systems

    • Abstract: ARPHA Conference s 4: e65521
      DOI : 10.3897/aca.4.e65521
      Authors : Florian Altermatt : Current local to global threats to biodiversity and anthropogenic changes of the environment call for rapid and effective conservation and management of ecosystems and the services they provide. In this context, the use of environmental DNA to assess biodiversity and conduct biomonitoring has been established as a novel, potentially revolutionizing approach over the last decade, especially in aquatic ecosystems. Rapid initial success, broad applicability and advances in sequencing technologies have raised high expectations about its potential. However, as with any revolution, true success requires formal implementation and establishing and integration of routines. In this talk, I will exemplify challenges and opportunities for implementation of eDNA biomonitoring in riverine systems, addressing both scientists and stakeholders. I will discuss potential pitfalls and misunderstandings caused by different targets, inference, and possible conclusions when comparing traditional sampling approaches with eDNA. I postulate that the focus should be on the strengths of new approaches, and not their matching to existing techniques. Finally, I will exemplify a strategy and the necessary steps of how a new order in biomonitoring can be established. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 21:09:26 +0200
  • Evaluating eDNA-based monitoring of fire salamander larvae under field

    • Abstract: ARPHA Conference s 4: e65291
      DOI : 10.3897/aca.4.e65291
      Authors : Bianca Spitzl, Daniela Sint, Florian Glaser, Gerda Ludwig, Michael Traugott : Amphibians globally belong to the most threatened animal groups and monitoring their populations is of critical importance for their conservation. The fire salamander (Salamandra salamandra) is one of the European amphibian species which has been experiencing drastic regional population declines due to the spread of the invasive chytrid fungus Batrachochytrium salamandrivorans, making it a key species for monitoring efforts. Here, we evaluated how the sampling and analysis of eDNA can aid the monitoring of larval salamander populations in small streams under field conditions. Nine small streams with known adult and larval fire salamander populations were investigated in Tirol (Austria). Per steam a stretch of 30 m downstream from its source was divided into 10 m sections where salamander larvae were counted. Water samples were taken at the end of each section and filtered on site. The DNA extracted from these filters was tested by a new PCR assay developed for the detection of mitochondrial DNA of S. salamandra. This assay combines endpoint PCR with capillary electrophoresis, allowing to relatively quantify the amount of fire salamander eDNA present in the water samples. In two of the nine streams no eDNA of S. Salamandra could be detected. The outcomes of an analysis of how larval densities, discharge and volume of filtered water affected the detection of salamander eDNA will be presented. Finally, we will conclude on the practical implications of the current findings for eDNA-based monitoring of fire salamander populations. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 18:42:37 +0200
  • Amplicon Sequencing in the Era of Highly-Accurate Long Reads

    • Abstract: ARPHA Conference s 4: e65405
      DOI : 10.3897/aca.4.e65405
      Authors : Benjamin Callahan : An important advance in DNA sequencing has been the development of long-read sequencing technologies that produce sequencing reads of tens to hundreds of kilobases in length. However, these technologies typically have high (~8%) per-base error rates. Recently, an effectively new technology I call highly-accurate long-read sequencing has been developed, that allows for the generation of multi-kilobase reads with extremely high per-base accuracies (>99.9%). I will present and evaluate two such technologies, PacBio HiFi and LoopSeq SLR sequencing, and discuss potential metabarcoding applications of highly-accurate long-read amplicon sequencing in general. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • The challenges we've overcome and the new opportunities for using
           environmental DNA in biomonitoring aquatic resources

    • Abstract: ARPHA Conference s 4: e65502
      DOI : 10.3897/aca.4.e65502
      Authors : Kristy Deiner : Since the first kick-off meeting of the DNAqua-Net until now, the interest and use of environmental DNA in scientific studies, management and even the start of companies has exponentially grown. It is often said that this is how we will monitor biodiversity in the future. Together, with over 400 members from 39 countries making up the COST Action, we have honed our DNA detection methods, determined the many trade-offs, identified and filled gaps in our knowledge, and made steps towards standardization. In my seminar, I will highlight some of the major challenges we’ve overcome and propose the opportunities and research directions I see for the future of using eDNA in biodiversity monitoring. We are poised at the start of the UN’s Decade on Ecosystem Restoration and there is no better time to cease DNA-based monitoring technology for making the best informed decisions for the future of life on our planet. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • 101 lakes - lentic fish community monitoring using eDNA metabarcoding

    • Abstract: ARPHA Conference s 4: e65438
      DOI : 10.3897/aca.4.e65438
      Authors : Bernd Hänfling, Nigel Willby, Lori Lawson-Handley, Graham Sellers : We have recently developed and deployed methods for environmental DNA (eDNA) based monitoring of lake fish communities in the UK. This approach combines eDNA with modern High-Throughput-Sequencing technology, so-called eDNA metabarcoding. This non-invasive method has proven to be more effective at detecting elusive species than established invasive surveying techniques such as electro fishing or fyke netting and can provide meaningful semi-quantitative abundance estimates. The UK Environment Agencies have funded the collection of an eDNA meta-barcoding data set of vertebrates from 101 UK lakes covering a broad spectrum of environmental conditions Fig. 1. This dataset is based on analysing 20 water samples per lake and has successfully been used to develop a WFD compatible water quality assessment tool. In its current form this tool is suitable for reporting the status of fish in water bodies where eutrophication is the dominant pressure.DNA is not homogeneously distributed in lentic environments and hence the detection of species relies on the collection of an adequate number of samples from a water body to capture the eDNA signal. Previous analyses on a subset of lakes have indicated that the number of samples used for the 101 lake fish data set is more than sufficient to reliably estimate species richness of lakes, but it is unclear how exactly reduced sampling effort affects other biodiversity estimates and inferences made about water quality. As the number of samples determines the cost of monitoring programmes it is essential that the sampling effort is optimised for a specific monitoring objective. The objective of this study was to explore the effect a reduced sampling effort would have on various biological inferences using algorithmic and statistical resampling techniques. with a much lower number of samples. For example, almost 90% of lakes achieved a sample coverage of 95% with only 10 samples. However, rare species are more often missed with fewer samples, with implications for monitoring programs of invasive or endangered species. Estimates of community composition and the ecological quality ratio (EQR) responded slowly to decreasing sampling effort. For example, subsets of 10 samples were in most cases much more closely related to each other than to sample sets from other lakes and showed very similar Ecological Quality Ratios. These results are able to inform the design of eDNA sampling strategies, so that these can be optimised to achieve specific monitoring goals. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Towards a simple way to collect eDNA using a 3D-printed passive sampler

    • Abstract: ARPHA Conference s 4: e65499
      DOI : 10.3897/aca.4.e65499
      Authors : Héloïse Verdier, Lara Konecny, Christophe Marquette, Tristan Lefebure : Environmental DNA has emerged as a revolutionary approach to monitor aquatic biodiversity. The study of the DNA released by macro-organisms in their habitat offers a fast, non-invasive and sensitive approach to monitor their presence. Despite its many advantages, methodological challenges limit the widespread use of eDNA. Among them, eDNA sampling represents one of the most challenging step. Often based on the filtration of a large volume of water, this process can be long and tedious, requiring human intervention and special care, and which is not applicable to a wide range of habitats. As an alternative to filtration, passive eDNA sampling using natural substrates appears to be a promising solution. This approach uses the natural properties of some minerals (eg. silica), organisms (eg. sponges) or even communities (e.g. biofilms) to collect and preserved eDNA. Yet, such approaches are difficult to standardize and may not be applicable in many habitats. To circumvent that problem, we have designed 3D-printed samplers made of hydroxyapatite (HAp samplers), a mineral known for its high binding affinity with DNA. The shape of the samplers has been designed to facilitate their handling in laboratory and field experiments. Here we describe and test the ability of HAp samplers to recover freshwater eDNA. We show that HAp samplers recover DNA with high efficiency and are effective even on small amounts of waterlouse eDNA. However, the eDNA recovery is also highly variable across experiments. We show that by understanding the physico-chemical interactions between DNA and the HAp sampler surface, we could improve the replicability of the process and provide a robust alternative to filtration. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • DNA barcoding reveals an unknown Chironomidae diversity from the
           freshwater biodiversity hot-spot: comparison between local and the
           European datasets

    • Abstract: ARPHA Conference s 4: e65498
      DOI : 10.3897/aca.4.e65498
      Authors : Piotr Gadawski, Matteo Montagna, Bruno Rossaro, Wojciech Giłka, Vladimir Pesic, Michal Grabowski, Giulia Magoga : In the present study, we developed and evaluated the first reference barcode library for non-biting midges (Diptera: Chironomidae) as flagship taxa of freshwater ecology from Skadar Lake system (Montenegro and Albania), a well-known hotspot of freshwater biodiversity composed by the young lake Skadar (originated 1200 before present) and by its old system of springs (originated during Pliocene). Using an expanded reference library and records deposited in Barcode of Life Database (BOLD), we estimated DNA barcoding efficiency for the European Chironomidae. Study provides COI barcodes for 770 Chironomidae individuals assigned, based on morphology, to 77 species collected in the Skadar Lake basin. Molecular analyses assigned sequences to 100 BINs and 104 OTUs (all records from this area are new for online repositories) and confirms the usefulness of DNA barcoding for the identification of non-biting midges. Additionally, we explored chironomid species distribution patterns in Europe using universal Barcode Index Number (BIN) with a discussion of problematic species groups, both for traditional taxonomy and DNA barcoding. The results of our study provide the first insight into the factual chironomid species diversity of the Lake Skadar basin, in comparison with chironomid fauna at the European scale. The results fill a significant gap in knowledge of biodiversity in the Balkan region. Based on the results of Chironomidae fauna investigation, we conclude that the Skadar Lake basin is now well sampled and such a high representation of species from various sampling sites provides reliable estimation of the non-biting midges fauna. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • DNA barcode library revealed unknown diversity of chironomid midges in

    • Abstract: ARPHA Conference s 4: e65497
      DOI : 10.3897/aca.4.e65497
      Authors : Piotr Gadawski, Bruno Rossaro, Wojciech Giłka, Tomasz Rewicz, Giulia Magoga, Paul Hebert, Matteo Montagna, Michal Grabowski : We present the first results of the study aiming to investigate the diversity of the non-biting midges (Diptera: Chironomidae) fauna of the Skadar Lake system (Montenegro and Albania), a well-known hotspot of freshwater biodiversity composed by the young lake Skadar (originated 1200 before present) and by its old system of springs (originated during Pliocene). During the study, 8,147 COI barcodes were obtained and revealed the presence of 474 BINs and 586 OTUs assigned to 148 species. Our results provide the first insight into the factual molecular diversity among chironomids inhabiting Skadar Lake basin and fill a significant gap in the knowledge of the biodiversity in the Balkan region. With 219 (46.2%) unique BINs from the Skadar Lake basin new for BOLD, we can expect that further development of barcode reference libraries will help to bind unidentified developmental stages with those identified based on morphology and will reveal hidden Chironomidae species diversity. Further studies should be focused on sampling developmental stages which provides the best species-level resolution, such as mature males. It will help to develop a reliable reference barcode library - fundamental during further assessments. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • A tufA metabarcoding approach for Ulva and related seaweeds

    • Abstract: ARPHA Conference s 4: e65495
      DOI : 10.3897/aca.4.e65495
      Authors : Florian Weinberger, Sophie Steinhagen, Rolf Karez, Guido Bonthond : Ulva-like green algae are notoriously difficult to distinguish due to their morphological variability and/or similarity. DNA barcoding approaches are therefore currently essential for their reliable identification. However, such approaches often fail when rare or inconspicuous species are to be detected in large mixed populations of Ulva species, for example, at early stages following the introduction of species into new habitats. We therefore developed a detection method based on next-generation DNA sequencing. The approach is suitable for the analysis of DNA traces in preserved water samples or in particles enriched by filtration from such samples. A new pair of primers was designed to amplify a 475 bp segment within the tufA marker gene. The primers were relatively group specific. 68.5% of all reads obtained after quality filtering represented the genus Ulva, 11.1% other Ulvophyceae, and only 20% other Chlorophyta, despite their relatively higher abundance in phytoplankton. The relatively short target amplicon still allows good differentiation of Ulvales and Ulothrichales at the species level. Using a database containing tufA sequences of 879 species - 281 of which were Ulvophyceae and 35 Ulva - we were able to detect mostly Ulvophyceae that had been previously detected in our study area in northern Germany using Sanger sequencing. However, the number of species detected at individual sites was generally higher than in previous studies, which could be due to drifting DNA: Analysis of samples collected at different distances from shore suggests that a sample collected at a given site may be influenced by Ulvophyceae within a radius of up to about 1 km in winter. In summer, this radius is reduced to less than 100 m, possibly due to the less frequent occurrence of strong wind events. Nonetheless, rare species may be detected with this new approach: At one site, an undescribed Blidingia species that was not previously known from our study area was repeatedly detected. Based on these findings, the species was searched for and found, and its identity confirmed by traditional tufA barcoding. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • DNA Barcoding approach reveals 17 exploited fish species, including an
           unrecognised species of the yellow-lined snapper complex

    • Abstract: ARPHA Conference s 4: e65494
      DOI : 10.3897/aca.4.e65494
      Authors : ADIBAH ABU BAKAR, Siti Mohd Nor : Management of wild fisheries resources requires accurate knowledge on which species are being routinely exploited, but it can be hard to identify fishes to species level, especially in speciose fish groups where colour patterns vary with age. Snappers of the genus Lutjanus represent one such group, where fishes can be hard to identify and as a result fisheries statistics fail to capture species-level taxonomic information. This study employs DNA barcoding approaches to identify adult and juvenile Lutjanus species harvested in Malaysian wild-capture fishery. We uncovered two divergent groups of bigeye snapper ('Lutjanus lutjanus') distributed on either side of the Malay Peninsula, displaying a biogeographical pattern similar to distributions observed for many co-occurring reef distributed fish groups. One of these bigeye snapper groups almost certainly represents an unrecognized species in need of taxonomic description. The study demonstrates the utility of DNA barcoding in identifying overlooked diversity and for assessing species catch composition in a complicated but economically important taxonomic group. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Labelling strategies in metabarcoding studies & how to ensure that
           nucleotide tags stay in place

    • Abstract: ARPHA Conference s 4: e65450
      DOI : 10.3897/aca.4.e65450
      Authors : Kristine Bohmann, Christian Carøe : Labelling strategies in metabarcoding studies & how to ensure that nucleotide tags stay in place Metabarcoding of environmental DNA (eDNA) and DNA extracted from bulk specimen samples is a powerful tool in studies of ecological interactions, diet and biodiversity, as its labelling of amplicons allows high-throughput sequencing of taxonomically informative DNA sequences from many samples in parallel. The backbone of metabarcoding is the addition of sample-specific nucleotide identifiers to amplicons and then following sequencing using these to assign metabarcoding sequences to the samples they originated from. This allows the pooling of hundreds to thousands of samples before sequencing and thereby full utilisation of the capacity of high-throughput sequencing platforms. The nucleotide identifiers can be added both during the metabarcoding PCR and during library preparation, i.e. when amplicons are prepared for sequencing. There are three main strategies with which to achieve nucleotide labelling in metabarcoding studies. One commonly used strategy is the so-called tagged PCR approach in which DNA extracts are individually amplified with metabarcoding primers that carry sample-specific nucleotide tags at the 5’ end. The uniquely tagged products are then pooled and a library prepared on the pool of amplicons. However, tag‐jumps have been documented in this commonly used metabarcoding approach (Schnell et al. 2015).Tag-jumps cause nucleotide tags to switch between amplicons, resulting in occurrence of amplicons that carry different tags than originally applied. Sequences in the sequencing output that carry tag combinations not used in the study design are easily identified and excluded. However, sequences carrying incorrect, but already used, tag combinations will cause incorrect assignments of sequences to samples. This can - much to the detriment of metabarcoding studies - lead to false positives and artificial inflation of diversity in the samples (Schnell et al. 2015). The occurrence of tag-jumps has led to recommendations to only carry out metabarcoding PCR amplifications with primers carrying twin-tags to ensure that tag‐jumps cannot result in false assignments of sequences to samples (Schnell et al. 2015). However, this increases both cost and workload of metabarcoding studies.In a recently published article, we demonstrate a tag-jump free single-tube library preparation protocol for Illumina sequencing specifically designed for 5’ nucleotide tagged amplicons, the Tagsteady protocol (Carøe & Bohmann 2020). We designed the Tagsteady protocol to circumvent the two steps during library preparation of pools of 5ʹ nucleotide-tagged amplicons that had previously been suggested to cause tag-jumps; i) T4 DNA polymerase blunt-ending in the end-repair step, and ii) post-ligation PCR amplification of amplicon libraries. We used pools of twin‐tagged amplicons to investigate the effect of these two steps on the occurrence of tag‐jumps. Doing this, we demonstrated that blunt‐ending and post-ligation PCR, alone or together, can result in high proportions of tag-jumps, in our study up to ca. 49% of total sequences. The Tagsteady protocol where both these steps were left out resulted in tag‐jump levels comparable to background contamination (Carøe & Bohmann 2020). In our study, we encourage practitioners to avoid using T4 DNA polymerase blunt‐ending and post-ligation PCR in library preparation of 5’ nucleotide tagged amplicon pools, for example by using the Tagsteady protocol (Carøe & Bohmann 2020). This will enable efficient and cost-effective generation of metabarcoding data with correct assignment of sequences to samples.ReferencesCarøe C, Bohmann K (2020) Tagsteady: A metabarcoding library preparation protocol to avoid false assignment of sequences to samples. Molecular Ecology Resources, 20, 1620–1631.Schnell IB, Bohmann K, Gilbert MTP (2015) Tag jumps illuminated - reducing sequence-to-sample misidentifications in metabarcoding studies. Molecular Ecology Resources, 15, 1289–1303. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Applicability of DNA-based identifications for the WFD-guided monitoring
           using macroinvertebrates: a large-scale DNA metabarcoding study for
           implementing routine ecological status assessments in Iberian rivers

    • Abstract: ARPHA Conference s 4: e65490
      DOI : 10.3897/aca.4.e65490
      Authors : Raquel González, Juan Antonio Villaescusa, Antonio Picazo, Ana M. Pujante, Antonio Camacho : Over the last decade, remarkable improvements have been made in the field of metabarcoding-based tools for routine ecological status assessments. However, important issues are yet to be solved to fulfil the European Water Framework Directive (WFD) requirements and standards. These limitations, which include problems related to e.g. the lack of a complete COI macroinvertebrate barcode database available for the Iberian Peninsula Murria 2020, or the scarce recovery of specific taxa due to DNA extraction and/or PCR amplification bias, are especially difficult to overcome for routine freshwater macroinvertebrate monitoring. For that purpose, a large-scale study is on going to test how metabarcoding data can infer existing macroinvertebrate morphotaxonomy-based biotic indexes and ecological status of Iberian rivers. Freshwater macroinvertebrates were selected as a Biological Quality Element and identified by using both morphological and metabarcoding approaches. The mitochondrial gene for cytochrome c oxidase subunit I (COI) was used as a DNA Barcode. Taxonomic coverage, taxonomic composition metrics and ecological status obtained from both approaches were analysed. Physical and chemical variables obtained during the routine biomonitoring, as well as other ecological parameters including biodiversity indexes, were also assessed. Multivariate data analysis of these environmental and biotic data obtained from both approaches were compared. Results seem to support the hypothesis Kuntke 2019 that the DNA-metabarcoding approach might deliver similar quality assessments results to the morphological approach, though some refinement must be done at the different steps of the process prior to establish a reliable procedure allowing the alternative use of both methods giving similar results for the ecological status classes marked by the WFD. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Autonomous in situ sampling and analysis of eDNA using an Environmental
           Sample Processor (ESP)

    • Abstract: ARPHA Conference s 4: e65171
      DOI : 10.3897/aca.4.e65171
      Authors : Magnus Jacobsen : Monitoring of marine offshore biodiversity is expensive and has traditionally relied on invasive techniques like net fishing or direct observations that can only be conducted in calm seas by experts. Analysis of environmental DNA (eDNA) is a non-invasive method and can easily be collected from sea water by water filtration followed by DNA extraction. Due to the fast degradation time in sea water it is considered a good proxy for present living biodiversity. It allows direct identification of species based on their unique DNA sequence and is cheaper compared to traditional methods, which often are carried out from dedicated fishing or research vessels. However, while eDNA collection may reduce operational cost of offshore sampling, it still relies on boat time. Thus, traditional eDNA sampling still presents substantial costs for offshore biodiversity monitoring. This may reduce the number of samples that can be collected and analysed, limiting the sampling to single ‘time shots’, which may not give an adequate picture of the present biodiversity. The 2nd generation Environmental Sample Processor (2G-ESP) is an autonomous sampler/analyser of eDNA. It can collect, extract and analyse eDNA samples in situ using quantitative PCR (qPCR) or store filters for subsequent laboratory analysis after deployment. The instrument can be deployed directly on the seabed or in pelagic configuration where it can operate for several months depending on power supply and power consumption, while it is controlled by, and sends back results to scientists on land. These unique features make the 2G-ESP an interesting candidate for offshore monitoring of marine biodiversity, as well as a potential early warning/detection system e.g. for invasive species. Moreover, the possibility to preserve filters aboard makes it possible to investigate temporal changes of full biological communities by applying metabarcoding techniques on the collected samples.Here we present the major results of three years of work testing the potential use of a second generation Environmental Sample Processor (2G-ESP) for marine monitoring. These include both practical and analytical issues that we have encountered along the way, as well as results on target species detection and temporal analysis using qPCR and metabarcoding methods. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Alcohol keeps eDNA at the party longer

    • Abstract: ARPHA Conference s 4: e65015
      DOI : 10.3897/aca.4.e65015
      Authors : Sarah Licul, Rachael Impey, Andrew Weeks : For a typical eDNA water study, water will be filtered on site, before prompt transfer to a laboratory for DNA extraction and required scientific analysis. In a setting where transport is quick and available, this is a straightforward process. However, many of our studies can occur in remote Australia where sample preservation presents many logistical challenges. Typically, we advise clients to store eDNA water filters after sampling below 4 °C to ensure minimal DNA degradation. For many clients however, field studies often occur in an isolated setting without adequate refrigeration facilities, and as such present challenges for this process. Rather than compromise on sample integrity, EnviroDNA conducted a pilot study into the use of alternate preservation methods on our most commonly used 0.22 mm Sterivex filters. With help from our friendly neighbourhood goldfish tank, our standard 4 °C protocol was compared to a variety of conditions including filled ethanol filters, flushed ethanol filters, lysis buffer and silica bead storage conditions at both 4 °C and room temperature. The study, conducted at various time points over 14-days, used qPCR to quantify the amount of DNA extracted from the filter. Our results revealed that storage within or using flushed ethanol, allowed the samples to be stored for longer time intervals at room temperature, with similar, or in some cases, improved DNA elutions. This protocol optimisation has allowed us to offer an alternate sample storage protocol for clients, expanding the availability and accessibility of eDNA biodiversity assessments around Australia. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Application of environmental DNA for monitoring and management of aquatic
           biological invasions: Emerging trends and advancements towards best

    • Abstract: ARPHA Conference s 4: e65476
      DOI : 10.3897/aca.4.e65476
      Authors : Emily Chen : IntroductionAquatic Invasive Species (AIS) are a growing concern for global biodiversity as humans continue to accelerate the transport of non-indigenous species beyond their natural range. These species may possess traits that allow them to thrive in new environmental conditions such as non-selective feeding and high reproductive output, causing ecological harm through competition with native species for limited local resources. Consequently, environmental DNA (eDNA) has come to the forefront of AIS management in recent years as a promising method to detect or monitor invasive species using rapid and non-invasive sampling to complement traditional surveying. As eDNA’s potential is explored and beginning to be adopted for a variety of applications around the world, it is increasingly important to synthesize the trends in field and laboratory protocols from different working groups to establish guidelines that will allow greater comparability between studies and improve experimental design. Methodology and ResultsThis meta-analytic study collated and reviewed information from previously published eDNA studies that targeted AIS in freshwater and marine environments to recognize current patterns in sampling techniques, laboratory protocols, and potential geographic or taxonomic biases. A total of 492 records from 192 full-text articles were used in the analysis, composed of 408 species-specific and 84 metabarcoding records. With regards to sampling procedures, many studies were not explicit enough for true replicability, lacking critical information such as the volume of filtered water and details of storage conditions. There was no observable trend for eDNA extraction methods in either species-specific or metabarcoding approaches, with choice of extraction method being mostly arbitrary among laboratories as well as influenced by the recent emergence of dedicated commercial kits . DiscussionThis analysis revealed a wide variety of choices for collecting and processing eDNA samples, so it is recommended that there should be some sort of standardized workflow diagram or decision tree for every stage of the experimental design in order for researchers to determine what approaches best meet their research objectives. There is also a clear need for improving metadata reporting guidelines; although the relevance of some criteria depends on the goals and limitations of specific projects, there should be a standardized minimum set of parameters to be reported for each eDNA study, from environmental variables to decontamination practices to PCR conditions. This will increase consistency and transparency through all stages of eDNA research, which is key to collectively improving methodologies and moving forward in this field. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Mind the gap-analysis! – How complete are DNA barcode reference
           libraries for monitoring-relevant aquatic species in Europe'

    • Abstract: ARPHA Conference s 4: e65473
      DOI : 10.3897/aca.4.e65473
      Authors : Hannah Weigand : Molecular species identification with DNA metabarcoding can potentially accelerate, streamline and standardise biomonitoring routines. Currently, it is tested how this new technique can be implemented for the European Water Framework Directive (WFD) and the European Marine Strategy Framework Directive (MSFD). To connect the results from DNA metabarcoding with the current monitoring routines, an extensive, high-quality DNA barcode reference database is required. Hence, a gap-analysis of the Barcode of Life Data Systems (BOLD) was performed as part of the EU-COST Action DNAqua-Net (Weigand et al. 2019), which was updated in 2021. It aimed to analyse the completeness of BOLD for species on the national WFD monitoring lists and for marine species on the ERMS (European Register of Marine Species) and AMBI (AZTI Marine Biotic Index) lists. The data were supplemented by MitoFish for freshwater fish and Diat.barcode for diatoms.Several thousands of species were included in the gap-analysis, although not all countries currently apply species-level data for all WFD biological quality elements. The barcode coverage of the different taxonomic groups varied strongly, with high levels (> 80%) for fish and freshwater vascular plants, and low levels for diatoms and freshwater plathelminths (< 15%). As a general pattern, species monitored by several countries had a higher coverage compared to those monitored only by a single country.The gap-analysis focused additionally on the availability of metadata (e.g., geographical origin of the specimen or determiner name) for the barcodes. Hence, we analysed if the data were stored public (with access to metadata) or private (without access to metadata) in BOLD or if the data were mined from GenBank (metadata are potentially available but not easy to access). Although public data were stored for many species (43% of freshwater macroinvertebrates and 21% of AMBI marine species), the proportion of species without public metadata was not neglectable (22% of freshwater macroinvertebrates and 22% of AMBI marine species).Another issue that emerged from the gap-analysis was that several deposited barcodes were identified by reverse taxonomy (RT), i.e., specimens were molecularly identified via its DNA barcode and the barcode itself is stored in BOLD with the associated species name. This can be problematic as originally misidentified samples can lead to false RT-identifications, making the data appear more trustworthy than it actually is. For the analysed freshwater macroinvertebrates, 39% of all barcodes and 65% of all public data originated from RT, impacting 11% of all monitored species. As the information about RT is only available for publicly stored data, the real impact of RT might even be higher. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Ichtyological diversity in a tropical nature reserve (Etang de Saint-Paul,
           Réunion Island) from environmental DNA (eDNA) metabarcoding

    • Abstract: ARPHA Conference s 4: e65462
      DOI : 10.3897/aca.4.e65462
      Authors : Natacha Nikolic, Emmanuel Corse, Nicolas Juillet : The aim of this study was to test whether the ichthyological diversity of one natural reserve in Reunion Island (Réserve de l'Etang de Saint-Paul) could be established with a molecular tool, environmental DNA (eDNA). We hence filtrated the water (2L) at 10 different areas around the reserve. For each sampling area, 12 PCR replicas were performed and the identification of fish species was carried out by metabarcoding through a primer over a mitochondrial region (12S). This study showed the importance of reference sequencing databases as well as improvements through phylogenetic analyses. This first fish study by eDNA in La Réunion also revealed the coherence of the distribution of species and their habitat.See the Poster in Suppl. material 1.Collection : 1st DNAQUA International Conference - poster session. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Optimization of eDNA metabarcoding methodology for the biomontoring of the
           ichthyofauna in the Eastern Mediterranean Sea

    • Abstract: ARPHA Conference s 4: e65460
      DOI : 10.3897/aca.4.e65460
      Authors : Panagiotis Kasapidis, Christina Karli : Environmental DNA (eDNA) metabarcoding is a relatively new methodology for the detection of organisms in an environmental sample, with emerging applications in the fields of ecology, conservation, invasive biology, biomonitoring and more. Several studies are using eDNA nowadays, yet in the Mediterranean marine ecosystems, its use is limited mostly on plankton studies so far. The icthyofauna of the Eastern Mediterranean is undergoing major changes due to biological invasions, mainly form the Red Sea, in combination with the climate change, and a reliable high-throughput biomonitoring tool is essential to monitor these changes.The main goal of this study was to develop a reliable eDNA metabarcoding protocol to study and monitor fish biodiversity in the oligotrophic ecosystems of the Eastern Mediterranean. The study had two parts: a) standardization of the method by testing two different sets of primers in aquaria with known fish species assemblages, and b) estimation of the heterogeneity of fish eDNA distribution in different habitats within a coastal area, in order to determine the most efficient sampling strategy. In both cases, samples were analysed through Next Generation Sequencing on a Illumina MiSeq platform.To standardize the method, we sampled and filtered water from two tanks of the 'Cretaquarium'. We tested two different sets of primers, one for 16S rRNA and the MiFish primers of Miya et al. (2015) for 12S rRNA, in order to estimate their efficiency in assessing species' composition both qualitatively and semi-quantitatively. Both primer sets performed well and most taxa in both tanks were detected up to species level, with 16S marker exhibiting higher resolution. A rather weak correlation was also detected between actual fish biomass and relative abundance as estimated by eDNA metabarcoding.To estimate the eDNA heterogeneity in natural ecosystems, we sampled water in a coastal ecosystem over three distinct types of habitats: hard substrate, soft substrate, Posidonia meadows, as well as in the mid of the water column. Three samples per habitat were collected, two PCRs per DNA extract were performed and results were obtained only for the 16S marker. A total of 69 taxa were detected, with 55 of them distinguished at the species level, while in each sample the number of taxa detected ranged from 13 to 27. Posidonia meadows and the water column samples showed the greatest heterogeneity, in contrast to the hard and soft substrate samples that showed little differentiation both within and between habitat type. Based on these results, an improved protocol should include more technical PCR replicates per sample (at least 3 PCRs), at least one sample per habitat in each area, and a larger volume of water filtered per sample or alternatively, more samples mixed together in order to achieve better representation of the community.Moreover, it was apparent the need of a more complete and curated reference database for the Mediterranean fishes for the aforementioned markers, in order to be able to reliably identify fishes of the Mediterranean ecosystems at species level.In conclusion, the method seems to work well, and with some small improvements, as well as with the complementation of the respective reference databases, it can be used as a reliable tool for the study of biodiversity and biomonitoring of fish communities of the oligotrophic ecosystems of the Eastern Mediterranean Sea. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Monitoring a loss: Detection of the semi‐aquatic crocodile lizard

    • Abstract: ARPHA Conference s 4: e65457
      DOI : 10.3897/aca.4.e65457
      Authors : Patrick Fink, Timm Reinhardt, Mona van Schingen, Heidrun Windisch, Truong Nguyen, Thomas Ziegler : Assessing the conservation status of a species is strongly dependent upon data on species distribution and abundance. With the emergence of novel methods for species monitoring – such as the use of environmental DNA (eDNA) – monitoring success can be improved at reduced expenditure in the field, particularly in remote regions and terrains where access is difficult or dangerous. The highly endangered crocodile lizard (Shinisaurus crocodilurus Ahl, 1930) inhabits fragmented sites of the remaining evergreen forest with running water systems in a narrow distribution range in southern China and north‐east Vietnam. Crocodile lizards spend most of the day within or above water bodies, which are commonly remote and inaccessible. To monitor recent spatial occurrences, and to confirm the persistence or extinction of previously reported populations (especially in heavily altered habitats), the suitability of using eDNA and quantitative polymerase chain reaction (qPCR) was tested as an alternative method for monitoring this semiaquatic lizard. To assess the accuracy and limitations of this method, eDNA results from the field were compared with eDNA data from mesocosms and census data on the actual abundance of this species in the field. Environmental DNA of the crocodile lizard was detected in all of the positive controls, and in four of six natural sites; thus, all data collected using traditional field surveys were confirmed with eDNA results. eDNA monitoring was found to be a reliable method for assessing the viability of populations; we suggest that it should be developed as a tool for efficient wildlife management, particularly under difficult field and funding conditions. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Scanning ferry routes: looking for eDNA traces of marine mammals and their

    • Abstract: ARPHA Conference s 4: e65448
      DOI : 10.3897/aca.4.e65448
      Authors : Elena Valsecchi : Marine environmental DNA (eDNA) surveys are becoming a promising approach to monitor biodiversity status and its variation over time. However, monitoring offshore areas could be extremely costly when using dedicated vessels, beside the impossibility to sample simultaneously geographically distant (even if adjacent) areas. The unexplored possibility of availing on operating ferries as an opportunistic platform for eDNA sampling offers several advantages besides opening limitless opportunities for systematic surveys on marine biodiversity.We present the results of both metabarcoding and barcoding approaches obtained from the analysis of water samples collected on board of a ferry boat along a pilot Mediterranean route crossing the Pelagos Sanctuary for Mediterranean Marine Mammals. The recently described MarVer primer sets (12SrDNA and 16SrDNA regions), specifically designed for the simultaneous detection of marine mammals and other marine vertebrates, were employed. The High Throughput Sequencing (HTS) outcome showed that the markers successfully detected most trophic levels of vertebrate marine communities, and classes, including bony fish, rays, cetaceans and birds. Ferry-based sampling allow to collect sample at any time of the day, and we indeed found diel differences in both quantitative and qualitative distribution of read counts. For instances, we observed an increased abundance of lantern fish amplicons in night-time collect samples (50%), reflecting nocturnal migration through the water column. In general, the number of read counts was significantly higher in nocturnal samples. Such diel differences within our sample indirectly provides evidence of the efficiency of the eDNA approach to detect contemporary signals in the sampled environment. Similarly, cetaceans were detected in correspondence of visual sightings (when these occurred, supplementary samples were collected). Rare species, such as the monk seal, are difficult to be detected in metabarcoding surveys, thus we opted to side the screening of the ferry-samples with a panel of species-specific qPCR assays, which were able to detect DNA traces of the endangered pinniped in the Tuscany archipelago (Tyrrhenian Sea) long before visual observations witnessed its presence in the same area. The study demonstrates the feasibility of using commercial shipping as a platform for eDNA marine sampling without dedicated survey cruises. Commercial shipping routes have potential to act as regular systematic sampling transects which can contribute to evaluating and monitoring marine biodiversity. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Cray Benchmark – calibrating different eDNA methods for crayfish and
           crayfish plague

    • Abstract: ARPHA Conference s 4: e65359
      DOI : 10.3897/aca.4.e65359
      Authors : Patrik Bohman : Many countries are on the verge to integrate eDNA analyses into environmental monitoring. There are no standards yet for this kind of monitoring, and many different methods are used. Still, various methods and protocols have previously proven to be very precise and credible. If we accept that the way to monitor species’ eDNA will vary, different methods and actors can be used. To be able to rely on different types of protocols, benchmarking is much needed. We have initiated a project to perform benchmarking on crayfish and crayfish plague in 2021. Previous year a meeting was held in Stockholm where different methods were discussed with researchers, managers and stakeholders from 15 different organisations in 8 countries. The meeting pointed out the necessity for benchmarking to successfully monitor eDNA from crayfish and crayfish plague. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Improving the detection of rare native fish species in environmental DNA
           metabarcoding surveys

    • Abstract: ARPHA Conference s 4: e65387
      DOI : 10.3897/aca.4.e65387
      Authors : Jack Rojahn, Dianne Gleeson, Elise Furlan, Tim Haeusler, Jonas Bylemans : The presence of threatened or endangered species often strongly influences management and conservation decisions. Within the Murray–Darling Basin (MDB), Australia, the presence of threatened native fish affects the management and allocation of water resources. These decisions are currently based on traditional fisheries data and a predictive MaxEnt model. However, it is important to verify the model's predictive power given the implication it may have, but this requires methods with a high detection sensitivity for rare species. Although the use of environmental DNA (eDNA) metabarcoding achieves a higher detection sensitivity compared with traditional methods, earlier surveys in the MDB have shown that the highly abundant and invasive common carp (Cyprinus carpio) can reduce detection probabilities for rare species. Consequently, a polymerase chain reaction (PCR) blocking primer designed to block the amplification of carp eDNA could increase the detection probabilities for rare native species while simultaneously reducing the required sampling effort and survey costs. Although PCR blocking primers are often used in ancient DNA and dietary studies, no aquatic eDNA metabarcoding study to date has evaluated the potential benefits of using PCR blocking primers. A laboratory and field‐based pilot study was used to address this knowledge gap and assess the impact of a blocking primer on the detection probabilities of native species and the minimum sampling effort required. Results showed that the inclusion of the blocking primer increased the detection probabilities for native species by 10–20% and reduced the minimum required sampling effort by 25–50%. These findings provide important insights into possible methods for optimizing eDNA metabarcoding surveys for the detection of rare aquatic species. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Development of species-specific Cichla species eDNA primers for alien
           invasive species (AIS) monitoring in Malaysia

    • Abstract: ARPHA Conference s 4: e65449
      DOI : 10.3897/aca.4.e65449
      Authors : O. Nurul Fizatul Nabilah, A. R. Ramizah, A. B. Adibah, S. Syazwan, A.G. Intan Faraha, A. Amirrudin, M. N. Siti Azizah : Peacock bass or the cichlids are known locally as top predator fishes which are invasive in Malaysia freshwater system. Detection probabilities for these fishes are typically low, especially using conventional capture-survey method due to the fish’s behaviour of hiding beneath the water’s surface. Hence, the environmental DNA (eDNA) monitoring is a relatively new approach that can be used to assess the distribution of these invasive fishes. Here, we report the strategy to develop small fragment (280- 400 bp) specific-specific primers for three selected invasive Cichla species namely, C. ocellaris, C. monoculus, and C. kelberi based on mitochondrial DNA (mtDNA) sequences. Current research showed that the developed species-specific primers from cytochrome oxidase I (COI) gene has high resolution at species level. Species-specific amplification tests also proved the specificity of the developed primers, securing the high- level species identification potential which may help in controlling the spread of alien invasive fish species. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Bacteria Communities in Swiss Fish Farms with Recirculating Aquaculture

    • Abstract: ARPHA Conference s 4: e65444
      DOI : 10.3897/aca.4.e65444
      Authors : Jessica Rieder, Adamantia Kapopoulou, Nicolas Zürcher, Claudia Bank, Irene Adrian-Kalchhauser : Recirculating aquaculture systems (RAS), often used in fish farming, rely on microorganisms to maintain healthy water quality, nutrient cycling, animal welfare, and disease control. However, many daily operations in fish farms (e.g., stocking) may negatively affect the microorganisms' community composition and create a favorable environment for opportunistic pathogens. Currently, understanding microorganisms' communities within RAS is scarce, which presents an obstacle for pro-active system management. To better understand microorganism communities' spatial and temporal structure within fish farms using a RAS, we collected samples of filtered water and biofilm swabs from two different Swiss fish farms and two different locations within each farm. Water was collected from within one tank and the biofilter, while biofilm swabs were collected from the same tank's wall where the water sample was collected. DNA was extracted using the Purelink Microbial DNA Purification kit, and then each sample was prepared with three different primer pairs, 341F/805R (V3V4 region), 515F/806R (V4), 27F/534R (V1-3), and ran on the MiSeq platform (v3 600 cycles). The pilot study aimed to understand 1) how the microbiota composition changes regarding spatial and temporal scales within and between farms, 2) the primer effect on detected taxa, and 3) the difference between commonly-used 16s pipelines. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • The European eel Anguilla anguillain Cyprus - Investigating the role of
           freshwaters for eel conservation using integrated monitoring methods

    • Abstract: ARPHA Conference s 4: e65417
      DOI : 10.3897/aca.4.e65417
      Authors : Nathan Griffiths, Rosalind Wright, Bernd Hänfling, Jonathan Bolland, Katerina Drakou, Graham Sellers, Stamatis Zogaris, Marlen Vasquez : The European eel (Anguilla anguilla) is a catadromous fish species, with population trends presenting significant declines over the last four decades. These declines throughout their range have resulted in their classification as ‘critically endangered’ by the IUCN. In addition, the European Union has implemented specific legislation surrounding A. anguilla, requiring member states to develop eel management plans [The EC Eel Regulation (1100/2007)]. Aimed to facilitate increased recruitment, these regulations state>40% of historic eel biomass should be allowed safe passage between inland waters and the sea. Cyprus however, applied and were granted an exemption from this, on the basis that there are no rivers on the island of suitable habitat and flow regimes which naturally host A. anguilla (2009/310/EC). Following this decision, recent findings have suggested that historically eels were more widespread in Cyprus than previously recognised. Indeed, a study by Zogaris et al. (2012) indicated that eels are likely the island’s most widespread native species. Cyprus’ freshwater lotic systems are dominated by intermittent rivers and streams, however the natural state and fish populations of these systems are poorly understood. The freshwaters of the island are now heavily fragmented, and with an estimated 108 dams retaining water are host to one of the highest densities of dam reservoirs in Europe. These interruptions to longitudinal connectivity lead to degraded freshwater systems in the lowlands, but despite this the island does have perennial freshwaters, particularly in the western side of the island and at higher elevations. If A. anguilla are indeed present in inland Cyprus, one key deterministic factor on their survival could be access to perennial summer refugia. Here, multiple monitoring methods were applied to build knowledge on present day eel distribution in Cyprus. By increasing knowledge regarding distribution, we can re-evaluate whether conservation initiatives are in fact justified and worthwhile. In 2020 environmental DNA metabarcoding was applied, 130 samples were taken across 26 freshwater sites to provide an up-to-date snapshot of eel distribution. In addition to this, temporal trends were considered based on an island wide fish monitoring programme spanning 2009 - 2019 which predominantly used electric fishing. Overall the results suggest that A. anguilla is widespread in western lowland Cyprus; 11/26 study sites (31/130 samples) tested positive for eel using eDNA metabarcoding, while eels were captured in 61/299 surveys (355 individuals) over the 10 year fish monitoring programme. The trends in eel distribution are highly concordant across methods, although not all sites were monitored with both methods (Fig. 1). These data indicate widespread eel recruitment in lowland freshwaters, but a lack of eels at higher elevation perennial areas. Lowland perennial areas are few and far between, however results here suggest they are abundant in A. anguilla. While higher elevation areas had higher overall freshwater fish species richness suggesting good habitat quality, the lack of the migratory eel in upper reaches indicates that barriers including dam structures may be preventing access for such migratory fish species. Environmental DNA detected eels in intermittent outlet flows of dams, but only in 2/9 reservoirs surveyed. We provide evidence for present day widespread eel recruitment in Cyprus’ inland freshwaters, however highlight the need to increase connectivity to inland perennial systems in order to enhance survival of this critically endangered species at its eastern most range. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Environmental genomics for benthic monitoring of North Sea oil and gas
           offshore platforms

    • Abstract: ARPHA Conference s 4: e65440
      DOI : 10.3897/aca.4.e65440
      Authors : Jan Pawlowski, Florian Mauffrey, Tristan Cordier, Laure Apothéloz-Perret-Gentil, Kristina Cermakova, Thomas Merzi, Mathieu Delefosse, Philippe Blanc : During the last decade, considerable efforts have been undertaken to achieve a “good ecological status” of European coastal waters and ensuring the development of methodological standards for the evaluation of this status. However, the current routine biomonitoring implicates time-consuming and costly manual sorting and morphological identification of benthic macrofauna. In our study, we tested the performance of environmental DNA metabarcoding targeting microbial communities and meiofauna as an alternative to traditional macrofauna-based monitoring. We focused on environmental impact assessment of offshore oil and gas industry. We used three genetic markers (18S V1V2, 18S V9 and COI) to assess the environmental pressures induced by the platforms. All markers showed patterns of alpha and beta diversity consistent with morphology-based macrofauna analyses, significantly changing along distance gradients from the platforms. The impact of the operational discharges was also detected by the variation of biotic indices values, AMBI index showing the best correlation between morphological and eDNA datasets. Finally, the sediment physicochemical parameters were used to build a local de novo pressure index that served as benchmark to test the potential of a taxonomy-free approach. Our study demonstrates that metabarcoding approach outperforms morphology-based approach and can be used as a cost and time-saving alternative solution to the traditional morphology-based monitoring in order to monitor more efficiently the impact of industrial activities on marine biodiversity. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Developing DNA Based Methods for Environmental Regulation and Management-
           UK Insights

    • Abstract: ARPHA Conference s 4: e65436
      DOI : 10.3897/aca.4.e65436
      Authors : Willie Duncan : DNA assessments are revolutionising biomonitoring opportunities across the globe, including the monitoring of rare and invasive species, creating biodiversity inventories, and developing pollution diagnostic and ecosystem resilience assessment methods. To date pollution and ecosystem resilience assessments have been based on assessing the diversity of familiar taxonomic groups but the introduction of DNA based methods will significantly increase the opportunities to exploit groups not previously used for this work.Environmental regulators and managers can derive many benefits from the adoption of these methods, such as improved understanding of environmental conditions, cost effective sample processing, overcoming taxonomic bottlenecks, either through shortages in trained taxonomists or utilising biota with challenging taxonomies. In addition to creating diversity based metrics DNA monitoring also allows for the assessment of functional attributes such as those that support important ecosystem services. The UK has been an early adopter of this technology and this paper will explore how the alignment of scientific advances have coincided with operational needs to create a fertile arena for the development of DNA based assessment methods that will be used in environmental regulation and management. Development projects advanced in the UK will be examined to identify the common and specific issues associated with them that have led to early engagement and adoption. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Quantitative eDNA estimates show a strong correlation with Northern Pike
           biomass in a controlled environment

    • Abstract: ARPHA Conference s 4: e65426
      DOI : 10.3897/aca.4.e65426
      Authors : Erik Karlsson, Göran Sundblad, Martin Ogonowski, Josefin Sundin, Ofir Svensson, Anti Vasemägi : Northern pike, Esox Lucius, is one of the most important species for recreational fisheries in Sweden and as a top-predator; it holds an important role in the food web. Despite its ecological and socioeconomic significance, pike is largely neglected in current monitoring programs because it is seldom caught by conventional monitoring methods. As a result, there is not sufficient data for management authorities to employ biologically justified management strategies. To be able to further understand pike ecology and monitor populations, new monitoring methods are needed. Recent developments in environmental DNA (eDNA) has shown the potential to produce (semi-)quantitative estimates of fish biomass. However, the amount of eDNA in water may vary depending on temperature, fish size, age and density, and it is therefore important to first evaluate the effect of biotic and abiotic factors on eDNA biomass relationships. Here, we evaluated the relationship between eDNA concentration and individual biomass of pike by keeping adult fish of varying size (0.75 – 3.41 kg, n = 11) individually in outdoor mesocosms (~7000 L) filled with water from the adjacent Lake Mälaren. Samples were collected by filtering water through a combination of cellulose nitrate and glass microfiber filter membranes. After a comparison of different extraction methods, the eDNA was extracted using Chelex 100 resin, a low-cost and fast method. Samples were subsequently diluted in 1:8 to alleviate problems from inhibition. A TaqMan assay targeting Cytochrome Oxidase I developed by Olsen et al.(Olsen et al., 2016, 2015) was used to quantify pike eDNA. The results show a strong positive relationship between pike biomass and pike eDNA indicating that the latter represents a promising tool for estimating pike biomass in natural waters.References:Olsen, J.B., Lewis, C.J., Massengill, R.L., Dunker, K.J., Wenburg, J.K., 2016. Erratum to: An evaluation of target specificity and sensitivity of three qPCR assay for detecting environmental DNA from Northern Pike (Esox lucius). Conservation Genetics Resources 8.Olsen, J.B., Lewis, C.J., Massengill, R.L., Dunker, K.J., Wenburg, J.K., 2015. An evaluation of target specificity and sensitivity of three qPCR assays for detecting environmental DNA from Northern Pike (Esox lucius). Conservation Genetics Resources 7. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Ichthyoplankton metabarcoding as a tool for studying fish reproductive

    • Abstract: ARPHA Conference s 4: e65404
      DOI : 10.3897/aca.4.e65404
      Authors : Daniel Teixeira, Heron Hilário, Gustavo Rosa, Guilherme Santos, Gilmar Santos, Daniel Carvalho : The study of ichthyoplankton composition, abundance and distribution is paramount to understand the reproductive dynamics of local fish assemblages. The analysis of these parameters allows the identification of spawning sites, nursery areas and migration routes. However, due to the lack of characters in early life stages, the morphological identification of ichthyoplankton is often impractical and many studies identify only fish larvae. Additionally, its accuracy shows great variation between taxonomists and laboratories according to their experience and specialty. DNA barcoding emerged as an alternative to provide assertive identification of fish eggs and larvae, but it becomes too expensive and laborious when the study demands the processing of huge amounts of organisms. DNA metabarcoding can overcome these limitations as a rapid, cost-effective, broad and accurate taxonomy tool, allowing the identification of multiple individuals simultaneously. Here, we present the identification of a sample containing 68 fish eggs and another containing 293 fish larvae from a single site in the São Francisco River Basin, Eastern Brazil, through DNA metabarcoding. We used a low-cost saline DNA extraction followed by PCR amplification with three primer sets targeting the 12S rRNA gene: MiFish (~170bp), Teleo_1 (~60bp), and NeoFish (~190bp). The latter was recently developed by our research group specifically for the identification of Neotropical fishes. All the amplified samples were sequenced in a single multiplexed Illumina MiniSeq run. We performed the filtering steps and assigned Amplicon Sequence Variants (ASVs) using a DADA2/Phyloseq based pipeline and a custom 12S reference sequence database including 101 species and 70 genera from the Jequitinhonha and São Francisco basins. The species Cyphocharax gilbert, Leporinus taeniatus, Megaleporinus elongatus, Prochilodus argenteus, P. costatus and Psalidodon fasciatus were detected by all three primer sets in the larva pool, while Pterygoplichthys etentaculatus was detected solely by NeoFish (Fig. 1). Within the egg pool, all three markers detected the species Characidium zebra, Curimatella lepidura, M. elongatus, Pimelodus fur and P. costatus, but Brycon orthotaenia was detected only by NeoFish, P. maculatus only by Teleo, and P. pohli by MiFish and Teleo (Fig. 1). The consistency in species detection among all three markers underpins the credibility of this method to accurately describe the sample composition. Considering that most of species were exclusive to the larvae or egg pool, our experiment highlights the importance of including the identification of fish eggs in reproduction studies, as it can provide additional information about which species are spawning in an area. Furthermore, the application of DNA metabarcoding to the study of ichthyoplankton can help decision makers create more informed guidelines for conservation of economically and ecologically important fish species. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • A validated protocol for fish farm monitoring using environmental DNA

    • Abstract: ARPHA Conference s 4: e65421
      DOI : 10.3897/aca.4.e65421
      Authors : Xavier Pochon, Susie Wood, Javier Atalah, Lauren Fletcher, Olivier Laroche, John Pearman, Deanna Elvines, Anastasija Zaiko, Laura Biessy, Georgia Thomson-Laing, Nigel Keeley : Sea-based fish farms are associated with strong benthic enrichment gradients and routine monitoring is usually required by regulation. Internationally a wide range of approaches exist for measuring the degree of benthic deterioration around fish farming activities, ranging from simple visual or odour assessments to the calculation of secondary indices that combine multiple biological and/or physico-chemical metrics (e.g., AZTI Marine Biotic Index; Invertebrate Species Index; Norwegian Quality Index; Infauna Trophic Index).In New Zealand, the health of marine benthic ecosystems around coastal salmon farms is currently measured using an Enrichment State (ES) index. This index incorporates physico-chemical (redox, organic matter, sulphates, etc.) and benthic macrofaunal measurements, which requires taxonomic expertise, is time consuming and expensive.Supported by a range of private/government agencies and industry partners, we have developed and tested the robustness of bacterial, eukaryotic, and multi-trophic Metabarcoding Biotic Indices (b-MBI, e-MBI, and mt-MBI, respectively) using a molecular Eco-Group approach. The indices were calculated via automatic computer pipelines using data collected over a period of nine years from a range of high- and low-flow salmon farms (12 farms and 60 stations) from three distinct regions in New Zealand. The MBIs were compared against the established ES index. All MBIs yielded strong and highly significant relationships with the ES index. The strongest relationships (R2> 0.9) were obtained with the b-MBI. A refinement of the b-MBI (2019-2020) was supported by highly prolific microbes throughout the ES spectrum, and in particular in the upper end of the organic enrichment scale where traditional benthic indices tend to fail. This resulted in ES values of both (molecular-based versus morphology-based) indices to follow a near one-to-one relationship, performing consistently across water flow environments and considered sub-regions. Station-averaged results were also used to compare regulated compliance outcomes between the two indices, based on the current key compliance criteria for farms within each flow regime. Of the 67 seabed monitoring stations that were subsequently classified as compliant or non‑compliant, 62 stations had identical compliance outcomes (i.e. 92% of instances). Furthermore, the b-MBI showed consistently narrower (~50%) confidence interval bands when compared to the traditional ES index. The b-MBI offers unprecedented precision for determining subtle changes along enrichment gradients, constituting a valuable asset for triggering timely management responses and improving compliance.The protocols developed in this project enable rapid, standardised, and cost-effective eDNA isolation and extraction, followed by automatic b-MBI calculation. The affordability and versatility of the b-MBI tool suggests that it could be immediately integrated into current monitoring strategies as the primary benthic assessment tool for assessing benthic impacts of salmon farms in New Zealand. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Evolution of a high-throughput environmental DNA sampling platform

    • Abstract: ARPHA Conference s 4: e65412
      DOI : 10.3897/aca.4.e65412
      Authors : Austen Thomas, Caren Goldberg : With the rise of environmental DNA as a surveillance tool for aquatic species, a need has also arisen for professionally engineered research tools specifically designed for eDNA applications. We created the first portable, purpose-built eDNA sampling system in the form of a backpack smart-pump filtration apparatus and custom made eDNA filter packets for each sample. The eDNA-Sampler (previously ANDe) enables both point-location sampling and mobile sampling over a spatial distance, with the ability to standardize filtration parameters (e.g. flow rate, pressure, water volume, etc.). In this presentation we will describe the evolution of the eDNA sampling backpack and associated components, each designed to help streamline the eDNA sampling process and increase sampling efficiency. We have optimized the platform for mobile sampling by integrating GPS and data logging capabilities, in addition to modifying the chemistry of the eDNA filter packets to minimize the effort required for sample preservation. Results will be presented from a series of pilot studies in which the eDNA-Sampler capabilities were evaluated. Combined, the innovations described herein should help remove barriers to entry for potential eDNA practitioners and also improve overall eDNA data quality. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • eDNA reveals estuarine benthic community response to nutrient enrichment
           – evidence from an in-situ experiment

    • Abstract: ARPHA Conference s 4: e65403
      DOI : 10.3897/aca.4.e65403
      Authors : Dana Clark, Conrad Pilditch, Joanne Ellis, Angel Borja, Javier Atalah, John Pearman, Anastasija Zaiko : Nutrient loading is a major threat to estuaries and coastal environments worldwide, therefore, it is critical that we have good monitoring tools to detect early signs of degradation in these ecologically important and vulnerable ecosystems. We carried out a seven-month manipulative experiment in two estuaries to assess the effects of nutrient loading on benthic communities. Environmental DNA metabarcoding was used to examine the response of eukaryotic (18S rRNA), diatom (rbcL), and bacterial (16S rRNA) communities to two levels of nutrient enrichment (150 and 600 g N m-2). Multivariate analyses demonstrated consistent changes in eukaryotic, diatom, and bacterial communities in response to enrichment, despite differing environmental conditions between sites (Fig. 1). These patterns aligned with changes in macrofaunal communities identified using traditional morphological techniques, confirming concordance between disturbance indicators detected by eDNA and current monitoring approaches. Clear shifts in eukaryotic and bacterial indicator taxa were seen in response to nutrient loading while changes in diatom communities were more subtle. Community changes were discernable between nutrient levels, suggesting that estuary health assessment tools could be developed to detect early signs of degradation. Existing eDNA-based biotic indices (microgAMBI and mtMBI) were able to detect these community shifts, suggesting transferability of these indices to other regions and systems. This work represents a first step towards the development of molecular-based estuary monitoring tools, which could provide a more holistic and sensitive approach to ecosystem health assessment with faster turn-around times and lower costs. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • High-throughput DNA barcoding of oligochaetes for abundance-based indices
           to assess the biological quality of sediments in streams and lakes

    • Abstract: ARPHA Conference s 4: e65394
      DOI : 10.3897/aca.4.e65394
      Authors : Régis Vivien, Laure Apothéloz-Perret-Gentil, Jan Pawlowski, Inge Werner, Michel Lafont, Benoit J.D. Ferrari : Aquatic oligochaete communities are valuable indicators of the biological quality of sediments in streams and lakes, but identification of specimens to the species level based on morphological features requires solid expertise in taxonomy and is possible only for a fraction of specimens present in a sample. The identification of aquatic oligochaetes using DNA barcodes would facilitate their use in biomonitoring and allow a wider use of this taxonomic group for ecological diagnoses. Previous approaches based on DNA metabarcoding of samples composed of total sediments or pools of specimens have been proposed for assessing the biological quality of ecosystems, but such methods do not provide precise information on species abundance, which limits the value of resulting ecological diagnoses. Here, we tested how a DNA barcoding approach based on high-throughput sequencing of sorted and genetically tagged specimens performed to assess oligochaete species diversity and abundance and the biological quality of sediments in streams and lakes. We applied both molecular and morphological approaches at 13 sites in Swiss streams and at 7 sites in Lake Geneva. We genetically identified 33 or 66 specimens per site. For both approaches, we used the same index calculations. We found that the ecological diagnoses derived from the genetic approach matched well with those of the morphological approach and that the genetic identification of only 33 specimens per site provided enough ecological information for correctly estimating the biological quality of sediments in streams and lakes. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Is the ecological status assessment result the same using genomic- and
           morphology-based benthic biotic indices'

    • Abstract: ARPHA Conference s 4: e65393
      DOI : 10.3897/aca.4.e65393
      Authors : Ángel Borja, Iñigo Muxika, Anders Lanzén : Monitoring and assessment of benthic communities have been routinely undertaken using morphology-based benthic indices, among which AZTI’s Marine Biotic Index (AMBI) and multivariate-AMBI (M-AMBI), are the most successful worldwide (Borja et al., 2019). They are used officially in routine monitoring in many European countries, where they have been intercalibrated (European Commission, 2018). AMBI has been mirrored by metabarcoding, and the genomic version (gAMBI; Aylagas et al., 2014) has demonstrated to yield results comparable to the morphological version. However, we have so far failed to develop a reliable genomic version of M-AMBI (M-gAMBI), which includes gAMBI, richness and diversity (Aylagas et al., 2018). This is because richness and diversity present very different results in morphological and genomic analyses. Since the multivariate method needs reference conditions, these must be set specifically for M-gAMBI to make its results comparable to those obtained with M-AMBI. To this aim, we started annual surveys in 2018, in 22 sampling locations, in Basque estuaries and coast. We present the results from the first three years of these surveys and discuss the problems faced when developing genomic reference conditions for M-gAMBI. The findings are of paramount importance for managers, since any new method or modification of an existing assessment method, needs to demonstrate that the results obtained when assessing the status are similar to those morphological-based methods, already approved and intercalibrated.Aylagas, E., Á. Borja, N. Rodríguez-Ezpeleta, 2014. Environmental Status Assessment Using DNA Metabarcoding: Towards a Genetics Based Marine Biotic Index (gAMBI). PLoS ONE, 9: e90529.Aylagas, E., Á. Borja, I. Muxika, N. Rodríguez-Ezpeleta, 2018. Adapting metabarcoding-based benthic biomonitoring into routine marine ecological status assessment networks. Ecological Indicators, 95: 194-202.Borja, A., G. Chust, I. Muxika, 2019. Chapter Three - Forever young: The successful story of a marine biotic index. Advances in Marine Biology, 82: 93-127.European Commission, 2018. Commission Decision (EU) 2018/229 of 12 February 2018 establishing, pursuant to Directive 2000/60/EC of the European Parliament and of the Council, the values of the Member State monitoring system classifications as a result of the intercalibration exercise and repealing Commission Decision 2013/480/EU. Official Journal of the European Communities, L47: 1-91. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Development of eDNA tools for the detection of marine invasive non-native
           species to support European flat oyster (Ostrea edulis) restoration

    • Abstract: ARPHA Conference s 4: e65383
      DOI : 10.3897/aca.4.e65383
      Authors : Sean Markus, Iveta Matejusova, Alex Douglas, William Sanderson : The European flat oyster (Ostrea edulis) is an important keystone species in Scottish coastal waters. However due to anthropogenic pressures, significant reductions to oyster beds have been observed across Europe. In Scotland, several projects are currently aiming to restore European flat oyster habitats through the translocation of juvenile oysters from various sources including hatcheries and aquaculture. However, translocation of shellfish is not risk free and can increase the risk of accidental translocation of invasive non-native species (INNS). If INNS become established outside of their native range they can cause irreversible harm to native organisms and habitats. This study aims to develop molecular tools to detect environmental DNA of INNS which can be potentially associated with the translocation of live shellfish stocks. We have developed a species-specific real-time PCR assay for detection of Pacific oyster (Crassostrea gigas) and tested its sensitivity in a large-scale replicated mesocosm based experiment with varying densities of C.gigas. A secondary objective of the experiment was to assess the detection of another invasive species, the carpet sea squirt Didemnum vexillum which was cohabited with C. gigas. We aim to quantify the detection probability of increasing densities of C. gigas from repeat water samples and qPCR replicates. This project also aims to investigate the feasibility of using portable, real-time sequencing technologies such as the Oxford Nanopore MinION to develop robust tools to support native oyster restoration programmes. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Macroinvertebrate community assessment and biomonitoring of European water
           bodies. Is a multimarker approach necessary'

    • Abstract: ARPHA Conference s 4: e65390
      DOI : 10.3897/aca.4.e65390
      Authors : Juan Antonio Villaescusa, Mª José Villena, Raquel González, Antonio Picazo, Verónica Rojo, Marko Nieminem, Silke Classen, Ana Pujante, Antonio Camacho : The present work, included in the European Project BIOWAT, aims to develop and validate the use of genomic tools or metabarcoding for its utilization as a routine method for river biomonitoring in different European Biogeographical regions. The project included sampling points in three biogeographic regions, Mediterranean (Spain), Continental (Germany) and Boreal (Finland). The current development of the study was designed using mock communities obtained from the three mentioned areas and different aspects were tested: DNA extraction methods, selection of informative region (16S vs COI), design and performance of primers, bioinformatic pipeline, etc…Although the use of COI has become very popular, and its barcode database is more complete, the use of mitochondrial 16S as taxonomic marker can provide similar or even better results when accompanied by a rich local barcode database Elbrecht 2016. In this presentation, the results and conclusions obtained for the biomonitoring of nine rivers (3 for each of the biogeographic regions) using 16S as DNA marker and a local barcode database are shown. The results of ecological status assignment using 16S marker were promising, showing a good correlation between morphological determinations and metabarcoding data for most of the studied rivers. However, in some cases, the metabarcoding data showed a jump in the ecological status class (to better or worst status), highlighting the existence of some problems related with primers (for some taxonomic groups) or missing taxa in the barcode database that still need to be solved prior the utilization of this method on a routine basis.Additionally, for the studied Mediterranean rivers, a complementary analysis using COI as marker was made, using the universal primers developed by Elbrecht and Leese 2017. In general, this marker showed better results in the identification for some taxa, whereas other included in the mock communities were not identified showing important problems that could be related with primers (sometimes not well covering characteristic taxa present in other biogeographic regions) or the lack of a complete COI macroinvertebrate barcode databases in the Iberian Peninsula for the case of Spain Múrria 2020.Our results show that both markers have the potential to produce a good identification of benthic macroinvertebrates, showing an acceptable correlation between morphology and metabarcoding approaches. However, none of them is able to amplify all of the present groups, so the parallel use of both markers (mitochondrial 16S and COI) in a multimarker approach could solve some of the problems, giving a more complete profile of the macroinvertebrate community. This approach has already been proposed and can lead the future of macroinvertebrate community assessment Ficetola 2020, Martins 2020. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • New trends in bioassessment of aquatic ecosystems: from organisms to
           DNA-based metrics

    • Abstract: ARPHA Conference s 4: e65382
      DOI : 10.3897/aca.4.e65382
      Authors : Jan Pawlowski, Maria Kahlert : Traditionally, the biological quality of aquatic ecosystems is assessed using selected groups of organisms that can be identified morphologically. Recent advances in high-throughput genomic approaches offered new opportunities to monitor biodiversity and assess ecological status using DNA barcoding and metabarcoding. The DNA-based tools have been used in three different ways: (1) to replace morphological identification of biological quality elements in existing biotic indices, (2) to develop new molecular indices based on morphologically inconspicuous groups of potential environmental indicators, and (3) to predict biotic indices from environmental DNA datasets using machine learning methods (Pawlowski et al. 2018). The next steps need to take advantages and challenges of these different approaches into account in view of their future application in routine bioassessment.The Working Group 2 of DNAqua-Net, Biotic Indices & Metrics, has worked with several task forces tackling different organism groups (fish, macroinvertebrates, diatoms, bacteria, protists, meiofauna), because challenges have been shown to be quite different dependent on the target organisms Kahlert et al. 2019. For the fish the eDNA-metabarcoding methods are well developed and give very good results in terms of species detection. The important question is to see if the semi-quantitative data retrieved from the metabarcoding (proportion in eDNA sequences) could be translated to proportions in biomass/numbers that are now used in many indices. The fish researchers are trying to fit these data in, but some correction factors might be needed to correct for differences between molecular and conventional methodsRegarding the macroinvertebrates, much discussion regarding index development was focusing on the importance of abundance measurements, and it was tested how existing indices would perform if barcoding data would be used instead of morphological data. Still discussion is ongoing on several technical issues, including the use of preservative for DNA extraction from bulk samples, the choice of primers for PCR amplification and the incompleteness of reference databases which impedes the correct assignment of eDNA sequences. Also minimum standards for routine operation are still missing.The diatom group has worked much on practical issues, starting a large initiative to compare diatom metabarcoding protocols used in routine freshwater biomonitoring for standardization (Bailet et al. 2019, Keck et al. 2018, Vasselon et al. 2017). With diatoms, all three approaches to develop molecular indices have been tested and seem promising, i.e. using existing indices with taxa names derived by matching sequences with reference databases, developing new indices based on molecular data only with traditional fixed scores, and using machine-learning techniques (Bailet et al. 2020, Vasselon et al. 2018, Tapolczai et al. 2019, Keck et al. 2018) The micro- and meiobiota group has worked towards an inclusion of microorganisms into aquatic assessment, because the microbial community dynamics are a missing link important for our understanding of rapid changes in the structure and function of aquatic ecosystems, and should be addressed in the future environmental monitoring of freshwater ecosystems (Sagova-Mareckova et al. 2021). Another focus was on how sediment DNA analysis can be integrated into stated goals of routine monitoring applications. It has been an interesting journey, and we WG2 coordinators would like to thank all the people for their engagement! Keep up the good work! HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Replicate DNA metabarcoding can discriminate seasonal and spatial
           abundance shifts in river macroinvertebrate assemblages

    • Abstract: ARPHA Conference s 4: e65379
      DOI : 10.3897/aca.4.e65379
      Authors : Alex Bush, Zacchaeus Compson, Matilda Kattilakoski, Natalie Rideout, Brianna Levenstein, Mehrdad Hajibabaei, Wendy Monk, Donald Baird : Metabarcoding is capable of delivering consistent and accurate fine-resolution biodiversity data, and offers great promise for improving aspects of environmental assessment and research. Even so, many ecologists are keen to make further inferences about species’ abundances and the number of sequence reads has proven to be a poor proxy for abundance. The conservative interpretation has been to treat metabarcoding data as presence/absence, and although such data are less rich, occurrence and abundance are only different expressions of the same phenomenon. Interestingly if we assume the probability of detecting individuals is constant, it should be possible to use changes in the frequency of detection to infer changes in the underlying abundance. We tested the possibility that changes in the abundance structure of benthic macroinvertebrate communities could be recovered using replicated metabarcoding.We conducted 5 monthly surveys from Jun-Nov 2019 at the Catamaran Brook, a small tributary of the Little Southwest Miramichi River in New Brunswick, Canada. Each survey collected 30 benthic samples divided between control and treatment cages that excluded predatory fish. A further 6 samples were taken for traditional microscopic identification and counting.Analysis of the metabarcoding data demonstrated that we could recover plausible changes in abundance from occurrence data, including significant responses to both seasonal dynamics and the experimental exclusion of predators. The microscopy samples merely confirmed that count data are highly stochastic, and therefore while specific estimates of expected abundance from our model are highly uncertain, they capture those differences we could validate. In summary, while we confirmed that occurrence data are more robust for routine bioassessment, it is possible to recover fine-resolution changes in abundance that can inform ecological studies using metabarcoding. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • The use of machine learning predictive models to assess rivers quality
           with molecular data

    • Abstract: ARPHA Conference s 4: e65380
      DOI : 10.3897/aca.4.e65380
      Authors : Maria Feio : Many tests have been made so far to assess the biological quality of rivers with molecular data. Most often HTS-related eDNA metabarcoding sequences clustered into Operational Taxonomic Units (OTUs) are assigned to taxa, using reference barcode databases. From there, the existing biotic indices, developed for morphological data are calculated. However, this approach has several drawbacks that may justify their lower performances compared to traditional ones, or not extracting the maximum potential from the molecular data. The first is the incompleteness of reference databases (despite their continuous evolution) - avoiding the conversion of molecular into taxonomic may overcome this issue. Yet, another likely source of bias in the assessments is at the basis of existing classification systems: a possible poor correspondence between the biological reference conditions developed based on species morphology and on molecular data. In other words, molecular-based assemblages from different rivers may not group similarly or respond to the same environmental variables as taxa. Correcting this would require rebuilding the whole systems and establishing new typological-based molecular reference values, which are then used to calculate Ecological Quality Ratios (EQR) and determine the Ecological Quality Status (EQS) of river sites. One alternative to the grouping step inherent to the typological approach, and that may be viewed as artificial (nature is a continuum), is the prediction of site-specific reference conditions based on abiotic characteristics of sites. Thus, we tested a combination of machine-learning modelling techniques to build a taxonomic-free site-specific index to assess rivers based on diatom assemblages, from 81 sites located in Portugal (Feio et al. (2020)Feio et al. 2020). The models are trained to predict diatom OTUs expected under reference conditions, from environmental data. Then, for each test site, an OTU EQR is calculated based on the deviation between the observed OTUs in the field samples and the expected OTUs under reference conditions, which is finally converted into a quality class (after the construction of a new classification system). The molecular-based model was accurate and sensitive to global anthropogenic disturbance (such as, changes in land use and habitat quality), which gives promising insights to its use for bioassessment of rivers. Further work consists of testing this approach with invertebrate data as well as investigating the potential of ISO or ESV in alternative to OTUs. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Lake sedimentary DNA to reveal long-term changes in aquatic biodiversity
           and ecosystem functioning

    • Abstract: ARPHA Conference s 4: e65378
      DOI : 10.3897/aca.4.e65378
      Authors : Isabelle Domaizon : The emergence of molecular analyses based on the sequencing of sedimentary DNA has opened up many new areas of inquiry in paleolimnology. DNA preserved in sediments (SedDNA) offers the possibility to consider taxa that were traditionally not accessible because they do not leave distinct morphological fossils.Recent applications that considered a diversity of biological groups (including bacteria, protists, zooplankton, fish) illustrate how efficiently SedDNA-based methods complement both classical paleolimnology proxies and limnological data. The knowledge gained from this approach is very diverse in scope, ranging from quantifying natural variability in population and community dynamics to understanding how these biological variables respond to anthropogenic disturbances and climatic change. The use of lake sedimentary DNA to track long-term changes in aquatic biota is a rapidly advancing field of research. Based on recent applications, this presentation illustrates (i) the potential and challenges associated with the study of SedDNA to address critical research questions in lacustrine ecology (ii) the main methodological precautions to be taken into account for implementing these types of DNA analyses (i.e. best practices) and (iii) the emerging topics that could be addressed using sedimentary DNA, in particular to reconstruct the temporal dynamics of lacustrine biodiversity. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • DNA-based monitoring for assessing the effect of invasive species on
           aquatic communities in the Amazon basin of Ecuador

    • Abstract: ARPHA Conference s 4: e65376
      DOI : 10.3897/aca.4.e65376
      Authors : Christine Van der Heyden, Lenin Riascos, Andrea Carrera-Gonzalez, Katherine Elizabeth Apunte Ramos, Marcela Carbrera, Rodrigo Espinosa, Pieter Boets, Tom Moens, Wim Vanden Berghe, Erik Verbruggen, Filip Volckaert, Francisco Villamarin, Peter Goethals, Julio Bonilla, Mauricio Ortega, Jorge Celi : Ecuador is well-known as one of the most biodiverse countries, but this species richness is being threatened by invasive alien species. The early detection of these invasive species is crucial for their fast and successful eradication and for limiting their effects on aquatic communities. Therefore, a Belgian VLIR-UOS project was started that aims at the development of a fast detection method to monitor the Ecuadorian Amazon river basin for the presence of invasive fishes, macroinvertebrates and amphibians. An (e)DNA field lab, equipped with miniaturized and portable DNA-processing equipment, such as centrifuges, thermal cyclers, and electrophoresis equipment (MiniPCR), was developed. In the next phase, the Nanopore Next-Generation sequencing (NGS) technique (MinION) will be optimized to enable the eDNA-based biomonitoring of tropical aquatic environments in the field. The fast detection of invasive species may help to prevent their further spread and perhaps even facilitate their eradication, and will promote more effective actions for the conservation of aquatic ecosystems. Furthermore, new DNA-sequences of amphibians, macroinvertebrates, and fishes are being incorporated into the newly developed Ecuadorian DNA database. We also focus on building and strengthening the capacities of staff and students (Ecuadorian as well as Belgian) through theses, practical courses, field work, trainings and internships. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Developing DNA-Based Ecological Classifications for Shallow Streams in
           Northeastern United States

    • Abstract: ARPHA Conference s 4: e65377
      DOI : 10.3897/aca.4.e65377
      Authors : Alison Watts, Devin Thomas : Several regions in the Unites States have developed algal bioassessment methods for wadeable streams (e.g. Maine, Connecticut). Algal communities and indicator species are correlated with land use, water chemistry, and other watershed metrics to identify ecosystem indicators relevant to local conditions. Taxonomic analysis has historically been performed by microscopic examination and identification of species within a sample. A pilot survey was conducted to assess the use of DNA-based taxonomic methods in algal bioassesments of stream condition in New Hampshire and Maine in the northeastern United States. Algae samples were collected at 60 wadeable streams throughout the region in the summer of 2019. Samples were collected at sites included in long term water quality monitoring networks, to allow comparison with longer term water quality and bioassesment data. Samples were extracted and sequenced with primers targeting 18S for eukaryote species, rbcl for diatoms, and 12S for fish. Algal features were correlated with stream parameters including nutrient concentration, historic Benthic IBI indices, and other water quality metrics. Our results support previous studies indicating that molecular-based methods are a viable approach to water quality assessment. We found that:DNA-derived algal communities can be correlated to nutrient categories, and indices developed from multiyear data are reflected in the community.DNA-derived algal communities can be correlated to Benthic IBI ratios, and to traditional algal bioassesment categories.18S and rbcl primers were both effective at amplifying target species to identify distinguishable community assemblages. Fish were detected in water samples at all sites, and the species identified represent those that are likely to be present based on previous electro-fishing surveys. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Complexity matters: Evaluating the impact of bioinformatics parameters on
           eukaryotic MOTU delimitation and taxonomy assignment

    • Abstract: ARPHA Conference s 4: e65374
      DOI : 10.3897/aca.4.e65374
      Authors : Holly Bik : Microbial metazoans (e.g. nematodes, copepods, tardigrades and other 'minor' animal phyla < 1mm in size) are ubiquitous and abundant across most ecosystems on earth. In marine sediment habitats, microbial metazoa exhibit high biodiversity but suffer from poor taxonomy and an ongoing lack of reference DNA sequences in public databases. Environmental DNA metabarcoding thus represents an increasingly critical tool for rapidly assessing the global biodiversity and phylogeographic patterns of such neglected metazoan groups. However, there are significant bioinformatics hurdles facing the study of microbial eukaryotes. Most software pipelines and databases have been designed and optimized for smaller (e.g. bacteria/archaea) or larger (e.g. vertebrate) taxa, and emphasize "standard" metabarcoding loci such as COI which are not useful for groups such as nematodes which lack universal COI primer binding regions. In addition, the sparsity of public reference barcodes for microbial metazoa often precludes accurate taxonomy assignments for unknown MOTUs in metabarcoding datasets. Here, I will present recent work focused on the refinement of bionformatics workflows for microbial metazoan groups, including efforts to account for intragenomic variation observed in rRNA loci, discrepancies in results across OTU vs. ASV generation pipelines, and biases in sequence-based taxonomhy assignment methods. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Characterization of marine eukaryotic biofilms at offshore wind farm
           sites: assessment of DNA extraction methods and marker gene used for
           metabarcoding approaches

    • Abstract: ARPHA Conference s 4: e65369
      DOI : 10.3897/aca.4.e65369
      Authors : Aurelie Portas, Gérald Culicoli, Jean-françois Briand, Nolwenn Quillien : Among marine lifestyles, biofilms are considered as diversified communities embedded in complex exopolymers whose development depends on several factors, related to both environmental conditions and physical-chemical characteristics of substrates (Antunes et al. 2019, Bellou et al. 2012). For the maritime industry, bio-colonization and its impact on human activities were well-described (Schultz et al. 2011). However, this phenomenon represents a new challenge in Renewable Marine Energies (RME) due to their specificities (materials, structures, localization…). In particular, macro-organism assemblages appeared to include a wide variety of eukaryotic groups but the literature is sparse considering the sequencing of eukaryotic diversity in comparison to those of bacterial communities (Briand et al. 2018, Dang and Lovell 2000, Salta et al. 2013). As a matter of fact, the very small size of some of the eukaryotes and/or their insufficient morphological discernible features appear to considerably limit their detection and identification, leading to underestimate their diversity (Carugati et al. 2015). When talking about molecular approaches, analysis of eukaryotes also represents a challenge because such organisms possess resilient cellular structures which can give poor DNA extraction yield (Hermans et al., 2018Hermans et al. 2018). In addition, SSU rRNA in eukaryotes fails to be as universal as for prokaryotes (Bik et al. 2012, Medinger et al. 2010). However, the use of marker genes from environmental DNA, when focused on the targeted eukaryotic community, remains critical to decoding the complexity of marine biofilms diversity.In this study, four extraction methods, including a preliminary mechanic cell lysis, both soil and biofilm kits, and global approaches, have been compared. We also examined the coverage and the identification capability of several primers to characterize eukaryotic communities colonizing three plastic surface types (polyvinyl chloride, HD polyethylene, and polyamide) which have been immersed in several locations along the French Mediterranean and Atlantic coasts. Sequence quality and number remain the same whatever the extraction method. However, the richness and community structure were clearly affected regardless of the sample type (Figure 1). Finally, two kits (PowerMaxSoil, and PowerBiofilm kits) evaluated in this study were considered as the most powerful overall.Secondly, we amplified and sequenced short fragments of two genes: one region of the mitochondrial Cytochrome Oxidase subunit I (COI) and five variable regions of the 18S small subunit ribosomal DNA (rDNA) gene (V1V2, V4TAR, V4UNI, V7, and V9). The Chao1 index was considerably lower for the CO1 gene compared to those of the 18S rDNA regions. The V4TAR and V7 regions showed a significant highest richness, followed closely by the V1V2 and V9 regions. The 18S rDNA gene sequences were dominated by microeukaryotes whereas the COI sequences were dominated by macro-organisms. Each of the 18rDNA primer pairs also exhibited dissimilar community structures although the dominant taxa seemed to be common.To conclude, our results provided a global assessment of tools dedicated to the description of the diversity of marine eukaryotes biofilms from three surfaces used in the design of RME. Among the four extraction methods described here, PowerMaxSoil and PowerBiofilm kits allowed recovering the highest diversity. COI and 18S rDNA gene sequencing covered different groups including at high taxonomic levels. Despite limitations, metabarcoding will help in the characterization of marine biofilms diversity on RME. Especially, it may be relevant to use primers targeting these two genes to better cover the eukaryotic diversity. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Metabarcoding to establish freshwater indicators of environmental
           degradation in the Indo-Burmese biodiversity hotspot

    • Abstract: ARPHA Conference s 4: e65364
      DOI : 10.3897/aca.4.e65364
      Authors : Alfried Vogler, Md. Mizanur Rahman, Alfred Burian, Thomas Creedy : Biodiversity hotspots of the world are increasingly exposed to anthropogenic pressures and resulting ecosystem breakdowns. However, biotic surveys for ecological status assessment are rarely conducted in poorly characterised, yet highly diverse ecosystems in the tropics and subtropics. Here, we addressed the challenge of developing a monitoring system for the highland streams of the Indo-Burmese biodiversity hotspot in Bangladesh, using a meta-barcoding approach to investigate the impacts of growing anthropogenic pressures on poorly studied invertebrate communities. Species richness and beta diversity in the region were correlated with anthropogenic stressors that varied greatly between sampling sites. A partial-network approach allowed us to identify potential indicator species for either a good or poor ecological status. Overall, our results document high species richness and pronounced responses to disturbance in these unexplored, but threatened habitats. In combination with classical taxonomy approaches, metabarcoding can therefore serve as a valuable tool to rapidly generate lacking baseline information facilitating the conservation of vulnerable ecosystems. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • DNA barcodes combined with geometric morphometry challenge species
           hypothesis in palaemonid shrimp

    • Abstract: ARPHA Conference s 4: e65363
      DOI : 10.3897/aca.4.e65363
      Authors : Aleksandra Jablonska, Nicolas Navarro, Remi Laffont, Remi Wattier, Vladimir Pesic, Andrzej Zawal, Jasna Vukic, Michal Grabowski : Although the Mediterranean Region is known as a hotspot for biodiversity and endemism its freshwater fauna is still greatly unexplored, and even the emblematic taxa such as decapods require in-depth integrative investigation. In our research we used integrative approach composed of various geometric morphometric and molecular methods to challenge the taxonomic status of  two  freshwater shrimps representing Palaemonidae: Palaemon antennarius and Palaemon minos. Basing on 352 COI sequences, three Molecular Operational Taxonomic Units (MOTUs) were defined. Two of them belonged to P. antennarius: first inhabiting Apennine Peninsula and Sicily, the second one from the Balkan Peninsula. The third MOTU corresponded to Palaemon minos from Crete. The Balkan MOTU of P. antennarius was closer to P. minos in terms of genetics, than to the other conspecific MOTU. The carapace shape variation, studied on 180 individuals, was mainly explained by the geographic distribution. Balkan and Cretan groups were clearly distinguished, while other samples were distributed along the shape gradient from Sicily and southern Apennine Peninsula to the Balkans. The results of our study showed that, either the MOTU assigned to the Apennine Peninsula and Sicily constitutes a separate species or, alternatively, P. minos should be synonymised with P. antennarius. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Establishement of a national monitoring program based on environmental DNA
           for amphibians and the chytrid fungus Batrachochytrium dendrobatidis   

    • Abstract: ARPHA Conference s 4: e65362
      DOI : 10.3897/aca.4.e65362
      Authors : Omneya Osman, Johan Andersson, Tomas Larsson, Mats Töpel, Alexander Eiler : National monitoring programs provide the basis for evaluating the integrity of ecosystems, their responses to disturbances, and the success of actions taken to conserve or recover biodiversity. In this study, we successfully established a national program for the invasive chytrid fungus Batrachochytrium dendrobatidis (Bd) based on dual TaqMan assays. Amphibian diversity based on metabarcoding of the mitochondrial 12S rRNA gene was also performed. Assays were optimized for sensitive detection of target species from a wide range of amphibian ponds with variable potential of inhibitions for eDNA based detection. An amphibian mock community of 5 species was used to validate the metabarcoding approach while internal standards were used in the case of the dual TaqMan assays. First sampling of over 170 ponds in Norway resulted in Bd detection in 12 environmental samples and one swab sample taken over multiple years indicating the establishment of Bd in Norway. Five amphibian species Bufo bufo, Lissotriton vulgaris, Triturus cristatus, Rana arvalis and Rana temporaria as predicted from data in long-term citizen science reporting systems were widely detected in the collected eDNA samples. Our large scale-monitoring program indicates a low risk of a Bd outbreak and amphibian decline caused by chytridiomycosis but continued monitoring is recommended in the future. These findings indicate that eDNA is an effective method to detect invasive species, and to monitor endangered amphibian species. Still, several shortcomings (such as PCR inhibitors and sample volume) were identified that need to be addressed to improve eDNA-based monitoring at the national level. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Field and Lab Protocols: Achievements of the DNAqua-Net Working Group 3

    • Abstract: ARPHA Conference s 4: e65306
      DOI : 10.3897/aca.4.e65306
      Authors : Kat Bruce, Emre Keskin : We review the progress made in building capacity and consensus around field and laboratory methods during the course of the DNAqua-net programme, including short-term scientific missions, training courses, workshops and ring tests.  We highlight the challenge of methodological standardisation, where in some cases the same written protocol employed by different laboratories may not always give the same results, but in other cases almost identical results can be obtained using different methodological workflows. As the field of DNA-based biomonitoring moves increasingly into practical application, we consider how end-users can best ensure quality and consistency of results, while also enabling continued technical innovation, which will continue to move the field forwards. We also reflect on the benefits of multi-stakeholder forums such as DNAqua-net for bridging the research-policy gap and accelerating the application and impact of research. Finally, we introduce a major output from the working group: A Practical Guide to DNA-based Methods for Biodiversity Assessment, which synthesises current knowledge to help end-users or those new to the field to understand the methodological choices that have to be made, and the trade-offs implicit in those choices.  HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Challenges in data analyses and data storage for metabarcoding

    • Abstract: ARPHA Conference s 4: e65361
      DOI : 10.3897/aca.4.e65361
      Authors : Diego Fontaneto, Alain Franc : The working group of the DNAquanet COST Action dealing with data analyses and data storage for the use of metabarcoding approaches in biodiversity assessment, namely Working Group 4, had two main goals. On the one hand the comparison of the available analytical pipelines, while keeping track of new advances in bioinformatics, and on the other hand to deal with potential issues in data storage and sharing in the era of big data. Such activities were carried out through discussions at meetings of the COST Action, organisations of workshops, online surveys, and meta-analyses.The main achievements of the first line of activity, comparing pipelines, will be summarised in the first of the talks of the session, dealing with differences in clustering algorithms to obtain clusters of sequences that are then used for subsequent analyses and inference. The other talks in the session will introduce different pipelines and approaches that are currently developed to improve the way biological monitoring can be performed.The main achievement of the second line of activity, on data storage and sharing, will be summarised in the first flash talk of the session, dealing with the current scenario and potential pitfalls related to sharing the raw data from massive sequencing.The other flash talks in the session will provide examples on the applications of different approaches to analyse biodiversity using DNA sequence data.We are confident that the pluralism in approaches and applications that will be presented in the session will provide supporting discussions and interactions for a convergence towards the optimisation of the pipelines and the best use of data from metabarcoding. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Mapping biodiversity hotspots of fish communities in subtropical streams
           through environmental DNA

    • Abstract: ARPHA Conference s 4: e65352
      DOI : 10.3897/aca.4.e65352
      Authors : Rosetta Blackman, Maslin Osathanunkula, Jeanine Brantschen, Cristina Di Muri, Lynsey Harper, Elvira Mächler, Bernd Hänfling, Florian Altermatt : Tropical and subtropical freshwater habitats are among the most biodiverse ecosystems worldwide, containing a characteristic fauna and high numbers of endemic species. However, exploitation of organisms, global climate change, pollution and the introduction of invasive species are severely threatening this diversity. Implementation of appropriate conservation and protection measures in tropical freshwater systems depends on comprehensive knowledge of state and change in biodiversity, which however, has been barely feasible due to logistic, technical and taxonomic challenges abound in tropical and subtropical ecosystems. Here we use a single environmental DNA (eDNA) multi-site sampling campaign distributed evenly through the 200,000 km2 Chao Phraya river basin, Thailand, to provide key information on freshwater fish diversity. We found a total of 108 fish taxa and identified key biodiversity patterns within the river network with respect to alpha- and beta-diversity patterns. By using a hierarchical clustering, we grouped the fish communities of all sites across the catchment into distinct clusters. Mapping these clusters over the catchment not only accurately matched the topology of the river network, but also revealed distinct groups of sites which should each be considered of high conservation value. Our study demonstrates a key application of large-scale monitoring (via eDNA) to identify distinct areas within a catchment for conservation and habitat protection. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • A large-scale ecological assessment of Swiss rivers using environmental
           DNA for the monitoring of macroinvertebrates 

    • Abstract: ARPHA Conference s 4: e65307
      DOI : 10.3897/aca.4.e65307
      Authors : Jeanine Brantschen, Rosetta Blackman, Jean-Claude Walser, Florian Altermatt : Anthropogenic activities are changing the state of ecosystems worldwide, affecting community composition and often resulting in loss of biodiversity. Riverine ecosystems are among the most impacted ecosystems. Recording their current state with regular biomonitoring is important to assess the future trajectory of biodiversity. However, traditional monitoring methods for ecological assessments are costly and time-intense. Here, we compare environmental DNA (eDNA) to traditional kick-net sampling in a standardized framework of surface water quality assessment. We use surveys of macroinvertebrate communities to assess biodiversity and the biological state of riverine systems. Both methods were employed to monitor aquatic macroinvertebrate indicator groups at 92 sites across major Swiss river catchments. The eDNA data were taxonomically assigned using a customised reference database. All zero-radius Operational Taxonomic Units (zOTUs) mapping to one of the 142 traditionally used indicator taxon levels were used for subsequent diversity analyses (n = 205). At the site level, eDNA detected less indicator taxa than the kick-net method and alpha diversity correlated only weakly between the methods. However, the methods showed a strong congruence in the overall community composition (gamma diversity), as the same indicator groups were commonly detected. In order to set the community composition in relation to the biotic index, the ecological states of the sampling sites were predicted by a random forest approach. Using all zOTUs mapping to macroinvertebrate indicator groups (n = 693) as predictive features, the random forest models successfully predicted the ecological status of the sampled sites. The majority of the  predictions (71%) resulted in the same classification like the kick-net based scores. Thus, the sampling of eDNA enabled the detection of indicator communities and provided valuable classifications of the ecological state, when combined with machine learning. Overall, eDNA based sampling has the potential to complement traditional surveys of macroinvertebrate communities in routine large-scale assessments in a non-invasive and scalable approach.  HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Novel DNA-based test for the identification of benthic diatoms of European
           freshwater waterbodies (WAT-DIMON)

    • Abstract: ARPHA Conference s 4: e65203
      DOI : 10.3897/aca.4.e65203
      Authors : Panayiota Pissaridou, Athina Papatheodoulou, Gregoris Notarides, Juan Antonio Villaescusa, Maria Villena, Antonio Picazo, Rosa Trobajo, David Mann, Antonio Camacho, Marlen Vasquez Hadjilyra : Diatoms are unicellular eukaryotic organisms, which have been exploited over the years for effective freshwater bioassessment. Therefore, they are excellent bioindicators, routinely used in national environmental monitoring programs all over Europe within the Water Framework Directive (WFD) 2000/60 /EC (Foster et al., 2000) and CEN standards (CEN, 2018).Over the years, new technologies have been introduced to this field to maximise and improve the time and cost required for freshwater bioassessment. The application of DNA metabarcoding for the characterisation of benthic diatom communities for WFD was recently introduced (Vasselon et al. 2017, Kelly et al. 2018). Through this technique, the identification of the species present in one environmental sample, is established using genetic variability and is characterised by a short DNA fragment called a barcode (Vasselon et al., 2019).The Wat-Dimon Eurostars project aim at creating a novel genomic test for the identification of European benthic diatoms. This new DNA-based test could be routinely implemented in national environmental monitoring programs all over Europe within the Water Framework Directive (WFD) 2000/60 /EC and CEN standards. In the near future, metabarcoding can complement and/or replace the traditional ecological assessments based on the morpho-taxonomy methodology approach needing taxonomic expertise and been subjected to scientific bias. Additionally, the project aims at developing a complementary bioinformatics tool for the biotechnological interpretation of the results. Such product will allow the prompt response to the environmental needs, the early assessment of environmental quality and early treatment response. The study will be developed and validated along a longitudinal gradient in the south part of Europe (Portugal, Spain, Cyprus), including four different biogeographical regions (Macaronesia, Atlantic, Alpine and Mediterranean). The method will cover all steps, from sampling and DNA extraction of diatom assemblages and amplification of DNA barcodes using universal primers for diatoms. The amplified products will be sequenced using Illumina MiSeq. Then, existing bioinformatic pipelines will be adjusted to quality-filter the high number of sequences from the samples and identify them by comparison with reference databases (Diat.Barcode, BOLD, GenBank). Enhancing these databases with diatom species prevalent in the different biogeographical regions assayed will be essential as existing databases are biased to more northerly regions and do not take into consideration harsh, extreme climatic conditions which are prominent in the Mediterranean and Macaronesia regions (Fig. 1). The project focuses on the rbcL gene and will used 18S gene only as an alternative or complementary tool if any problematic taxa appear. CEN, 2018. CEN/TR 17245: Water quality – Technical report for the routine sampling of benthic diatoms from rivers and lakes adapted for metabarcoding analyses. CEN/TC 230/WG 23 – Aquat. Macrophytes Algae 1–8. https://doi.org/CEN/TR 17245:2018Foster, D., Wood, A., Griffiths, M., 2000. THE WATER FRAMEWORK DIRECTIVE (2000/60/EC) – AN INTRODUCTION Dave Foster – Policy Advisor (Europe), Aram Wood EP Scientist (Water), Dr Martin Griffiths – Head of Water Quality, Environment Agency, Head Office, Rio House, Waterside Drive, Aztec West, Almon 7–9.Kelly et al. (2018). A DNA based diatom metabarcoding approach for Water Framework Directive classification of rivers. Environment Agency.Vasselon et al. (2017). Assessing ecological status with diatoms DNA metabarcoding: Scaling-up on a WFD monitoring network (Mayotte island, France). Ecological Indicators. 82:1-12Vasselon, V., Rimet, F., Domaizon, I., Monnier, O., Reyjol, Y., Bouchez, A., 2019. Assessing pollution of aquatic environments with diatoms’ DNA metabarcoding: Experience and developments from France Water Framework Directive networks. Metabarcoding and Metagenomics 3, 101–115. https://doi.org/10.3897/mbmg.3.39646 HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Sediment eDNA Metabarcoding for monitoring impacts from offshore oil

    • Abstract: ARPHA Conference s 4: e65034
      DOI : 10.3897/aca.4.e65034
      Authors : Anders Lanzén, Jon Thomassen Hestetun, Andrea Bagi, Thomas Dahlgren : Routine biological monitoring of the areas affected by offshore oil drilling and extraction is critical for ensuring proper environmental management. In addition to sufficient knowledge of the ecosystem affected, formalised e.g. as biotic indices of indicator species, adequate temporal and spatial resolution is also required, to provide accurate information. As already demonstrated in several types of environments, environmental DNA (eDNA) metabarcoding offers an attractive alternative to current morphology-based assessments, including for impacts of oil extraction or spills.We have recently studied the influence of different experimental strategies on the accuracy of marine sediment metabarcoding, suggesting minimum criteria for technical and spatial replication (Hestetun et al. 2020). Here, we aim to evaluate the predictive power of this strategy, through agreement with assessments based on physicochemical measurements and current bioindicators. To this end, we targeted the metazoan, and total eukaryotic benthic communities, using COI and 18S V1-V2 markers, respectively. Sampled sites ranged from high to low impacts. The studied areas were located near active production installations and reference sites on the Norwegian continental shelf, in the North Sea and Barents Sea. As a proxy for accumulated impact, we developed a simple physicochemical pressure index (PI) based on total hydrocarbons, PAH16, barium and copper, all of which agreed well with impact reported from recent routine monitoring. Alpha diversity of both molecular datasets, as well as of morphology data, correlated strongly with this PI. However, the correlation was stronger yet with the macroinvertebrate-based Norwegian Sensitivity Index (NSI) derived from COI metabarcoding data, which also agreed well with NSI values derived from morphology-based monitoring. We also identified a set of bioindicator taxa from each of the two metabarcoding datasets, used to develop two novel metabarcoding-based biotic indeces. Using cross-validation, we demonstrated that predictions based on these indeces agreed well with PI. Predictive performance was better, and similar to NSI, for the COI-based index, but also high for the 18S-based version. In conclusion, this study demonstrates how de novo biotic indices can be developed, that perform comparably to existing biotic indices. We are confident that, using a larger set of samples, performance can be improved beyond that of current monitoring practices. Thanks to the reduced costs of eDNA analysis in comparison to morphological identifiation, this would also pave the way for improved spatial and temporal resolution employed in routine environmental monitoring. In doing so, it can also provide valuable raw data for improving our understanding of benthic ecology, biodiversity and its sensitivity to anthropogenic pressures. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • The use of eDNA and DNA metabarcoding in monitoring the ecological
           condition of Norwegian lakes

    • Abstract: ARPHA Conference s 4: e65309
      DOI : 10.3897/aca.4.e65309
      Authors : Sara Atienza Casas, Markus Majaneva, Thomas Jensen, Marie Davey, Frode Fossøy, Knut Bækkelie, Karl Øystein Gjelland, Ann Schartau, Torbjørn Ekrem : Biodiversity assessments using molecular identification of organisms through high-throughput sequencing techniques have been a game changer in ecosystem monitoring, providing increased taxonomic resolution, more objective identifications, potential cost reductions, and reduced processing times. The use of DNA metabarcoding of bulk samples and environmental DNA (eDNA) is now widespread but is not yet universally implemented in national monitoring programs. While bulk sample metabarcoding involves extraction of DNA from organisms in a sample, eDNA analysis involves obtaining DNA directly from environmental samples, which can include microorganisms, meiofauna-size taxa and macrofauna traces such as larval stages, skin and hair cells, gametes, faeces and free DNA bound to particles.In Norway, freshwater biomonitoring in compliance with the EU Water Framework Directive (WFD) is conducted on several administrative levels, including national monitoring programs for running water, small and large lakes. These programs typically focus on a fraction of the actual biodiversity present in the monitored habitats (Weigand 2019). DNA metabarcoding of both bulk samples and eDNA samples are relevant tools for future freshwater biomonitoring in Norway.The aim of this PhD project is to develop assessment protocols based on DNA-metabarcoding and eDNA of benthic invertebrates, microcrustaceans and fish that can be used as standard biomonitoring tools to assess the ecological condition of lakes. The main topics addressed will be:- Development of protocols throughout the eDNA-metabarcoding workflow (i.e. sampling, filtration, preservation, extraction, amplification and sequencing) suitable to execute biodiversity assessments and determine the ecological status of lakes.- Comparison of the results obtained using molecular tools and traditional morphology-based approaches in order to assess the feasibility of such techniques to be incorporated as standard biomonitoring tools, such as the ones implemented under the provisions of the WFD.- Evaluate the effect of improved taxonomic resolution from molecular techniques on determining the ecological status of lakes, both by broadening the number of taxa analyzed and by identifying more taxa to species level.- Assess the feasibility of using eDNA extracted from water samples, taken at different depths and fish densities, to measure fish abundance/biomass as a proxy to calculate the ecological quality indices regulated in the WFD.- Analyze the coverage and resolution provided by reference libraries for certain taxa, such as crustacea, in order to assess the reliability and precision of taxonomic assignments. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • DNA-based biomonitoring in the tropics: Detection and control of
           Batrachochytrium dendrobatidis in Ecuadorian ecosystem

    • Abstract: ARPHA Conference s 4: e65304
      DOI : 10.3897/aca.4.e65304
      Authors : Lenin Riascos-Flores, Andrea Carrera, Leopoldo Naranjo, Jomira Yanez, Peter Goethals, Julio Bonilla, Jorge Celi, Christine Van der heyden, Mauricio Ortega : Batrachochytrium dendrobatidis (Bd) is a fungus that parasites vertebrates, and is associated with population declines worldwide in endemic amphibian species. As such, it is one of several invasive species which pose a serious threat to a variety of vertebrate hosts, in casu: amphibians. Detection of such invasive species is generally based on DNA-based methods where, for instance, swabs or tissue samples of candidate hosts are analysed for their presence. Any management strategy of these invasive species would greatly benefit from sensitive and rapid detection methods which can be applied at a large scale. The analysis of eDNA from the habitat of candidate host organisms may hold significant potential for this purpose. In this study, we compare the ability of eDNA from habitat samples with that of swab and/or tissue samples of candidate hosts to detect the presence of Bd in Ecuador.We collected individuals from the amphibians: Pristimantis (Anura: Craugastoridae), Rhinella (Anura: Bufonidae), Gastroteca (Anura: Hemiphractidae), from the endangered toad species of the genus Atelopus (Anura: Bufonidae) as well as water samples from different water bodies in Andean and coastal Ecuadorian areas. Samples were processed using a portable field molecular laboratory. Commercial primers for the internal transcribed spacer (ITS), in combination with a new set of primers designed from Bd sequences from tropical countries, were used. Positive PCR results from both types of samples were obtained within eight hours after sampling.Prevalence of BD was detected in eDNA, swab and tissue samples in four of the six ecosystems monitored -14 out of 26 water samples and 27 out of 43 amphibian of in total 12 species- including three endangered toad species (Atelopus balios, A. nanay, and the rediscovered A. bomolochos). Our results highlight the potential of eDNA-based monitoring to assess the presence and prevalence of Bd in Ecuadorian aquatic ecosystems, in accordance with the National Action Plan for the Conservation of Ecuadorian Amphibians. Furthermore, our field lab approach leads to reliable and fast results for the monitoring of invasive species in a tropical context of a pandemic. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • An eDNA-based method for monitoring a salmonid infectious disease:
           Development and application

    • Abstract: ARPHA Conference s 4: e64797
      DOI : 10.3897/aca.4.e64797
      Authors : Eloïse Duval, Simon Blanchet, Erwan Quéméré, Lisa Jacquin, Charlotte Veyssière, Armand Lautraite, Laurent Garmendia, Allan Yotte, Nathalie Parthuisot, Jessica Côte, Géraldine Loot : In the current context of global change, freshwater species are increasingly exposed to emerging infectious diseases (Okamura and Feist 2011). As an example, the Proliferative Kidney Disease (PKD) has emerged in salmonid fish during the last two decades, both in Europe and North America, causing important losses in aquaculture and worrying declines of several wild salmonid populations (Sudhagar et al. 2019). It is caused by Tetracapsuloides bryosalmonae, a myxozoan parasite with a complex life cycle involving two hosts: salmonids (intermediate host) and bryozoans (primary host). As PKD development strongly depends upon water temperature and quality, it is expected that global change could lead to more outbreaks (Okamura et al. 2011). Current monitoring of fish parasite load and infection status relies on histological observation or T. bryosalmonae DNA amplification out of kidney samples, involving fish euthanasia, and thus relatively small sample sizes when inferring infection prevalence. As large-scale screening of this parasite infections are required to better understand PKD dynamics, we have developed a non-lethal method for T. bryosalmonae detection in fish host based on the biological fact that T. bryosalmonae spores can be excreted from infected fish into the water through urine (Hedrick et al. 2004). This novel approach based on the detection of T. bryosalmonae DNA in fish urine was developed on wild brown trout (Salmo trutta), a species known to be an intermediate host of T. bryosalmonae and for releasing infective spores (only towards bryozoan host) through urine (Okamura et al. 2011). Applying this method, we have been able to map wild brown trout infection prevalence across 50 sites at the foothill of French Pyrenees and to identify the main environmental drivers of this disease. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • The use of environmental DNA in monitoring aquatic biodiversity for
           conservation: a review of challenges and opportunities

    • Abstract: ARPHA Conference s 4: e65283
      DOI : 10.3897/aca.4.e65283
      Authors : PJ Stephenson : Evidence-based decision-making in conservation and natural resource management is often constrained by lack of robust biodiversity data. Technology offers opportunities for enhanced data collection, with satellite-based remote sensing increasingly complemented by Earth-based sensors such as camera traps, acoustic recording devices and drones. In aquatic as well as terrestrial systems, environmental DNA is increasingly promoted as a tool to monitor species diversity and community composition. But if conservationists and natural resource managers are to know when to use eDNA, they need to understand its relative advantages and disadvantages, and when it can be used with or instead of other tools. In this paper, I expand on two recent publications (Stephenson 2020; Stephenson et al. 2020) to review lessons learned from the application of eDNA, especially metabarcoding, to the monitoring of aquatic biodiversity for conservation and to identify factors affecting its relevance and applicability.Over the past decade there have been many advances in technological solutions for biodiversity monitoring. eDNA and various remote sensing tools offer opportunities to create the enabling conditions for enhanced biodiversity monitoring, and are becoming cheaper and easier to use for scientists, public and private sector resource managers, and citizen scientists. Nonetheless, a number of challenges need to be addressed to, for example, improve the standardisation of tool use and to enhance capacity for the use, storage, sharing and analysis of huge volumes of data, especially in high-biodiversity countries. More studies comparing the relative efficiency and cost-effectiveness of different tools with different species in different habitats would help managers choose the right tools for their needs and capacity and better integrate them into monitoring schemes.eDNA is becoming the go-to option for the monitoring of aquatic species diversity and community composition and has also proven successful in some terrestrial settings. eDNA is especially useful for monitoring species that are in low densities or difficult to observe with traditional observer-based methods; indeed, several studies show eDNA metabarcoding techniques have a much better detection probability overall for taxa such as amphibians and fish. In some cases, eDNA has been shown to complement other tools when used together, by either increasing animal detection probabilities or increasing the number of indicators that can be measured at one site. This suggests that, in future, more effort should be made to test the effectiveness of integrating eDNA with one or more other tools to enhance the efficiency and effectiveness of measuring indicators and to increase the diversity of species detected. For example, eDNA could be combined with camera traps for monitoring vertebrates visiting waterholes. Testing multiple tools would also provide better opportunity to quantify when and how traditional observer-based methods can complement the technological solutions and when they are more cost-effective. However, it is noteworthy that, in general, the taxa for which data are most lacking, such as invertebrates, plants and fungi, are still those less easily monitored by eDNA and other new technologies. This suggests a focus only on technological solutions for biodiversity monitoring may perpetuate existing taxonomic data biases.I conclude by discussing the international policy context and the relevance of eDNA for monitoring global biodiversity indicators. Several opportunities exist to integrate eDNA into monitoring programmes to measure government, business and civil society contributions towards delivery of the post-2020 global biodiversity framework and the Sustainable Development Goals. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • OTU picking on large datasets: comparing methods on a diversity of

    • Abstract: ARPHA Conference s 4: e65027
      DOI : 10.3897/aca.4.e65027
      Authors : Jean-Marc Frigerio, Ester Eckert, Emre Keskin, Frédéric Mahé, Michael Monaghan, Matteo Montagna, Franck Salin, Paul Schmidt Yáñez, Douglas Yu, Diego Fontaneto, Alain Franc : De novo OTU picking from large metabarcoding read datasets is at the same time a current and a complex task, and several methods coexist to perform it. We present here the outcome of a collective project developed within Working Group on « Data Analysis and Storage » in DNAqua.net. Our aim has been to organize a thorough comparison of OTU composition according to some selected methods called by the wrappers, in a diversity of situations. This has been done by disposing of a set of different datasets, and a set of different methods, applying each method on each dataset, and comparing the results. We have deliberately chosen to work with cleaned datasets only, and not to include cleaning in the process.We have worked with a set of about 60 different datasets, some environmental, some as mock communities, produced by six teams, in different countries (D, F, I, T, UK), each with specific markers for different organisms. All datasets have been cleaned beforehand by the team proposing it. We have installed four different tools for building OTUs by unsupervised clustering : Swarm (Mahé et al. 2015), Vsearch (Rognes et al. 2016) with the same receipe for all datasets, usearch (Edgar 2010) with a unique command, the same for all datasets, and yapotu, which computes pairwise Smith-Waterman distances between all reads of a given dataset, and then clusters them with graph based techniques. Yapotu approach is expected to be the most accurate one, as there are no heuristics in the calculations.We have harmonized common input/output format for the four methods, to make comparisons. Here is a summary of the indicators selected for comparing results.We have first computed basic indicators per sample and method, like the number of OTU, the number of singletons, the number of OTUs with ten reads or more (after dereplication), and the fraction of reads that have been allocated to an OTU. The four methods displayed a great variety of counts, with highest number of OTUs and singletons for Swarm, then slighltly equivalent figures (but a smaller number of singletons) for yapotu, and significantly smaller counts for Vsearch and Usearch. However, the counts for the number of OTUs with 10 reads or more are much more convergent between the four methods.We have then compared rank-size curves, which have been computed for all pairs (sample by method). Here again, yapotu and swarm results are very similar, whereas Vsearch and Usearch sometimes are close to the former pattern, sometimes very different (I attach a figure')We then have computed 10 different diversity indices, like OTU richness, Shannon, Chao, eveness. Here again, results provided by Swarm and Yapotu are very similar, with very strong correlations between indices over all samples by method, whereas the correlations with Vsearch and Usearch is very poor.Finally, we have computed all contingency tables (in a sparse format) between all pairs of methods (hence, 6 pairs) for all samples, which accurately describe whether OTUs composition are similar or dissimilar between methods. We have observed that swarm OTUs are systematically nested within yapotu OTUs, and most often, there is a one to one correspondence between a Swarm and a Yapotu OTU.As a conclusion, we show that- Swarm and yapotu yield very similar results including for fine details, the only diffrence being a larger number of singletons provided by Swarm ;- This shows that Swarm OTUs are very close to OTUs built by single linkage Clustering on Smith-Waterman pairwise distances, and consolidates these approaches.- Very often, both Vsearch and Usearch diverge from those convergent results, but not always, and it is not easy to understand when and why. Some further investigations are needed therefore.All datasets will be publicly available for further benchmarking of a wider set of methods and datasets. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Implementation and legal issues of DNA-based monitoring

    • Abstract: ARPHA Conference s 4: e65277
      DOI : 10.3897/aca.4.e65277
      Authors : Pieter Boets, Daniel Hering, Patricia Mergen : DNA-based methods are at the edge of being implemented into routine monitoring systems. WG5 aimed to develop implementation options for DNA-based methods under a range of environmental directives and legal frameworks, in particular the Water Framework Directive (WFD), the EU Marine Strategy Framework Directive, the UN SDGs, the Global Biodiversity Assessment under the IPBES, the CBD Nagoya Protocol on Access and Benefit Sharing, the digital sequence information on genetic resources (DSI), the Biodiversity Indicator Partnership, and the Essential Biodiversity Variables. It further aimed at starting the standardisation process for DNA-based methods.In the talk, we will give an overview of all WG5 activities, with a focus on the options to use DNA-based methods for the implementation of the WFD. Overall, suitability of DNA-based identification is particularly high for fish, as eDNA is a well-suited sampling approach which can replace expensive and potentially harmful methods. For invertebrates and phytobenthos, the main challenges include the modification of indices and completing barcode libraries. For phytoplankton, the barcode libraries are even more problematic, due to the high taxonomic diversity in plankton samples. If current assessment concepts are kept, DNA-based identification is least appropriate for macrophytes (rivers, lakes) and angiosperms/macroalgae (transitional and coastal waters), which are surveyed rather than sampled. We discuss the challenges and opportunities of implementing DNA-based identification into standard ecological assessment, in particular considering any adaptations to existing legislation that may be required to facilitate the transition to using molecular data. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • A Feasibility Study on a Monitoring for Seagrass beds Using Environmental

    • Abstract: ARPHA Conference s 4: e65262
      DOI : 10.3897/aca.4.e65262
      Authors : Maiko Akatsuka : In order to protect marine resources listed in the SDGs, it is important to conduct surveys to understand the current status and transition of aquatic species such as fish and seagrass. However, the area and frequency of surveys are often limited due to the lack of manpower and cost. Environmental DNA (eDNA) analysis is a method to obtain information of aquatic species in the sea or rivers. The aquatic species are identified by analyzing DNA contained in the sampled water. The eDNA analysis can be utilized as a new and efficient method for investigating aquatic species.The author is developing a monitoring method for seagrass beds using eDNA.Seagrasses are sessile organisms and don’t move like a fish, the areas where eDNA is generated are fixed. Therefore, eDNA path in a seagrass beds area can be predicted by flow prediction simulation. The monitoring carries out in the path of eDNA released from seagrass.When there is a correlation between the amount of DNA and the amount of biomass, it is possible to gain knowledge of the amount of biomass from sampling water in the path of eDNA. For example, the changes in the amount of biomass due to growth and withering of seagrass beds can be studied by observing the changes in the amount of DNA. The purpose of this study is to examine the feasibility of the monitoring method using experiments, field surveys and numerical simulations for eDNA.The water tank experiments for 15 months, suggested that the amount of seagrass eDNA is related to the seasonal changes in the biomass of seagrass, which has been grown in the water tank. In the conducted four field surveys, the amount of eDNA tended to be high during times of spring with high amounts of seagrasses. In addition, the decomposition process of eDNA were examined by laboratory experiments using sea water. As a result, seagrass DNA contained in the sampling sea water was degraded and undetected within around 5 days. This result suggests that information obtained from the sampling water reflects biological information within a few days. These results suggest the possibility to estimate the changes in biomass by using eDNA in the sea.And, in the field surveys, it was shown that the amount of eDNA is small, so we have recognized that increasing the amount of sampling water and improvement of the DNA analysis method is an issue.This monitoring method is feasible when the collected eDNA is related to a specific seagrass bed. The feasibility of the method was considered using numerical simulation.In the numerical simulation, a particle tracking method using 10 types of simple bay shapes was used to trace the path of eDNA regarded as a particle.When the eDNA started from multiple positions, it was searched for position where the point where DNA starts and arrives is uniquely determined.As a result, at some observation points, the particles that departed from a specific position was observed without being mixed with particles departing from other starting positions, and this was true for all bay types. In addition, the same tendency could be obtained in calculations using the bathymetry of Ago bay, in Mie prefecture.These suggests that it may be possible to monitor seagrass beds by performing fixed-point observations according to the seagrass beds distribution and bay flow.Since few cases in the sea were conducted, the authors intend to continue the survey for the upcoming years and continue the study under various conditions including numerical analysis. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • A roadmap for integrating eDNA in Australian marine park monitoring

    • Abstract: ARPHA Conference s 4: e65257
      DOI : 10.3897/aca.4.e65257
      Authors : Maarten De Brauwer : Australia has one of the world’s largest marine park estates. At 3.3 million km2, it spans an area three times larger than Germany, France, and the UK combined. Managing and monitoring such a vast and often remote area is logistically challenging and expensive. Current monitoring of Australian parks is decentralised and depends on traditional survey methods. As a result, real-time data on the state of Australia’s marine parks is incomplete, hampering effective management. Environmental DNA has been suggested as a potential solution to some of these challenges, but practical large-scale applications remain largely lacking in Australia. To overcome this, we are developing a roadmap towards integrating eDNA methods in marine park monitoring. We present an overview of the current state of marine monitoring in Australia marine, identify the aspects of bio-monitoring that eDNA can best contribute to, and suggest pathways towards best practice use of eDNA for resource managers in Australia and globally. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • DNA barcodes for UK freshwater arthropod species: coverage, quality and

    • Abstract: ARPHA Conference s 4: e65255
      DOI : 10.3897/aca.4.e65255
      Authors : Liz Davidson : DNA-based identification methods have been shown to have high detection capability and reduced costs compared to traditional methods and can also enable the detection of species that might be missed using traditional methods (e.g. rare species, cryptic species, larval stages). The success of DNA-based identification is dependent on the ‘DNA barcodes’ of target species being present in a barcode reference database. In order to use DNA-based identification methods to assess and monitor UK freshwater arthropods for biodiversity and ecological quality assessments, it is vital that comprehensive reference databases are available. Incomplete reference databases result in many sequences derived from metabarcoding not being assigned to species.Two current projects aim to create collections of high-quality sequences from expertly identified specimens of UK species. The Darwin Tree of Life project aims to sequence the genomes of all the eukaryotic species in Britain and Ireland and FreshBase aims to create a genomic reference collection for UK freshwater invertebrates. The Barcode of Life Data System (BOLD) is one of the main reference databases for animal barcodes. Prioritising the sequencing of UK freshwater arthropod species that are not yet represented in BOLD, would enable more complete identification of UK freshwater biodiversity using metabarcoding and would enable the development of primers to target specific arthropod groups or species.We analysed the coverage of UK freshwater arthropod species in BOLD. Our analyses show that coverage varies between taxonomic groups and large proportions of sequences in some orders are only represented by privately stored sequences in BOLD. Analyses of intra- and inter-specific variation in sequences stored in BOLD show that misidentifications or errors can reduce the barcode gap in some species which could cause difficulties in accurately identifying sequences derived from metabarcoding. Representation in BOLD by specimens from the UK is extremely low and analyses show that high geographic variation in sequences in some species could be important for accurate DNA-based identification of UK species. Our results have implications for prioritising the sequencing of UK freshwater arthropods and for the quality control of stored sequences in order to reduce the occurrence of misidentifications and errors that could impact the accuracy of DNA-based identification. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • New insights into Danube’s macroinvertebrate communities from DNA
           metabarcoding as part of the Joint Danube Survey 4 (JDS4)

    • Abstract: ARPHA Conference s 4: e65206
      DOI : 10.3897/aca.4.e65206
      Authors : Arne Beermann, Dominik Buchner, Florian Leese, Till-Hendrik Macher, Miroslav Ocadlik, Alexander Weigand : The Joint Danube Survey (JDS) is a multinational effort in monitoring Danube’s water quality, including its major tributaries. The Danube river stretches over a distance of 2,800 km and flows through or borders 10 different countries to which it is of utter importance as a source of potable water and hydrodynamic power. The JDS is conducted every 6 years and provides a unique opportunity to collect comprehensive data on both abiotic parameters and organisms and to raise awareness of the importance of water as a natural resource. As part of JDS and as a biological quality element in many monitoring programs worldwide, macroinvertebrates are monitored as indicators for various environmental conditions. However, due to their diverse taxonomic composition, associated difficulties with their morphology-based identification as well as their sheer abundance, macroinvertebrates are often analysed with a low taxonomic resolution (i.e., above species level). As an alternative, DNA metabarcoding offers a promising approach to capture this species diversity more accurately.Here, we used DNA metabarcoding to investigate the macrozoobenthic diversity of 46 sites from the latest JDS sampling campaign in 2019. To analyse macroinvertebrate diversity, bulk samples were taken by kick-net sampling and analysed using two different approaches, analysing the bulk sample fixative and analysing homogenised organisms from complete bulk samples. DNA metabarcoding of the sample fixative revealed 1,146 Operational Taxonomic Units (OTUs) and 231 species compared to 833 OTUs and 333 species from homogenised sample analysis. While more dipterans, in particular Chironomidae, were detected in fixative (136 species) than homogenised bulk (90 species) analyses, the latter picked up more Trichoptera (19 vs. 2), Amphipoda (10 vs. 4) and Bivalvia species (13 vs. 5). Even though these results of a DNA-based assessment deliver new insights into species richness and composition of Danube’s macroinvertebrate communities from the Danube source to its delta already, it is evident that the majority of OTUs was not assigned to species. While filling this lack of reference sequences poses a major challenge, the JDS consortium also offers a unique opportunity to complement reference databases in a multinational effort towards a more comprehensive Danube assessment and monitoring. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Morphotaxonomy- and metabarcoding-based ecological assessment of Cyprus
           streams’ diatom communities and correlation with environmental and
           anthropogenic influences

    • Abstract: ARPHA Conference s 4: e64962
      DOI : 10.3897/aca.4.e64962
      Authors : Panayiota Pissaridou, Agnès Bouchez, Marlen Vasquez Hadjilyra, Valentin Vasselon, Andreas Christou, Teofana Chonova, Katerina Drakou, Frederic Rimet, Marco Cantonati, Gerald Dörflinger, Iakovos Tziortzis : In freshwater ecosystems, periphytic biofilms include diatom assemblages that depend on environmental conditions (e.g., nutrient concentrations, salinity, temperature etc.). These assemblages respond rapidly to environmental changes, which makes diatoms valuable bioindicators. For this reason, they are currently used in freshwater biomonitoring programs (e.g., EU Water Framework Directive - WFD) (Foster et al., 2000). To date, diatom taxonomic identification is based on morphological criteria, which requires high taxonomic expertise to identify them to the species level needed for biomonitoring. Having this in mind, new strategies have been examined for the development of high-throughput, non-biased identification approaches. Human activities are the leading cause of environmental impairments and appropriate biomonitoring of ecosystems is needed to effectively assess the impact of their activities. In the last ten years, DNA metabarcoding combined with next-generation sequencing and bioinformatics, have been proposed as a complementary approach to morphological identification. In the past ten years, DNA metabarcoding coupled with next-generation sequencing and bioinformatics represents a complementary approach for diatom biomonitoring (Vasselon et al., 2019). In this study, this approach was used for the first time in Cyprus considering the association of environmental and anthropogenic pressures to diatom assemblages using the rbcL 312 bp barcode, next-generation sequencing (MiSeq Illumina), and bioinformatic evaluation (Mothur Software). Statistical analysis was then applied to identify the environmental (i.e., river types, geo-morphological) and anthropogenic (i.e., physical, chemical, human land-use pressures) variables' role in the observed diatom diversity. The Indice de Polluosensibilité Spécifique (IPS) index was used as it was shown to better respond to pressures that affect water quality in Cyprus rivers (WDD, 2014). Results indicate differences in diatom assemblages between intermittent and perennial rivers. Achnanthidium minutissimum was more abundant in intermittent rivers; whereas Amphora pediculus and Planothidium victorii (P. caputium) in perennial ones. Furthermore, we could demonstrate the correlation between nutrients (e.g., nitrogen, phosphorus), characteristics of the individual sampling sites (e.g., elevation), and land use activities on the observed differences in diatom diversity (Pissaridou, 2021). Additionally, results were compared to the morphotaxonomy-based approach which was conducted microscopically. Results show a positive correlation between morphological and molecular IPS scores. Points deviating from the norm are influenced by the limitations of both techniques. Fistulifera saprophila had a key role in this observation, as it negatively influences IPS scores. All in all, we conclude that DNA metabarcoding complements the morphological methodology for the ecological quality assessment of freshwaters in Cyprus. Multi-stressors and anthropogenic pressures have a significant statistical relationship to the observed diatom diversity and play a pivotal role in determining Cyprus' rivers' ecological status (Fig. 1).Foster, D., Wood, A., Griffiths, M., 2000. The Water Framework Directive (2000/60/EC) – An introduction Dave Foster – Policy Advisor (Europe), Aram Wood EP Scientist (Water), Dr Martin Griffiths – Head of Water Quality, Environment Agency, Head Office, Rio House, Waterside Drive, Aztec West, Almon 7–9.Pissaridou, P., Vasselon V., Christou A., Chonova T., Lacroix S., Papatheodoulou A., Drakou K., Tziortzis I., Dörflinger G., Rimet F., Bouchez A. and Vasquez MI. 2021 Deciphering Cyprus’ diatom diversity and the effects of environmental and anthropogenic influences for ecological assessment of rivers using DNA metabarcoding.Chemosphere (In Press)Vasselon, V., Frédéric, R., Isabelle, D., Olivier, M., Yorick, R., Agnès, B., 2019. Assessing pollution of aquatic environments with diatoms’ DNA metabarcoding: Experience and developments from France Water Framework Directive networks. Metabarcoding and Metagenomics 3, 101–115. https://doi.org/10.3897/mbmg.3.39646WDD, 2014. Review and update of article 5 of Directive 2000/60/EC (Water reservoirs) & Classification of water status (Rivers, natural lakes and water reservoirs), That will establish baseline information and data for the 2nd cyprus river basin management plan. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • There's always a better way: The application of eDNA to effectively assess

    • Abstract: ARPHA Conference s 4: e65156
      DOI : 10.3897/aca.4.e65156
      Authors : Prabir Roy, Mary Thiess : Our ecosystem monitoring methodologies focus on data collection for reporting purposes that may not serve to identify the systematic causes of ecological change. Managers need precise and timely information at appropriate scales to build ecosystem resilience.Traditional species detection methodologies offer little information when species abundance are low, especially in large water ecosystems such as the Great Lakes. Species not found during monitoring doesn’t necessarily mean that species are absent. Moreover, even if a change in the ecosystem is detected, it is often not possible to determine its cause at a spatiotemporal scale or a trophic cascade level. As a result, we often find ourselves being reactive in our mitigation measures. Before irreversible change occurs, we must be guided by a better understanding of the actual ecological landscape which Environmental DNA (eDNA) may help provide.eDNA is a potential tool to effectively overcome traditional species survey limitations currently in use at many Parks Canada sites. As various organisms interact with the environment, DNA is expelled and accumulates in their surroundings. Such samples can be analyzed by high-throughput DNA sequencing methods for rapid measurement and monitoring of biodiversity. Access to this genetic information makes a critical contribution to the understanding of population size, species distribution, and population dynamics for species not well documented. Despite the increasing use of eDNA in conservation practice, it requires further methodological improvement for greater influence on management decisions. The tool requires standardized protocols based on site-specific covariates and objectives.We’re working to tackle the challenge with 2 objectives: (1) to combine traditional biomonitoring knowledge and metagenomics to further develop eDNA as a reliable sampling tool for Parks Canada and (2) to support site-specific monitoring objectives for species-at-risk, invasive species, aquatic species inventories, and/or culturally significant species. The overall goal is to increase our capacity to make more informed, timely, regionally-coordinated conservation decisions through the rapid and sensitive species detection methods offered by eDNA. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Functional traits link anthropogenic impact and disturbance regimes
           driving ecosystem function in a floodplain wetland complex

    • Abstract: ARPHA Conference s 4: e65144
      DOI : 10.3897/aca.4.e65144
      Authors : Natalie Rideout, Zacchaeus Compson, Wendy Monk, Mehrdad Hajibabaei, Teresita Porter, Michael Wright, Donald Baird : Floodplains are disturbance-driven ecosystems with high spatial and temporal habitat diversity, making them both highly productive and hosts to high biodiversity. The unpredictable timing of flood and drought years creates a mosaic of habitat patches at different stages of succession, while water level fluctuation directly influences macrophyte community dynamics, and thus habitat structure. This habitat complexity and diversity of disturbance regimes makes floodplains an ideal ecosystem in which to examine the links between biodiversity, traits and ecosystem function. With up to 90% of floodplains in North America and Europe altered to the point of functional extinction, it is particularly imperative to study and conserve those that remain intact, such as the Lower Saint John River and its associated floodplain, including the Grand Lake Meadows and Portobello Creek wetland complex. Despite the rise in trait-based science, taxonomic resolution has imposed limitations, especially in wetland and floodplain ecosystems where communities are vastly understudied compared to their riverine counterparts. Compared to traditional biomonitoring, DNA-based biomonitoring from high-throughput genomics sequencing methods is powerful in that it can reliably characterize community composition in unprecedented detail, allowing us to assess how disturbance and environmental filters interact with invertebrate traits and ecosystem function. Using structural equation analysis, we take a whole ecosystem approach to examine ecosystem health across a floodplain disturbance gradient. We focus chiefly on how anthropogenic alteration within watersheds affects downstream floodplain wetlands, how the resulting patch diversity shapes communities and, finally, how those communities influence ecosystem function through trait diversity metrics. We also examine and compare which traits are associated with crucial ecosystem gradients. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • The Fellowship of the Ring Test: DNAqua-Net WG2 initiative to compare
           diatom metabarcoding protocols used in routine freshwater biomonitoring
           for standardisation

    • Abstract: ARPHA Conference s 4: e65142
      DOI : 10.3897/aca.4.e65142
      Authors : Valentin Vasselon, Éva Ács, Salomé Almeida, Karl Andree, Laure Apothéloz-Perret-Gentil, Bonnie Bailet, Ana Baricevic, Kevin Beentjes, Juliane Bettig, Agnès Bouchez, Camilla Capelli, Cécile Chardon, Mónika Duleba, Tina Elersek, Clémence Genthon, Melanie Hurtz, Louis Jacas, Maria Kahlert, Martyn Kelly, Matthieu Lewis, Jan Macher, Federica Mauri, Marina Moletta-Denat, Andreia Mortágua, Jan Pawlowski, Javier Pérez Burillo, Martin Pfannkuchen, Erik Pilgrim, Panayiota Pissaridou, Jonathan Porter, Frederic Rimet, Karmen Stanic, Kálmán Tapolczai, Susanna Theroux, Rosa Trobajo, Berry van der Hoorn, Marlen Vasquez Hadjilyra, Kerry Walsh, David Wanless, Jonathan Warren, Jonas Zimmermann, Maša Zupančič : During the past decade genetic approaches have been developed to monitor biodiversity in aquatic ecosystems. These enable access to taxonomic and genetic information from biological communities using DNA from environmental samples (e.g. water, biofilm, soil) and methods based on high-throughput sequencing technologies, such as DNA metabarcoding. Within the context of the Water Framework Directive (WFD), such approaches could be applied to assess Biological Quality Elements (BQE). These are used as indicators of the ecological status of aquatic ecosystems as part of national monitoring programs of the european network of 110,000 surface water monitoring sites with 79.5% rivers and 11% lake sites (Charles et al. 2020). A high-throughput method has the potential to increase our spatio-temporal monitoring capacity and to accelerate the transfer of information to water managers with the aim to increase protection of aquatic ecosystems.Good progress has been made with developing DNA metabarcoding approaches for benthic diatom assemblages. Technological innovation and protocol optimization have allowed robust taxonomic (species) and genetic (OTU, ESV) information to be obtained from which diatom quality indices can be calculated to infer ecological status to rivers and lakes. Diatom DNA metabarcoding has been successfully applied for biomonitoring at the scale of national river monitoring networks in several countries around the world and can now be considered technically ready for routine application (e.g. Apothéloz-Perret-Gentil et al. 2017, Bailet et al. 2019, Mortágua et al. 2019, Vasselon et al. 2019, Kelly et al. 2020, Pérez-Burillo et al. 2020, Pissaridou et al. 2021). However, protocols and methods used by each laboratory still vary between and within countries, limiting their operational transferability and the ability to compare results. Thus, routine use of DNA metabarcoding for diatom biomonitoring requires standardization of all steps of the metabarcoding procedure, from the sampling to the final ecological status assessment in order to define good practices and standards. Following previous initiatives which resulted in a CEN technical report for biofilm sampling and preservation (CEN 2018), a set of experiments was initiated during the DNAqua-Net WG2 diatom workshop (Cyprus, 2019) to focus on DNA extraction and PCR amplification steps in order to evaluate: i) the transferability and reproducibility of a protocol between different laboratories; ii) the variability introduced by different protocols currently applied by the scientific community. 19 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using i) the same fixed protocol and ii) their own protocol. Experiments were performed by each participant on a set of standardized DNA and biofilm samples (river, lake, mock community). In order to specifically test the variability of DNA extraction and PCR amplification steps, all other steps of the metabarcoding process were fixed and the preparation of the Miseq sequencing was performed by only one laboratory. The variability within and between participants will be evaluated on DNA extracts quantity, taxonomic (genus, species) and genetic richness, community structure comparison and diatom quality index scores (IPS). We will also evaluate the variability introduced by different DNA extraction and PCR amplification protocols on diatom quality index scores and the final ecological status assessment. The results from this collaborative work will not serve to define “one protocol to rule them all”, but will provide valuable information to define guidelines and minimum requirements that should be considered when performing diatom metabarcoding for biomonitoring. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • The Wild Rivers Program: Development of a “Wild Rivers” Conservation
           Label to be used by River Management

    • Abstract: ARPHA Conference s 4: e65141
      DOI : 10.3897/aca.4.e65141
      Authors : Denis Caudron, Lucie Galland, Melanie Taquet, Valentin Vasselon : A wild river is a living river, which is at baseline, well-preserved, and which runs freely and is home to a rich biodiversity in its high quality waters and on its banks. In Europe there are very few rivers which could be considered “wild”, which function at a high ecological level, since wild rivers, in the true sense of the term, no longer exist.Based on the fact that these rivers remain threatened, and that the existing tools (technical, regulatory, and financial) are insufficient and not adapted to ensure their preservation over the long term, the Wild Rivers project was founded in 2007, through a meeting of environmental defenders, scientists, fishermen, managers of land and river natural resources, and elected officials, all of whom were anxious to save the last of the French rivers which were still preserved, with a human impact that would be compatible with the conservation of the ecosystem.In 2014 the “Wild Rivers Site” label was created in France, as a conservation tool for rivers, both voluntary and non-regulatory, which allows the support necessary to enable the territorial players to preserve their rivers in harmony with the activity in the surrounding valleys. It also identifies and highlights these unique watercourses.The Valserine in the Ain region was the first river to obtain the Wild Rivers Site label. Today 28 rivers in France are labelled “Wild Rivers Sites” and the 22 management structures of these rivers are members of the Wild Rivers Site Network.To obtain the label, a river must fulfill two sets of criteria1. The criteria grid: The watercourse must obtain a mark over 70/100. The grid is composed of 47 criteria evaluating the quality of the area, of which 12 are eliminatory, 8 are unrated, and 9 are under a bonus/penalty scheme2. The program of actions taken by local players: The local managers must put in place a system of governance built around actions to be taken over a period of years, shared among them, and ambitious, going beyond the regulatory objectives of the European Directive Framework. It allows for the restoration of penalty points and the establishment of innovative conservation activities.The Wild Rivers Sites are also an open air laboratory for the development and use of innovative methods in order to provide new information on aquatic environments, and to improve their management and conservation. Numerous steps have already been taken within the network, such as the Ecosystem Services Study (Costa and Hernandez 2019); on the study of the genetic makeup of the brown trout population. Recently, the use of genetic study using environmental DNA to complete biodiversity inventories has also been deployed to study benthic diatoms (DNA of Diatoms Project 2020-2022). This project seeks to use DNA metabarcoding to respond to a number of objectives: i) inventory of the species of diatoms and their community structure in these watercourses which are generally seldom studied; ii) complete ecological status studies; iii) develop new genetic metrics and taxonomies adapted to the conservation of wild river watercourses.It is in this spirit that the Wild Rivers program was developed, and has received numerous positive responses on the behalf of watercourse management in France. Thanks to this impetus, work has been conducted to extend this conservation label to water sources in other countries (Switzerland, Ireland, Spain), with the future plan of building a European network dedicated to the conservation of Wild Rivers. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Establishing the first DNA barcode reference library for freshwater
           decapod species (Crustacea: Decapoda) in Republic of North Macedonia

    • Abstract: ARPHA Conference s 4: e65138
      DOI : 10.3897/aca.4.e65138
      Authors : Valentina Slavevska Stamenkovic, Jelena Hinic, Michal Grabowski, Tomasz Mamos, Leona Lovrenčić, Mišel Jelić, Goran Klobučar, Ivana Maguire : The freshwater ecosystems in the Republic of North Macedonia are considered as biodiversity hotspot on the European level since they provide diverse habitats that correspond with the complex of ecological preferences that many species require. This specially applies to the freshwater crustaceans that, unfortunately, have never been in the focus of a continuous research. In R. North Macedonia, freshwater crustaceans usually inhabit ecosystems exposed to negative anthropogenic impact. Thus, some species may become extinct presenting an irreversible loss for the Macedonian natural heritage. Although DNA barcoding, as a highly effective tool for fast species detection, is already a routine protocol in many taxonomical studies all over the world, there is still no official national DNA barcoding initiative in Republic of North Macedonia. This study employs DNA barcoding based on the ca. 650-bp long standard fragment of the mt COI gene of Astacus astacus, Austropotamobius torrentium, Potamon fluviatile and Potamon ibericum previously identified based on morphological characters, collected from different parts in R. North Macedonia. The ability of the DNA barcoding to rapidly identify all species has been proven. Our research presents the first comprehensive study that employs DNA barcoding as a molecular tool in decapod taxonomy in Republic of North Macedonia, giving the first attempt to establish DNA barcode reference library for freshwater decapod species in the country. We hope that further application of this approach will lead to the construction of DNA barcode reference library for different aquatic biota in the country. Such a library will find purpose in effective and modern bioassessment protocols as well as in phylogenetic research detecting interpopulation genetic variability. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Application of eDNA method in the detection of Cordulegaster (Insecta:
           Odonata) species

    • Abstract: ARPHA Conference s 4: e65041
      DOI : 10.3897/aca.4.e65041
      Authors : Judit Fekete, Dominik Buchner, Florian Leese, Judit Padisák, Gábor Várbíró : The aim of this pilot study was to investigate the potential of eDNA techniques to detect the presence of the two dragonfly species Cordulegaster heros and Cordulegaster bidentata. Both species are classified as “near threatened” according to the IUCN Red List and are strictly protected in several countries. Monitoring these species with traditional sampling methods is often difficult, time-consuming and invasive. In this pilot study, we first collected tissue samples from C. heros and C. bidentata to sequence the traditional DNA-barcode gene fragment COI. We then collected further dragonfly COI sequences from BOLD to design species-specific primers. This, however, was impossible given the enormous variability of COI. Therefore, we refrained from species-specific eDNA assays and followed eDNA metabarcoding protocol using universal (BF2/BF2) and a newly designed dragonfly specific primer. For the evaluation of the method, we took water samples from places where Cordulegaster specimens are known to occur. After the extraction, we used two sequential PCR steps for obtaining the desired amplicon (two-step PCR) using universal primers in the first step, and group (dragonfly) specific primers or universal primers. Amplicons were sequenced on an Illumina MiSeq platform and then analysed the data with the JAMP pipeline. With the newly designed primers and we could effectively detect the targeted dragonfly species from tissue samples, and also from filtered environmental samples. The detection of the species with the traditional method is time consuming and involves the destruction of the specimens. In comparison, with the eDNA method we could easily detect these near threatherned odonates and other dragonfly species in a non-invasive way. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Revisiting global biogeography of freshwater diatoms: new insights from
           molecular data

    • Abstract: ARPHA Conference s 4: e65129
      DOI : 10.3897/aca.4.e65129
      Authors : Teofana Chonova, Frédéric Rimet, Agnès Bouchez, François Keck : The high-dispersal rates of microorganisms have driven to the expectation of their cosmopolitan geographic distribution. However, recent studies demonstrate that microorganisms instead show particular biogeography. Despite the existence of cosmopolitan species, geographically limited microbial groups have been found in aquatic and terrestrial environments worldwide.Diatoms are long time used model to study the biogeography of microorganisms. They are unicellular eukaryotic microalgae that contribute significantly to the aquatic primary production and have huge taxonomic diversity and marked species-specific ecological preferences. Several authors considered that diatoms have no limits in dispersion and are ubiquitously present. On the other hand, recent studies have shown that endemism exists for several genera, and species may have low dispersal capacity. However, all these studies are based on data obtained by microscopy and therefore suffer from the many well-identified biases associated with the optical identification of microorganisms at large scale.Metabarcoding technologies provide an access to taxonomic precision with a higher resolution compared to microscopy and open therefore the possibility of analyzing microbial diversity at genetic level. Recent bioinformatics tools allow reliable and standardized comparison of large datasets originating from distant geographic regions, overcoming issues related to biases in species identification.In this study we used metabarcoding data to revisit central questions in freshwater diatom biogeography. We assembled a large dataset of samples of benthic diatoms collected from rivers in seven different geographic regions. These regions cover the subpolar (Fennoscandia), temperate (France Mainland) and tropical (West Africa, French Guyana, New Caledonia, Tahiti island and Mayotte island) climate zones. The selected geographic regions can also be classified into four continental areas (Fennoscandia, France Mainland, West Africa, and French Guyana) and three islands (New Caledonia, Tahiti and Mayotte).We analyzed diatom alpha, beta and gamma diversity patterns in this dataset to address two main questions: 1) the presence of a latitudinal gradient in diatom diversity and 2) the cosmopolitanism of diatoms.Similarly to results previously reported by Soininen et al. 2016, our data showed a decrease in diatom richness with a decrease in latitude. However, testing the effect of land type (island vs. mainland) showed that this factor explains the actual variability of richness along the climatic gradient and the effect of latitude is not significant. Differences in community structure between regions and climate zones were significant. In multivariate analysis, tropical samples did not overlap with any of the other climate zones, suggesting the specificity of these communities. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Environmental DNA variability in lake sediment cores

    • Abstract: ARPHA Conference s 4: e65128
      DOI : 10.3897/aca.4.e65128
      Authors : John Pearman, Georgia Thomson-Laing, Jamie Howarth, Marcus Vandergoes, Lucy Thompson, Andrew Rees, Susie Wood : Lake sediments are natural archives that accumulate information about biological communities and their surrounding catchments. Paleolimnology has traditionally focussed on identifying fossilized organisms to reconstruct past environments. In the last decade, the application of molecular methodologies has increased in paleolimnological studies, but further studies investigating factors such as sample heterogeneity and DNA degradation are required. Here we investigated bacterial community heterogeneity (16S rRNA metabarcoding) within depth slices. Sediment cores were collected from three lakes with differing sediment compositions. Samples were collected from a variety of depths (1-cm width) which represent a period of time of approximately 1,200 years. Triplicate samples were collected from each slice and bacterial 16S rRNA metabarcoding was undertaken on each sample. Rarefaction curves showed that except for the deepest (oldest) slices, the combination of three replicate samples were insufficient to characterise the entire bacterial diversity. However, shared Amplicon Sequence Variants (ASVs) accounted for the majority of the reads in each slice (max. shared proportional read abundance 96%, 86%, 65% in the three lakes). Within slice similarity was higher than between slice similarity. No general trend was observed in variability among replicates with depth amongst the lakes. In one core. there was a higher community dissimilarity in older sediment, which may be due to laminae not being horizontal. These results highlight the fact that microbial communities can be differentiated with depth however it is critical to interpret these results in the context of the stratigraphic data of the core. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • To blend or not to blend' The role of morphological traits for the
           detection of marine macrobenthos in bulk DNA and eDNA from the ethanol

    • Abstract: ARPHA Conference s 4: e65127
      DOI : 10.3897/aca.4.e65127
      Authors : Sofie Derycke, Sara Maes, Laure Van den Bulcke, Joran Vanhollebeke, Jan Wittoeck, Hans Hillewaert, Bart Ampe, Annelies Haegeman, Kris Hostens, Annelies De Backer : The impact of methodological choices on the reliability and reproducibility of DNA metabarcoding need to be well understood to allow successful implementation in routine monitoring frameworks. For macrobenthos communities, the metabarcoding protocol focuses on a fragment of the mitochondrial COI gene and depending on the primer set used for amplification of COI, different taxa can be detected. To identify the primer set that allows the best diversity estimates for macrobenthos in the North Sea region, we sampled four distinct and well characterised communities and identified macrobenthos using traditional morpho-taxonomy before molecular processing. Of the five primer sets tested, the Leray primer set yielded the highest number of non-chimeric reads, detected the highest number of macrobenthos species and best recovered beta diversity patterns. Despite the availability of a nearly complete reference database, 19 out of the 59 morphological species were not picked up with DNA metabarcoding. Next to primer choice, the DNA source used in metabarcoding studies can affect whether or not a species is detected. DNA can be extracted from bulk specimens or from the ethanol preservative in which the macrobenthos sample was preserved. The latter DNA source would greatly speed up processing time of samples in the laboratory. We therefore compared species detection in bulk DNA and eDNA from the ethanol preservative from the four macrobenthos communities in the North Sea. Our results show that community composition differed significantly between bulk DNA and eDNA samples, but both sample types are able to differentiate the four macrobenthos communities from the North Sea. Of the 49 species that are detected in both sample types, 27 are also found in the morphological dataset. The 14 species that are exclusively detected in the ethanol preservative are mainly pelagic species. In view of the low read numbers allocated to these species (at most 153 reads) they most likely represent “contaminant” DNA molecules that are attached to the specimens or the organic debris. To better understand the different results between bulk DNA and eDNA from the ethanol preservative, we investigated the importance of four categorical traits in explaining the probability of detecting a species in the two sample types: body, larval stage (benthic or pelagic), longevity and body skeleton (chitin, CaCO3 or soft tissue). A generalized linear mixed effects model approach shows that the probability of detecting a species in the eDNA from the ethanol preservative is significantly lower than for bulk DNA for macrobenthos species having small to medium body size and for species having chitine or CaCO3 in their skeleton. In contrast, detection in the bulk DNA samples is not affected by the investigated traits. Although the ethanol preservative can be used to characterize beta diversity patterns, our results show that monitoring of macrobenthos species will be most robust when using bulk DNA as template for metabarcoding. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Strategy for Successful Integration of eDNA-based Methods in Aquatic

    • Abstract: ARPHA Conference s 4: e65125
      DOI : 10.3897/aca.4.e65125
      Authors : Philippe Blancher, Estelle Lefrançois, Frederic Rimet, Agnès Bouchez : Recent developments in the use of environmental DNA are opening up new horizons for the assessment of the quality of aquatic environments. These rapid and cost-effective methods, in very swift progress, will potentially offer the opportunity to identify all the taxa present in an environmental sample (water or biota) by the use of complementary markers. The produced inventories can then be used for the assessment of biodiversity and ecological quality. However, the inclusion of these new DNA-based methods in monitoring practices is not straightforward and requires harmonised actions in the coming years at national and international levels.In order to foresee and stimulate such a harmonised implementation, the European network DNAqua-Net (COST Action CA15219) brought together some of its members, experts of ECOSTAT and other environmental biomonitoring stakeholders from different European countries. Through workshops, bringing together 51 participants in 7 sub-groups in April 2020, an implementation roadmap was designed. The coordinated actions to be taken in the different countries, and the possible collaborations and steps to be taken at the EU level were identified.This presentation will give an overview of all discussions (Lefrançois et al. 2020) reflecting the diversity of situations in Europe, as well as common views. We will highlight important actions required for a successful implementation of DNA-based biomonitoring of aquatic ecosystems to the horizon of 2030. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • First steps towards a barcoding database for the Iberian cladocerans

    • Abstract: ARPHA Conference s 4: e65122
      DOI : 10.3897/aca.4.e65122
      Authors : Charo López-Blanco, Antonio García-Alix, Yi Wang, Laura S. Epp : Due to similarities in morphological features together with strong dispersal abilities, it was thought that some groups of zooplankton (e.g. rotifers, copepods, and cladocerans) have cosmopolitan distributions. In the particular case of cladocerans, recent molecular studies using DNA barcode regions have indicated a different picture, including the existence of multiple regional endemic species and geographical phylogroups; even at very small geographical scales. This has demostrated that cladocera species are less widely distributed than assumed. Morphological identifications of these animals require expertise and high taxonomic specialization. Even so, species identifications are hampered by the small size of the organisms (especially from the littoral zone) and by the sampling cost for obtaining rare species and both parthenogenetic and gamogenetic specimens. The use of molecular techniques can provide new tools to identify cryptic diversity, and by being added to taxonomical approaches, provide more precise data of biodiversity. However, the accuracy of species assignment in metabarcoding relies on the availability of a DNA reference library, which is challenging in areas with high endemicity rates such as the Iberian Peninsula. A preliminary compilation of the available molecular data for the cytochrome (COI) in public repositories (Barcode of Life Data Systems and NCBI GenBank) shows that the available sequences only cover ~60% of the Iberian freshwater cladocerans. The family Daphniidae is very well represented, while the family Chydoridae, which contained most of the Iberian endemism, is underrepresented. We have identified the gaps, and are now focusing on collecting the target organisms to fill the missing taxa of the Iberian library. A compendium of the sampling points, species recovered so far, pitfalls, and future strategy is presented here. The effective completion of a DNA database for cladocerans will have applications not only in biomonitoring programs but to develop DNA-based methods in paleolimnology. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Tracing the almost extinct mayfly Prosopistoma pennigerum (Müller, 1785)
           – an eDNA approach

    • Abstract: ARPHA Conference s 4: e65115
      DOI : 10.3897/aca.4.e65115
      Authors : Jan Martini, Florian Altermatt, Emil Birnstiel, Wolfram Graf, Vyacheslav Kuzovlev, Rebecca Oester, Tamara Schenekar, Martin Schletterer, Franziska Walther, Steven Weiss, Olivia Wilfling, Remo Wüthrich, Bernadette Schindelegger, Gabriel Singer, Simon Vitecek : The rare mayfly Prosopistoma pennigerum was once widely distributed across Europe and occurred virtually in every large river. Today, it holds fast against the ever-growing destruction of its habitat with a few relic populations remaining. Preliminary data and information on its congeners suggest that free-flowing rivers with near-natural hydrodynamics are its primary habitat. Rivers where the species currently occurs should be primary targets for large-scale landscape conservation and protection. According to the Water Framework Directive, achieving longitudinal and lateral connectivity is a priority target, and occurrence and viability of Prosopistoma pennigerum populations could be an indicator of restoration success.We developed and validated a targeted qPCR protocol to detect this mayfly and applied it to standardized water samples filtered for eDNA analysis from the Vjosë (Albania) and Volga (Russia), two rivers with extant populations of P. pennigerum. In the Vjosë river, 45 sampling sites were sampled three times in 2018 and 2019. In the Volga river, we focused on a site with>15 years of continuous records of P. pennigerum at Rzhev and two downstream locations, where eDNA samples were collected in 2017. At each sampling site in the Vjosë, eDNA samples were collected by filtering 0.5 L of stream water through each of two 0.45 µm Sterivex filters. In the Volga, 2 L of stream water were filtered through a total of eight 0.7 µm glass fibre filters. Filters were stored and shipped at -20°C until further processing. Environmental DNA extraction was performed using the DNeasy® PowerWater® Sterivex™ Kit following the Experienced User protocol for Vjosë samples and via a Phenol-Chloroform Isoamyl extraction for the Volga samples. A Taqman qPCR assay was developed using a newly designed primer and probe set. Standard curves obtained from an amplicon dilution series yielded a reaction efficiency of 88% and an R2-value of 0.995, with a calculated limit of quantification of 1801 copies/µL and a limit of detection of 59 copies/µL. Each eDNA sampling replicate was tested using five qPCR replicates, yielding ten qPCR reactions per sampling site in the Vjosë river and 40 qPCR reactions in the Volga river. With each batch of eDNA samples, four negative and two positive controls were analysed. At each site, we also collected benthic MHS samples with a 25x25 cm 500 µm net where 20 samples were taken to reflect microhabitat distribution and dominance. Benthos samples were subsampled in the lab and all zoobenthos hand-picked. Prosopistoma pennigerum occurrence was assessed as areal density (number of specimens per m2).While P. pennigerum occured in high frequencies and abundances in benthic samples along the Vjosë river main stem in all sampling seasons, qPCR of eDNA samples suggested slightly different occurrence patterns. Conversely, P. pennigerum was detected with high consistency at two sites in the Volga river (at Rzhev and a site 99 km downstream), where its frequency is much lower than in the Vjosë river. The success of detecting a benthic species in eDNA samples depends on a variety of factors that may have affected DNA quality and prevented better detection of P. pennigerum in the Vjosë river eDNA samples.We demonstrate the principal applicability of molecular methods to search for rare species in hot-spots of biodiversity in Central Europe. The remnant populations of P. pennigerum in the hydrodynamically minimally impaired Vjosë and the Volga highlight the conservation and protection needs in Eastern and Southern Europe. At the European scale, restoration efforts should be geared towards creating viable habitat conditions for large-river species such as P. pennigerum. Here, our qPCR assay can deliver crucial data for better management of Europe’s large rivers. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • A web-based tool to standardise reporting and interpret results of eDNA
           qPCR assays

    • Abstract: ARPHA Conference s 4: e65104
      DOI : 10.3897/aca.4.e65104
      Authors : Kirsten Harper, Cuong Tang, Kat Bruce, Andrea Ross-Gillespie, Vere Ross-Gillespie, Bastian Egeter : Environmental DNA, or eDNA, methodologies can enable the rapid detection of a target species without either visual or physical confirmation of the species presence. Over the last decade, targeted quantitative PCR (qPCR) assays have become an increasingly useful method employed by government and non-government agencies alike for purposes such as protecting and preserving ecosystems from invasive species, or for the conservation of endangered species. As the application of eDNA to answer ecological questions pushes the limits of qPCR-based detection, there is a pressing need to standardise the way qPCR results are reported and interpreted, as well as the way qPCR assays are evaluated for use outside of the remit of the original study.Natural England is one such government agency who have begun to use eDNA methodologies more widely to answer ecological questions. However, while some qPCR assays available for detecting the presence or absence of species such as the great crested newt (Triturus cristatus) have been specified, validated and quality assured to a high degree (Biggs et al. 2014), existing qPCR assays for other species are generally less well developed and validated. Additionally, for some species there are multiple qPCR assays available, with each being developed and validated to different stages. As such, Natural England identified a need to understand how the data derived from eDNA should be interpreted dependent on the level of qPCR assay development, and ultimately the confidence they can have in the accuracy of resulting data, including the associated risk of false positives or false negatives.NatureMetrics has developed a prototype web-based tool and protocol (European Technical Readiness Level 5) which would enable end users such as Natural England to inform their interpretation of qPCR results. The prototype is currently in the beta testing stage and is expected to be available in the coming months. The web-based tool will simplify qPCR assay evaluation, enabling end users to select the most appropriate qPCR assay for their needs, as well as standardise the reporting and interpretation of their qPCR results by generating a report at both the sample and site level from the inputted qPCR data. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Application of microfluidic high-throughput quantitative PCR systems for
           the monitoring and surveillance of aquatic species

    • Abstract: ARPHA Conference s 4: e65100
      DOI : 10.3897/aca.4.e65100
      Authors : Luca Mirimin, Dennis van der Pouw Kraan : Quantitative PCR (qPCR) has been increasingly used for the detection of target organisms in environmental DNA (eDNA) studies, and this is thanks to high sensitivity and ability to quantify DNA targets copy number. However, prior to their implementation, qPCR species-specific assays must be developed and validated and, when implemented, they are limited to relatively low number of targets that can be screened as a multiplex or in parallel. Thanks to recent technological advances, several qPCR-based platforms have become available to increase the throughput capability of qPCR systems as well as lowering time of execution and costs associated to sample processing. The present study describes the use of a microfluidic high-throughput qPCR/dPCR system (Biomark HD, Fluidigm) for the screening of species of ecologic and economic importance in bulk plankton environmental samples from marine coastal areas around the Irish coast.Data was generated using the configuration enabling the highest throughput (in terms of data points) of the system, including Integrated Fluidic Control (IFC) units capable of producing 96 x 96 sample/assay combinations in each run (9,216 individual qPCR tests in a single run). Thanks to such a capability, it was possible to execute the following three main development and implementation phases in a relatively short period of time (weeks as opposed to months/years): (i) development of a panel of species-specific assays targeting a range of crustacean and bivalve species; (ii) assessment of Limits of Detection (LOD), Limits of Quantification (LOQ), and enzymatic inhibition control for selected assays; and (iii) screening of environmental time-series samples (n = 242) obtained from a citizen-like sampling effort that involved a range of stakeholders and locations throughout the Irish marine coastal territory between 2019 and 2020.During the assay-development phase, the IFC system configuration (whereby all assays are tested in parallel against all samples) enabled the rapid screening of species-specific assays against a wide range of (genomic DNA of) non-target organisms, hence enabling for rapid specificity testing. LOD/LOQ experiments showed high levels of sensitivity and thanks to the large number of assays that could be accommodated in a single run, it was possible to include up to four distinct Internal Positive Controls (IPCs) at different concentrations in each run (hence controlling for potential inhibition at different target concentration levels). The inclusion of inhibitor-removal reagents in a pre-amplification step as well as the dilution factor of conducting reactions in small volumes (6.7 nL reaction volumes, hence comparable to a “digital PCR” effect) proved to be an effective strategy to reduce the effect of inhibitors in control experiments (humic acid and EDTA), as well as in actual environmental samples from a range of marine environments. Combining such a high-throughput screening platform with a nation-wide citizen science-like sampling programme enabled the acquisition of large datasets that are being used to monitor occurrence and (spawning) activity of important species that are of conservation concern, commercial value, or non-indigenous and invasive to Irish waters. The Biomark HD system provides a remarkable flexibility to modify existing and/or incorporate new assays because IFCs are customizable just prior to usage (i.e. are not pre-loaded or spotted with primers/probes), thus current work is focussing on increasing the number of species targeted in a single run, and (thanks to the quantitative nature of data) discriminating between different fractions of DNA in heterogeneous bulk samples (e.g. gametes and larvae vs intra- and extracellular eDNA).Thanks to low sample processing cost, assay flexibility and high-throughput capability, microfluidic qPCR platforms behold the potential to significantly advance biomonitoring of aquatic ecosystems. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Challenges in assessing the Neotropical fishes using DNA metabarcoding

    • Abstract: ARPHA Conference s 4: e65093
      DOI : 10.3897/aca.4.e65093
      Authors : Izabela Mendes, Heron Hilário, Daniel Teixeira, Daniel Cardoso de Carvalho : Species richness is a metric of biodiversity usually used in fish community assessment for monitoring programs. This metric is often obtained using traditional fisheries methods that rely on capture of target organisms, resulting in underestimation of fish species. DNA metabarcoding has been recognized as a powerful noninvasive alternative tool for fish biomonitoring and management. Despite the increasing popularity of this method for the assessment of aquatic megadiverse ecosystems, its implementation for studying the highly diverse Neotropical ichthyofauna still presents some challenges. One of them is to devise what primer set could reliably amplify the DNA of all fish species from a megadiverse river basin and have enough resolution to identify them. In order to identify and overcome these drawbacks, we have investigated the efficiency of the metabarcoding approach on Neotropical fishes using a mock sample containing genomic DNA of 18 fish species from the Jequitinhonha River basin, Eastern Brazil. We compared three primer sets targeting the 12S rRNA gene: two universal and widely used markers for fish metabarcoding [MiFish (~170bp) and Teleo_1 (~60bp)], and NeoFish (~190bp), recently developed by our research group specifically for the identification of Neotropical fishes (Milan et al., 2020). Two samples amplified using three primers were sequenced in a single multiplexed Illumina MiniSeq run, using normalized and non-normalized pools. Bioinformatic analyses were performed using a DADA2/Phyloseq based pipeline to perform filtering steps and to assign Amplicon Sequence Variants (ASVs). We used a custom 12S reference sequence database that included 190 specimens representing 101 species and 70 genera from the Jequitinhonha and São Francisco river basins. A total of 187 ASVs were recovered: 79, 66 and 42 for NeoFish, MiFish and Teleo_1, respectively. ASVs of unexpected species were identified for both pools (Fig. 1), though each of these ASVs had an abundance of less than 50 copies. In addition, species of the Hoplias and Prochilodus genera could not be identified at the species level, due to identical sequences within each genus, possibly because of the insufficient variation within the 12S region recovered by these primers’ amplicons. Unexpectedly, although a single individual of each species was placed in the pools, more than one ASV was identified for some species, likely caused by PCR biases. Overall, all primer sets displayed similar taxonomic resolution for the DNA pools and recovered all species, except for NeoFish, which could not detect Steindachneridion amblyurum due to an incompatibility in the 3’ of the NeoFish forward primer and Teleo_1, which could not identify Steindachnerina elegans. These results highlight the need of reliable databases in order to enable the full assignment of ASVs and OTUs to species level, and the importance of calibrating the DNA metabarcoding approach with mock samples to identify weaknesses and pivotal steps prior to the application on large scale DNA based biodiversity evaluation, that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Impacts of mountain forest dieback on aquatic insect communities: A DNA
           metabarcoding analysis of samples from the Bavarian Forest

    • Abstract: ARPHA Conference s 4: e65070
      DOI : 10.3897/aca.4.e65070
      Authors : Erik Rohe, Paul Schmidt Yáñez, Michael Monaghan : Mountain forests are increasingly affected by changes in rainfall and pest outbreaks, and the way forests are managed can have direct consequences for the streams flowing through forests. Aquatic macroinvertebrate communities are great bioindicators and changes to their ecosystem likely translates to changes in their overall composition and abundance. The Bavarian Forest National Park (SE Germany) is dominated by the Norway spruce (Picea abies) which, weakened by storms and other stressors, is susceptible to infestation by the European spruce bark beetle (Ips typographus).The result is large scale forest dieback in some areas, and forest management practices that lead to a predominance of three different forest types (hereafter habitats):Intact forest that is healthy and not impacted by Ips typographus;Disturbed forest that was impacted by Ips typographus, left to regenerate naturally, and from which deadwood was not removed; andSalvaged forest that was heavily impacted by Ips typographus with the same consequences, but from which deadwood was removed, creating a treeless forest meadow.To analyze the impacts these different forest management strategies have on the aquatic insect communities, 30 samples from 11 different streams were taken using kick-sampling. Operational taxonomic units (OTUs) were identified by bulk metabarcoding of dried, ground samples. A mock community was used to verify the setup and a DNA spike-in with three foreign OTUs was added to each sample to measure the biases introduced by PCR amplification and sequencing. Biases varied across samples, but spike-in OTUs produced a pattern indicating predictable biases which could lead to quantifiable metabarcoding results in the future. In total, 260 macroinvertebrate OTUs were identified.In comparison, a morphological study by Bojková et al. (2018) in the same region with twice the number of sampling sites collected 194 taxa in the same month as our samples. This underlines the potential for metabarcoding in evaluating species richness. Species richness was high across all habitats. A significant difference between the forest conditions was detected: The number of detected Diptera OTUs was lowest in disturbed habitats (55) and highest in salvaged areas (73). A permutational multivariate analysis of variance (PERMANOVA) indicated that habitat (i.e., intact, disturbed, salvaged) had an effect on the observed OTU distribution (9.2%), but that the stream catchment had a much larger effect (39.3%) regardless of the habitat.Our findings indicate that forest management can affect stream macroinvertebrate communities, and that this was most pronounced for the Diptera, a group for which DNA metabarcoding is particularly well suited because of their small size and high diversity. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
  • Targeted Next Generation Sequencing improves detection and quantification
           of rare species from eDNA

    • Abstract: ARPHA Conference s 4: e65075
      DOI : 10.3897/aca.4.e65075
      Authors : Kristen Westfall, Thomas Therriault, Cathryn Abbott : Targeted species detection from eDNA is central to identifying and quantifying rare (i.e. invasive or endangered) species to inform conservation and resource management. Here we introduce a new targeted Next Generation Sequencing (tNGS) assay that shows improved detection relative to quantitative (q)PCR at low eDNA concentrations and increased precision to detect spatial variation in eDNA concentration related to species abundance. We compare the tNGS and qPCR methods using invasive European green crab (Carcinus maenas) in the northeast Pacific Ocean as a test case, and find that crab abundance measured by traditional trapping is significantly correlated with eDNA concentration across multiple sites for both methods. However, the tNGS assay outperformed qPCR in all tests: (1) increased precision of eDNA concentration estimation; (2) a 7-10% increase in detection probability at low abundance sites; and (3) greater power to detect spatial variation in eDNA concentration. The accuracy of predicting green crab abundance from eDNA concentration increased with the number of field replicates sampled and did not change appreciably over a tidal cycle. Green crab eDNA concentration behaving similarly to abundance measured from trapping demonstrates great promise for this tool to be implemented for early detection and routine monitoring surveys. The tNGS assay is easily accessible for surveying other species with existing qPCR assays and can thus be potentially important for detection and quantification of any species of high interest to management. HTML XML PDF
      PubDate: Thu, 4 Mar 2021 16:00:00 +0200
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