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  • 1
    In: ARPHA Conference Abstracts, Pensoft Publishers, Vol. 4 ( 2021-03-04)
    Abstract: 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 rbc L 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.3.39646 WDD, 2014. Review and update of article 5 of Directive 2000/60/EC (Water reservoirs) & amp; 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.
    Type of Medium: Online Resource
    ISSN: 2603-3925
    Language: Unknown
    Publisher: Pensoft Publishers
    Publication Date: 2021
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  • 2
    In: ARPHA Conference Abstracts, Pensoft Publishers, Vol. 4 ( 2021-03-04)
    Abstract: 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 rbc L 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 17245:2018 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. 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. 82:1-12 Vasselon, 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
    Type of Medium: Online Resource
    ISSN: 2603-3925
    Language: Unknown
    Publisher: Pensoft Publishers
    Publication Date: 2021
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  • 3
    In: Research Ideas and Outcomes, Pensoft Publishers, Vol. 2 ( 2016-11-29), p. e11321-
    Type of Medium: Online Resource
    ISSN: 2367-7163
    Language: Unknown
    Publisher: Pensoft Publishers
    Publication Date: 2016
    detail.hit.zdb_id: 2833254-4
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  • 4
    In: ARPHA Conference Abstracts, Pensoft Publishers, Vol. 4 ( 2021-03-04)
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 2603-3925
    Language: Unknown
    Publisher: Pensoft Publishers
    Publication Date: 2021
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