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  • 1
    In: Metabarcoding and Metagenomics, Pensoft Publishers, Vol. 5 ( 2021-08-25)
    Abstract: Environmental conditions, such as nutrient concentrations, salinity, elevation etc., shape diatom assemblages of periphytic biofilms. These assemblages respond rapidly to environmental changes, a fact which makes diatoms valuable bioindicators. Hence, freshwater biomonitoring programmes currently use diatom indices (e.g. EU Water Framework Directive - WFD). To date, microscopy-based assessments require high taxonomic expertise for diatom identification at the species level. High-throughput technologies now provide cost-effective identification approaches that are promising, complementary or alternative tools for bioassessment. The suitability of the metabarcoding method is evaluated for the first time in the Cyprus streams WFD monitoring network, an eastern Mediterranean country with many endemic species and results are compared to the results acquired from the morphotaxonomic analysis. Morphotaxonomic identification was conducted microscopically, using the most updated taxonomic concepts, literature and online resources. At the same time, DNA metabarcoding involved the use of the rbc L 312 bp barcode, high-throughput sequencing and bioinformatic analysis. The ecological status was calculated using the IPS Index. Results show a positive correlation between morpho-taxonomic and molecular IPS scores. Discrepancies between the two methodologies are related to the limitations of both techniques. This study confirmed that Fistulifera saprophila can have a crucial role in key differences observed, as it negatively influences IPS scores and microscopy methods frequently overlook it. Importantly, gaps in the DNA barcoding reference databases lead to a positive overestimation in IPS scores. Overall, we conclude that DNA metabarcoding offsets the morphotaxonomic methodology for the ecological quality assessment of freshwaters.
    Type of Medium: Online Resource
    ISSN: 2534-9708
    Language: Unknown
    Publisher: Pensoft Publishers
    Publication Date: 2021
    detail.hit.zdb_id: 2913203-4
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  • 2
    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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  • 3
    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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  • 4
    In: Chemosphere, Elsevier BV, Vol. 272 ( 2021-06), p. 129814-
    Type of Medium: Online Resource
    ISSN: 0045-6535
    RVK:
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2021
    detail.hit.zdb_id: 1496851-4
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  • 5
    In: Ecotoxicology and Environmental Safety, Elsevier BV, Vol. 232 ( 2022-03), p. 113213-
    Type of Medium: Online Resource
    ISSN: 0147-6513
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2022
    detail.hit.zdb_id: 1466969-9
    SSG: 24,1
    SSG: 12
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  • 6
    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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  • 7
    Online Resource
    Online Resource
    Proceedings of the National Academy of Sciences ; 2018
    In:  Proceedings of the National Academy of Sciences Vol. 115, No. 49 ( 2018-12-04), p. 12519-12524
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 115, No. 49 ( 2018-12-04), p. 12519-12524
    Abstract: The type VI secretion system (T6SS) is a supramolecular complex involved in the delivery of potent toxins during bacterial competition. Pseudomonas aeruginosa possesses three T6SS gene clusters and several hcp and vgrG gene islands, the latter encoding the spike at the T6SS tip. The vgrG1b cluster encompasses seven genes whose organization and sequences are highly conserved in P. aeruginosa genomes, except for two genes that we called tse7 and tsi7 . We show that Tse7 is a Tox-GHH2 domain nuclease which is distinct from other T6SS nucleases identified thus far. Expression of this toxin induces the SOS response, causes growth arrest and ultimately results in DNA degradation. The cytotoxic domain of Tse7 lies at its C terminus, while the N terminus is a predicted PAAR domain. We find that Tse7 sits on the tip of the VgrG1b spike and that specific residues at the PAAR–VgrG1b interface are essential for VgrG1b-dependent delivery of Tse7 into bacterial prey. We also show that the delivery of Tse7 is dependent on the H1-T6SS cluster, and injection of the nuclease into bacterial competitors is deployed for interbacterial competition. Tsi7, the cognate immunity protein, protects the producer from the deleterious effect of Tse7 through a direct protein–protein interaction so specific that toxin/immunity pairs are effective only if they originate from the same P. aeruginosa isolate. Overall, our study highlights the diversity of T6SS effectors, the exquisite fitting of toxins on the tip of the T6SS, and the specificity in Tsi7-dependent protection, suggesting a role in interstrain competition.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    RVK:
    RVK:
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2018
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
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  • 8
    In: Scientific Reports, Springer Science and Business Media LLC, Vol. 6, No. 1 ( 2016-09-30)
    Abstract: Type III secretion systems (T3SSs) are essential devices in the virulence of many Gram-negative bacterial pathogens. They mediate injection of protein effectors of virulence from bacteria into eukaryotic host cells to manipulate them during infection. T3SSs involved in virulence (vT3SSs) are evolutionarily related to bacterial flagellar protein export apparatuses (fT3SSs), which are essential for flagellar assembly and cell motility. The structure of the external and transmembrane parts of both fT3SS and vT3SS is increasingly well-defined. However, the arrangement of their cytoplasmic and inner membrane export apparatuses is much less clear. Here we compare the architecture of the cytoplasmic regions of the vT3SSs of Shigella flexneri and the vT3SS and fT3SS of Salmonella enterica serovar Typhimurium at ~5 and ~4 nm resolution using electron cryotomography and subtomogram averaging. We show that the cytoplasmic regions of vT3SSs display conserved six-fold symmetric features including pods, linkers and an ATPase complex, while fT3SSs probably only display six-fold symmetry in their ATPase region. We also identify other morphological differences between vT3SSs and fT3SSs, such as relative disposition of their inner membrane-attached export platform, C-ring/pods and ATPase complex. Finally, using classification, we find that both types of apparatuses can loose elements of their cytoplasmic region, which may therefore be dynamic.
    Type of Medium: Online Resource
    ISSN: 2045-2322
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2016
    detail.hit.zdb_id: 2615211-3
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  • 9
    Online Resource
    Online Resource
    Elsevier BV ; 2023
    In:  Journal of Environmental Chemical Engineering Vol. 11, No. 1 ( 2023-02), p. 109195-
    In: Journal of Environmental Chemical Engineering, Elsevier BV, Vol. 11, No. 1 ( 2023-02), p. 109195-
    Type of Medium: Online Resource
    ISSN: 2213-3437
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2023
    detail.hit.zdb_id: 2710047-9
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  • 10
    In: Journal of Biological Chemistry, Elsevier BV, Vol. 293, No. 23 ( 2018-06), p. 8829-8842
    Type of Medium: Online Resource
    ISSN: 0021-9258
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2018
    detail.hit.zdb_id: 2141744-1
    detail.hit.zdb_id: 1474604-9
    SSG: 12
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