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Traditional biodiversity assessment methods rely on morphological identification of bioindicators species such as benthic macroinvertebrates. This is the case of the Water Framework Directive (WFD), the key legislation for the protection and sustainable use of European freshwater resources (2000/60/EC). The possibility of identifying all life stages is particularly relevant, but it requires high levels of specialization and expertise and entail procedures that are expensive and time consuming. Also, the majority of these bioindicator taxa are in their larval stage, which imposes constrains on their morphological identification at lower taxonomic levels (e.g. genus or species). Advanced molecular tools, such as metabarcoding, allow the processing of complex multi-species assemblages at greater resolution (up to species level), by combining DNA taxonomy with high-throughput DNA sequencing. Here, we demonstrate the potential use of preservative ethanol from macroinvertebrate bulk and metabarcoding tools to assess macroinvertebrate communities and biological quality of surface water bodies. We examined etanol samples collected from five macroinvertebrate bulks of Tua subbasin (Douro) and morphologically identified organisms down to the lowest practical taxonomic level. Our results showed that 60% of the taxa found in ethanol were macroinvertebrate taxa targeted by WFD, while the remaining percentage was identified as e.g. Bacteria, Stramenopiles, terrestrial invertebrates, amphibians and fishes. In comparison with morphological identification, molecular methods detected in average 70% of the WFD families and 86% of the EPTO (Ephemeroptera, Plecoptera, Trichoptera and Odonata) families. Yet, the percentage of families matching between etanol metabarcoding and morphological identification increased when rare families (<5 individuals) were excluded (80 and 92.2% respectively) and was maximum on groups well represented in DNA reference databases, such as the order Trichoptera. Also, 113 species were successfully identified from ethanol samples but only half was detected morphologically. Ethanol metabarcoding can potentially be a faster, low-priced and more refined approach for assessing biological quality than traditional methods, by increasing taxonomic resolution and thus sensitivity of metrics to fine variations in stream Ecosystem functioning. Nevertheless, further validation is needed as well as expanding the existing DNA reference databases. ; info:eu-repo/semantics/publishedVersion

DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring. ; This article has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 668981 (ERA Chair in Environmental metagenomics, EnvMetaGen) and was supported by EDP‐Biodiversity Chair (EDP/FCT) and the project FRESHING funded by the Portuguese Foundation for Science and Technology (FCT) and COMPETE (PTDC/ AAGMAA/2261/2014—POCI‐01‐0145‐FEDER‐356 016824). FMSM was supported by FCT PhD grant SFRH/BD/104703/2014. PB was supported by EDP‐Biodiversity Chair, MG and JP by the EnvMetaGen, and AFF by FRESHING/FCT. We thank B. Maia‐ Carvalho for his invaluable support on the development of the BEAD protocol, and M. Monaghan for suggestions on DNA quality assessment. ; info:eu-repo/semantics/publishedVersion

We present the most updated list of non-native freshwater fauna established in Portugal, including the Azores and Madeira archipelagos. This list includes 67 species at national level but corresponds to 84 species records, of which 53 are in the mainland, 23 in the Azores and 8 in Madeira archipelagos. We also discuss the progression of the cumulative number of introductions since 1800 and identify the most probable vectors of introduction, main taxonomic groups and their regions of origin. Furthermore, we review the existing knowledge about ecological and economic impacts, invasion risk and potential distribution of invaders, under present and future climatic conditions, and the applied management actions, including the production of legislation. Along the 20th century the number of successful introductions increased at an approximate rate of two new species per decade until the beginning of 1970s. Since then, this rate increased to about 14 new species per decade. These introductions were mainly a result of fisheries, as contaminants or for ornamental purposes. Fish and mollusks are the taxonomic groups with more established species, representing more than half of the total. Most species (>70%) are native from other regions of Europe and North America. Studies about ecological or socioeconomic impacts are more common for fish, crustaceans and mollusks. Impacts for most amphibians, reptiles and mammals are not thoroughly studied. A few studies on the impacts and management actions of health-threatening mosquitoes are also available. The potential distribution in the Portuguese territory was modelled for 26 species. Only a minority of these models provides projections of distributions under scenarios of future climate change. A comparison of the Portuguese and EU legislation shows large discrepancies in the invasive species lists. Using the EU list and a ranking procedure for the national context, we identify freshwater species of high national concern for which actions are urgently needed. ; This work was ...

High quality water is vital for human life, and ensuring its availability is a basic requirement and a major societal aim. The Water Framework Directive (WFD; 2000/60/EC) is a key piece of legislation for the protection and sustainable use of water in the European Union. In this work we briefly review the WFD directive and the current status of European inland surface waters. Additionally, we summarize major challenges and threats for the biological assessment of inland surface waters under climate change effects and invasion by alien species, and highlight the emerging tools and approaches that might help improve biological assessments, including molecular indices based on environmental DNA (eDNA), to new data from the Earth Observation programmes, and data-sharing platforms. Finally, we present recommendations to improve monitoring systems and assessments in the context of the WFD. Developments in this field may increase the likelihood of assuring high quality water for society ; FRESHING Project funded by the Portuguese Foundation for Science and Technology (FCT) and COMPETE (PTDC/AAG-MAA/ 2261/2014 – POCI-01-0145-FEDER-356 016824). AFF, AGR, and JPR were supported by FRESHING. FMSM was supported by FCT grant SFRH/BD/104703/2014. MJF was supported by the strategic project UID/MAR/04292/2013 granted to ...

The Mediterranean Basin is considered one of the world's biodiversity hotspots, harbouring particularly high species richness and endemicity of taxonomic groups such as plants. This hotspot is unique at the global scale, because it has a history of intense anthropogenic influences that dates back to thousands of years, and where the current high-levels of biodiversity have thus been able to coexist with humans for millennia. Despite this long history of coexistence, biodiversity in the Mediterranean Basin is at risk due to a number of old and new anthropogenic stressors, including fast land use changes, overexploitation of natural resources, and global climate change. To tackle these problems, researchers should concentrate their efforts in answering questions that can have a true impact on the success of conservation programs, but there is at present considerable uncertainty on what these questions might be. To identify these questions, a group of scientists from Portugal, Spain, France, Greece, Italy and Israel have worked to identify priority questions that, if answered, would have a high probability of increasing the success of actions targeted at the conservation of Mediterranean biological diversity. This was part of a larger initiative covering the five Mediterranean regions of the world (Mediterranean Basin, California, Australia, Chile and South Africa), which was organised under the scope of the Society for Conservation Biology (SCB) – Europe Section and the International Society of Mediterranean Ecologists (ISOMED). Here we present the first results of this exercise, which is expected to be extended in the near future to other countries in the Mediterranean Basin. The study was based on enquiries targeted at individuals from a number of stakeholder types, including research institutions, environmental non-governmental organizations, environmental consultancy companies, organizations linked to land management (e.g., farmers, hunters), governmental agencies, and large business corporations. We obtained replies from 92 respondents, which suggested a total of 830 questions, divided in 11 major topics. After eliminating questions that were out of scope given the objectives of the study, the three topics most referred to by respondents were related to governance, species management, and farming and forestry, while other important topics were public participation and social sciences, climate change, and impact assessment. The results obtained highlight the importance of interdisciplinary research linking natural and social scientists, which is needed to understand how environmental and socioeconomic drivers interact to shape biodiversity patterns and trends, and to develop and optimise the models of governance and public engagement that are required to preserve biodiversity in the face of such drivers. ; peerReviewed

The global plan to eradicate hepatitis C virus (HCV) led by the World Health Organization outlines the use of highly effective direct‐acting antiviral drugs (DAAs) to achieve elimination by 2030. Identifying individuals with active disease and investigation of the breadth of diversity of the virus in sub‐Saharan Africa (SSA) is essential as genotypes in this region (where very few clinical trials have been carried out) are distinct from those found in other parts of the world. We undertook a population‐based, nested case‐control study in Uganda and obtained additional samples from the Democratic Republic of Congo (DRC) to estimate the prevalence of HCV, assess strategies for disease detection using serological and molecular techniques, and characterize genetic diversity of the virus. Using next‐generation and Sanger sequencing, we aimed to identify strains circulating in East and Central Africa. A total of 7,751 Ugandan patients were initially screened for HCV, and 20 PCR‐positive samples were obtained for sequencing. Serological assays were found to vary significantly in specificity for HCV. HCV strains detected in Uganda included genotype (g) 4k, g4p, g4q, and g4s and a newly identified unassigned g7 HCV strain. Two additional unassigned g7 strains were identified in patients originating from DRC (one partial and one full open reading frame sequence). These g4 and g7 strains contain nonstructural (ns) protein 3 and 5A polymorphisms associated with resistance to DAAs in other genotypes. Clinical studies are therefore indicated to investigate treatment response in infected patients. Conclusion: Although HCV prevalence and genotypes have been well characterized in patients in well‐resourced countries, clinical trials are urgently required in SSA, where highly diverse g4 and g7 strains circulate. ; Supported by the Medical Research Council (MRC) (MC_UU_12014/1) and Wellcome Trust (102789/Z/13/A) (to E.T.). M.S. is funded by the Wellcome Trust Sanger Institute (WT098051), the National Institute for Health Research Cambridge Biomedical Research Centre, the African Partnership for Chronic Disease Research (MRC UK partnership grant number MR/K013491/1), and the UK MRC (G0901213‐92157, G0801566). P.K. is funded by the UK MRC and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement. J.S. is funded by the MRC Confidence in Concept award to the University of Glasgow (MC PC 16045). G.M. is a Gates Cambridge Scholar supported by the Gates Cambridge Trust.

The global plan to eradicate hepatitis C virus (HCV) led by the World Health Organization outlines the use of highly effective direct‐acting antiviral drugs (DAAs) to achieve elimination by 2030. Identifying individuals with active disease and investigation of the breadth of diversity of the virus in sub‐Saharan Africa (SSA) is essential as genotypes in this region (where very few clinical trials have been carried out) are distinct from those found in other parts of the world. We undertook a population‐based, nested case‐control study in Uganda and obtained additional samples from the Democratic Republic of Congo (DRC) to estimate the prevalence of HCV, assess strategies for disease detection using serological and molecular techniques, and characterize genetic diversity of the virus. Using next‐generation and Sanger sequencing, we aimed to identify strains circulating in East and Central Africa. A total of 7,751 Ugandan patients were initially screened for HCV, and 20 PCR‐positive samples were obtained for sequencing. Serological assays were found to vary significantly in specificity for HCV. HCV strains detected in Uganda included genotype (g) 4k, g4p, g4q, and g4s and a newly identified unassigned g7 HCV strain. Two additional unassigned g7 strains were identified in patients originating from DRC (one partial and one full open reading frame sequence). These g4 and g7 strains contain nonstructural (ns) protein 3 and 5A polymorphisms associated with resistance to DAAs in other genotypes. Clinical studies are therefore indicated to investigate treatment response in infected patients. Conclusion: Although HCV prevalence and genotypes have been well characterized in patients in well‐resourced countries, clinical trials are urgently required in SSA, where highly diverse g4 and g7 strains circulate.

Background and rationale for the study: The global plan to eradicate hepatitis C (HCV) led by the World Health Organisation (WHO) outlines the use of highly effective direct‐acting antiviral drugs (DAAs) to achieve elimination by 2030. Identifying individuals with active disease and investigation of the breadth of diversity of the virus sub‐Saharan Africa (SSA) is essential as genotypes in this region (where very few clinical trials have been carried out) are distinct from those found in other parts of the world. We undertook a population‐based nested case‐control study in Uganda and obtained additional samples from the Democratic Republic of Congo (DRC), to estimate the prevalence of HCV, assess strategies for disease detection using serological and molecular techniques, and characterise genetic diversity of the virus. Using next generation (NGS) and Sanger sequencing, we aimed to identify strains circulating in East and Central Africa. Main results: 7751 Ugandan patients were initially screened for HCV and 20 PCR positive samples obtained for sequencing. Serological assays were found to vary significantly in specificity for HCV. HCV strains detected in Uganda included genotypes (g) 4k, 4p, 4q and 4s and a new unassigned genotype 7 HCV strain. Two additional unassigned g7 strains were identified in patients originating from DRC (one partial and one full ORF sequence). These g4 and 7 strains contain NS3 and NS5A polymorphisms associated with resistance to DAAs in other genotypes. Clinical studies are therefore indicated to investigate treatment response in infected patients. Conclusion: While HCV prevalence and genotypes have been well characterised in patients in well‐resourced countries, clinical trials are urgently required in SSA where highly diverse g4 and 7 strains circulate.

The global plan to eradicate hepatitis C virus (HCV) led by the World Health Organization outlines the use of highly effective direct-acting antiviral drugs (DAAs) to achieve elimination by 2030. Identifying individuals with active disease and investigation of the breadth of diversity of the virus in sub-Saharan Africa (SSA) is essential as genotypes in this region (where very few clinical trials have been carried out) are distinct from those found in other parts of the world. We undertook a population-based, nested case-control study in Uganda and obtained additional samples from the Democratic Republic of Congo (DRC) to estimate the prevalence of HCV, assess strategies for disease detection using serological and molecular techniques, and characterize genetic diversity of the virus. Using next-generation and Sanger sequencing, we aimed to identify strains circulating in East and Central Africa. A total of 7,751 Ugandan patients were initially screened for HCV, and 20 PCR-positive samples were obtained for sequencing. Serological assays were found to vary significantly in specificity for HCV. HCV strains detected in Uganda included genotype (g) 4k, g4p, g4q, and g4s and a newly identified unassigned g7 HCV strain. Two additional unassigned g7 strains were identified in patients originating from DRC (one partial and one full open reading frame sequence). These g4 and g7 strains contain nonstructural (ns) protein 3 and 5A polymorphisms associated with resistance to DAAs in other genotypes. Clinical studies are therefore indicated to investigate treatment response in infected patients. Conclusion: Although HCV prevalence and genotypes have been well characterized in patients in well-resourced countries, clinical trials are urgently required in SSA, where highly diverse g4 and g7 strains circulate.