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Current freshwater biomonitoring with diatoms is based on microscopic examination of the morphology of their silica skeleton. This standardized approach is time consuming and requires a high degree of taxonomic expertise. Metabarcoding combined with high-throughput sequencing (HTS) has great potential for next-generation biomonitoring applications but requires standardization. Molecular inventories are strongly influenced by the DNA extraction method used, but the effect of extraction protocols has not been tested to enable selection of the best DNA extraction method for HTS metabarcoding. We used 5 DNA extraction methods combining various types of cell lysis and DNA purification to extract DNA from 8 pure diatom cultures and 8 samples from streams and lakes with differing water quality. We compared the methods based on: 1) quality and purity of the extracted DNA, 2) community inventories obtained from HTS targeting the ribulose-1, 5-bisphosphate carboxylase (rbcL) barcode, and 3) similarity between molecular and microscopy-based inventories of community composition and the Specific Pollution-sensitivity Index [SPI]. A method based on GenElute™-LPA had higher extraction efficiency than the 4 commercial kits but had the highest polymerase chain reaction inhibition level. All 5 methods were efficient for HTS, and method did not affect operational taxonomic unit richness. We observed variations in the relative abundance of some taxa within Nitzschia, Amphora, Encyonema, Gomphonema, and Navicula between 2 of the 5 methods, but method did not affect global diatom community composition or SPI values. SPI values calculated from microscopy-based inventories and molecular inventories based on all 5 extraction methods were strongly correlated. For convenience purposes (high DNA quantity and low cost), we encourage standardization of HTS diatom biomonitoring based on the SA-Gen method.

Sea turtles are distributed in tropical and subtropical seas worldwide. They play several ecological roles and are considered important indicators of the health of marine ecosystems. Studying epibiotic diatoms living on turtle shells suggestively has great potential in the study of turtle behavior because diatoms are always there. However, diatom identification at the species level is time consuming, requires well-trained specialists, and there is a high probability of finding new taxa growing on turtle shells, which makes identification tricky. An alternative approach based on DNA barcoding and high throughput sequencing (HTS), metabarcoding, has been developed in recent years to identify species at the community level by using a DNA reference library. The suitabilities of morphological and molecular approaches were compared. Diatom assemblages were sampled from seven juvenile green turtles (Chelonia mydas) from Mayotte Island, France. The structures of the epibiotic diatom assemblages differed between both approaches. This resulted in different clustering of the turtles based on their diatom communities. Metabarcoding allowed better discrimination between turtles based on their epibiotic diatom assemblages and put into evidence the presence of a cryptic diatom diversity. Microscopy, for its part, provided more ecological information of sea turtles based on historical bibliographical data and the abundances of ecological guilds of the diatom species present in the samples. This study shows the complementary nature of these two methods for studying turtle behavior.

Classical biomonitoring techniques have focused primarily on measures linked to various biodiversity metrics and indicator species. Next-generation biomonitoring (NGB) describes a suite of tools and approaches that allow the examination of a broader spectrum of organisational levels - from genes to entire ecosystems. Here, we frame ten key questions that we envisage will drive the field of NGB over the next decade. While not exhaustive, this list covers most of the key challenges facing NGB, and provides the basis of the next steps for research and implementation in this field. These questions have been grouped into current- and outlook-related categories, corresponding to the organization of this paper.

The white‐clawed crayfish (Austropotamobius pallipes) is an emblematic taxon of European rivers, found mainly in oxygenated streams, known to be an excellent indicator of river quality. Since several decades, the population of A. pallipes declined in relation to anthropogenic pressure, habitat loss, and competition with pests (invasive crayfish, crayfish plague). This endangered species is now submitted to conservation strategies by freshwater managers in order to survey and protect the remaining populations. In France, traditional surveys in freshwater environments were performed by electric fishing, kick‐net fishing, or trapping, particularly disruptive for the environment and very time‐consuming. However, with the rise of molecular genetic technology, new methods based on the detection of environmental DNA (eDNA) have emerged. We present here the results of an optimized study for the detection of the endangered crayfish Austropotamobius pallipes in France, considering certain environmental co‐factors and comparing two PCR methods (qPCR and ddPCR). After improving laboratory procedures, we were able to detect the presence of the crayfish up to 2 km downstream from a known point of presence and unfortunately highlight the disappearance of a historical population, after sampling two consecutive years. Such a level of precision is interesting because it makes it possible to precise the presence of specimens in a relatively restricted area and to orient traditional prospecting, necessary for certain additional studies. During our study, we observed better probabilities of detection during the summer period, but in a growing context of climate change, we advise to adapt the sampling year by year. That said, this methodology is a very useful tool for the detection of rare and/or endemic species and we did not observe any difference between the two PCR methods used.

Ecological status assessment of watercourses is based on the calculation of quality indices using pollution sensitivity of targeted biological groups, including diatoms. The determination and quantification of diatom species is generally based on microscopic morphological identification, which requires expertise and is time-consuming and costly. In Europe, this morphological approach is legally imposed by standards and regulatory decrees by the Water Framework Directive (WFD). Over the past decade, a DNAbased molecular biology approach has newly been developed to identify species based on genetic criteria rather than morphological ones (i.e. DNA metabarcoding). In combination with high throughput sequencing technologies, metabarcoding makes it possible both to identify all species present in an environmental sample and to process several hundred samples in parallel. This article presents the results of two recent studies carried out on the WFD networks of rivers of Mayotte (2013-2018) and metropolitan France (2016-2018). These studies aimed at testing the potential application of metabarcoding for biomonitoring in the context of the WFD. We discuss the various methodological developments and optimisations that have been made to make the taxonomic inventories of diatoms produced by metabarcoding more reliable, particularly in terms of species quantification. We present the results of the application of this DNA approach on more than 500 river sites, comparing them with those obtained using the standardised morphological method. Finally, we discuss the potential of metabarcoding for routine application, its limits of application and propose some recommendations for future implementation in WFD.

Parkinson's disease (PD) is a disorder characterized by dopaminergic neuron programmed cell death. The etiology of PD remains uncertain-some cases are due to selected genes associated with familial heredity, others are due to environmental exposure to toxic components, but over 90% of cases have a sporadic origin. are Actinobacteria that can cause human diseases like nocardiosis. This illness can lead to lung infection or central nervous system (CNS) invasion in both immunocompromised and immunocompetent individuals. The main species involved in CNS are , and . Some studies have highlighted the ability of to induce Parkinson's disease-like symptoms in animals. Actinobacteria are known to produce a large variety of secondary metabolites, some of which can be neurotoxic. We hypothesized that neurotoxic secondary metabolite production and the onset of PD-like symptoms in animals could be linked. Here we used a method to screen bacteria that could induce dopaminergic neurodegeneration before performing mouse experiments. The nematode allowed us to demonstrate that strains belonging to and species can induce dopaminergic neurodegeneration. Strains of interest involved with the nematodes in neurodegenerative disorders were then injected in mice. Infected mice had behavioral disorders that may be related to neuronal damage, thus confirming the ability of strains to induce neurodegeneration. These behavioral disorders were induced by species ( GUH-2 and 44484) and 10152. We conclude that is a good model for detecting strains involved in neurodegeneration. This model allowed us to detect bacteria with high neurodegenerative effects and which should be studied in mice to characterize the induced behavioral disorders and bacterial dissemination.

Benthic diatoms are relevant indicators of the ecological status of the littoral zone of lakes. Their use as bio-indicators is based on their morphological identification at species level using microscopy which is time consuming, requires taxonomic expertise, and is consequently expensive. To overcome these limitations, a molecular approach for diatom identification has been tested with success in rivers. DNA metabarcoding enables species identification from a standardized DNA barcode and high-throughput sequencing (HTS), using DNA reference library. The suitability of the morphological and molecular approaches to assess the diatom community structure and the ecological status of the littoral zone of the largest deep lake in France (Lake Bourget) was compared. 66 sites were sampled in August 2015 along the shoreline, all around the lake. The composition of diatom assemblages was similar with both morphological and molecular approaches, and diatom assemblages were structured by the same environmental factors. However, the ecological status of Lake Bourget differed significantly among approaches since floristic inventories to species level also differed significantly. The main source of this difference was the incompleteness of the DNA reference library. Nevertheless, in a near future, when this constraint will be solved, the use of DNA metabarcoding for biomonitoring purposes seems promising.

Freshwaters worldwide face serious threats, making their protection increasingly important. Freshwater monitoring has historically produced valuable data and continues to develop. Rapid improvements to biomolecular techniques are revolutionizing the way scientists describe biological communities and are bringing about major changes in biomonitoring. Combined with high-throughput sequencing, DNA metabarcoding is fast and cost-effective, generating massive amounts of data. In a world with numerous ecological threats, \"big data\" constitute a tremendous opportunity to improve the efficiency of biological monitoring. These fundamental changes in biomonitoring will require freshwater ecologists and environmental managers to reconsider how they handle large amounts of data.

Stenotrophomonas maltophilia is found ubiquitously in the environment and is an important emerging nosocomial pathogen. S. maltophilia has been recently described as an Amoebae-Resistant Bacteria (ARB) that exists as part of the microbiome of various free-living amoebae (FLA) from waters. Co-culture approaches with Vermamoeba vermiformis demonstrated the ability of this bacterium to resist amoebal digestion. In the present study, we assessed the survival and growth of six environmental and one clinical S. maltophilia strains within two amoebal species: Acanthamoeba castellanii and Willaertia magna. We also evaluated bacterial virulence properties using the social amoeba Dictyostelium discoideum. A co-culture approach was carried out over 96 hours and the abundance of S. maltophilia cells was measured using quantitative PCR and culture approach. The presence of bacteria inside the amoeba was confirmed using confocal microscopy. Our results showed that some S. maltophilia strains were able to multiply within both amoebae and exhibited multiplication rates up to 17.5 and 1166 for A. castellanii and W. magna, respectively. In contrast, some strains were unable to multiply in either amoeba. Out of the six environmental S. maltophilia strains tested, one was found to be virulent. Surprisingly, this strain previously isolated from a soil amoeba, Micriamoeba, was unable to infect both amoebal species tested. We further performed an assay with a mutant strain of S. maltophilia BurA1 lacking the efflux pump ebyCAB gene and found the mutant to be more virulent and more efficient for intra-amoebal multiplication. Overall, the results obtained strongly indicated that free-living amoebae could be an important ecological niche for S. maltophilia.

Environmental DNA (eDNA) metabarcoding revolutionized the biodiversity monitoring in aquatic ecosystems, giving access to taxonomic lists in a non-disruptive way. Although the method has limits, such as reduced taxonomic resolution for certain groups and difficulties in estimating species abundance, it has proven its effectiveness in many contexts. In Martinique, a Caribbean island, traditional methods like electrofishing (TEF) are known to be stressful for organisms, non-selective and disruptive for the ecosystem, and have been progressively abandoned for routine monitoring. The aim of this project was to explore the possibility of using the eDNA-based metabarcoding method for the detection of fish and decapods in Martinique streams, by first validating it with TEF. We selected 14 stations, a representative panel of the river diversity, and performed TEF and eDNA-based monitoring to compare both, based on the species richness. Then, from eDNA taxonomic inventories, we assessed the ecological state of the studied stations, using Simpson index and investigated how stations abiotic characteristics shape assemblages. Here, we confirmed the eDNA metabarcoding method is a reliable tool for monitoring fish and decapods, confirming most of the taxa caught by TEF and revealing the presence of additional (native and/or invasive) species. We faced some issues in discriminating some genetically close species (e.g. Sicydium sp.) potentially leading to under-representation in community assemblages, but not in functional diversity. Additional efforts are needed to raise standardized protocols, but we encourage stakeholders to join such an initiative to shed light on the rich biodiversity in sometimes poorly studied regions and to face invasions.