Recommendations for the preservation of environmental samples in diatom metabarcoding studies

Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa

Recommendations for the preservation of environmental samples in diatom metabarcoding studies

Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa

Recommendations for the preservation of environmental samples in diatom metabarcoding studies

Implementation of DNA metabarcoding for diatoms for environmental monitoring is now moving from a research to an operational phase, requiring rigorous guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies and now need to be rationalised. The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design used samples from four freshwater and two marine sites of diverse ecological characteristics. The impact of the sample preservation and storage duration was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, diatom assemblage composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflected the site origin. Only rare taxa (< 100 reads) differed among preservation methods and storage durations. For biomonitoring purposes, freshwater ecological index values were not affected by the preservation method and storage duration tested (including ethanol preservation), all treatments returning the same ecological status for a site. This study contributes to consolidating diatom metabarcoding. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring

New 16S rRNA primers to uncover Bdellovibrio and like organisms diversity and abundance

Appropriate use and specific primers are important in assessing the diversity and abundance of microbial groups of interest. Bdellovibrio and like organisms (BALOs), that refer to obligate Gram-negative bacterial predators of other Gram-negative bacteria, evolved in terms of taxonomy and classification over the past two decades. Hence, some former primers have become inadequate while others are yet to be designed, for both PCR (especially with the advent of NGS) and qPCR approaches. Thus, to study BALOs' abundance and diversity in a variety of aquatic ecosystems, we designed in silico specific primer sets for each BALO genera and tested them in vitro on a variety of cultures and environmental samples. Also, we performed Sanger and Nano Miseq sequencing to reveal the exact degree of specificity of the most promising primers set. Here we report our success in designing specific primers for some BALOs genera, i.e. Bdellovibrio (PCR), Bacteriovorax (qPCR), Peredibacter (PCR)

Comparaison de la qPCR et de la ddPCR pour détecter et surveiller la dynamique de la petite crevette rouge sang Hemimysis anomala et de son interaction avec la perche : une étude appliquée au Léman

Les espèces exotiques envahissantes constituent une menace majeure pour les systèmes aquatiques en raison de leurs impacts potentiels sur la biodiversité endémique, le fonctionnement des écosystèmes, les infrastructures ou encore en raison d'éventuels problèmes sanitaires. L'obtention d'informations sur leur présence, leur abondance et leur répartition est toujours un enjeu clé. Récemment, la petite crevette rouge sang Hemimysis anomala, originaire de la région de Ponto Caspienne, s'est installée dans des lacs d'Europe occidentale tels que le Léman. Bien que les plongeurs aient signalé, à de nombreuses reprises, sa présence et son développement au cours de la dernière décennie, aucun suivi n'a encore été proposé pour ce petit crustacé. Nous avons testé et optimisé une approche ADNe en utilisant et en comparant deux techniques de PCR (PCR quantitative et PCR digitale) pour évaluer la présence, l'abondance et la dynamique de l'animal ainsi que celle d'un prédateur potentiel, la perche (Perca fluviatilis) pendant 2,5 ans. Nous montrons et discutons l'efficacité des méthodes et révélons pour la première fois la dynamique saisonnière d'Hemimysis anomala sur un site sélectionné dans le Léman. Nous soulignons, en accord avec les observations effectuées en plongée, que l'abondance de l'animal est élevée en hiver et diminue rapidement au début du printemps, de manière concomitante à l'augmentation de la température de l'eau et au « retour » visible des perches proches de la surface

A quantitative eDNA-based method to monitor spawning activity of two emblematic fish species in lake Geneva

Anthropogenic pressures and more recently climatic change have increased the interest to study the impact of environmental changes on the key stages of fish life cycle. In lake Geneva, a deep peri-alpine lake, climate change and phosphorous level are known to have consequences on salmonid and percid populations, including key species for recreational and commercial fisheries, whose stocks are subject to significant fluctuations. To follow these stock variations, the spawning activity of European perch (Perca fluviatilis) and whitefish (Coregonus lavaretus) is monitored in this lake since several years using traditional methods, unfortunately mostly destructive or damaging (e.g. gillnetting and collection of fertilized eggs).DNA isolated from the environment (eDNA) has been widely developed for the detection of specific species or whole biological communities, and this non-invasive method offers an alternative to conventional surveying tools. Until recently, the methods used for eDNA analysis (e.g. qPCR, metabarcoding) could be limited by their sensitivity, quantification limit or price, but the emergence of new methods, such as the droplet digital PCR (ddPCR), offers the possibility to quantify an absolute eDNA signal in a very sensitive way and at a lower cost.Here, we show for the first time the applicability of an eDNA method to monitor the spawning activity of two fish species in a lake by using ddPCR.During two spawning seasons for whitefish and one spawning season for European perch, water samples were collected every week from the subsurface, simultaneously to traditional monitoring sampling, and filtered through sterile cartridges. The eDNA was then extracted and analyzed using ddPCR, targeting the mitochondrial DNA of the two fish species.The results demonstrate the efficiency of eDNA coupled with ddPCR to identify the timing and duration of the spawning periods, as well as the peak of the spawning activity for whitefish and European perch in Lake Geneva. This study shows that we have reached an operational level to use this non-invasive eDNA monitoring of the spawning activity of these fish species in lakes

Contrasting temporal patterns in ammonia oxidizing archaeal community dynamics in two peri-alpine lakes with different trophic status

International audienceWe studied the spatiotemporal dynamics of ammonia-oxidizing archaea (AOA) in 2 deep and large peri-alpine lakes (Annecy and Bourget; oligotrophic and mesotrophic, respectively) over 2 years. Monthly, we characterized the structure, richness, and abundance of AOA populations in the epi- and hypolimnion using DGGE and qPCR of the archaea-specific amoA gene. Clear vertical patterns were observed in both lakes, with greater values for AOA richness and amoA gene abundance in the hypolimnetic layers. AOA community composition and structure was much more stable throughout the year in Lake Bourget than in Lake Annecy. In the upper layers, AOA communities displayed seasonal succession patterns and had greater abundance in winter. The temporal structure showed more pronounced seasonal patterns in Lake Annecy than in Lake Bourget. In the deeper layers of both lakes, AOA relative abundance showed no clear temporal pattern. Temporal changes in amoA gene composition were correlated with changes in the archaeal 16S rRNA gene in surface waters. Changes in the structure of both genes were not significantly correlated in the hypolimnion suggesting that the temporal changes in the structure of archaeal communities in the deeper waters might be globally driven by the dynamics of heterotrophic archaea and not archaeal ammonia oxidizers. None of the many environmental variables we measured explained significant amounts of variation in AOA community structure and richness; thus, other factors and processes may exert selective pressure on the structure of these communities. Nevertheless, archaeal amoA gene abundance varies with water temperature (negative correlation) and with nitrate concentration (positive correlation). Our study is the first to show that archaeal amoA gene abundance is correlated positively with silica and negatively with total nanoflagellate abundance. This suggests that AOA could play a significant role in silica dissolution and regeneration, and points to the possible influence of predation on AOA abundance

A quantitative eDNA-based approach to monitor fish spawning in lakes: Application to European perch and whitefish

International audienceThere is an urgent need to evaluate the effects of anthropogenic pressures and climatic change on fish pop-ulations' dynamics. When monitored in lakes, fish spawning is generally assessed using traditional, mostly destructive or damaging, methods as gillnetting and collection of fertilized eggs. Over the last decade, envi-ronmental DNA (eDNA) based methods have been widely developed and now offer a non-invasive alternative method to these traditional biomonitoring tools. In particular, the emergence of new methods as the droplet digital PCR (ddPCR) offers the possibility to quantify an absolute eDNA signal in a very sensitive way and at a low cost. Here, we developed and implemented a quantitative eDNA approach to monitor the spawning activity in a lentic environment for two non-migratory fish species, European perch and whitefish. The ddPCR protocols were formalized based on existing and newly designed COI primers, and were applied during four spawning periods in Lake Geneva. The results demonstrate the effectiveness of weekly eDNA sampling coupled with ddPCR to identify the timing and duration of the spawning periods, as well as the peak of the spawning activity for the target species. In addition, we highlight that the use of a control species (i.e., quantification of the eDNA signal of a fish that does not reproduce during the monitoring period) helps to clearly discriminate the eDNA signal associated to the spawning activity. The filtration of 2 L of subsurface water was shown to be effective for the monitoring of spawning activity; however, using discrete sampling strategy we observed a spatial variability in the eDNA signal. For future implementation, we recommend (i) using an integrative sampling strategy to smooth the local variability of the eDNA signal, and (ii) to cover a sampling period long enough to clearly distinguish the spawning signal from the basal signal of the target species. These results show that we reached an operational level to use these non-invasive eDNA approaches to monitor the spawning periods of these two fish species in large lakes

First Order Design by Optimization Method: Application to an Interleaved Buck Converter and Validation

International audienceThis paper presents a design methodology of powerconverter and its components using deterministic algorithmsassociated with differentiable models. This “first order” designby optimization method is illustrated in the case of an InterleavedBuck Converter used in a stratospheric airship. Therequirements and the models are briefly presented as well as theoptimization results in the imaginary space of continuousvariables. The method to come back in the power electronicsworld with its discrete variables is presented and validated with aprototype of Interleaved Buck Converte

Design by Optimization Methodology: Application to a Wide Input and Output Voltage Ranges Interleaved Buck Converter

International audienc