Evaluation of sequential filtration and centrifugation to capture environmental DNA and survey microbial eukaryotic communities in aquatic environments.

Sequential membrane filtration of water samples is commonly used to monitor the diversity of aquatic microbial eukaryotes. This capture method is efficient to focus on specific taxonomic groups within a size fraction, but it is time-consuming. Centrifugation, often used to collect microorganisms from pure culture, could be seen as an alternative to capture microbial eukaryotic communities from environmental samples. Here, we compared the two capture methods to assess diversity and ecological patterns of eukaryotic communities in the Thau lagoon, France. Water samples were taken twice a month over a full year and sequential filtration targeting the picoplankton (0.2-3 µm) and larger organisms (>3 µm) was used in parallel to centrifugation. The microbial eukaryotic community in the samples was described using an environmental DNA approach targeting the V4 region of the 18S rRNA gene. The most abundant divisions in the filtration fractions and the centrifugation pellet were Dinoflagellata, Metazoa, Ochrophyta, Cryptophyta. Chlorophyta were dominant in the centrifugation pellet and the picoplankton fraction but not in the larger fraction. Diversity indices and structuring patterns of the community in the two size fractions and the centrifugation pellet were comparable. Twenty amplicon sequence variants were significantly differentially abundant between the two size fractions and the centrifugation pellet, and their temporal patterns of abundance in the two fractions combined were similar to those obtained with centrifugation. Overall, centrifugation led to similar ecological conclusions as the two filtrated fractions combined, thus making it an attractive time-efficient alternative to sequential filtration.

Do microbial planktonic communities reflect the ecological changes of Glorieuses coral reefs (Iles Eparses, Western Indian Ocean)?

Ecological baselines for the structure and functioning of ecosystems in the absence of human activity can provide essential information on their health status. The Glorieuses islands are located in the Western Indian Ocean (WIO) and can be considered as "pristine" ecosystems that have not been subjected to anthropogenic pressure. Their nutrient context and the microbial assemblages were assessed by determining the abundance of heterotrophic prokaryotes (archaea and bacteria), picocyanobacteria, picoeukaryotes, microphytoplankton and protozooplankton communities in five stations, during two contrasted periods (November 2015 and May 2016). Chlorophyll-a concentrations were always under 1 µg/L and associated to very low levels in orthophosphates, nitrate and dissolved organic carbon, revealing an ultra-oligotrophic status for the Glorieuses waters. Picocyanobacteria confirmed the ultra-oligotrophic status with a predominance of Synechococcus. Zeaxanthin associated with the presence of picocyanobacteria represented the major pigment in both surveys. Three indices of diversity (species richness, Shannon and Pielou indexes) from microscopy observations highlighted the difference of diversity in microphytoplankton between the surveys. A focus on a 16S metabarcoding approach showed a high dominance of picocyanobacteria, Alpha- and Gammaproteobacteria, regardless of station or period. Multivariate analyses (co-inertia analyses) revealed a strong variability of ecological conditions between the two periods, with (i) high nutrient concentrations and heterotrophic nanoflagellate abundance in November 2015, and (ii) high heterotrophic prokaryote and picoeukaryote abundance in May 2016. The impact of a category 5 tropical cyclone (Fantala) on the regional zone in April 2016 is also advanced to explain these contrasted situations. Relative importance of top-down factors between bacterial and heterotrophic nanoflagellates was observed in November 2015 with an active microbial food web. All the results indicate that three microbial indexes potentially can be considered to assess the ecological change in Glorieuses marine waters.

In situ characterisation of pathogen dynamics during a Pacific oyster mortality syndrome episode.

Significant mortality of Crassostrea gigas juveniles is observed systematically every year worldwide. Pacific Oyster Mortality Syndrome (POMS) is caused by Ostreid Herpesvirus 1 (OsHV-1) infection leading to immune suppression, followed by bacteraemia caused by a consortium of opportunistic bacteria. Using an in-situ approach and pelagic chambers, our aim in this study was to identify pathogen dynamics in oyster flesh and in the water column during the course of a mortality episode in the Mediterranean Thau lagoon (France). OsHV-1 concentrations in oyster flesh increased before the first clinical symptoms of the disease appeared, reached maximum concentrations during the moribund phase and the mortality peak. The structure of the bacterial community associated with oyster flesh changed in favour of bacterial genera previously associated with oyster mortality including Vibrio, Arcobacter, Psychrobium, and Psychrilyobacter. During the oyster mortality episode, releases of OsHV-1 and opportunistic bacteria were observed, in succession, in the water surrounding the oyster lanterns. These releases may favour the spread of disease within oyster farms and potentially impact other marine species, thereby reducing marine biodiversity in shellfish farming areas.

Fifty years of ecological changes: Regime shifts and drivers in a coastal Mediterranean lagoon during oligotrophication.

Thau lagoon is a large Mediterranean coastal lagoons and it supports traditional shellfish farming activities. It has been subject to eutrophication leading to major anoxic events associated with massive mortalities of shellfish stocks. Since the 1970s, improvements have been made to wastewater treatment systems, which have gradually led to oligotrophication of the lagoon. The aim of our study was to determine how the decrease in nutrient inputs resulted in major ecological changes in Thau lagoon, by analysing five decades of time-series (1970-2018) of observations on pelagic and benthic autotrophic communities. We were able to identify two periods during the oligotrophication process. Period 1 (1970-1992) was considered a eutrophic period, characterised by the shift from seagrass dominance to dominance of red macroalgae. Period 2 (1993-2018), characterised by improved eutrophication status, was further divided into three: a transition phase (1993-2003) during which the water column continued to recover but the benthic community lagged behind in recovery and in partial resilience; a regime shift (2003-2006), after which the water column became oligotrophic and seagrass began to recover (2007-2018). Considering anoxia crises as indicators of ecosystem resilience and resistance, we used a generalised linear model to analyse meteorological and environmental data with the aim of identifying the triggers of summer anoxia over the study period. Among the meteorological variables studied, air temperature had the strongest positive effect, followed by the period and wind intensity (both negative effects) and by rainfall in July (positive effect). The risk of triggering anoxia was lower in period 2, evidence for the increasing resistance of the ecosystem to climatic stress throughout the oligotrophication process. At the ecosystem scale and in the long term perspective, the ecological gains related to oligotrophication are especially important in the context of climate change, with more frequent and severe heat waves predicted.

Evaluation of FluoroProbe® performance for the phytoplankton-based assessment of the ecological status of Mediterranean coastal lagoons.

The European Water Framework Directive and several other legislations worldwide have selected phytoplankton for monitoring the ecological status of surface waters. This assessment is a complicated task in coastal lagoons due to their intrinsic variability, prompting moves to use real-time measurements. Here, we tested the ability of the submersible spectrofluorometer FluoroProbe® to accurately estimate the phytoplankton biomass and to efficiently discriminate spectral groups in Mediterranean coastal lagoons, by using sub-surface water samples (n = 107) collected at Biguglia lagoon (Corsica) in different environmental situations (salinity and trophic state) from March 2012 to December 2014. We compared the estimates of biomass and phytoplankton group composition obtained with the FluoroProbe® (in situ and lab measurements) with the spectrofluorimetrically measured biomass and HPLC-derived quantifications of pigment concentrations. FluoroProbe® provided good estimates of the total phytoplankton biomass (particularly, the lab measurements). The FluoroProbe® data were significantly correlated with the HPLC results, except for the in situ measurements of very weak concentrations of blue-green and red algae. Our findings indicate that factory-calibrated FluoroProbe® is an efficient and easy-to-use real-time phytoplankton monitoring tool in coastal lagoons, especially as an early warning system for the detection of potentially harmful algal blooms. Practical instructions dedicated to non-specialist field operators are provided. A simple and efficient method for discarding in situ measurement outliers is also proposed.

Insights into the harmful algal flora in northwestern Mediterranean coastal lagoons revealed by pyrosequencing metabarcodes of the 28S rRNA gene.

This study investigated the genetic diversity of phytoplankton communities in six shallow lagoons located on the French coast of the northwestern Mediterranean Sea that represented a trophic gradient ranging from oligotrophic to hypereutrophic. The phytoplankton communities were sampled once a month from spring (May) to the beginning of autumn (September/early October) in 2012 and fractionated by size. Metabarcodes were generated from cDNAs by targeting the D1-D2 region of the 28S rRNA gene and pyrosequenced using Roche 454 technology. Examination of the annotated barcodes revealed harmful algal species not previously documented in these lagoons. Three ichthyotoxic species belonging to Pfiesteriaceae were detected: Luciella masanensis was relatively widespread and abundant in many samples, whereas Pfiesteria piscicida and Stoeckeria changwonensis were found as single barcode sequences. Furthermore, a phylogenetic analysis of barcodes annotated as belonging to Pfiesteriaceae suggested the existence of two previously undescribed clades. The other toxic or potentially harmful dinoflagellates detected through rare barcodes were Dinophysis acuminata, Vulcanodinium rugosum, Alexandrium andersonii and A. ostenfeldii. The two most abundant dinoflagellate taxa were Gymnodinium litoralis and Akashiwo sanguinea with respect to sequence numbers. Four diatom species from the genus Pseudo-nitzschia that potentially produce domoic acid were identified (P. galaxiae, P. delicatissima, P. brasiliana and P. calliantha). These observations are discussed in terms of the literature and monitoring records related to the identified taxa in this Mediterranean area.