Environmental DNA metabarcoding for benthic monitoring: A review of sediment sampling and DNA extraction methods.

Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring.

Expanding ecological assessment by integrating microorganisms into routine freshwater biomonitoring.

Bioindication has become an indispensable part of water quality monitoring in most countries of the world, with the presence and abundance of bioindicator taxa, mostly multicellular eukaryotes, used for biotic indices. In contrast, microbes (bacteria, archaea and protists) are seldom used as bioindicators in routine assessments, although they have been recognized for their importance in environmental processes. Recently, the use of molecular methods has revealed unexpected diversity within known functional groups and novel metabolic pathways that are particularly important in energy and nutrient cycling. In various habitats, microbial communities respond to eutrophication, metals, and natural or anthropogenic organic pollutants through changes in diversity and function. In this review, we evaluated the common trends in these changes, documenting that they have value as bioindicators and can be used not only for monitoring but also for improving our understanding of the major processes in lotic and lentic environments. Current knowledge provides a solid foundation for exploiting microbial taxa, community structures and diversity, as well as functional genes, in novel monitoring programs. These microbial community measures can also be combined into biotic indices, improving the resolution of individual bioindicators. Here, we assess particular molecular approaches complemented by advanced bioinformatic analysis, as these are the most promising with respect to detailed bioindication value. We conclude that microbial community dynamics are a missing link important for our understanding of rapid changes in the structure and function of aquatic ecosystems, and should be addressed in the future environmental monitoring of freshwater ecosystems.

Sensitive vs. tolerant Nitzschia palea (Kützing) W. Smith strains to atrazine: a biochemical perspective.

Organic contaminants, and herbicides in particular, represent a risk for aquatic ecosystems. The primary target of herbicides are producers, the base of food webs, but frequently they end up far from the application point affecting non-target species. Its presence can work as sub-lethal stimulus, which sort the genetic and phenotypic differences within a species. Intraspecific variation allows adaptation to changes in the environment but also to new niches due to variations in species' sensitivity and biochemical response to a certain chemical. A better understanding of these variations can lead to the development of improved strategies for ecosystem protection. This research aimed to compare a sensitive and a tolerant strain of the freshwater diatom Nitzschia palea to atrazine. Strains were exposed to three concentrations within their tolerance range, during 96 h. The activity of the antioxidant enzymes superoxide dismutase, catalase, glutathione-S-transferases and glutathione peroxidases was determined. In addition, chlorophylls a and c, carotenoids, reduced glutathione, proteins and lipid peroxidation were quantified. Both strains displayed different strategies to deal with atrazine toxicity: while the sensitive strain decreased the oxidative stress, increasing the activity of antioxidant enzymes such as superoxide dismutase, the tolerant strain invested in conjugation pathways and carotenoids' maintenance.

Comparative sensitivity to the fungicide tebuconazole of biofilm and plankton microbial communities in freshwater ecosystems.

Stream and lake ecosystems in agricultural watersheds are exposed to fungicide inputs that can threaten the structure and functioning of aquatic microbial communities. This research analyzes the impact of the triazole fungicide tebuconazole (TBZ) on natural biofilm and plankton microbial communities from sites presenting different degrees of agricultural contamination. Biofilm and plankton communities from less-polluted (LP) and polluted (P) sites were exposed to nominal concentrations of 0 (control), 2 and 20 µg TBZ L(-1) in 3-week microcosm experiments. Descriptors of microbial community structure (bacterial density and chlorophyll-a concentration) and function (bacterial respiration and production and photosynthesis) were analyzed to chart the effects of TBZ and the kinetics of TBZ attenuation in water during the experiments. The results showed TBZ-induced effects on biofilm function (inhibition of substrate-induced respiration and photosynthetic activity), especially in LP-site communities, whereas plankton communities experienced a transitory stimulation of bacterial densities in communities from both LP and P sites. TBZ attenuation was stronger in biofilm (60-75%) than plankton (15-18%) experiments, probably due to greater adsorption on biofilms. The differences between biofilm and plankton responses to TBZ were likely explained by differences in community structure (presence of extracellular polymeric substances (EPS) matrix) and microbial composition. Biofilm communities also exhibited different sensitivity levels according to their in-field pre-exposure to fungicide, with P-site communities demonstrating adaptation capacities to TBZ. This study indicates that TBZ toxicity to non-targeted aquatic microbial communities essentially composed by microalgae and bacteria was moderate, and that its effects varied between stream and lake microbial communities.

Next-generation sequencing to inventory taxonomic diversity in eukaryotic communities: a test for freshwater diatoms.

The recent emergence of barcoding approaches coupled to those of next-generation sequencing (NGS) has raised new perspectives for studying environmental communities. In this framework, we tested the possibility to derive accurate inventories of diatom communities from pyrosequencing outputs with an available DNA reference library. We used three molecular markers targeting the nuclear, chloroplast and mitochondrial genomes (SSU rDNA, rbcL and cox1) and three samples of a mock community composed of 30 known diatom strains belonging to 21 species. In the goal to detect methodological biases, one sample was constituted directly from pooled cultures, whereas the others consisted of pooled PCR products. The NGS reads obtained by pyrosequencing (Roche 454) were compared first to a DNA reference library including the sequences of all the species used to constitute the mock community, and second to a complete DNA reference library with a larger taxonomic coverage. A stringent taxonomic assignation gave inventories that were compared to the real one. We detected biases due to DNA extraction and PCR amplification that resulted in false-negative detection. Conversely, pyrosequencing errors appeared to generate false positives, especially in case of closely allied species. The taxonomic coverage of DNA reference libraries appears to be the most crucial factor, together with marker polymorphism which is essential to identify taxa at the species level. RbcL offers a high resolving power together with a large DNA reference library. Although needing further optimization, pyrosequencing is suitable for identifying diatom assemblages and may find applications in the field of freshwater biomonitoring.

Comparison of the effects of two herbicides and an insecticide on tropical freshwater plankton in microcosms.

Natural plankton communities from a tropical freshwater reservoir (Combani Reservoir, Mayotte Island, Mozambique Channel) were exposed, in 20-l nutrient-enriched microcosms, to two nominal concentrations of three pesticides: the herbicides diuron (2.2 and 11 µg/l) and paraquat (10 and 40.5 µg/l) and the insecticide fenitrothion (10 and 100 µg/l), commonly used in the tropics for agriculture and disease vector control. Bacterioplankton, phytoplankton, and zooplankton communities were monitored for 5 days after exposure, and the concentrations of toxicant and major nutrients were measured. Bacterioplankton growth was noticeable in all systems and was slightly affected by pesticide at any concentration. A transitory increase in thymidine-based bacterial production was observed in diuron- and fenitrothion-treated microcosms, followed by a marked decrease in all microcosms after 5 days. The functional diversity of bacterioplankton, evaluated using BIOLOG ECO(®) microplates, was reduced by exposure to the highest pesticide concentrations. Phytoplankton was affected by pesticides in different ways. Chlorophyll biomass and biovolumes were increased by diuron addition and decreased by paraquat, whereas fenitrothion-treated microcosms remained unaffected relative to controls. Phytoplankton taxonomic diversity was decreased by paraquat and high doses of fenitrothion but was unaffected by addition of diuron. The decrease in diversity was due to a reduction in the number of species, whereas the density of small cells increased, especially after addition of paraquat. Heterotrophic flagellates were sensitive to paraquat and to the highest diuron concentration; a reduction in biomass of up to 90% was observed for 40.5 µg/l paraquat. Zooplankton, dominated by Thermocyclops decipiens and Diaphanosoma excisum, was slightly sensitive to diuron, and very sensitive to paraquat. High concentrations of the insecticide fenitrothion were effective only on young stages. The potential direct and indirect effects of pesticide contamination on such a simplified plankton food web, typical of newly constructed reservoirs, appear to differ significantly depending on the biological compartment considered. The overall sensitivity of tropical plankton is comparable to the sensitivity for temperate systems, and direct and indirect effects appeared rapidly, within 5 days of exposure.

Genotoxic pressure of vineyard pesticides in fish: field and mesocosm surveys.

The present study deals with the genotoxicity assessment of vineyard pesticides in fish exposed in the field or in mesocosm conditions. Primary DNA damage was quantified as strand breaks using the single cell gel electrophoresis assay (Comet assay) applied to fish erythrocytes. In a first experiment, a significant genotoxic effect was observed following an upstream-downstream gradient in early life stages of brown trout (Salmo trutta fario) exposed in the Morcille River contaminated by a mixture of vineyard pesticides during three consecutive years. The pronounced response in terms of DNA damage reported in the present study could argue for a high sensitivity of fish early life stage and/or a high level of exposure to genotoxic compounds in the Morcille River. This stresses the interest in using trout larvae incubated in sediment bed to assess genotoxic compounds in the field. In a second experiment, adult European topminnow (Phoxinus phoxinus) were exposed in water running through artificial channels to a mixture of diuron and azoxystrobin, two of the main pesticides detected in the Morcille watershed. As compared with the unexposed channel, a 3-5-fold increase in the DNA damage was observed in fish exposed to chronic environmental pesticide concentrations (1-2 microg L(-1) for diuron and 0.5-1 microg L(-1) for axoxystrobin). A single 6h pulse of pesticide (14 microg L(-1) of diuron and 7 microg L(-1) of azoxystrobin) was applied to simulate transiently elevated chemical concentrations in the river following storm conditions. It did not increase genotoxicity. After a 1-month recovery period, DNA damage in exposed fish erythrocytes recovered to unexposed level, suggesting possible involvement of both repair mechanisms and cellular turnover in this transient response. This work highlights that vineyard treatment by pesticides and in particular diuron and azoxystrobin can represent a genotoxic threat to fish from contaminated watershed rivers.

The use of MapPop1.0 for choosing a QTL mapping sample from an advanced backcross population.

QTL detection is a good way to assess the genetic basis of quantitative traits such as the plant response to its environment, but requires large mapping populations. Experimental constraints, however, may require a restriction of the population size, risking a decrease in the quality level of QTL mapping. The purpose of this paper was to test if an advanced backcross population sample chosen by MapPop 1.0 could limit the effect of size restriction and improve the QTL detection when compared to random samples. We used the genotypic and phenotypic data obtained for 280 genotypes, considered as the reference population. The "MapPop sample" of 100 genotypes was first compared to the reference population, and genetic maps, genotypic and phenotypic data and QTL results were analysed. Despite the increase in donor allele frequency in the MapPop sample, this did not lead to an increase of the genetic map length or a biased phenotypic distribution. Three QTL among the 10 QTL found in the reference population were also detected in the MapPop sample. Next, the MapPop sample results were compared to those from 500 random samples of the same size. The main conclusion was that the MapPop software avoided the selection of biased samples and the detection of false QTL and appears particularly interesting to select a sample from an unbalanced population.

No oceans on Titan from the absence of a near-infrared specular reflection.

With its substantial atmosphere of nitrogen, hydrocarbons and nitriles, Saturn's moon Titan is a unique planetary satellite. Photochemical processing of the gaseous constituents produces an extended haze that obscures the surface. Soon after the Voyager fly-bys in 1980 and 1981 photochemical models led to the conclusion that there should be enough liquid methane/ethane/nitrogen to cover the surface to a depth of several hundred metres. Recent Earth-based radar echoes imply that surface liquid may be present at a significant fraction of the locations sampled. Here we present ground-based observations (at near-infrared wavelengths) and calculations showing that there is no evidence thus far for surface liquid. Combined with the specular signatures from radar observations, we infer mechanisms that produce very flat solid surfaces, involving a substance that was liquid in the past but is not in liquid form at the locations we studied.

The response of Jupiter's magnetosphere to an outburst on Io.

A 6-month-long monitoring campaign of the Io plasma torus and neutral cloud was conducted to determine the characteristics of their interaction. During the observations, a large outburst of material from Io-inferred to be caused by the eruption of a volcanic plume on Io-caused a transient increase in the neutral cloud and plasma torus masses. The response of the plasma torus to this outburst shows that the interaction between Io and Jupiter's magnetosphere is stabilized by a feedback mechanism in which increases in the plasma torus mass cause a nonlinear increase in loss from the plasma torus, limiting plasma buildup.

Evaluation of selection index: application to the choice of an indirect multitrait selection index for soybean breeding.

Selection indices can be used to predict one trait from information available on several traits in order to improve the prediction accuracy. Plant or animal breeders are interested in selecting only the best individuals, and need to compare the efficiency of different trait combinations in order to choose the index ensuring the best prediction quality for individual values. As the usual tools for index evaluation do not remain unbiased in all cases, we propose a robust way of evaluation by means of an estimator of the mean-square error of prediction (EMSEP). This estimator remains valid even when parameters are not known, as usually assumed, but are estimated. EMSEP is applied to the choice of an indirect multitrait selection index at the F5 generation of a classical breeding scheme for soybeans. Best predictions for precocity are obtained by means of indices using only part of the available information.