Morphological vs. DNA metabarcoding approaches for the evaluation of stream ecological status with benthic invertebrates: Testing different combinations of markers and strategies of data filtering.

Macroinvertebrate assemblages are the most common bioindicators used for stream biomonitoring, yet the standard approach exhibits several time-consuming steps, including the sorting and identification of organisms based on morphological criteria. In this study, we examined if DNA metabarcoding could be used as an efficient molecular-based alternative to the morphology-based monitoring of streams using macroinvertebrates. We compared results achieved with the standard morphological identification of organisms sampled in 18 sites located on 15 French wadeable streams to results obtained with the DNA metabarcoding identification of sorted bulk material of the same macroinvertebrate samples, using read numbers (expressed as relative frequencies) as a proxy for abundances. In particular, we evaluated how combining and filtering metabarcoding data obtained from three different markers (COI: BF1-BR2, 18S: Euka02 and 16S: Inse01) could improve the efficiency of bioassessment. In total, 140 taxa were identified based on morphological criteria, and 127 were identified based on DNA metabarcoding using the three markers, with an overlap of 99 taxa. The threshold values used for sequence filtering based on the "best identity" criterion and the number of reads had an effect on the assessment efficiency of data obtained with each marker. Compared to single marker results, combining data from different markers allowed us to improve the match between biotic index values obtained with the bulk DNA versus morphology-based approaches. Both approaches assigned the same ecological quality class to a majority (86%) of the site sampling events, highlighting both the efficiency of metabarcoding as a biomonitoring tool but also the need for further research to improve this efficiency.

Assessing the effects of silver nanoparticles on the ecophysiology of Gammarus roeseli.

Being part of the macrobenthic fauna, gammarids are efficient indicators of contamination of aquatic ecosystems by nanoparticles that are likely to sediment on the bottom. The present study investigates the effects of silver nanoparticles (nAg) on ecophysiological functions in Gammarus roeseli by using a realistic scenario of contamination. Indeed, an experiment was conducted during 72 h, assessing the effects of 5 silver nAg from 10 to 100 nm diluted at concentrations of maximum 5 µg L-1 in a natural water retrieved from a stream and supplemented with food. The measured endpoints in gammarids were survival, silver concentrations in tissues, consumption of oxygen and ventilation of gills. Additionally, a set of biomarkers of the energetic metabolism was measured. After a 72-h exposure, results showed a concentration-dependent increase of silver levels in G. roeseli that was significant for the smallest nAg size (10 nm). Ecophysiological responses in G. roeseli were affected and the most striking effect was a concentration-dependent increase in oxygen consumption especially for the smallest nAg (10 to 40 nm), whereas ventilation of gills by gammarids was not changed. The potential mechanisms underlying these findings are discussed. Thus, we demonstrated the very low exposure concentration of 0.5 µg L-1 for the small nAg size led to significant ecophysiological effects reinforcing the need to further investigate subtle effects on nanoparticles on aquatic organisms.

Cross-effects of nickel contamination and parasitism on zebra mussel physiology.

Aquatic organisms are exposed to pollution which may make them more susceptible to infections and diseases. The present investigation evaluated effects of nickel contamination and parasitism (ciliates Ophryoglena spp. and intracellular bacteria Rickettsiales-like organisms), alone and in combination, on biological responses of the zebra mussel Dreissena polymorpha, and also the infestation abilities of parasites, under laboratory controlled conditions. Results showed that after 48 h, more organisms were infected in nickel-exposed groups, which could be related to weakening of their immune system. Acting separately, nickel contamination and infections were already stressful conditions; however, their combined action caused stronger biological responses in zebra mussels. Our data, therefore, confirm that the parasitism in D. polymorpha represents a potential confounding factor in ecotoxicological studies that involve this bivalve.

The combination of chemical, structural, and functional indicators to evaluate the anthropogenic impacts on agricultural stream ecosystems.

Freshwater contamination by pesticides in agricultural landscapes is of increasing concern worldwide, with strong pesticide impacts on biodiversity, ecosystem functions, and ultimately human health (drinking water, fishing). In addition, the excessively large number of substances, as well as their low - and temporally variable - concentrations in water, make the chemical monitoring by grab sampling very demanding and not fully representative of the actual contamination. Tools that integrate temporal variations and that are ecologically relevant are clearly needed to improve the monitoring of freshwater contamination and assess its biological effects. Here, we studied pesticide contamination and its biological impacts in 10 stream sections (sites) belonging to 3 agricultural catchments in France. In each site, we deployed a combination of pesticide integrative samplers, biocenotic indicators based on benthic macroinvertebrates, and functional indicators based on leaf litter decomposition and associated fungal communities. The 3 approaches largely proved complementary: structural and functional indicators did not respond equally to different agricultural impacts such as pesticide contamination (as revealed by integrative samplers), nutrients, or oxygen depletion. Combining chemical, structural, and functional indicators thus seems an excellent strategy to provide a comprehensive picture of agricultural impacts on stream ecosystems.

Organic-matter decomposition as a bioassessment tool of stream functioning: A comparison of eight decomposition-based indicators exposed to different environmental changes.

Organic-matter decomposition has long been proposed as a tool to assess stream functional integrity, but this indicator largely depends on organic-matter selection. We assessed eight decomposition-based indicators along two well-known environmental gradients, a nutrient-enrichment gradient (0.2-1.4 mg DIN/L) in central Portugal and an acidification gradient (pH: 4.69-7.33) in north-eastern France to identify the most effective organic-matter indicator for assessing stream functional integrity. Functional indicators included natural leaf litter (alder and oak) in 10-mm and 0.5-mm mesh bags, commercial tea (Lipton green and rooibos teas in 0.25-mm mesh bags), wood sticks (wood tongue depressors) and cotton strips. Biotic indices based on benthic macroinvertebrates (IPtIN for Portugal and IBGN for France) were calculated to compare the effectiveness of structural and functional indicators in detecting stream impairment and to assess the relationship between both types of indicators. The effectiveness of organic-matter decomposition rates as a functional indicator depended on the stressor considered and the substrate used. Decomposition rates generally identified nutrient enrichment and acidification in the most acidic streams. Decomposition rates of alder and oak leaves in coarse-mesh bags, green and rooibos teas and wood sticks were positively related with pH. Only decomposition rates of rooibos tea and wood sticks were related with DIN concentration; decomposition rates along the nutrient-enrichment gradient were confounded by differences in shredder abundance and temperature among streams. Stream structural integrity was good to excellent across streams; the IPtIN index was unrelated to DIN concentration, while the IBGN index was positively related with pH. The relationships between decomposition rates and biotic indices were loose in most cases, and only decomposition rates of alder leaves in coarse-mesh bags and green tea were positively related with the IBGN. Commercial substrates may be a good alternative to leaf litter to assess stream functional integrity, especially in the case of nutrient enrichment.