Freshwater Biomonitoring Research Articles

Cyprus’ diatom diversity and the association of environmental and anthropogenic influences for ecological assessment of rivers using DNA metabarcoding

Human activities are the leading cause of environmental impairments. Appropriate biomonitoring of ecosystems is needed to assess these activities effectively. In freshwater ecosystems, periphytic and epilithic biofilms have diatom assemblages. These assemblages respond rapidly to environmental changes, making diatoms valuable bioindicators. For this reason, freshwater biomonitoring programs are currently using diatoms (e.g., Water Framework Directive). In the past ten years, DNA metabarcoding coupled with next-generation sequencing and bioinformatics represents a complementary approach for diatom biomonitoring. In this study, this approach is used for the first time in Cyprus by considering the association of environmental and anthropogenic pressures to diatom assemblages. Statistical analysis was then applied to identify the environmental (i.e., river types, geo-morphological) and anthropogenic (i.e., physicochemical, human land-use pressures) variables’ role in the observed diatom diversity. Results indicate differences in diatom assemblages between intermittent and perennial rivers. Achnanthidium minutissimum was more abundant in intermittent rivers; whereas Amphora pediculus and Planothidium caputium in perennial ones. Additionally, we could demonstrate the correlation between nutrients (e.g., nitrogen, phosphorus), stations’ local characteristics (e.g., elevation), and land use activities on the observed differences in diatom diversity. Finally, we conclude that multi-stressors and anthropogenic pressures together as multiple stressors have a significant statistical relationship to the observed diatom diversity and play a pivotal role in determining Cyprus’ rivers’ ecological status.

Ethanol eDNA Reveals Unique Community Composition of Aquatic Macroinvertebrates Compared to Bulk Tissue Metabarcoding in a Biomonitoring Sampling Scheme

Freshwater ecosystems provide essential ecosystem services and support biodiversity; however, their water quality and biological communities are influenced by adjacent agricultural land use. Aquatic macroinvertebrates are commonly used as bioindicators of stream conditions in freshwater biomonitoring programs. Sorting benthic samples for molecular identification is a time-consuming process, and this study investigates the potential of ethanol-collected environmental DNA (eDNA) for metabarcoding macroinvertebrates, especially for common bioindicator groups. The objective of this study was to compare macroinvertebrate composition between paired bulk tissue and ethanol eDNA samples, as eDNA could provide a less time-consuming and non-destructive method of sampling macroinvertebrates. We collected benthic samples from streams in Ontario, Canada, and found that community composition varied greatly between sampling methods and that few taxa were shared between paired tissue and ethanol samples, suggesting that ethanol eDNA is not an acceptable substitute. It is unclear why we did not detect all the organisms that were preserved in the ethanol, or the origin of the DNA we did detect. Furthermore, we also detected no difference in community composition for bioindicator taxa due to surrounding land use or water chemistry, suggesting sites were similar in ecological condition.

Invasive species influence macroinvertebrate biomonitoring tools and functional diversity in British rivers

Abstract Biological invasions could have major implications for the management and conservation of freshwater systems if they lead to a misclassification of waterbodies. However, there is limited understanding of the sensitivity of existing biomonitoring tools to invasive species in rivers; and even less known regarding how they influence community taxonomic and functional measures. This research explores the response of freshwater macroinvertebrate communities to biological invasion using taxonomic and functional indices. Utilising a long‐term dataset (spanning 2000–2019, 5,988 samples) from rivers in England, the performance of four biomonitoring tools (WHPT, WHPT‐ASPT, LIFE and PSI) and two community functional indices (functional richness and redundancy) was examined before and after the colonisation of the invasive species, Dikerogammarus haemobaphes (Eichwald, 1841; Crustacea: Gammaridae). This species represents a recent (first record 2012) and highly successful invader, allowing its range expansion within waterbodies to be examined in detail. Spatial (national and basin level) and seasonal (spring and autumn) effects were investigated using a before–after control–impact (BACI) experimental framework and linear mixed effects models. Results indicated that invasion by D. haemobaphes resulted in significant reductions to the WHPT index and functional diversity metrics (richness and redundancy) while more subtle patterns were observed for other metrics. Analysis of seasonal and individual river basins (River Trent and R. Thames) identified largely consistent responses. The establishment of D. haemobaphes also resulted in some modifications to the functional composition of aquatic communities primarily associated with voltinism and resistance features. Synthesis and applications. Our findings indicate that Dikerogammarus haemobaphes should be considered a significant pressure to riverine communities. These results have implications for biomonitoring, which informs managerial actions as effects may not be detected using a single taxonomic index. Community functional measures are useful in characterising the effects of invasive species and may form a valuable part of the ‘toolbox’ used for studying biological invasions in rivers. The research illustrates the need to consider the wider threats posed by invasive species on the long‐term integrity of freshwaters and the efficacy of freshwater biomonitoring tools.

Freshwater diatom biomonitoring through benthic kick-net metabarcoding.

Biomonitoring is an essential tool for assessing ecological conditions and informing management strategies. The application of DNA metabarcoding and high throughput sequencing has improved data quantity and resolution for biomonitoring of taxa such as macroinvertebrates, yet, there remains the need to optimise these methods for other taxonomic groups. Diatoms have a longstanding history in freshwater biomonitoring as bioindicators of water quality status. However, multi-substrate periphyton collection, a common diatom sampling practice, is time-consuming and thus costly in terms of labour. This study examined whether the benthic kick-net technique used for macroinvertebrate biomonitoring could be applied to bulk-sample diatoms for metabarcoding. To test this approach, we collected samples using both conventional multi-substrate microhabitat periphyton collections and bulk-tissue kick-net methodologies in parallel from replicated sites with different habitat status (good/fair). We found there was no significant difference in community assemblages between conventional periphyton collection and kick-net methodologies or site status, but there was significant difference between diatom communities depending on site (P = 0.042). These results show the diatom taxonomic coverage achieved through DNA metabarcoding of kick-net is suitable for ecological biomonitoring applications. The shift to a more robust sampling approach and capturing diatoms as well as macroinvertebrates in a single sampling event has the potential to significantly improve efficiency of biomonitoring programmes that currently only use the kick-net technique to sample macroinvertebrates.

Characterization of Cu/Zn-SODs in sympatric species: A comparison of zebra and quagga mussels

Zebra mussels (Dreissena polymorpha) are commonly used as sentinel organisms in freshwater biomonitoring. The displacement of this species by quagga mussels (Dreissena rostriformis bugensis) has raised the issue of their discrimination after field sampling. Even if those two species exhibit specific morphological characteristics, confusion can occur. However, behind the visible phenotypes lie important physiological and metabolic differences that may affect the interpretation of biomarkers in risk assessment. In this study, we present a characterization of Cu/Zn-SODs (superoxide dismutases) in Dreissena spp. with a comparison of activity profiles from individuals originating from North America and Europe. A phylogenetic tree was constructed and dreissenid SOD sequences appear to be closely related to clam species sequences. A specific isoform at an isoelectric point value of 4.95 was observed only for quagga mussels independently of their origin, while zebra mussels exhibited several polymorphic profiles, even for a single sampling site. A comparison of transcript sequences was conducted and the observed differences were highlighted. Alignment of amino acid sequences shows a high level of identity between the two species associated with minor changes in the predicted secondary structures.

Assessment of stream macroinvertebrate communities with eDNA is not congruent with tissue-based metabarcoding.

Freshwater biomonitoring programmes routinely sample aquatic macroinvertebrates. These samples are time-consuming to collect, as well as challenging and costly to identify reliably genus or species. Environmental DNA (eDNA) metabarcoding has emerged as a surrogate to traditional collection techniques and has been used in whole-community approaches across several taxa and ecosystems. However, the usefulness of eDNA-based detection of freshwater macroinvertebrates has not been extensively explored. Few studies have directly compared bulk sample and eDNA metabarcoding at a local scale to assess how effective each method is at characterizing aquatic macroinvertebrate communities. Here, we collected both eDNA and kicknet samples at the same sample transect locations across nine different streams in southern Ontario, Canada. We observed minimal overlap in community composition between these paired samples. Bulk tissue metabarcoding resulted in a greater proportion of sequences belonging to metazoan taxa (over 99%) than eDNA (12%) and had higher OTU richness for macroinvertebrate taxa. We suggest that degenerate primers are not effective for eDNA metabarcoding due to the high degree of nontarget amplification and subsequently low yield of target DNA. While both bulk sample and eDNA metabarcoding had the power to detect differences between stream communities, eDNA did not represent local communities. Bulk tissue metabarcoding thus provides a more accurate representation of local stream macroinvertebrate communities and is the preferred method if smaller-scale spatial resolution is an important factor in data analyses.

Abruptly and irreversibly changing Arctic freshwaters urgently require standardized monitoring

Abstract Arctic regions support a wide variety of freshwater ecosystems. These naturally oligotrophic and cold‐water streams, rivers, ponds and lakes are currently being impacted by a diverse range of anthropogenic pressures, such as accelerated climate change, permafrost thaw, land‐use change, eutrophication, brownification and the replacement of northern biota with the range expansion of more southern species. Multiple stressors are rapidly changing Arctic freshwater systems as aquatic habitats are becoming more suitable for species originating from more southerly regions and thereby threatening biota adapted to cold waters. The livelihoods of Indigenous Peoples of the north will be altered when ecosystem services associated with changes in biodiversity are affected. Unfortunately, monitoring of biodiversity change in Arctic freshwaters is currently inadequate, making it difficult, if not impossible, to predict changes in ecosystem services. Synthesis and applications. We propose a three‐step approach to better address and facilitate monitoring of the rapid ecological changes that Arctic freshwater ecosystems are currently experiencing as a result of climate change. First, we should increase our efforts in the monitoring of freshwaters across all Arctic countries by setting up a network of monitoring sites and devoting more effort to a broad‐scale baseline survey using standardized methods. Second, we should enhance modelling efforts to include both ecological change and socio‐economic development. These models should help pinpoint species, ecosystems and geographical areas that are likely to show abrupt changes in response to any changes. Third, we should increase interaction among scientists, policymakers and different stakeholder groups. In particular, Indigenous Peoples must be involved in the leadership, planning and execution of monitoring and assessment activities of Arctic freshwaters. The proposed approach, which is critical to detecting the effects of climate change in the circumpolar region, has broader applications for global coordination of Arctic freshwater biomonitoring. Through routine monitoring, standardization of methods, enhanced modelling of integrated scientific and socio‐economic change, and increased collaboration within and among sectors, more effective monitoring and management of climate change impacts on freshwater biodiversity will be possible in the Arctic and globally.

Use of Aquatic Biota to Detect Ecological Changes in Freshwater: Current Status and Future Directions

Freshwater ecosystems have been severely damaged worldwide by a multitude of human pressures, such as pollution, nutrient enrichment, damming or overexploitation, and this has been more intense over the past five decades. It is therefore important that the impacts of such stressors can be effectively detected, monitored and assessed in order to provide adequate legislative tools and to protect and restore freshwater ecosystems. The use of aquatic biota to detect, measure and track changes in the environment is often known as freshwater biomonitoring and is based on the premise that the presence or absence of biotic assemblages at a given site reflects its degree of environmental quality. For over a century, since the early pollution-oriented indicators, freshwater monitoring has been developing and testing progressively more complex indicator systems, and increasing the plethora of pressures addressed, using different biological groups, such as benthic macroinvertebrates, macrophytes, fish, phytoplankton and phytobenthos. There is an increasing demand for precision and accuracy in bioassessment. In this Special Issue, five high-quality papers were selected and are briefly presented herein, that cover a wide range of issues and spatial contexts relevant to freshwater biomonitoring.

Behavioural and biochemical alterations in gammarids as induced by chronic metallic exposures (Cd, Cu and Pb): Implications for freshwater biomonitoring

In freshwater species, metal toxicity is usually assessed through short-term exposures, hence limiting the practical usefulness of biomarkers for monitoring long-term impacts on wildlife populations. This study investigates the biological alterations elicited by chronic metallic exposures in Gammarus fossarum using multi-level biomarkers. In aquaria, gammarids were exposed for 10 weeks to field-realistic concentrations of Cd, Cu or Pb (0.25, 1.5 or 5.0 μg/L). At the individual level, behavioural traits (respiration, locomotion and feeding) were compared between naive and chronically-exposed gammarids. At the cellular level, enzymatic activities involved in digestion, moult and cell stress were monitored after 2, 6 and 10 weeks of exposure in males and females to consider the temporal feature of their responses. Results showed that the inhibitory effects of Cd and Pb on respiration and locomotion disappeared in chronically-exposed gammarids, reflecting acclimation to maintain these processes, unlike Cu. Chronic Cu- and Pb-elicited feeding inhibition was associated with the inhibitions of digestion enzymes. Chitobiase was inhibited by Cu in males and, by Cd and Pb in females, suggesting gender-dependent disturbances in moulting. In both genders, Cd generated cellular stress by stimulating acidic phosphatase and peroxidase activities. To conclude, such cellular impairments and alterations in individual performances are likely to disturb individual growth, population dynamics and litter decomposition in the long-term. Besides, obtaining biological responses, common to metals or specific to a metal or a gender, supports the development of biomarkers highlighting long-term impacts of metals on the health of organisms and their associated ecological functions in natural environments.

Macroinvertebrate community responses to land use: a trait-based approach for freshwater biomonitoring in Mongolia

Land-use practices in Mongolia can lead to environmental degradation and consequently affect the structure and function of biological communities. The main aim of this study was to determine land-use effects on freshwater macroinvertebrate communities based on their response to grazing and mining, using a trait-based approach (TBA). The functional structure of macroinvertebrate communities was examined using 86 categories of 16 traits. A total of 13 physical and chemical variables were significantly different among the levels of land-use intensity. Significant declines in functional diversity were observed with increased land-use intensity. The community weighted mean of 19 trait categories for 11 traits varied significantly among different levels of land-use intensity. Traits were significantly explained by environmental variables across a land-use intensity gradient. Water temperature, gravel, nitrate, silt, and cobble were the main predictor variables and explained 28% of the total variance of the trait variation. The functional structure of the macroinvertebrate community was strongly related to environmental conditions. The TBA is an important method in assessing disturbance responses in freshwater communities of steppe and taiga regions, such as in Mongolia and other countries in Central Asia and will be useful in finding best management practices for conserving aquatic ecosystems.

Macroinvertebrate and Fish Community Metrics: Confounding Effects and Consistency over Time

Macroinvertebrate and fish metrics are an essential tool in freshwater biomonitoring. Yet, many well-known stream metrics do not perform well as indicators of anthropogenic stress in lakes and wetlands, and there is a need to better understand the role of potential confounding factors such as latitude or environmental variables acting at regional scales. We attempt to untangle the relationships among metrics and watershed stressors while accounting for potential confounding factors using hierarchical partitioning, and test metric consistency across time, focusing on the Laurentian Great Lakes wetland monitoring programs conducted 10 years apart. Our results show that many frequently used metrics have high temporal variability and are significantly affected by spatial factors, most notably richness-type metrics used in numerous regional monitoring studies. Only a few metrics (invasive fish species richness and relative abundance, mayfly family richness and the relative abundance of Ephemeroptera, Trichoptera, Sphaeriidae and Odonata) had slightly better and more consistent correlations with watershed land use. We suggest that explicit consideration of confounding factors is essential in the context of large-scale monitoring programs, and focus on the less general (such as habitat-specific) metrics may be a more promising approach.

First development and characterization of 27 novel microsatellite markers in the dobsonfly Neoneuromus ignobilis (Megaloptera: Corydalidae) at genome-scale level

The dobsonfly species Neoneuromus ignobilis Navas (Megaloptera: Corydalidae) is endemic to but widely distributed from eastern and southeastern Asia, being an important insect indicator for freshwater biomonitoring. At present, there is no report on the development of microsatellites of Megaloptera. Here, we developed 27 novel microsatellite markers of N. ignobilis from 850,920 candidate microsatellites with the stringent screening criteria considering the amplification success rate, the presence or absence of stutter peaks, the peak intensity, the polymorphism of the loci, the heterozygosity, and the number of alleles. The allele number of 27 microsatellite markers ranges from 3 to 12 with an average value of 6.19 per locus. The observed heterozygosity (HO) and expected heterozygosity (HE) revealed a range from 0.000 to 0.947 and 0.000 to 0.842, respectively. We constructed three panels (MP panel, most polymorphic; SS panel, most stringent strategy; ALL panel, total 27 microsatellite markers) and compared the analyses on population genetic diversity and structure. The result showed that the MP panel can significantly improve the analyses of individual assignment and genetic diversity. Accordingly, we advocate selecting the most polymorphic microsatellite marker for analyzing population genetics based on microsatellite data. The present work represents the first study on the microsatellite development of Megaloptera.

DNA damage detection by Comet Assay on Daphnia magna: Application in freshwater biomonitoring

Monitoring of water genotoxicity still remains underexploited in risk assessment. The present study aimed at standardizing and evaluating the sensitivity and applicability of the Comet Assay adapted for Daphnia magna in genotoxicological investigations in freshwater environments. Two sampling campaigns (2014–2015) were performed in the watercourses of a pilot basin located in the Parma district (Italy). Fourteen sampling stations with different Ecological Status and/or EBI values were selected, all with a good Chemical Status according to the EU–Water Framework Directive 2000/60. The Alkaline Comet Assay was performed on 48 h-aged daphnids exposed (24 h) to 23 water samples. In parallel, the acute toxicity test was carried out. Daphnids exposed to samples, collected upstream the main watercourses in non-impacted areas, showed low DNA migration (Tail Intensity percentage – TI% - in the range 2.97–13.21), similar to laboratory controls. An increase in genotoxicity (TI% in the range 20–40) proceeding from the mountain towards the plain area was observed, in agreement with the land uses and the ES/EBI values of the stations. The highest genotoxic damage was observed after exposure to samples from watercourses of the minor hydrographic network in the plain area and waterbodies receiving wastewater treatment plant outflows. A modified version of the Comet Assay able to identify the presence of genotoxins inducing DNA oxidative damage, after standardization, was applied to daphnids treated with waters from 4 selected monitoring stations. The presence of oxidative contaminants was detected downstream a wastewater treatment plant outflow.The Comet Assay on D. magna has proven to be sensitive and able to discriminate among differently impacted areas and might be applied routinely. The FPG-Comet proved to be able to highlight the presence of contaminants causing oxidative stress.In our knowledge, this is the first time that Comet Assay on Daphnia magna is successfully applied for freshwater monitoring.

Steps towards a more efficient use of chironomids as bioindicators for freshwater bioassessment: Exploiting eDNA and other genetic tools

• We present a synthesis of improving bioindication using chironomids. • Morphological approaches are needed to accurately delimit taxa. • Molecular biological approaches can help biomonitoring using chironomids. • Environmental DNA can inform on the diversity and distribution of chironomids. • Expression analysis can be used to detect non-lethal stressors. Chironomids are a formidable indicator taxon, as they are globally ubiquitous, can tolerate a wide range of environmental conditions, and can occur in high numbers in anthropogenically impaired environments where other indicator taxa are not encountered. Biomonitoring is frequently performed through analyzing species composition, abundance and morphology. However, these traditional approaches may be problematic, since indicator taxon morphology is often cryptic, and inadequately assigned species complexes may lead to generalized ecological tolerances. We argue that the use of chironomids in freshwater biomonitoring could be much improved if physiological responses to a given pollutant were measured in conjunction with precisely determined species, aided by molecular approaches such as is already done for some well known mesophilic and extremophile species in certain regions of the world. We explore molecular biological approaches that may prove useful for this purpose.

COI metabarcoding primer choice affects richness and recovery of indicator taxa in freshwater systems.

Mixed community or environmental DNA marker gene sequencing has become a commonly used technique for biodiversity analyses in freshwater systems. Many cytochrome c oxidase subunit I (COI) primer sets are now available for such work. The purpose of this study is to test whether COI primer choice affects the recovery of arthropod richness, beta diversity, and recovery of target assemblages in the benthos kick-net samples typically used in freshwater biomonitoring. We examine six commonly used COI primer sets on samples collected from six freshwater sites. Biodiversity analyses show that richness is sensitive to primer choice and the combined use of multiple COI amplicons recovers higher richness. Thus, to recover maximum richness, multiple primer sets should be used with COI metabarcoding. In ordination analyses based on community dissimilarity, samples consistently cluster by site regardless of amplicon choice or PCR replicate. Thus, for broadscale community analyses, overall beta diversity patterns are robust to COI marker choice. Recovery of traditional freshwater bioindicator assemblages such as Ephemeroptera, Trichoptera, Plectoptera, and Chironomidae as well as Arthropoda site indicators were differentially detected by each amplicon tested. This work will help future biodiversity and biomonitoring studies develop not just standardized, but optimized workflows that either maximize taxon-detection or the selection of amplicons for water quality or Arthropoda site indicators.

How freshwater biomonitoring tools vary sub‐seasonally reflects temporary river flow regimes

AbstractCharacterizing temporary river ecosystem responses to flow regimes is vital for conserving their biodiversity and the services they provide to society. However, freshwater biomonitoring tools rarely reflect community responses to hydrological variations or flow cessation events, and those available have not been widely tested within temporary rivers. This study examines two invertebrate biomonitoring tools characterizing community responses to different flow‐related properties: the “Drought Effect of Habitat Loss on Invertebrates” (DEHLI) and “Lotic‐invertebrate Index for Flow Evaluation” (LIFE), which, respectively reflect community responses to habitat and hydraulic properties associated with changing flow conditions. Sub‐seasonal (monthly) variations of LIFE and DEHLI were explored within two groundwater‐fed intermittent rivers, one dries sporadically (a flashy, karstic hydrology—River Lathkill) and the other dries seasonally (a highly buffered flow regime—South Winterbourne). Biomonitoring tools were highly sensitive to channel drying and also responded to reduced discharges in permanently flowing reaches. Biomonitoring tools captured ecological recovery patterns in the Lathkill following a supra‐seasonal drought. Some unexpected results were observed in the South Winterbourne where LIFE and DEHLI indicated relatively high‐flow conditions despite low discharges occurring during some summer months. This probably reflected macrophyte encroachment, which benefitted certain invertebrates (e.g., marginal‐dwelling taxa) and highlights the importance of considering instream habitat conditions when interpreting flow regime influences on biomonitoring tools. Although LIFE and DEHLI were positively correlated, the latter responded more clearly to drying events, highlighting that communities respond strongly to the disconnection of instream habitats as flows recede. The results highlighted short‐term ecological responses to hydrological variations and the value in adopting sub‐seasonal sampling strategies within temporary rivers. Findings from this study indicate the importance of establishing flow response guilds which group taxa that respond comparably to flow cessation events. Such information could be adopted within biomonitoring practices to better characterize temporary river ecosystem responses to hydrological variations.

Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring.

DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.

Differential activities of the multixenobiotic resistance mechanism in freshwater fishes inhabiting environments of Patagonia Argentina

Environmental impairment resulted from urbanizations can produce damage on freshwater species including strong physiological effects at individual or population level. The multixenobiotic resistance (MXR) is a defence mechanism which has been demonstrated in several aquatic organisms. The key mediators of MXR activity are ATP-binding cassette (ABC) proteins like P-glycoprotein (P-gp). This system protects aquatic organisms against the accumulation of xenobiotics by extruding them from cells in an energy-dependent manner. MXR has been pointed out as relevant in the ecotoxicological context and has been proposed as a biomarker for pollution assessment. Since fish species are common target in freshwater biomonitoring programs, the purpose of the study was to evaluate the MXR mechanism in native Hatcheria macraei (Patagonian catfish) and exotics Salmo trutta (brown trout), Oncorhynchus mykiss (rainbow trout) and Oncorhynchus tshawytscha (Chinook salmon) freshwater fishes widespread in Argentine Patagonia. We characterized the MXR mechanism using a combination of functional assays and Western blot analysis. Our results in different tissues such as liver, gills, muscle and epidermis indicate that the fishes studied have different species-specific levels of MXR activity, being gills and liver the tissues with greater detoxifying activity. Induction of MXR transport activity was also identified in liver tissue from rainbow trout from urban stream suggesting their suitability in the biomonitoring of aquatic environments subjected to urban contaminants.

Development and implementation of eco-genomic tools for aquatic ecosystem biomonitoring: the SYNAQUA French-Swiss program.

The effectiveness of environmental protection measures is based on the early identification and diagnosis of anthropogenic pressures. Similarly, restoration actions require precise monitoring of changes in the ecological quality of ecosystems, in order to highlight their effectiveness. Monitoring the ecological quality relies on bioindicators, which are organisms revealing the pressures exerted on the environment through the composition of their communities. Their implementation, based on the morphological identification of species, is expensive because it requires time and experts in taxonomy. Recent genomic tools should provide access to reliable and high-throughput environmental monitoring by directly inferring the composition of bioindicators' communities from their DNA (metabarcoding). The French-Swiss program SYNAQUA (INTERREG France-Switzerland 2017-2019) proposes to use and validate the tools of environmental genomic for biomonitoring and aims ultimately at their implementation in the regulatory bio-surveillance. SYNAQUA will test the metabarcoding approach focusing on two bioindicators, diatoms, and aquatic oligochaetes, which are used in freshwater biomonitoring in France and Switzerland. To go towards the renewal of current biomonitoring practices, SYNAQUA will (1) bring together different actors: scientists, environmental managers, consulting firms, and biotechnological companies, (2) apply this approach on a large scale to demonstrate its relevance, (3) propose robust and reliable tools, and (4) raise public awareness and train the various actors likely to use these new tools. Biomonitoring approaches based on such environmental genomic tools should address the European need for reliable, higher-throughput monitoring to improve the protection of aquatic environments under multiple pressures, guide their restoration, and follow their evolution.

In-field and in-vitro study of the moss Leptodictyum riparium as bioindicator of toxic metal pollution in the aquatic environment: Ultrastructural damage, oxidative stress and HSP70 induction.

This study evaluates the effects of toxic metal pollution in the highly contaminated Sarno River (South Italy), by using the aquatic moss Leptodictyum riparium in bags at 3 representative sites of the river. Biological effects were assessed by metal bioaccumulation, ultrastructural changes, oxidative stress, as Reactive Oxygen Species (ROS) production and Glutathione S-transferase (GST) activity, as well as Heat Shock Proteins 70 (HSP70s) induction. The results showed that L. riparium is a valuable bioindicator for toxic metal pollution of water ecosystem, accumulating different amounts of toxic metals from the aquatic environment. Toxic metal pollution caused severe ultrastructural damage, as well as increased ROS production and induction of GST and HSP70s, in the samples exposed at the polluted sites. To assess the role and the effect of toxic metals on L. riparium, were also cultured in vitro with Cd, Cr, Cu, Fe, Ni, Pb, Zn at the same concentrations as measured at the 3 sites. Ultrastructure, ROS, GST, and HSP70s resulted severely affected by toxic metals. Based on our findings, we confirm L. riparium as a model organism in freshwater biomonitoring surveys, and GST and HSP70s as promising biomarkers of metal toxicity.