Fungal identity mediates the impacts of multiple stressors on freshwater ecosystems.

Predicting how multiple anthropogenic stressors affect natural ecosystems is a major challenge in ecology. Freshwater ecosystems are threatened worldwide by multiple co-occurring stressors, which can affect aquatic biodiversity, ecosystem functioning and human wellbeing. In stream ecosystems, aquatic fungi play a crucial role in global biogeochemical cycles and food web dynamics, therefore, assessing the functional consequences of fungal biodiversity loss under multiple stressors is crucial. Here, a microcosm approach was used to investigate the effects of multiple stressors (increased temperature and nutrients, drying, and biodiversity loss) on three ecosystem processes: organic matter decomposition, fungal reproduction, and fungal biomass accrual. Net effects of stressors were antagonistic for organic matter decomposition, but additive for fungal reproduction and biomass accrual. Net effects of biodiversity were mainly positive for all processes, even under stress, demonstrating that diversity assures the maintenance of ecosystem processes. Fungal species displayed distinct contributions to each ecosystem process. Furthermore, species with negligible contributions under control conditions changed their role under stress, either enhancing or impairing the communities' performance, emphasizing the importance of fungal species identity. Our study highlights that distinct fungal species have different sensitivities to environmental variability and have different influence on the overall performance of the community. Therefore, preserving high fungal diversity is crucial to maintain fungal species with key ecosystem functions within aquatic communities in face of environmental change.

Current trends and mismatches on fungicide use and assessment of the ecological effects in freshwater ecosystems.

Despite increasing evidence of off-site ecological impacts of pesticides and policy efforts worldwide, pesticide use is still far from being ecologically sustainable. Fungicides are among the most sold classes of pesticides and are crucial to ensure global food supply and security. This study aimed to identify potential gaps of knowledge and mismatches between research and usage data of fungicides by: (i) systematizing the current trends in global sales of fungicides, focusing on the European context in particular (where they are proportionally important); (ii) reviewing the scientific literature on the impacts of synthetic fungicides on non-target freshwater organisms. Sales data revealed important global and regional asymmetries in the relative importance of fungicides and the preferred active ingredients. The literature review on the ecological effects of fungicides disclosed a mismatch between the most studied and the most sold substances, as well as a bias towards the use of single species assays with standard test organisms. To ensure a proper evaluation, risk scenarios should focus on a regional scale, and research agendas must highlight sensitive aquatic ecorreceptors and improve the crosstalk between analytical and sales data.

Land Cover Implications on Ecosystem Service Delivery: a Multi-Scenario Study of Trade-offs and Synergies in River Basins.

Land cover change scenarios hold far-reaching implications for ecosystem services (ES), highlighting the need for understanding the trade-offs and synergies underlying the provision of multiple ES. The insufficient knowledge of the mechanisms governing the relationships among multiple ES, along with the lack of information on trade-offs among ES under different scenarios, restricts the ability to provide effective information for decision-makers. To fill this gap, we assessed the interplay among six ES: climate regulation, habitat creating and maintaining species diversity, cultivated crops, regulation of the chemical condition of freshwaters by living processes (water quality), water yield, and control of erosion rates, within three river basins in northwest Portugal. We employed the InVEST to map the state of these ES in 2018, along with three projected land cover scenarios for 2050: business-as-usual, farmland return, and afforestation. Our findings indicated the business-as-usual scenario could lead to detrimental impacts on climate regulation, habitat creating and maintaining species diversity, and control of erosion rates. In contrast, the farmland return scenario showed less drastic decreases in habitat-creating and maintaining species diversity and control of erosion rates compared to the business-as-usual scenario. Afforestation emerged as the most favorable scenario, with a 13.6% increase in climate regulation and a 1.3% improvement in habitat-creating and maintaining species diversity. Cluster analysis allowed the identification of six levels of spatial synergies between ES, with regions of high forest cover showing extreme synergy and populated areas exhibiting the lowest levels of synergy, suggesting that a well-planned combination of these practices could yield substantial benefits for future ES provision. These results provide crucial insights for decision-makers to enhance ecosystem management and promote societal well-being. Importantly, our findings underscore the significance of considering multiple ES and their interrelationships in land use planning to achieve sustainable development objectives.

Eco-physiological Responses of Aquatic Fungi to Three Global Change Stressors Highlight the Importance of Intraspecific Trait Variability.

Anthropogenic change at a global scale is affecting life on Earth with impacts on freshwaters. Aquatic hyphomycetes are fungi that drive organic matter decomposition in freshwaters and mediate energy transfer to higher trophic levels. Intraspecific trait variability affects ecological processes and can account for species adaptations to environmental change. To ascertain how aquatic hyphomycetes respond to global change related stressors, we selected 20 strains (7 species), based on their co-occurrence in streams and phylogenetic relatedness. We measured fungal growth rates at different temperatures (7 levels), nutrient concentrations (6 levels) and medium moisture (6 levels). Our results indicate that all stressors affected fungal growth, and responses to nutrient enrichment and moisture were strain specific. Fungal responses to the stressors were not explained by their phylogenetic relatedness. In the absence of stressors, interspecific diversity best explained the variance in fungal traits, while the increase in the stress gradient increased the importance of intraspecific diversity.

ITS rDNA Barcodes Clarify Molecular Diversity of Aquatic Hyphomycetes.

Aquatic hyphomycetes are key microbial decomposers of allochthonous organic matter in freshwater ecosystems. Although their importance in carbon flow and food webs in streams is widely recognized, there are still gaps in our understanding of their molecular diversity and distribution patterns. Our study utilized the growing database of ITS rDNA barcodes of aquatic hyphomycetes (1252 sequences) and aimed to (i) produce new barcodes for some lesser-known taxa; (ii) clarify the taxonomic placement of some taxa at the class or order level, based on molecular data; and (iii) provide insights into the biogeographical origins of some taxa. This study increased the number of aquatic hyphomycete species with available ITS barcodes from 119 (out of ~300 species described) to 136. Phylogenetically, the 136 species were distributed between 2 phyla, 6 classes, and 10 orders of fungi. Future studies should strive to increase the database of ITS sequences, especially focusing on species with unclear phylogenetic relationships (incertae sedis) and with few sequences available. The geographical distribution of species with available ITS sequences included 50 countries from five continents, but 6 countries had more than 20 species associated, showing a bias toward the northern hemisphere, likely due to sampling bias.

Plastic Interactions with Pollutants and Consequences to Aquatic Ecosystems: What We Know and What We Do Not Know.

Plastics are a group of synthetic materials made of organic polymers and some additives with special characteristics. Plastics have become part of our daily life due to their many applications and uses. However, inappropriately managed plastic waste has raised concern regarding their ecotoxicological and human health risks in the long term. Due to the non-biodegradable nature of plastics, their waste may take several thousands of years to partially degrade in natural environments. Plastic fragments/particles can be very minute in size and are mistaken easily for prey or food by aquatic organisms (e.g., invertebrates, fishes). The surface properties of plastic particles, including large surface area, functional groups, surface topography, point zero charge, influence the sorption of various contaminants, including heavy metals, oil spills, PAHs, PCBs and DDT. Despite the fact that the number of studies on the biological effects of plastic particles on biota and humans has been increasing in recent years, studies on mixtures of plastics and other chemical contaminants in the aquatic environment are still limited. This review aims to gather information about the main characteristics of plastic particles that allow different types of contaminants to adsorb on their surfaces, the consequences of this adsorption, and the interactions of plastic particles with aquatic biota. Additionally, some missing links and potential solutions are presented to boost more research on this topic and achieve a holistic view on the effects of micro- and nanoplastics to biological systems in aquatic environments. It is urgent to implement measures to deal with plastic pollution that include improving waste management, monitoring key plastic particles, their hotspots, and developing their assessment techniques, using alternative products, determining concentrations of micro- and nanoplastics and the contaminants in freshwater and marine food-species consumed by humans, applying clean-up and remediation strategies, and biodegradation strategies.

Evidence of micro and macroplastic toxicity along a stream detrital food-chain.

Freshwater ecosystems are subjected to plastic extensive pollution because they are the direct link between plastic wastes and marine ecosystems. The aim of this study was to assess the impacts of different sizes of polyethylene plastics (micro: µPs and macroplastics: PBs) on freshwater decomposers of plant litter. We exposed leaf associated microbial assemblages to µPs (0.5 or 1.5 g L-1) and discs of PBs as follows: green plastic bags (PB-G) alone or in mixtures with transparent plastic bags (PB-Mix). Then, we conducted a feeding preference experiment with the invertebrate shredder Limnephilus sp. to assess their capacity to distinguish leaf discs from PB discs of the same size (12 mm). Leaf decomposition, activities of fungal enzymes and sporulation were inhibited by µPs and PB-Mix, and shifts in fungal community composition were observed. The invertebrate shredders preferred to feed on leaves treated with µPs avoiding those exposed to PB-G/PB-Mix. Our results demonstrated that plastics can have a direct effect on stream-dwelling microbial decomposers and an indirect effect on higher trophic levels (shredders), highlighting that trophic transfer is a route of plastic exposure. The plastic properties (size, concentration, colour) appear to influence plastic toxicity to microbes and shredders, indicating the importance of considering physicochemical properties when assessing their risks to freshwater ecosystems.

Antiparasitic potential of agrochemical fungicides on a non-target aquatic model (Daphnia × Metschnikowia host-parasite system).

Pesticides are a major anthropogenic threat to the biodiversity of freshwater ecosystems, having the potential to affect non-target aquatic organisms and disrupt the processes in which they intervene. Important knowledge gaps have been recognised concerning the ecological effects of synthetic fungicides on non-target symbiotic aquatic fungi and the ecological processes where they intervene. The goal of this work was to assess the influence of three commonly used fungicides (myclobutanil, metalaxyl and cymoxanil), which differ in their mode of action, on a host (the crustacean Daphnia magna) × parasite (the yeast Metschnikowia bicuspidata) experimental model. Using a set of life history experiments, we evaluated the effect of each fungicide on the outcome of this relationship (disease) and on the fitness of both host and parasite. Contrasting results were observed: (i) cymoxanil and metalaxyl were overall innocuous to host and parasite at the tested concentrations, although host reproduction was occasionally reduced in the simultaneous presence of parasite and fungicide; (ii) on the contrary, myclobutanil displayed a clear antifungal effect, decreasing parasite prevalence and alleviating infection signs in the hosts. This antiparasitic effect of myclobutanil was further investigated with a follow-up experiment that manipulated the timing of application of the fungicide, to understand which stage of parasite development was most susceptible: while myclobutanil did not interfere in the early stages of infection, its antifungal activity was clearly observable at a later stage of the disease (by impairing the production of transmission stages of the parasite). More research is needed to understand the broader consequences of this parasite-clearance effect, especially in face of increasing evidence that parasites are ecologically more important than their cryptic nature might suggest.

Can microplastics from personal care products affect stream microbial decomposers in the presence of silver nanoparticles?

Microplastics (MPs) are emerging contaminants of great concern due to their abundance and persistence over time in aquatic environments. However, studies on their impacts on freshwater organisms are scarce. In resemblance, silver nanoparticles (Ag-NPs) are incorporated into textiles and personal care products and are also classified as emerging contaminants. We used the leaf litter decomposition model system to investigate the effects of MPs from a commercially used personal care product, alone or in mixture with Ag-NPs, on the diversity and activities of freshwater microbial decomposers. We exposed stream microbial communities associated with leaf litter to increasing concentrations of MPs (polyethylene extracted from a personal care product; 100 µg L-1 up to 1 g L-1 5 concentrations plus 1 control) for 27 days in the absence or presence of Ag-NPs (0.1 mg L-1 and 1 mg L-1). The exposure to MPs, alone or in mixture with Ag-NPs, negatively affected fungal diversity and sporulation, with a reduction in leaf litter decomposition (Cohen's d > 1.5; r> 0.8; Bonferroni, P < 0.01). Shifts in community structure of sporulating fungi were observed, and effects were more pronounced in mixtures with Ag-NPs at the highest concentration. Mixtures of MPs with Ag-NPs (at the higher concentration) had the strongest impacts on extracellular enzymatic (ß-glucosidase, Cohen's d > 1; r > 0.5; phenol oxidase, Cohen's d > 1; r > 0.4) activities (ANOVAs, P < 0.05). Apart from sporulation rates, observed toxicity in mixtures was lower than that expected based on individual toxicity effects, mainly for higher concentrations (Bonferroni, P < 0.05). Our study provided evidence of the potential harmful effects of MPs, alone or in mixtures with Ag-NPs, on the activities of aquatic fungi and on a key ecosystem process, determinant to organic matter turnover in streams.

Elevated temperature may reduce functional but not taxonomic diversity of fungal assemblages on decomposing leaf litter in streams.

Mounting evidence points to a linkage between biodiversity and ecosystem functioning (B-EF). Global drivers, such as warming and nutrient enrichment, can alter species richness and composition of aquatic fungal assemblages associated with leaf-litter decomposition, a key ecosystem process in headwater streams. However, effects of biodiversity changes on ecosystem functions might be countered by the presumed high functional redundancy of fungal species. Here, we examined how environmental variables and leaf-litter traits (based on leaf chemistry) affect taxonomic and functional α- and ß-diversity of fungal decomposers. We analysed taxonomic diversity (DNA-fingerprinting profiles) and functional diversity (community-level physiological profiles) of fungal communities in four leaf-litter species from four subregions differing in stream-water characteristics and riparian vegetation. We hypothesized that increasing stream-water temperature and nutrients would alter taxonomic diversity more than functional diversity due to the functional redundancy among aquatic fungi. Contrary to our expectations, fungal taxonomic diversity varied little with stream-water characteristics across subregions, and instead taxon replacement occurred. Overall taxonomic ß-diversity was fourfold higher than functional diversity, suggesting a high degree of functional redundancy among aquatic fungi. Elevated temperature appeared to boost assemblage uniqueness by increasing ß-diversity while the increase in nutrient concentrations appeared to homogenize fungal assemblages. Functional richness showed a negative relationship with temperature. Nonetheless, a positive relationship between leaf-litter decomposition and functional richness suggests higher carbon use efficiency of fungal communities in cold waters.

Aquatic Hyphomycete Taxonomic Relatedness Translates into Lower Genetic Divergence of the Nitrate Reductase Gene.

Aquatic hyphomycetes are key microbial decomposers in freshwater that are capable of producing extracellular enzymes targeting complex molecules of leaf litter, thus, being crucial to nutrient cycling in these ecosystems. These fungi are also able to assimilate nutrients (e.g., nitrogen) from stream water, immobilizing these nutrients in the decomposing leaf litter and increasing its nutritional value for higher trophic levels. Evaluating the aquatic hyphomycete functional genetic diversity is, thus, pivotal to understanding the potential impacts of biodiversity loss on nutrient cycling in freshwater. In this work, the inter- and intraspecific taxonomic (ITS1-5.8S-ITS2 region) and functional (nitrate reductase gene) diversity of 40 aquatic hyphomycete strains, belonging to 23 species, was evaluated. A positive correlation was found between the taxonomic and nitrate reductase gene divergences. Interestingly, some cases challenged this trend: Dactylella cylindrospora (Orbiliomycetes) and Thelonectria rubi (Sordariomycetes), which were phylogenetically identical but highly divergent regarding the nitrate reductase gene; and Collembolispora barbata (incertae sedis) and Tetracladium apiense (Leotiomycetes), which exhibited moderate taxonomic divergence but no divergence in the nitrate reductase gene. Additionally, Tricladium chaetocladium (Leotiomycetes) strains were phylogenetically identical but displayed a degree of nitrate reductase gene divergence above the average for the interspecific level. Overall, both inter- and intraspecific functional diversity were observed among aquatic hyphomycetes.

Moniliophthora perniciosa, the Causal Agent of Cacao Witches' Broom Disease Is Killed in vitro by Saccharomyces cerevisiae and Wickerhamomyces anomalus Yeasts.

Cacao plantations from South America have been afflicted with the severe fungal disease known as Witches' Broom Disease (WBD), caused by the basidiomycete Moniliophthora perniciosa. Yeasts are increasingly recognized as good fungal biocides, although their application is still mostly restricted to the postharvest control of plant and fruit decay. Their possible utilization in the field, in a preharvest phase, is nevertheless promising, particularly if the strains are locally adapted and evolved and if they belong to species considered safe for man and the environment. In this work, a group of yeast strains originating from sugarcane-based fermentative processes in Brazil, the cacao-producing country where the disease is most severe, were tested for their ability to antagonize M. perniciosa in vitro. Wickerhamomyces anomalus LBCM1105 and Saccharomyces cerevisiae strains LBCM1112 from spontaneous fermentations used to produce cachaça, and PE2 widely used in Brazil in the industrial production of bioethanol, efficiently antagonized six strains of M. perniciosa, originating from several South American countries. The two fastest growing fungal strains, both originating from Brazil, were further used to assess the mechanisms underlying the yeasts' antagonism. Yeasts were able to inhibit fungal growth and kill the fungus at three different temperatures, under starvation, at different culture stages, or using an inoculum from old yeast cultures. Moreover, SEM analysis revealed that W. anomalus and S. cerevisiae PE2 cluster and adhere to the hyphae, push their surface, and fuse to them, ultimately draining the cells. This behavior concurs with that classified as necrotrophic parasitism/mycoparasitism. In particular, W. anomalus within the adhered clusters appear to be ligated to each other through roundish groups of fimbriae-like structures filled with bundles of microtubule-sized formations, which appear to close after cells detach, leaving a scar. SEM also revealed the formation of tube-like structures apparently connecting yeast to hypha. This evidence suggests W. anomalus cells form a network of yeast cells connecting with each other and with hyphae, supporting a possible cooperative collective killing and feeding strategy. The present results provide an initial step toward the formulation of a new eco-friendly and effective alternative for controlling cacao WBD using live yeast biocides.

Individual and mixed effects of anticancer drugs on freshwater rotifers: A multigenerational approach.

Human population growth has led to an increased release of chemical contaminants into aquatic environments. Emerging chemical contaminants (ECCs) are of increasing concern because they can affect non-target organisms in aquatic ecosystems. The application of anticancer drugs is increasing because of enhanced cancer rates and use of chemotherapy. We assessed the impacts of two widely used anticancer drugs known for their distinct modes of action, namely 5-fluorouracil (5-FU) and doxorubicin (DOX), on the freshwater rotifer Brachionus calyciflorus across generations. Rotifer mortality (24 h) and population growth (48 h) were assessed to determine initial lethal and sub-lethal effects. Exposure of rotifers to 5-FU (up to 200 mg L-1) did not cause mortality, while DOX caused mortality at high concentrations (EC50 = 15.6 mg L-1). Effects of 5-FU on population growth rate was higher than DOX (5-FU EC50 =10.49 µg L-1, DOX EC50 = 8.78 mg L-1). The effects of the drugs in binary mixture on population growth rates were dose dependent; significant antagonistic effects were found when 5-FU was present in the mixture at high concentrations. Finally, a transgenerational assay for five generations revealed that rotifers were able to recover their population growth rate after fourth generation when exposed to 5-FU; however, population became non-viable after the second generation of exposure to DOX. At the cellular level, accumulation of reactive oxygen species and plasma membrane damage were observed at EC10 and increased at EC50 for both drugs. After exposure of rotifers to 5-FU across generations, there were signs of oxidative stress recovery, as shown by a decrease in ROS accumulation and plasma membrane damage. Our results showed for the first time that the adverse effects of anticancer drugs on freshwater rotifer populations are drug and dose dependent and can persist or be attenuated along generations.

Importance of exposure route in determining nanosilver impacts on a stream detrital processing chain.

The commercial use and spread of silver nanoparticles (AgNPs) in freshwaters have greatly increased over the last decade. Both AgNPs and ionic silver (Ag+) released from nanoparticles are toxic to organisms and compromise ecosystem processes such as leaf litter decomposition. Yet little is known about how AgNPs affect multitrophic systems of interacting species. Furthermore, past work has focused on waterborne exposure with scarce attention given to effects mediated by the consumption of contaminated food. We assessed the importance of direct (via water) and indirect (via diet) AgNP exposure to a processing chain comprising leaf litter, fungi, a shredder (Gammarus pulex) and a collector (Habroleptoides confusa) in microcosms. Direct exposure to contaminated water for 15 days impaired microbial leaf decomposition by ∼50% and leaf-associated fungal biomass by ∼10%. Leaf consumption was reduced by ∼20% but only when G. pulex was exposed to silver via contaminated leaves. There was no effect on FPOM production. Ag + could impose oxidative stress on the shredders and collectors independent of exposure route, as indicated by increased catalase and glutathione S-transferase activities and decreased superoxide dismutase activity. The activity of a neuronal enzyme (cholinesterase) in collectors, but not shredders, also decreased by almost 50% when the animals were indirectly exposed to AgNP. Our results show that AgNPs and Ag+ may disrupt detrital processing chains through direct and indirect exposure routes, even at low concentrations. This highlights the importance of AgNP exposure pathways to interconnected stream biota and ecosystem processes for realistic assessments of risks to freshwater ecosystems.

Priority effects of stream eutrophication and assembly history on beta diversity across aquatic consumers, decomposers and producers.

Priority effects are stochastic processes that consider the influence of the order of arrival of species on community dynamics and structure. We evaluated the short-term effects of stream eutrophication and colonization time in freshwater benthic communities (primary producers - periphytic algae, decomposers - fungi, and consumers - macroinvertebrates) to test whether (i) beta diversity is higher in eutrophic streams due to priority effects driven by stochastic community formation processes (ecological drift or random dispersal), and (ii) in the early stages of colonization, priority effects drive the history of the formation and the initial establishment of the community in the stream, resulting in higher beta diversity. The present study was conducted in situ over 28 days in temperate streams along a trophic gradient, with colonization being evaluated every seven days. The study identified 84 species of alga, 43 families of macroinvertebrates, and 44 species of aquatic fungi. Our results demonstrated that deterministic processes were responsible for the formation of aquatic producers, while priority effects (stochasticity) were more important for the aquatic decomposers and consumers. In the case of the producers, beta diversity was highest in the hypertrophic stream, but did not vary significantly over colonization time. The beta diversity of the decomposers was highest in the hypertrophic stream and in the later stages of succession, due primarily to mechanisms of facilitation. The beta diversity of the consumers was lowest in the hypertrophic stream due primarily to the priority and inhibitory effects of the predominant groups, and highest at seven and 21 days of colonization. As these three taxonomic groups differ in their intrinsic biological characteristics, and in their functional role in the ecosystem, our short-term field study demonstrated that both stochastic and deterministic processes combine to influence the configuration of the community, and that the relative importance of the two processes varies systematically along a trophic gradient.

Can photocatalytic and magnetic nanoparticles be a threat to aquatic detrital food webs?

Freshwaters are likely to serve as reservoirs for engineered nanomaterials (ENMs) due to their accelerated unintentional release, increasing the relevance of assessing their impacts on aquatic biota and the ecosystem processes they drive. Stream-dwelling microbes, particularly fungi, and invertebrate shredders play an essential role in the decomposition of organic matter and transfer of energy to higher trophic levels. We assessed the impacts of two photocatalytic (nano-TiO2 and erbium doped nano-TiO2) and one magnetic (nano-CoFe2O4) ENMs on detrital-based food webs in freshwaters by exposing chestnut leaves, colonized by stream-dwelling microbes, to a series of concentrations (0.25-150 mg L-1) of these ENMs. Microbial decomposition and biomass of fungal communities, associated with leaves, were not affected by the ENMs. However, the activities of antioxidant enzymes of microbial decomposers were significantly (P < 0.05) stimulated by ENMs in a concentration-dependent way, suggesting oxidative stress in stream microbial communities. The stronger responses of these stress biomarkers against nano-TiO2 (increase upto 837.5% for catalase, 1546.8% for glutathione peroxidase and 1154.6% for glutathione S-transferase) suggest a higher toxicity of this ENM comparing to the others. To determine whether the effects could be transferred across trophic levels, the invertebrate shredder Sericostoma sp. was exposed to ENMs (1 and 50 mg L-1) for 5 days either via contaminated water or contaminated food (leaf litter). Leaf consumption rate by shredders decreased significantly (P < 0.05) with increasing concentrations of ENMs via food or water; the effects were more pronounced when exposure occurred via contaminated food (up to 99.3%, 90.7% and 90.3% inhibition by nano-Er:TiO2, nano-CoFe2O4 and nano-TiO2, respectively). Overall, the tested photocatalytic and magnetic ENMs can be harmful to microbial decomposers and invertebrate shredders further compromising detrital-based food webs in streams.

Transcriptomics reveals the action mechanisms and cellular targets of citrate-coated silver nanoparticles in a ubiquitous aquatic fungus.

Silver nanoparticles (AgNPs) are among the major groups of contaminants of emerging concern for aquatic ecosystems. The massive application of AgNPs relies on the antimicrobial properties of Ag, raising concerns about their potential risk to ecologically important freshwater microbes and the processes they drive. Moreover, it is still uncertain whether the effects of AgNPs are driven by the same mechanisms underlying those of Ag ions (Ag+). We employed transcriptomics to better understand AgNP toxicity and disentangle the role of Ag+ in the overall toxicity towards aquatic fungi. To that end, the worldwide-distributed aquatic fungus Articulospora tetracladia, that plays a central role in organic matter turnover in freshwaters, was selected and exposed for 3 days to citrate-coated AgNPs (∼20 nm) and Ag+ at concentrations inhibiting 20% of growth (EC20). Responses revealed 258 up- and 162 down-regulated genes upon exposure to AgNPs and 448 up- and 84 down-regulated genes under exposure to Ag+. Different gene expression patterns were found after exposure to each silver form, suggesting distinct mechanisms of action. Gene ontology (GO) analyses showed that the major cellular targets likely affected by both silver forms were the biological membranes. GO-based biological processes indicated that AgNPs up-regulated the genes involved in transport, nucleobase metabolism and energy production, but down-regulated those associated with redox and carbohydrate metabolism. Ag+ up-regulated the genes involved in carbohydrate and steroid metabolism, whereas genes involved in localization and transport were down-regulated. Our results showed, for the first time, distinct profiles of gene expression in aquatic fungi exposed to AgNPs and Ag+, supporting different modes of toxicity of each silver form. Also, our results suggest that Ag+ had a negligible role in the toxicity induced by AgNPs. Finally, our study highlights the power of transcriptomics in portraying the stress induced by different silver forms in organisms.

Effects of metal nanoparticles on freshwater rotifers may persist across generations.

Nanotechnology has become one of the fastest growing industries in the current century because nanomaterials (NMs) are present in an ever-expanding range of consumer products increasing the chance of their release into natural environments. In this study, the impacts of two metal nanoparticles (Ag-NPs and CuO-NPs) and their equivalent ionic forms (Ag+ and Cu2+) were assessed on the lentic freshwater rotifer Brachionus calyciflorus and on its ability to adapt and recover through generations. In our study, Ag-NPs and CuO-NPs inhibited the rotifer population growth rate and caused mortality at low concentrations (< 100 µg L-1). Ag-NPs and CuO-NPs decreased in the medium when organisms were present (48 h exposure: 51.1 % and 66.9 %, respectively), similarly Ag+ and Cu2+ also decreased from medium in presence of the organisms (48 h: 35.2 % and 47.3 %, respectively); although the metal concentrations removed from the medium were higher for nanoparticles than metal ions, metal ions showed higher effects then their respective nanoparticle forms. Rotifer populations exposed for 4 generations to the toxicants were able to recover the population growth rate, but some rotifers showed developmental delay and inability to reproduce even after the removal of the toxicants. Intracellular accumulation of reactive oxygen species as well as plasma membrane damage were found in the rotifers at concentrations corresponding to EC10 (Ag-NPs = 1.7 µg L-1, Ag+ = 4.5 µg L-1, CuO-NPs = 46.9 µg L-1, Cu2+ = 35 µg L-1) of the population growth rate. Our results showed, for the first time, that effects of metal nanoparticles and metal ions on rotifer populations may persist along several generations. This should be taken into account when assessing risks of metal nanoparticles in freshwaters.

Biochemical and functional responses of stream invertebrate shredders to post-wildfire contamination.

Forests in Mediterranean Europe including Portugal are highly susceptible to wildfires. Freshwaters are often exposed to post-wildfire contamination that contains several toxic substances, which may impose risk to freshwater organisms and ecosystem functions. However, knowledge on the impacts of post-wildfire runoffs from different origins on freshwater biota is scarce. In forest streams, invertebrate shredders have a major contribution to aquatic detrital-based food webs, by translocating energy and nutrients from plant-litter to higher trophic levels. We investigated the leaf consumption behaviour and the responses of oxidative and neuronal stress enzymatic biomarkers in the freshwater invertebrate shredder Allogamus ligonifer after short-term exposure (96 h) to post-wildfire runoff samples from Pinus and Eucalyptus plantation forests and stream water from a burnt catchment in Portugal. Chemical analyses indicated the presence of various metals and PAHs at considerable concentrations in all samples, although the levels were higher in the runoff samples from forests than in the stream water. The shredding activity was severely inhibited by exposure to increased concentrations of post-wildfire runoff samples from both forests. The dose-response patterns of enzymatic biomarkers suggest oxidative and neuronal stress in the shredders upon exposure to increasing concentrations of post-wildfire runoffs. The impacts were more pronounced for the runoffs from the burnt forests. Moreover, the response patterns suggest that the energy from the feeding activity of shredders might have contributed to alleviate the stress in A. ligonifer. Overall, the outcomes suggest that the post-wildfire contamination can induce sublethal effects on invertebrate shredders with impacts on key ecological processes in streams.