Towards early-warning gene signature of Chlamydomonas reinhardtii exposed to Hg-containing complex media.

The potential of using gene expression signature as a biomarker of toxicants exposure was explored in the microalga Chlamydomonas reinhardtii exposed 2 h to mercury (Hg) as inorganic mercury (IHg) and methyl mercury (MeHg) in presence of copper (Cu) and Suwannee River Humic Acid (SRHA). Total cellular Hg (THg = IHg + MeHg) decreased in presence of SRHA for 0.7 nM IHg and 0.4 nM MeHg, but increased for 70 nM IHg exposure. In mixtures of IHg + MeHg and (IHg or MeHg) + Cu, SRHA decreased THg uptake, except for 0.7 nM IHg + 0.4 nM MeHg which was unchanged (p-value>0.05). In the absence of SRHA, 0.5 µM Cu strongly decreased intracellular THg concentration for 70 nM IHg, while it had no effect for 0.7 nM IHg and 0.4 nM MeHg. The expression of single transcripts was not correlated with measured THg uptake, but a subset of 60 transcripts showed signatures specific to the exposed metal(s) and was congruent with exposure concentration. Notably, the range of fold change values of this subset correlated with THg bioaccumulation with a two-slope pattern in line with [THg]intra/[THg]med ratios. Gene expression signature seems a promising approach to complement chemical analyses to assess bioavailability of toxicants in presence of other metals and organic matter.

Comparative study of Cu uptake and early transcriptome responses in the green microalga Chlamydomonas reinhardtii and the macrophyte Elodea nuttallii.

Microalgae are widely used as representative primary producers in ecotoxicology, while macrophytes are much less studied. Here we compared the bioavailability and cellular toxicity pathways of 2 h-exposure to 10-6 mol L-1 Cu in the macrophyte Elodea nuttallii and the green microalga Chlamydomonas reinhardtii. Uptake rate was similar but faster in the algae than in the macrophyte, while RNA-Sequencing revealed a similar number of regulated genes. Early-regulated genes were congruent with expected adverse outcome pathways for Cu with Gene Ontology terms including gene regulation, energy metabolism, transport, cell processes, stress, antioxidant metabolism and development. However, the gene regulation level was higher in E. nuttallii than in C. reinhardtii and several categories were more represented in the macrophyte than in the microalga. Moreover, several categories including oxidative pentose phosphate pathway (OPP), nitrate metabolism and metal handling were only found for E. nuttallii, whereas categories such as cell motility, polyamine metabolism, mitochondrial electron transport and tricarboxylic acid cycle (TCA) were unique to C. reinhardtii. These differences were attributed to morphological and metabolic differences and highlighted dissimilarities between a sessile and a mobile species. Our results highlight the efficiency of transcriptomics to assess early molecular responses in biota, and the importance of studying more aquatic plants for a better understanding on the impact and fate of environmental contaminants.

Molecular Effects, Speciation, and Competition of Inorganic and Methyl Mercury in the Aquatic Plant Elodea nuttallii.

Mercury (Hg) remains hazardous in aquatic environments because of its biomagnification in food webs. Nonetheless, Hg uptake and impact in primary producers is still poorly understood. Here, we compared the cellular toxicity of inorganic and methyl Hg (IHg and MeHg, respectively) in the aquatic plant Elodea nuttallii. IHg and MeHg regulated contigs involved in similar categories (e.g., energy metabolism, development, transport, secondary metabolism), but MeHg regulated more contigs, supporting a higher molecular impact than IHg. At the organism level, MeHg induced antioxidants, while IHg decreased chlorophyll content. The uptake of Hg and expression of a subset of contigs was subsequently studied in complex media. Measured uptake pointed to a contrasted impact of cell walls and copper (Cu) on IHg and MeHg. Using a speciation modeling, differences in uptake were attributed to the differences in affinities of IHg and MeHg to organic matter in relation to Cu speciation. We also identified a distinct gene expression signature for IHg, MeHg, and Cu, further supporting different molecular toxicity of these trace elements. Our data provided fundamental knowledge on IHg and MeHg uptake in a key aquatic primary producer and confirmed the potential of transcriptomics to assess Hg exposure in environmentally realistic systems.

Effects of two-hour exposure to environmental and high concentrations of methylmercury on the transcriptome of the macrophyte Elodea nuttallii.

The effects of two methylmercury (CH3Hg+, MeHg) concentrations, representative of environmental level and extreme contamination, were investigated on the macrophyte Elodea nuttallii during a 2h-exposure combining transcriptomic (RNA-Seq), physiological endpoints (pigment contents, activity of anti-oxidative stress enzymes) and bioaccumulation. Exposure to MeHg induced the up- and down-regulation of numerous genes (4389 and 16853 for 10ngL-1 and 10µgL-1 MeHg exposure, respectively) involved in sugar, amino acid and secondary metabolism (e.g. cinnamic acid, flavonoids) at both concentrations. Genes coding for photosynthesis, membrane integrity, metal homeostasis, water transport and anti-oxidative enzymes were additionally up- and down-regulated at the higher concentration. At the physiological level, exposure to both MeHg concentrations resulted in a strong increase of anthocyanin content in shoots. Chlorophyll content and antioxidant enzyme activities were unchanged. The data suggest that the macrophyte was able to efficiently cope with the stress resulting from MeHg exposure, possibly by using anthocyanin as anti-oxidant and S-rich amino acids (such as cysteine and methionine) as chelators. Transcriptomics analysis enabled gaining novel insights on molecular effects of MeHg in primary producers, which are one of the main entry pathway of hazardous MeHg in aquatic food webs.

Cellular toxicity pathways of inorganic and methyl mercury in the green microalga Chlamydomonas reinhardtii.

Contamination by mercury (Hg) is a worldwide concern because of Hg toxicity and biomagnification in aquatic food webs. Nevertheless, bioavailability and cellular toxicity pathways of inorganic (IHg) and methyl-Hg (MeHg) remain poorly understood. We analyzed the uptake, transcriptomic, and physiological responses in the microalga Chlamydomonas reinhardtii exposed to IHg or MeHg. Bioavailability of MeHg was up to 27× higher than for IHg. Genes involved in cell processes, energy metabolism and transport were dysregulated by both Hg species. Physiological analysis revealed an impact on photosynthesis and reduction-oxidation reaction metabolism. Nevertheless, MeHg dysregulated a larger number of genes and with a stronger fold-change than IHg at equivalent intracellular concentration. Analysis of the perturbations of the cell's functions helped to derive a detailed mechanistic understanding of differences in cellular handling of IHg and MeHg resulting in MeHg having a stronger impact. This knowledge is central for the prediction of impact of toxicants on organisms.

Transcriptomic approach for assessment of the impact on microalga and macrophyte of in-situ exposure in river sites contaminated by chlor-alkali plant effluents.

Water quality degradation is a worldwide problem, but risk evaluation of chronic pollution in-situ is still a challenge. The present study aimed to evaluate the potential of transcriptomic analyses in representative aquatic primary producers to assess the impact of environmental pollution in-situ: the microalga Chlamydomonas reinhardtii and the macrophyte Elodea nuttallii were exposed 2 h in the Babeni Reservoir of the Olt River impacted by chlor-alkali plant effluent release resulting in increased concentrations of Hg and NaCl in receiving water. The response at the transcriptomic level was strong, resulting in up to 5485, and 8700 dysregulated genes (DG) for the microalga and for the macrophyte exposed in the most contaminated site, respectively. Transcriptomic response was congruent with the concentrations of Hg and NaCl in the water of the impacted reservoir. Genes involved in development, energy metabolism, lipid metabolism, nutrition, and RedOx homeostasis were dysregulated during in-situ exposure of both organisms. In addition, genes involved in the cell motility of C. reinhardtii and development of the cell wall of E. nuttallii were affected. DG were in line with adverse outcome pathways and transcriptomic studies reported after exposure to high concentrations of Hg and NaCl under controlled conditions in the laboratory. Transcriptomic response provided a sensitive measurement of the exposure as well as hints on the tolerance mechanisms of environmental pollution, and is thus promising as an early-warning tool to assess water quality degradation.

Trophic fate of inorganic and methyl-mercury in a macrophyte-chironomid food chain.

Dietary transfer of mercury (Hg) is central for its effects on higher trophic animals, nonetheless, its driving parameters and characteristics are not well understood. Here we measured Hg species transfer (uptake) from the macrophyte Elodea nuttallii -mimicking tissues incorporation in sediments after decay- to Chironomus riparius. Methyl-Hg (MMHg) was more transferable than inorganic Hg (IHg) from plant's intracellular and cell wall compartments. After 10-d-long exposure, MMHg was predominantly found in MMHg form in the cytosolic compartment (S) of chironomids, while IHg showed similar concentrations in S and insoluble debris (P) compartments. After cessation of Hg species exposure (depuration), only MMHg resulted in a bioaccumulation factor >1. Toxicokinetics modelling indicated a demethylation of MMHg in the S fraction and its concomitant storage in the P fraction as IHg during both uptake and depuration, revealing an elimination and detoxification mechanism. Our data support that MMHg is more transferable than IHg to sensitive subcellular targets as well as bioavailable fraction in chironomids, in line with field studies showing higher MMHg transfer than IHg in food webs. Hence our data point out macrophytes as a potential Hg source to benthic food webs to be considered for enhancing aquatic environment protection during phytoremediation programs.

Elodea nuttallii exposure to mercury exposure under enhanced ultraviolet radiation: Effects on bioaccumulation, transcriptome, pigment content and oxidative stress.

The hypothesis that increased UV radiation result in co-tolerance to Hg toxicity in aquatic plants was studied at the physiological and transcriptomic level in Elodea nuttallii. At the transcriptomic level, combined exposure to UV+Hg enhanced the stress response in comparison with single treatments, affecting the expression level of transcripts involved in energy metabolism, lipid metabolism, nutrition, and redox homeostasis. Single and combined UV and Hg treatments dysregulated different genes but with similar functions, suggesting a fine regulation of the plant to stresses triggered by Hg, UV and their combination but lack of co-tolerance. At the physiological level, UV+Hg treatment reduced chlorophyll content and depleted antioxidative compounds such as anthocyanin and GSH/GSSG in E. nuttallii. Nonetheless, combined exposure to UV+Hg resulted in about 30% reduction of Hg accumulation into shoots vs exposure to Hg alone, which was congruent with the level of expression of several transporter genes, as well as the UV effect on Hg bioavailability in water. The findings of the present work underlined the importance of performing experimentation under environmentally realistic conditions and to consider the interplay between contaminants and environmental variables such as light that might have confounding effects to better understand and anticipate the effects of multiple stressors in aquatic environment.

Role of cellular compartmentalization in the trophic transfer of mercury species in a freshwater plant-crustacean food chain.

Mercury (Hg) represents an important risk for human health through the food webs contamination. Macrophytes bioaccumulate Hg and play a role in Hg transfer to food webs in shallow aquatic ecosystems. Nevertheless, the compartmentalization of Hg within macrophytes, notably major accumulation in the cell wall and its impact on trophic transfer to primary consumers are overlooked. The present work focusses on the trophic transfer of inorganic Hg (IHg) and monomethyl-Hg (MMHg) from the intracellular and cell wall compartments of the macrophyte Elodea nuttallii - considered a good candidate for phytoremediation - to the crustacean Gammarus fossarum. The results demonstrated that Hg accumulated in both compartments was trophically bioavailable to gammarids. Besides IHg from both compartments were similarly transferred to G. fossarum, while for MMHg, uptake rates were ∼2.5-fold higher in G. fossarum fed with the cell wall vs the intracellular compartment. During the depuration phase, Hg concentrations in G. fossarum varied insignificantly suggesting that both IHg and MMHg were strongly bound to biological ligands in the crustacean. Our data imply that cell walls have to be considered as an important source of Hg to consumers in freshwater food webs when developing procedures for enhancing aquatic environment protection during phytoremediation programs.

Environmental quality assessment of reservoirs impacted by Hg from chlor-alkali technologies: case study of a recovery.

Mercury (Hg) pollution legacy of chlor-alkali plants will be an important issue in the next decades with the planned phase out of Hg-based electrodes by 2025 within the Minamata convention. In such a context, the present study aimed to examine the extent of Hg contamination in the reservoirs surrounding the Oltchim plant and to evaluate the possible improvement of the environmental quality since the closure of its chlor-alkali unit. This plant is the largest chlor-alkali plant in Romania, which partly switched to Hg-free technology in 1999 and definitely stopped the use of Hg electrolysis in May 2012. Total Hg (THg) and methylmercury (CH3Hg) concentrations were found to decrease in the surface waters and sediments of the reservoirs receiving the effluents of the chlor-alkali platform since the closure of Hg units. Hence, calculated risk quotients (RQ) indicated no adverse effect of Hg for aquatic organisms from the ambient water exposure. RQ of Hg in sediments were mostly all higher than 1, showing important risks for benthic organisms. However, ecotoxicity testing of water and sediments suggest possible impact of other contaminants and their mixtures. Hg hotspots were found in soils around the platform with RQ values much higher than 1. Finally, THg and CH3Hg concentrations in fish were below the food safety limit set by the WHO, which contrasts with previous measurements made in 2007 revealing that 92 % of the studied fish were of high risk of consumption. Discontinuing the use of Hg electrodes greatly improved the surrounding environment of chlor-alkali plants within the following years and led to the decrease environmental exposure to Hg through fish consumption. However, sediment and soil still remained highly contaminated and problematic for the river reservoir management. The results of this ecological risk assessment study have important implications for the evaluation of the benefits as well as limits of the Minamata Convention implementation.

Transcriptomic and Physiological Responses of the Green Microalga Chlamydomonas reinhardtii during Short-Term Exposure to Subnanomolar Methylmercury Concentrations.

The effects of short-term exposure to subnanomolar methyl-mercury (MeHg) concentrations, representative of contaminated environments, on the microalga Chlamydomonas reinhardtii were assessed using both physiological end points and gene expression analysis. MeHg bioaccumulated and induced significant increase of the photosynthesis efficiency, while the algal growth, oxidative stress, and chlorophyll fluorescence were unaffected. At the molecular level, MeHg significantly dysregulated the expression of genes involved in motility, energy metabolism, lipid metabolism, metal transport, and antioxidant enzymes. Data suggest that the cells were able to cope with subnanomolar MeHg exposure, but this tolerance resulted in a significant cost to the cell energy and reserve metabolism as well as ample changes in the nutrition and motility of C. reinhardtii. The present results allowed gaining new insights on the effects and uptake mechanisms of MeHg at subnanomolar concentrations in aquatic primary producers.