Differential reaction norms to ocean acidification in two oyster species from contrasting habitats.

Ocean acidification (OA), a consequence of the increase in anthropogenic emissions of carbon dioxide, causes major changes in the chemistry of carbonates in the ocean with deleterious effects on calcifying organisms. The pH/PCO2 range to which species are exposed in nature is important to consider when interpreting the response of coastal organisms to OA. In this context, emerging approaches, which assess the reaction norms of organisms to a wide pH gradient, are improving our understanding of tolerance thresholds and acclimation potential to OA. In this study, we deciphered the reaction norms of two oyster species living in contrasting habitats: the intertidal oyster Crassostrea gigas and the subtidal flat oyster Ostrea edulis, which are two economically and ecologically valuable species in temperate ecosystems. Six-month-old oysters of each species were exposed in common garden tanks for 48 days to a pH gradient ranging from 7.7 to 6.4 (total scale). Both species were tolerant down to a pH of 6.6 with high plasticity in fitness-related traits such as survival and growth. However, oysters underwent remodelling of membrane fatty acids to cope with decreasing pH along with shell bleaching impairing shell integrity and consequently animal fitness. Finally, our work revealed species-specific physiological responses and highlights that intertidal C. gigas seem to have a better acclimation potential to rapid and extreme OA changes than O. edulis. Overall, our study provides important data about the phenotypic plasticity and its limits in two oyster species, which is essential for assessing the challenges posed to marine organisms by OA.

Pacific oysters do not compensate growth retardation following extreme acidification events.

Ocean acidification caused by anthropogenic carbon dioxide emissions alters the growth of marine calcifiers. Although the immediate effects of acidification from global ocean models have been well studied on calcifiers, their recovery capacity over a wide range of pH has never been evaluated. This aspect is crucial because acidification events that arise in coastal areas can far exceed global ocean predictions. However, such acidification events could occur transiently, allowing for recovery periods during which the effects on growth would be compensated, maintained or amplified. Here we evaluated the recovery capacity of a model calcifier, the Pacific oyster Crassostrea gigas. We exposed juveniles to 15 pH conditions between 6.4 and 7.8 for 14 days. Oyster growth was retarded below pH 7.1 while shells were corroded at pH 6.5. We then placed the oysters under ambient pH > 7.8 for 42 days. Growth retardation persisted at pH levels below pH 7.1 even after the stress was removed. However, despite persistent retardation, growth has resumed rapidly suggesting that the oysters can recover from extreme acidification. Yet we found that the differences in individual weight between pH conditions below 7.1 increased over time, and thus the growth retardation cannot be compensated and may affect the fitness of the bivalves.

Molecular and phenotypic effects of early exposure to an environmentally relevant pesticide mixture in the Pacific oyster, Crassostrea gigas.

Early life stages are crucial for organism development, especially for those displaying external fertilization, whose gametes and early stages face environmental stressors such as xenobiotics. The pacific oyster, Crassostrea gigas, is considered a model species in ecotoxicology because of its ecological characteristics (benthic, sessile, filter feeding). So far studies have investigated the impact of xenobiotics at embryotoxic, genotoxic and physiological endpoints, sometimes at the multigenerational scale, highlighting the role of epigenetic mechanisms in transmitting alterations induced by exposure to single xenobiotics. However, to date, little is known about the impact of environmentally-mimicking contaminants cocktails. Thus, we examined the impact of an early exposure to environmentally relevant mixture on the Pacific oyster life history. We studied transcriptomic, epigenetic and physiological alterations induced in oysters exposed to 18 pesticides and metals at environmental concentration (nominal sum concentration: 2.85 µg.L-1, measured sum concentration: 3.74 ± 0.013 µg.L-1) during embryo-larval stage (0-48 h post fertilization, hpf). No significant differences in embryo-larval abnormalities at 24 hpf were observed during larval and spat rearing; the swimming behaviour of exposed individuals was disturbed, while they were longer and heavier at specific time points, and exhibited a lower epinephrine-induced metamorphosis rate as well as a higher survival rate in the field. In addition, RNA-seq analyses of gastrula embryos revealed the differential expression of development-related genes (e.g. Hox orthologues and cell cycle regulators) between control and exposed oysters. Whole-genome DNA methylation analyses demonstrated a significant modification of DNA methylation in exposed larvae marked by a demethylation trend. Those findings suggest that early exposure to an environmentally relevant pesticide mixture induces multi-scale latent effects possibly affecting life history traits in the Pacific oyster.

Responses of early life stages of European abalone (Haliotis tuberculata) to ocean acidification after parental conditioning: Insights from a transgenerational experiment.

CO2 absorption is leading to ocean acidification (OA), which is a matter of major concern for marine calcifying species. This study investigated the effects of simulated OA on the reproduction of European abalone Haliotis tuberculata and the survival of its offspring. Four-year-old abalone were exposed during reproductive season to two relevant OA scenarios, ambient pH (8.0) and low pH (7.7). After five months of exposure, abalone were induced to spawn. The gametes, larvae and juveniles were then exposed for five months to the same pH conditions as their parents. Several biological parameters involved in adult reproduction as well as in larval, post-larval and juvenile fitness were measured. No effects on gametes, fertilisation or larval oxidative stress response were detected. However, developmental abnormalities and significant decreases in shell length and calcification were observed at veliger stages. The expression profile of a GABA A receptor-like gene appeared to be regulated by pH, depending on larval stage. Larval and post-larval survival was not affected by low pH. However, a lower survival and a reduction of growth were recorded in juveniles at pH 7.7. Our results confirm that OA negatively impacts larval and juvenile fitness and suggest the absence of carry-over effects on abalone offspring. This may compromise the survival of abalone populations in the near future.

Revisiting tolerance to ocean acidification: Insights from a new framework combining physiological and molecular tipping points of Pacific oyster.

Studies on the impact of ocean acidification on marine organisms involve exposing organisms to future acidification scenarios, which has limited relevance for coastal calcifiers living in a mosaic of habitats. Identification of tipping points beyond which detrimental effects are observed is a widely generalizable proxy of acidification susceptibility at the population level. This approach is limited to a handful of studies that focus on only a few macro-physiological traits, thus overlooking the whole organism response. Here we develop a framework to analyze the broad macro-physiological and molecular responses over a wide pH range in juvenile oyster. We identify low tipping points for physiological traits at pH 7.3-6.9 that coincide with a major reshuffling in membrane lipids and transcriptome. In contrast, a drop in pH affects shell parameters above tipping points, likely impacting animal fitness. These findings were made possible by the development of an innovative methodology to synthesize and identify the main patterns of variations in large -omic data sets, fitting them to pH and identifying molecular tipping points. We propose the broad application of our framework to the assessment of effects of global change on other organisms.

Combined effects of ocean warming and acidification on the larval stages of the European abalone Haliotis tuberculata.

This study examined the physiological responses of the larval stages of Haliotis tuberculata, an economically important abalone, to combined temperature (17 °C and 19 °C) and pH (ambient pH and -0.3 units, i.e., +200% increase in seawater acidity) in a full factorial experiment. Tissue organogenesis, shell formation, and shell length significantly declined due to low pH. High temperature significantly increased the proportion of fully shelled larvae at 24 h post-fertilization (hpf), but increased the proportion of unshelled larvae at 72 hpf. Percentage of swimming larvae at 24 hpf, 72 hpf and 96 hpf significantly declined due to high temperature, but not because of low pH. Larval settlement increased under high temperature, but was not affected by low pH. Despite the fact that no interaction between temperature and pH was observed, the results provide additional evidence on the sensitivity of abalone larvae to both low pH and high temperature. This may have negative consequences for the persistence of abalone populations in natural and aquaculture environments in the near future.

Impact of magnetic fields generated by AC/DC submarine power cables on the behavior of juvenile European lobster (Homarus gammarus).

The number of submarine power cables using either direct or alternating current is expected to increase drastically in coming decades. Data concerning the impact of magnetic fields generated by these cables on marine invertebrates are scarce. In this context, the aim of this study was to explore the potential impact of anthropogenic static and time-varying magnetic fields on the behavior of recently settled juvenile European lobsters (Homarus gammarus) using two different behavioral assays. Day-light conditions were used to stimulate the sheltering behavior and facilitate the video tracking. We showed that juvenile lobsters did not exhibit any change of behavior when submitted to an artificial magnetic field gradient (maximum intensity of 200 µT) compared to non-exposed lobsters in the ambient magnetic field. Additionally, no influence was noted on either the lobsters' ability to find shelter or modified their exploratory behavior after one week of exposure to anthropogenic magnetic fields (225 ±â€¯5 µT) which remained similar to those observed in control individuals. It appears that static and time-varying anthropogenic magnetic fields, at these intensities, do not significantly impact the behavior of juvenile European lobsters in daylight conditions. Nevertheless, to form a complete picture for this biological model, further studies are needed on the other life stages as they may respond differently.

Nanoplastics impaired oyster free living stages, gametes and embryos.

In the marine environment, most bivalve species base their reproduction on external fertilization. Hence, gametes and young stages face many threats, including exposure to plastic wastes which represent more than 80% of the debris in the oceans. Recently, evidence has been produced on the presence of nanoplastics in oceans, thus motivating new studies of their impacts on marine life. Because no information is available about their environmental concentrations, we performed dose-response exposure experiments with polystyrene particles to assess the extent of micro/nanoplastic toxicity. Effects of polystyrene with different sizes and functionalizations (plain 2-µm, 500-nm and 50-nm; COOH-50 nm and NH2-50 nm) were assessed on three key reproductive steps (fertilization, embryogenesis and metamorphosis) of Pacific oysters (Crassostrea gigas). Nanoplastics induced a significant decrease in fertilization success and in embryo-larval development with numerous malformations up to total developmental arrest. The NH2-50 beads had the strongest toxicity to both gametes (EC50 = 4.9 µg/mL) and embryos (EC50 = 0.15 µg/mL), showing functionalization-dependent toxicity. No effects of plain microplastics were recorded. These results highlight that exposures to nanoplastics may have deleterious effects on planktonic stages of oysters, presumably interacting with biological membranes and causing cyto/genotoxicity with potentially drastic consequences for their reproductive success.

Toxicity assessment of five emerging pollutants, alone and in binary or ternary mixtures, towards three aquatic organisms.

Despite a growing scientific attention on ecological impact of emerging pollutants (EPs) such as pharmaceuticals, personal care products, and pesticides, knowledge gaps remain regarding mixture toxicity and effects on aquatic organisms. Several EPs were screened in seawater (Normandy, France), and the ecotoxicity of five compounds, chosen on their occurrence in ecosystems and use worldwide, was assessed and were the biocides methylparaben (MP) and triclosan (TCS), a pesticide degradation product (AMPA), and the pharmaceuticals venlafaxine (VEN) and carbamazepine (CBZ). The acute or sub-chronic toxicity, alone or in binary/ternary mixtures of three of them (CBZ, AMPA, and MP), was assessed on one marine and two freshwater organisms: Crassostrea gigas, Pseudokirchneriella subcapitata, and Daphnia magna. TCS and AMPA were, respectively, the most (EC50 < 1 mg L-1) and the least (EC50 > 50 mg L-1) toxic chemicals for the four endpoints (algal growth inhibition, daphnia immobilization, oyster embryotoxicity, and metamorphosis). The anxiolytic VEN (EC50 < 1 mg L-1) was particularly toxic to oyster larvae showing sensitivity difference between freshwater and marine organisms. If all the mixtures appeared to be in the same range of toxicity, the joint-toxic effects mainly led to synergistic or antagonistic interactions compared to single-compound toxicity. The data also highlighted species-dependent differing models of toxicity and underscored the need for an awareness of cocktail effects for better ecological risk assessment.

The antidepressant venlafaxine may act as a neurodevelopmental toxicant in cuttlefish (Sepia officinalis).

The Serotonin/Norepinephrine Reuptake Inhibitor (SNRI) antidepressant venlafaxine (VEN, Effexor(®)) has become one of the most common antidepressants detected in North American and European streams. Mammalian research has established that VEN exposure is associated with a range of structural, neurochemical, and functional alterations of the brain in adults and newborns. However, the neurodevelopmental effects of VEN on non-target organisms have never been investigated. The aim of our research was to decrease this gap in knowledge by characterizing the effects of VEN exposure on a cephalopod mollusk, the common cuttlefish Sepia officinalis. This species inhabits VEN-contaminated waters and possesses an unusually sophisticated brain. These characteristics render it a unique invertebrate species for studying the neurodevelopmental effects of VEN. Cuttlefish were exposed to environmentally-relevant concentrations of VEN (Measured concentrations ≈5 and 100ngL(-)(1)) or to filtered natural seawater (control) in a closed-loop system with regular water changes during the first 20days after hatching. We evaluated brain maturation as well as neurochemical changes and behavioral performances during this critical period of development. Our results show that both VEN-exposed groups exhibited a decrease in norepinephrine levels, along with a reduction in the relative number of glutamate NMDA-like receptors binding sites in the group exposed to 5ngL(-1) of VEN after 20days of exposure. Brain regional changes in cellular proliferation were observed in VEN-exposed groups in the vertical lobe (i.e. a key structure involved in cognitive processes) and in the optic lobes (i.e. main visual processing centers) in the absence of significant change in their volume. Along with these neurodevelopmental changes, 20days of exposure to 100ngL(-1) of VEN was associated with a decrease in camouflage ability. Overall, our study suggests that VEN is a neurodevelopmental toxicant in non-target aquatic organisms at environmentally-relevant concentrations.

Receptors rather than signals change in expression in four physiological regulatory networks during evolutionary divergence in threespine stickleback.

The molecular mechanisms underlying behavioural evolution following colonization of novel environments are largely unknown. Molecules that interact to control equilibrium within an organism form physiological regulatory networks. It is essential to determine whether particular components of physiological regulatory networks evolve or if the network as a whole is affected in populations diverging in behavioural responses, as this may affect the nature, amplitude and number of impacted traits. We studied the regulation of four physiological regulatory networks in freshwater and marine populations of threespine stickleback raised in a common environment, which were previously characterized as showing evolutionary divergence in behaviour and stress reactivity. We measured nineteen components of these networks (ligands and receptors) using mRNA and monoamine levels in the brain, pituitary and interrenal gland, as well as hormone levels. Freshwater fish showed higher expression in the brain of adrenergic (adrb2a), serotonergic (htr2a) and dopaminergic (DRD2) receptors, but lower expression of the htr2b receptor. Freshwater fish also showed higher expression of the mc2r receptor of the glucocorticoid axis in the interrenals. Collectively, our results suggest that the inheritance of the regulation of these networks may be implicated in the evolution of behaviour and stress reactivity in association with population divergence. Our results also suggest that evolutionary change in freshwater threespine stickleback may be more associated with the expression of specific receptors rather than with global changes of all the measured constituents of the physiological regulatory networks.

Sub-chronic exposure to fluoxetine in juvenile oysters (Crassostrea gigas): uptake and biological effects.

The bioconcentration potential of fluoxetine (FLX) and its biological effects were investigated in juvenile Pacific oyster exposed for 28 days to environmentally relevant concentrations of FLX (1 ng L(-1), 100 ng L(-1) and up to 10 µg L(-1)). FLX bioaccumulated in oyster flesh resulting in 28-day bioconcentration factors greater than 2,000 and 10,000 by referring to wet and dry weights, respectively. Nevertheless, FLX did not induce oyster mortality, delayed gametogenesis, or lead to adverse histopathological alterations. At the two highest concentrations, despite non-optimal trophic conditions, FLX stimulated shell growth but only in a transient manner, suggesting a role of serotonin in the regulation of feeding and metabolism in bivalves. Those high concentrations seemed to drive bell-shaped responses of catalase and glutathione S-transferase activities throughout the exposure period, which may indicate the activation of antioxidant enzyme synthesis and then an enhanced catabolic rate or direct inhibition of those enzymes. However, no clear oxidative stress was detected because no strong differences in thiobarbituric acid-reactive substance (TBARS) content (i.e. lipid peroxidation) were observed between oyster groups, suggesting that cellular defence mechanisms were effective. These results demonstrate the importance of considering additional biomarkers of oxidative stress to obtain a comprehensive overview of the FLX-induced changes in marine bivalves exposed under realistic conditions. Considering the battery of biomarkers used, FLX appears to induce little or no effects on oyster physiology even at a concentration of 10 µg L(-1). These results do not confirm the lowest observed effect concentration (LOEC) values reported by some authors in other mollusc species.

Pre-hatching fluoxetine-induced neurochemical, neurodevelopmental, and immunological changes in newly hatched cuttlefish.

Embryonic and early postembryonic development of the cuttlefish Sepia officinalis (a cephalopod mollusk) occurs in coastal waters, an environment subject to considerable pressure from xenobiotic pollutants such as pharmaceutical residues. Given the role of serotonin in brain development and its interaction with neurodevelopmental functions, this study focused on fluoxetine (FLX), a selective serotonin reuptake inhibitor (SSRI, antidepressant). The goal was to determine the effects of subchronic waterborne FLX exposure (1 and 10 µg L(-1)) during the last 15 days of embryonic development on neurochemical, neurodevelopmental, behavioral, and immunological endpoints at hatching. Our results showed for the first time that organic contaminants, such as FLX, could pass through the eggshell during embryonic development, leading to a substantial accumulation of this molecule in hatchlings. We also found that FLX embryonic exposure (1 and 10 µg L(-1)) (1) modulated dopaminergic but not serotonergic neurotransmission, (2) decreased cell proliferation in key brain structures for cognitive and visual processing, (3) did not induce a conspicuous change in camouflage quality, and (4) decreased lysozyme activity. In the long term, these alterations observed during a critical period of development may impair complex behaviors of the juvenile cuttlefish and thus lead to a decrease in their survival. Finally, we suggest a different mode of action by FLX between vertebrate and non-vertebrate species and raise questions regarding the vulnerability of early life stages of cuttlefish to the pharmaceutical contamination found in coastal waters.

Comparison of the sensitivity of seven marine and freshwater bioassays as regards antidepressant toxicity assessment.

The hazards linked to pharmaceutical residues like antidepressants are currently a major concern of ecotoxicology because they may have adverse effects on non-target aquatic organisms. Our study assesses the ecotoxicity of three antidepressants (fluoxetine, sertraline and clomipramine) using a battery of marine and freshwater species representing different trophic levels, and compares the bioassay sensitivity levels. We selected the following bioassays: the algal growth inhibition test (Skeletonema marinoi and Pseudokirchneriella subcapitata), the microcrustacean immobilization test (Artemia salina and Daphnia magna), development and adult survival tests on Hydra attenuata, embryotoxicity and metamorphosis tests on Crassostrea gigas, and in vitro assays on primary cultures of Haliotis tuberculata hemocytes. The results showed high inter-species variability in EC50-values ranging from 43 to 15,600 µg/L for fluoxetine, from 67 to 4,400 µg/L for sertraline, and from 4.70 µg/L to more than 100,000 µg/L for clomipramine. Algae (S. marinoi and P. subcapitata) and the embryo-larval stages of the oyster C. gigas were the most sensitive taxa. This raises an issue due to their ecological and/or economic importance. The marine crustacean A. salina was the least sensitive species. This difference in sensitivity between bioassays highlights the importance of using a test battery.

Extensive behavioural divergence following colonisation of the freshwater environment in threespine sticklebacks.

Colonisation of novel environments means facing new ecological challenges often resulting in the evolution of striking divergence in phenotypes. However, little is known about behavioural divergence following colonisation, despite the predicted importance of the role of behavioural phenotype-environment associations in adaptive divergence. We studied the threespine stickleback (Gasterosteus aculeatus), a model system for postglacial colonisation of freshwater habitats largely differing in ecological conditions from the ones faced by the descendants of the marine ancestor. We found that common-environment reared freshwater juveniles were less social, more active and more aggressive than their marine counterparts. This behavioural divergence could represent the result of natural selection that acted on individuals following freshwater colonisation, with predation as a key selection agent. Alternatively, the behavioural profile of freshwater juveniles could represent the characteristics of individuals that preferentially invaded freshwater after the glacial retreat, drawn from the standing variation present in the marine population.

Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine.

Antidepressants released in the environment have the potential to generate neural disrupting effects in non-target organisms, yet their putative effects on behaviors have never been studied in cephalopod molluscs. This study assessed the impact of the antidepressant fluoxetine (FLX) on the efficiency of cryptic behaviors (body patterns on uniform, checkerboard and sandy substrates), locomotor activity, and brain chemistry in young cuttlefish exposed to environmental concentrations (1 and 100ngL(-1) of FLX) during the perinatal period. Behavioral responses of cuttlefish were monitored at hatching and two weeks later, and brain monoamine contents were quantified at one month of age. FLX significantly altered the camouflage efficiencies on uniform and sandy backgrounds only at the lowest concentration, but not at 100ngL(-1). Hatchlings exposed to 1ngL(-1) of FLX exhibited a duration exposure-dependent decrease in the uniform camouflage. They also showed a significant increase of the frequency of sand digging behaviors which might make them highly visible to predators in nature. When tested again two weeks later, cuttlefish seemed to have recovered and no more behavioral alterations were observed showing a transitory effect of the antidepressant. FLX did not affect the levels of serotonin, norepinephrine and their metabolites; however, it seemed to influence dopaminergic activity between the two FLX-exposed groups. The results show for the time that environmentally realistic concentrations of a single SSRI significantly impair the cryptic performances of newly hatched cuttlefish, and may ultimately reduce their chance for survival.

Effects of perinatal exposure to waterborne fluoxetine on memory processing in the cuttlefish Sepia officinalis.

Recent ecotoxicological studies highlight the increasing presence of pharmaceuticals discharged in the aquatic environment. Amongst them is the antidepressant fluoxetine (FLX), a selective serotonin reuptake inhibitor, primarily indicated for treatment of depression. The effect of chronic exposure to FLX on memory processing in 1-month-old cuttlefish Sepia officinalis was evaluated. Three groups of new-borns were reared in different conditions: one control group (no FLX) and two groups exposed to environmental concentrations of FLX (1 and 100ng/L) from 15 days pre-hatching to 1 month post-hatching. Acquisition and retention performances were assessed using the 'prawn-in-the-tube' procedure. Perinatal exposure to fluoxetine led to significant changes in memory processing of the animals. The lowest observed effect concentration of this antidepressant on learning and retention was 1ng/L which is under the range of environmental contamination. Cuttlefish exposed at low FLX concentration had impaired acquisition capabilities and animals exposed at high FLX concentration displayed a deficit of memory retention compared to the control group that had nonimpaired initial acquisition and retention performances. The results subsequently suggested that FLX-induced changes in cognitive capacities could potentially lead to inappropriate predatory behaviors in the natural environment. The study provides the basis for future studies on how pharmaceutical contaminants disrupt cognition in ecologically and economically relevant marine invertebrates.

Demand feeding and welfare in farmed fish.

Following the development of demand-feeding systems, many experiments have been conducted to explore feeding motivation and feed intake in farmed fish. This work aims to review a selection of studies in the field, focusing on three key factors, related to demand feeding and fish welfare. Firstly, we outline how demand feeders should be considered when developing feed management strategies for improving welfare in production conditions. Secondly, via laboratory demand-feeding experiments, we show self-feeding activities depend not only on feeding motivation and social organisation, but also on individual learning capacity and risk-taking behaviour. Thirdly, we report encouraging results demonstrating that when presented with two or more self-feeders containing complementary foods, fish select a diet according to their specific nutritional requirements, suggesting that demand feeders could be used to improve welfare by allowing fish to meet their nutritional needs.

Biodiversity matters in a changing world.

It is now widely accepted that the climate of our planet is changing, but it is still hard to predict the consequences of these changes on ecosystems. The impact is worst at the poles, with scientists concerned that impacts at lower latitudes will follow suit. Canada has a great responsibility and potential for studying the effects of climate changes on the ecological dynamics, given its geographical location and its scientific leadership in this field. The 5th annual meeting of the Canadian Society for Ecology and Evolution was held in the International Year of Biodiversity, to share recent advances in a wide variety of disciplines ranging from molecular biology to behavioural ecology, and to integrate them into a general view that will help us preserve biodiversity and limit the impact of climate change on ecosystems.

Mercury immune toxicity in harbour seals: links to in vitro toxicity.

BACKGROUND: Mercury is known to bioaccumulate and to magnify in marine mammals, which is a cause of great concern in terms of their general health. In particular, the immune system is known to be susceptible to long-term mercury exposure. The aims of the present study were (1) to determine the mercury level in the blood of free-ranging harbour seals from the North Sea and (2) to examine the link between methylmercury in vitro exposure and immune functions using seal and human mitogen-stimulated peripheral blood mononuclear cells (T-lymphocytes). METHODS: Total mercury was analysed in the blood of 22 harbour seals. Peripheral blood mononuclear cells were isolated from seals (n = 11) and from humans (n = 9). Stimulated lymphocytes of both species were exposed to functional tests (proliferation, metabolic activity, radioactive precursor incorporation) under increasing doses of methylmercury (0.1 to 10 microM). The expression of cytokines (IL-2, IL-4 and TGF-beta) was investigated in seal lymphocytes by RT-PCR and by real time quantitative PCR (n = 5) at methylmercury concentrations of 0.2 and 1 microM. Finally, proteomics analysis was attempted on human lymphocytes (cytoplasmic fraction) in order to identify biochemical pathways of toxicity at concentration of 1 microM (n = 3). RESULTS: The results showed that the number of seal lymphocytes, viability, metabolic activity, DNA and RNA synthesis were reduced in vitro, suggesting deleterious effects of methylmercury concentrations naturally encountered in free-ranging seals. Similar results were found for human lymphocytes. Functional tests showed that a 1 microM concentration was the critical concentration above which lymphocyte activity, proliferation and survival were compromised. The expression of IL-2 and TGF-beta mRNA was weaker in exposed seal lymphocytes compared to control cells (0.2 and 1 microM). Proteomics showed some variation in the protein expression profile (e.g. vimentin). CONCLUSION: Our results suggest that seal and human PBMCs react in a comparable way to MeHg in vitro exposure with, however, larger inter-individual variations. MeHg could be an additional cofactor in the immunosuppressive pollutant cocktail generally described in the blood of seals and this therefore raises the possibility of additional additive effects in the marine mammal immune system.