Latitudinal influence on gametogenesis and host-parasite ecology in a marine bivalve model.

Reproduction and parasites have significant impacts on marine animal populations globally. This study aimed to investigate the associative effects of host reproduction and a host-parasite interplay on a marine bivalve, along a geographic gradient of latitude. Cockles Cerastoderma edule were sampled from five European sites (54°N to 40°N), between April 2018 and October 2019. A histological survey provided data on trematode (metacercaria and sporocyst life stages), prevalence, and cockle stage of gametogenesis to assess the influence of a latitudinal gradient on both interplays. Sex ratios at the northernmost sites were skewed toward females, and spawning size was reduced at the lower latitudes. Trematode infection did not follow a latitudinal gradient. Localized site-related drivers, namely seawater temperature, varied spatially, having an impact on cockle-trematode interactions. Spawning was related to elevated temperatures at all sites. Prolonged spawning occurred at southern latitudes, where seawater temperatures were warmer. Trematode prevalence and the impact of trematodes on gametogenesis were found to be spatially variable, but not latitudinally. Therefore, it is not possible to determine the likelihood of boom and bust events in cockles, based on the latitudinal location of a population. In terms of sublethal impacts, it appeared that energy was allocated to reproduction rather than somatic growth in southern populations, with less energy allocated to reproduction in the larger, northern cockles. The demonstrated spatial trend of energy allocation indicates the potential of a temporal trend of reduced cockle growth at northern sites, as a result of warming sea temperatures. This awareness of the spatially varying drivers of populations is crucial considering the potential for these drivers/inhibitors to be exacerbated in a changing marine environment.

Sensitivity to cadmium of the endangered freshwater pearl mussel Margaritifera margaritifera from the Dronne River (France): experimental exposure.

Margaritifera margaritifera is a critically endangered species in Europe. Among the causes explaining its decline, metal pollution had never been deeply studied. Thus, an ecotoxicological investigation was developed on this species which comes from the Dronne River (South-West of France). Cadmium (Cd) exposure of mussels at 2 and 5 µg/L for 7 days was conducted to test their vulnerability to this metal, and also the potential endocrine disruption power of Cd. Morphometric analyses, gonad histological observations, metal bioaccumulation, metallothionein (MTs) production, measures of malondialdehyde (MDA), and finally quantitative relative expression analysis of genes involved in various metabolic functions were performed.The main results showed Cd accumulation increasing in a dose-dependent manner, especially in the gills. The same trend was observed for gene expression relative to oxidative stress. Histological analysis of the gonads highlighted a predominance of hermaphrodite individuals, but after 7 days of exposure to Cd, the percentage of female was largely increased compared with controls, from 17 to 33%. These results demonstrate the endocrine disruption effect of Cd on freshwater pearl mussels.The pearl mussel Margaritifera margaritifera is sensitive to cadmium since the metallothioneins are poorly induced, gene expression reveals oxidative stress, and gonads tend to be feminized.

Mercury contamination levels in the bioindicator piscivorous fish Hoplias aïmara in French Guiana rivers: mapping for risk assessment.

In French Guiana, native populations present high level of mercury contamination, which has been linked to the consumption of contaminated fishes. The goal of this study is to undertake a cartography of mercury contamination levels in fishes from the six main Guiana rivers. The selected species for this study is the ubiquitous piscivorous fish Hoplias aimara. A total number of 575 fishes from 134 discrete fishing sites are regrouped into 51 river sectors. Results from this study permits to rank the six main Guiana rivers by their mean level of contamination: Oyapock (0.548 mg kg-1), Comté (0.624 mg kg-1), Maroni (0.671 mg kg-1), Approuague (0.684 mg kg-1), Mana (0.675 mg kg-1), and Sinnamary (1.025 mg kg-1). The contamination is however not spatially homogenous along each river, and a map of the different levels of mercury contamination in fishes is provided. Sectors of low mean Hg contamination are observed both upstream (0.471 mg kg-1) and downstream (0.424 mg kg-1), corresponding to areas without any influence of gold mining activities and areas under the influence of estuarine dilution, respectively. Anoxia and gold mining activities are found to be the two main factors responsible for the high mercury concentration in fish muscles. While mean levels of mercury contaminations are higher in anoxia areas (1.029 mg kg-1), contaminations induced by gold mining activities (0.717 mg kg-1) present the most harmful consequences to human populations. No significant differences in Hg concentrations are observed between 2005 and 2014 for neither a pristine nor a gold mining area, while Hg concentration differences are observed between former (0.550 mg kg-1) and current gold mining sites (0.717 mg kg-1).

Trophic transfer and effects of gold nanoparticles (AuNPs) in Gammarus fossarum from contaminated periphytic biofilm.

This work addressed the trophic transfer and effects of functionalized gold nanoparticles (AuNPs) from periphytic biofilms to the crustacean Gammarus fossarum. Biofilms were exposed for 48 h to 10 nm positively charged functionalized AuNPs at two concentrations, 4.6 and 46 mg/L, and crustaceans G. fossarum grazed on these for 7 days, with daily biofilm renewal. Gold bioaccumulation in biofilm and crustacean were measured to estimate the trophic transfer ratio of these AuNP, and, for the first time, a transcriptomic approach and transmission electron microscopy observations in the crustacean were made. These two approaches showed cellular damage caused by oxidative stress and, in particular, an impact of these AuNPs on mitochondrial respiration. Modulation of digestive enzyme activity was also observed, suggesting modifications of digestive functions. The damage due to these nanoparticles could then have vital consequences for the organisms during chronic exposure.

Biosynthesis of gold nanoparticles by the living freshwater diatom Eolimna minima, a species developed in river biofilms.

Testing biotransformation capacities of living aquatic microalgae diatoms to naturally synthetize gold nanoparticles (AuNP) from gold salts and assessing aftereffects on their viability by microscope observations is a great challenge. In this work, a laboratory experiment was conducted, which aimed to observe (i) directly by transmission electronic and light microscopy and (ii) through indirect measurements (UV-visible spectroscopy) the periphytic freshwater diatom Eolimna minima exposed to gold salts. This work revealed the capacity of E. minima to intracellularly biosynthetize AuNP and to tolerate it. AuNP synthesis appears as a mechanism of detoxification to protect diatom from gold salt contamination. We also pointed out the risks associated with the spread of diatoms full of AuNP, through the trophic web of freshwater ecosystems. The preponderant part of the diatoms in natural biofilms associated with their position at the basis of the trophic webs in rivers could then make them responsible for the contamination of their consumers (grazer animals) and consequently for the potential release of AuNP through the entire food web.

Specific Effects of Dietary Methylmercury and Inorganic Mercury in Zebrafish (Danio rerio) Determined by Genetic, Histological, and Metallothionein Responses.

A multidisciplinary approach is proposed here to compare toxicity mechanisms of methylmercury (MeHg) and inorganic mercury (iHg) in muscle, liver, and brain from zebrafish (Danio rerio). Animals were dietary exposed to (1) 50 ng Hg g(-1), 80% as MeHg; (2) diet enriched in MeHg 10000 ng Hg g(-1), 95% as MeHg; (3) diet enriched in iHg 10000 ng Hg g(-1), 99% as iHg, for two months. Hg species specific bioaccumulation pathways were highlighted, with a preferential bioaccumulation of MeHg in brain and iHg in liver. In the same way, differences in genetic pattern were observed for both Hg species, (an early genetic response (7 days) for both species in the three organs and a late genetic response (62 days) for iHg) and revealed a dissimilar metabolization of both Hg species. Among the 18 studied genes involved in key metabolic pathways of the cell, major genetic responses were observed in muscle. Electron microscopy revealed damage mainly because of MeHg in muscle and also in liver tissue. In brain, high MeHg and iHg concentrations induced metallothionein production. Finally, the importance of the fish origin in ecotoxicological studies, here the seventh descent of a zebrafish line, is discussed.

Specific Pathways of Dietary Methylmercury and Inorganic Mercury Determined by Mercury Speciation and Isotopic Composition in Zebrafish (Danio rerio).

An original approach is proposed to investigate inorganic (iHg) and methylmercury (MeHg) trophic transfer and fate in a model fish, Danio rerio, by combining natural isotopic fractionation and speciation. Animals were exposed to three different dietary conditions: (1) 50 ng Hg g(-1), 80% as MeHg; (2) diet enriched in MeHg 10,000 ng Hg g(-1), 95% as MeHg, and (3) diet enriched in iHg 10,000 ng Hg g(-1), 99% as iHg. Harvesting was carried out after 0, 7, 25, and 62 days. Time-dependent Hg species distribution and isotopic fractionation in fish organs (muscle, brain, liver) and feces, exhibited different patterns, as a consequence of their dissimilar metabolization. The rapid isotopic re-equilibration to the new MeHg-food source reflects its high bioaccumulation rate. Relevant aspects related to Hg excretion are also described. This study confirms Hg isotopic fractionation as a powerful tool to investigate biological processes, although its deconvolution and fully understanding is still a challenge.

Methylmercury effects on migratory behaviour in glass eels (Anguilla anguilla): an experimental study using isotopic tracers.

The effect of methylmercury (MeHg) on glass eels' propensity to migrate, mitochondrial activity and antioxidative defence systems was investigated. Marine glass eels were first sorted in an experimental flume according to their response to dusk. Fish responding to the decrease in light intensity by ascending in the water column and moving with or against the flow were considered as having a high propensity to migrate (migrant). Glass eels still sheltering at the end of the 24 h catching period were considered as having a low propensity to migrate and were called non-migrant. Migrant and non-migrant glass eels were then individually tagged and exposed to isotopically enriched (201)MeHg (50 ng L(-1)) for 11 days. The effect of contamination was studied on muscle fibre structure, and the expression level of genes involved in mitochondrial activity and antioxidative defence systems. To investigate the effect of MeHg on glass eel behaviour, migrant and non-migrant glass eels were sorted again and the bioaccumulation of (201)MeHg and its demethylation product ((201)Hg(II)) were determined for each individual. MeHg exposure increased activity in non-migrant glass eels but not migratory behaviour. Contamination affected mitochondrial structure and metabolism and suggests a higher oxidative stress and activation of antioxidative defence systems in non-migrant glass eels. Overall, our results suggest that exposure to MeHg might induce an increase in energy expenditure and a higher vulnerability to predation in non-migrant glass eels in the wild.

Effects of methylmercury contained in a diet mimicking the Wayana Amerindians contamination through fish consumption: mercury accumulation, metallothionein induction, gene expression variations, and role of the chemokine CCL2.

Methylmercury (MeHg) is a potent neurotoxin, and human beings are mainly exposed to this pollutant through fish consumption. We addressed the question of whether a diet mimicking the fish consumption of Wayanas Amerindians from French Guiana could result in observable adverse effects in mice. Wayanas adult men are subjected to a mean mercurial dose of 7 g Hg/week/kg of body weight. We decided to supplement a vegetarian-based mice diet with 0.1% of lyophilized Hoplias aimara fish, which Wayanas are fond of and equivalent to the same dose as that afflicting the Wayanas Amerindians. Total mercury contents were 1.4 ± 0.2 and 5.4 ± 0.5 ng Hg/g of food pellets for the control and aimara diets, respectively. After 14 months of exposure, the body parts and tissues displaying the highest mercury concentration on a dry weight (dw) basis were hair (733 ng/g) and kidney (511 ng/g), followed by the liver (77 ng/g). Surprisingly, despite the fact that MeHg is a neurotoxic compound, the brain accumulated low levels of mercury (35 ng/g in the cortex). The metallothionein (MT) protein concentration only increased in those tissues (kidney, muscles) in which MeHg demethylation had occurred. This can be taken as a molecular sign of divalent mercurial contamination since only Hg(2+) has been reported yet to induce MT accumulation in contaminated tissues. The suppression of the synthesis of the chemokine CCL2 in the corresponding knockout (KO) mice resulted in important changes in gene expression patterns in the liver and brain. After three months of exposure to an aimara-containing diet, eight of 10 genes selected (Sdhb, Cytb, Cox1, Sod1, Sod2, Mt2, Mdr1a and Bax) were repressed in wild-type mice liver whereas none presented a differential expression in KO Ccl2(-/-) mice. In the wild-type mice brain, six of 12 genes selected (Cytb, Cox1, Sod1, Sod2, Mdr1a and Bax) presented a stimulated expression, whereas all remained at the basal level of expression in KO Ccl2(-/-) mice. In the liver of aimara-fed mice, histological alterations were observed for an accumulated mercury concentration as low as 32 ng/g, dw, and metal deposits were observed within the cytoplasm of hepatic cells.

Morphological evidence of neurotoxicity in retina after methylmercury exposure.

The visual system is particularly sensitive to methylmercury (MeHg) exposure and, therefore, provides a useful model for investigating the fundamental mechanisms that direct toxic effects. During a period of 70 days, adult of a freshwater fish species Hoplias malabaricus were fed with fish prey previously labeled with two different doses of methylmercury (0.075 and 0.75 µgg(-1)) to determine the mercury distribution and morphological changes in the retina. Mercury deposits were found in the photoreceptor layer, in the inner plexiform layer and in the outer plexiform layer, demonstrating a dose-dependent bioaccumulation. The ultrastructure analysis of retina revealed a cellular deterioration in the photoreceptor layer, morphological changes in the inner and outer segments of rods, structural changes in the plasma membrane of rods and double cones, changes in the process of removal of membranous discs and a structural discontinuity. These results lead to the conclusion that methylmercury is able to cross the blood-retina barrier, accumulate in the cells and layers of retina and induce changes in photoreceptors of H. malabaricus even under subchronic exposure.

Effects of dietary methylmercury on the zebrafish brain: histological, mitochondrial, and gene transcription analyses.

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for wildlife and human health through fish consumption. To study the neurotoxic impact of MeHg on piscivorous fish, we contaminated the model fish species Danio rerio for 25 and 50 days with food containing 13.5 µg/g dry weight (dw) of MeHg (0.6 µg MeHg/fish/day), an environmentally relevant dose leading to brain mercury concentrations of 30 ± 4 µg of Hg g(-1) (dw) after 25 days of exposure and 46 ± 7 µg of Hg g(-1) (dw) after 50 days. Brain mitochondrial respiration was not modified by exposure to MeHg, contrary to what happens in skeletal muscles. A 6-fold increase in the expression of the sdh gene encoding the succinate dehydrogenase Fe/S protein subunit was detected in the contaminated brain after 50 days of exposure. An up regulation of 3 genes, atp2b3a, atp2b3b, and slc8a2b, encoding for calcium transporters was noticed after 25 days of exposure but the atp2b3a and atp2b3b were repressed and the slc8a2b gene expression returned to its basal level after 50 days, suggesting a perturbation of calcium homeostasis. After 50 days, we detected the up regulation of glial fibrillary acidic protein and glutathione S-transferase genes (gfap and gst), along with a repression of the glutathione peroxidase gene gpx1. These results match well with a MeHg-induced onset of oxidative stress and inflammation. A transmission electron microscopic observation confirmed an impairment of the optical tectum integrity, with a decrease of the nucleal area in contaminated granular cells compared to control cells, and a lower density of cells in the contaminated tissue. A potential functional significance of such changes observed in optical tectum when considering wild fish contaminated in their natural habitat might be an impaired vision and therefore a lowered adaptability to their environment.

Methylmercury localization in Danio rerio retina after trophic and subchronic exposure: a basis for neurotoxicology.

Methylmercury is a known neurotoxic organometal which affects visual functions and few studies concerns to wild fish are available. The autometallography mercury distribution in the retina of Danio rerio was mapped using light and electron microscopy. Abundant mercury deposits were found in the photoreceptor layer (outer and inner segments of the photoreceptors) and in the inner and outer nuclear layers. Occasionally, the presence of mercury deposits in plexiform layers was observed and very rarely in the ganglion cell layer. Also the occurrence of mercury deposits in cells from the disc region was observed, but not in the nerve fiber layer. An interesting difference was found between mercury accumulation in the central and peripheral regions of the retina. These results demonstrate that mercury after trophic exposure to Danio rerio is able to cross the blood-retina barrier and accumulate in the cells of the retina even under subchronic exposure.

Brown muscle disease (BMD), an emergent pathology affecting Manila clam Ruditapes philippinarum in Arcachon Bay (SW France).

We describe an emerging pathology, brown muscle disease (BMD), which specifically affects the Manila clam Ruditapes philippinarum in Arcachon Bay (France). BMD induces a transformation of the posterior adductor muscle, which becomes infused by conchiolin and calcified, reducing the ability of clams to bury. The disease affects both types of muscular tissue, with striated muscle becoming affected to a higher degree than smooth muscle. Two indices were created to quantify the symptoms: the Muscle Print Index, used for empty and live shells, and the Final Disease Index, utilized for live clams only. Histological sections were made and observed under light microscopy to examine the muscular damage and to investigate a causal agent. Sections revealed an important inflammatory response with a large invasion of hemocytes into tissues and a heavy necrosis of muscular fibers. Additionally, molecular biology analyses were carried out to search for bacteria and protozoan agents using generic primers. In both histological and molecular assays, bacteria and protozoans were discounted. We monitored 4 sites scattered around the bay over 2 yr. The mean prevalence was <12% without seasonal variation in 3 sites against 30% and a winter peak in 1 site. The latter site was accurately surveyed and revealed that clams at the sediment surface (abnormal position) were affected 3 times more frequently than buried clams (normal position).

How cadmium could compromise the completion of the European eel's reproductive migration.

The European eel (Anguilla anguilla L.) is severely threatened with extinction. Surprisingly, even though their unusual life cycle makes them particularly vulnerable to pollution, the possible contribution of contamination remains especially poorly known. Here we have investigated the possible effect of cadmium (Cd), a widespread nonessential metal, on eel reproductive capacities. Both control and Cd precontaminated female silver eels were experimentally matured and forced to swim in metal-free conditions to mimic their reproductive migration. Cd pre-exposure was found to strongly stimulate the pituitary-gonad-liver axis of maturing female silver eels leading to early and enhanced vitellogenesis. This was followed by a strong phenomenon of oocyte atresia and eel mortality. These phenomena occurred before oocytes could reach full maturation and were associated with a large entry of both vitellogenin and Cd into the ovaries. Indeed, a redistribution of previously stored cadmium, even from the low Cd levels of control eels, was observed during sexual maturation. Atresia and mortality phenomena were also associated with an overexpression of the pituitary gene encoding the growth hormone, a marker of physiological stress and energy reserves exhaustion. Significantly, these devastating effects of Cd were observed in organisms that presented liver and kidney Cd concentrations still below those observed in eels from Cd contaminated hydrosystems. Our research shows how common levels of cadmium contamination could disrupt endocrine pathways implicated in gonad maturation and subsequently impair reproductive capacity of eel future genitors.

Role of metallothioneins in superoxide radical generation during copper redox cycling: defining the fundamental function of metallothioneins.

In order to demonstrate the in vivo antioxidant properties of metallothioneins (MTs), the bacteria Escherichia coli was used as a cell reactor in which we compared the metal binding and antioxidative functions of MTs from different species, with different structures and polypeptide lengths. No protective effects of cytoplasmic MTs from cadmium (Cd) or zinc (Zn) contamination were observed in a wild-type E. coli strain, although these MTs can efficiently bind both Cd and Zn. To test their antioxidant properties, MTs were expressed within the cytoplasm of a sodA sodB deficient mutated strain (QC1726). However, a paradoxical MT toxicity was found when this strain was contaminated with Cd and Zn, suggesting that in a wild-type strain, superoxide dismutase counteracts MT toxicity. The most toxic MT was the one with the strongest Cd and Zn binding capacities. This toxic effect was linked to the generation of superoxide radicals, since a Cd-contaminated QC1726 strain expressing oyster MT isoforms produced 75-85% more O(2)*(-) than the control QC1726 strain. Conversely, under anaerobiosis or in the presence of a copper chelator, MTs protected QC1726 strain from Cd and Zn contamination. A model is proposed to explain the observed MT toxicity.

Cadmium bioaccumulation in Tubificidae from the overlying water source and effects on bioturbation.

Cadmium bioaccumulation in tubificid oligochaetes in relation to metal vertical distribution in sediment and bioturbation intensity was studied during a 56-day experiment with a constant contamination source in the overlying water (20 microg L(-1)). The indoor microcosms simulate a two-compartment biotope with three experimental treatments based on metal exposure and faunal composition: contaminated water column with or without worms and uncontaminated water column with worms. Cadmium bioaccumulation in worms was studied after 7, 14, 21, 28, and 56 days. Bioturbation was analyzed as a functional parameter representative of organisms' activity and using conservative particulate tracers: luminophores (phi = 63-100 microm and 100-315 microm) and microspheres (phi = 1 microm). The results show no significant effects of cadmium exposure on bioturbation, despite high bioaccumulation levels in worms (50 microg g(-1) dry wt.), suggesting the existence of detoxification/sequestration processes.