Effects of glyphosate and a commercial formulation Roundup® exposures on maturation of Xenopus laevis oocytes.

Pesticides are often found at high concentrations in small ponds near agricultural field where amphibians are used to live and reproduce. Even if there are many studies on the impacts of phytopharmaceutical active ingredients in amphibian toxicology, only a few are interested in the earlier steps of their life cycle. While their populations are highly threatened with extinction. The aim of this work is to characterize the effects of glyphosate and its commercial formulation Roundup® GT Max on the Xenopus laevis oocyte maturation which is an essential preparation for the laying and the fertilization. Glyphosate is an extensively used herbicide, not only known for its effectiveness but also for its indirect impacts on non-target organisms. Our results showed that exposures to both forms of glyphosate delayed this hormone-dependent process and were responsible for spontaneous maturation. Severe and particular morphogenesis abnormalities of the meiotic spindle were also observed. The MAPK pathway and the MPF did not seem to be affected by exposures. The xenopus oocyte is particularly affected by the exposures and appears as a relevant model for assessing the effects of environmental contamination.

Litter breakdown as a tool for assessment of the efficiency of afforestation and ash-aided phytostabilization on metal-contaminated soils functioning in Northern France.

The main objective of the study was to assess the efficiency of phytoremediation methods implemented for 14 years on highly metal-contaminated soils. The different experimental strategies were plots planted with a tree mix or with a single tree species coupled or not with the use of fly-ashes as an amendment to limit metals mobility in soil. The breakdown of poplar litter on the four plots was monitored during 10 months. In parallel, colonization of litter bags by functional groups of mesofauna (Collembola and Acari) was followed. Two mesh-sized litter bags were used to allow distinguishing microbial and mesofaunal actions on the litter breakdown. We observed the breakdown of litter in four studied plots. Litter breakdown occurred faster in 3-mm litter bags than 250 µm ones during summer demonstrating the importance of mesofauna. Mixed plantation allowed faster litter breakdown than mono-specific plantation. A higher abundance of mesofauna and/or better abiotic conditions (moisture, shading…) could explain this result. Regarding litter breakdown and mesofauna, no significant difference was observed between the amended plots and those subjected to soil phytomanagement. However, communities of the studied area are disturbed since a low abundance of detritivores was observed. This could explain also the slower litter breakdown than expected in our study. To conclude, among the phytomanagement methods tested, mixed plantations could provide a benefit for the restoration of degraded soils. By contrast, the use of fly-ashes does not seem to have any effect on the functionality of ecosystem neither on the litter breakdown process nor on the abundance of mesofauna.

Maturation of Xenopus laevis oocytes under cadmium and lead exposures: Cell biology investigations.

Since amphibians are recognised as good models to assess the quality of environments, only few studies have dealt with the impacts of chemical contaminants on their gametes, while toxic effects at this stage will alter all the next steps of their life cycle. Therefore, we propose to investigate the oocyte maturation of Xenopus laevis in cadmium- and lead-contaminated conditions. The impacts of cadmium and lead ions were explored on events involved in the hormone-dependent process of maturation. In time-course experiments, cadmium, at the highest concentration, delayed and prevented the germinal vesicle breakdown. Even in the absence of progesterone this ion could also induce it. No such spontaneous maturation was observed after lead exposures. An acceleration of the process at the highest tested concentration of lead (90µM), in presence of progesterone, was recorded. Cytological observations highlighted that cadmium exposures drove severe disturbances of meiotic spindle morphogenesis. At last, cadmium exposures altered the MAPK pathway, regarding the activation of ERK2 and RSK, but also the activation and the activity of the MPF, by disturbing the state of phosphorylation of Cdc2 and histone H3. Xenopus laevis oocytes were affected by these metal ion exposures, notably by Cd2+. Signatures of these metal exposures on the oocyte maturation were detected. This germ cell appeared to be a relevant model to assess the effects of environmental contaminants such as metals.

Cadmium but not lead exposure affects Xenopus laevis fertilization and embryo cleavage.

Among the toxicological and ecotoxicological studies, few have investigated the effects on germ cells, gametes or embryos, while an impact at these stages will result in serious damage at a population level. Thus, it appeared essential to characterize consequences of environmental contaminant exposures at these stages. Therefore, we proposed to assess the effects of exposure to cadmium and lead ions, alone or in a binary mixture, on early stages of Xenopus laevis life cycle. Fertilization and cell division during segmentation were the studied endpoints. Cadmium ion exposures decreased in the fertilization rates in a concentration-dependent manner, targeting mainly the oocytes. Exposure to this metal ions induced also delays or blockages in the embryonic development. For lead ion exposure, no such effect was observed. For the exposure to the mixture of the two metal ions, concerning the fertilization success, we observed results similar to those obtained with the highest cadmium ion concentration.

Comparative avoidance behaviour of the earthworm Eisenia fetida towards chloride, nitrate and sulphate salts of Cd, Cu and Zn using filter paper and extruded water agar gels as exposure media.

We studied the avoidance behaviour of the earthworm Eisenia fetida towards Cd, Cu, and Zn, trace elements (TEs) tested as chloride, nitrate and sulphate salts. Sub adults were exposed individually using dual-cell chambers at 20+2°C in the dark. Recordings were realised at different dates from 2h to 32h. We used filter paper and extruded water agar gel as exposure media to evaluate the contribution of the dermal and the digestive exposure routes on the avoidance reactions. Exposures to Cu or Cd (10mgmetal ionL(-1)) resulted in highly significant avoidance reactions through the exposure duration. Worms avoided Zn poorly and reactions towards Zn salts varied along the exposure. Worm sensitivity towards TEs differed between salts and this could result from differential toxicity or accessibility of these TE salts to earthworms. The anion in itself was not the determinant of the avoidance reactions since exposures to similar concentrations of these anions using calcium salts did not result in significant avoidance worm behaviour. Avoidance responses towards TEs were higher in the case of water agar exposures than in filter paper exposures. Thus, dermal contacts with TE solutions would elicit worm avoidance but signals from receptors located inside the digestive tract could reinforce this behaviour. The use of extruded water agar gels as the substrate allows checking the real sensitivity of earthworm species towards TEs since the TE concentrations leading to significant avoidance reactions were below those reported in the literature when using TE-spiked soils.

Earthworm populations of highly metal-contaminated soils restored by fly ash-aided phytostabilisation.

Highly metal contaminated soils found in the North of France are the result of intense industrial past. These soils are now unfit for the cultivation of agricultural products for human consumption. Solutions have to be found to improve the quality of these soils, and especially to reduce the availability of trace elements (TEs). Phytostabilisation and ash-aided phytostabilisation applied since 2000 to an experimental site located near a former metallurgical site (Metaleurop-Nord) was shown previously as efficacious in reducing TEs mobility in soils. The aim of the study was to check whether this ten years trial had influenced earthworm communities. This experimental site was compared to plots located in the surroundings and differing by the use of soils. Main results are that: (1) whatever the use of soils, earthworm communities are composed of few species with moderate abundance in comparison with communities found in similar habitats outside the TEs-contaminated area, (2) the highest abundance and specific richness (4-5 species) were observed in afforested plots with various tree species, (3) ash amendments in afforested plots did not increase the species richness and modified the communities favoring anecic worms but disfavoring epigeic ones. These findings raised the questions of when and how to perform the addition of ashes firstly, to avoid negative effects on soil fauna and secondly, to keep positive effects on metal immobilization.

Effects of field metal-contaminated soils submitted to phytostabilisation and fly ash-aided phytostabilisation on the avoidance behaviour of the earthworm Eisenia fetida.

The earthworm Eisenia fetida avoidance behaviour test was used to assess the quality recovery of metal-contaminated soils from lands submitted for 10 years to remediation. Soils were from plots located in the surroundings of a former lead smelter plant of Northern France. Metal concentrations in the soils ranged from 93 to 1231, 56 to 1424, 0.3 to 20 and 15 to 45.5mg metal/kg dry soil for Pb, Zn, Cd and Cu, respectively. Several former agricultural plots were treated either by a single phytostabilisation process involving the plantation of a tree mix or by fly ash aided-phytostabilisation. Silico-aluminous or sulfo-calcic ashes used were ploughed up to a 25- to 30-cm soil depth at a rate of 23.3kg/m(2) (i.e., 6 percent W/W). E. fetida was shown to avoid significantly the 10 years ash-treated soils whose habitat function has to be considered as limited. This avoidance would relate to a change of the texture of soils induced by the addition of ashes and consisting in an increased level of fine silts together with a decreased level of clays. By contrast, afforested metal-contaminated soils appeared for E. fetida as more attractive than unplanted ones. Regarding the influence of the metal contamination of the soils on E. fetida, none of the soils tested even the highest contaminated one was significantly avoided by worms. This lack of reaction would result from the low bioavailability of the metals in the soils tested. At the lights of our results and those previously published on both these ashes and these ash-treated soils, the usefulness of these soil treatments is discussed.

Mycosubtilin and surfactin are efficient, low ecotoxicity molecules for the biocontrol of lettuce downy mildew.

The use of surfactin and mycosubtilin as an eco-friendly alternative to control lettuce downy mildew caused by the obligate pathogen Bremia lactucae was investigated. Preliminary ecotoxicity evaluations obtained from three different tests revealed the rather low toxicity of these lipopeptides separately or in combination. The EC50 (concentration estimated to cause a 50 % response by the exposed test organisms) was about 100 mg L(-1) in Microtox assays and 6 mg L(-1) in Daphnia magna immobilization tests for mycosubtilin and 125 mg L(-1) and 25 mg L(-1) for surfactin, respectively. The toxicity of the mixture mycosubtilin/surfactin (1:1, w/w) was close to that obtained with mycosubtilin alone. In addition, the very low phytotoxic effect of these lipopeptides has been observed on germination and root growth of garden cress Lepidium sativum L. While a surfactin treatment did not influence the development of B. lactucae on lettuce plantlets, treatment with 100 mg L(-1) of mycosubtilin produced about seven times more healthy plantlets than the control samples, indicating that mycosubtilin strongly reduced the development of B. lactucae. The mixture mycosubtilin/surfactin (50:50 mg L(-1)) gave the same result on B. lactucae development as 100 mg L(-1) of mycosubtilin. The results of ecotoxicity as well as those obtained in biocontrol experiments indicated that the presence of surfactin enhances the biological activities of mycosubtilin. Mycosubtilin and surfactin were thus found to be efficient compounds against lettuce downy mildew, with low toxicity compared to the toxicity values of chemical pesticides. This is the first time that Bacillus lipopeptides have been tested in vivo against an obligate pathogen and that ecotoxic values have been given for surfactin and mycosubtilin.

Long-term phenological shifts in raptor migration and climate.

Climate change is having a discernible effect on many biological and ecological processes. Among observed changes, modifications in bird phenology have been widely documented. However, most studies have interpreted phenological shifts as gradual biological adjustments in response to the alteration of the thermal regime. Here we analysed a long-term dataset (1980-2010) of short-distance migratory raptors in five European regions. We revealed that the responses of these birds to climate-induced changes in autumn temperatures are abrupt and synchronous at a continental scale. We found that when the temperatures increased, birds delayed their mean passage date of autumn migration. Such delay, in addition to an earlier spring migration, suggests that a significant warming may induce an extension of the breeding-area residence time of migratory raptors, which may eventually lead to residency.

Growth and metal accumulation in Porcellio scaber exposed to poplar litter from Cd-, Pb-, and Zn-contaminated sites.

This study aimed at determining, in the laboratory, the effects of poplar litter collected in woody habitats contaminated by heavy metals on growth and metal accumulation in the terrestrial isopod Porcellio scaber. Cd, Pb, and Zn pseudototal and CaCl(2)-extractable concentrations in litter types were determined using AAS. Juveniles were fed ad libitum, individually, for 28 days with four litter types presenting an increasing gradient of metal contamination. Individuals were weighed every week and metal body burdens were determined at the end of the experiment. From the first week until the end of the experiment, a decrease in P. scaber growth related to the increase of metal concentration in litter types was recorded. Significant correlations were observed between metal body burdens and metal concentrations in litter types. However, Cd accumulation in woodlice appeared to be related to the Cd/Zn concentration ratio in litter types. All these results showed the potential of weight gain in P. scaber as a suitable indicator for litter quality assessment with ecological relevance.

Assessing the effects of FBC ash treatments of metal-contaminated soils using life history traits and metal bioaccumulation analysis of the earthworm Eisenia andrei.

Earthworms (Eisenia andrei) were exposed, in controlled conditions, to metal-contaminated soils previously treated in situ with two types of fluidized bed combustion ashes. Effects on this species were determined by life history traits analysis. Metal immobilizing efficiency of ashes was indicated by metal bioaccumulation. Ashes-treated soils reduced worm mortality compared to the untreated soil. However, these ashes reduced both cocoon hatching success and hatchlings numbers compared to the untreated soil. In addition, sulfo-calcical ashes reduced or delayed worm maturity and lowered cocoon production compared to silico-alumineous ones. Metal immobilizing efficiency of ashes was demonstrated for Zn, Cu and to a lesser extent Pb. Only silico-alumineous ashes reduced Cd bioaccumulation, although Cd was still bioconcentrated. Thus, although ash additions to metal-contaminated soils may help in immobilizing metals, their use might result, depending on the chemical nature of ashes, to severe detrimental effects on earthworm reproduction with possible long term consequences to populations.

Identification and expression profile of gene transcripts differentially expressed during metallic exposure in Eisenia fetida coelomocytes.

The aim of this work was to identify in Eisenia fetida genes whose expression are regulated following exposure to a complex mixture of metallic trace elements (MTE) representative of a highly polluted smelter soil. Suppression subtractive hybridization (SSH) was used to construct cDNA libraries enriched in up- or down-regulated transcripts in the immune-circulating cells of the coelomic cavities, namely coelomocytes, from worms exposed to metallic pollution. Among 1536 SSH-derived cDNA clones sequenced, we identified 764 unique ESTs of which we selected 18 candidates on the basis of their redundancy. These selected candidates were subjected to a two-step validation procedure based on the study of their expression level by real-time PCR. The first step consisted in measuring the expression of the 18 candidates in worms exposed to artificial contaminated soil. The second step consisted in measuring the expression in animals exposed to a "naturally" contaminated soil sampled close to a smelter. Both steps allowed us to highlight 3 candidates that are strongly induced in worms exposed to a smelter polluted soil. These candidates are: the well-known MTE-induced Cd-metallothionein and 2 original biomarkers, lysenin, and a transcript, which cloning of the complete coding sequence identified as the coactosin-like protein.

cDNA cloning and expression analysis of Eisenia fetida (Annelida: Oligochaeta) phytochelatin synthase under cadmium exposure.

Metallothioneins (MTs) are central to trace metal homeostasis and detoxification throughout biological systems. Prokaryotes, plants, and fungi utilize both gene encoded cysteine-rich polypeptides (classically designated Class I and II MTs) and enzymatically synthesized cysteine-rich peptides (classically designated Class III MTs or phytochelatins). In contrast, although gene encoded MTs are ubiquitous in animal species the identification of a functional phytochelatin synthase in the nematode Caenorhabditis elegans, a representative member of the Ecdysozoa, provided the first evidence for these metal-binding peptides in animals. By exploiting the conservation observed between species we have been able to clone and transcriptionally characterize a phytochelatin synthase from the immune cells of the earthworm Eisenia fetida, the first evidence for its presence in a phylum belonging to the Lophototrochozoa. The complete coding sequence of this enzyme was determined and the phylogenetic relationship to plant, yeast and nematode enzymes elucidated. Temporal- and dose-profiling of the transcriptional regulation of phytochelatin synthase and MT in response to cadmium was performed by using real-time PCR.

Metallothionein response following cadmium exposure in the oligochaete Eisenia fetida.

We studied the metallothionein (MT) response in cadmium-exposed worms (Eisenia fetida) both at the protein level by Dot Immunobinding Assay (DIA) with a polyclonal antibody raised against the most immunogenic part of this protein and at the expression level by Northern blotting using a specific probe. MT appeared as two close isoforms. DIA results clearly demonstrated significant differences in MT level of whole worm heat-treated supernatants between E. fetida exposed to Cd concentrations as low as 8 mg Cd kg(-1) of dry soil compared to controls. Northern blotting analysis performed on whole bodies of worms revealed that a single exposure to 8 mg Cd kg(-1) of dry soil for 1 day resulted in the production of MT mRNA. This response was maintained for exposure of at least 1 month. Clear differences of MT gene expression were also observed between worms exposed to different Cd concentrations (8, 80 or 800 mg Cd kg(-1) of dry soil). Immunocytochemistry demonstrated that MT was located in the chloragogenous tissue surrounding the gut where metals are known to be accumulated. This work revealed that E. fetida MT is a sensitive and relevant biomarker of Cd exposure and especially when considering gene expression response. Further experiments have now to prove its usefulness in natural metal-contaminated soil toxicity assessments.

The human H19 gene is frequently overexpressed in myometrium and stroma during pathological endometrial proliferative events.

We studied the patterns of H19 expression in normal, hyperplastic and neoplastic human uterine tissues. H19 RNAs were detected by an in situ hybridisation technique (ISH). In both normal and pathological conditions, H19 was expressed in stromal and myometrial cells, but never in epithelial cells. 34/48 carcinomas overexpressed H19 compared with the expression in normal tissues. This high expression was frequently observed in the vicinity of malignant epithelial cells. This suggests that the level of H19 RNA synthesis could be the result of epithelium/stroma interactions. We also demonstrated that several cancerous or immortalised breast epithelial cells release factors into the culture medium, which in turn stimulate H19 expression in stromal cells. The level of H19 expression, estimated by ISH, was not significantly correlated with histological type when all types were considered together (P = 0.108), but was highly correlated to one type of cancer, i.e. carcinomas with an epidermoid component (P = 0.0015). The level of H19 expression was also strongly correlated with tumour invasion of the reproductive organs (P = 0.006) and significantly correlated with neoplastic cell invasion of the myometrium (P = 0.048). In conclusion, our results indicate that H19 overexpression is correlated with the progression of the disease and we propose that this frequent overexpression of the gene in the myometrium and in stroma is a reaction to pathological cell proliferation.

Stimulation by cadmium of myohemerythrin-like cells in the gut of the annelid Nereis diversicolor.

Isolated guts of Nereis diversicolor revealed the existence of a cadmium-binding protein, the MPII, belonging to the group of hemerythrins and myohemerythrins. The presence of MPII in the cells of the intestine was demonstrated by immunocytochemistry, using anti-MPII, a monoclonal antibody. In addition, using in situ hybridization and northern blotting, it was shown that MPII-cells are the site of synthesis of this molecule. Exposure of the worms to cadmium led to the cellular activation process of MPII-cells (i.e. transformation of the nucleolus, development of the endoplasmic reticulum and the Golgi apparatus), although MPII mRNA transcript levels were unchanged. Enzyme-linked immunosorbent assay (ELISA) of gut extracts revealed that MPII levels were increased after exposure to Cd, so it appears that this protein is synthesized as a response to Cd exposure without any new synthesis of mRNA. This mechanism of regulation is quite similar to that reported in the case of mammalian ferritin and may be involved in the regulation of Cd levels in this worm.