Development of metallic exposure biomarkers on physiological functions of Eisenia fetida (Annelida ; Oligochaeta)

Les activités métallurgiques entrainent l'accumulation d'éléments traces métalliques dans les couches . superficielles des sols, où l'on peut observer des concentrations largement supérieures au fond pédogéochimique et aux normes en vigueur. La contamination des sols est une menace pour la santé publique et la présence de grandes quantités d'éléments traces métalliques peut générer un stress susceptible d'affecter les organismes exposés à des sols pollués. Les Annélides Oligochètes vivent en contact étroit avec ces sols pollués et sont parmi les organismes vivants présentant une sensibilité exacerbée aux métaux. Très peu de choses sont connues quant à l'identification et la mise en place des mécanismes de réponse à ces métaux au niveau moléculaire. En exploitant, la conservation phylogénique observée entre espèces nous avons été capables de cloner et de caractériser un ensemble de biomarqueurs de pollution potentiels à partir des cœlomocytes de l'Annélide Oligochètes Eisenia fetida. Deux approches ont été mises en place. Premièrement, une approche qualifiée de ciblée, consistant à identifier tous les effecteurs parmi des protéines fortement conservées pour lesquels une variation lors d'une exposition métallique était reportée dans la littérature. Deuxièmement, nous avons entrepris une approche qualifiée de globale consistant en la construction de banques soustractives pour identifier chez Eisenia fetida, les gènes dont l'expression est affectée lors d'une exposition à un mélange complexe de métaux, représentatif d'un site naturel fortement contaminé. Ces deux approches ont permis l'identification de 4 candidats biomarqueurs de pollution métallique.Metallurgical activities lead to accumulation of metal trace elements in the topsoils, where one typically observes concentrations largely exceeding the natural background. Contaminations of soils are a threat for public health and the presence of high amount of metals can generate an environmental stress likely to affect the organisms exposed to polluted soils. Earthworms living in close contact with polluted soils were studied in order to better understand the physiological changes, the mechanisms of acclimation and of detoxification caused by metals. By exploiting the conservation observed between species and using molecular biology techniques, we have been able to clone and transcriptionally characterise potential biomarkers form the immune ceIls of the ecotoxicologically important earthworm species Eisenia fetida. During this Ph. D. we developed two approaches. First, a targeted approach based on bibliographie work, enabled us to identify and select effectors among highly preserved proteins for which variations following metal exposure were reported in invertebrates. Second, an open strategy was undertaken by performing a Suppression Subtractive Hybridization in order to identify genes which are differentially expressed following exposure to a complex mixture of metals representative of a strongly polluted soil. These approaches allowed the description of a group of genes which expression varies following metallic exposure as weIl as the analysis of their expression profiles may be used to analyse mechanistically the immune response to a metallic stress. Moreover, expression profiles of selected genes may constitute a signature of changes due to MTE

Développement de biomarqueurs d'exposition des métaux sur les fonctions physiologiques de l'annélide oligochète Eisenia fetida

Les activités métallurgiques entrainent l'accumulation d'éléments traces métalliques dans les couches . superficielles des sols, où l'on peut observer des concentrations largement supérieures au fond pédogéochimique et aux normes en vigueur. La contamination des sols est une menace pour la santé publique et la présence de grandes quantités d'éléments traces métalliques peut générer un stress susceptible d'affecter les organismes exposés à des sols pollués. Les Annélides Oligochètes vivent en contact étroit avec ces sols pollués et sont parmi les organismes vivants présentant une sensibilité exacerbée aux métaux. Très peu de choses sont connues quant à l'identification et la mise en place des mécanismes de réponse à ces métaux au niveau moléculaire. En exploitant, la conservation phylogénique observée entre espèces nous avons été capables de cloner et de caractériser un ensemble de biomarqueurs de pollution potentiels à partir des cœlomocytes de l'Annélide Oligochètes Eisenia fetida. Deux approches ont été mises en place. Premièrement, une approche qualifiée de ciblée, consistant à identifier tous les effecteurs parmi des protéines fortement conservées pour lesquels une variation lors d'une exposition métallique était reportée dans la littérature. Deuxièmement, nous avons entrepris une approche qualifiée de globale consistant en la construction de banques soustractives pour identifier chez Eisenia fetida, les gènes dont l'expression est affectée lors d'une exposition à un mélange complexe de métaux, représentatif d'un site naturel fortement contaminé. Ces deux approches ont permis l'identification de 4 candidats biomarqueurs de pollution métallique.Metallurgical activities lead to accumulation of metal trace elements in the topsoils, where one typically observes concentrations largely exceeding the natural background. Contaminations of soils are a threat for public health and the presence of high amount of metals can generate an environmental stress likely to affect the organisms exposed to polluted soils. Earthworms living in close contact with polluted soils were studied in order to better understand the physiological changes, the mechanisms of acclimation and of detoxification caused by metals. By exploiting the conservation observed between species and using molecular biology techniques, we have been able to clone and transcriptionally characterise potential biomarkers form the immune ceIls of the ecotoxicologically important earthworm species Eisenia fetida. During this Ph. D. we developed two approaches. First, a targeted approach based on bibliographie work, enabled us to identify and select effectors among highly preserved proteins for which variations following metal exposure were reported in invertebrates. Second, an open strategy was undertaken by performing a Suppression Subtractive Hybridization in order to identify genes which are differentially expressed following exposure to a complex mixture of metals representative of a strongly polluted soil. These approaches allowed the description of a group of genes which expression varies following metallic exposure as weIl as the analysis of their expression profiles may be used to analyse mechanistically the immune response to a metallic stress. Moreover, expression profiles of selected genes may constitute a signature of changes due to MTE.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Transcriptomic underpinning of toxicant-mediated physiological function alterations in three terrestrial invertebrate taxa: A review.

Diverse anthropogenic activities often lead to the accumulation of inorganic and organic residues in topsoils. Biota living in close contact with contaminated soils may experience stress at different levels of biological organisation throughout the continuum from the molecular-genetic to ecological and community levels. To date, the relationship between changes at the molecular (mRNA expression) and biochemical/physiological levels evoked by exposures to chemical compounds has been partially established in a limited number of terrestrial invertebrate species. Recently, the advent of a family of transcriptomic tools (e.g. Real-time PCR, Subtractive Suppressive Hybridization, Expressed Sequence Tag sequencing, pyro-sequencing technologies, Microarray chips), together with supporting informatic and statistical procedures, have permitted the robust analyses of global gene expression changes within an ecotoxicological context. This review focuses on how transcriptomics is enlightening our understanding of the molecular-genetic responses of three contrasting terrestrial macroinvertebrate taxa (nematodes, earthworms, and springtails) to inorganics, organics, and agrochemicals

Differential expression of genes involved in immunity and biomineralization during Brown Ring Disease development and shell repair in the Manila clam, Ruditapes philippinarum.

International audienceSevere drop in Manila clams production in French aquacultured fields since the end of the 1980's is associated to Brown Ring Disease (BRD). This disease, caused by the bacteria Vibrio tapetis, is characterized by specific symptoms on the inner face of the shell. Diseased animals develop conchiolin deposit to enrobe bacteria and form new calcified layers on the shell. Suppression subtractive hybridization was performed to identify genes differentially expressed during the early interaction of V. tapetis and Ruditapes philippinarum. Results revealed 301 unique genes differentially expressed during V. tapetis challenge. Several candidates involved in immune and biomineralization processes were selected from libraries. Transcriptional expression of selected candidates was determined in hemolymph and mantle tissues and revealed spatial and temporal variations. At 56days after infection, when clams were in phase of shell repair, transcripts of galectin and ferritin in hemocytes showed higher expression. Ca-like and serpin transcripts were specifically expressed in mantle and could contribute to defense against BRD

Identification of suitable qPCR reference genes in leaves of Brassica oleracea under abiotic stresses

International audienc

Transcriptomic analysis of Ruditapes philippinarum hemocytes reveals cytoskeleton disruption after in vitro Vibrio tapetis challenge.

International audienceThe Manila clam, Ruditapes philippinarum, is an economically-important, commercial shellfish; harvests are diminished in some European waters by a pathogenic bacterium, Vibrio tapetis, that causes Brown Ring disease. To identify molecular characteristics associated with susceptibility or resistance to Brown Ring disease, Suppression Subtractive Hybridization (SSH) analyzes were performed to construct cDNA libraries enriched in up- or down-regulated transcripts from clam immune cells, hemocytes, after a 3-h in vitro challenge with cultured V. tapetis. Nine hundred and ninety eight sequences from the two libraries were sequenced, and an in silico analysis identified 235 unique genes. BLAST and "Gene ontology" classification analyzes revealed that 60.4% of the Expressed Sequence Tags (ESTs) have high similarities with genes involved in various physiological functions, such as immunity, apoptosis and cytoskeleton organization; whereas, 39.6% remain unidentified. From the 235 unique genes, we selected 22 candidates based upon physiological function and redundancy in the libraries. Then, Real-Time PCR analysis identified 3 genes related to cytoskeleton organization showing significant variation in expression attributable to V. tapetis exposure. Disruption in regulation of these genes is consistent with the etiologic agent of Brown Ring disease in Manila clams

A two years field experiment to assess the impact of two fungicides on earthworm communities and their recovery

International audienceRecent EFSA (European Food Safety Authority) reports highlighted that the ecological risk assessment of pesticides needed to go further by taking more into account the impacts of chemicals on biodiversity under field conditions. We assessed the effects of two commercial formulations of fungicides separately and in mixture, i.e., Cuprafor Micro® (containing 500 g kg−1 copper oxychloride) at 4 (C1, corresponding to 3.1 mg kg−1 dry soil of copper) and 40 kg ha−1 (C10), and Swing® Gold (50 g L−1 epoxiconazole EPX and 133 g L−1 dimoxystrobin DMX) at one (D1, 5.81 10−2 and 1.55 10−1 mg kg−1 dry soil of EPX and DMX, respectively) and ten times (D10) the recommended field rate, on earthworms at 1, 6, 12, 18 and 24 months after the application following the international ISO standard no. 11268-3 to determine the effects on earthworms in field situations. The D10 treatment significantly reduced the species diversity (Shannon diversity index, 54% of the control), anecic abundance (29% of the control), and total biomass (49% of the control) over the first 18 months of experiment. The Shannon diversity index also decreased in the mixture treatment (both fungicides at the recommended dose) at 1 and 6 months after the first application (68% of the control at both sampling dates), and in C10 (78% of the control) at 18 months compared with the control. Lumbricus terrestris, Aporrectodea caliginosa, Aporrectodea giardi, Aporrectodea longa, and Allolobophora chlorotica were (in decreasing order) the most sensitive species to the tested fungicides. This study not only addressed field ecotoxicological effects of fungicides at the community level and ecological recovery, but it also pinpointed some methodological weaknesses (e.g., regarding fungicide concentrations in soil and statistics) of the guideline to determine the effects on earthworms in field situations

Earthworm indicators as tools for soil monitoring, characterization and risk assessment. An example from the national Bioindicator programme (France)

International audienceConsidering the limited number of bioindicators available to assess soil quality, a national research programme was set up in France to develop such indicators (2006-2012), the "Bioindicator" programme. This programme tested 47 biological parameters (i.e. microorganisms, fauna, flora) including earthworms, in several sites differing in terms of land use, contamination type - PAHs or metals - and pollution levels. The present study proposes some study objectives for bioindicator approaches, based on the earthworm results from the programme. Therefore, different earthworm descriptors were tested at the community level (e.g. abundance, biomass, species and functional structures, and ecological traits) as well as the organism level (i.e. measuring the metallothionein coding gene expression level in earthworms). The present results, obtained from the programme's spring 2009 sampling campaign, discriminated among the different descriptors and showed that earthworm and endogeic abundance as well as the individual weight of endogeics seem to be good indicators in non-contaminated (cultivated) sites, while the ecological structure, namely the proportion of anecic vs. endogeic species, and the proportion of non-vulnerable species should be used as indicators of contaminated soils. Furthermore, the first results obtained for Lumbricus terrestris and L. rubellus rubellus are encouraging as they show that metallothionein expression increases in metal-contaminated soils. The relevance of these descriptors, which have to be considered in study objectives, requires the analysis of 2010 results