Behavioural and Physiological Responses of Gammarus pulex Exposed to Cadmium and Arsenate at Three Temperatures: Individual and Combined Effects

This study aimed at investigating both the individual and combined effects of cadmium (Cd) and arsenate (AsV) on the physiology and behaviour of the Crustacean Gammarus pulex at three temperatures (5, 10 and15°C). G. pulex was exposed during 96 h to (i) two [Cd] alone, (ii) two [AsV] alone, and (iii) four combinations of [Cd] and [AsV] to obtain a complete factorial plane. After exposure, survival, [AsV] or [Cd] in body tissues, behavioural (ventilatory and locomotor activities) and physiological responses (iono-regulation of [Na+] and [Cl−] in haemolymph) were examined. The interactive effects (antagonistic, additive or synergistic) of binary mixtures were evaluated for each tested temperature using a predictive model for the theoretically expected interactive effect of chemicals. In single metal exposure, both the internal metal concentration in body tissues and the mortality rate increased along metallic gradient concentration. Cd alone significantly impaired both [Na+] and [Cl−] while AsV alone had a weak impact only on [Cl−]. The behavioural responses of G. pulex declined with increasing metal concentration suggesting a reallocation of energy from behavioural responses to maintenance functions. The interaction between AsV and Cd was considered as ‘additive’ for all the tested binary mixtures and temperatures (except for the lowest combination at 10°C considered as “antagonistic”). In binary mixtures, the decrease in both ventilatory and locomotor activities and the decline in haemolymphatic [Cl−] were amplified when respectively compared to those observed with the same concentrations of AsV or Cd alone. However, the presence of AsV decreased the haemolymphatic [Na+] loss when G. pulex was exposed to the lowest Cd concentration. Finally, the observed physiological and behavioural effects (except ventilation) in G. pulex exposed to AsV and/or Cd were exacerbated under the highest temperature. The discussion encompasses both the toxicity mechanisms of these metals and their interaction with rising temperature

Single or combined metallic exposure effect at different temperatures in Gammarus pulex : multi-markers approach and perspectiv in bioevaluation

Les contaminations multiples par des métaux et métalloïdes menacent la structure et le fonctionnement des écosystèmes aquatiques. Pourtant, les mécanismes d'action des polluants, leurs interactions et leur toxicité dans un contexte de réchauffement climatique sont encore peu abordés. Parallèlement, nous avons un besoin urgent de marqueurs d'alerte (i.e., précoces, sub-létaux) des effets de ces substances. Par ailleurs, une attention croissante est actuellement portée au protéome des êtres vivants, considéré comme une entité dynamique, modifié par les conditions du milieu. Dans ce contexte, nous avons travaillé en laboratoire avec comme espèce modèle le crustacé amphipode Gammarus pulex avec trois objectifs: (1) évaluer les effets de deux éléments métalliques (cadmium et arséniate) seuls ou en interaction sur des organismes exposés en conditions contrôlées, (2) appréhender les effets de la température en tant que facteur confondant potentiel des réponses mesurées, et (3) rechercher l'existence d'une réponse singulière potentielle du protéome d'individus exposés aux éléments métalliques seuls ou en mélange ; à plusieurs températures. Il s'agissait donc à la fois de réaliser une analyse diagnostique susceptible de fournir des marqueurs d'intérêt aptes à révéler précocement un stress chimique et de dresser les bases d'une approche pronostique permettant de mieux prédire les conséquences à l'échelle individuelle d'une dégradation anthropique du milieuMultiple metal and metalloid contaminations impair aquatic ecosystem community structure and functioning. However, the mechanistic understanding of individual pollutant toxic effects, but also of their interaction effects in a "global warning" context are still rarely tackled. However, aquatic ecosystem managers have an urgent need of early warning indicators (i.e. early, sub-lethal) of toxicant effects. Recently, an increasing attention has been turned to studies of the proteome of species/populations, which is considered as a dynamic entity modified by environmental conditions. In this context, we have worked with a model organism, Gammarus pulex (crustacea, amphipoda), under laboratory controlled conditions. Three objectives have been defined: (1) assessing the single and combined effects of two metals (i.e. arsenate and/or cadmium), (2) understanding the potential confounding effect of temperature on measured physiological and behavioural responses of gammarids and (3) investigating the existence of a singular response of the proteome of individuals exposed to different single or combined metal concentrations, at several tested temperatures. The major goals were first applying a diagnostic analysis to provide valuable biomarkers early indicating metallic stress, and second developing the foundations for a prognostic approach better predicting the organismal consequences of toxic contaminations due to the anthropogenic degradation of aquatic ecosystem

Effets d'expositions métalliques simples ou multiples à différentes températures chez Gammarus pulex : approche multi-marqueurs et perspectives en bioévaluation

Multiple metal and metalloid contaminations impair aquatic ecosystem community structure and functioning. However, the mechanistic understanding of individual pollutant toxic effects, but also of their interaction effects in a "global warning" context are still rarely tackled. However, aquatic ecosystem managers have an urgent need of early warning indicators (i.e. early, sub-lethal) of toxicant effects. Recently, an increasing attention has been turned to studies of the proteome of species/populations, which is considered as a dynamic entity modified by environmental conditions. In this context, we have worked with a model organism, Gammarus pulex (crustacea, amphipoda), under laboratory controlled conditions. Three objectives have been defined: (1) assessing the single and combined effects of two metals (i.e. arsenate and/or cadmium), (2) understanding the potential confounding effect of temperature on measured physiological and behavioural responses of gammarids and (3) investigating the existence of a singular response of the proteome of individuals exposed to different single or combined metal concentrations, at several tested temperatures. The major goals were first applying a diagnostic analysis to provide valuable biomarkers early indicating metallic stress, and second developing the foundations for a prognostic approach better predicting the organismal consequences of toxic contaminations due to the anthropogenic degradation of aquatic ecosystemsLes contaminations multiples par des métaux et métalloïdes menacent la structure et le fonctionnement des écosystèmes aquatiques. Pourtant, les mécanismes d'action des polluants, leurs interactions et leur toxicité dans un contexte de réchauffement climatique sont encore peu abordés. Parallèlement, nous avons un besoin urgent de marqueurs d'alerte (i.e., précoces, sub-létaux) des effets de ces substances. Par ailleurs, une attention croissante est actuellement portée au protéome des êtres vivants, considéré comme une entité dynamique, modifié par les conditions du milieu. Dans ce contexte, nous avons travaillé en laboratoire avec comme espèce modèle le crustacé amphipode Gammarus pulex avec trois objectifs: (1) évaluer les effets de deux éléments métalliques (cadmium et arséniate) seuls ou en interaction sur des organismes exposés en conditions contrôlées, (2) appréhender les effets de la température en tant que facteur confondant potentiel des réponses mesurées, et (3) rechercher l'existence d'une réponse singulière potentielle du protéome d'individus exposés aux éléments métalliques seuls ou en mélange ; à plusieurs températures. Il s'agissait donc à la fois de réaliser une analyse diagnostique susceptible de fournir des marqueurs d'intérêt aptes à révéler précocement un stress chimique et de dresser les bases d'une approche pronostique permettant de mieux prédire les conséquences à l'échelle individuelle d'une dégradation anthropique du milie

Effects of Sublethal Cadmium Exposure on Antipredator Behavioural and Antitoxic Responses in the Invasive Amphipod Dikerogammarus villosus

Amphipods are recognised as an important component of freshwater ecosystems and are frequently used as an ecotoxicological test species. Despite this double interest, there is still a lack of information concerning toxic impacts on ecologically relevant behaviours. The present study investigated the influence of cadmium (Cd), a non-essential heavy metal, on both antipredator behaviours and antitoxic responses in the invasive amphipod Dikerogammarus villosus under laboratory conditions. Amphipod behaviour (i.e. refuge use, aggregation with conspecifics, exploration and mobility) was recorded following a 4-min test-exposure to 500 µg Cd/L with or without a 24-h Cd pre-exposure and in the presence or absence of a high perceived risk of predation (i.e. water scented by fish predators and injured conspecifics). Following behavioural tests, malondialdehyde (MDA) levels, a biomarker for toxic effect, and energy reserves (i.e. lipid and glycogen contents) were assessed. Cd exposures induced (1) cell damage reflected by high MDA levels, (2) erratic behaviour quantified by decreasing refuge use and exploration, and increasing mobility, and (3) a depletion in energy reserves. No significant differences were observed between 4-min test-exposed and 24-h pre-exposed individuals. Gammarids exposed to Cd had a disturbed perception of the alarm stimuli, reflected by increased time spent outside of refuges and higher mobility compared to gammarids exposed to unpolluted water. Our results suggest that Cd exposure rapidly disrupts the normal behavioural responses of gammarids to alarm substances and alters predator-avoidance strategies, which could have potential impacts on aquatic communities

Antagonistic toxicity of arsenate and cadmium in a freshwater amphipod (Gammarus pulex)

International audienc

Le problème de l'évaluation des risques toxiques associés à des facteurs de stress multiples: le cas d'un amphipode d'eau douce, Gammarus pulex, soumis à des expositions métalliques simples et combinées dans un contexte de changements climatiques

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Investigating the emerging role of comparative proteomics in the search for new biomarkers of metal contamination under varying abiotic conditions

13 pagesInternational audienceThis study aims at investigating the potential use of comparative proteomics as a multi-marker approach of metal contamination, taking into account the potential confounding effect of water temperature. The major objective was to identify combinations of proteins specifically responding to a given metal, even if included in a metal mixture. The diagnostic approach was performed via the comparative analysis of protein expression on spot mapping provided by adult males of Gammarus pulex (Amphipoda, Crustacea) respectively exposed to arsenate (As), cadmium (Cd) or a binary mixture of these metals (AsCd) at three realistic temperatures (5, 10 and 15 °C). Proteomic expression analysis was performed by Differential in-Gel Electrophoresis (2D-DiGE), and completed by an adapted inferential statistical approach. Combinations of under/over-expressed protein spots discriminated the metal identity. However, none of these spots discriminated both the individual metal effect (As or Cd) and its effect in metal mixture (AsCd) whatever the tested temperature. Some limits of the two-dimensional analysis of protein spot maps in G. pulex have been highlighted: (i) the presence of contaminating peptides and/or abundant “déja-vu” proteins which can mask the responses of other proteins of interest or (ii) the presence of post-translational modifications. An optimization of the experimental design (especially during the sample preparation) has been described for future investigations.This study has also highlighted (i) the importance of precisely identifying the protein spots of interest to avoid erroneous interpretations in terms of action mechanisms of chemicals and (ii) the importance of working under controlled laboratory conditions with a temperature close to 10 °C. In such conditions, we have demonstrated a higher impact of As than Cd on the energetic metabolism of Gammarus. This As impact is reduced in AsCd mixture confirming the antagonistic interaction of this binary mixture previously observed on G. pulex mortality at 10 °C