Patrice Couture

Effects of chronic metal exposure on wild fish populations revealed by high-throughput cDNA sequencing

Given the inherent variability of aquatic systems, predicting the in situ effects of contaminants on such ecosystems still represents a major challenge for ecotoxicology. In this context, transcriptomic tools can help identify and investigate the mechanisms of toxicity beyond the traditional morphometric, physiological and population-level endpoints. In this study, we used the 454 sequencing technology to examine the in situ effects of chronic metal (Cd, Cu) exposure on the yellow perch (Perca flavescens) transcriptome. Total hepatic mRNA from fish sampled along a polymetallic gradient was extracted, reverse transcribed, labeled with unique barcode sequences and sequenced. This approach allowed us to identify correlations between the transcription level of single genes and the hepatic concentrations of individual metals; 71% of the correlations established were negative. Chronic metal exposure was thus associated with a decrease in the transcription levels of numerous genes involved in protein biosynthesis, in the immune system, and in lipid and energy metabolism. Our results suggest that this marked decrease could result from an impairment of bile acid metabolism by Cd and energy restriction but also from the recruitment of several genes involved in epigenetic modifications of histones and DNA that lead to gene silencing.

RAD sequencing reveals within‐generation polygenic selection in response to anthropogenic organic and metal contamination in North Atlantic Eels

Measuring the effects of selection on the genome imposed by human‐altered environment is currently a major goal in ecological genomics. Given the polygenic basis of most phenotypic traits, quantitative genetic theory predicts that selection is expected to cause subtle allelic changes among covarying loci rather than pronounced changes at few loci of large effects. The goal of this study was to test for the occurrence of polygenic selection in both North Atlantic eels (European Eel, Anguilla anguilla and American Eel, A. rostrata), using a method that searches for covariation among loci that would discriminate eels from ‘control’ vs. ‘polluted’ environments and be associated with specific contaminants acting as putative selective agents. RAD‐seq libraries resulted in 23 659 and 14 755 filtered loci for the European and American Eels, respectively. A total of 142 and 141 covarying markers discriminating European and American Eels from ‘control’ vs. ‘polluted’ sampling localities were obtained using the Random Forest algorithm. Distance‐based redundancy analyses (db‐RDAs) were used to assess the relationships between these covarying markers and concentration of 34 contaminants measured for each individual eel. PCB153, 4′4′DDE and selenium were associated with covarying markers for both species, thus pointing to these contaminants as major selective agents in contaminated sites. Gene enrichment analyses suggested that sterol regulation plays an important role in the differential survival of eels in ‘polluted’ environment. This study illustrates the power of combining methods for detecting signals of polygenic selection and for associating variation of markers with putative selective agents in studies aiming at documenting the dynamics of selection at the genomic level and particularly so in human‐altered environments.

Transcriptional responses to environmental metal exposure in wild yellow perch (Perca flavescens) collected in lakes with differing environmental metal concentrations (Cd, Cu, Ni)

To investigate the mechanisms involved in metal stress in wild fish, yellow perch (Perca flavescens) were collected in eight lakes of the Rouyn-Noranda and Sudbury regions (Canada). Due to mining and smelting activities, these two regions indeed present a broad contamination gradient in metal concentrations (Cd, Cu, Zn and Ni; water, sediment and prey) and offer a unique research opportunity to investigate relationships between metal bioaccumulation and resulting deleterious effects in indigenous biota chronically exposed to metal mixtures. The expression level of genes encoding for proteins involved in metal detoxification (metallothioneins, mts), protein protection (heat shock protein-70, hsp-70), growth (insulin-like growth factor-1, igf-1), aerobic energy metabolism (cytochrome c oxydase, cco-1) and protection against oxidative stress (Cu/Zn superoxide dismutase, sod-1) were assessed in fish liver and muscle in association with protein and enzymatic assays for cytochrome c oxidase (CCO). Bioaccumulation of both Cd and Cu increased in response to higher ambient metal concentrations, but the two metals clearly have different modes of action. For Cd, changes in gene expression levels were more marked in the liver than in the dorsal muscle, whereas for Cu the opposite trend was observed. Hepatic Cd accumulation was linked to decreased cco-1 and sod-1 gene expression, whereas Cu accumulation was associated with a decrease in CCO enzymatic activity and an increase in total protein concentration and in cco-1, mts and hsp-70 gene expression levels. For Ni, no significant correlations were observed at the transcriptional level, but increasing hepatic Ni concentrations were significantly and positively correlated with protein concentrations and CCO activity. By coupling gene expression to biochemical and physiological endpoints, this work provides new insights into the mechanisms involved in metal stress and the adaptive response of fish chronically exposed to metal mixtures.

Individual and combined effects of heat stress and aqueous or dietary copper exposure in fathead minnows (Pimephales promelas)

Despite its role as an essential micronutrient, copper (Cu) can be present in aquatic ecosystems at concentrations able to cause adverse health effects on aquatic organisms. Although Cu is acquired by fish by either water or diet, studies that have investigated Cu impacts in fish have mainly focused on the toxicity of waterborne Cu. Moreover, as the majority of experiments were carried out under simplified conditions, little is known about the effects of natural factors other than competitive ions on Cu toxicity in fish. As temperature is a primary factor that affects the physiological state of poikilotherm organisms, we investigated the individual and combined effects of temperature and waterborne or dietary Cu on fathead minnows (Pimephales promelas). Fish were exposed to environmentally realistic concentrations of waterborne or dietary Cu at 20 °C and 32 °C. Transcriptional and enzymatic responses of various indicators of metabolic capacities as well as indicators of heat, oxidative and metal stresses were measured in fish muscle. Under our experimental conditions, temperature was the most important factor affecting the general condition of fish. Although no significant Cu accumulation was observed in the muscle of Cu-exposed fish, at 20 °C, waterborne and dietary Cu triggered significant changes in the transcription level of genes encoding for proteins involved in energy metabolism, metal detoxification and protein protection. Moreover, the response was quantitatively more important for dietary Cu than for waterborne Cu. Combined exposure to heat and Cu triggered the most significant changes in gene transcription levels and enzyme activities. During combined exposure to heat and Cu, in addition to synergistic effects of the two factors, both waterborne and dietary Cu impaired the adaptive response developed by fish to curb heat stress. Reciprocally, temperature impaired the adaptive response developed by fish to combat Cu toxicity. These results suggest that wild fish populations subjected to elevated temperatures due to seasonal warming or global climate change may become more susceptible to Cu pollution, and vice versa.

Seasonal and regional variations of metal contamination and condition indicators in yellow perch (Perca flavescens) along two polymetallic gradients. I. Factors influencing tissue metal concentrations

ABSTRACT This study examined relationships among water, sediment, diet, and fish tissue metal (Cd, Cu, Ni, Se, and Zn) concentrations in yellow perch from metal gradients in two regions (Sudbury (S), Ontario, and Rouyn-Noranda (RN), Québec, Canada) in two seasons (spring and summer). The objectives of this study were (1) to examine the influences of aqueous and dietary metal contamination on yellow perch liver and kidney metal accumulation; (2) to compare the seasonal and regional variations in gut content and tissue metal concentrations along the two gradients studied; and (3) to investigate the potential of metals for tissue accumulation under conditions of life-long chronic exposure. Our results suggest a greater aqueous than dietary influence on tissue metal concentrations for all metals examined except Cd, where the opposite was observed. Metals did not accumulate in older fish, except for Cd that accumulated with age in RN, but not S, fish. Regional, but also metal-specific differences in metal handling capacities are proposed. Fish from neither region appeared capable of regulating tissue Cd concentrations, but fish from both regions regulated Zn tightly. Sudbury fish appeared better at regulating tissue Cu, Ni, and perhaps also Se concentrations than RN fish, suggesting acclimation or selection for metal tolerance. There were several significant seasonal effects on tissue metal concentrations. However, close examination of the dataset does not allow proposing the presence of a season-linked mechanism explaining these variations, precluding a modeling approach and implying that repeat sampling within and among years is required for proper ecological risk assessment.

Physiological effects of temperature and a herbicide mixture on the soft‐shell clam Mya arenaria (Mollusca, Bivalvia)

The aim of the current study was to investigate effects of temperature and a mixture of herbicides on the physiological status of the bivalve Mya arenaria. Bivalves acclimated to two temperatures (7 and 18°C) were exposed for 28 d to 0.01 mg/L of a pesticide formulation containing dichlorophenoxyacetic acid (2,4-D), 2-(2-methyl-4-chlorophenoxy) propionic acid (mecoprop), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). At days 7, 14, and 28, mortality, immune parameters (hemocyte number, phagocytic activity, and efficiency), biomarkers of oxidative stress (catalase [CAT] and superoxide dismutase [SOD] activities and malondialdehyde [MDA] content), the metabolic enzyme cytochrome C oxidase (CCO), a biomarker of pesticide exposure (acetylcholinesterase [AChE]), and the activity of an enzyme related to gametogenesis (aspartate transcarbamylase [ATCase]) were monitored in clam tissues. Gonadosomatic index (GSI), condition factor (CF), and sex were also assessed. In clams acclimated to 7°C, exposure to pesticide enhanced CCO activity and CF and decreased MDA content, hemocyte number, CAT, and SOD activities. In clams kept at 18°C, pesticide effects appeared minor compared with samples kept at 7°C. In bivalves acclimated to 18°C, CCO, SOD, and ATCase activity and MDA content were enhanced, and hemocyte number, CAT, and AchE activities and phagocytosis were suppressed. In samples exposed to pesticides, increased temperature enhanced MDA content and CCO and SOD activity and suppressed hemocyte number and CAT and AchE activity. A gradual sexual maturation was observed in both sexes through experimental time, but females had a higher sensitivity to temperature and pesticides compared to males. Increased temperature altered the ability of the sentinel species Mya arenaria to respond to pesticide exposures. Further work is needed to understand the impacts of increasing temperature on the whole St. Lawrence estuary ecosystem.

Live Fast and Die Young: Metal Effects on Condition and Physiology of Wild Yellow Perch from along Two Metal Contamination Gradients

ABSTRACT This review summarizes some of the main findings of our work with the Metals in the Environment Research Network examining seasonal and regional effects on metal accumulation, growth, condition, and physiology in wild yellow perch (Perca flavescens) from 10 lakes comprising two metal contamination gradients in the industrial regions of Sudbury, Ontario and Rouyn-Noranda, Québec, Canada. The specific objectives of this review are: (1) to propose threshold tissue metal concentrations to discriminate between fish from contaminated and reference sites; (2) to identify factors that can influence metal accumulation and fish condition; and (3) to define an experimental approach for measuring metal effects in wild yellow perch. Using tissue thresholds appeared useful not only for discriminating fish from clean or contaminated environments, but also provided a simple approach to examine metabolic consequences of tissue metal accumulation. Overall, fish from Sudbury grew faster, expressed higher aerobic capacities, and died younger, but also appeared better at limiting accumulation of some metals than Rouyn-Noranda fish. The condition of the latter fish was clearly more affected by metals than Sudbury fish. Finally, our dataset allows us to propose that yellow perch are highly suitable for ecological risk assessment studies of metal effects in wild fish, but that fish size, season, and region must be considered in sampling design and that several reference sites must be studied for meaningful conclusions to be reached.

Inhibition of goldfish mitochondrial metabolism by in vitro exposure to Cd, Cu and Ni

Although impairment of aerobic capacities has been reported in metal-contaminated wild fish, little is known about the direct toxicity of the metals themselves at the low concentrations found in the field compared to indirect consequences mediated by metal effects on ecological variables such as prey type and abundance, predation and competition. This study examined the in vitro effects of Cd, Cu and Ni on mitochondrial enzyme activity and maximal (State 3) mitochondrial oxygen consumption rate in goldfish (Carassius auratus) tissues at concentrations representative of values reported in wild metal-contaminated fish. There was little effect of adding metals to liver or muscle homogenates on the activity of citrate synthase (CS), although a slight inhibition of liver CS was observed at the highest Cd concentration tested. In contrast, adding high concentrations of Ni to muscle homogenates increased muscle CS activity. Unlike CS, the metalloenzyme cytochrome C oxidase (CCO) was quite sensitive to metal additions; its activity was consistently enhanced by all three metals tested. When added to liver mitochondrial preparations, both Cd and Cu strongly inhibited State 3 respiration. In contrast, Ni did not affect mitochondrial respiration even at the highest concentration tested. Taken together, these results demonstrate that low concentrations of Cd, Cu and Ni have toxic effects on mitochondrial metabolism and enzyme activities and suggest that the inhibition of aerobic capacities frequently reported for wild metal-contaminated fish is at least partly due to metal effects on mitochondrial function, although the mechanisms probably do not involve direct enzyme inhibition.

How does exposure to nickel and cadmium affect the transcriptome of yellow perch (Perca flavescens) – Results from a 1000 candidate-gene microarray

The molecular mechanisms underlying nickel (Ni) and cadmium (Cd) toxicity and their specific effects on fish are poorly understood. Documenting gene transcription profiles offers a powerful approach toward identifying the molecular mechanisms affected by these metals and to discover biomarkers of their toxicity. However, confounding environmental factors can complicate the interpretation of the results and the detection of biomarkers for fish captured in their natural environment. In the present study, a 1000 candidate-gene microarray, developed from a previous RNA-seq study on a subset of individual fish from contrasting level of metal contamination, was used to investigate the transcriptional response to metal (Ni and Cd) and non metal (temperature, oxygen, and diet) stressors in yellow perch (Perca flavescens). Specifically, we aimed at (1) identifying transcriptional signatures specific to Ni and Cd exposure, (2) investigating the mechanisms of their toxicity, and (3) developing a predictive tool to identify the sublethal effects of Ni and Cd contaminants in fish sampled from natural environments. A total of 475 genes displayed significantly different transcription levels when temperature varied while 287 and 176 genes were differentially transcribed at different concentrations of Ni and Cd, respectively. These metals were found to mainly affect the transcription level of genes involved in iron metabolism, transcriptional and translational processes, vitamin metabolism, blood coagulation, and calcium transport. In addition, a linear discriminant analysis (LDA) made using gene transcription levels yielded 94% correctly reassigned samples regarding their level of metal contamination, which indicates the potential of the microarray to detect perch response to Cd or Ni effects.

Physiological correlates of growth and condition in the yellow perch (Perca flavescens)

This study on yellow perch (Perca flavescens) examines a series of enzymatic markers and the relative weights of pyloric caeca and visceral lipids, their response to changes in feeding regime and their potential use to infer recent changes in growth rate and fish condition. Fish were exposed to four different feeding regimes for 12 weeks resulting in specific growth rates ranging from 0.3% to 3.5% (%/day). Growth and condition responded rapidly to changes in ration and the weight of pyloric caeca and visceral lipids reflected increased feed intake. Growth rate was correlated with muscle citrate synthase and caecal nucleoside-diphosphate kinase activities, whereas condition was correlated with muscle citrate synthase and lactate dehydrogenase activities and with caecal glucose-6-phosphate dehydrogenase activity. Results showed that enzyme activities and biometric parameters responded rapidly to increased feed intake, but the response was slower when food intake decreased. Plateaus were attained for both condition and visceral lipid index, but the relative weight of pyloric caeca continued to increase throughout the experimental period. Results from this study could, in principle, be used to infer recent growth and energy status in wild yellow perch and thus provide an indicator of food availability in their environment.