Réponses de trois espèces sentinelles aquatiques à la contamination en métaux traces dans deux rivières de l'Abitibi, Québec

La métallothionéine (MT) est un ligand protéinique intracellulaire important à cause de son implication dans les processus de régulation des métaux traces essentiels et non-essentiels. Bon nombre de travaux ont été conduits en laboratoire pour bien comprendre les mécanismes de synthèse de cette protéine, mais les études réalisées en milieu aquatique sont beaucoup plus rares, particulièrement dans les rivières (milieu lotique). Dans ce contexte, la présente étude avait deux objectifs principaux : (1) identifier en milieu lotique les métaux traces (argent "Ag", cadmium "Cd", cuivre "Cu", nickel "Ni", plomb "Pb" et zinc "Zn") susceptibles d'induire la synthèse de la MT chez trois espèces sentinelles, soit le mollusque bivalve Pyganodon grandis, la larve d'éphémère Hexagenia limbata et le grand brochet du nord Esox lucius ; (2) définir les variables physico-chimiques ou toxicologiques pouvant influencer la prise en charge des métaux traces et conséquemment les concentrations en MT chez le mollusque P. grandis et l'éphémère H. limbata. Deux rivières de la région de l'Abitibi ont été échantillonnées à l'été 2001 afin de récolter les espèces sentinelles à 21 stations. Les concentrations en métaux traces, ainsi qu'en métallothionéine, ont été mesurées dans les branchies des mollusques, dans le rein des brochets et dans le corps entier des éphémères. La métallothionéine a été mesurée à l'aide d'une technique de saturation au mercure. D'après nos résultats, le cadmium tissulaire semble être le meilleur prédicteur de la concentration de MT chez les trois espèces sentinelles. Cependant, l'implication d'autres métaux est également possible. Dans le cas de P. grandis, la concentration de manganèse (Mn) dans les sédiments pourrait influencer la prise en charge du Cd et la synthèse de MT.Metallothionein (MT), an intracellular metal binding protein, is recognized for its role in the regulation of essential and non-essential trace metals. The mechanisms of synthesis of this protein have been studied in the laboratory but much less work has been carried out in the field, particularly in lotic environments. According to the literature, metallothionein protects tissues from metal damage, and may be used as a direct measure of the biochemical state of the affected tissue. Metallothionein shows high affinity for group IB and IIB metals ions (e.g. Cd, Cu, Zn), and consequently can sequester and detoxify metals. The use of MT as a biomarker for elevated levels of trace metals in aquatic environments has therefore been proposed as a tool in ecotoxicological studies. Changes at the biochemical level offer distinct advantages as biomarkers, since molecular alterations are normally the first detectable responses to environmental changes.The present study focused on two objectives, the first of which was to identify which trace metals (silver "Ag", cadmium "Cd", copper "Cu", nickel "Ni", lead "Pb" and zinc "Zn") may induce MT synthesis in three sentinel species collected in two northern Quebec rivers (Colombière River, close to Val d'Or, and Matagami River close to the town of Matagami) in a mining region (Abitibi, Québec). The three sentinel species were the adult bivalve Pyganodon grandis, the larval mayfly Hexagenia limbata, and the adult great northern pike Esox lucius. The second objective was to define variables that could influence the uptake of these trace metals and consequently the concentrations of MT in the sentinel species. The selection of these sentinel species was based on the following criteria: their relative abundance in the aquatic systems studied; their relative immobility; the ease of sampling; metal tolerance; metal bioaccumulation capacity; dynamics of metal accumulation; capacity to synthesize metallothionein; and availability of physiological and behavioural data.In 2001, physico-chemical conditions (e.g.: temperature, oxygen, transparency, phosphorus, chlorophyll a, etc.) were monitored during two sampling campaigns at 21 stations. Trace metals were measured in water and sediment in all stations, and in tissues of the sentinel species that were collected when present at the station (P. grandis - gills; H. limbata - whole body; E. lucius - kidney). Dialysis cells were used to collect water samples for the determination of concentrations of total dissolved metals, major anions and cations, humic and fulvic acids, and dissolved organic carbon. At each sampling site, divers collected three replicate sub-samples from two sediment cores for metal analyses. The cores were extruded in the boat and samples taken from the uppermost 0.5 cm, i.e. from the oxidized layer.The two rivers differ in their morphometry, their physico-chemistry, and their biology. The Allard River is larger (200 m vs. 7 m) and deeper (30 m vs. 3 m) than the Colombière River. The Colombière River is slightly more acid (pH: 6.2 - 6.7) and less productive (14 - 32 µg total P L-1) than the Allard River (pH: 6.7 - 7.4; 24 - 45 µg total P L-1). However, the rivers present some similarities such as slow current conditions and limited water transparencies. The range of Ag, Cu and Ni concentrations in water and sediment was greater in the Colombière River, whereas Pb concentrations were higher in the Allard River and Cd concentrations were similar in the two rivers. Zinc was the dominant metal in both rivers, but variations were more significant in water in the Colombière River. Although trace metal and MT concentrations were highly variable among species, stations and rivers, no consistent spatial gradients were detected in animals collected downstream from mining activities.As was the case for the ambient environment, Zn was also the dominant metal in organisms of both rivers. Zinc concentrations were similar in mayflies from the two rivers, but higher in molluscs and lower in pike collected from the Colombière River than in comparable animals from the Allard River. Following Zn, Cu was the second metal in importance in species from the two rivers, whereas the concentrations of Ag were low in all organisms and were undetectable in pike. Cadmium was more concentrated in mayflies and molluscs from the Colombière River than in those from Allard River, and also higher in the pike collected in the contaminated zone of the Colombière River. The concentrations of Ni in all species were comparable between rivers, but were more elevated in mayflies than in the other sentinel species.The range of Pb concentrations was comparable between rivers for all species, but concentrations were lower in the pike (kidney). The concentration of MT was higher in the pike than in the two other species. Mayflies had higher MT levels in the Allard River, while MT concentrations were higher in molluscs of the Colombière River. The relationships between metals and MT were very variable between rivers and species. Nevertheless, MT concentrations were most strongly related to those of accumulated Cd in the three sentinel species. The relationships between MT and Cd for all three species were stronger in the Allard River than in the Colombière River. In the case of P. grandis, because the number of stations where the bivalves could be collected from the Colombière River was low (n=4), no statistically significant relationship was observed between MT and Cd in the tissues, in spite of the high correlation coefficient (r=0.98). For the bivalves, manganese (Mn) concentrations in the sediment were negatively correlated with accumulated Cd concentrations and with MT levels. In H. limbata, in addition to Cd, other metals such as Cu (-) and Zn (+) also exhibited weak relationships with MT. Finally, in bivariate analyses, Pb concentrations in pike liver were significantly correlated with those of MT in this species, but based on multiple regression models, no variable other than Cd affected the level of MT in E. lucius

Adrenocorticotropic Hormone Suppresses Gonadotropin-Stimulated Estradiol Release from Zebrafish Ovarian Follicles

While stress is known to impact reproductive performance, the pathways involved are not entirely understood. Corticosteroid effects on the functioning of the hypothalamus-pituitary-gonadal axis are thought to be a key aspect of stress-mediated reproductive dysfunction. A vital component of the stress response is the pituitary secretion of adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. We recently reported MC2R mRNA abundance in fish gonads leading to the hypothesis that ACTH may be directly involved in gonadal steroid modulation. Using zebrafish (Danio rerio) ovarian follicles, we tested the hypothesis that acute ACTH stimulation modulates cortisol and estradiol (E2) secretion. ACTH neither affected cortisol nor unstimulated E2 release from ovarian follicles. However, ACTH suppressed human chorionic gonadotropin (hCG)-stimulated E2 secretion in a dose-related manner, with a maximum decrease of 62% observed at 1 I.U. ACTH mL−1. This effect of ACTH on E2 release was not observed in the presence of either 8-bromo-cAMP or forskolin, suggesting that the mechanism(s) involved in steroid attenuation was upstream of adenylyl cyclase activation. Overall, our results suggest that a stress-induced rise in plasma ACTH levels may initiate a rapid down-regulation of acute stimulated E2 biosynthesis in the zebrafish ovary, underscoring a novel physiological role for this pituitary peptide in modulating reproductive activity

Active behaviour during early development shapes glucocorticoid reactivity

TGlucocorticoids are the final effectors of the stress axis, with numerous targets in the central nervous system and the periphery. They are essential for adaptation, yet currently it is unclear how early life events program the glucocorticoid response to stress. Here we provide evidence that involuntary swimming at early developmental stages can reconfigure the cortisol response to homotypic and heterotypic stress in larval zebrafish (Danio rerio), also reducing startle reactivity and increasing spontaneous activity as well as energy efficiency during active behaviour. Collectively, these data identify a role of the genetically malleable zebrafish for linking early life stress with glucocorticoid function in later life

Species-specific sensitivity to selenium-induced impairment of cortisol secretion in adrenocortical cells of rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis

Species differences in physiological and biochemical attributes exist even among closely related species and may underlie species-specific sensitivity to toxicants. Rainbow trout (RT) are more sensitive than brook trout (BT) to the teratogenic effects of selenium (Se), but it is not known whether all tissues exhibit this pattern of vulnerability. In this study, primary cultures of RT and BT adrenocortical cells were exposed to selenite (Na 2 SO 3 ) and selenomethionine (Se-Met) to compare cell viability and ACTH-stimulated cortisol secretion in the two fish species. Cortisol, the primary stress hormone in fish, facilitates maintenance of homeostasis when fish are exposed to stressors, including toxicants. Cell viability was not affected by Se, but selenite impaired cortisol secretion, while Se-Met did not (RT and BT EC 50 N 2000 mg/L). RT cells were more sensitive (EC 50 = 8.7 mg/L) to selenite than BT cells (EC 50 = 90.4 mg/L). To identify the targets where Se disrupts cortisol synthesis, selenite-impaired RT and BT cells were stimulated with ACTH, dbcAMP, OH-cholesterol, and pregnenolone. Selenite acted at different steps in the cortisol biosynthesis pathway in RT and BT cells, confirming a species-specific toxicity mechanism. To test the hypothesis that oxidative stress mediates Seinduced toxicity, selenite-impaired RT cells were exposed to NAC, BSO and antioxidants (DETCA, ATA, Vit A, and Vit E). Inhibition of SOD by DETCA enhanced selenite-induced cortisol impairment, indicating that oxidative stress plays a role in Se toxicity; however, modifying GSH content of the cells did not have an effect. The results of this study, with two closely related salmonids, provided additional evidence for species-specific differences in sensitivity to Se which should be considered when setting thresholds and water quality guidelines

Behavioural and physiological responses of brook trout Salvelinus fontinalis to midwinter flow reduction in a small ice-free mountain stream

This study presents an experimental analysis of the effects of midwinter flow reduction (50-75%, reduction in discharge in 4 h daily pulses) on the physical habitat and on behaviour and physiology of overwintering brook trout Salvelinus fontinalis in a small mountain stream. Flow reduction did not result in significant lowering of temperature or formation of surface or subsurface ice. The main findings were (1) daily movement by S. fontinalis increased (c. 2·5-fold) during flow reduction, but was limited to small-scale relocations (<10 m). (2) Undercut banks were the preferred habitat and availability of these habitats was reduced during flow reduction. (3) Although both experimental and reference fish did lose mass and condition during the experiment, no effects of flow reduction on stress indicators (blood cortisol or glucose) or bioenergetics (total body fat, water content or mass loss) were detected, probably because access to the preferred type of cover remained available. Like other salmonids, S. fontinalis moves little and seeks physical cover during winter. Unlike many of the more studied salmonids, however, this species overwinters successfully in small groundwaterrich streams that often remain ice-free, and this study identifies undercut banks as the critical winter habitat rather than substratum cover