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Transcriptomic and physiological responses of the green microalga Chlamydomonas reinhardtii during short-term exposure to sub-nanomolar methylmercury concentrations Rebecca Beauvais-Flück, Vera I Slaveykova, and Claudia Cosio Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00403 • Publication Date (Web): 02 Jun 2016 Downloaded from http://pubs.acs.org on June 4, 2016
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Environmental Science & Technology
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Transcriptomic and physiological responses of the green microalga
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Chlamydomonas reinhardtii during short-term exposure to sub-nanomolar
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methylmercury concentrations
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Rebecca Beauvais-Flück, Vera I. Slaveykova, Claudia Cosio*
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Institute F.-A. Forel, Earth and Environmental Sciences, Faculty of Sciences, University of
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Geneva, 66, boulevard Carl-Vogt, 1211 Genève 4, Switzerland.
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*corresponding author:
[email protected], Phone: +41 22 379 03 10, Fax: +41 22 379
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03 29.
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ABSTRACT:
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The effects of short-term exposure to sub-nanomolar methyl-mercury (MeHg) concentrations,
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representative of contaminated environments, on the microalga Chlamydomonas reinhardtii
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were assessed using both physiological endpoints and gene expression analysis. MeHg
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bioaccumulated and induced significant increase of the photosynthesis efficiency, while the
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algal growth, oxidative stress and chlorophyll fluorescence were unaffected. At the molecular
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level, MeHg significantly dysregulated the expression of genes involved in motility, energy
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metabolism, lipid metabolism, metal transport and antioxidant enzymes. Data suggest that the
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cells were able to cope with sub-nanomolar MeHg exposure, but this tolerance resulted in a
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significant cost to the cell energy and reserve metabolism as well as ample changes in the
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nutrition and motility of C. reinhardtii. The present results allowed gaining new insights on
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the effects and uptake mechanisms of MeHg at sub-nanomolar concentrations in aquatic
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primary producers.
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INTRODUCTION
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Mercury (Hg) is a naturally occurring toxic metal which environmental cycling is strongly
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affected by anthropogenic activities 1,2. It is particularly problematic in aquatic systems where
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inorganic Hg (IHg) is transformed to methyl-Hg (MeHg), which strongly biomagnifies in
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food webs 3. Aquatic primary producers, like phytoplankton, are central for Hg fate in the
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aquatic environment, as they are the entry pathway of MeHg into food webs 4. Indeed, Hg
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bioconcentration step from water to primary producers represents the largest increase of Hg in
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the food chain reaching 104-fold and greater
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understand the effects, uptake and fate of Hg in primary producers.
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Nevertheless, the effects of Hg on aquatic primary producers have been less studied than on
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other organisms, e.g. fish
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concentrations affected photosynthesis and induced the production of antioxidant enzymes
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(e.g. GSH), whereas MeHg influenced the algal growth but not photosynthesis 7-10. However,
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most studies were performed using Hg concentrations 103-106 fold higher (µM) than those
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commonly found in the environment 2, which limits their relevance. Moreover, a precise
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mechanistic understanding of the intracellular targets and effects of MeHg on aquatic primary
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producers, such as microalgae is not yet available. For this aim, new sequencing technology,
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such as RNA-Seq offers a sensitive, specific and promising approach
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years, transcriptomic helped assess the toxicity of Hg in animals, invertebrates or terrestrial
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plant models (e.g.
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nanoparticles
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microalgae to MeHg has been yet published.
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The present study explores the effects of MeHg at sub-nanomolar concentrations in the green
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microalga Chlamydomonas reinhardtii
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short-term exposure to 3.6×10-11 and 3.7×10-10 M MeHg - concentrations below the European
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. It is therefore of the upmost importance to
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. Studies on phytoplankton revealed that nanomolar IHg
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. In the last ten
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). Transcriptomic on algae focused on metals like Cu, Cd, Ag or
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. To our knowledge, no study exploring the transcriptomic responses of
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. More specifically, we investigated the effects of
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Environmental Quality Standard for Hg (2.5×10-9 M)
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growth rate, oxidative stress and photosynthesis efficiency of the microalga.
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- on the transcriptome response,
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EXPERIMENTAL
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Lab-ware and reagents
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Material was soaked in 10% v/v HNO3 followed by 10% v/v HCl baths for 1 week, rinsed
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with MilliQ·water (Millipore, Darmstadt, Germany) and dried under a laminar flow hood.
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Reagents were of analytical grade.
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Exposure of C. reinhardtii to MeHg
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Exposures and analysis were performed on three biological replicates. Wild-type C.
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reinhardtii (CPCC11, Canadian Phycological Culture Centre, Waterloo, Canada) was grown
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axenically at 20.2±0.5°C, 115 rpm and 110 µmol·phot·m-2·s-1. Cells were pre-cultured in 4×
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diluted Tris-Acetate-Phosphate medium
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inoculation), centrifuged (10min, 1300g), rinsed and re-suspended (~106 cells·mL-1) in the
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exposure medium (8.2×10-4 M CaCl2·2H2O, 3.6×10-4 M MgSO4·7H2O, 2.8×10-4 M NaHCO3,
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1.0×10-4 M KH2PO4 and 5.0×10-6 M NH4NO3, pH 6.9±0.1) and exposed 2h to 3.6×10-11 or
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3.7×10-10 M MeHg using MeHgCl standard solution (Alfa Aesar, Ward Hill, MA, USA).
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These concentrations are a compromise between unambiguous MeHg bioaccumulation in the
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microalga in our experimental conditions and concentrations reached in contaminated sites
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2,29
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(Figure S1). Besides, this short-term exposure allows targeting specific stress response at the
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transcriptomic level prior general stress response and acclimation occur. Measured MeHg and
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total Hg (THg=IHg+MeHg) concentrations in control medium (
