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Title:
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Characterizing the effects of heavy metal and Vibrio exposure on hsp70 in Crassostrea gigas.
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Short running title: (must be no more than 48 characters)
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Hsp70 expression in Crassostrea gigas
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Authors:
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David C. Metzger, Paul Pratt, and Steven B. Roberts*
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*Corresponding author: e-mail: sr320@uw.edu; phone: 206-685-3742
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Affiliations:
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School of Aquatic and Fishery Sciences, University of Washington, 1122 NE Boat Street,
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Seattle, Washington, USA
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Key Words:
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Crassostrea gigas, Pacific oyster, hsp70, gene expression, protein expression, copper, Vibrio
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Abstract:
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The Pacific oyster, Crassostrea gigas, is an intertidal bivalve mollusc inhabiting several
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continents. Similar to other shellfish, the Pacific oyster is considered an important bioindicator
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species. One way to assess environmental perturbations is to examine an organism’s stress
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response. Molecular chaperones, including heat shock proteins, are common targets when
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evaluating an organism’s response to environmental stress. In this study, oysters were exposed
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to copper and the bacteria Vibrio tubiashii to examine how these environmental stressors
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influence Hsp70 gene and protein expression. Bacterial exposure did not effect Hsp70
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expression, while copper exposure significantly changed both transcript and protein level.
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Interestingly, copper exposure increased gene expression and decreased protein levels when
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compared to controls. The dynamics of Hsp70 regulation observed here provide important
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insight into heavy metal exposure, heat shock protein activity, and highlight considerations that
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should be made when using Hsp70 as an indicator of an organism’s general stress response.
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Introduction
Perturbations in environmental conditions can alter physiological processes and
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measuring these changes can provide information with respect to an organism’s response to
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stress. A common means to characterize a physiological response to environmental change is
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by measuring changes in gene expression levels, since mRNA expression is considered directly
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related to corresponding protein levels (Bierkens, 2000). However, protein expression does not
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always correlate with messenger RNA (mRNA) levels (Gygi et al., 1999; Piña et al., 2007).
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Consequently, analysis of stress response pathways that do not account for both transcriptional
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and translational mechanisms may not accurately represent the organism’s response
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(Greenbaum et al., 2003).
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Heat shock proteins (Hsp) are a common focus in assays used to establish
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measurements of physiological and environmental stress (Franzellitti et al., 2010; Roberts et al.,
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2010; Piña et al., 2007; Downs, 2001). Heat shock proteins are involved in a variety of stress-
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related processes including thermal stress, immune response, and apoptosis (Feder &
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Hofmann, 1999; Roberts et al., 2010). The most highly conserved family of heat shock proteins
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is the Hsp70 family(Sanders 1994). Hsp70s are molecular chaperones involved in the folding
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and unfolding of newly translated proteins, and proteins that have been damaged from various
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forms of cellular stress (Feder & Hofmann, 1999). Hsp70 was originally characterized in C.
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gigas by Gourdon et al. (2000), followed by further sequence identification and characterization
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by Boutet et al. (2003). Hsp70 is one of the most targeted proteins for studying the stress
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response in shellfish (Fabbri et al., 2008).
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Oysters, like other bivalves, are sessile organisms that are continually filtering water and
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often bioaccumulate toxins, making them ideal bioindicator species (Rittschof et al., 2005;
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Fabbri et al., 2008; Morley, 2010). While studies have documented Hsp70 accumulation in
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response to individual stressors such as temperature and heavy metals (Clegg et al.,1998:
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Boutet et al., 2003), fewer studies have characterized Hsp70 in organisms that are subjected to
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multiple stressors. Combinations of environmental contaminants can act synergistically, the
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effects of which can be greater than the sum of the individual stressors combined (Anderson et
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al., 1998). As combinations of stressors are more representative of natural ecosystems,
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characterizing physiological stress response in the presence of multiple stressors is relevant to
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understanding stress response physiology (Anderson et al., 1998; Gupta et al., 2010).
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The primary objective of the current study was to characterize Hsp70 activity at the
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transcript and protein levels in C. gigas when exposed to copper and bacteria. Copper is one of
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several heavy metal contaminants found in aquatic environments (O’Connor & Lauenstein,
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2005). The bacterial pathogen Vibrio tubiashii (Vt), is a re-emergent bacterial pathogen of
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shellfish on the west coast of North America (Elston et al., 2008). This study provides insight
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into heavy metal exposure in shellfish, heat shock protein activity, and highlights considerations
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that should be made when using Hsp70 as an indicator for an organism’s general stress
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response.
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Materials and Methods
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Experimental design
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Pacific oysters were collected from the University of Washington’s field station at Big
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Beef Creek, WA in November 2010. A total of 32 oysters were collected with a mean length of
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112.3mm. Following four days of acclimation in 12oC seawater, oysters were randomly assigned
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to four treatment groups (n=8). One group received a dose of copper(II) sulfate (33mg/L copper
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ion concentration) for 72 hours. A second group of oysters were subjected to a Vibrio tubiashii
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(Strain ATTC 19106) bath exposure at 7.5x105 CFU/ml for 24 hours. A third group of oysters
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was subjected to both the copper and Vt exposure. The fourth group of oysters served as a
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control and was held in seawater for the duration of the experiment. Vt was cultured overnight at
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37°C in 1 liter LB broth, plus an additional 1% NaCl. Cells were harvested by centrifugation at
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4,200 rpm for 20min. Pelleted bacteria were then re-suspended in 0.22um filter sterilized
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seawater. Immediately following treatments, gill tissue was removed and stored at -80°C for
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subsequent RNA and protein extraction.
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cDNA synthesis and quantitative PCR analysis
RNA from all gill tissue samples (~25mg) was extracted using TRI Reagent (Molecular
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Research Center, Inc., Cincinnati, OH) following the manufacturer’s protocol. RNA samples
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were treated with the Turbo DNA-free Kit (Ambion Inc., Foster City, CA) per manufacturer’s
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recommended standard protocol to remove potential genomic DNA carryover. RNA (1ug) was
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reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, Wisconsin)
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according to the manufacturer’s protocol. For gene expression analysis, primers were designed
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for heat shock protein 70 (GenBank Accession # AJ318882)
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(hsp70fw:TGGCAACCAATCGCAAGGTGAG; hsp70rv: CCTGAGAGCTTGAGGACAAGGT)
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using Primer3 software (Rozen & Skaletsky, 2000). Quantitative PCR (qPCR) reactions were
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carried out in a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Each 25-µl
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reaction contained 1X Immomix Master Mix (Bioline USA Inc., Boston, MA), 0.2 µM of each
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primer, 0.2 µM Syto-13 (Invitrogen, Carlsbad, CA), 2 µl of cDNA, and sterile water.
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Thermocycling conditions included an initial denaturation (10 min at 95ºC), followed by 40
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cycles of 15 sec at 95ºC, 15 sec at 55ºC, and 30 sec at 72ºC, with fluorescence measured at
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the end of annealing and extension steps. Following qPCR, melting curve analysis was
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performed by increasing the temperature from 65ºC to 95ºC at a rate of 0.2ºC per second,
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measuring fluorescence every 0.5ºC. All samples were run in duplicate. Analysis of qPCR data
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was carried out based upon the kinetics of qPCR reactions (Zhao & Fernald, 2005)) and
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normalized to elongation factor 1 alpha expression (ef1fw: AAGGAAGCTGCTGAGATGGG;
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ef1rv: CAGCACAGTCAGCCTGTGAAGT (GenBank Accession # AB122066). DNased RNA
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was used as template to insure the no genomic DNA carryover was present. Data are
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expressed as fold increases over the minimum.
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Protein isolation and Western Blot Analysis
Protein was extracted from gill tissue using CellLytic Cell Lysis Reagent tissue (Sigma,
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St Louis, MO) containing protease inhibitor cocktail (Sigma) at a ratio of 1:20 (1g tissue/20ml
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reagent). The levels of Hsp70 protein were assessed by Western blot analysis using anti-heat
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shock protein 70 monoclonal antibody (3A3) (Product # MA3-006) (Pierce, Thermo Fisher
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Scientific, Rockford, IL). Total protein was subjected to gel electrophoresis using 4-20% Precise
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Protein Gels (Precise, Thermo Fisher Scientific, Rockford, IL). Gels were transferred to
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nitrocellulose membranes, blocked and incubated with diluted (1:3000) primary hsp70 antibody.
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Membrane incubations and visualization were carried using the WesternBreeze Chromogenic
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Kit - Anti-Mouse (Invitrogen, Carlsbad, CA). Integrated density values were calculated using
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Image J (Rasband, W.S.). Values of background densities on SDS-PAGE gels were calculated
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and used to normalize values based on protein band density.
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Statistical analysis
Two-way ANOVA analyses were carried out to determine significant differences in
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Hsp70 mRNA expression and protein levels (p<0.05) (SPSS v18). As no effect of Vt was
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detected for either hsp70 mRNA expression or protein levels, data from Vt exposed oysters was
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combined with control oysters while data from Vt and copper exposed oysters was combined
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with the copper exposed oysters and a t-test was conducted to determine the effect of copper
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exposure on Hsp70 expression (p<0.05).
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Results
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Vibrio Exposure
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Heat shock protein 70 gene and protein levels were not significantly different in gill tissue
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from oysters exposed to only Vt when compared to controls (data not shown). Furthermore,
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mRNA expression and protein levels in gill tissue from oysters exposed to Vt in combination
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with copper were not significantly different from oysters exposed to copper alone. As there was
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no influence of the Vt exposure on Hsp70 mRNA expression or protein levels, subsequent
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analyses and data presented from copper exposed oysters will refer to those oysters exposed to
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copper only (n=8), and the group of oysters exposed to copper and Vt (n=8).
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Copper Exposure
Copper exposure significantly altered both gene and protein expression. Specifically,
expression of hsp70 mRNA was significantly higher in copper treated oysters compared to
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control individuals (Figure 1a). Protein expression was significantly decreased in gill tissue from
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oysters exposed to copper compared to controls (Figure 1b).
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Discussion
While Hsp70 expression has received considerable attention as an indicator of stress in
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shellfish, there are limited studies that characterize both gene and protein Hsp70 expression.
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This study demonstrates that exposure to copper influences Hsp70 gene and protein expression
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in oysters. Furthermore, exposure to copper resulted in discordant expression of Hsp70 mRNA
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and protein.
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Oysters used in this study were exposed to 33mg/L of copper and this resulted in a
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significant increase in hsp70 mRNA expression. This result is similar to what has been observed
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in other shellfish. In Argopecten purpuratus, Zapata et al. (2009) observed that exposure of
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copper as low as 2.5 μg/L for eight days showed significant increases in hsp70 mRNA.
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Driessena polymorpha exposed to copper had significantly increased expression of hsp70
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mRNA in gill tissue after 24 hours of exposure that returned to control levels after 7 days
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(Navarro et al., 2011). A study on Fenneropenaeus chinensis showed a significant increase in
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hsp70 mRNA in shrimp exposed to 20μg/L copper after 24 hours of exposure, however 72
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hours of exposure actually decreased expression levels (Luan et al., 2010). The decrease in
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gene expression level in Fenneropenaeus chinensis could be attributed the toxicity threshold
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being exceeded, impairing general biological processes including transcription. In the current
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study, the increased expression of hsp70 mRNA following 72 hours of a relatively high dose of
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copper could indicate resilience in the ability to respond high levels of copper exposure.
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Assuming that hsp70 gene expression would no longer be affected by a specific environmental
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stressor, an interesting future research direction would be to examine this hypothesis across
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numerous stressors.
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Changes in Hsp70 protein levels in shellfish exposed to copper appear to be taxa and
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dose dependent. The concentration of Hsp70 protein in this study was significantly lower in the
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gill tissue of copper exposed oysters compared to controls. These results are consistent with
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other studies of C. gigas in which decreased levels of Hsp70 protein were observer in oysters
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exposed to lower levels of copper (Boutet et al., 2003). However, in in other bivalves, copper
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exposure has resulted in an increase in Hsp70 protein concentration. Zebra mussels exposed to
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100-500ug/L copper showed increased Hsp70 protein levels while no change was detected in
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lower doses (Clayton et al., 2000). Protein levels for Hsp70 were also elevated in Mytilus edulis
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after 7 days exposure to copper (Sanders, 1994). In Chamelea gallina, a concentration
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dependent regulation of protein expression was observed in which protein concentrations
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increased in the presence of low copper concentrations (<1mg/ml) and decreased in high
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copper treatments (>5mg/ml) (Rodriguez-Ortega et al., 2003). This could indicate that,
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independent of species, Hsp70 protein can perform appropriate biological functions related to
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stress response and protein damage. However, in the oyster, Hsp70 protein integrity appears to
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be more sensitive to a range of copper concentrations compared to other shellfish species
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despite the expression of hsp70 mRNA.
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Copper is known to cause cytopathological damage to gill tissue (Spicer & Weber, 1991;
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Sanders, 1994; Pawert et al., 1996; Triebskorn and Köhler, 1996; Quig, 1998) as the result of
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peroxidation reactions that produce free radicals that damage lipids and proteins (Donato,1981).
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The observed discordant expression of hsp70 mRNA with Hsp70 protein levels could therefore
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be due to the rate of Hsp70 protein degradation in the cytoplasm exceeding the rate of hsp70
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mRNA transcription. Copper exposure could be damaging Hsp70 proteins themselves or the
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overall translational machinery (Lewis et al., 2001), which would result in lower protein levels.
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Copper has also been shown to interfere with RNA transcriptional machinery and RNA integrity
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(Viarengo, 1985). Results from this study suggest that the transcriptional machinery in oysters is
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quite robust as expression of hsp70 mRNA was observed at levels above that of control
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animals.
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This study illustrates that environmental stressors can influence Hsp70 gene and protein
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expression in a discordant fashion. We did not observe an effect of Vt exposure on hsp70
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expression, and presumably the bacterial exposure did not significantly impact the physiological
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response to copper. The influence of copper on Hsp70 regulation in oysters appears to be
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different compared to closely related species, which could offer an interesting system to study
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Hsp70 dynamics in greater detail. Understanding the mechanisms responsible for this apparent
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difference in the sensitivity of Hsp70 regulation between species could help us better
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understand mechanisms underlying species resilience. These findings highlight important
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considerations that should be taken when using Hsp70 as an indicator of environmental stress
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the associated physiological response.
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Figure 1.
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Figure 1:
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Quantification of hsp70 mRNA (A) and protein (B) in oysters held in sea water or in 33mg/l
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copper. Expression values are presented as fold change over the minimum expression value +/-
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standard error. “No copper” expression values include both control and Vt exposed animals
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while “copper exposed” include both copper only and coppter and Vt treatesd animals as Vt
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exposure was shown to have no significant effect on hsp70 expression by ANOVA analysis.
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Asterisks denote significant difference in hsp70 expression value compared to “no copper”
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treatment (p<0.05).
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