Accepted Article
Received Date : 10-Oct-2014 Revised Date : 14-Jan-2015 Accepted Date : 27-Jan-2015 Article type : Original Article
Differential proteomic responses of selectively bred and wild Sydney rock oyster populations exposed to elevated CO2
Running head: Impact of CO2 on two Sydney rock oyster populations
Thompson, E. L.1,2, O’Connor, W.3 , Parker, L.4, Ross, P.4, Raftos, D. A1,2.
1. Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia 2. Sydney Institute of Marine Science, Building 19 Chowder Bay Rd, Mosman NSW 2088, Australia 3. NSW Department of Primaries Industries, Port Stephens Fisheries Research Institute, Taylors Beach, NSW 2316, Australia 4. School of Science and Health, University of Western Sydney, Hawkesbury Bldg K12, Locked Bay 1797, Penrith South DC 1797, NSW, Australia
Corresponding author Emma Thompson. Tel: +61 (2) 9435 4620. Department of Biological Sciences Macquarie University, North Ryde, NSW 2109, Australia. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/mec.13111 This article is protected by copyright. All rights reserved.
Accepted Article
Email: [email protected]
List of symbols and abbreviations CO2, carbon dioxide; pCO2, partial pressure of carbon dioxide; OA, ocean acidification; 2-DE,
two-dimensional electrophoresis; mRNA, messenger ribonucleic acid; DNA, deoxyribonucleic acid; NH4HCO3, ammonium bicarbonate; SDS, sodium dodecyl sulphate; ACN, acetonitrile; HCl, hydrochloric acid; LC-MS/MS, liquid chromatography tandem mass spectrometry; µatm, micro atmosphere; IEF, isoelectric focussing; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis
Abstract Previous work suggests that larvae from Sydney rock oysters that have been selectively bred for fast growth and disease resistance are more resilient to the impacts of ocean acidification than non-selected, wild type oysters. In the current study, we used proteomics to investigate the molecular differences between oyster populations in adult Sydney rock oysters, and to identify if these form the basis for observations seen in larvae. Adult oysters from a selective breeding line (B2) and non-selected wild types (WT) were exposed for four weeks to elevated pCO2 (856
µatm) before their proteomes were compared to those of oysters held under ambient conditions (375 µatm pCO2). Exposure to elevated pCO2 resulted in substantial changes in the proteomes of oysters from both the selectively bred and wild type populations. When biological functions were assigned, these differential proteins fell into five broad, potentially interrelated categories of subcellular functions, in both oyster populations. These functional categories were energy production, cellular stress responses, the cytoskeleton, protein synthesis and cell signaling. In the wild type population, proteins were predominantly upregulated. However, unexpectedly, these cellular systems were downregulated in the selectively bred oyster population, indicating
This article is protected by copyright. All rights reserved.
Accepted Article
cellular dysfunction. We argue that this reflects a tradeoff, whereby an adaptive capacity for enhanced mitochondrial energy production in the selectively bred population may help to protect larvae from the effects of elevated CO2, whilst being deleterious to adult oysters.
Keywords Environmental proteomics, Sydney rock oyster, Saccostrea glomerata, CO2, carbon dioxide, selective breeding
Introduction The capacity of marine species for rapid, heritable adaptation to increasing ocean acidification and temperature remains an outstanding question in climate change research. Some evidence suggests that such evolution may be possible (Franks and Hoffmann, 2012). However, we still understand very little about the genes and subcellular processes that might be involved in the necessary adaptations. The current study addresses this question by analysing two distinct populations of Sydney rock oysters (Saccostrea glomerata) from the east coast of Australia.
The coastal estuaries of eastern Australia are highly productive and biodiverse ecosystems. However, they are under threat from a range of natural and anthropogenic stressors including extremes of temperature, salinity, and chemical contamination. These impacts are likely to be exacerbated by ocean acidification (OA), which is a global environmental threat resulting from increasing atmospheric CO2 (Hoegh-Guldberg and Smith, 1989, Kroeker et al., 2010, Hendriks et al., 2010). Much of the anthropogenic CO2 released into the atmosphere from the burning of
fossil fuels is being absorbed into the oceans, causing acidification. It is predicted that the
This article is protected by copyright. All rights reserved.
Accepted Article
Materials and Methods Oysters and exposure to elevated pCO2
Two populations of S. glomerata, (aged 1.5-2 years) were collected at the beginning of their reproductive conditioning. One population (B2 line), were 5th generation oysters selectively bred
for fast growth and disease resistance by the NSW Department of Primary Industry (NSW DPI).
They were collected from a NSW DPI lease on the Clyde River, NSW (34°78" S, 150°69" E). The second population comprised wild type (WT) oysters that had never been subjected to selective breeding. They were collected from leases in Port Stephens, NSW (32°45" S, 152°10" E). Oysters were transferred to the NSW DPI’s Port Stephens Fisheries Centre, cleaned and left to acclimate for 2 weeks (Thompson et al., 2012a) in 40L trays supplied with re-circulating seawater from 750 L tanks containing water collected from Little Beach, Port Stephens (salinity 34.6, temperature ~24ºC). The oysters were fed daily with an algal diet of 50% Chaetoceros muelleri, 25% Pavlova lutheri and 25% Tahitian Isochrysis aff. galbana (2 x 109 cells oyster-1).
After acclimation, oysters in three trays (10 oysters population-1 tray-1; each tray supplied by a
separate 750 L header tank) were held under ambient conditions (375 µatm pCO2; 8.20 ± 0.01 pH) whilst oysters in the other three trays were exposed to elevated conditions (856 µatm pCO2;
7.84 ± 0.01 pH) for four weeks. A pH negative-feedback system (Aqua Medic; accuracy ±0.001 pH units) was used to maintain 856 µatm pCO2, CO2 was bubbled into tanks via a reactor and
regulated with a solenoid valve. pCO2 levels were determined using data for pH, total alkalinity, temperature and salinity of seawater in a CO2 calculation system (CO2 sys) (Lewis and Wallace,
1998). Complete water changes were performed every two days throughout the duration of the experiment and oysters were fed daily as per the acclimation the period.
This article is protected by copyright. All rights reserved.
Accepted Article
Protein quantification Protein concentrations of each sample were determined using Amersham 2DE Quant Kits with a modification of the manufacturer’s instructions (GE Healthcare, Buckinghamshire, UK). Briefly, 2 µl of each sample were added in triplicate to a 96-well microtitre plate followed by 10 µl of Cu solution, 40 µl of Milli Q water and 100 µl of colour reagent. Plates were incubated at room temperature for 20 minutes, after which absorbance was measured at 490nm on an M550 spectrophotometer (Bio-Rad). Protein concentrations were interpolated from a standard curve generated with bovine serum albumin. Five randomly selected oysters were pooled per tray to run each individual gel. This gave three biological replicates per treatment (with a total of 15 oysters used per treatment), and 12 gels in total across the four treatments. Each pooled gill sample contained 150 µg of protein based on relative protein concentrations of the individual oysters.
Two-dimensional electrophoresis Two-dimensional electrophoresis (2-DE) was performed on each pooled sample. A total of three gels were run per treatment (three gels each for B2 and wild type oysters held at ambient CO2
and three each for B2 and wild type oysters exposed to elevated CO2, 12 gels in total). In the first dimension, immobilized pH linear gradient gel strips (7cm, pH 4-7; GE Healthcare) were passively re-hydrated overnight with 150 µg of extracted proteins in 125 µL rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 0.002% bromophenol blue and 0.5% carrier ampholytes; GE Healthcare). Isoelectrofocusing (IEF) was undertaken using an IPGphor IEF system (GE Healthcare) at 100V for 2 h, 500V for 20 min, a gradient up to 5000V for 2 hours and then 5000V for 2 h. Gel strips were then reduced for 20 min (1% DTT in equilibration
This article is protected by copyright. All rights reserved.
Accepted Article
solution: 6M urea, 30% glycerol, 2% SDS, 0.002% bromophenol) and alkylated for 20 min (2.5% iodoacetamide in equilibration solution). Second dimension separation was undertaken using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) with TGX precast (Bio-Rad) 12% Tris-HCl polyacrylamide gels (1.5 M Tris-HCl, 10% SDS, 12% acrylamide) in a Mini PROTEAN system (Bio-Rad). Gels were stained with Lava Purple (The Gel Company, San Francisco, CA, USA) and visualised using a Pharos UV scanner (Bio-Rad). PDQuest proteomic analysis software (Bio-Rad) was used to determine the relative intensities of protein spots in each of the 2-DE gels.
Statistical analysis We used one-way non-parametric multivariate analyses of variance (PERMANOVA; Anderson, 2001); n = 3 replicates per treatment) with matrices of Bray-Curtis dissimilarity on untransformed, unstandardised normalised intensities of all matched protein spots to test for multivariate differences in the proteomes of WT compared to B2 oysters and differences resulting from pCO2 exposure (Primer v.6, Plymouth, UK). The PERMANOVA partitioned sources of variation in a similar way to ANOVA and used unrestricted permutation of raw data to assess statistical significance. Proteins spots were considered as being differentially expressed between treatments when P-values were < 0.05.
Similarity of percentage analyses (SIMPER) identified protein spots with the greatest contribution to multivariate dissimilarity between pCO2 treatments or populations (Clarke,
1993). Protein spots were considered to be good discriminators when they showed dissimilarity to the standard deviation ratio > 1.3 (Clarke and Warwick, 1994). These proteins were then
This article is protected by copyright. All rights reserved.
Accepted Article
further analysed using separate analysis of variance (ANOVA) and considered to be significantly different in relative intensity if p
Received Date : 10-Oct-2014 Revised Date : 14-Jan-2015 Accepted Date : 27-Jan-2015 Article type : Original Article
Differential proteomic responses of selectively bred and wild Sydney rock oyster populations exposed to elevated CO2
Running head: Impact of CO2 on two Sydney rock oyster populations
Thompson, E. L.1,2, O’Connor, W.3 , Parker, L.4, Ross, P.4, Raftos, D. A1,2.
1. Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia 2. Sydney Institute of Marine Science, Building 19 Chowder Bay Rd, Mosman NSW 2088, Australia 3. NSW Department of Primaries Industries, Port Stephens Fisheries Research Institute, Taylors Beach, NSW 2316, Australia 4. School of Science and Health, University of Western Sydney, Hawkesbury Bldg K12, Locked Bay 1797, Penrith South DC 1797, NSW, Australia
Corresponding author Emma Thompson. Tel: +61 (2) 9435 4620. Department of Biological Sciences Macquarie University, North Ryde, NSW 2109, Australia. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/mec.13111 This article is protected by copyright. All rights reserved.
Accepted Article
Email: [email protected]
List of symbols and abbreviations CO2, carbon dioxide; pCO2, partial pressure of carbon dioxide; OA, ocean acidification; 2-DE,
two-dimensional electrophoresis; mRNA, messenger ribonucleic acid; DNA, deoxyribonucleic acid; NH4HCO3, ammonium bicarbonate; SDS, sodium dodecyl sulphate; ACN, acetonitrile; HCl, hydrochloric acid; LC-MS/MS, liquid chromatography tandem mass spectrometry; µatm, micro atmosphere; IEF, isoelectric focussing; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis
Abstract Previous work suggests that larvae from Sydney rock oysters that have been selectively bred for fast growth and disease resistance are more resilient to the impacts of ocean acidification than non-selected, wild type oysters. In the current study, we used proteomics to investigate the molecular differences between oyster populations in adult Sydney rock oysters, and to identify if these form the basis for observations seen in larvae. Adult oysters from a selective breeding line (B2) and non-selected wild types (WT) were exposed for four weeks to elevated pCO2 (856
µatm) before their proteomes were compared to those of oysters held under ambient conditions (375 µatm pCO2). Exposure to elevated pCO2 resulted in substantial changes in the proteomes of oysters from both the selectively bred and wild type populations. When biological functions were assigned, these differential proteins fell into five broad, potentially interrelated categories of subcellular functions, in both oyster populations. These functional categories were energy production, cellular stress responses, the cytoskeleton, protein synthesis and cell signaling. In the wild type population, proteins were predominantly upregulated. However, unexpectedly, these cellular systems were downregulated in the selectively bred oyster population, indicating
This article is protected by copyright. All rights reserved.
Accepted Article
cellular dysfunction. We argue that this reflects a tradeoff, whereby an adaptive capacity for enhanced mitochondrial energy production in the selectively bred population may help to protect larvae from the effects of elevated CO2, whilst being deleterious to adult oysters.
Keywords Environmental proteomics, Sydney rock oyster, Saccostrea glomerata, CO2, carbon dioxide, selective breeding
Introduction The capacity of marine species for rapid, heritable adaptation to increasing ocean acidification and temperature remains an outstanding question in climate change research. Some evidence suggests that such evolution may be possible (Franks and Hoffmann, 2012). However, we still understand very little about the genes and subcellular processes that might be involved in the necessary adaptations. The current study addresses this question by analysing two distinct populations of Sydney rock oysters (Saccostrea glomerata) from the east coast of Australia.
The coastal estuaries of eastern Australia are highly productive and biodiverse ecosystems. However, they are under threat from a range of natural and anthropogenic stressors including extremes of temperature, salinity, and chemical contamination. These impacts are likely to be exacerbated by ocean acidification (OA), which is a global environmental threat resulting from increasing atmospheric CO2 (Hoegh-Guldberg and Smith, 1989, Kroeker et al., 2010, Hendriks et al., 2010). Much of the anthropogenic CO2 released into the atmosphere from the burning of
fossil fuels is being absorbed into the oceans, causing acidification. It is predicted that the
This article is protected by copyright. All rights reserved.
Accepted Article
Materials and Methods Oysters and exposure to elevated pCO2
Two populations of S. glomerata, (aged 1.5-2 years) were collected at the beginning of their reproductive conditioning. One population (B2 line), were 5th generation oysters selectively bred
for fast growth and disease resistance by the NSW Department of Primary Industry (NSW DPI).
They were collected from a NSW DPI lease on the Clyde River, NSW (34°78" S, 150°69" E). The second population comprised wild type (WT) oysters that had never been subjected to selective breeding. They were collected from leases in Port Stephens, NSW (32°45" S, 152°10" E). Oysters were transferred to the NSW DPI’s Port Stephens Fisheries Centre, cleaned and left to acclimate for 2 weeks (Thompson et al., 2012a) in 40L trays supplied with re-circulating seawater from 750 L tanks containing water collected from Little Beach, Port Stephens (salinity 34.6, temperature ~24ºC). The oysters were fed daily with an algal diet of 50% Chaetoceros muelleri, 25% Pavlova lutheri and 25% Tahitian Isochrysis aff. galbana (2 x 109 cells oyster-1).
After acclimation, oysters in three trays (10 oysters population-1 tray-1; each tray supplied by a
separate 750 L header tank) were held under ambient conditions (375 µatm pCO2; 8.20 ± 0.01 pH) whilst oysters in the other three trays were exposed to elevated conditions (856 µatm pCO2;
7.84 ± 0.01 pH) for four weeks. A pH negative-feedback system (Aqua Medic; accuracy ±0.001 pH units) was used to maintain 856 µatm pCO2, CO2 was bubbled into tanks via a reactor and
regulated with a solenoid valve. pCO2 levels were determined using data for pH, total alkalinity, temperature and salinity of seawater in a CO2 calculation system (CO2 sys) (Lewis and Wallace,
1998). Complete water changes were performed every two days throughout the duration of the experiment and oysters were fed daily as per the acclimation the period.
This article is protected by copyright. All rights reserved.
Accepted Article
Protein quantification Protein concentrations of each sample were determined using Amersham 2DE Quant Kits with a modification of the manufacturer’s instructions (GE Healthcare, Buckinghamshire, UK). Briefly, 2 µl of each sample were added in triplicate to a 96-well microtitre plate followed by 10 µl of Cu solution, 40 µl of Milli Q water and 100 µl of colour reagent. Plates were incubated at room temperature for 20 minutes, after which absorbance was measured at 490nm on an M550 spectrophotometer (Bio-Rad). Protein concentrations were interpolated from a standard curve generated with bovine serum albumin. Five randomly selected oysters were pooled per tray to run each individual gel. This gave three biological replicates per treatment (with a total of 15 oysters used per treatment), and 12 gels in total across the four treatments. Each pooled gill sample contained 150 µg of protein based on relative protein concentrations of the individual oysters.
Two-dimensional electrophoresis Two-dimensional electrophoresis (2-DE) was performed on each pooled sample. A total of three gels were run per treatment (three gels each for B2 and wild type oysters held at ambient CO2
and three each for B2 and wild type oysters exposed to elevated CO2, 12 gels in total). In the first dimension, immobilized pH linear gradient gel strips (7cm, pH 4-7; GE Healthcare) were passively re-hydrated overnight with 150 µg of extracted proteins in 125 µL rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 0.002% bromophenol blue and 0.5% carrier ampholytes; GE Healthcare). Isoelectrofocusing (IEF) was undertaken using an IPGphor IEF system (GE Healthcare) at 100V for 2 h, 500V for 20 min, a gradient up to 5000V for 2 hours and then 5000V for 2 h. Gel strips were then reduced for 20 min (1% DTT in equilibration
This article is protected by copyright. All rights reserved.
Accepted Article
solution: 6M urea, 30% glycerol, 2% SDS, 0.002% bromophenol) and alkylated for 20 min (2.5% iodoacetamide in equilibration solution). Second dimension separation was undertaken using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) with TGX precast (Bio-Rad) 12% Tris-HCl polyacrylamide gels (1.5 M Tris-HCl, 10% SDS, 12% acrylamide) in a Mini PROTEAN system (Bio-Rad). Gels were stained with Lava Purple (The Gel Company, San Francisco, CA, USA) and visualised using a Pharos UV scanner (Bio-Rad). PDQuest proteomic analysis software (Bio-Rad) was used to determine the relative intensities of protein spots in each of the 2-DE gels.
Statistical analysis We used one-way non-parametric multivariate analyses of variance (PERMANOVA; Anderson, 2001); n = 3 replicates per treatment) with matrices of Bray-Curtis dissimilarity on untransformed, unstandardised normalised intensities of all matched protein spots to test for multivariate differences in the proteomes of WT compared to B2 oysters and differences resulting from pCO2 exposure (Primer v.6, Plymouth, UK). The PERMANOVA partitioned sources of variation in a similar way to ANOVA and used unrestricted permutation of raw data to assess statistical significance. Proteins spots were considered as being differentially expressed between treatments when P-values were < 0.05.
Similarity of percentage analyses (SIMPER) identified protein spots with the greatest contribution to multivariate dissimilarity between pCO2 treatments or populations (Clarke,
1993). Protein spots were considered to be good discriminators when they showed dissimilarity to the standard deviation ratio > 1.3 (Clarke and Warwick, 1994). These proteins were then
This article is protected by copyright. All rights reserved.
Accepted Article
further analysed using separate analysis of variance (ANOVA) and considered to be significantly different in relative intensity if p
