Shotgun proteomics reveals physiological response to ocean acidification in Crassostrea gigas
- Subject Areas
- Aquaculture, Fisheries and Fish Science, Marine Biology, Molecular Biology
- Pacific oyster, proteomics, ocean acidification
- © 2014 Timmins-Schiffman et al.
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ PrePrints) and either DOI or URL of the article must be cited.
- Cite this article
- 2014. Shotgun proteomics reveals physiological response to ocean acidification in Crassostrea gigas. PeerJ PrePrints 2:e388v1 https://doi.org/10.7287/peerj.preprints.388v1
Background. Ocean acidification as a result of increased anthropogenic CO2 emissions is occurring in marine and estuarine environments worldwide. The coastal ocean experiences additional daily and seasonal fluctuations in pH that can be lower than projected end of century open ocean pH reductions. Projected and current ocean acidification have wide-ranging effects on many aquatic organisms, however the exact mechanisms of the impacts of ocean acidification on many of these animals remains to be characterized.
Methods. In order to assess the impact of ocean acidification on marine invertebrates, Pacific oysters (Crassostrea gigas) were exposed to one of four different pCO2 levels for four weeks: 400 µatm (pH 8.0), 800 µatm (pH 7.7), 1000 µatm (pH 7.6), or 2800 µatm (pH 7.3). At the end of 4 weeks a variety of physiological parameters were measured to assess the impacts of ocean acidification: tissue glycogen content and fatty acid profile, shell micromechanical properties, and response to acute heat shock. To determine the effects of ocean acidification on the underlying molecular physiology of oysters and their stress response, some of the oysters from 400 µatm and 2800 µatm were exposed to an additional mechanical stress and shotgun proteomics were done on oysters from high and low pCO2 and from with and without mechanical stress.
Results. At the end of the four week exposure period, oysters in all four pCO2 environments deposited new shell, but growth rate was not different among the treatments. However, micromechanical properties of the new shell were compromised by elevated pCO2. Elevated pCO2 affected neither whole body fatty acid composition, nor glycogen content, nor mortality rate associated with acute heat shock. Shotgun proteomics revealed that several physiological pathways were significantly affected by ocean acidification, including antioxidant response, carbohydrate metabolism, and transcription and translation. Additionally, the proteomic response to a second stress differed with pCO2, with numerous processes significantly affected by mechanical stimulation at high versus low pCO2 (all proteomics data are available in the ProteomeXchange under the identifier PXD000835).
Discussion. Oyster physiology is significantly altered by exposure to elevated pCO2, indicating changes in energy resource use. This is especially apparent in the assessment of the effects of pCO2 on the proteomic response to a second stress. The altered stress response illustrates that ocean acidification may impact how oysters respond to other changes in their environment. These data contribute to an integrative view of the effects of ocean acidification on oysters as well as physiological trade-offs during environmental stress.
This manuscript is being published as a preprint in PeerJ in order to make the proteomics data accessible to a broad community of scientists. It is being considered for publication in another journal.
Representative indents made during micromechanical testing for the (A) 400 µatm and (B) 2800 µatm treatment. The radius of a circle radiating from the center of the indent enclosing all visible cracks was used to calculate fracture toughness, a portion of which is shown for each treatment. Arrow denotes the longest crack found for each indent. Radius length is shown on the image in µm. Mean crack radius was similar between the 400 and 1000 µatm treatments.
Glycogen content (µg glycogen per mg tissue) for oysters from the pCO2 treatments of 400, 800, and 2800 µatm. There is no difference in glycogen content among treatment groups.
Representation of key metabolic pathways that are significantly affected by ocean acidification (A), mechanical stress at low pCO2 (B), and mechanical stress at high pCO2 (C). Red lines are proteins/pathways that are differentially expressed at higher levels in the stress treatments and blue lines are those that are differentially expressed at lower levels. In the key, the different colored lines represent different metabolic pathways that are affected by oyster exposure to ocean acidification and/or mechanical stimulation. Figures are also available on FigShare with input files for iPath2 to allow for interactive exploration of the data .
Heat maps of differentially expressed proteins annotated with protein names. Protein expression values have been log-transformed. The dendrograms on the left of the heat maps represent the clustering of proteins according to expression profile.
Raw and normalized (proportion) fatty acid data for 8 oysters each from 3 treatments: 400, 800, and 2800 µatm.
ProteinProphet output for each technical replicate. Information for each protein includes percent coverage by sequenced peptides, total number unique peptides, total independent spectra (spectral count), and peptide sequences.
Protein expression values (NSAF) for each oyster for the 1 616 proteins identified. Also included are average expression values across treatments (i.e. 2800 avg NSAF is the average expression across all four high pCO2-exposed oysters); fold change for treatment/control oysters (i.e. Fold Diff OA is [2800 avg NSAF]/[400 avg NSAF]); columns for each of the three treatment comparisons with an asterisk indicating if the protein is >5-fold up- or down-regulated; SwissProt annotation, e-value, and gene description; proteins responsible for enrichment and the treatment comparisons in which they are enriched; a column indicating in which stress treatment proteins are differentially expressed (q-value < 0.1). In the fold difference columns “up” signifies that the protein was only expressed in oysters from the 2800 µatm treatment (versus the 400 µatm , Fold Diff OA), mechanical stress at 400 µatm treatment (versus 400 µatm, Fold Diff 400 MechS), or in the mechanical stress at 2800 µatm (versus 2800 µatm, Fold Diff 2800 MechS); “down” represents proteins that were only expressed in the other treatment for each comparison.
C. gigas proteins with associated SwissProt/UniProt-KB, Gene Ontology (GO), and GO Slim annotations.
Enriched biological processes for proteins >2-fold differentially expressed in the stress responses to elevated pCO2 of 2800 µatm (“OA”), mechanical stress after a one month exposure to 400 µatm (“Mech Stress 400 µatm”), and mechanical stress after a one month exposure to 2800 µtam (“Mech Stress 2800 µatm”). Table includes enriched GO term, number of proteins contributing to that GO term, p-value indicating degree of enrichment, the SwissProt accession numbers for those proteins, the fold enrichment for each GO term, and the false discovery rate (FDR).