Emma Timmins-Schiffman

Shotgun proteomics reveals physiological response to ocean acidification in Crassostrea gigas.

BackgroundOcean 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. 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).ResultsAt 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).ConclusionsOyster 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.

Proteomics of Colwellia psychrerythraea at subzero temperatures - a life with limited movement, flexible membranes and vital DNA repair.

The mechanisms that allow psychrophilic bacteria to remain metabolically active at subzero temperatures result from form and function of their proteins. We present first proteomic evidence of physiological changes of the marine psychrophile Colwellia psychrerythraea 34H (Cp34H) after exposure to subzero temperatures (-1, and -10°C in ice) through 8 weeks. Protein abundance was compared between different treatments to understand the effects of temperature and time, independently and jointly, within cells transitioning to, and being maintained in ice. Parallel [3H]-leucine and [3H]-thymidine incubations indicated active protein and DNA synthesis to -10°C. Mass spectrometry-based proteomics identified 1763 proteins across four experimental treatments. Proteins involved in osmolyte regulation and polymer secretion were found constitutively present across all treatments, suggesting that they are required for metabolic success below 0°C. Differentially abundant protein groups indicated a reallocation of resources from DNA binding to DNA repair and from motility to chemo-taxis and sensing. Changes to iron and nitrogen metabolism, cellular membrane structures, and protein synthesis and folding were also revealed. By elucidating vital strategies during life in ice, this study provides novel insight into the extensive molecular adaptations that occur in cold-adapted marine organisms to sustain cellular function in their habitat.

Critical decisions in metaproteomics: achieving high confidence protein annotations in a sea of unknowns

Critical decisions in metaproteomics: achieving high confidence protein annotations in a sea of unknowns

Genomic resource development for shellfish of conservation concern

Effective conservation of threatened species depends on the ability to assess organism physiology and population demography. To develop genomic resources to better understand the dynamics of two ecologically vulnerable species in the Pacific Northwest of the United States, larval transcriptomes were sequenced for the pinto abalone, Haliotis kamtschatkana kamtschatkana, and the Olympia oyster, Ostrea lurida. Based on comparative species analysis the Ostrea lurida transcriptome (41 136 contigs) is relatively complete. These transcriptomes represent the first significant contribution to genomic resources for both species. Genes are described based on biological function with particular attention to those associated with temperature change, oxidative stress and immune function. In addition, transcriptome‐derived genetic markers are provided. Together, these resources provide valuable tools for future studies aimed at conservation of Haliotis kamtschatkana kamtschatkana, Ostrea lurida and related species.

Shotgun proteomics as a viable approach for biological discovery in the Pacific oyster

The oyster gill proteome (expressed proteins) was sequenced using shotgun proteomics. This effort represents the first time that a global, non-gel based approach has been used to characterize proteins from oyster gill. The data provide insight into the dynamic functions of this tissue and demonstrate the viability of this approach.

Human-mediated evolution in a threatened species? Juvenile life-history changes in Snake River salmon

Evaluations of human impacts on Earths ecosystems often ignore evolutionary changes in response to altered selective regimes. Freshwater habitats for Snake River fall Chinook salmon (SRFCS), a threatened species in the US, have been dramatically changed by hydropower development and other watershed modifications. Associated biological changes include a shift in juvenile life history: Historically essentially 100% of juveniles migrated to sea as subyearlings, but a substantial fraction have migrated as yearlings in recent years. In contemplating future management actions for this species should major Snake River dams ever be removed (as many have proposed), it will be important to understand whether evolution is at least partially responsible for this life‐history change. We hypothesized that if this trait is genetically based, parents who migrated to sea as subyearlings should produce faster‐growing offspring that would be more likely to reach a size threshold to migrate to sea in their first year. We tested this with phenotypic data for over 2,600 juvenile SRFCS that were genetically matched to parents of hatchery and natural origin. Three lines of evidence supported our hypothesis: (i) the animal model estimated substantial heritability for juvenile growth rate for three consecutive cohorts; (ii) linear modeling showed an association between juvenile life history of parents and offspring growth rate; and (iii) faster‐growing juveniles migrated at greater speeds, as expected if they were more likely to be heading to sea. Surprisingly, we also found that parents reared a full year in a hatchery produced the fastest growing offspring of all—apparently an example of cross‐generational plasticity associated with artificial propagation. We suggest that SRFCS is an example of a potentially large class of species that can be considered to be “anthro‐evolutionary”—signifying those whose evolutionary trajectories have been profoundly shaped by altered selective regimes in human‐dominated landscapes.

Characterization of genes involved in ceramide metabolism in the Pacific oyster (Crassostrea gigas)

BackgroundThe lipid signaling molecule, ceramide, is a key component of the vertebrate stress response, however, there is limited information concerning its role in invertebrate species. In order to identify genes involved in ceramide metabolism in bivalve molluscs, Pacific oyster genomic resources were examined for genes associated with ceramide metabolism and signaling.ResultsSeveral genes were identified including full-length sequences characterized for serine palmitoyltransferase-1, 3-ketodihydrosphingosine reductase, acid ceramidase, and ceramide glucosyltransferase. Genes involved in ceramide synthesis and metabolism are conserved across taxa in both form and function. Expression analysis as assessed by quantitative PCR indicated all genes were expressed at high levels in gill tissue. The role of the ceramide pathway genes in the invertebrate stress response was also explored by measuring expression levels in adult oysters exposed to Vibrio vulnificus. Two genes demonstrated increased expression during the bacterial challenge: a gene involved in hydrolytic breakdown of ceramide (acid ceramidase) and a gene involved in de novo generation of ceramide (3-ketodihydrosphingosine reductase), suggesting a possible role of ceramide in the invertebrate stress and immune responses.ConclusionsIn silico and laboratory results support that Pacific oysters have the basic components of the ceramide metabolism pathway. These results also indicate that ceramide may have analogous functions in vertebrates and invertebrates. The gene expression pattern of acid ceramidase and 3-kethodihydrosphingosine reductase in response to bacterial exposure especially supports that ceramide and sphingolipid metabolism may be involved in the oyster’s stress and/or immune responses.

Pacific geoduck (Panopea generosa) resilience to natural pH variation

Pacific geoduck aquaculture is a growing industry, however little is known about how geoduck respond to varying environmental conditions, or how production might be impacted by low pH associated with ocean acidification. Ocean acidification research is increasingly incorporating multiple environmental drivers and natural pH variability into biological response studies for more complete understanding of the effects of projected ocean conditions. In this study, eelgrass habitats and environmental heterogeneity across four estuarine bays were leveraged to examine low pH effects on geoduck under different natural regimes, using proteomics to assess physiology. Juvenile geoduck were deployed in eelgrass and adjacent unvegetated habitats for 30 days while pH, temperature, dissolved oxygen, and salinity were monitored. Across the four bays pH was lower in unvegetated habitats compared to eelgrass habitats, however this did not impact geoduck growth, survival, or proteomic expression patterns. However, across all sites temperature and dissolved oxygen corresponded to growth and protein expression patterns. Specifically, three protein abundance levels (trifunctional-enzyme β-subunit, puromycin-sensitive aminopeptidase, and heat shock protein 90-α) and shell growth positively correlated with dissolved oxygen variability and inversely correlated with mean temperature. These results demonstrate that geoduck are resilient to low pH in a natural setting, and other abiotic factors (i.e. temperature, dissolved oxygen variability) may have a greater influence on geoduck physiology. In addition this study contributes to the understanding of how eelgrass patches influences water chemistry.

MS analysis of a dilution series of bacteria:phytoplankton to improve detection of low abundance bacterial peptides

Assigning links between microbial activity and biogeochemical cycles in the ocean is a primary objective for ecologists and oceanographers. Bacteria represent a small ecosystem component by mass, but act as the nexus for both nutrient transformation and organic matter recycling. There are limited methods to explore the full suite of active bacterial proteins largely responsible for degradation. Mass spectrometry (MS)-based proteomics now has the potential to document bacterial physiology within these complex systems. Global proteome profiling using MS, known as data dependent acquisition (DDA), is limited by the stochastic nature of ion selection, decreasing the detection of low abundance peptides. The suitability of MS-based proteomics methods in revealing bacterial signatures outnumbered by phytoplankton proteins was explored using a dilution series of pure bacteria (Ruegeria pomeroyi) and diatoms (Thalassiosira pseudonana). Two common acquisition strategies were utilized: DDA and selected reaction monitoring (SRM). SRM improved detection of bacterial peptides at low bacterial cellular abundance that were undetectable with DDA from a wide range of physiological processes (e.g. amino acid synthesis, lipid metabolism, and transport). We demonstrate the benefits and drawbacks of two different proteomic approaches for investigating species-specific physiological processes across relative abundances of bacteria that vary by orders of magnitude.

MetaGOmics: A Web-Based Tool for Peptide-Centric Functional and Taxonomic Analysis of Metaproteomics Data

Metaproteomics is the characterization of all proteins being expressed by a community of organisms in a complex biological sample at a single point in time. Applications of metaproteomics range from the comparative analysis of environmental samples (such as ocean water and soil) to microbiome data from multicellular organisms (such as the human gut). Metaproteomics research is often focused on the quantitative functional makeup of the metaproteome and which organisms are making those proteins. That is: What are the functions of the currently expressed proteins? How much of the metaproteome is associated with those functions? And, which microorganisms are expressing the proteins that perform those functions? However, traditional protein-centric functional analysis is greatly complicated by the large size, redundancy, and lack of biological annotations for the protein sequences in the database used to search the data. To help address these issues, we have developed an algorithm and web application (dubbed “MetaGOmics”) that automates the quantitative functional (using Gene Ontology) and taxonomic analysis of metaproteomics data and subsequent visualization of the results. MetaGOmics is designed to overcome the shortcomings of traditional proteomics analysis when used with metaproteomics data. It is easy to use, requires minimal input, and fully automates most steps of the analysis—including comparing the functional makeup between samples. MetaGOmics is freely available at https://www.yeastrc.org/metagomics/.