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Reproductive development in geoduck clams
Collaborators: Steven Roberts, Brent Vadopalas Geoduck clams (Panopea generosa) are an increasingly important fishery and aquaculture product in the Pacific Northwest of the United States while also holding essential ecological roles in the ecosystem. These long-lived clams are highly fecund, but sustainable hatchery production of genetically diverse larvae is hindered by the lack of sexual dimorphism, resulting in asynchronous spawning of broodstock, unequal sex ratios, and low numbers of breeders. Proteomic profiles were determined for three reproductive maturation stages in both male and female clams using data dependent acquisition (DDA), or whole proteome profiling, of gonad proteins. Gonad proteomes became increasingly divergent between males and females as maturation progressed. The DDA data was used to develop targets analyzed with selected reaction monitoring (SRM) in gonad tissue as well as hemolymph. The SRM assay yielded a suite of indicator peptides that can be used as an efficient assay to non-lethally determine geoduck sex and maturation stage pre-spawning. These results lay the foundation for the development of field based tools to assess reproductive status in marine mollusks. |
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Development of peptide assays for probing biologically complex ocean samples
Collaborator: H. Rodger Harvey
Bacteria transform nutrients and degrade organic matter, which are essential roles for healthy ecosystems. By documenting bacterial physiology within a complex system, the status of the whole ecosystem can be investigated. Proteins are the dynamic molecules that control essential cellular physiological responses; characterizing an organism’s proteome can therefore provide information on its interaction with the environment. Data dependent proteomic analysis (DDA) is a global approach to assay the entire proteome, but sample complexity and the stochastic nature of mass spectrometry can make it difficult to detect low abundance proteins. We explored the development of targeted proteomic (selected reaction monitoring, SRM) assays in complex ocean samples in order to detect specific bacterial proteins of interest and to assess new tools for metaproteomic exploration. As a starting point, a mixed community of known composition was created from a dilution series of pure bacteria (Ruegeria pomeroyi) and diatom (Thalassiosira pseudonana). Using SRM, we were able to select and detect bacterial peptides from a wide range of physiological processes (e.g. amino acid synthesis, lipid metabolism, and transport) from the community that were undetectable with the standard DDA approach. We demonstrate benefits and drawbacks of two different proteomic approaches that can be used to probe for and resolve nuances of bacterial physiological processes in complex environmental systems.
Collaborator: H. Rodger Harvey
Bacteria transform nutrients and degrade organic matter, which are essential roles for healthy ecosystems. By documenting bacterial physiology within a complex system, the status of the whole ecosystem can be investigated. Proteins are the dynamic molecules that control essential cellular physiological responses; characterizing an organism’s proteome can therefore provide information on its interaction with the environment. Data dependent proteomic analysis (DDA) is a global approach to assay the entire proteome, but sample complexity and the stochastic nature of mass spectrometry can make it difficult to detect low abundance proteins. We explored the development of targeted proteomic (selected reaction monitoring, SRM) assays in complex ocean samples in order to detect specific bacterial proteins of interest and to assess new tools for metaproteomic exploration. As a starting point, a mixed community of known composition was created from a dilution series of pure bacteria (Ruegeria pomeroyi) and diatom (Thalassiosira pseudonana). Using SRM, we were able to select and detect bacterial peptides from a wide range of physiological processes (e.g. amino acid synthesis, lipid metabolism, and transport) from the community that were undetectable with the standard DDA approach. We demonstrate benefits and drawbacks of two different proteomic approaches that can be used to probe for and resolve nuances of bacterial physiological processes in complex environmental systems.
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Community response to climate change
Collaborators: Philip Boyd
Climate change does and will continue to affect species assemblages and ecosystems, but many studies to date have limited investigations to a single species in isolation from its community. These single-species studies may overlook important community-scale interactions that may mitigate or enhance climate change impacts. Natural communities in the Southern Ocean were exposed to future ocean conditions in mesocosms to assess how climate change will impact the entire prokaryotic-eukaryotic plankton community. We will use data independent acquisition (DIA) and metagenomics to characterize the physiological impacts of climate change on these planktonic communities.
Collaborators: Philip Boyd
Climate change does and will continue to affect species assemblages and ecosystems, but many studies to date have limited investigations to a single species in isolation from its community. These single-species studies may overlook important community-scale interactions that may mitigate or enhance climate change impacts. Natural communities in the Southern Ocean were exposed to future ocean conditions in mesocosms to assess how climate change will impact the entire prokaryotic-eukaryotic plankton community. We will use data independent acquisition (DIA) and metagenomics to characterize the physiological impacts of climate change on these planktonic communities.
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Oyster larval response to temperature
Collaborators: Steven Roberts Larval rearing temperature can have a significant impact on oyster larval cohort success or failure. Higher temperatures can allow for faster larval growth, but require heating large quantities of water. However, mortality events can be common at lower temperatures, making extended rearing at low temperatures relatively risky. In this experiment, Pacific oyster larvae were raised at 23°C and 29°C. Pooled larvae were analyzed using data independent acquisition (DIA) to achieve a deep sequencing of the proteome. This technique will allow us to detect proteins that occur at low abundances in the larval proteome. From these data, we will reconstruct the oyster larval physiological response to different hatchery rearing temperatures to reveal how that response may contribute to cohort success or failure. |
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Bivalve proteomic response to environmental stress
Collaborators: Steven Roberts, Department of Natural Resources
Naturally occurring shellfish populations occur across heterogeneous environments that offer differing degrees of protection from different environmental impacts. We outplanted Pacific oysters and geoduck in different pH environments throughout the Puget Sound region and then routinely sampled them to assess their physiological responses to their environment. We will use DIA methods to uncover their proteomic acclimations to each environment.
Collaborators: Steven Roberts, Department of Natural Resources
Naturally occurring shellfish populations occur across heterogeneous environments that offer differing degrees of protection from different environmental impacts. We outplanted Pacific oysters and geoduck in different pH environments throughout the Puget Sound region and then routinely sampled them to assess their physiological responses to their environment. We will use DIA methods to uncover their proteomic acclimations to each environment.
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Influence of the hatchery microbiome on oyster larvae
Collaborator: Steven Roberts One of the major bottlenecks in bivalve aquaculture is the unpredictable mass mortality events that occur during larval production. These mortality events occur after considerable resources have been used to raise a cohort of larvae. The causes of these events are rarely known and thus cannot be avoided in the future. We hypothesized that the intimate relationship between oyster larvae and their ambient microbiome may influence the success or failure of larval cohorts. We followed multiple cohorts of larvae at two different temperatures over the time period that usually includes the mass mortality events. We concurrently sampled the larvae and their rearing tank microbiome. We will explore the proteomic responses of both the larvae and their microbiome to find biomarkers of larval outcomes. |
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