Project description:Global climate change is rapidly altering coastal marine ecosystems important for food production. A comprehensive understanding of how organisms will respond to these complex environmental changes can come only from observing and studying species within their natural environment. To this end, the effects of environmental drivers — pH, dissolved oxygen content, salinity, and temperature — on Pacific oyster (Crassostrea gigas) physiology were evaluated in an outplant experiment. Sibling juvenile oysters were outplanted to eelgrass and unvegetated habitat at five different estuarine sites within the Acidification Nearshore Monitoring Network in Washington State, USA to evaluate how regional environmental drivers influence molecular physiology. Within each site, we also determined if eelgrass presence that buffered pH conditions changed the oysters’ expressed proteome. A novel, two-step, gel-free proteomic approach was used to identify differences in protein abundance in C. gigas ctenidia tissue after a 29 day outplant by 1) identifying proteins in a data independent acquisition survey step and 2) comparing relative quantities of targeted environmental response proteins using selected reaction monitoring. While there was no difference in protein abundance detected between habitats or among sites within Puget Sound, C. gigas outplanted at Willapa Bay had significantly higher abundances of antioxidant enzymes and molecular chaperones. Environmental factors at Willapa Bay, such as higher average temperature, may have driven this protein abundance pattern. These findings generate a suite of new hypotheses for lab and field experiments to compare the effects of regional conditions on physiological responses of marine invertebrates.
Project description:DNA oligonucleotide microarrays were designed with 307 probes for 96 internal transcribed spacer (ITS1, located between 18S and 26S rRNA genes) sequences of known species and strains from the genus Pseudo-nitzschia (Bacillariophyceae). In addition, microarrays also carried 1893 probes targeting ITS1 aequences of marine Crenarchaeota and Alphaproteobacteria of SAR11 clade. In order to assign microarray profiles to Pseudo-nitzschia ribotypes and species and to 'train' the data analysis system, we grew cultures of Pseudo-nitzschia in the laboratory with identities confirmed through rDNA sequence analysis. In total, 9 cultures and 35 environmental water samples were hybridized to microarrays, in some cases, in duplicate or triplicate. Analysis of microarray data allowed us to identify and map Pseudo-nitzschia spp. in the coastal waters along Washington and Oregon coast of the Eastern Pacific Ocean, and to observe seasonal changes in diatom community composition. Total DNA was isolated from 9 Pseudo-nitzschia laboratory cultures and 35 environmental water samples collected during 7 field campaigns in 2007-2009. The environmental samples were collected at distances of 5 to 55 km from the coast, along the following transects in the Pacific Ocean covering over 300 km of the coastline: La Push (LP), Grays Harbor (GH), Columbia River (CR), and Newport Hydroline (NH). The DNA samples were subjected to PCR amplification with the primers specific for ITS1 sequences. The resultant biotin-labeled target samples were analyzed using microarray hybridization with the CombiMatrix ElectraSense 4X2K format. Out of 44 analyzed samples, 40, 2, and 2 were used for single, duplicate and triplicate hybridizations, respectively.
Project description:DNA oligonucleotide microarrays were designed with 307 probes for 96 internal transcribed spacer (ITS1, located between 18S and 26S rRNA genes) sequences of known species and strains from the genus Pseudo-nitzschia (Bacillariophyceae). In addition, microarrays also carried 1893 probes targeting ITS1 aequences of marine Crenarchaeota and Alphaproteobacteria of SAR11 clade. In order to assign microarray profiles to Pseudo-nitzschia ribotypes and species and to 'train' the data analysis system, we grew cultures of Pseudo-nitzschia in the laboratory with identities confirmed through rDNA sequence analysis. In total, 9 cultures and 35 environmental water samples were hybridized to microarrays, in some cases, in duplicate or triplicate. Analysis of microarray data allowed us to identify and map Pseudo-nitzschia spp. in the coastal waters along Washington and Oregon coast of the Eastern Pacific Ocean, and to observe seasonal changes in diatom community composition.