MTD submitter_name Grace Crandall MTD submitter_email graceac9@uw.edu MTD submitter_affiliation University of Washington MTD submitter_pride_login graceac9@uw.edu MTD lab_head_name Steven Roberts MTD lab_head_email sr320@washington.edu MTD lab_head_affiliation School of Aquatic and Fishery Sciences, University of Washington, Seattle MTD project_title PROTEOMIC RESPONSE OF EARLY JUVENILE PACIFIC OYSTERS TO TEMPERATURE MTD project_description The shellfish aquaculture industry provides a sustainable food source and jobs for a growing population. Oysters are the primary aquaculture species produced in the United States and account for a significant portion of seafood exports. Shellfish hatcheries have been experiencing frequent mass mortality events over the last couple of decades that occur approximately 10-14 days after oyster settlement. Settlement is a process that shellfish such as oysters undergo in which they transform from a free-swimming pelagic larvae to a sessile juvenile oyster. In order for this energy-intensive process to be successful, the oyster has to undergo behavioral and morphological changes. This is a vulnerable period in the oyster life cycle and conditions need to be such that they aren’t creating added stress. However, due to the oysters’ vulnerability, this is often a time when bacterial infections can occur, which when occurring with environmental conditions that are unfavorable, can prove to be fatal. In order to help oysters survive this process, scientists at the Taylor Shellfish Hatchery in Quilcene, WA has experimented with altering abiotic and biotic factors such as algal diet densities, pH, water flow rate, among others. At this hatchery, Pacific oysters are typically reared at 23˚C, however preliminary research results have suggested that oysters may have a higher survival rate when held at 29˚C during the settlement period. This pilot experiment attempts to identify differences in protein expression between oyster seed held at 23˚C and 29˚C during the settlement period using novel proteomic technology. Our proteomic results, paired with survival data, suggest that holding oyster seed at 29˚C during the settlement period results in higher survival rates. MTD keywords pacific oyster, temperature, juvenile, proteomic MTD sample_processing_protocol To the four pooled larval samples (~250 µl larvae), 50mM NH4HCO3 + 6M urea (500ul) was added to each sample and larvae were homogenized using a pestle. Samples were centrifuged at 2000 x g for 5 minutes. Supernatant (150 µl) was pipetted from each sample and placed into new tubes. The supernatant samples were sonicated three times each for 5 seconds, cooling samples in between sonication rounds using an ethanol/dry ice bath for 5 seconds. Protein concentration was determined using a Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA USA). Samples were digested and desalted for mass spectrometry as described in 5. The dried peptides were reconstituted in 100 µl 3% acetonitrile + 0.1% formic acid and stored at -80°C. Data Independent Acquisition (DIA) was performed to assess protein abundance patterns via liquid chromatography tandem mass spectrometry (LC-MS/MS) with a Q-Exactive mass spectrometer. Samples were analyzed in MS1 over 400–900 m/z range with 30k resolution in four separate injections with ranges of 400-525 m/z, 535-650 m/z, 650-775 m/z, and 775-900 m/z, and in 4m/z isolation windows from 450 to 850 m/z with 60 K resolution in MS2. MTD data_processing_protocol Raw mass spectrometry files were converted to .mzML format using MSConvert from the ProteoWizard Toolkit version 3.0 6. Resulting files and the C. gigas deduced proteome (Supplemental materials) were used to create a chromatogram library using EncyclopeDIA with Walnut version 0.6.14 7. Specific details are provided = EncyclopeDIA. The chromatogram library, C. gigas proteome, and .mzML files were imported into Skyline Daily version 4.1.9.18271 8, which provides a means of setting filters, viewing spectral data for quality inspection, and exporting the data for downstream analyses (specific details are provided = Skyline Daily). The Skyline Daily report includes spectral data as well as the proteins detected, and was exported using the built-in MS Stats report. Using the MS Stats report file, differentially abundant proteins were identified using MS Stats version 3.12.3 9 in RStudio version 1.1.453 10. Specific details are provided = MS Stats. R scripts and data analysis protocols (project repository), raw data (link to raw data published on PRIDE), and the Skyline document file (PanoramaWeb) are publicly available. DAVID version 6.8 11,12 was used to identify the enriched genes from a list of differentially abundant proteins. The DAVID output was then used in REViGO, accessed 04/15/2019 13, to create visualizations of the GO terms (Figures 1-3). MTD experiment_type [PRIDE, PRIDE:0000451, MSE, ] MTD submission_type PARTIAL MTD species [NEWT, 29159, Crassostrea gigas, ] MTD tissue [BTO, BTO:0001489, whole body, ] MTD instrument [MS, MS:1001911, Q Exactive, ] MTD modification [PRIDE, PRIDE:0000398, No PTMs are included in the dataset, ] FMH file_id file_type file_path file_mapping FME 0 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2015_December_30_DIA_oysterseed1.mzML.elib 16 FME 1 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2015_December_30_DIA_oysterseed11.mzML.elib 17 FME 2 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2015_December_30_DIA_oysterseed12.mzML.elib 18 FME 3 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2015_December_30_DIA_oysterseed13.mzML.elib 19 FME 4 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2015_December_30_DIA_oysterseed3.mzML.elib 20 FME 5 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2015_December_30_DIA_oysterseed4.mzML.elib 21 FME 6 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2015_December_30_DIA_oysterseed5.mzML.elib 22 FME 7 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2015_December_30_DIA_oysterseed7.mzML.elib 23 FME 8 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2015_December_30_DIA_oysterseed8.mzML.elib 24 FME 9 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2015_December_30_DIA_oysterseed9.mzML.elib 25 FME 10 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2015_December_30_oysterseed15.mzML.elib 26 FME 11 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2015_December_30_oysterseed16.mzML.elib 27 FME 12 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2016_Feb_01_DIA_oyster5.mzML.elib 28 FME 13 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2016_Feb_01_DIA_oyster6.mzML.elib 29 FME 14 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2016_Feb_01_DIA_oyster7.mzML.elib 30 FME 15 SEARCH /Volumes/web/scaphapoda/grace/2015-Cgseed-DIA/EncyclopeDIA-Walnut-outputs/intermediary-files_elib/2016_Feb_01_DIA_oyster8.mzML.elib 31 FME 16 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2015_December_30_DIA_oysterseed1.raw FME 17 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2015_December_30_DIA_oysterseed11.raw FME 18 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2015_December_30_DIA_oysterseed12.raw FME 19 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2015_December_30_DIA_oysterseed13.raw FME 20 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2015_December_30_DIA_oysterseed3.raw FME 21 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2015_December_30_DIA_oysterseed4.raw FME 22 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2015_December_30_DIA_oysterseed5.raw FME 23 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2015_December_30_DIA_oysterseed7.raw FME 24 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2015_December_30_DIA_oysterseed8.raw FME 25 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2015_December_30_DIA_oysterseed9.raw FME 26 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2015_December_30_oysterseed15.raw FME 27 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2015_December_30_oysterseed16.raw FME 28 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2016_Feb_01_DIA_oyster5.raw FME 29 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2016_Feb_01_DIA_oyster6.raw FME 30 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2016_Feb_01_DIA_oyster7.raw FME 31 RAW /Volumes/web/phainopepla/C_gigas/2015-12-30/2016_Feb_01_DIA_oyster8.raw