Coastal habitats fluctuate with the 12.4 h tidal and 24 h light/dark cycle to predictably alter conditions such as air exposure, temperature, and food availability. Intertidal sessile bivalves exhibit behavioral and physiological adjustments to minimize the challenges of this environment. We investigated a high-resolution time course of the changes in protein abundance in the gill tissue of the intertidal mussel Mytilus californianus in a simulated tidal environment of 12:12 h light:dark cycles and a matching 6:6 h high:low tide cycle within each 12 h period. Approximately 38% of detected proteins showed significant rhythms in their abundances, with diversity in the phases of rhythmic isoforms. The circadian rhythm was dominant in protein abundance changes, particularly with oxidative metabolism. A tidal cycle elicited changes within functional groups, including in cytoskeletal proteins, chaperones, and oxidative stress proteins. In addition to protein abundance changes, we found the possibility for post-translational modifications driving rhythms, including methylation, mitochondrial peptide processing (proteolysis), and acylation. Dynamic changes in the proteome across functional categories demonstrate the importance of the tidal environment in entraining cellular processes, confirming that differential expression studies should not assume a static baseline of cellular conditions in intertidal organisms.

M. californianus were acclimated for four weeks in a tidal simulator with square-wave 12:12 h full-spectrum L:D cycles and 6:6 h high/low tidal cycles (Fig. 1A). Mussels were fully exposed to the air during simulated low tide and a low unidirectional flow of filtered seawater approximately 25 cm above the mussels during high tide. Every hour during high tide a 15 mL dose of 1:10 Shellfish Diet 1800 (4 – 20 µm algal size; 40% Isochrysis, 15% Pavlova, 25% Tetraselmis, and 20% Thalassiosira pseudonana; Reed Mariculture, Inc., San Jose, CA) was automatically delivered during the 6 h high tide period to ensure a consistent feeding regime. Gill tissue was dissected during continued acclimation conditions from six haphazardly-selected individuals every two hours over a 48 h period and frozen on dry ice. Samples were then processed using the proteomic workflow outlined in Tomanek and Zuzow (Tomanek & Zuzow, 2010). Rhythmic proteins of period lengths between 4 – 28 h were detected using JTK_CYCLE algorithm (Hughes, Hogenesch, & Kornacker, 2010) and RAIN (Thaben & Westermark, 2014). Protein abundance changes were significantly rhythmic when both JTK_CYCLE and RAIN false discovery rate q values < 0.05 (Benjamini & Hochberg, 1995). Period and phase attributes of significant rhythms were generated by JTK_CYCLE (Hughes et al., 2010). Unique IDs (not including isoforms) for significantly rhythmic proteins were compared to putative human orthologs using NCBI tBLASTn (Altschul, Gish, Miller, Myers, & Lipman, 1990) and the resulting 82 proteins with matches were used to find enriched gene ontology (GO) categories using DAVID (Huang et al., 2009b; Huang, Sherman, & Lempicki, 2009a).

The file CircadianTidal_AllSpots_Raw.csv contains the raw normalized spot volume output from Delta2D image analysis. Digitized gel images were processed using Delta2D (version 4.3, Decodon, Greifswald, Germany). Gel images were warped to each other using a chained-group warping strategy and a fusion image was generated from the 144 gels to show the average spot volume for overlapping protein spots. This fusion image was used to generate spot boundaries and labels which were then transferred to all gel images. After background subtraction, spot volumes were normalized against the total spot volume of all proteins in the gel image. Each row is an individual gel image from one gill tissue sample, along with the time point for the collection. Each column is a spot number for a protein from the gel image.

CircadianTidal_Raw_Spectra.zip contains the raw output from each protein spot run through the MALDI TOF/TOF mass spectrometer (Ultraflex II, Bruker Daltonics Inc.). Included are the peaklists for peptide mass fingerprints (PMFs) and peptide fragment fingerprints (PFFs) for plated proteins using a +1 charge state and a mass range of 900 – 3300 Da. FlexAnalysis (version 3.0, Bruker Daltonics Inc.) was used to detect peptide peaks with a SNAP peak detection algorithm using a signal-to-noise ratio threshold of 4 for MS and 5 for MS/MS.

Also included are summary files for the proteins (Dataset_S1_Proteins.xlsx) and for the DAVID enrichment analysis results (Dataset_S2_DAVID.xlsx) that were both included as supplementary information in the publication. The protein summary file includes information for each individual spot, including the protein ID and match information and the output from JTK_CYCLE and RAIN analysis of rhythmicity.