DOI: 10.5061/dryad.qrfj6q5gj
title: Circadian and circatidal rhythms of protein abundance in the California mussel (Mytilus californianus)

description: 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.
description: <i>M. californianus </i>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%<i> Isochrysis, </i>15%
 <i>Pavlova</i>, 25%<i> Tetraselmis, </i>and
 20%<i> Thalassiosira pseudonana</i>; 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 &lt; 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).
description: 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.