Fig 1.
Phylogenetic analysis based on clock and cycle/Bmal sequences.
The tree was generated by Maximum Likelihood method using Mega 6 program and based on the multiple alignments performed with Clustal Omega. Percentage of bootstraps based on 1,000 replicates were indicated with only values > 50%. Hypoxia-inducible factor (HIF) from C. gigas was used as outgroup to root the tree. See S2 Table for sequence details and accession numbers.
Fig 2.
Phylogenetic analysis based on period sequences.
The tree was generated by Maximum Likelihood method using Mega 6 program and based on the multiple alignments performed with Clustal Omega. Percentage of bootstraps based on 1,000 replicates were indicated with only values > 50%. Neuronal PAS domain-containing protein (NPas) from C. gigas was used as outgroup to root the tree. See S2 Table for sequence details and accession numbers.
Fig 3.
Phylogenetic analysis based on timeless sequences.
The tree was generated by Maximum Likelihood method using Mega 6 program and based on the multiple alignments performed with Clustal Omega. Percentage of bootstraps based on 1,000 replicates were indicated with only values > 50%. Swi1 from Schizosaccharomyces. pombe was used as outgroup to root the tree. See S2 Table for sequence details and accession numbers.
Fig 4.
Phylogenetic analysis based on cryptochrome / photolyase sequences.
The tree was generated by Maximum Likelihood method using Mega 6 program and based on the multiple alignments performed with Clustal Omega. Percentage of bootstraps based on 1,000 replicates were indicated with only values > 50%. CPD photolyase (CPD) from P. dumerilii was used as outgroup to root the tree. See S2 Table for sequence details and accession numbers.
Fig 5.
Schematic representation of putative functional domains and motifs in photolyase / cryptochrome proteins in oyster, Arabidopsis thaliana (AtCRY), fruit fly (Dm6-4PHOTOLYASE and DmCRY), marine worm (Platynereis dumerilii, PdCRY2) and human (HsCRY1).
The number at the end of each diagram indicated protein size in amino acid residues. Numbers below domains indicated sequence identity / similarity with oyster ortholog. Inset showed multiple sequence alignments of a putative nuclear localization signal (NLS) in the RD2b domain and sequences referred to oyster (CgpCRY, CgCRY1, CgCRY2, Cg6-4PHOTOLYASE), human (HsCRY2), fruit fly (DmCRY1 and Dm6-4), zebrafish Danio rerio (DrCRY1a), butterfly Danaus plexippus (DpCRY1, DpCRY2 and Dp6-4) and marine worm P. dumerilii (PdCRY2).
Fig 6.
Variation of expression levels of clock genes.
Relative transcription levels (RQ, mean ± SEM, n = 9 oysters) of Cg6-4photolyase, CgCry1, CgpCry, CgCry2, CgClock, CgBmal, CgPeriod, CgTim, CgRev-Erb and CgROR RNA in gill tissues of oysters exposed for 26 h to L:D 10:14 (□) and for 26 h to constant darkness (■). Gray areas referred to scotophase during L:D cycles. Significant differences at p < 0.05 between light regimes are indicated and asterisks denote significant time-specific differences between light regime treatments.
Table 1.
Spearman analysis of clock gene expression under L:D regime (n = 72 oysters).
Table 2.
Spearman analysis of clock gene expression under D:D regime (n = 72 oysters).
Fig 7.
Oysters expressed circadian cycles of valve activity behavior.
A. Group level analysis. Left panel (A1): Double-plotted actograms of mean hourly opening duration (%) of the group (n = 15 oysters) submitted to 15 days of L:D (10:14) and 15 days of D:D. Right panel (A2): Period determined by spectral analysis (Lomb and Scargle periodogram; dotted line for p-value = 0.05) and percent rhythm (PR) of the Cosinor model. B. Individual analysis. Distribution of rhythmic (R) and arrhythmic (AR) oysters among LD and DD conditions. Details of periods in rhythmic oysters (circadian: 20-28h and ultradian <20h) are provided.
Fig 8.
Schematic representation of the hypothetic molecular clockwork of the oyster Crassostrea gigas.
Simplified molecular oscillators from butterfly (A) and vertebrate (mouse) (B) were presented in comparison to the putative model in C. gigas (C). Red lines indicated inhibition. Adapted from Chaves et al. [38] and Emery and Reppert [49].