Metabolic molecular markers of the tidal clock in the marine crustacean Eurydice pulchra

Figure 1. Molecular readouts of the circatidal clock. (A) Section of representative actogram depicting mean spontaneous swimming activity of E. pulchra (n = 21, period = 12.5 ± 0.1 h; see [2] for methods) HW: predicted high water on home beach. (B) Representative western blot of overoxidised peroxiredoxin and loading control. (C) Relative intensity of overoxidised peroxiredoxin signal from E. pulchra heads collected May 2014 (Group data, mean ± SEM, n = 4 collections, 10 heads per sample) (ANOVA time effect p < 0.005; cosinor period = 14.5 h). See [1] for methods. Arrows indicate peak swimming activity in parallel sample. (D,E) Relative abundance of mitochondrially encoded mRNAs from E. pulchra heads (10 per sample), collected over two tidal cycles under free-running laboratory conditions (April 2013). ND, subunits of NADH dehydrogenase; COX, cytochrome oxidase subunits; CYT B, cytochrome B. All profi les were signifi cantly (p < 0.05) rhythmic by cosinor analysis (mean period ± SEM: mRNAs in D = 15.2 ± 0.6 h, E = 15.7 ± 0.8 h). Three mRNAs encoding large and small ribosomal RNA subunits, and ATP synthase subunit had comparable profi les but failed to reach statistical signifi cance. RNA was extracted using Trizol (Life Technologies) and DNAse treated with Turbo DNAfree (Ambion). Libraries for RNAseq were prepared with Illumina TruSeq sample preparation kit, transcriptome was assembled with Trinity (release 2014-07-17), read counts calculated with RSEM 1.9, data annotated in Trinotate (release 2014-07-08), assessed by Transrate 0.3.1 and CEGMA 2.5 and validated with BLAST+2.2.28 against the mitochondrial genome. Metabolic molecular markers of the tidal clock in the marine crustacean Eurydice pulchra