Growth history of Atlantic cod juveniles reared under continuous light (LD 24:0, red bars) or natural photoperiod conditions (LDN, blue bars) for six months.

<p>Details of the light regimes are shown by red diamonds or green triangles for LD 24:0 and LDN groups, respectively. Sea water temperature is also indicated by blue circles. Significant differences in mean weight between the two light groups at a particular time point (two-tailed t-test, n = 123) are highlighted by an asterisk.</p

Phylogenetic tree of the seven <i>mll</i> and <i>setd1</i> paralogues found in vertebrates.

<p>Numbers at the nodes indicate posterior probability and approximate likelihood-ratio values obtained from Bayesian (left) and maximum likelihood (right) methods, respectively. Species abbreviations are as follows: <i>Bt, Bos Taurus; Ce, Caenorhabditis elegans; Cl, Canis lupus familiaris; Dr, Danio rerio; Gg, Gallus gallus; Gm, Gadus morhua; Hs, Homo sapiens; Mm, Mus musculus; On, Oreochromis niloticus; Pt, Pan troglodytes; Rn, Rattus norvegicus; Xt, Xenopus tropicalis.</i> GenBank accession numbers for <i>mll</i> sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036908#pone.0036908.s010" target="_blank">Table S1</a>.</p

Representative tissue distribution of <i>mll</i> paralogues in adult Atlantic cod.

<p>cDNAs from various tissues (brain, gill, heart, head kidney, kidney, liver, spleen, stomach, mid gut, gas bladder, testis, ovary, fast skeletal muscle, skin and blood) were used for semi-quantitative RT-PCR. <i>Actb</i> and <i>eef1a</i> were used as endogenous references. Expression patterns were determined using three biological replicates.</p

Quantification of <i>mll</i> paralogues and key myogenic genes (<i>myog</i>, <i>myf5</i> and <i>pax7</i>) in fast muscle of Atlantic cod juveniles reared under continuous light (red bars, LD 24:0) or natural photoperiod conditions (blue bars, LDN) for 6 months.

<p>In general, <i>mll</i> genes were differentially expressed between the two light groups and there was a decrease in <i>mll</i> transcript levels with continuous illumination as early as 12 hours, compared to the natural photoperiod group. Myog transcript levels were consistently higher in the constant light group compared to natural photoperiod. Asterisks * and ** indicate significant differences at p<0.05 and p<0.01, respectively (n = 6).</p

Gene name, GenBank accession number, primer sequences (5′ to 3′), amplicon sizes (bp) and PCR efficiency (%) of <i>mll,</i> s<i>et1d</i> and myogenic genes cloned in Atlantic cod.

<p>Reference genes used are also indicated.</p

Partial synteny map of the genomic region surrounding <i>mll2</i>.

<p>Synteny was disrupted between teleosts and tetrapods. Orthologous genes in <i>Gadus morhua</i>, <i>Oryzias latipes</i>, <i>Gasterosteus aculeatus</i>, <i>Takifugu rubripes</i>, <i>Tetraodon nigroviridis</i> and <i>Danio rerio</i> are colour coded and represented by block arrows that show their orientation in the genome. <i>Mll2</i> paralogues are indicated by the arrow. Additional synteny results for other <i>mll</i> paralogues can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036908#pone.0036908.s001" target="_blank">Figures S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036908#pone.0036908.s009" target="_blank">9</a>.</p

Daily Rhythmicity of Clock Gene Transcripts in Atlantic Cod Fast Skeletal Muscle

<div><p>The classical notion of a centralized clock that governs circadian rhythmicity has been challenged with the discovery of peripheral oscillators that enable organisms to cope with daily changes in their environment. The present study aimed to identify the molecular clock components in Atlantic cod (<i>Gadus morhua</i>) and to investigate their daily gene expression in fast skeletal muscle. Atlantic cod clock genes were closely related to their orthologs in teleosts and tetrapods. Synteny was conserved to varying degrees in the majority of the 18 clock genes examined. In particular, <i>aryl hydrocarbon receptor nuclear translocator-like 2</i> (<i>arntl2), RAR-related orphan receptor A</i> (<i>rora</i>) and <i>timeless</i> (<i>tim</i>) displayed high degrees of conservation. Expression profiling during the early ontogenesis revealed that some transcripts were maternally transferred, namely <i>arntl2</i>, <i>cryptochrome 1b</i> and <i>2</i> (<i>cry1b</i> and <i>cry2</i>), and <i>period 2a</i> and <i>2b</i> (<i>per2a</i> and <i>per2b</i>). Most clock genes were ubiquitously expressed in various tissues, suggesting the possible existence of multiple peripheral clock systems in Atlantic cod. In particular, they were all detected in fast skeletal muscle, with the exception of neuronal PAS (Per-Arnt-Single-minded) domain-containing protein (<i>npas1</i>) and <i>rora</i>. Rhythmicity analysis revealed 8 clock genes with daily rhythmic expression, namely <i>arntl2</i>, circadian locomotor output cycles kaput (<i>clock</i>), <i>npas2</i>, <i>cry2</i>, <i>cry3 per2a</i>, nuclear receptor subfamily 1, group D, member 1 (<i>nr1d1</i>), and <i>nr1d2a</i>. Transcript levels of the myogenic genes <i>myogenic factor 5</i> (<i>myf5</i>) and <i>muscleblind-like 1</i> (<i>mbnl1</i>) strongly correlated with clock gene expression. This is the first study to unravel the molecular components of peripheral clocks in Atlantic cod. Taken together, our data suggest that the putative clock system in fast skeletal muscle of Atlantic cod has regulatory implications on muscle physiology, particularly in the expression of genes related to myogenesis.</p></div

Partial synteny map of <i>arntl2</i> of Atlantic cod showing high syntenic conservation.

<p>Orthologous genes in <i>G. morhua</i>, <i>O. latipes</i>, <i>T. rubripes</i>, <i>T. nigroviridis</i>, <i>O. niloticus</i> and <i>X. maculatus</i>, are indicated by block arrows showing their position and orientation. Additional synteny results can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099172#pone.0099172.s002" target="_blank">Figure S2A</a>-O. (un: unidentified gene).</p

Daily expression of muscle-related genes.

<p>The values presented here are mean<u>+</u>SEM (n = 6) of normalized expression. Statistical differences between time points are indicated by different letter notations. The broken line is the periodic sinusoidal function of the gene expression in a daily cycle constructed from the rhythmicity parameters provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099172#pone.0099172.s005" target="_blank">Table S3</a>. An asterisk (*) beside the gene name indicates that the expression is daily rhythmic. The photoperiod regime is represented by the composite block above the graph. White, black and gray represent the light phase, the dark phase and the light-dark transition phase, respectively.</p

List of clock gene pairs showing either positive or negative expression correlation in fast muscle during a daily cycle.

<p>List of clock gene pairs showing either positive or negative expression correlation in fast muscle during a daily cycle.</p