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Identification, Characterization, and Diel Pattern of Expression of Canonical Clock Genes in Nephrops norvegicus (Crustacea: Decapoda) Eyestalk.

Sbragaglia V, Lamanna F, M Mat A, Rotllant G, Joly S, Ketmaier V, de la Iglesia HO, Aguzzi J - PLoS ONE (2015)

Bottom Line: We found a list of candidate clock genes and focused our attention on canonical ones: timeless, period, clock and bmal1.We also found a vertebrate-like cryptochrome 2.Our data are in accordance with the current knowledge of the crustacean circadian clock, reinforcing the idea that the molecular clockwork of this group shows some differences with the established model in Drosophila melanogaster.

View Article: PubMed Central - PubMed

Affiliation: Marine Science Institute, (ICM-CSIC), Barcelona, Spain.

ABSTRACT
The Norway lobster, Nephrops norvegicus, is a burrowing decapod with a rhythmic burrow emergence (24 h) governed by the circadian system. It is an important resource for European fisheries and its behavior deeply affects its availability. The current knowledge of Nephrops circadian biology is phenomenological as it is currently the case for almost all crustaceans. In attempt to elucidate the putative molecular mechanisms underlying circadian gene regulation in Nephrops, we used a transcriptomics approach on cDNA extracted from the eyestalk, a structure playing a crucial role in controlling behavior of decapods. We studied 14 male lobsters under 12-12 light-darkness blue light cycle. We used the Hiseq 2000 Illumina platform to sequence two eyestalk libraries (under light and darkness conditions) obtaining about 90 millions 100-bp paired-end reads. Trinity was used for the de novo reconstruction of transcriptomes; the size at which half of all assembled bases reside in contigs (N50) was equal to 1796 (light) and 2055 (darkness). We found a list of candidate clock genes and focused our attention on canonical ones: timeless, period, clock and bmal1. The cloning of assembled fragments validated Trinity outputs. The putative Nephrops clock genes showed high levels of identity (blastx on NCBI) with known crustacean clock gene homologs such as Eurydice pulchra (period: 47%, timeless: 59%, bmal1: 79%) and Macrobrachium rosenbergii (clock: 100%). We also found a vertebrate-like cryptochrome 2. RT-qPCR showed that only timeless had a robust diel pattern of expression. Our data are in accordance with the current knowledge of the crustacean circadian clock, reinforcing the idea that the molecular clockwork of this group shows some differences with the established model in Drosophila melanogaster.

No MeSH data available.


Related in: MedlinePlus

Canonical clock genes expression (RT-qPCR).Canonical clock genes expression in Nephrops eyestalk. Measurements (n = 3 each time point) were normalized to α-act and 18S and expressed as fold change respect to a control time point (7:30). Vertical bars represent the confidence limits. Black and white bars represent darkness and light hours, respectively. Timeless shows a significant pattern of expression (ANOVA, P < 0.05). Letters indicate the output of the Tukey’s post hoc test (a>b).
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pone.0141893.g005: Canonical clock genes expression (RT-qPCR).Canonical clock genes expression in Nephrops eyestalk. Measurements (n = 3 each time point) were normalized to α-act and 18S and expressed as fold change respect to a control time point (7:30). Vertical bars represent the confidence limits. Black and white bars represent darkness and light hours, respectively. Timeless shows a significant pattern of expression (ANOVA, P < 0.05). Letters indicate the output of the Tukey’s post hoc test (a>b).

Mentions: The melting curves of the RT-qPCR indicated the presence of a single peak, suggesting no signs of contamination by DNA (as also supported by gel electrophoresis and absorbance ratio, see above). Among the canonical Nephrops clock genes, only timeless has a significant (F = 10.470, P < 0.01) pattern of expression with a peak just before the light-OFF (late day, Fig 5). The other transcripts did not show significant differences of expression among the different sampling time points: period, (F = 2.020, p = 0.19); clock, (F = 1.354; p = 0.32); bmal1, (F = 1.342, p = 0.33). Despite its lack of significant rhythmicity, period expression pattern appeared to be similar to timeless. The 2ΔCT values were used to assess differences among sampling times and are available in the Appendix C in the S1 File.


Identification, Characterization, and Diel Pattern of Expression of Canonical Clock Genes in Nephrops norvegicus (Crustacea: Decapoda) Eyestalk.

Sbragaglia V, Lamanna F, M Mat A, Rotllant G, Joly S, Ketmaier V, de la Iglesia HO, Aguzzi J - PLoS ONE (2015)

Canonical clock genes expression (RT-qPCR).Canonical clock genes expression in Nephrops eyestalk. Measurements (n = 3 each time point) were normalized to α-act and 18S and expressed as fold change respect to a control time point (7:30). Vertical bars represent the confidence limits. Black and white bars represent darkness and light hours, respectively. Timeless shows a significant pattern of expression (ANOVA, P < 0.05). Letters indicate the output of the Tukey’s post hoc test (a>b).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4629887&req=5

pone.0141893.g005: Canonical clock genes expression (RT-qPCR).Canonical clock genes expression in Nephrops eyestalk. Measurements (n = 3 each time point) were normalized to α-act and 18S and expressed as fold change respect to a control time point (7:30). Vertical bars represent the confidence limits. Black and white bars represent darkness and light hours, respectively. Timeless shows a significant pattern of expression (ANOVA, P < 0.05). Letters indicate the output of the Tukey’s post hoc test (a>b).
Mentions: The melting curves of the RT-qPCR indicated the presence of a single peak, suggesting no signs of contamination by DNA (as also supported by gel electrophoresis and absorbance ratio, see above). Among the canonical Nephrops clock genes, only timeless has a significant (F = 10.470, P < 0.01) pattern of expression with a peak just before the light-OFF (late day, Fig 5). The other transcripts did not show significant differences of expression among the different sampling time points: period, (F = 2.020, p = 0.19); clock, (F = 1.354; p = 0.32); bmal1, (F = 1.342, p = 0.33). Despite its lack of significant rhythmicity, period expression pattern appeared to be similar to timeless. The 2ΔCT values were used to assess differences among sampling times and are available in the Appendix C in the S1 File.

Bottom Line: We found a list of candidate clock genes and focused our attention on canonical ones: timeless, period, clock and bmal1.We also found a vertebrate-like cryptochrome 2.Our data are in accordance with the current knowledge of the crustacean circadian clock, reinforcing the idea that the molecular clockwork of this group shows some differences with the established model in Drosophila melanogaster.

View Article: PubMed Central - PubMed

Affiliation: Marine Science Institute, (ICM-CSIC), Barcelona, Spain.

ABSTRACT
The Norway lobster, Nephrops norvegicus, is a burrowing decapod with a rhythmic burrow emergence (24 h) governed by the circadian system. It is an important resource for European fisheries and its behavior deeply affects its availability. The current knowledge of Nephrops circadian biology is phenomenological as it is currently the case for almost all crustaceans. In attempt to elucidate the putative molecular mechanisms underlying circadian gene regulation in Nephrops, we used a transcriptomics approach on cDNA extracted from the eyestalk, a structure playing a crucial role in controlling behavior of decapods. We studied 14 male lobsters under 12-12 light-darkness blue light cycle. We used the Hiseq 2000 Illumina platform to sequence two eyestalk libraries (under light and darkness conditions) obtaining about 90 millions 100-bp paired-end reads. Trinity was used for the de novo reconstruction of transcriptomes; the size at which half of all assembled bases reside in contigs (N50) was equal to 1796 (light) and 2055 (darkness). We found a list of candidate clock genes and focused our attention on canonical ones: timeless, period, clock and bmal1. The cloning of assembled fragments validated Trinity outputs. The putative Nephrops clock genes showed high levels of identity (blastx on NCBI) with known crustacean clock gene homologs such as Eurydice pulchra (period: 47%, timeless: 59%, bmal1: 79%) and Macrobrachium rosenbergii (clock: 100%). We also found a vertebrate-like cryptochrome 2. RT-qPCR showed that only timeless had a robust diel pattern of expression. Our data are in accordance with the current knowledge of the crustacean circadian clock, reinforcing the idea that the molecular clockwork of this group shows some differences with the established model in Drosophila melanogaster.

No MeSH data available.


Related in: MedlinePlus