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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

Gene ontology annotation of the two transcriptomes (NEP-L and NEP-D).Gene ontology (GO) annotation of the assembled transcriptomes. A: The percentage distribution of functional categories between the two transcriptomes together with the proportion on no hits. B: The percentage of sequences distributed among 62 different functional groups of both samples (black columns: photophase; grey columns: scotophase).
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pone.0141893.g003: Gene ontology annotation of the two transcriptomes (NEP-L and NEP-D).Gene ontology (GO) annotation of the assembled transcriptomes. A: The percentage distribution of functional categories between the two transcriptomes together with the proportion on no hits. B: The percentage of sequences distributed among 62 different functional groups of both samples (black columns: photophase; grey columns: scotophase).

Mentions: The sequencing of libraries produced a total of approximately 88 and 92 millions of paired-end reads for NEP-L and NEP-D respectively (Table 1; SRA accession SRP063649). The de novo assembly of NEP-Comb produced 108,599 contigs with a N50 (i.e. the size at which half of all assembled bases reside in contigs of this size or longer) of 1810. The de novo assembly of NEP-L produced 106,256 contigs with a N50 of 1,796, while for NEP-D the number of contigs was 114,235 with an N50 of 2,055. The species distribution of the annotated hits of transcripts of NEP-Comb against the NCBI non-redundant protein sequence database is presented in Fig 2. At about 73% of the first 30 species in order of number of annotated hits were insects, while the second species was the crustacean Daphnia pulex. The annotation of transcript sequences of NEP-L and NEP-D against the GO vocabulary produced 81,711 (77%) of no hits in NEP-L and 87,312 (76%) in NEP-D. The positive hits and following assignment of functional categories were distributed as follows: biological processes 8,522 (8%), cellular component 6,944 (6%), molecular function 9,079 (9%) for NEP-L; biological process 9,586 (8%), cellular component 7,583 (7%), molecular function 9,754 (9%) for NEP-D (Fig 3). The Fisher's exact test indicated that the 62 functional groups are equally represented among the two transcriptomes, so we reported the detailed percentage of GO annotation for both samples (Fig 3).


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)

Gene ontology annotation of the two transcriptomes (NEP-L and NEP-D).Gene ontology (GO) annotation of the assembled transcriptomes. A: The percentage distribution of functional categories between the two transcriptomes together with the proportion on no hits. B: The percentage of sequences distributed among 62 different functional groups of both samples (black columns: photophase; grey columns: scotophase).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141893.g003: Gene ontology annotation of the two transcriptomes (NEP-L and NEP-D).Gene ontology (GO) annotation of the assembled transcriptomes. A: The percentage distribution of functional categories between the two transcriptomes together with the proportion on no hits. B: The percentage of sequences distributed among 62 different functional groups of both samples (black columns: photophase; grey columns: scotophase).
Mentions: The sequencing of libraries produced a total of approximately 88 and 92 millions of paired-end reads for NEP-L and NEP-D respectively (Table 1; SRA accession SRP063649). The de novo assembly of NEP-Comb produced 108,599 contigs with a N50 (i.e. the size at which half of all assembled bases reside in contigs of this size or longer) of 1810. The de novo assembly of NEP-L produced 106,256 contigs with a N50 of 1,796, while for NEP-D the number of contigs was 114,235 with an N50 of 2,055. The species distribution of the annotated hits of transcripts of NEP-Comb against the NCBI non-redundant protein sequence database is presented in Fig 2. At about 73% of the first 30 species in order of number of annotated hits were insects, while the second species was the crustacean Daphnia pulex. The annotation of transcript sequences of NEP-L and NEP-D against the GO vocabulary produced 81,711 (77%) of no hits in NEP-L and 87,312 (76%) in NEP-D. The positive hits and following assignment of functional categories were distributed as follows: biological processes 8,522 (8%), cellular component 6,944 (6%), molecular function 9,079 (9%) for NEP-L; biological process 9,586 (8%), cellular component 7,583 (7%), molecular function 9,754 (9%) for NEP-D (Fig 3). The Fisher's exact test indicated that the 62 functional groups are equally represented among the two transcriptomes, so we reported the detailed percentage of GO annotation for both samples (Fig 3).

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