title: Identification, Characterization, and Diel Pattern of Expression of Canonical Clock Genes in Nephrops norvegicus (Crustacea: Decapoda) Eyestalk.
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.
authors.affiliation: Marine Science Institute, (ICM-CSIC), Barcelona, Spain.
authors.forename: Valerio
authors.initials: V
authors.lastname: Sbragaglia
authors.affiliation: Unit of Evolutionary Biology/Systematic Zoology, Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany.
authors.forename: Francesco
authors.initials: F
authors.lastname: Lamanna
authors.affiliation: Department of Biology, University of Washington, Seattle, WA, United States of America.
authors.forename: Audrey M
authors.initials: AM
authors.lastname: Mat
authors.affiliation: Marine Science Institute, (ICM-CSIC), Barcelona, Spain.
authors.forename: Guiomar
authors.initials: G
authors.lastname: Rotllant
authors.affiliation: Marine Science Institute, (ICM-CSIC), Barcelona, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Joly
authors.affiliation: Department of Biology and Biotechnology "Charles Darwin", University of Rome "La Sapienza", Rome, Italy.
authors.forename: Valerio
authors.initials: V
authors.lastname: Ketmaier
authors.affiliation: Department of Biology, University of Washington, Seattle, WA, United States of America.
authors.forename: Horacio O
authors.initials: HO
authors.lastname: de la Iglesia
authors.affiliation: Marine Science Institute, (ICM-CSIC), Barcelona, Spain.
authors.forename: Jacopo
authors.initials: J
authors.lastname: Aguzzi
mesh_terms: D000818:Animals; D056926:CLOCK Proteins / Q000235:genetics*; D002940:Circadian Rhythm; D033364:Decapoda / Q000033:anatomy & histology / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D008297:Male; D017423:Sequence Analysis, RNA / Q000379:methods; D012689:Sequence Homology, Nucleic Acid
chemical_list: D056926:CLOCK Proteins