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.
Highlights
The Norway lobster, Nephrops norvegicus, is a burrowing decapod distributed in the European Atlantic Ocean and in the Mediterranean Sea, from 10 to 800 m depth [1]
The de novo assembly of NEP-Comb produced 108,599 contigs with a N50 of 1810
The Fisher's exact test indicated that the 62 functional groups are represented among the two transcriptomes, so we reported the detailed percentage of Gene Ontology (GO) annotation for both samples (Fig 3)
Summary
The Norway lobster, Nephrops norvegicus (hereafter referred as to Nephrops), is a burrowing decapod distributed in the European Atlantic Ocean and in the Mediterranean Sea, from 10 to 800 m depth [1]. It is one of the most important resources for European fisheries with landed tons of about 70000 per year (http://www.fao.org). Stock assessment and fishery management are influenced by its rhythmic pattern of burrow emergence, promoting research on its circadian regulation both in the field and in the laboratory. Molecular mechanisms governing the rhythmic behavior are not known
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