Identification and temporal expression of putative circadian clock transcripts in the amphipod crustaceanTalitrus saltator

Abstract

BackgroundTalitrus saltatoris an amphipod crustacean that inhabits the supralittoral zone on sandy beaches in the Northeast Atlantic and Mediterranean.T. saltatorexhibits endogenous locomotor activity rhythms and time-compensated sun and moon orientation, both of which necessitate at least one chronometric mechanism. Whilst their behaviour is well studied, currently there are no descriptions of the underlying molecular components of a biological clock in this animal, and very few in other crustacean species.MethodsWe harvested brain tissue from animals expressing robust circadian activity rhythms and used homology cloning and Illumina RNAseq approaches to sequence and identify the core circadian clock and clock-related genes in these samples. We assessed the temporal expression of these genes in time-course samples from rhythmic animals using RNAseq.ResultsWe identified a comprehensive suite of circadian clock gene homologues inT. saltatorincluding the ‘core’ clock genesperiod(Talper),cryptochrome 2(Talcry2),timeless(Taltim),clock(Talclk), andbmal1(Talbmal1). In addition we describe the sequence and putative structures of 23 clock-associated genes including two unusual, extended isoforms of pigment dispersing hormone (Talpdh). We examined time-course RNAseq expression data, derived from tissues harvested from behaviourally rhythmic animals, to reveal rhythmic expression of these genes with approximately circadian period inTalperandTalbmal1. Of the clock-related genes,casein kinase IIβ(TalckIIβ),ebony(Talebony),jetlag(Taljetlag),pigment dispensing hormone(Talpdh),protein phosphatase 1(Talpp1),shaggy(Talshaggy),sirt1(Talsirt1), sirt7 (Talsirt7) and supernumerary limbs (Talslimb) show temporal changes in expression.DiscussionWe report the sequences of principle genes that comprise the circadian clock ofT. saltatorand highlight the conserved structural and functional domains of their deduced cognate proteins. Our sequencing data contribute to the growing inventory of described comparative clocks. Expression profiling of the identified clock genes illuminates tantalising targets for experimental manipulation to elucidate the molecular and cellular control of clock-driven phenotypes in this crustacean.