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

Abstract

Background: Talitrus saltator is an amphipod crustacean that inhabits the supralittoral zone on sandy beaches in the Northeast Atlantic and Mediterranean. T. saltator exhibits 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.

Methods: We 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.

Results: We identified a comprehensive suite of circadian clock gene homologues in T. saltator including the 'core' clock genes period (Talper), cryptochrome 2 (Talcry2), timeless (Taltim), clock (Talclk), and bmal1 (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 in Talper and Talbmal1. 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.

Discussion: We report the sequences of principle genes that comprise the circadian clock of T. saltator and 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.

Keywords: Circadian; Crustacean; Rhythms; Talitrus saltator; Transcriptome.

Figure 1. Talitrus saltator exhibits free-running circadian locomotor activity rhythms.

(A) Plots of activity in five representative animals removed from the shore and held in DD over 9 days. Grey and black bars show time of subjective night and day, respectively. (B) Chi Square Periodogram analysis of activity data of five representative animals recorded in DD over nine days. The period of activity (τ) is shown inside the plot. Red line represents significance at P < 0.001.

Figure 2. Cartoon representing conserved domains of core clock gene proteins.

Putative Talitrus saltator core clock protein amino acid sequences aligned with other arthropod circadian clock protein sequences. SMART identified domains are coloured (see key for colours corresponding to domains). Tal, Talitrus saltator; Dp, Daphnia pulex; Ep, Eurydice pulchra; Drom, Drosophila melanogaster; Dplex, Danaus plexippus; Tc, Tribolium castaneum; Ae, Aedes aegypti.

Figure 3. Talitrus saltator brain tissue transcriptome BLAST2GO analysis.

Ontology level 2 data showing functional classification of the brain transcripts from T. saltator for three main gene ontology categories.

Figure 4. Temporal expression profiles of gene transcripts in the brains of free-running Talitrus saltator.

Plots show TPM values of each identified core clock and clock-associated genes over a 24 h period. Data points represent mean TPM values +/− SEM (N = 4 biological replicates, except CT18 where N = 3). Significance values are shown in red for JTK_YCLE and in blue for ANOVA. Grey and black bars indicate expected daytime and nighttime, respectively.