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

Abstract

BackgroundTalitrus 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.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 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.DiscussionWe 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.

In silico identification of a molecular circadian system with novel features in the crustacean model organism Parhyale hawaiensis

“…b - peptide is a construct of multiple separate de novo assembled contigs. Both Tal-CRY2 and Tal-CLK have been previously reported in a shorter form (O’Grady et al , 2016).…”

“…A small number of reads mapped to these fragments, indicating very low levels of expression in the head. Through iterative searches of the P. hawaiensis and H. azteca genomes using the CLK protein reported for the amphipod T. saltator (O’Grady et al , 2016), and informed by read mapping evidence, this was further extended to a putative final sequence encoding a 2,065 aa peptide. A putative Ha-CLK was also identified in this manner (Figure 1B).…”

“…This system is found in vertebrates, hymenopterans and now in crustaceans. The gene reported as T. saltator timeless (O’Grady et al , 2016) appears to be a timeout (Figure 2B) and no sequence encoding a CRY1 or true TIM could be found in the various transcriptomic and genomic resources available for T. saltator and H. azteca , indicating that this system may be common in amphipods. This contrasts with other crustaceans studied so far – Daphnia pulex possesses the full complement of core clock proteins including a CRY1 and CRY2 (Tilden et al , 2011), as does Calanus finmarchicus (Christie et al , 2013), Trigriopus californicus (Nesbit and Christie, 2014), Limulus polyphemus (Chesmore et al , 2016) and Euphausia superba (Hunt et al , 2017).…”

“…RNA-seq reads for H. azteca (PRJNA312414) were downloaded, aligned and assembled as above. P. hawaiensis sequences obtained from from BLAST+ searches and contigs identified as circadian genes in T. saltator (O’Grady et al , 2016) were used to search the genomes/transcriptomes of all three species, with results expanded using read mapping evidence and then these extended sequences used to search all resources again until no further extension was possible. Sequences returned by these processes were translated to peptides and subject to validation using the NCBI BLAST web interface (blastp, Database ‘nr’, Entrez Query ‘all[filter] NOT predicted[title] NOT hypothetical[title] NOT putative[title]’) to ascertain their closest match in other species.…”

“…In crustaceans, the first core clock gene to be identified was clock in the prawn Macrobrachium rosenbergii (Yang et al , 2006). The falling cost of next generation sequencing has subsequently brought a wave of such data in crustacean species including the water flea Daphnia pulex (Tilden et al , 2011), the copepods Calanus finmarchicus (Christie et al , 2013) and Tigriopus californicus (Nesbit and Christie, 2014), the lobster Nephrops norvegicus (Sbragaglia et al , 2015), the amphipod Talitrus saltator (O’Grady et al , 2016) and the Antarctic krill Euphausia superba (Hunt et al , 2017), in some cases including identification of rhythmic transcription. The clock of E. superba has been further characterised through analysis of the interactions of its protein components (Biscontin et al , 2017), as has that of the isopod Eurydice pulchra (Zhang et al , 2013), while tissue-specific identification of clock components has been performed in the American lobster Homarus americanus (Christie et al , 2018a,b).…”