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

Abstract

The amphipod Parhyale hawaiensis is a model organism of growing importance in the fields of evolutionary development and regeneration. A small, hardy marine crustacean that breeds year-round with a short generation time, it has simple lab culture requirements and an extensive molecular toolkit including the ability to generate targeted genetic mutant lines. Here we identify canonical core and regulatory clock genes using genomic and transcriptomic resources as a first step in establishing this species as a model in the field of chronobiology. The molecular clock of P. hawaiensis lacks orthologs of the canonical circadian genes cryptochrome 1 and timeless, in common with the mammalian system but in contrast to many arthropods including Drosophila melanogaster. Furthermore the predicted CLOCK peptide is atypical and CRY2 shows an extended 5' region of unknown function. These results appear to be shared by two other amphipod species. Introduction 1The subphylum Crustacea has a rich history of chronobiological research. One of the earliest recorded 2 examples of a persistent circadian phenomenon in animals was the colour changes seen in the prawn Hip-3 polyte varians (Gamble and Keeble, 1900), while investigations on the circadian and circatidal rhythms 4 of the fiddler crab Uca pugnax helped lay the basis for modern chronobiology (Palmer, 1991), with pub-5 lished works on the persistence of chromatophore and locomotor activity rhythms in constant conditions; 6 temperature compensation; persistance of rhythms in isolated tissue; and the phase-shifting effect of light 7 pulses, this latter observation coming a decade before the phase response curve (PRC) was incorporated 8 into circadian concepts (De Coursey, 1960). A variety of endogenously controlled rhythmic phenomena 9 have been documented in wide range of crustaceans, not only circadian rhythms but also tidal, lunar 10 and semi-lunar (Strauss and Dircksen, 2010), under the control of zeitgebers such as light, hydrostatic 11 pressure, immersion, wave motion and salinity. Where the clade has historically proved lacking, how-12 ever, is in providing an organism with a molecular toolkit comparable to that of the fruit fly Drosophila 13 melanogaster, in which transgenics, reporter genes and binary expression systems have been used to 14 characterise, locate and dissect the endogenous biological clock at a neurological level 15 2005). 16At the centre of the Drosophila clock are the bHLH-PAS proteins CLOCK (Allada et al., 1998; 17 Gekakis et al., 1998, CLK) and CYCLE (Rutila et al., 1998, CYC), which form a heterodimer that drives 18 transcription of clock-controlled genes including period (Konopka and Benzer, 1971, per) and timeless 19 (Sehgal et al., 1994, tim), whose protein products dimerise and interact with CLK:CYC to inhibit its 20 transcription activity and thus their own expression. TIM is degraded by the light-activated CRYP-21 TOCHROME (Emery et al., 1998, CRY), exposing PER to degradation in turn and ending CLK:CYC 22 repression. The transcriptio...

Analysis of the genetically tractable crustacean Parhyale hawaiensis reveals the organisation of a sensory system for low-resolution vision

“…Opsins determine the spectral sensitivity of photoreceptors. To identify which opsins are expressed in Parhyale compound eyes, we searched for opsin homologues in Parhyale embryo and adult head transcriptomes and in the Parhyale genome [37, 47, 48] (see the “Materials and methods” section). We identified two opsins, named PhOpsin1 and PhOpsin2 .…”

“…The protein sequence of Drosophila Rh1 was used as a query to search the Parhyale embryonic and head transcriptomes and the Parhyale genome by BLAST [37, 47, 48]. The best matches were confirmed to be opsins based on BLAST searches on the NCBI GenBank database and their predicted transmembrane structure (performed on PRALINE [79]).…”

In silico Identification of a Molecular Circadian System With Novel Features in the Crustacean Model Organism Parhyale hawaiensis

“…In Drosophila melanogaster a number of rhythmic phenomena have been identified including locomotor activity, eclosion, egg deposition, eye sensitivity, and oxygen consumption (Helfrich-Förster, 1996), while as a model organism P. hawaiensis has been largely exploited at a molecular level and often at early developmental stages, and behavioral observations are lacking. We have gathered encouraging preliminary locomotor activity results (Hunt, 2016), albeit compromised by issues with temperature control, hunger, age-matching and water evaporation, aeration and fouling. These results align with the observations of Poovachiranon et al (1986) in indicating a nocturnal peak in activity that persists in constant darkness, with some individuals exhibiting 12 h periodic components, a finding that may be of interest to researchers studying circa-tidal rhythms given that P. hawaiensis is found in inter-tidal zones.…”

“…Thanks are also due to Damian Kao, Aziz Aboobaker, Anastasios Pavlopoulos and all other authors of Kao et al (2016) for granting us access to the then unpublished P. hawaiensis genome. Parts of this research first appeared in the thesis of BH (Hunt, 2016). This research used the ALICE High Performance Computing Facility at the University of Leicester.…”

The Crustacean Model <i>Parhyale hawaiensis</i>

“…Expression and chromatin profiling: Genome-wide expression profiling by RNAseq has been applied in a number of projects to identify transcriptional dynamics and responses to treatments in Parhyale, including the timecourses of embryonic development (D. Sun, H. Bruce and N. Patel, unpublished) and limb development and regeneration (C. Sinigaglia, M. Paris and M. Averof, unpublished), the study of circadian rhythms (Hunt et al 2019), the maternal to zygotic transition (S. Rawas, W. Wei, M. Jara Espejo and A. Aboobaker, unpublished), innate immune responses to bacterial infection (L. Li and A. Aboobaker, unpublished), and responses to environmental toxicants (Artal et al 2020). Single-cell or single-nucleus RNAseq has been applied to embryos (L. Blondel and C. Extavour, unpublished) Besides single-cell sequencing, cell-or tissue-specific transcriptional profiling can be carried out with the thiouracil/TU tagging approach, whereby transcripts expressed in specific cells over a chosen period of time can be tagged, isolated and sequenced (Miller et al 2009, Gay et al 2014).…”