Sequences of Circadian Clock Proteins in the Nudibranch Molluscs Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea

Abstract

While much is known about the genes and proteins that make up the circadian clocks in vertebrates and several arthropod species, much less is known about the clock genes in many other invertebrates, including nudibranchs. The goal of this project was to identify the RNA and protein products of putative clock genes in the central nervous system of three nudibranchs, Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea. Using previously published transcriptomes (Hermissenda and Tritonia) and a new transcriptome (Melibe), we identified nudibranch orthologs for the products of five canonical clock genes: brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1, circadian locomotor output cycles kaput, non-photoreceptive cryptochrome, period, and timeless. Additionally, orthologous sequences for the products of five related genes—aryl hydrocarbon receptor nuclear translocator like, photoreceptive cryptochrome, cryptochrome DASH, 6-4 photolyase, and timeout—were determined. Phylogenetic analyses confirmed that the nudibranch proteins were most closely related to known orthologs in related invertebrates, such as oysters and annelids. In general, the nudibranch clock proteins shared greater sequence similarity with Mus musculus orthologs than Drosophila melanogaster orthologs, which is consistent with the closer phylogenetic relationships recovered between lophotrochozoan and vertebrate orthologs. The suite of clock-related genes in nudibranchs includes both photoreceptive and non-photoreceptive cryptochromes, as well as timeout and possibly timeless. Therefore, the nudibranch clock may resemble the one exhibited in mammals, or possibly even in non-drosopholid insects and oysters. The latter would be evidence supporting this as the ancestral clock for bilaterians.

Localization and expression of putative circadian clock transcripts in the brain of the nudibranch Melibe leonina

“…In the Mus genus bmal , a homolog of cycle , binds to clock and regulates the transcription of period and npcry (Shearman et al, 2000). Although not included in this study, Cook et al , (in press) have demonstrated the presence of bmal in the M. leonina transcriptome. Therefore, the molecular mechanisms involved in the M. leonina circadian clock(s) contains the same genes present in the mammalian system and the connection/pathway of these genes is also similar.…”

“…Sequence-specific RNA probes were developed by Biosearch Technologies for clock , period , and photoreceptive and non-photoreceptive cryptochrome (pcry and npcry , respectively), based on sequences derived from RNA transcriptomes (Cook et al , in press). In brief, 48 non-overlapping, 20-nucleotide RNA probes were designed for each transcript, with each probe being conjugated to a fluorophore.…”

“…M. leonina moves more at night (Newcomb et al , 2004) and this pattern of activity is controlled by an endogenous circadian clock (Newcomb et al , 2014). Furthermore, transcripts of clock genes in M. leonina have been recently sequenced (Cook et al , in press), making it possible to carry out experiments that were previously not possible. The goal of this project was to use these transcript sequences to accomplish two objectives: 1) determine the location of putative clock neurons that express these clock transcripts using RNA probes with fluorescent in situ hybridization (FISH); and 2) investigate the temporal expression profile of some of these clock genes.…”

cAMP, Ca2+, pHi, and NO Regulate H-like Cation Channels That Underlie Feeding and Locomotion in the Predatory Sea Slug Pleurobranchaea californica

“…Tamvacakis et al identified genes related to learning and memory in the CNS transcriptome of Hermissenda crassicornis . 40 Cook et al 41 fingered sequences of circadian clock proteins Hermissenda crassicornis , Melibe leonina , and Tritonia diomedea . Such results enable identification of genes encoding cyclic nucleotide-gated channels, proton channels, NOS, components of the cyclic AMP and cGMP cascades, a diversity of putative 5HT receptors and many neuropeptides.…”

Differential Impacts of the Head on Platynereis dumerilii Peripheral Circadian Rhythms

“…Molecular data on rhythms and clocks in marine invertebrates have become increasingly available over the last decade, now including the bivalves Mytilus californianus (Connor and Gracey, 2011) and Crassostrea gigas (Perrigault and Tran, 2017), the sea slugs Hermissenda crassicornis , Melibe leonina , and Tritonia diomedea (Cook et al, 2018; Duback et al, 2018), the isopod Eurydice pulchra (Wilcockson et al, 2011; Zhang et al, 2013; O’Neill et al, 2015), the amphipod Talitrus saltator (Hoelters et al, 2016), the lobsters Nephrops norvegicus (Sbragaglia et al, 2015) and Homarus americanus (Christie et al, 2018), the mangrove cricket Apteronemobius asahinai (Takekata et al, 2012), the copepods Calanus finmarchicus (Häfker et al, 2017), and Tigriopus californicus (Nesbit and Christie, 2014), the Antarctic krill Euphausia superba (Mazzotta et al, 2010; Teschke et al, 2011; Pittà et al, 2013; Biscontin et al, 2017), the Northern krill Meganyctiphanes norvegica (Christie et al, 2018), the marine midge C. marinus (Kaiser and Heckel, 2012; Kaiser et al, 2016), and the marine polychaete P. dumerilii (Zantke et al, 2013; Schenk et al, 2019). On the marine vertebrate side, especially teleost fish species have been investigated (Park et al, 2007; Sánchez et al, 2010; Hur et al, 2011; Watanabe et al, 2012; Vera et al, 2013; Rhee et al, 2014; Toda et al, 2014; Mogi et al, 2015; Okano et al, 2017).…”