RNA sequencing indicates widespread conservation of circadian clocks in marine zooplankton

Abstract

Zooplankton are important eukaryotic constituents of marine ecosystems characterized by limited motility in the water. These metazoans predominantly occupy intermediate trophic levels and energetically link primary producers to higher trophic levels. Through processes including diel vertical migration (DVM) and production of sinking pellets they also contribute to the biological carbon pump which regulates atmospheric CO₂ levels. Despite their prominent role in marine ecosystems, and perhaps, because of their staggering diversity, much remains to be discovered about zooplankton biology. In particular, the circadian clock, which is known to affect important processes such as DVM has been characterized only in a handful of zooplankton species. We present annotated de novo assembled transcriptomes from a diverse, representative cohort of 17 marine zooplankton representing six phyla and eight classes. These transcriptomes represent the first sequencing data for a number of these species. Subsequently, using translated proteomes derived from this data, we demonstrate in silico the presence of orthologs to most core circadian clock proteins from model metazoans in all sequenced species. Our findings, bolstered by sequence searches against publicly available data, indicate that the molecular machinery underpinning endogenous circadian clocks is widespread and potentially well conserved across marine zooplankton taxa.

Figure 1.

Bioinformatics workflow used to assemble de novo and annotate transcriptomes of the marine zooplankton, and subsequently identify circadian clock protein candidates from translated protein sequences. Black solid arrows indicate the direction of data flow. Text adjacent to arrows indicate an action (e.g., assembly) and the tooling used to achieve it (e.g., Trinity for assembly). Tools with ‘rscript’ in their names are custom scripts written in the R programming language. For details see Section ‘Methods’. P.E.: paired-end. Colors of the horizontal bars in figure sections (A)–(F) indicate different genes. In figure subsection (C) the multiple horizontal bars grouped together (top half of the figure subsection) are translated protein sequences of transcript isoforms originating from the same gene; in the bottom half of that subsection only the protein sequences of the selected isoforms (one per gene) are indicated. Figure subsection (F) retains this coloring scheme for the protein sequences of one input species (Species 1) as an example for the process described by it, namely clock protein identification by means of orthology to a known clock protein sequence from a reference proteome; here sequence sets representing the other species are not colored in this manner, and are instead colored uniformly with a single color.

Figure 2.

Taxonomic tree of the species investigated in this study. Species marked with an asterisk were sampled on 30.07.2020. All other species were sampled on 21.06.2018. Values indicated in parentheses are the number of RNA-seq data sets and the number of individuals sacrificed for those data sets, respectively.

Figure 3.

Quality assessment statistics for the initial (top) and final (bottom panels) de novo transcriptome assemblies. The vertical dashed lines indicate the mean completeness levels (single-copy + duplicated) in the BUSCO panels, and the mean read support across all assemblies in the Bowtie2 panels. Initial and final refer to the status of the assemblies prior to – and after – an assembly optimization process. Phylum affiliations are indicated in parentheses after the species name. Phylum abbreviations: Ar - Arthropoda, An - Annelida, Ph - Phoronida, Ch - Chordata, Ec - Echinodermata, Cn - Cnidaria.

Figure 4.

Sequence annotation statistics for the de novo assembled transcriptomes. The inset table refers to aggregated sequences from all transcriptomes. Phylum affiliations are indicated in parentheses after the species name. Phylum abbreviations: Ar - Arthropoda, An - Annelida, Ph - Phoronida, Ch - Chordata, Ec - Echinodermata, Cn - Cnidaria.

Figure 5.

Domain structure diagram of all CLK protein candidates found. The bait sequence used to identify these candidates is the topmost sequence. Annotations in first set of parentheses: cmp = complete sequence, 3p = 3’-partial sequence, 5p = 5’-partial sequence, int = internal fragment sequence. Phylum abbreviations in second set of parentheses: Ar - Arthropoda, An - Annelida, Ph - Phoronida, Ch - Chordata, Ec - Echinodermata, Cn - Cnidaria.

Figure 6.

Domain structure diagram of all CRY1 protein candidates found. The bait sequence used to identify these candidates is the topmost sequence. In parentheses: cmp = complete sequence, 3p = 3’-partial sequence, 5p = 5’-partial sequence, int = internal fragment sequence. Phylum abbreviations in second set of parentheses: Ar - Arthropoda, An - Annelida, Ph - Phoronida, Ch - Chordata, Ec - Echinodermata, Cn - Cnidaria.

Figure 7.

Domain structure diagram of all CRY2 protein candidates found. The bait sequence used to identify these candidates is the topmost sequence. In parentheses: cmp = complete sequence, 3p = 3’-partial sequence, 5p = 5’-partial sequence, int = internal fragment sequence. Phylum abbreviations in second set of parentheses: Ar - Arthropoda, An - Annelida, Ph - Phoronida, Ch - Chordata, Ec - Echinodermata, Cn - Cnidaria.