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Many marine animals, ranging from corals to fishes, synchronise reproduction to lunar cycles. In the annelid Platynereis dumerilii, this timing is orchestrated by an endogenous monthly (circalunar) clock entrained by moonlight. Whereas daily (circadian) clocks cause extensive transcriptomic and proteomic changes, the quality and quantity of regulations by circalunar clocks have remained largely elusive. By establishing a combined transcriptomic and proteomic profiling approach, we provide first systematic insight into the molecular changes in Platynereis heads between circalunar phases, and across sexual differentiation and maturation. Whereas maturation elicits large transcriptomic and proteomic changes, the circalunar clock exhibits only minor transcriptomic, but strong proteomic regulation. Our study provides a versatile extraction technique and comprehensive resources. It corroborates that circadian and circalunar clock effects are likely distinct and identifies key molecular brain signatures for reproduction, sex and circalunar clock phase. Examples include prepro-whitnin/proctolin and ependymin-related proteins as circalunar clock targets., Competing Interests: SS, FS, DA, BM, AB, HM, Av, CG, FR, KT No competing interests declared, SB is affiliated with Lexogen GmbH. The author has no other competing interests to declare., (© 2019, Schenk et al.)

Background: The presence of photoreceptive molecules outside the eye is widespread among animals, yet their functions in the periphery are less well understood. Marine organisms, such as annelid worms, exhibit a 'shadow reflex', a defensive withdrawal behaviour triggered by a decrease in illumination. Herein, we examine the cellular and molecular underpinnings of this response, identifying a role for a photoreceptor molecule of the G o -opsin class in the shadow response of the marine bristle worm Platynereis dumerilii., Results: We found Pdu-Go-opsin1 expression in single specialised cells located in adult Platynereis head and trunk appendages, known as cirri. Using gene knock-out technology and ablation approaches, we show that the presence of Go-opsin1 and the cirri is necessary for the shadow reflex. Consistently, quantification of the shadow reflex reveals a chromatic dependence upon light of approximately 500 nm in wavelength, matching the photoexcitation characteristics of the Platynereis Go-opsin1. However, the loss of Go-opsin1 does not abolish the shadow reflex completely, suggesting the existence of a compensatory mechanism, possibly acting through a ciliary-type opsin, Pdu-c-opsin2, with a Lambda max of approximately 490 nm., Conclusions: We show that a Go-opsin is necessary for the shadow reflex in a marine annelid, describing a functional example for a peripherally expressed photoreceptor, and suggesting that, in different species, distinct opsins contribute to varying degrees to the shadow reflex.

The advent of high-throughput sequencing technology facilitates the exploration of a variety of reference species outside the few established molecular genetic model systems. Bioinformatic and gene expression analyses provide new ways for comparative analyses between species, for instance, in the field of evolution and development. Despite these advances, a critical bottleneck for the exploration of new model species remains the establishment of functional tools, such as the ability to experimentally express genes in specific cells of an organism. We recently established a first transgenic strain of the annelid Platynereis, using a Tc1/mariner-type Mos1 transposon vector. Here, we compare Mos1 with Tol2, a member of the hAT family of transposons. In Platynereis, Tol2-based constructs showed a higher frequency of nuclear genome insertion and sustained gene expression in the G0 generation. However, in contrast to Mos1-mediated transgenes, Tol2-mediated insertions failed to retain fluorescence in the G1 generation, suggesting a germ line-based silencing mechanism. Furthermore, we present three novel expression constructs that were generated by a simple fusion-PCR approach and allow either ubiquitous or cell-specific expression of a reporter gene. Our study indicates the versatility of Tol2 for transient transgenesis, and provides a template for transgenesis work in other emerging reference species.

The Cryptochrome/Photolyase Family (CPF) represents an ancient group of widely distributed UV-A/blue-light sensitive proteins sharing common structures and chromophores. During the course of evolution, different CPFs acquired distinct functions in DNA repair, light perception and circadian clock regulation. Previous phylogenetic analyses of the CPF have allowed reconstruction of the evolution and distribution of the different CPF super-classes in the tree of life. However, so far only limited information is available from the CPF orthologs in aquatic organisms that evolved in environments harboring great diversity of life forms and showing peculiar light distribution and rhythms. To gain new insights into the evolutionary and functional relationships within the CPF family, we performed a detailed study of CPF members from marine (diatoms, sea urchin and annelid) and freshwater organisms (teleost) that populate diverse habitats and exhibit different life strategies. In particular, we first extended the CPF family phylogeny by including genes from aquatic organisms representative of several branches of the tree of life. Our analysis identifies four major super-classes of CPF proteins and importantly singles out the presence of a plant-like CRY in diatoms and in metazoans. Moreover, we show a dynamic evolution of Cpf genes in eukaryotes with various events of gene duplication coupled to functional diversification and gene loss, which have shaped the complex array of Cpf genes in extant aquatic organisms. Second, we uncover clear rhythmic diurnal expression patterns and light-dependent regulation for the majority of the analyzed Cpf genes in our reference species. Our analyses reconstruct the molecular evolution of the CPF family in eukaryotes and provide a solid foundation for a systematic characterization of novel light activated proteins in aquatic environments., (Copyright © 2014. Published by Elsevier B.V.)

Life is controlled by multiple rhythms. Although the interaction of the daily (circadian) clock with environmental stimuli, such as light, is well documented, its relationship to endogenous clocks with other periods is little understood. We establish that the marine worm Platynereis dumerilii possesses endogenous circadian and circalunar (monthly) clocks and characterize their interactions. The RNAs of likely core circadian oscillator genes localize to a distinct nucleus of the worm's forebrain. The worm's forebrain also harbors a circalunar clock entrained by nocturnal light. This monthly clock regulates maturation and persists even when circadian clock oscillations are disrupted by the inhibition of casein kinase 1δ/ε. Both circadian and circalunar clocks converge on the regulation of transcript levels. Furthermore, the circalunar clock changes the period and power of circadian behavior, although the period length of the daily transcriptional oscillations remains unaltered. We conclude that a second endogenous noncircadian clock can influence circadian clock function., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Neurosecretory control centers form part of the forebrain in many animal phyla, including vertebrates, insects, and annelids. The evolutionary origin of these centers is largely unknown. To identify conserved, and thus phylogenetically ancient, components of neurosecretory brain centers, we characterize and compare neurons that express the prohormone vasotocin (vasopressin/oxytocin)-neurophysin in the developing forebrain of the annelid Platynereis dumerilii and of the zebrafish. These neurons express the same tissue-restricted microRNA, miR-7, and conserved, cell-type-specific combinations of transcription factors (nk2.1, rx, and otp) that specify their identity, as evidenced by the specific requirement of zebrafish rx3 for vasotocin-neurophysin expression. MiR-7 also labels another shared population of neurons containing RFamides. Since the vasotocinergic and RFamidergic neurons appear to be directly sensory in annelid and fish, we propose that cell types with dual sensory-neurosecretory properties were the starting point for the evolution of neurosecretory brain centers in Bilateria.

The circadian clock controls behavior and metabolism in various organisms. However, the exact timing and strength of rhythmic phenotypes can vary significantly between individuals of the same species. This is highly relevant for rhythmically complex marine environments where organismal rhythmic diversity likely permits the occupation of different microenvironments. When investigating circadian locomotor behavior of Platynereis dumerilii, a model system for marine molecular chronobiology, we found strain-specific, high variability between individual worms. The individual patterns were maintained for several weeks. A diel head transcriptome comparison of behaviorally rhythmic versus arrhythmic wild-type worms showed that 24-h cycling of core circadian clock transcripts is identical between both behavioral phenotypes. While behaviorally arrhythmic worms showed a similar total number of cycling transcripts compared to their behaviorally rhythmic counterparts, the annotation categories of their transcripts, however, differed substantially. Consistent with their locomotor phenotype, behaviorally rhythmic worms exhibit an enrichment of cycling transcripts related to neuronal/behavioral processes. In contrast, behaviorally arrhythmic worms showed significantly increased diel cycling for metabolism- and physiology-related transcripts. The prominent role of the neuropeptide pigment-dispersing factor (PDF) in Drosophila circadian behavior prompted us to test for a possible functional involvement of Platynereis pdf. Differing from its role in Drosophila, loss of pdf impacts overall activity levels but shows only indirect effects on rhythmicity. Our results show that individuals arrhythmic in a given process can show increased rhythmicity in others. Across the Platynereis population, rhythmic phenotypes exist as a continuum, with no distinct "boundaries" between rhythmicity and arrhythmicity. We suggest that such diel rhythm breadth is an important biodiversity resource enabling the species to quickly adapt to heterogeneous or changing marine environments. In times of massive sequencing, our work also emphasizes the importance of time series and functional tests. Behavioral rhythmicity is often used as a readout for organismal rhythmicity. This study reports high individual diversity in circadian behavioral patterns in a marine annelid worm, showing that behavioral rhythmicity is not correlated with the relative fraction of cycling transcripts or with oscillations in core clock genes. [ABSTRACT FROM AUTHOR]

The advent of high-throughput sequencing technology facilitates the exploration of a variety of reference species outside the few established molecular genetic model systems. Bioinformatic and gene expression analyses provide new ways for comparative analyses between species, for instance, in the field of evolution and development. Despite these advances, a critical bottleneck for the exploration of new model species remains the establishment of functional tools, such as the ability to experimentally express genes in specific cells of an organism. We recently established a first transgenic strain of the annelid Platynereis, using a Tc1/mariner-type Mos1 transposon vector. Here, we compare Mos1 with Tol2, a member of the hAT family of transposons. In Platynereis, Tol2-based constructs showed a higher frequency of nuclear genome insertion and sustained gene expression in the G0 generation. However, in contrast to Mos1-mediated transgenes, Tol2-mediated insertions failed to retain fluorescence in the G1 generation, suggesting a germ line-based silencing mechanism. Furthermore, we present three novel expression constructs that were generated by a simple fusion-PCR approach and allow either ubiquitous or cell-specific expression of a reporter gene. Our study indicates the versatility of Tol2 for transient transgenesis, and provides a template for transgenesis work in other emerging reference species. [ABSTRACT FROM AUTHOR]

Introduction: In Annelida two types of photoreceptor cells (PRCs) are regarded as generally present, rhabdomeric and ciliary PRCs. In certain taxa, however, an additional type of PRC may occur, the so called phaosomal PRC. Whereas the former two types of PRCs are always organized as an epithelium with their sensory processes projecting into an extracellular cavity formed by the PRCs and (pigmented) supportive cells, phaosomes are seemingly intracellular vacuoles housing the sensory processes. Phaosomal PRCs are the only type of PRC found in one major annelid group, Clitellata. Several hypotheses have been put forward explaining the evolutionary origin of the clitellate phaosomes. To elucidate the evolution of clitellate PRC and eyes the leech Helobdella robusta, for which a sequenced genome is available, was chosen. Results: TEM observations showed that extraocular and ocular PRCs are structurally identical. Bioinformatic analyses revealed predictions for four opsin genes, three of which could be amplified. All belong to the rhabdomeric opsin family and phylogenetic analyses showed them in a derived position within annelid opsins. Gene expression studies showed two of them expressed in the eye and in the extraocular PRCs. Polychaete eye-typic key enzymes for ommochromme and pterin shading pigments synthesis are not expressed in leech eyes. Conclusions: By comparative gene-expression studies we herein provide strong evidence that the phaosomal PRCs typical of Clitellata are derived from the rhabdomeric PRCs characteristic for polychaete adult eyes. Thus, they represent a highly derived type of PRC that evolved in the stem lineage of Clitellata rather than another, primitive type of PRC in Metazoa. Evolution of these PRCs in Clitellata is related to a loss of the primary eyes and most of their photoreceptive elements except for the rhabdomeric PRCs. Most likely this happened while changing to an endobenthic mode of life. This hypothesis of PRC evolution is in accordance with a recently published phylogeny of Annelida based on phylogenomic data. The data provide a nice example how morphologically highly divergent light sensitive structures emerged from a standard type of photoreceptor cell. [ABSTRACT FROM AUTHOR]

Annelids and arthropods share a similar segmented organization of the body whose evolutionary origin remains unclear. The Hedgehog signaling pathway, prominent in arthropod embryonic segment patterning, has not been shown to have a similar function outside arthropods. We show that the ligand Hedgehog, the receptor Patched, and the transcription factor Gli are all expressed in striped patterns before the morphological appearance of segments in the annelid Platynereis dumerilii. Treatments with small molecules antagonistic to Hedgehog signaling disrupt segment formation. Platynereis Hedgehog is not necessary to establish early segment patterns but is required to maintain them. The molecular similarity of segment patterning functions of the Hedgehog pathway in an annelid and in arthropods supports a common origin of segmentation in protostomes. [ABSTRACT FROM AUTHOR]

The advent of high-throughput sequencing technology facilitates the exploration of a variety of reference species outside the few established molecular genetic model systems. Bioinformatic and gene expression analyses provide new ways for comparative analyses between species, for instance, in the field of evolution and development. Despite these advances, a critical bottleneck for the exploration of new model species remains the establishment of functional tools, such as the ability to experimentally express genes in specific cells of an organism. We recently established a first transgenic strain of the annelid Platynereis, using a Tc1/mariner-type Mos1 transposon vector. Here, we compare Mos1 with Tol2, a member of the hAT family of transposons. In Platynereis, Tol2-based constructs showed a higher frequency of nuclear genome insertion and sustained gene expression in the G0 generation. However, in contrast to Mos1-mediated transgenes, Tol2-mediated insertions failed to retain fluorescence in the G1 generation, suggesting a germ line-based silencing mechanism. Furthermore, we present three novel expression constructs that were generated by a simple fusion-PCR approach and allow either ubiquitous or cell-specific expression of a reporter gene. Our study indicates the versatility of Tol2 for transient transgenesis, and provides a template for transgenesis work in other emerging reference species. [ABSTRACT FROM AUTHOR]