Jérôme Delroisse

Opsin evolution in the Ambulacraria.

Opsins--G-protein coupled receptors involved in photoreception--have been extensively studied in the animal kingdom. The present work provides new insights into opsin-based photoreception and photoreceptor cell evolution with a first analysis of opsin sequence data for a major deuterostome clade, the Ambulacraria. Systematic data analysis, including for the first time hemichordate opsin sequences and an expanded echinoderm dataset, led to a robust opsin phylogeny for this cornerstone superphylum. Multiple genomic and transcriptomic resources were surveyed to cover each class of Hemichordata and Echinodermata. In total, 119 ambulacrarian opsin sequences were found, 22 new sequences in hemichordates and 97 in echinoderms (including 67 new sequences). We framed the ambulacrarian opsin repertoire within eumetazoan diversity by including selected reference opsins from non-ambulacrarians. Our findings corroborate the presence of all major ancestral bilaterian opsin groups in Ambulacraria. Furthermore, we identified two opsin groups specific to echinoderms. In conclusion, a molecular phylogenetic framework for investigating light-perception and photobiological behaviors in marine deuterostomes has been obtained.

High opsin diversity in a non-visual infaunal brittle star

BackgroundIn metazoans, opsins are photosensitive proteins involved in both vision and non-visual photoreception. Echinoderms have no well-defined eyes but several opsin genes were found in the purple sea urchin (Strongylocentrotus purpuratus) genome. Molecular data are lacking for other echinoderm classes although many species are known to be light sensitive.ResultsIn this study focused on the European brittle star Amphiura filiformis, we first highlighted a blue-green light sensitivity using a behavioural approach. We then identified 13 new putative opsin genes against eight bona fide opsin genes in the genome of S. purpuratus. Six opsins were included in the rhabdomeric opsin group (r-opsins). In addition, one putative ciliary opsin (c-opsin), showing high similarity with the c-opsin of S. purpuratus (Sp-opsin 1), one Go opsin similar to Sp-opsins 3.1 and 3.2, two basal-branch opsins similar to Sp-opsins 2 and 5, and two neuropsins similar to Sp-opsin 8, were identified. Finally, two sequences from one putative RGR opsin similar to Sp-opsin 7 were also detected. Adult arm transcriptome analysis pinpointed opsin mRNAs corresponding to one r-opsin, one neuropsin and the homologue of Sp-opsin 2. Opsin phylogeny was determined by maximum likelihood and Bayesian analyses. Using antibodies designed against c- and r-opsins from S. purpuratus, we detected putative photoreceptor cells mainly in spines and tube feet of A. filiformis, respectively. The r-opsin expression pattern is similar to the one reported in S. purpuratus with cells labelled at the tip and at the base of the tube feet. In addition, r-opsin positive cells were also identified in the radial nerve of the arm. C-opsins positive cells, expressed in pedicellariae, spines, tube feet and epidermis in S. purpuratus were observed at the level of the spine stroma in the brittle star.ConclusionLight perception in A. filiformis seems to be mediated by opsins (c- and r-) in, at least, spines, tube feet and in the radial nerve cord. Other non-visual opsin types could participate to the light perception process indicating a complex expression pattern of opsins in this infaunal brittle star.

Discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms

Neuropeptides are a diverse class of intercellular signalling molecules that mediate neuronal regulation of many physiological and behavioural processes. Recent advances in genome/transcriptome sequencing are enabling identification of neuropeptide precursor proteins in species from a growing variety of animal taxa, providing new insights into the evolution of neuropeptide signalling. Here, detailed analysis of transcriptome sequence data from three brittle star species, Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea, has enabled the first comprehensive identification of neuropeptide precursors in the class Ophiuroidea of the phylum Echinodermata. Representatives of over 30 bilaterian neuropeptide precursor families were identified, some of which occur as paralogues. Furthermore, homologues of endothelin/CCHamide, eclosion hormone, neuropeptide-F/Y and nucleobinin/nesfatin were discovered here in a deuterostome/echinoderm for the first time. The majority of ophiuroid neuropeptide precursors contain a single copy of a neuropeptide, but several precursors comprise multiple copies of identical or non-identical, but structurally related, neuropeptides. Here, we performed an unprecedented investigation of the evolution of neuropeptide copy number over a period of approximately 270 Myr by analysing sequence data from over 50 ophiuroid species, with reference to a robust phylogeny. Our analysis indicates that the composition of neuropeptide ‘cocktails’ is functionally important, but with plasticity over long evolutionary time scales.

De novo adult transcriptomes of two European brittle stars: spotlight on opsin-based photoreception

Next generation sequencing (NGS) technology allows to obtain a deeper and more complete view of transcriptomes. For non-model or emerging model marine organisms, NGS technologies offer a great opportunity for rapid access to genetic information. In this study, paired-end Illumina HiSeqTM technology has been employed to analyse transcriptomes from the arm tissues of two European brittle star species, Amphiura filiformis and Ophiopsila aranea. About 48 million Illumina reads were generated and 136,387 total unigenes were predicted from A. filiformis arm tissues. For O. aranea arm tissues, about 47 million reads were generated and 123,324 total unigenes were obtained. Twenty-four percent of the total unigenes from A. filiformis show significant matches with sequences present in reference online databases, whereas, for O. aranea, this percentage amounts to 23%. In both species, around 50% of the predicted annotated unigenes were significantly similar to transcripts from the purple sea urchin, the closest species to date that has undergone complete genome sequencing and annotation. GO, COG and KEGG analyses were performed on predicted brittle star unigenes. We focused our analyses on the phototransduction actors involved in light perception. Firstly, two new echinoderm opsins were identified in O. aranea: one rhabdomeric opsin (homologous to vertebrate melanopsin) and one RGR opsin. The RGR-opsin is supposed to be involved in retinal regeneration while the r-opsin is suspected to play a role in visual-like behaviour. Secondly, potential phototransduction actors were identified in both transcriptomes using the fly (rhabdomeric) and mammal (ciliary) classical phototransduction pathways as references. Finally, the sensitivity of O.aranea to monochromatic light was investigated to complement data available for A. filiformis. The presence of microlens-like structures at the surface of dorsal arm plate of O. aranea could potentially explain phototactic behaviour differences between the two species. The results confirm (i) the ability of these brittle stars to perceive light using opsin-based photoreception, (ii) suggest the co-occurrence of both rhabdomeric and ciliary photoreceptors, and (iii) emphasise the complexity of light perception in this echinoderm class.

De novo transcriptome of the European brittle star Amphiura filiformis pluteus larvae

BACKGROUND In non-classical model species, Next Generation Sequencing increases the ability to analyze the expression of transcripts/genes. In this study, paired-end Illumina HiSeq sequencing technology has been employed to describe a larval transcriptome generated from 64 h post-fertilization pluteus larvae of the brittle star Amphiura filiformis. We focused our analysis on the detection of actors involved in the opsin based light perception, respectively the opsins and the phototransduction actors. METHODS & RESULTS In this research, about 47 million high quality reads were generated and 86,572 total unigenes were predicted after de novo assembly. Of all the larval unigenes, 18% show significant matches with reference online databases. 46% of annotated larval unigenes were significantly similar to transcripts from the purple sea urchin. COG, GO and KEGG analyses were performed on predicted unigenes. Regarding the opsin-based photoreception process, even if possible actors of ciliary and rhabdomeric phototransduction cascades were detected, no ciliary or rhabdomeric opsin was identified in these larvae. Additionally, partial non-visual RGR (retinal G protein coupled receptor) opsin mRNAs were identified,possibly indicating the presence of visual cycle reaction in early pluteus larvae. The eye morphogene Pax 6 was also identified in the pluteus transcriptome. CONCLUSIONS Contrary to sea-urchin larvae, brittle star larvae appear to be characterized by an absence of visual-like opsins. These RNA-seq data also provide a useful resource for the echinoderm research community and researchers with an interest in larval biology.

Cytological changes during luminescence production in lanternshark (Etmopterus spinax Linnaeus, 1758) photophores

Studying an organism’s photogenic structures at the ultrastructural level is a key step in the understanding of its light-emission process. Recently, the photophore ultrastructure of the deep-sea lanternshark Etmopterus spinax Linnaeus, 1758 was described. The photocytes appeared to be divided into three areas including an apical granular area, which contains inclusions and was hypothesized to be the light-producing reaction site. In this study, we investigated the morphological changes occurring within the granular area during the bioluminescent emissions induced by two hormones: prolactin and melatonin. Prolactin provoked the formation of new structures in the granular area, the “grey particles”, whose number was proportional to the amount of light produced by the reaction. An increase in the number of granular inclusions was also detected at the end of the prolactin-induced light emission. Conversely, melatonin induced a decrease in the number of granular inclusions and an increase in their diameter. An effect of hormones was also observed on the iris-like structure where they triggered pigment retraction and hence an increase in the iris aperture diameter. This is consistent with previous findings and is shown for the first time at the cellular level. The possible role of grey particles in E. spinax light-emission mechanism is discussed, while granular inclusion is considered to be E. spinax’s intracellular luminescence site. Regarding typical shark long-lasting glows, a new term (“glowon”) is proposed to characterize this novel membrane-free microsource.

Ultrastructural organization of lantern shark ( Etmopterus spinax Linnaeus, 1758) photophores

Etmopterus spinax Linnaeus, 1758 is a deep-sea lantern shark that emits blue light thanks to thousands of tiny cup-shaped organs made of a pigmented sheath enclosing light-emitting cells topped by an iris-like structure and a lens. In this study, we investigate the ultrastructure of these photophores in order to improve our understanding of the light emission process. The presence of a novel layer, a putative reflector upholstering the pigmented sheath, is highlighted. The intracellular organization of the photocytes is addressed. They appear as regionalized cells: their basal area is occupied by an ovoid nucleus, their medial area is highly vesiculated and their apical area, oriented toward the photophore center, displays small granular inclusions. We hypothesize this granular area to be the intracellular site of photogenesis in E. spinax, as it is also the most fluorescent part of the photocyte.

A puzzling homology: A brittle star using a putative cnidarian-type luciferase for bioluminescence

Bioluminescence relies on the oxidation of a luciferin substrate catalysed by a luciferase enzyme. Luciferins and luciferases are generic terms used to describe a large variety of substrates and enzymes. Whereas luciferins can be shared by phylogenetically distant organisms which feed on organisms producing them, luciferases have been thought to be lineage-specific enzymes. Numerous light emission systems would then have co-emerged independently along the tree of life resulting in a plethora of non-homologous luciferases. Here, we identify for the first time a candidate luciferase of a luminous echinoderm, the ophiuroid Amphiura filiformis. Phylogenomic analyses identified the brittle star predicted luciferase as homologous to the luciferase of the sea pansy Renilla (Cnidaria), contradicting with the traditional viewpoint according to which luciferases would generally be of convergent origins. The similarity between the Renilla and Amphiura luciferases allowed us to detect the latter using anti-Renilla luciferase antibodies. Luciferase expression was specifically localized in the spines which were demonstrated to be the bioluminescent organs in vivo. However, enzymes homologous to the Renilla luciferase but unable to trigger light emission were also identified in non-luminous echinoderms and metazoans. Our findings strongly indicate that those enzymes, belonging to the haloalkane dehalogenase family, might then have been convergently co-opted into luciferases in cnidarians and echinoderms. In these two benthic suspension-feeding species, similar ecological pressures would constitute strong selective forces for the functional shift of these enzymes and the emergence of bioluminescence.

Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome

Neuropeptides are diverse and evolutionarily ancient regulators of physiological/behavioural processes in animals. Here we have investigated the evolution and comparative physiology of luqin-type neuropeptide signalling, which has been characterised previously in protostomian invertebrates. Phylogenetic analysis indicates that luqin-type receptors and tachykinin-type receptors are paralogous and probably originated in a common ancestor of the Bilateria. In the deuterostomian lineage, luqin-type signalling has been lost in chordates but interestingly it has been retained in ambulacrarians. Therefore, here we characterised luqin-type signalling for the first time in an ambulacrarian – the starfish Asterias rubens (phylum Echinodermata). A luqin-like neuropeptide with a C-terminal RWamide motif (ArLQ; EEKTRFPKFMRW-NH2) was identified as the ligand for two luqin-type receptors in A. rubens, ArLQR1 and ArLQR2. Furthermore, analysis of the expression of the ArLQ precursor using mRNA in situ hybridisation revealed expression in the nervous system, digestive system and locomotory organs (tube feet) and in vitro pharmacology revealed that ArLQ causes dose-dependent relaxation of tube feet. Accordingly, previous studies have revealed that luqin-type signalling regulates feeding and locomotor activity in protostomes. In conclusion, our phylogenetic analysis combined with characterisation of luqin-type signalling in a deuterostome has provided new insights into neuropeptide evolution and function in the animal kingdom.

De novo Transcriptome Analyses Provide Insights into Opsin-based Photoreception in the Lantern shark Etmopterus spinax

The velvet belly lantern shark (Etmopterus spinax) is a small deep-sea shark commonly found in the Easter Atlantic and the Mediterranean Sea. In this study, paired-end illumina HiSeqTM technology has been employed to analyse transcriptome data from eye and ventral skin tissues of the lantershark species. About 64 and 49 million Illumina reads were generated from skin and eyetissues respectively. The assembly allowed us to predict 119,749 total unigenes including 94,569 for the skin transcriptome and 94,365 for the eye transcriptome while 74,753 were commonly found in both transcriptomes. Among unigenes, 60,322 sequences were annotated using classical public databases. The assembled and annotated transcriptomes provide a valuable resource for further understanding of the shark biology. We identified potential “light-interacting toolkit” genes including multiple genes related to ocular and extraocular light perception processes such as opsins. In particular, a single rhodopsin gene mRNA and its potentially associated peropsin were only detected in the eye transcriptome confirming a monochromatic vision of the lantern-shark. Conversely, an encephalopsin mRNA was mainly detected in the skin transcriptome. The encephalopsin was immunolocalized in various shark tissues confirming its wide expression in the shark skin and pinpointing a possible functional relation with the photophore, i.e. epidermal light organs. We hypothesize that extraocular photoreception might be involved in the bioluminescence control possibly acting on the shutter opening and/or the photocyte activity itself.