A functional characterization of a Go‑opsin and a ratio-chromatic depth gauge in Platynereis dumerilii

Light guides marine invertebrate larvae to their settlement places. A light guided behavior is phototaxis, which is mediated by opsins. Among the opsins, the Go‑opsins are ancient, but poorly characterized, because they only survived in marine invertebrates. A Go‑opsin is expressed with two rhabdomeric opsins in the adult eyes of the larva of Platynereis dumerilii. In the larva, the adult eyes mediate phototaxis. Here, I functionally characterized this Go‑opsin1, by generating a Go‑opsin1 knockout line with zinc-finger-nucleases. I designed several assays to study light guided behaviors of the larvae and to compare phototaxis of wild type and Go‑opsin1 knockout larvae. The Go‑opsin1 knockout larvae were phototactic but less phototactic to blue-cyan-green light, which is the spectral range that closely matches the in vitro spectrum of Go‑opsin1. Additionally, I found a new light guided behavior, which is as fast as phototaxis. When I stimulated the larvae with UV-light, the larvae swam down irrespective whether the light came from the top, the bottom or diffusely from all sides. This UV-response is a positive geotaxis induced by non-directional UV-light. The UV-response worked against phototaxis; the larvae swam down to certain ratios of UV and visible light. The ratios did not change when the light was dimmed. Therefore, the UV-response forms with phototaxis a ratio-chromatic depth gauge. The UV-response spectrum matched the absorption spectrum of c‑opsin1. C‑opsin1 is expressed in the ciliary photoreceptor cells, which have stacked membranes and so may be very sensitive. Therefore, the ciliary photoreceptor cells with c‑opsin1 may mediate the UV-response, while phototaxis is mediated by Go‑opsin1 and the rhabdomeric opsins. Go‑opsin1 seems to couple to a Gq‑protein in the rhabdomeric photoreceptor cells of the adult eyes. Therein, Go‑opsin1 differs from a scallop Go‑opsin, which seems to couple to a Go‑protein in ciliary photoreceptor cells. Ciliary photoreceptor cells as in Platynereis dumerilii are common among marine invertebrate larvae so that the depth gauge may be common among those larvae, too. The depth gauge may even trace back to the last common ancestor of all bilaterians. The depth gauge helps the larvae to find the right depth for settling on a global level, while positive and negative phototaxis helps the larvae to select locally a settlement site.Licht lenkt die Larven mariner Wirbellosen zu ihren Siedlungsorten. Ein lichtgelenktes Verhalten ist Phototaxis, welches durch Opsine vermittelt wird. Unter den Opsinen sind die Go‑Opsine sehr alt, aber schlecht charakterisiert, weil sie nur noch in marinen Wirbellosen existieren. Ein Go‑Opsin ist mit zwei rhabdomerischen Opsinen in den definitiven Augen der Larve von Platynereis dumerilii exprimiert. In der Larve vermitteln die definitiven Augen Phototaxis. Hier charakterisierte ich dieses Go‑Opsin1 funktionell, indem ich eine Go‑Opsin1-Knockout-Linie mit Zinkfingernukleasen erzeugte. Ich entwickelte verschiedene Versuche, um lichtgelenktes Verhalten der Larven zu untersuchen, und um Phototaxis von Wildtyp- und Go‑Opsin-Knockout-Larven zu vergleichen. Die Go‑Opsin-Knockout-Larven waren phototaktisch aber sie reagierten weniger phototaktisch auf blau-zyan-grünes Licht, welches den Spektralbereich abdeckt, der dem in vitro Absorptionsspektrum von Go‑Opsin1 entspricht. Außerdem fand ich ein neues lichtgelenktes Verhalten, das genauso schnell einsetzt wie Phototaxis. Als ich die Larven mit UV-Licht stimulierte, schwammen sie nach unten, egal ob das Licht von oben, unten oder diffus von allen Seiten kam. Diese UV-Antwort ist eine positive Geotaxis, die von UV-Licht aktiviert wird. Die UV-Antwort arbeitet gegen Phototaxis: Die Larven schwammen nach unten, wenn UV und sichtbares Licht in bestimmten Verhältnissen zueinanderstanden. Diese Verhältnisse änderten sich nicht, als das Licht gedimmt wurde. Daher bildet die UV-Antwort mit Phototaxis einen ratio-chromatischen Tiefenmesser. Das UV-Antwortspektrum stimmte mit dem Spektrum von C‑Opsin1 überein. C‑Opsin1 ist in den ziliären Photorezeptorzellen exprimiert, die Membranstapel besitzen, und somit sehr sensitiv sein könnten. Daher könnten die ziliären Photorezeptorzellen mit C‑Opsin1 die UV-Antwort vermitteln, während Phototaxis durch Go‑Opsin1 und den rhabdomerischen Opsinen vermittelt wird. Go‑Opsin1 scheint in den rhabdomerischen Photorezeptorzellen der definiten Augen an ein Gq‑Protein zu koppeln. Darin unterscheidet sich Go‑Opsin1 von einem Muschel-Go‑Opsin, welches an ein Go‑Protein in ziliären Photorezeptorzellen zu koppeln scheint. Ziliäre Photorezeptorzellen sind verbreitet unter marinen Wirbellosenlarven, so dass auch der Tiefenmesser verbreitet sein könnte, ja sogar, dass er bereits vom letzten gemeinsamen Vorfahren aller Zweiseitentiere verwendet worden sein könnte. Der Tiefenmesser hilft den Larven, die richtige Tiefe im Allgemeinen zu finden, während positive und negative Phototaxis den Larven vor Ort hilft, einen Siedlungsort zu wählen

Role of histamine as a putative inhibitory transmitter in the honeybee antennal lobe

BACKGROUND: Odors are represented by specific spatio-temporal activity patterns in the olfactory bulb of vertebrates and its insect analogue, the antennal lobe. In honeybees inhibitory circuits in the AL are involved in the processing of odors to shape afferent odor responses. GABA is known as an inhibitory transmitter in the antennal lobe, but not all interneurons are GABAergic. Therefore we sought to analyze the functional role of the inhibitory transmitter histamine for the processing of odors in the honeybee AL. RESULTS: We optically recorded the representation of odors before, during and after histamine application at the input level (estimated from a compound signal), and at the output level (by selectively measuring the projection neurons). For both, histamine led to a strong and reversible reduction of odor-evoked responses. CONCLUSION: We propose that histamine, in addition to GABA, acts as an inhibitory transmitter in the honeybee AL and is therefore likely to play a role in odor processing

Spectral Tuning of Phototaxis by a Go-Opsin in the Rhabdomeric Eyes of Platynereis

SummaryPhototaxis is characteristic of the pelagic larval stage of most bottom-dwelling marine invertebrates [1]. Larval phototaxis is mediated by simple eyes that can express various types of light-sensitive G-protein-coupled receptors known as opsins [2–8]. Since opsins diversified early during metazoan evolution in the marine environment [9], understanding underwater light detection could elucidate this diversification. Opsins have been classified into three major families, the r-opsins, the c-opsins, and the Go/RGR opsins, a family uniting Go-opsins, retinochromes, RGR opsins, and neuropsins [10, 11]. The Go-opsins form an ancient and poorly characterized group retained only in marine invertebrate genomes. Here, we characterize a Go-opsin from the marine annelid Platynereis dumerilii [3–5, 12–15]. We found Go-opsin1 coexpressed with two r-opsins in depolarizing rhabdomeric photoreceptor cells in the pigmented eyes of Platynereis larvae. We purified recombinant Go-opsin1 and found that it absorbs in the blue-cyan range of the light spectrum. To characterize the function of Go-opsin1, we generated a Go-opsin1 knockout Platynereis line by zinc-finger-nuclease-mediated genome engineering. Go-opsin1 knockout larvae were phototactic but showed reduced efficiency of phototaxis to wavelengths matching the in vitro Go-opsin1 spectrum. Our results highlight spectral tuning of phototaxis as a potential mechanism contributing to opsin diversity

A gonad-expressed opsin mediates light-induced spawning in the jellyfish Clytia

This is the final version of the article. Available from the publisher via the DOI in this record.Across the animal kingdom, environmental light cues are widely involved in regulating gamete release, but the molecular and cellular bases of the photoresponsive mechanisms are poorly understood. In hydrozoan jellyfish, spawning is triggered by dark-light or light-dark transitions acting on the gonad, and is mediated by oocyte maturation-inducing neuropeptide hormones (MIHs) released from the ectoderm. We determined in Clytia hemisphaerica that blue-cyan light triggers spawning in isolated gonads. A candidate opsin (Opsin9) was found co-expressed with MIH within specialised ectodermal cells. Opsin9 knockout jellyfish generated by CRISPR/Cas9 failed to undergo oocyte maturation and spawning, a phenotype reversible by synthetic MIH. Gamete maturation and release in Clytia is thus regulated by gonadal photosensory-neurosecretory cells that secrete MIH in response to light via Opsin9. Similar cells in ancestral eumetazoans may have allowed tissue-level photo-regulation of diverse behaviours, a feature elaborated in cnidarians in parallel with expansion of the opsin gene family.Funding was provided by the Marie Curie ITN NEPTUNE, French ANR grant OOCAMP, EMBRC-Fr infrastructure development funding and core CNRS funding to the LBDV. Microscopy was performed at the PIV imaging platform.European Commission: FP7-PEOPLE-2012-ITN 317172 (NEPTUNE); Gaspar Jekely, Evelyn HoulistonAgence Nationale de la Recherche: ANR- 13-BSV2-0008-01 ("OOCAMP"), Evelyn HoulistonCentre National de la Recherche Scientifique: Evelyn Houlisto

The Gluopsins : Opsins without the Retinal Binding Lysine

Opsins allow us to see. They are G-protein-coupled receptors and bind as ligand retinal, which is bound covalently to a lysine in the seventh transmembrane domain. This makes opsins light-sensitive. The lysine is so conserved that it is used to define a sequence as an opsin and thus phylogenetic opsin reconstructions discard any sequence without it. However, recently, opsins were found that function not only as photoreceptors but also as chemoreceptors. For chemoreception, the lysine is not needed. Therefore, we wondered: Do opsins exists that have lost this lysine during evolution? To find such opsins, we built an automatic pipeline for reconstructing a large-scale opsin phylogeny. The pipeline compiles and aligns sequences from public sources, reconstructs the phylogeny, prunes rogue sequences, and visualizes the resulting tree. Our final opsin phylogeny is the largest to date with 4956 opsins. Among them is a clade of 33 opsins that have the lysine replaced by glutamic acid. Thus, we call them gluopsins. The gluopsins are mainly dragonfly and butterfly opsins, closely related to the RGR-opsins and the retinochromes. Like those, they have a derived NPxxY motif. However, what their particular function is, remains to be seen

A Go-type opsin mediates the shadow reflex in the annelid Platynereis dumerilii

Abstract 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 Go-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 Lambdamax 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