Marine Biology (2019) 166:21 https://doi.org/10.1007/s00227-019-3473-9

SHORT NOTE

Embryonic expression of encephalopsin supports bioluminescence perception in lanternshark photophores

Laurent Duchatelet1 · Julien M. Claes1 · Jérôme Mallefet1

Received: 6 October 2018 / Accepted: 10 January 2019 / Published online: 18 January 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract Counterilluminating animals produce a ventral light to hide their silhouette in the water column. This midwater camoufage technique requires a fne and dynamic control of the wavelength, angular distribution, and intensity of their luminescence, which needs to continuously match ambient downwelling light. Recently, extraocular opsins have been suggested to play a role in the bioluminescence control of several organisms, such as squids, comb jellies, or brittle stars, providing a way for photogenic structures to perceive their own light output. By analysing a growing embryonic series of the velvet belly lanternshark, Etmopterus spinax, we show that the development of lanternshark luminescence competence is associated with the expression of encephalopsin within epidermal cells and in the light-regulating structure of the photogenic organs. Such an intra-uterine expression of encephalopsin strongly supports this blue-sensitive extraocular opsin to allow biolumi- nescence perception in lanternshark photophores and suggests a clear physiological interaction between photoemission and photoperception.

Introduction 2004)] and/or complex physiological control mechanisms, i.e., the use of neuromodulators, vasodilation, or regulation Counterillumination is an active camoufage method by of oxygen supply (Nealson and Hastings 1979; Young and which a midwater animal produces a ventral light to match Mencher 1980; Latz 1995; Krönström et al. 2005, 2007, the wavelength, intensity, and angular distribution of the 2009). Counterillumination typically requires simultaneous background downwelling light to cloak its silhouette from perception of bioluminescence and ambient light via visual both predators and prey passing below (Clarke 1963; Warner (e.g., the eyes) or non-visual organs (e.g., pineal organs et al. 1979; Denton et al. 1985). Widespread in non-trans- in bony fishes, photoreceptive vesicles in cephalopods) lucent mesopelagic organisms such as crustaceans, mol- closely associated with light organs (photophores) (Young lusks (cephalopods), and fshes, this fascinating camoufage et al. 1979). In some cases, light perception may occur strategy requires a fne and dynamic bioluminescence tun- within the photophore/photocyte itself via an extraocular ing to be efective, since any deviation from background opsin (Tong et al. 2009; Schnitzler et al. 2012; Delroisse light may attract the attention of upward-looking predators et al. 2014). The recent discovery in ventral photophores (Harper and Case 1999; Johnsen et al. 2004; Haddock et al. of adult Etmopterus spinax specimens, of an extraocular 2010). This control is performed via sophisticated opti- opsin, encephalopsin (opsin 3), that is primarily controlled cal structures [e.g., refectors, wavelength-specifc flters, by hormones produced by the pineal gland and the retina lenses, etc. (Denton et al. 1972, 1985; Jones and Nishiguchi (Claes and Mallefet 2009), suggested that bioluminescence and auto-perception mechanisms may concomitantly occur Responsible Editor: J. Carlson. within photogenic tissues in a single species (Delroisse et al. 2018). Interestingly, E. spinax is a bioluminescent ovovi- Reviewed by Undisclosed experts. viparous species, whose developing embryos are known to * produce light ventrally, laterally, and dorsally in utero (Claes Laurent Duchatelet E. spi- [email protected] et al. 2010a, 2015; Claes and Mallefet 2008, 2014). nax emits a blue–green glow [486 nm; Claes et al. (2010a)] 1 Marine Biology Laboratory, Earth and Life Institute, from lanternshark-specifc photophores, distributed mainly Université Catholique de Louvain, Place Croix du Sud 3, within the ventral epidermis. These structures are composed 1348 Louvain‑La‑Neuve, Belgium

Vol.:(0123456789)1 3 21 Page 2 of 5 Marine Biology (2019) 166:21 of multilayer cup-shaped pigmented cells that form a dark fns were dissected from each embryo and females with a granular sheath enclosing the emitting cells, the photocytes, metal cap driller (0.6 cm diameter) following the method which are capped by one or several lens cells. A cell zone described in Claes and Mallefet (2009), fxed in phosphate called the iris-like structure (ILS) is present between the bufer saline (PBS) with 4% paraformaldehyde for 12 h, and lens cells and photocytes (Claes and Mallefet 2009; Ren- stored in PBS prior to experimentation. wart et al. 2014). As mentioned previously, shark light emis- sion is under a hormonal control. Melatonin and prolactin Encephalopsin localization trigger light emission, while alpha melanocyte stimulating hormone inhibits it (Claes and Mallefet 2009). Modulation Preserved ventral shark skin patches were immersed in of bioluminescence also involves GABA and NO in lantern- a series of PBS baths with increasing concentrations of shark (Claes et al. 2010b, 2011). Currently, the biochemical sucrose (10% for 1 h, 20% for 1 h and 30% overnight) for compound underlying the bioluminescent reaction occurring cryoprotection. Each tissue was then embedded in optimal within photocytes of lanternsharks is not known (Renwart cutting temperature compound (OCT compound, Tissue- and Mallefet 2013; Oba et al. 2016). Tek, The Netherlands) and rapidly frozen at − 80 °C. Cry- This study set out to characterize the development of bio- ostat microtome (CM3050 S, Leica, Solms, Germany) was luminescence and encephalopsin photoreception during the used to perform 10 µm sections that were laid on coated morphogenesis of photophores from lanternshark E. spinax. Superfrost slides (Thermo Scientifc) and left overnight to We have assumed a close interaction between photoemission dry. To visualize the encephalopsin expression and localiza- (i.e., bioluminescence) and photoperception (i.e., through tion within the photophore, an immunofuorescence tech- encephalopsin) in photophores based on observations from nique was applied on E. spinax embryo and female ventral adult E. spinax (Delroisse et al. 2018). We used fuorescence skin sections. Slides were rinsed 15 min with Tris bufer microscopy and immuno-histochemistry to frst demonstrate saline 1% Tween [TTBS: Trizma base (Sigma) 20 mM, NaCl that light perception is present when light emission appeared 150 mM, pH 7.5 + 1% Tween 20 (Sigma)], then blocked during the embryogenesis of E. spinax. with TTBS containing 10% bovine albumin serum (BSA, Amresco). Following Delroisse et al. (2018), slides were incubated overnight with the anti-encephalopsin primary Materials and methods antibody (anti-encephalopsin Pab in Homo sapiens, Gene- tex, GTX 70609, lot number 821 400 929) at a dilution of Specimen collection 1/500 in TTBS 5% BSA. Slides were bathed again in TTBS during 30 min before to be incubated in the dark with the Gravid females of E. spinax were collected during feld ses- secondary antibody Alexa Fluor­ ® 594 Goat Anti-Rabbit sion in January 2017 by longlines lowered at 200 m depth in IgG (Goat Anti-Rabbit, Alexa ­Fluor® 594, Life Technolo- the Rauneford, Norway (60°15′54″N; 05°07′46″E). Living gies Limited) at a dilution of 1/200 in TTBS 5% BSA and specimens were brought to Espeland marine station (Espe- rinsed 15 min in TTBS. Finally, slides were subjected to grend, Norway), kept in a tank flled with 6 °C sea water 15 min DAPI (DAPI nucleic acid stain, Invitrogen) nucleus and placed in a dark cold room. Sharks were weighed and staining, rinsed 10 min in TTBS, and mounted with Mowiol sized before to be euthanized following techniques outlines (Mowiol­ ® 4-88, Sigma). Sections were observed under Poly- in Claes and Mallefet (2009). Female uteri were excised var SC epifuorescence microscope (Leica Reichter Jung) to access the embryos. Embryos from each uterus were equipped with a Nikon DS-UI digital camera coupled with counted and measured to the nearest millimeter before being NIS elements FW software. Control sections (i.e., omis- euthanized by a quick incision in the spinal cord. Embryo sion of the primary antibody as well as immunodetection in measurements were used to assign life stages. A total of ten dorsal skin and retina; immunoblot) were made following embryos (50–127 mm TL) from four diferent litters [two Delroisse et al. (2018). Immunodetection experiments were embryos from litter 1; two embryos from litter 2 (both were performed on each embryo and applied on at least ten sec- unpigmented embryos and lacking photogenic organs); three tions for each embryo. embryos from litter 3; and three embryos from litter 4 (both were pigmented embryos with photogenic organs)] form- ing a growing embryonic series as well as six adult females Results (stage V) were analysed for this study (Fig. 1). The yolk sac of each embryo was removed before daylight as well Figure 1 summarizes the diferent embryonic developmen- as bioluminescence photographs captured thanks to a Sony tal stages (stages I–IV) leading to the adult (stage V) in E. alpha 7S II camera (Sony Corporation, Japan) (Fig. 1a, b). spinax (Fig. 1a). Spontaneous luminescence (ventral, lateral, Ventral skin patches (1 cm2) between the pectoral and pelvic and dorsal) was observed only in stage IV embryos and in

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Fig. 1 Joint appearance of encephalopsin and photophores during presence and the ontogeny of photophore structures, when present. d Etmopterus spinax photophore embryogenesis within the ventral Histological section of the ventral epidermis under UV stimulation skin epidermis. a Developmental series of E. spinax in natural light highlighting the presence of photophore structures, when present. The (stages I–IV come from four distinct litters collectively constituting autofuorescence of the photocyte vesicles (green fuorescence, only an embryonic series, while stage V is an adult female specimen). present in mature photocytes) and encephalopsin immunodetection Arrowheads depict the ventral location sampled for histological and (red labelling) is shown. Dapi blue staining associated with to the cell immunodetection experiments. b Images taken in the dark high- nucleus. c connective tissue, cp pigmented cells, e epidermis, i iris- lighting spontaneous luminescence, when present. Contour dot line like structure cells, l lens cells, p photocytes, pp protophotocytes, s depicted the non-luminous embryo from stage I to III. c Histologi- pigmented cell layer. Scale bars represent 5 cm in a and b; 50 µm in cal section of the ventral epidermis under bright light highlighting the c and d stage V adult specimens (Fig. 1b). Ventral skin morphogen- ventral epidermis of E. spinax adult specimens (Fig. 1d). esis (Fig. 1c) shows pigmented cells dispersed within the Immunodetections were not observed within photocytes, epidermis layer (stages I, II), but lacks photogenic struc- while weaker labelings were detected at the level of the tures or encephalopsin immunoreactivity (Fig. 1d). Stage III epidermis surrounding the photophore for these two former embryos displayed protophotophores, with protophotocytes stages (Fig. 1d). Moreover, observed immunolabelings were lacking fuorescent vesicles and hence without luminescent consistent through the repetition of the experimentation for capabilities (Fig. 1c, d). Protophotocytes were embedded in each stage. All controls (data not shown) produced results an integrated layer of pigmented cells that was capped by an consistent with those reported in Delroisse et al. (2018). iris-like pigmented structure (ILS) and a lens cell (Fig. 1c, d). This, in turn, was surrounded by epidermis cells that labelled positively for encephalopsin (Fig. 1d). Stage IV Discussion embryos showed functional photophores (i.e., photophores containing photocytes with autofuorescent vesicles). A The photophore development steps observed in this study strong encephalopsin labelling was observed within ILS and are similar than those described in Claes and Mallefet photophore-topping epidermis cell membranes from stage (2008), which supports the idea that lanternshark photo- IV embryos (Fig. 1d). Stage V encephalopsin labelling was phore development follows a stereotypical trajectory during similar to those observed in Delroisse et al. (2018) in the embryogenesis. The development of luminous competence

1 3 21 Page 4 of 5 Marine Biology (2019) 166:21 during embryogenesis is associated with the appearance improve the present manuscript. LD is PhD students under a FRIA of encephalopsin expression in the cell membranes of the fellowship, JC is scientifc collaborator of the Université Catholique de Louvain Marine Biology Laboratory and JM is Research Associate to light organ ILS. This structure is known to act as a dia- FRS–FNRS. This paper is a contribution to the Biodiversity Research phragm that regulates the amount of light emitted to the Center (BDIV) and the Center Interuniversitaire de Biologie Marine outside (Claes and Mallefet 2010; Claes et al. 2011). This (CIBIM). and the embryonic luminescence, that can only be detected Author contributions by encephalopsin, within the female’s uteri strongly suggests LD performed, analysed, and interpreted the immunodetection and was a major contributor in writing and revis- a close association between the development of photorecep- ing the manuscript. JC revised the manuscript. JM took live pictures, tion and bioluminescence in lanternshark photophores. The supervised the work, contributed to, and revised the manuscript. Both extraocular perception of bioluminescence could represent a authors read and approved the fnal manuscript. feedback control mechanism analogous to the one suggested Funding for squid Euprymna scolopes (Tong et al. 2009), which has This work was supported by a grant from the Fonds de la Recherche Scientifique (FRIA/FRS–FNRS, Belgium) to LD and to be present before the photophore starts to produce light or, an FRS–FNRS Grant (FRFC 2.4516.01) awarded to the Université at least, before embryos hatch and start to rely on counteril- Catholique de Louvain Marine Biology Laboratory and the Université lumination for predator evasion (Claes and Mallefet 2008). de Mons Biology of Marine Organisms and Biomimetics Laboratory. In addition, a recent research highlighted the blue light sen- sitivity and the regulatory role of melanocyte for the human Compliance with ethical standards homologous encephalopsin (Regazzetti et al. 2018), add- Conflict of interest ing further evidence of a potential link between extraocular All authors declare that they have no confict of interest. opsin and the blue–green light emitted, at 486 nm (Claes et al. 2010a, b), by this lanternshark. The link between pho- Ethical statement All applicable international, national, and/or insti- toreception and regulation of light by pigmentation might be tutional guidelines for the care and use of animals were followed. All mediated by encephalopsin (Delroisse et al. 2018) regulating procedures performed in studies involving animals were in accord- ance with the ethical standards of the institution or practice at which melanocytes dispersion in ILS (Renwart et al. 2014, 2015). the studies were conducted. The shark collection and experiments The pattern of encephalopsin expression observed in were performed under the “Experimental fsh care PERMIT” Number this study is very similar to the immunoreactivity of GABA 12/14048. Following the local instructions for experimental fsh care within the shark photogenic tissue (Claes et al. 2011). GABA (Permit 12/14048), captive animals were euthanized by a blow to the head followed by a full incision of the spinal cord at the back of the is known to play an inhibitory role on the light emission of head. Animal procedures were conducted in compliance with the Bel- E. spinax (Claes et al. 2011) and hence could act jointly with gian national guidelines and in agreement with the European directive diferent transduction pathways on the ILS cells to regu- 2010/63/UE, under the approval of the Animal Ethics Committee of late the light output (Bertolesi et al. 2015). All together, the Catholic University of Louvain in Louvain-la-Neuve. This article does not contain any studies with human participants performed by hormones and neuromodulators controlling the light emis- any of the authors. sion [melatonin, alpha melanocyte stimulating hormone and GABA (Claes and Mallefet 2009; Claes et al. 2010b, 2011), Data accessibility All data supporting the paper are presented in the as well as extraocular opsin (i.e., encephalopsin)] could act main manuscript. in concert to regulate the ILS cells’ pigmentation and refne the bioluminescence output. Simultaneous emission and perception of light by a single organ have only been sug- References gested for Hawaiian bobtail squid Euprymna scolopes, warty comb jellies Mnemiopsis leidyi, and brittle stars Amphiura Bertolesi GE, Vazhappilly ST, Herh CL, McFarlane S (2015) Phar- fliformis (Tong et al. 2009; Schnitzler et al. 2012; Delroisse macological induction of skin pigmentation unveils the neuroen- et al. 2014). The presence of a similar dual capability in docrine circuit regulated by light. Pigment Cell Melanoma Res. the photophores of a vertebrate supports the idea that light 29(2):186–198. https​://doi.org/10.1111/pcmr.12442​ Claes JM, Mallefet J (2008) Early development of bioluminescence perception might be common in counterilluminating photo- suggests camoufage by counter-illumination in the velvet belly phores. Further research is required to better understand the lantern shark Etmopterus spinax (Squaloidea: Etmopteridae). role of this extraocular opsin and confrm its implication in J Fish Biol 73(6):1337–1350. https​://doi.org/10.1111/j.10958​ the control mechanism of lanternshark photophores. 649.2008.02006​.x Claes JM, Mallefet J (2009) Hormonal control of luminescence from lantern shark (Etmopterus spinax) photophores. J Exp Biol Acknowledgements The authors would like to thank T. Sorlie from the 212(22):3684–3692. https​://doi.org/10.1242/jeb.03436​3 Espegrend Marine Biological Station (University of Bergen, Norway) Claes JM, Mallefet J (2010) The lantern shark’s light switch: turn- E. spinax for the help during collection. We also acknowledge Olivia ing shallow water crypsis into midwater camoufage. Biol Lett Vinolo for the help provided during immunodetection experiments. 6(5):685–687. https​://doi.org/10.1098/rsbl.2010.0167 The authors want to acknowledge Dr. G. Naylor for helpful reading Claes JM, Mallefet J (2014) Ecological functions of shark lumines- and corrections as well as the anonymous reviewers whose comments cence. Luminescence 29(1):13–15

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