Bioluminescence induction in the ophiuroid Amphiura filiformis (Echinodermata).

Bioluminescence is a widespread phenomenon in the marine environment. Among luminous substrates, coelenterazine is the most widespread luciferin, found in eight phyla. The wide phylogenetic coverage of this light-emitting molecule has led to the hypothesis of its dietary acquisition, which has so far been demonstrated in one cnidarian and one lophogastrid shrimp species. Within Ophiuroidea, the dominant class of luminous echinoderms, Amphiura filiformis is a model species known to use coelenterazine as substrate of a luciferin/luciferase luminous system. The aim of this study was to perform long-term monitoring of A. filiformis luminescent capabilities during captivity. Our results show (i) depletion of luminescent capabilities within 5 months when the ophiuroid was fed a coelenterazine-free diet and (ii) a quick recovery of luminescent capabilities when the ophiuroid was fed coelenterazine-supplemented food. The present work demonstrates for the first time a trophic acquisition of coelenterazine in A. filiformis to maintain light emission capabilities.

Bioluminescence in lanternsharks: Insight from hormone receptor localization.

As part of the study of their bioluminescence, the deep-sea lanternshark Etmopterus spinax and Etmopterus molleri (Chondrichthyes, Etmopteridae) received growing interest over the past ten years. These mesopelagic sharks produce light thanks to a finely tuned hormonal control involving melatonin, adrenocorticotropic hormone and α-melanocyte-stimulating hormone. Receptors of these hormones, respectively the melatonin receptors and the melanocortin receptors, are all members of the G-protein coupled receptor family i.e. coupled with specific G proteins involved in the preliminary steps of their transduction pathways. The present study highlights the specific localization of the hormonal receptors, as well as of their associated G-proteins within the light organs, the so-called photophores, in E. spinax and E. molleri through immunohistofluorescence technic. Our results allow gaining insight into the molecular actors and mechanisms involved in the control of the light emission in Etmopterid sharks.

In the intimacy of the darkness: Genetic polyandry in deep-sea luminescent lanternsharks Etmopterus spinax and Etmopterus molleri (Squaliformes, Etmopteridae).

Multiple paternity seems common within elasmobranchs. Focusing on two deep-sea shark species, the velvet belly lanternshark (Etmopterus spinax) and the slendertail lanternshark (Etmopterus molleri) we inferred the paternity in 31 E. spinax litters from Norway (three to 18 embryos per litter) and six E. molleri litters from Japan (three to six embryos), using 21 and 10 specific microsatellites, respectively. At least two E. spinax litters were sired from multiple fathers each, with highly variable paternal skew (1:1 to 9:1). Conversely, no clear signal of genetic polyandry was found in E. molleri.

Isolation and characterization of 29 and 19 microsatellite loci from two deep-sea luminous lanternsharks, Etmopterus spinax and Etmopterus molleri (Squaliformes, Etmopteridae).

Etmopterus spinax (Linnaeus, 1758) and Etmopterus molleri (Whitley, 1939) are two bioluminescent deep-sea sharks, usually caught in large numbers as bycatch by deep-water fisheries. Yet, no study has ever involved population status of these two species using genetic tools. In order to investigate population genetic structure, diversity and connectivity of these two lanternsharks, 29 and 19 microsatellite loci were isolated from E. spinax DNA library for E. spinax and E. molleri, respectively. These loci were tested on 32 E. spinax individuals from the North Sea and seven E. molleri from the East China Sea. The number of alleles per locus ranged from 2 to 13. The observed heterozygosity ranged from 0.031 to 0.839 for E. spinax and from 0.000 to 1.000 for E. molleri, while the expected heterozygosity ranged from 0.031 to 0.903 and from 0.143 to 0.821, respectively. Almost all loci (24 and 16, respectively) were at Hardy-Weinberg equilibrium for both species and no linkage disequilibrium among loci was detected. These loci represent useful tools to better understand the population structure of these two species. Besides, they could also be suitable for other lanternsharks in general, as these latter remain largely understudied, specially in terms of understanding the basic science that will serve into their conservation.

Deep-sea sharks: Relation between the liver's buoyancy and red aerobic muscle volumes, a new approach.

Shark's buoyancy depends on two types of force: (i) the hydrostatic force which is mainly provided by their liver filled with low density lipids and (ii) the hydrodynamic force which is provided by the morphology of their body and fins. Shallow-water shark species are usually negatively buoyant, whereas deep-sea shark species have been suggested to display neutral buoyancy. It has been suggested that species that are close to the neutrality would have less red aerobic muscle fibers. Here, we investigated several liver features (the hepatosomatic index, the oil content and the lipid composition) playing a major role regarding the buoyancy of three deep-sea shark species (Etmopterus molleri, Etmopterus spinax and Isistius brasiliensis) and one shallow-water counterpart (Galeus melastomus). We used FT-Raman and FT-MIR spectroscopy to qualify/quantify the lipid composition of their liver. Our results showed that most deep-sea shark species studied have liver features providing more buoyancy than their shallow-water counterparts, appart from E. molleri which shows liver's features that resemble more shallow-water shark species (e.g. G. melastomus). Finally, data regarding liver features of several deep-sea shark species from the literature were added and the red aerobic muscle distribution/proportion of nine species was measured, to reveal how these parameters might be related. Our results showed that sharks characterized by a liver providing more hydrostatic force possess proportionally less red aerobic muscles than sharks having a liver that contributes less to their buoyancy. Therefore, our results i.e. deep-sea shark displaying less red aerobic muscle with a liver providing more buoyancy, support low metabolic rates hence slow swimming speed.

Behavioural responses of the yellow emitting annelid Tomopteris helgolandica to photic stimuli.

In contrast to most mesopelagic bioluminescent organisms specialised in the emission and reception of blue light, the planktonic annelid Tomopteris helgolandica produces yellow light. This unusual feature has long been suggested to serve for intraspecific communication. Yet, this virtually admitted hypothesis has never been tested. In this behavioural study of spectral colour sensitivity, we first present an illustrated repertoire of the postures and action patterns described by captive specimens. Then video tracking and motion analysis are used to quantify the behavioural responses of singled out worms to photic stimuli imitating intraspecific (yellow) or interspecific (blue) bioluminescent signals. We show the ability of T. helgolandica to react and to contrast its responses to bioluminescent-like blue and yellow light signals. In particular, the attractive effect of yellow light and the variation of angular velocity observed according to the pattern of yellow stimuli (flashes versus glows) support the intraspecific communication hypothesis. However, given the behavioural patterns of T. helgolandica, including mechanically induced light emission, the possibility that bioluminescence may be part of escape/defence responses to predation, should remain an open question.

High opsin diversity in a non-visual infaunal brittle star.

BACKGROUND: In 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. RESULTS: In 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. CONCLUSION: Light 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.

A brittle star is born: Ontogeny of luminous capabilities in Amphiura filiformis.

Bioluminescence is the production of visible light by living organisms thanks to a chemical reaction, implying the oxidation of a substrate called luciferin catalyzed by an enzyme, the luciferase. The luminous brittle star Amphiura filiformis depends on coelenterazine (i.e., the most widespread luciferin in marine ecosystems) and a luciferase homologous to the cnidarian Renilla luciferase to produce blue flashes in the arm's spine. Only a few studies have focused on the ontogenic apparitions of bioluminescence in marine organisms. Like most ophiuroids, A. filiformis displays planktonic ophiopluteus larvae for which the ability to produce light was not investigated. This study aims to document the apparition of the luminous capabilities of this species during its ontogenic development, from the egg to settlement. Through biochemical assays, pharmacological stimulation, and Renilla-like luciferase immunohistological detection across different developing stages, we pointed out the emergence of the luminous capabilities after the ophiopluteus larval metamorphosis into a juvenile. In conclusion, we demonstrated that the larval pelagic stage of A. filiformis is not bioluminescent compared to juveniles and adults.

Systematic Distribution of Bioluminescence in Marine Animals: A Species-Level Inventory.

Bioluminescence is the production of visible light by an organism. This phenomenon is particularly widespread in marine animals, especially in the deep sea. While the luminescent status of numerous marine animals has been recently clarified thanks to advancements in deep-sea exploration technologies and phylogenetics, that of others has become more obscure due to dramatic changes in systematics (themselves triggered by molecular phylogenies). Here, we combined a comprehensive literature review with unpublished data to establish a catalogue of marine luminescent animals. Inventoried animals were identified to species level in over 97% of the cases and were associated with a score reflecting the robustness of their luminescence record. While luminescence capability has been established in 695 genera of marine animals, luminescence reports from 99 additional genera need further confirmation. Altogether, these luminescent and potentially luminescent genera encompass 9405 species, of which 2781 are luminescent, 136 are potentially luminescent (e.g., suggested luminescence in those species needs further confirmation), 99 are non-luminescent, and 6389 have an unknown luminescent status. Comparative analyses reveal new insights into the occurrence of luminescence among marine animal groups and highlight promising research areas. This work will provide a solid foundation for future studies related to the field of marine bioluminescence.

Maintain the light, long-term seasonal monitoring of luminous capabilities in the brittle star Amphiura filiformis.

The European brittle star Amphiura filiformis emits blue light, via a Renilla-like luciferase, which depends on the dietary acquisition of coelenterazine. Questions remain regarding luciferin availability across seasons and the persistence of luminous capabilities after a single boost of coelenterazine. To date, no study has explored the seasonal, long-term monitoring of these luminous capabilities or the tracking of luciferase expression in photogenic tissues. Through multidisciplinary analysis, we demonstrate that luminous capabilities evolve according to the exogenous acquisition of coelenterazine throughout adult life. Moreover, no coelenterazine storage forms are detected within the arms tissues. Luciferase expression persists throughout the seasons, and coelenterazine's presence in the brittle star diet is the only limiting factor for the bioluminescent reaction. No seasonal variation is observed, involving a continuous presence of prey containing coelenterazine. The ultrastructure description provides a morphological context to investigate the green autofluorescence signal attributed to coelenterazine during luciferin acquisition. Finally, histological analyses support the hypothesis of a pigmented sheath leading light to the tip of the spine. These insights improve our understanding of the bioluminescence phenomenon in this burrowing brittle star.

Physiological control of bioluminescence in a deep-sea planktonic worm, Tomopteris helgolandica.

Tomopteris helgolandica Greeff 1879 (Tomopteridae) is a transparent holoplanktonic polychaete that can emit a bright light. In this study, we investigated the emission pattern and control of this deep-sea worm's luminescence. Potassium chloride depolarisation applied on anaesthetised specimens triggered a maximal yellow light emission from specific parapodial sites, suggesting that a nervous control pathway was involved. Pharmacological screening revealed a sensitivity to carbachol, which was confirmed by a dose-light response associated with a change in the light emission pattern, where physiological carbachol concentrations induced flashes and higher concentrations induced glows. The light response induced by its hydrolysable agonist, acetylcholine, was significantly weaker but was facilitated by eserine pretreatment. In addition, a specific inhibitory effect of tubocurarine was observed on carbachol-induced emission. Lastly, KCl- and carbachol-induced light responses were significantly reduced when preparations were pre-incubated in Ca(2+)-free artificial seawater or in different calcium channel blockers (verapamil, diltiazem) and calmodulin inhibitor (trifluoperazine) solutions. All of these results strongly suggest that T. helgolandica produces its light flashes via activation of nicotinic cholinergic receptors and a calcium-dependent intracellular mechanism involving L-type calcium channels.

Catecholamine Involvement in the Bioluminescence Control of Two Species of Anthozoans.

Bioluminescence, the ability of living organisms to emit visible light, is an important ecological feature for many marine species. To fulfil the ecological role (defence, offence, or communication), bioluminescence needs to be finely controlled. While many benthic anthozoans are luminous, the physiological control of light emission has only been investigated in the sea pansy, Renilla koellikeri. Through pharmacological investigations, a nervous catecholaminergic bioluminescence control was demonstrated for the common sea pen, Pennatula phosphorea, and the tall sea pen, Funiculina quadrangularis. Results highlight the involvement of adrenaline as the main neuroeffector triggering clusters of luminescent flashes. While noradrenaline and octopamine elicit flashes in P. phosphorea, these two biogenic amines do not trigger significant light production in F. quadrangularis. All these neurotransmitters act on both the endodermal photocytes located at the base and crown of autozooids and specific chambers of water-pumping siphonozooids. Combined with previous data on R. koellikeri, our results suggest that a catecholaminergic control mechanisms of bioluminescence may be conserved in Anthozoans.

Species-specific metabolites mediate host selection and larval recruitment of the symbiotic seastar shrimp.

In marine environments, host selection, defining how symbiotic organisms recognize and interact with their hosts, is often mediated by olfactory communication. Although adult symbionts may select their hosts detecting chemosensory cues, no information is available concerning the recruitment of symbiotic larvae which is a crucial step to sustain symbioses over generations. This study investigates the olfactory recognition of seastar hosts by adult Zenopontonia soror shrimps and the recruitment of their larvae. We examine the semiochemicals that influence host selection using chemical extractions, behavioural experiments in olfactometers, and mass spectrometry analyses. After describing the symbiotic population and the embryonic development of shrimps, our results demonstrate that asterosaponins, which are traditionally considered as chemical defences in seastars, are species-specific and play a role in attracting the symbiotic shrimps. Adult shrimps were found to be attracted only by their original host species Culcita novaeguineae, while larvae were attracted by different species of seastars. This study provides the first chemical identification of an olfactory cue used by larvae of symbiotic organisms to locate their host for recruitment. These findings highlight the importance of chemical communication in the mediation of symbiotic associations, which has broader significant implications for understanding the ecological dynamics of marine ecosystems.

Control of luminescence from pygmy shark (Squaliolus aliae) photophores.

The smalleye pygmy shark (Squaliolus aliae) is a dwarf pelagic shark from the Dalatiidae family that harbours thousands of tiny photophores. In this work, we studied the organisation and physiological control of these photogenic organs. Results show that they are mainly situated on the ventral side of the shark, forming a homogeneous ventral photogenic area that appears well suited for counterillumination, a well-known camouflage technique of pelagic organisms. Isolated ventral skin patches containing photophores did not respond to classical neurotransmitters and nitric oxide but produced light after melatonin (MT) application. Prolactin and α-melanocyte-stimulating hormone inhibited this hormonally induced luminescence as well as the spontaneous luminescence from the photogenic tissue. The action of MT seems to be mediated by binding to the MT(2) receptor subtype, as the MT(2) receptor agonist 4P-PDOT inhibited the luminescence induced by this hormone. Binding to this receptor probably decreases the intracellular cAMP concentration because forskolin inhibited spontaneous and MT-induced luminescence. In addition, a GABA inhibitory tonus seems to be present in the photogenic tissue as well, as GABA inhibited MT-induced luminescence and the application of bicuculline provoked luminescence from S. aliae photophores. Similarly to what has been found in Etmopteridae, the other luminous shark family, the main target of the luminescence control appears to be the melanophores covering the photocytes. Results suggest that bioluminescence first appeared in Dalatiidae when they adopted a pelagic style at the Cretaceous/Tertiary boundary, and was modified by Etmopteridae when they started to colonize deep-water niches and rely on this light for intraspecific behaviours.

A Third Luminous Shark Family: Confirmation of Luminescence Ability for Zameus squamulosus (Squaliformes; Somniosidae).

Since recently, shark's bioluminescence has been recorded from two Squaliformes families, the Etmopteridae and Dalatiidae. Pictures of luminescence, light organ morphologies and physiology of the luminous control have been described for species of the Etmopteridae and Dalatiidae families. In 2015, a third luminous family, Somniosidae, was assumed to present a bioluminescent species, Zameus squamulosus. Up to now, confirmation of the luminous abilities of Z. squamulosus is lacking. Here, the luminescence of Z. squamulosus was in vivo recorded for the first time confirming the bioluminescence status of the third luminescent shark family. Additionally, photophore histology revealed the conservation of the light organ morphology across the luminous Squaliformes. Light transmittance analysis through the placoid scale added information on the luminescence efficiency and highlighted a new type of bioluminescent-like squamation. All these data reinforced the likelihood that the common ancestor of Dalatiidae, Etmopteridae and Somniosidae may already have been luminescent for counterillumination purpose.

Red and white muscle proportions and enzyme activities in mesopelagic sharks.

In the last decade, there has been an increase in the study of the ecology of deep-sea organisms. One way to understand an organism's ecology is the study of its metabolism. According to literature, deep-sea sharks possess a lower anaerobic enzyme activity than their shallow-water counterparts, but no difference has been observed regarding their aerobic enzyme activities. These studies have suggested deep-sea sharks should be slow and listless swimmers. However, other studies based on video observations have revealed differences in cruise swimming speed between different species. The present study examined muscles of squaliform sharks, including both luminous and non-luminous species. We combined measurements of the relative amounts of red and white muscle with assays of enzymes that are used as markers for aerobic (citrate synthase, malate dehydrogenase) and anaerobic (lactate dehydrogenase) metabolism, searching for a relationship with cruising speeds. Non-luminous deep-sea species displayed lower aerobic enzyme activities but similar anaerobic enzyme activities than the benthic shallow-water counterpart (Squalus acanthias). Conversely, luminous Etmopteridae species were found to have similar aerobic enzyme activities to S. acanthias but displayed lower anaerobic enzyme activities. Analyses revealed that red muscle proportion and aerobic enzyme activities were positively related to the cruise swimming speed. In contrast, Dalatias licha, which swims at the slowest cruise swimming speed ever recorded, presented a very low aerobic metabolic phenotype (lower aerobic marker enzymes and less red muscle). Finally, the values obtained for white muscle proportion and anaerobic metabolic phenotype suggested a high burst capacity for D. licha and non-luminous sharks.

Functional physiology of lantern shark (Etmopterus spinax) luminescent pattern: differential hormonal regulation of luminous zones.

Lantern sharks are small deep-sea sharks that harbour complex species-specific luminescent photophore patterns. The luminescent pattern of one of these sharks, Etmopterus spinax, is made up of nine luminous zones. Previous experiments revealed that in the largest of these zones (ventral zone), photophores are under hormonal control, light being triggered by both melatonin (MT) and prolactin (PRL). In this study, we analysed the luminescent responses to MT and PRL in five other luminous zones from 12 female and eight male E. spinax specimens. The results showed that all luminous zones respond to both hormones, with each zone having its own kinetic parameters (maximum light intensity, L(max); total light emitted, L(tot); time from stimulation to L(max), TL(max)), which confirms the multifunctional character of this shark's luminescence. L(tot) and L(max) were found to be directly dependent on the photophore density (P(D)) of the luminous zone, while TL(max) varied independently from P(D). In addition, we demonstrate a sexual dimorphism in the luminescent response to PRL, with male specimens having smaller L(tot) and TL(max) in the luminous zones from the pelvic region. As this region also harbours the sexual organs of this species, this strongly suggests a role for the luminescence from these zones in reproduction.

Nitric oxide in the control of luminescence from lantern shark (Etmopterus spinax) photophores.

Photophores (photogenic organs) of the lantern shark Etmopterus spinax are under hormonal control, with prolactin (PRL) and melatonin (MT) triggering the light emission. Differential sensitivity to these hormones in adult individuals suggests, however, that the luminescence of this shark is controlled by an additional mechanism. In this study, different techniques were used to investigate a potential modulator of E. spinax luminescence - nitric oxide (NO). NO synthase (NOS)-like immunoreactivity (IR) was found in the photocytes (photogenic cells) of the photophores. In addition, acetylated tubulin IR also supported the presence of nerves running through the photogenic tissue and innervating different structural elements of the photophores: photocytes, pigmented cells from the iris-like structure and lens cells. Pharmacological experiments confirmed a modulatory action of NO on the hormonally induced luminescence: NO donors sodium nitroprusside (SNP) and hydroxylamine decreased the time to reach the maximum amplitude (TL(max)) of MT-induced luminescence while these substances decreased the maximum amplitude of PRL-induced luminescence (and also the TL(max) in the case of SNP). The small impact of the NOS inhibitor l-NAME on hormonally induced luminescence suggests that NO is only produced on demand. The cGMP analogue 8BrcGMP mimicked the effects of NO donors suggesting that the effects of NO are mediated by cGMP.

The lantern shark's light switch: turning shallow water crypsis into midwater camouflage.

Bioluminescence is a common feature in the permanent darkness of the deep-sea. In fishes, light is emitted by organs containing either photogenic cells (intrinsic photophores), which are under direct nervous control, or symbiotic luminous bacteria (symbiotic photophores), whose light is controlled by secondary means such as mechanical occlusion or physiological suppression. The intrinsic photophores of the lantern shark Etmopterus spinax were recently shown as an exception to this rule since they appear to be under hormonal control. Here, we show that hormones operate what amounts to a unique light switch, by acting on a chromatophore iris, which regulates light emission by pigment translocation. This result strongly suggests that this shark's luminescence control originates from the mechanism for physiological colour change found in shallow water sharks that also involves hormonally controlled chromatophores: the lantern shark would have turned the initial shallow water crypsis mechanism into a midwater luminous camouflage, more efficient in the deep-sea environment.

From extraocular photoreception to pigment movement regulation: a new control mechanism of the lanternshark luminescence.

The velvet belly lanternshark, Etmopterus spinax, uses counterillumination to disappear in the surrounding blue light of its marine environment. This shark displays hormonally controlled bioluminescence in which melatonin (MT) and prolactin (PRL) trigger light emission, while α-melanocyte-stimulating hormone (α-MSH) and adrenocorticotropic hormone (ACTH) play an inhibitory role. The extraocular encephalopsin (Es-Opn3) was also hypothesized to act as a luminescence regulator. The majority of these compounds (MT, α-MSH, ACTH, opsin) are members of the rapid physiological colour change that regulates the pigment motion within chromatophores in metazoans. Interestingly, the lanternshark photophore comprises a specific iris-like structure (ILS), partially composed of melanophore-like cells, serving as a photophore shutter. Here, we investigated the role of (i) Es-Opn3 and (ii) actors involved in both MT and α-MSH/ACTH pathways on the shark bioluminescence and ILS cell pigment motions. Our results reveal the implication of Es-Opn3, MT, inositol triphosphate (IP3), intracellular calcium, calcium-dependent calmodulin and dynein in the ILS cell pigment aggregation. Conversely, our results highlighted the implication of the α-MSH/ACTH pathway, involving kinesin, in the dispersion of the ILS cell pigment. The lanternshark luminescence then appears to be controlled by the balanced bidirectional motion of ILS cell pigments within the photophore. This suggests a functional link between photoreception and photoemission in the photogenic tissue of lanternsharks and gives precious insights into the bioluminescence control of these organisms.