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 Here, we combined a comprehensive literature review with unpublished data to establish a catalogue of marine luminescent animals.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.

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.

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.

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.

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.

The megamouth shark, Megachasma pelagios, is not a luminous species.

Despite its five meters length, the megamouth shark (Megachasma pelagios Taylor, Compagno & Struhsaker, 1983) is one of the rarest big sharks known in the world (117 specimens observed and documented so far). This filter-feeding shark has been assumed to be a luminous species, using its species-specific white band to produce bioluminescence as a lure trap. Another hypothesis was the use of the white band reflectivity to attract prey or for social recognition purposes. However, no histological study has ever been performed to confirm these assumptions so far. Two hypotheses about the megamouth shark's luminescence arose: firstly, the light emission may be intrinsically or extrinsically produced by specific light organs (photophores) located either on the upper jaw white band or inside the mouth; secondly, the luminous appearance might be a consequence of the reflection of prey luminescence on the white band during feeding events. Aims of the study were to test these hypotheses by highlighting the potential presence of specific photophores responsible for bioluminescence and to reveal and analyze the presence of specialized light-reflective structures in and around the mouth of the shark. By using different histological approaches (histological sections, fluorescent in situ hybridization, scanning electron microscopy) and spectrophotometry, this study allows to unravel these hypotheses and strongly supports that the megamouth shark does not emit bioluminescence, but might rather reflect the light produced by bioluminescent planktonic preys, thanks to the denticles of the white band.

Histological evidence for secretory bioluminescence from pectoral pockets of the American Pocket Shark (Mollisquama mississippiensis).

The function of pocket shark pectoral pockets has puzzled scientists over decades. Here, we show that the pockets of the American Pocket Shark (Mollisquama mississippiensis) contain a brightly fluorescent stratified cubic epithelium enclosed in a pigmented sheath and in close contact with the basal cartilage of the pectoral fins; cells of this epithelium display a centripetal gradient in size and a centrifuge gradient in fluorescence. These results strongly support the idea that pocket shark's pockets are exocrine holocrine glands capable of discharging a bioluminescent fluid, potentially upon a given movement of the pectoral fin. Such capability has been reported in many other marine organisms and is typically used as a close-range defensive trick. In situ observations would be required to confirm this hypothesis.

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.

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.

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.

Adrenocorticotropic Hormone and Cyclic Adenosine Monophosphate are Involved in the Control of Shark Bioluminescence.

Among Etmopteridae and Dalatiidae, luminous species use hormonal control to regulate bioluminescence. Melatonin (MT) triggers light emission and, conversely, alpha melanocyte-stimulating hormone (α-MSH) actively reduces ongoing luminescence. Prolactin (PRL) acts differentially, triggering light emission in Etmopteridae and inhibiting it in Dalatiidae. Interestingly, these hormones are also known as regulators of skin pigment movements in vertebrates. One other hormone, the adrenocorticotropic hormone (ACTH), also members of the skin pigmentation regulators, is here pharmacologically tested on the light emission. Results show that ACTH inhibits luminescence in both families. Moreover, as MT and α-MSH/ACTH receptors are members of the G-protein coupled receptor (GPCR) family, we investigated the effect of hormonal treatments on the cAMP level of photophores through specific cAMP assays. Our results highlight the involvement of ACTH and cAMP in the control of light emission in sharks and suggest a functional similarity between skin pigment migration and luminescence control, this latter being mediated by pigment movements in the light organ-associated iris-like structure cells.

Distribution and quantification of bioluminescence as an ecological trait in the deep sea benthos.

Bioluminescence is a prominent functional trait used for visual communication. A recent quantification showed that in pelagic ecosystems more than 75% of individual macro-planktonic organisms are categorized as able to emit light. In benthic ecosystems, only a few censuses have been done, and were based on a limited number of observations. In this study, our dataset is based on observations from remotely operated vehicle (ROV) dives conducted from 1991-2016, spanning 0-3,972 m depth. Data were collected in the greater Monterey Bay area in central California, USA and include 369,326 pelagic and 154,275 epibenthic observations at Davidson Seamount, Guide Seamount, Sur Ridge and Monterey Bay. Because direct observation of in situ bioluminescence remains a technical challenge, taxa from ROV observations were categorized based on knowledge gained from the literature to assess bioluminescence status. We found that between 30-41% of the individual observed benthic organisms were categorized as capable of emitting light, with a strong difference between benthic and pelagic ecosystems. We conclude that overall variability in the distribution of bioluminescent organisms is related to the major differences between benthic and pelagic habitats in the deep ocean. This study may serve as the basis of future investigations linking the optical properties of various habitats and the variability of bioluminescent organism distributions.

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.

Etmopterus spinax, the velvet belly lanternshark, does not use bacterial luminescence.

Marine organisms are able to produce light using either their own luminous system, called intrinsic bioluminescence, or symbiotic luminous bacteria, called extrinsic bioluminescence. Among bioluminescent vertebrates, Osteichthyes are known to harbor both types of bioluminescence, while no study has so far addressed the potential use of intrinsic/extrinsic luminescence in elasmobranchs. In sharks, two families are known to emit light: Etmopteridae and Dalatiidae. The deep-sea bioluminescent Etmopteridae, Etmopterus spinax, has received a particular interest over the past fifteen years and its bioluminescence control was investigated in depth. However, the nature of the shark luminous system still remains enigmatic. The present work was undertaken to assess whether the light of this shark species originates from a bioluminescent bacterial symbiosis. Using fluorescent in situ hybridization (FISH) and transmission electron microscopy (TEM) image analyses, this study supports the conclusion that the bioluminescence in the deep-sea lanternshark, Etmopterus spinax, is not of bacterial origin.

Etmopteridae bioluminescence: dorsal pattern specificity and aposematic use.

BACKGROUND: In the darkness of the ocean, an impressive number of taxa have evolved the capability to emit light. Many mesopelagic organisms emit a dim ventral glow that matches with the residual environmental light in order to camouflage themselves (counterillumination function). Sharks use their luminescence mainly for this purpose. Specific lateral marks have been observed in Etmopteridae sharks (one of the two known luminous shark families) suggesting an inter/intraspecific recognition. Conversely, dorsal luminescence patterns are rare within these deep-sea organisms. RESULTS: Here we report evidence that Etmopterus spinax, Etmopterus molleri and Etmopterus splendidus have dorsal luminescence patterns. These dorsal patterns consist of specific lines of luminous organs, called photophores, on the rostrum, dorsal area and at periphery of the spine. This dorsal light seems to be in contrast with the counterilluminating role of ventral photophores. However, skin photophores surrounding the defensive dorsal spines show a precise pattern supporting an aposematism function for this bioluminescence. Using in situ imaging, morphological and histological analysis, we reconstructed the dorsal light emission pattern on these species, with an emphasis on the photogenic skin associated with the spine. Analyses of video footage validated, for the first time, the defensive function of the dorsal spines. Finally, we did not find evidence that Etmopteridae possess venomous spine-associated glands, present in Squalidae and Heterondontidae, via MRI and CT scans. CONCLUSION: This work highlights for the first time a species-specific luminous dorsal pattern in three deep-sea lanternsharks. We suggest an aposematic use of luminescence to reveal the presence of the dorsal spines. Despite the absence of venom apparatus, the defensive use of spines is documented for the first time in situ by video recordings.

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.

Luminescence control of Stomiidae photophores.

Nervous control of light emission from deep-sea mesopelagic fishes has been documented for several species. Studies on the nervous control of photophores from deep-sea luminescent fish, are mainly restricted to a pharmacological approach. For example, the light organs, called photophores, isolated from Argyropelecus hemygimnus and Maurolicus muelleri show a much higher sensitivity to adrenaline than to noradrenaline. According to these results and other information in different species, catecholamines are considered as main neurotransmitters triggering bioluminescence in deep-sea fishes. The present work is a study of the nervous control of the isolated photophores from two Stomiid fishes, Chauliodus sloani (the viperfish) and Stomias boa (the dragonfish) with the aim to determine the nature of the nervous control by pharmacological, biochemical and morphological approaches. Results show that, although the photophores of both species are sensitive to catecholamines, adrenaline is present in larger amount than noradrenaline in the light organs of C. sloani. Both catecholamines have different immunoreactive (IR) sites, noradrenaline showing a very diffuse localization as compared to adrenaline in C. sloani. On the contrary, only adrenaline is detected in the photocytes chamber and nerves innervating the photophore in S. boa. Knowing that the majority of dragonfishes exhibit a luminescent chin barbel, we also investigated the presence of catecholamines in this specific tissue in S. boa. Immunohistology reveals the presence of adrenaline within the tissue forming the chin barbel; adrenaline-IR is found in the connective tissue surroundings two group of muscle fibers and blood vessels in the stem but also around the multiple blood vessels located within the barbel bulb. Our results strongly support the adrenergic control of light emission in bioluminescent stomiid fishes.

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.