Unexpected diversity of bioluminescence in planktonic worms.

The vast majority of pelagic bioluminescent organisms emit a blue light with emission maxima (λmax ) ranging from 450 to 490 nm. Among the known outliers, the tomopterids (Annelida: Polychaeta) are usually described as yellow-emitters (λmax  = 565-570 nm) for which bioluminescence functions as a specific recognition signal. Here, we report the first data regarding the colours emitted by four different tomopterid species, Tomopteris pacifica, T. carpenteri, T. septentrionalis and T. planktonis. Surprisingly, T. planktonis is a blue-emitter (λmax  = 450 nm). Our pharmacological results on T. planktonis support cholinergic control, as recently demonstrated in the yellow-emitter, T. helgolandica. Moreover, as revealed by epifluorescence microscopy, the light seems to be produced in both species from the same yellow-pigmented parapodial glands. Despite these similarities, tomopterids express an unexpected diversity of bioluminescent colour patterns. This leads us to reassess the ecological value of bioluminescence within this group.

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.

Synergic effects of tryptamine and octopamine on ophiuroid luminescence (Echinodermata).

In ophiuroids, bioluminescence is under nervous control. Previous studies have shown that acetylcholine is the main neurotransmitter triggering light emission in Amphipholis squamata and Amphiura filiformis. By contrast, none of the neurotransmitters tested so far induced luminescence in two other ophiuroid species, Ophiopsila aranea and Ophiopsila californica. The aim of this work was thus to investigate the putative involvement of two biogenic amines, tryptamine and octopamine, in light emission of three ophiuroid species. A. filiformis responds to both tryptamine and octopamine, mainly on its arm segments, while O. californica only responds to tryptamine stimulation. By contrast, tryptamine and octopamine do not seem to be involved in O. aranea luminescence control since none of these substances induced light emission in this species. The synergic effects of several other drugs with tryptamine and octopamine were also tested.

Effect of beta-adrenergic antagonists on bioluminescence control in three species of brittlestars (Echinodermata: Ophiuroidea).

The role of adrenaline in the nervous control of bioluminescence in three brittlestar species, Amphiura filiformis, Amphipholis squamata, and Ophiopsila aranea, was assessed by testing two different beta-adrenergic antagonists (propranolol and labetalol) over a wide concentration range (10(-10)-10(-3)M). We compared the effects of analogues (active vs. inactive) of the same substance (L- and D-enantiomers of propranolol). Propranolol presented both specific and nonspecific effects: (i) nonspecific effects were observed at the higher concentrations tested (10(-4) and 10(-3)M) in all three species; (ii) specific effects were detected only at the lower concentrations tested (10(-6)-10(-5)M). In A. squamata, the involvement of adrenaline in the nervous control of luminescence is supported by propranolol and labetolol specific inhibition. The neuropharmacological implications of nonspecific effects, the involvement of adrenaline and the interspecific differences in the brittlestar nervous control of bioluminescence are discussed.

Screening of second messengers involved in photocyte bioluminescence control of three ophiuroid species (Ophiuroidea: Echinodermata).

We investigated the effects of cyclic nucleotides (cGMP and cAMP) and inositol triphosphate/diacylglycerol pathways on the KCl-induced luminescence control of the ophiuroid species Amphiura filiformis, Ophiopsila aranea and Ophiopsila californica. Results show that dibutyrylcGMP, the cGMP analogue, and sodium nitroprusside, the guanylyl cyclase activator, had no effect on the luminescence of O. aranea and O. californica. On the other hand, cGMP could be involved in an inhibitory control in A. filiformis. Dibutyryl-cAMP, the cAMP analogue, and forskolin, the adenylyl cyclase activator, had no effect on maximal light emission, but the adenylyl cyclase inhibitors MDL-12,330A and SQ22,536 affected the kinetics of light production in both Ophiopsila species and strongly reduced KCl-induced luminescence in A. filiformis and O. aranea, suggesting cAMP pathway involvement in photogenesis. The phospholipase C inhibitor U-73122 also strongly reduced KCl-induced luminescence in all three species but this effect seems to be unspecific since U-73343, the inactive analogue of U-73122, equally inhibited photogenesis. Therefore, the results suggest that luminescence control of A. filiformis, O. aranea and O. californica is mediated by cAMP in synergy with calcium.

Luminescence in ophiuroids (Echinodermata) does not share a common nervous control in all species.

Study of the control mechanisms of light emission in invertebrates shows the involvement of several neurotransmitters. In ophiuroids, only one species (Amphipholis squamata) has so far been characterized for luminescence control, which seems to be cholinergic, with an influence of several excitatory and inhibitory neuromodulators (amino acids, catecholamines, neuropeptides S1 and S2, purines). The aim of this work is to investigate the nature of control mechanisms of light emission in three luminous ophiuroid species, A. filiformis, O. aranea and O. californica, in order to see whether or not they share common mechanisms. Luminescence induced by general depolarisation of tissues using KCl (200 mmol l(-1)) shows different patterns, according to species. Only A. filiformis emits light in response to acetylcholine. In this species, the involvement of both muscarinic and nicotinic receptors is proposed, since atropine and tubocurarine (at 10(-3) mol l(-1)) inhibited 99 % and 71 %, respectively, of the light emitted. Study of the subtypes of cholinergic receptors involved in photogenesis revealed that several subtypes of muscarinic receptors might be involved. It was also clearly shown that ophiuroids did not share a common mechanism of nervous control of luminescence in all species.

Calcium involvement in the luminescence control of three ophiuroid species (Echinodermata).

Although it has been shown that calcium is involved in the control of the luminous reaction of many invertebrate phyla, its role in Echinoderms is poorly documented. The aim of this work was to carry out a comparative study of calcium requirement of KCl-induced light emission by arm segments and dissociated luminous cells from three ophiuroid species, Ophiopsila californica, O. aranea and Amphiura filiformis. Results show a gradual inhibition of the luminescence when preparations are incubated in artificial sea water with lowered calcium concentration. The calcium substitutes Ba(2+) and Sr(2+) could act either as blockers or as substitutes, depending on the ophiuroid species; while calcium blockers Co(2+), Ni(2+) and Cd(2+) inhibit light emission in A. filiformis and in O. californica, but not in O. aranea. The nature of putative calcium voltage-gated channel has been studied pharmacologically using 1,4-dihydropyridine, benzodiazepine, phenylalkylamine and trifluoroperazine. From our results, it is proposed that calcium could act via an L-type voltage-gated calcium channel in O. californica and A. filiformis but not in O. aranea. The precise role of calcium in luminescence control still remains unknown; it could act as a second messenger or as a co-factor of the luminous reaction.

Kinetics of light emission and oxygen consumption by bioluminescent bacteria.

Oxygen plays a key role in bacterial bioluminescence. The simultaneous and continuous kinetics of oxygen consumption and light emission during a complete exhaustion of the exogenous oxygen present in a closed system has been investigated. The kinetics are performed with Vibrio fischeri, V. harveyi, and Photobacterium phosphoreum incubated on respiratory substrates chosen for their different reducing power. The general patterns of the luminescence time courses are different among species but not among substrates. During steady-state conditions, substrates, which are less reduced than glycerol, have, paradoxally, a better luminescence efficiency. Oxygen consumption by luciferase has been evaluated to be approximately 17% of the total respiration. Luciferase is a regulatory enzyme presenting a positive cooperative effect with oxygen and its affinity for this final electron acceptor is about 4-5 times higher than the one of cytochrome oxidase. The apparent Michaelis constant for luciferase has been evaluated to be in the range of 20 to 65 nM O2. When O2 concentrations are as low as 10 nM, luminescence can still be detected; this means that above this concentration, strict anaerobiosis does not exist. By n-butyl malonate titration, it was clearly shown that electrons enter the luciferase pathway only when the cytochrome pathway is saturated. It is suggested that, in bioluminescent bacteria, luciferase acts as a free-energy dissipating valve when anabolic processes (biomass production) are impaired.

Evidence from polychromatism and bioluminescence that the cosmopolitan ophiuroid Amphipholis squamata might not represent a unique taxon.

Individuals of the cosmopolitan ophiuroid Amphipholis squamata were collected from eight stations. Eleven colour varieties were described and their distribution was non-random among stations. This suggests that the varieties differ in ecophysiologic tolerance and that their geographical distribution is modulated by environmental conditions. Varieties also differed in bioluminescence. Contrary to kinetics, intensity of light production varied among co-occurring varieties, meaning that they have similar bioluminescent reactions but a different amount of bioluminescent reagent. Light intensity differed in absolute value among stations but the rank position of each variety relative to others remained constant from one station to another. The 'colour-bioluminescence' link appeared clearly fixed (the same level of bioluminescence for the same variety) and is suggested to be of genetic origin. The species 'A. squamata' may then be a mosaic of genetically different entities (the varieties) rather than a unique cosmopolitan taxonomic entity.

Effects of catecholamines and purines on luminescence in the brittlestar Amphipholis squamata (Echinodermata).

The effects of catecholamines (dopamine, adrenaline, noradrenaline and its derivatives), 5-hydroxytryptamine and purines (adenosine, ATP and their derivatives) on the acetylcholine-induced luminescence of isolated arms and dissociated photocytes of the luminescent ophiuroid Amphipholis squamata were tested. The results showed that catecholamines and 5-hydroxytryptamine (10(-)(5) to 10(-)(3 )mol l(-)(1)) had a strong dose-dependent inhibitory effect on acetylcholine-induced luminescence. In contrast, purines (10(-)(4) and 10(-)(3 )mol l(-)(1)) triggered luminescence in the absence of acetylcholine and/or potentiated acetylcholine-induced luminescence. The results with specific purinergic agonists and antagonists indicated the involvement of P(1)- and P(2)-like purinoceptors in the control of luminescence. Our study suggests that, in addition to the previously described cholinergic system in Amphipholis squamata, there may be a purinergic system, acting in synergy with acetylcholine, and an inhibitory neuromodulatory catecholaminergic system, all associated with the control of luminescence.

Involvement of cyclic nucleotides and IP(3) in the regulation of luminescence in the brittlestar Amphipholis squamata (Echinodermata).

We investigated the effects of cyclic nucleotides (cGMP, cAMP) and the phosphoinositide IP(3) on the luminescence of the ophiuroid Amphipholis squamata. The cGMP analogue, dibutyryl-cGMP, and the guanylate cyclase activator, sodium nitroprusside, had no effect on the luminescence. The cAMP analogue, dibutyryl-cAMP, and the adenylate cyclase activator, forskolin, triggered luminescence. Moreover, the adenylate cyclase inhibitor, MDL-12330A, significantly reduced ACh-induced luminescence. The phospholipase C inhibitor, U-73122, also significantly reduced ACh-induced luminescence. The results suggest that ACh-induced luminescence is mediated by both cAMP and IP(3) pathways but not by cGMP. The effects of calcium-free ASW confirmed this hypothesis. A hypothetical scheme of the transduction mechanisms involved in the intracellular control of luminescence is presented.

Evidence of seasonal variation in bioluminescence of Amphipholis squamata (Ophiuroidea, Echinodermata): effects of environmental factors.

The bioluminescence of Amphipholis squamata was assessed from freshly collected individuals for 16 successive months, and from individuals maintained in the laboratory under various experimental conditions of salinity, temperature and photoperiodic regime. Field investigations showed that bioluminescence intensity and kinetics varied seasonally, with the light produced being brighter and faster in winter and summer. The seasonal variation was not correlated with changes of ambient salinity. However, it was correlated with changes in temperature, the luminescence being brighter and faster in coldest and warmest seasons, and with the changes of photoperiod, the luminescence being brighter and faster in seasons with shortest and longest day length. Laboratory investigations also demonstrated that luminescence was not affected by salinity conditions. Conversely, luminescence was affected by temperature, the light production being brighter and faster in warmer conditions (in agreement with field observations) and dimmer and slower in colder conditions (in disagreement with field observations). Light production was also affected by photoperiod since experimental changes of natural light:dark regime caused the bioluminescence to decrease. Considering that photoperiod guides the biology of A. squamata and that reproduction takes place during coldest months in the species, an endogenous factor of neurophysiological nature linked to the ophiuroid reproductive cycle is proposed to induce the luminescence to peak in winter. This was confirmed by the fact that seasonal variation of luminescence was different between adult and juveniles, the latter showing no winter peak of luminescence. It is suggested that the luminescence normally associated with defense could also be part of an intraspecific visual signal related to individuals aggregating for reproduction during winter.

Cytological changes during bioluminescence production in dissociated photocytes from the ophiuroid Amphipholis squamata (Echinodermata).

Bioluminescence in the ophiuroid Amphipholis squamata is produced by photocytes located within the spinal ganglia of arm spines. Ganglionic cells were dissociated (pronase digestion) and photocytes separated from other cell types by using a continuous density Percoll gradient. Aliquots from a stock suspension of photocytes in artificial sea water were stimulated to produce light by using KCl or acetylcholine and fixed for ultrastructural observation at different times of the luminous process. Preluminescent, luminescent, and postluminescent photocytes contained various intracytoplasmic structures, such as Golgi, flat and distended rough endoplasmic reticulum cisternae, bundles of fibrils, and up to six types of membrane-bounded vesicles. These structures either co-occurred or succeeded one another during the process of light production, indicating that they were most probably participating in the luminescence reaction. Two types of vesicles, sharing some ultrastructural features, probably represented the microsources of the photocytes. One type occurred almost exclusively in luminescent photocytes, and the other almost exclusively in postluminescent photocytes, suggesting that one may be transformed into the other. The latter type of vesicle contained densely packed fibro-tubular units, giving a characteristic paracrystalline appearance to postluminescent photocytes.

Modulatory effects of some amino acids and neuropeptides on luminescence in the brittlestar amphipholis squamata

Amphipholis squamata is a polychromatic luminescent ophiuroid. The effects of amino acids ( &ggr; -aminobutyric acid, GABA, taurine, glycine and glutamate), N-methyl-d-aspartate (NMDA) and the invertebrate neuropeptides Antho-RFamide, FMRFamide and SALMFamides S1 and S2 were tested on acetylcholine-induced luminescence from isolated arms of clear and black specimens of Amphipholis squamata. The results showed that GABA, glycine and Antho-RFamide inhibited ACh-induced luminescence of clear specimens and had no significant effect on black specimens. Glutamic acid had no significant effect on ACh-induced luminescence, but triggered luminescence in the absence of ACh in both types of specimen. Taurine, NMDA and FMRFamide showed no significant effects on either clear or black specimens. S1 potentiated ACh-induced luminescence of clear and black specimens, while S2 had no clear modulatory effect on luminescence. These results suggest that, in addition to the previously described cholinergic system in Amphipholis squamata, there is also a modulatory component to luminescence control. Moreover, we observed a difference in modulation of luminescence between clear and black specimens.

Determination by HPLC of adrenalin (E) and of noradrenalin (NE) in certain species of mesopelagic fish in the Strait of Messina.

The presence of adrenalin (E) and noradrenalin (NE) was found by HPLC both in the photophores and at other tissue levels of numerous species of mesopelagic fish in The Strait of Messina, with the aim of determining the incidence of these catecholamines in photophores, in light transmission and the eventual presence at other tissue levels.

Localization of S1- and S2-like immunoreactivity in the nervous system of the brittle star Amphipholis squamata (Delle Chiaje 1828).

The recent isolation and characterization of the SALMFanide neuropeptides S1 GFNSALMFamide; and S2 (SGPYSFNSGLTFamide) from the sea stars. Asterias rubens and Asterias forbesi have initiated numerous studies on their morphological localization and distribution within the phylum Echinodermata. It has been shown by immunocytochemistry and radioimmunoassay that these peptides are widely distributed in the nervous system of some asteroids, echinoids and ophiuroids. A physiological approach has also shown that S1 and S2 potentiate the luminescence of the small ophiuroid Amphipholis squamata. In the present study. S1- and S2-like immunoreactivity have been localized in A. squamata by immunocytochemistry on both wholemount preparation and histological sections. The results reveal a widespread neuronal distribution of S1-like immunoreactivity in the circumoral ring, radial nerve cord, and tube feet. S1-like immunoreactivity was found to be associated with axons and cell bodies in both the ectoneural and hyponeural components of the nervous. S2-like immunoreactivity was detected only in the ectoneural plenus of the circumoral ring and radial nerve cord.

Immunocytochemical and autoradiographic studies of the endocrine cells interacting with GABA in the rat stomach.

There are now increasing evidences suggesting that GABA is able of direct interaction with certain endocrine cells. In the present study, highly specific anti-GABA-glutaraldehyde antibodies and 3H-GABA uptake were used at the light and electron microscope levels to investigate the occurrence of cells containing endogenous GABA or taking up exogenous GABA in the mucosal antrum and corpus of the rat stomach. Only certain endocrine cell types of both regions were immunostained or grain-labelled. However, the morphology of their secretory granules did not allow to identify the nature of their hormone with certainty but suggested that somatostatin-like cells could interact with GABA. The combination of gastrin and somatostatin immunodetection with 3H-GABA uptake autoradiography at the light microscope level, revealed that a subpopulation of somatostatin-like cells and other still unidentified endocrine cells are able to take up GABA, while the gastrin-like cells are not. These results reinforce the hypothesis that certain endocrine cell types of the diffuse endocrine system of the digestive tract are able to directly interact with GABA.

Oxygen consumption and luminescence of Maurolicus photophores stimulated by adrenalin.

1. Abdominal photophores isolated from Maurolicus muelleri freshly collected in the Strait of Messina show a stable oxygen consumption rate of 149.8 +/- 10.1 nmoles g-1 min-1 (N = 31). 2. In the presence of adrenalin (5 X 10(-4) mol/l), the photophores that did not luminesce showed a 78% decrease of the resting oxygen uptake. When the photophores did luminesce, the oxygen consumption never increased above the resting level, instead, it decreased at a different rate according to the amount of light emitted. 3. Photophores pretreated with MnCl2 (40 mM) and stimulated with adrenalin (5 X 10(-4) mol/l), showed a rapid and large emission of light associated with a significant increase of the oxygen consumption above the resting level. 4. The results are discussed with reference to the control mechanism of luminescence in mesopelagic and epipelagic fish.

Oxygen consumption and luminescence of Porichthys photophores stimulated by potassium cyanide.

Isolated photophores of Porichthys notatus, maintained in saline at 20 degrees C, do not luminesce and show an oxygen consumption rate of 0.07 +/- 0.01 nmol min-1 photophore-1. In the presence of 10(-6) M-KCN, the photophores do not luminesce but the resting respiration decreases by about 50%. In the presence of 10(-5) M-KCN, some photophores do not luminesce and their respiration rate decreases by about 75%. Others show a response and resting oxygen consumption slowly increases. At high concentration (10(-4) and 10(-3)M), KCN induces a large light emission and increase in oxygen consumption. The stimulatory effect of KCN on the photophore oxygen consumption is tentatively explained by an activation of the luciferin-luciferase system by calcium ions.