Luminescence in ophiuroids (Echinodermata) does not share a common nervous control in all species
Laboratory of Animal Physiology, Catholic University of Louvain, Bâtiment Carnoy, 5 Place Croix du Sud, B-1348 Louvain-la-Neuve, Belgium
*e-mail: dewael{at}bani.ucl.ac.be
Accepted 2 January 2002
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Summary |
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Key words: echinoderm, ophiuroid, bioluminescence, nervous control, acetylcholine, muscarinic receptor, nicotinic receptor, Amphiura filiformis, Ophiopsila aranea, Ophiopsila californica.
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Introduction |
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The aim of this work was to investigate nervous control mechanisms of luminescence in three other ophiuroid species (Amphiura filiformis, Ophiopsila aranea and O. californica) and hence to find out whether they share common signalling pathways, leading to light emission.
A. filiformis (O. F. Müller 1776) is a rapidly growing suspension feeder brittlestar frequently found on sub-tidal bottoms off the coasts of Europe and of the Mediterranean Sea. This burrowing ophiuroid is a dominant species in the benthic shelf ecosystem, especially in the northeastern part of the North Atlantic region (Josefson, 1995). It has been shown that arms of this species represent an important food source for flatfishes (Duineveld and Van Noort, 1986
). Although it has a high predatory rate, A. filiformis has a surprisingly long life span (up to 25 years) according to Muus (1981
). This can be explained by its ability to rapidly regenerate chopped arms (Wilkie, 1978
; Bowner and Keegan, 1983
) (J. Mallefet, unpublished results).
Despite numerous eco-ethological investigations on A. filiformis (see Josefson, 1995; Loo et al., 1996
; Sköld and Rosenberg, 1996
; Nilsson and Sköld 1996
; Rosenberg and Selander, 2000
) nearly nothing is known about its capability to produce light. Emson and Herring (1985
) reported the first data on A. filiformis bioluminescence: light emission is blue in colour, it appears to be intracellular and the luminous cells, called photocytes, are restricted to the arm spines. No physiological data are available concerning the control of light emission of A. filiformis.
O. aranea (Forbes 1843) inhabits the encrusting coralline algae zone (coralligene) in the Mediterranean Sea. Some morphological studies described the luminescence sites as originating from glandular cells located on lateral and ventral plates, and in some spines of the arms, next to the disc (Mangold, 1907; Reichensperger, 1908
; Trojan, 1909
). Later, Harvey (1952
) mentioned a yellowish green fluorescence at the sites of luminescence. The exact nature of luminous cells remains unknown. Mallefet and Dubuisson (1995
) described the KCl-induced luminescence as a series of flashes whose maximal intensity increases as a function of KCl concentration.
O. californica (Clarck 1921) is a sand-dwelling ophiuroid found along the Californian coast. Previous work has shown that luminescence is used as an aposematic signal (Basch, 1988) and that photocytes, of nervous origin, are located in the arms. Light emission seems to be under nervous control (Brehm, 1977
; Brehm and Morin, 1977
) and requires the presence of calcium (Brehm, 1977
).
These ophiuroid species were chosen for this comparative study since they belong to two different families (Amphiuridae for A. filiformis, Ophiocomidae for O. aranea and O. californica) and they live in two different types of habitat (in mud or sand for A. filiformis and O. californica, in coralligene for O. aranea). Our results show that control mechanisms of light emission differ from species to species; a cholinergic system appears to be involved in light emission of A. filiformis, but the nature of luminous control in Ophiopsila sp. remains undetermined.
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Materials and methods |
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Experiments on arm segments
After anaesthesia of the animals by immersion in 3.5 % MgCl2 in ASW, arms were isolated from the disc and divided into segments of 8 articles (Ophiopsila) or 20 articles (Amphiura), which were then rinsed in ASW (NaCl 400.4 mmol l1, CaCl2 9.9 mmol l1, KCl 9.6 mmol l1, MgCl2 52.3 mmol l1, Na2SO4 27.7 mmol l1, Tris 20 mmol l1, pH 8.3).
Stimulations
Stimulations were performed by injection of drugs onto arm segments. Light emission was measured with a FB12 Berthold luminometer linked to a PC-type computer. For each experimental protocol, one arm segment was treated with the control stimulus (200 mmol l1 KCl or 103 mol l1 ACh), while the other preparations were stimulated with the tested drug.
Drugs
The following drugs were used in this study: acetylcholine chloride (Sigma), adenosine (Sigma), adenosine 5'-triphosphate (ATP; Sigma), L-adrenaline (Fluka), 4-aminobutyric acid (GABA; Aldrich), 2-aminoethylsulfonic acid (taurine; Fluka), atropine (Sigma), carbamylcholine (carbachol; Janssen Chimica), 4-diphenylacetoxy-N-methyl peperidine (4-DAMP methiodide; ICN), 1,1-dimethyl-4-phenyl piperazium iodide (DMPP; ICN) eserine (Sigma), L-glutamic acid hydrochloride (glutamate; Sigma), glycine hydrochloride (Sigma), hexamethonium dichloride (RBI), hydroxylamine hydrochloride (Sigma), 5-hydroxytryptamine (5-HT; Sigma), 5-hydroxytyramine hydrochloride (dopamine; Sigma), McN-A-343 (RBI), L-noradrenaline hydrochloride (Fluka), pirenzepine dihydrochloride (RBI), SALMFamide 1 and SALMFamide 2 (provided by M. Thorndykes laboratory), sodium nitroprusside (Sigma), tubocurarine chloride (Janssen Chimica). All solutions were diluted in ASW. The concentrations used ranged from 106 to 103 mol l1. These rather high concentrations are commonly used in echinoderms because of the heavy calcification of the ophiuroid arms, which impairs adsorption and penetration to the photocytes.
Statistics
Statistical analyses (ANOVA) were performed using SAS (Statistic Analysis System).
Photogenesis characterization
Different parameters were used in order to characterize the photogenesis. (1) Lmax, the maximum level of light emission expressed as a percentage of the control; (2) LT, latency time, the time between stimulation and the beginning of the light emission; (3) TLmax, the time between onset of light production and maximum light emission.
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Results |
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In our experiments, arm segments treated for 10 min with 103 and 104 mol l1 eserine were then stimulated by 103 and 104 mol l1 ACh, respectively. Photogenesis was compared to the control, without eserine. Treatment with eserine alone did not trigger luminescence. The results showed no effect of 104 mol l1 eserine on ACh-induced luminescence. However, treatment of the arm segments with 103 mol l1 eserine reduced the ACh-induced light emission by 74 % (data not shown).
Effects of cholinergic antagonists
To identify the receptors involved in the control of light emission, we tested the effects of different cholinergic antagonists, including atropine, which selectively blocks cholinergic muscarinic receptors, and tubocurarine and hexamethonium, which block cholinergic nicotinic receptors.
Arm segments were treated with cholinergic antagonists for 10 min, whereas the controls were immersed in normal ASW. All the segments were then stimulated with 103 mol l1 ACh. Fig. 4 shows the effect of atropine and tubocurarine on luminescence at concentrations ranging from 106 to 103 mol l1. The doseresponse curve with atropine showed a gradual decrease of ACh-induced light emission; at 104 mol l1, 26±13 % of the control luminescence remains (P<0.05; N=8), while a total inhibition of the response occurred at 103 mol l1 (P<0.01; N=23). With tubocurarine, the doseresponse curve showed also a gradual decrease, only 29±9 % remaining at 103 mol l1 (P<0.01; N=24). A similar decrease of ACh-induced luminescence was observed with 103 mol l1 hexamethonium (result not shown).
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Pirenzepine, an M1 muscarinic antagonist, and 4-DAMP, an M2-M3 muscarinic antagonist, were applied using the same experimental protocol as above. Fig. 5 shows the effects of pirenzepine at different concentrations on luminescence triggered by 103 mol l1 ACh; pre-treatment of the arm segments with pirenzepine did not induce spontaneous luminescence. Although a progressive inhibition of light emission was observed, only 103 mol l1 pirenzepine significantly inhibited photogenesis (P<0.01; N=30). In the case of 4-DAMP, luminescence was induced before ACh injection in all trials, from 106 mol l1 to 103 mol l1. As shown in Fig. 6, 103 mol l1 4-DAMP triggered a light emission not significantly different from the control (83.5±18.0 %; N=23) while 106 mol l1 4-DAMP-induced luminescence was only 7.66 % of the control (N=10). The further photogenesis induced by 103 mol l1 ACh was equally inhibited by 105103 mol l1 4-DAMP. With 106 mol l1 4-DAMP, 57.5±33.5 % of the control photogenesis was produced (N=10), but this decrease in amplitude was not significantly different from the control.
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Discussion |
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Differences in amplitude of light emission might reveal either a variation in the amount of luminescent tissue in the arms, or a variation in the quantity of substrate for the light reaction in the luminescent cells. Because light emission is measured from all the photocytes of the entire arm segment taken together, it is impossible to distinguish between these two hypotheses.
Screening of neuromediators
ACh triggered a significant amount of light in A. filiformis, but only very weak photogenesis was produced in O. californica and none in O. aranea. The difference in intensity between KCl- and ACh-induced luminescence suggests that other neuromediators or neuromodulators are involved in luminescence control of A. filiformis. Moreover, since ACh did not trigger systematic luminous responses in O. aranea and O. californica, we tested several neuromediators, commonly found in echinoderms, to try to identify the nature of the luminous control mechanisms. In A. filiformis, taurine, 5-HT and dopamine occasionally triggered a weak luminescence, whose intensity did not exceed 3 % of that induced by KCl. This might reinforce the hypothesis that ACh is not the only neuromediator and that some drugs could act as neuromodulators of the luminous response. These neuromodulators could either act directly on the photocyte, or lead to activation of ACh release from the nervous system. Similar observations have been made in the ophiuroid Amphipholis squamata, where ACh is the main transmitter and some neuromodulators (GABA, glycine, catecholamines, ATP, adenosine) either increase or decrease the light emission (De Bremaeker et al., 1999a,b
,c
). In Ophiopsila species, only taurine (for O. aranea) and ACh, carbachol, taurine and dopamine (for O. californica) triggered a weak light emission. Luminescence intensity is so low, compared to that induced by KCl, that these drugs could not be considered as main neuromediators, but perhaps act synergically with another still-unidentified compound. Further experiments are in progress to try to identify the main neurotransmitter(s) involved in the luminous control of O. aranea and O. californica.
Effects of acetylcholine
Only arm segments from A. filiformis responded to ACh stimulation by emitting light in nearly all trials. The pattern of the light emission evoked by ACh was different from that evoked by KCl: both latency time and time to reach maximal intensity of light were smaller with ACh. Moreover, the amplitude of the KCl response was much higher than the response to ACh. To explain these differences, it could be assumed that cholinergic luminescence is mediated through cholinergic receptors, whereas KCl luminescence is due to a general depolarisation of the photogenic cells and of the nervous tissue controlling the photocytes. This was also the case with A. squamata, another luminous ophiuroid belonging to the same family (Amphiuridae). In this species, 103 mol l1 ACh triggered light emission about 10- to 100-fold lower than with KCl, according to the colour variety of the ophiuroids (De Bremaeker et al., 1996). In A. filiformis, 103 mol l1 ACh triggered a light emission whose intensity reached 26 % of the KCl response. Moreover, ACh at concentrations of 106104 mol l1 also initiates luminescence in A. filiformis. At these lower concentrations, both the intensity and the number of arm segments responding decreased. The difference in intensity between KCl- and ACh-induced photogenesis could be explained by their mechanism of action. The KCl peak of light may result from simultaneous recruitment of a large number of photocytes, by general depolarisation of the photogenous tissues and of the nervous tissues controlling photocytes, while ACh may diffuse progressively through the surrounding tissues. The concentration of ACh reaching the photogenic tissue may be low and, as a consequence, trigger a weaker intensity of flashes. A similar phenomenon has been observed in the ophiuroid Amphipholis squamata (De Bremaeker et al., 1996
) and in the starfish Asterias rubens, where ACh contraction of the tube feet was about 1000-fold stronger on tube feet whose epithelium was removed, thus lowering diffusion distance, compared to intact tube feet (Protas and Muske, 1980
). Moreover, ACh acts through cholinergic receptors, which are subject to positive and negative neuromodulation. Another hypothesis to explain the low response to ACh compared to KCl, is that exogenous ACh might be quickly hydrolysed by endogenous acetylcholinesterase before reaching the photocytes. This phenomenon has been observed in A. squamata, where pre-treatment of the arm with the anticholinesterase drug eserine significantly increased, by up to 100-fold, the maximal amplitude of light emitted by ACh (De Bremaeker et al., 1996
). But this hypothesis was not supported by results in A. filiformis since pre-treatment of arm segments by eserine (at 104 mol l1) did not affect or even (at 103 mol l1) inhibit the ACh-induced luminescence. This inhibitory effect of 103 mol l1 eserine could be due to increased ACh availability for a putative inhibitory receptor. This unexpected effect of eserine has not been reported in the literature.
Effects of cholinergic drugs
Both muscarinic and nicotinic antagonists inhibited light emission of A. filiformis. Consequently, it seems that both cholinergic muscarinic and nicotinic receptors are involved in the photogenesis of A. filiformis. Muscarinic receptors might be predominant since light emission was inhibited more strongly by the muscarinic antagonist atropine than by tubocurarine and hexamethonium. Similar observations have been reported in other tissues of echinoderms: tube foot muscle of the starfish Asterias amurensis (Protas and Muske, 1980), viscosity of the body wall of the sea cucumber Holothuria leucospilota (Motokawa, 1987
) and longitudinal muscle of the body wall of the sea cucumber Sclerodactyla briareus (Devlin et al., 2000
) are controlled by both nicotinic and muscarinic drugs. On the contrary, only muscarinic receptors are involved in the luminescence control of the ophiuroid Amphipholis squamata (De Bremaeker et al., 1996
).
The results obtained with M1 muscarinic agonists and antagonists suggest that ACh luminescence is partially mediated through the activation of M1 subtype muscarinic receptors in A. filiformis. The systematically higher intensity of light emitted by 106103 mol l1 McN, compared to 106103 mol l1 ACh, seems to bring out the existence of an inhibitory modulation, using another subtype of cholinergic receptor. The M2/M3 muscarinic antagonist 4-DAMP gave unexpected results since it triggered photogenesis itself, at concentrations ranging from 106 to 103 mol l1. Although there is no mention in the literature of any agonist effect of 4-DAMP, some drugs can act either as agonists or antagonists, according to the animal species studied. Baguet and Marechal (1978) showed that propranolol, a common ß-adrenergic antagonist, triggered light emission by isolated photophores from Argyropelecus hemigymnus, and an antagonistic effect of synthetic
-adrenoceptor agonists has been shown on isolated artery strips from Gadus morhua (Johansson, 1979
). Moreover, 4-DAMP inhibited ACh-induced luminescence with the same efficiency at concentrations from 105 to 103 mol l1. This inhibition could be due to the former light emission triggered by 4-DAMP, leading to a partial exhaustion of the luminous capabilities, or it could suggest that 4-DAMP blocks all M2/M3 receptors, even at concentrations as low as 105 mol l1. The remaining light emitted may be produced by the stimulation through another subtype of cholinergic receptor. It appears then that M2/M3 muscarinic receptors might also be involved in the luminous control of A. filiformis. Further experiments are planned in order to confirm the inhibitory effect of 4-DAMP on ACh-induced luminescence, using another specific M2/M3 antagonist that does not trigger light during the pre-treatment.
It must be pointed out that specific muscarinic antagonists or agonists used in this study have been demonstrated to have specific effects on mammalian tissues. We cannot rule out the possibility that muscarinic receptors in invertebrates, such as ophiuroids, are somewhat different from those encountered in mammalian tissues. Onai et al. (1989) have shown that invertebrate genes from Drosophilia melanogaster coding for muscarinic receptors showed only 60 % homology with the five vertebrate subtypes. Therefore, we have to be cautious in extrapolating pharmacological results from mammalian to invertebrate tissues.
In conclusion, we propose that ACh is the main transmitter controlling the luminescence in A. filiformis. Both nicotinic and muscarinic receptors seem to be involved. In Ophiopsila species, other mechanisms might act to trigger light emission. Therefore, we can postulate the absence of a common signal transmission pathway, leading to luminescence in all ophiuroid species.
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Acknowledgments |
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References |
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