Research 2001, Féral & David (eds.) ©2003 Swets

Bioluminescence in the ophiuroidAmphiura filiformis (O.F. Müller, 1776) is not temperature dependant 3 7 2 4 4

O. Bruggeman, S. Dupont, J. Mallefet Laboratoire de Physiologie Animale & Centre de Recherche sur la Biodiversité, Université catholique de Louvain, Louvain-la-Neuve, Belgium

R. Bannister & M.C, Thorndyke Bourne Laboratory, Royal Halloway College, University o f London, UK

ABSTRACT: Influence of temperature on the of the ophiuroidAmphiura filiformis was experimentally tested. Amputated arms from individuals acclimated at 6° or 14°C were incubated at five dif­ ferent temperatures ranging from 6° to 14°C for 2 hours before KC1 stimulation. Light response comparisons do not reveal any difference neither for the two temperatures of acclimation nor for the five temperatures of incubation. Therefore, the temperature does not affect the studied parameters of the light emission.

KEYWORDS: filiformis, ophiuroid, bio luminescence, temperature.

1 INTRODUCTION natural environment of this species, the aim of this work was to characterize the short-term (few hours) Amphiura filiformis (O.F. Müller, 1776) is a and long-term (a month) effect of temperature on the with a total diameter of 15 cm. It lives in the mud of bio luminescence A.of filiformis. North European coasts and in the Mediterranean Sea. It constitutes an important fish food resources since it has been estimated that it provides up to 301 metric 2 MATERIAL AND METHODS tons of biomass per year (Nilsson & Sköld 1996). The species is also bioluminescent and arms emit blueSpecimens o fAmphiura filiformis were collected in the light when individuals are mechanically stimulatedGullmar Fjord (Fiskebackskill, Sweden) by Petersen (Emson & Herring 1985; Mallefet pers. obs.). When mud grab at 40 meters depth and brought to the present, b io luminescence is always highly adaptiveKristineberg Marine Research Station. The mud was for the luminous organism (Hastings & Morin 1991filtered ; on a sifter and non-regenerating specimens of Hastings 1995). Moreover, Herring (1995) suggestedAmphiura filiformis were kept. These were that bioluminescence in is always linkedplaced into the collected mud into deep water sup­ to an anti-predatory function. Therefore, one mightplied tanks. Brittle stars were taken to the laboratory suggest that luminous properties have an impact onof physiology (Louvain-la-Neuve, Belgium) the efficiency of the behavior. These prop­ where they W'ere kept into aquaria in aerated and fil­ erties could be modulated by environmental condi­tered seawater at 14°C for three months. In the ophi­ tions. For example, temperature is known to have anuroid natural environment, temperature varied from influence on bioluminescence (see Discussion, Table6°C in winter to 14°C in summer. In order to test the 3). As for other chemical reaction, the reaction rate is long-term effect of temperature on bioluminescence, proportional to the temperature (Eckert 1966; Tett individuals were then separated in two groups: ten 1969). Differences in the colour and in the intensityindividuals being acclimated at 6°C for one month of the emitted light according to the temperature have and ten were maintained at 14°C during this period. also been observed in other phyla (Young & Menscher After this period, ophiuroids were anaesthetized by 1980; Eckert 1966). Since temperature varies in thea three minutes immersion in 3.5% w/w MgCl2 in

177 artificial seawater (ASW). The 5 arms were measured and cut off. In order to test the short-term effect of the temperature of the seawater on the bioluminescence, each arm was incubated in ASW at a given temperature from 6° to 14°C, for two hours before stimulation with KC1 200 mM (fig. 1). Light responses (fig. 2) were recorded during 1 minute using an FBI2 Berthold luminometer linked to a personal computer. Several parameters were measured on each curve (1) Lmax, the maximal intensity of the luminous response expressed in megaquanta per second and per nun o f arm (in Mq/s.mm); and (2) Ltot (in Mq/tnm i is the total amount of light divide by the length ol Figure 1. Experimental set-up. arm. (3) T1 is the time expressed in second (s) elaps ing between the stimulation and the beginning ol the luminous response; (4) Tlmax (s) is the time between the beginning of the response and the maximal intensity. Statistical analyses (ANOVA) were performed using SAS/STAT® software’s capabilities (SAS Institute Inc , 1990).

smo 3 RESULTS

Tables 1 and 2 show the mean values of the four parameters of the amputated arms luminous responses from specimens acclimated at 6° and 14°C respec Figure 2. original recording o f luminous response inducedtively. In both case, an important variability seems b ■ by potassium chloride 200 mM (arrow indicates the KC1 be the rule for all the parameters and at all the tested stimulation, RLU/s: relative light unit per second). temperatures of incubation. However, no significant

Table 1. Effect o f temperature on the parameters of the luminous response o f isolated arms from specimensAmphiura Aliformis acclimated at 6°C (mean ± standard error; n = 10).

Ltot (Mq/mm) Lmax (Mq/s.mm) T l(s) Tlmax (s)

6°C 31,16 ± 10,76 4,30 ± 1,15 0,22 ± 0,06 9,08 ± 3,58 8“C 24,60 ± 9,48 3,19 ± 1,00 0,18 ± 0,06 20,00 ± 5,90 10°C 29,77 ±11,84 3,31 ± 1,20 0,18 ± 0,07 15,94 ± 4,06 12°C 30,67 ± 12,94 3,27 ± 1,12 3,00 ± 1,76 16,20 ± 3,10 14°C 25,35 ± 6,31 3,89 ± 0,98 0,16 ±0,05 21,38 ±4,46

Table 2. Effect of temperature on the parameters of the luminous response of isolated arms from specimensAmphiura filiformis acclimated at 14°C (mean ± standard error; n = 10 for each treatment).

Ltot (Mq/mm) Lmax (Mq/s.mm) Tl(s) Tlmax (s)

6°C 23,60 ± 5.26 4,56 ± 1,43 0,16 ± 0,06 11,12 ± 5,19 8°C 46,17 ± 24,45 3,35 ± 0,61 0,46 ± 0,22 14,18 ± 4(21 10aC 15,58 ± 2,25 2,14 ± 0,52 0,46 ± 021 27,22 ± 5,86 12°C 33,69 ± 9,62 1,98 ± 0,30 0,42 ± 0,21 21,30 ±4,85 14°C 26,55 ± 4,76 2,23 ± 0,50 0,28 ± 0,18 21,08 ± 3,29

178 Table 3. Comparison of the effect of temperature (short or long term effects) on the parameters of the light emission in various species (Nl, no information; 0, no effect; Î , increase;4-. decrease).

Species Temperatures Lmax Kinetic Color Reference

Noctiluca miliaris in vivo 17 or 25°C t T NI Eckert (1966) (Dinoflagellate) (Short term) Abraliopsis sp. in vivo 6-25°C Nl T Effect Young & Menscher Abralia trigoniura (Short term) (1980) (Cephalopod) Thysanoessa raschi in vivo 0-20°C NI 1 NI Tett (1969) (Euphausiacea) (Short term) Ophiopsila californica in vitro 10--35°C T ? NI Shimomura (1986) (Ophiuroidea) (photoprotein) (Short term) Amphipholis squamata in vivo 8-20°C 0 0 Nl Dubuisson (1995) (Ophiuroidea) (Short term) in vitro 8-20 °C T & i t & i Nl Dubuisson (1995) (dissociated cells) (Short term) in vitro 8-20°C 0 0 NI Dubuisson (1995) (dissociated cells) (15 days) Amphiura filiformis in vivo 6-14°C 0 0 NI Present work (Ophiuroidea) (Short term) in vivo 0 0 0 NI Present work

difference was observed between the two groups and Both these species seems to be well adapted to the five temperatures of incubation. variations of temperature since bioluminescent properties remain constant in the natural range of temperature variations. Since bioluminescence might probably be linked to an anti-predatory function in 4 DISCUSSION these species, this capability to minimize the effect of temperature on light emission provides an important Several authors have studied the effect of the temper­ selective advantage. ature on the parameters of the light emission (Table 3). As previously described within this species A short-term incubation of the dinoflagellateNoctiluca (Dupont et al. 2001 and this volume), an important miliaris induces a faster light response of higher variability was observed in all the tested conditions. intensity when temperature increases from 17 to 25°CThis work constitutes a first step on the study of envi­ (Eckert 1966). In cephalopods, short-term incubation ronmental parameters on the bioluminescence in of the individuals has an effect on the kinetic andA. the filiformis. Further works are in progress in order to color of the emitted light (Young & Menscher 1980). understand this variability. In the EuphausiaceaThysanoessa raschi, the rate of the light emission increase with temperature (Tett 1969). In the OphiuroidAmphipholis squamata, no effect of a ACKNOWLEDGEMENTS short-term incubation was observedin vivo (Dubuisson 1995). Nevertheless, in ophiuroids, a similar treatmentWe gratefully acknowledge support from European has variable effect in vitro on the light emission of Community Program Access to the Research dissociated photocytes (Dubuisson 1995) and on photo­Infrastructure (AR1) at Kristinberg Marine Research proteins (Shimomura 1986). This effect disappeared Station. This research was supported by a FRIA grant when a long-term acclimation was applied to dissociated to S. Dupont. J. Mallefet is a research associate of photocytes (Dubuisson, 1995). FNRS (Belgium). Contribution to CIBIM. InA. filiformis, short-term incubation (comparison of the 5 tested temperature) and long-term acclimation (comparison between the two groups of animals) REFERENCES have no effect on the parameters of the light emission in the range of tested temperatures. These results Eckert, R. 1966. Luminescence ExcitationNoctiluca. in In: confirmed the observations carried outin vivo on Bioluminescence in Progress (Johnson & Haneda, eds), A. squamata. Princeton University Press, pp. 271-300.

179 Emson, R H. & Herring, P.J. 1985. Bioluminescence in deepSAS Institute Inc. 1990. SAS/STAT User’s guide, Version 6, and shallow water brittlestars.Proc. Int. Echinoderm Fourth edition. Volume 1. SAS Institute Inc., North Conf. 5: 656. Rotterdam: Balkema. Carolina, United States. Hastings, J.W. 1995. Bioluminescence.Cell In: Physiology, Tett, P.B. 1969. The effect of temperature upon the flash- Academic Press, New York, pp.665-681. stimulated luminescence of the EuphasiidThysanoessa Hastings, J.W. & Morin, J.G. 1991. Bioluminescence. In: raschi. J. Mar. Biol. Ass. U.K. 49: 245-258. Neural and Integrative Physiology (Prosser, C.L., ed.). Young, R.E. & Menscher, F.M. 1980. Bioluminescence in Willey-Liss Inc, Chap. 3, pp. 131-170. mesopelagic squid: did color change during counter­ Herring, P.J. 1995. Bioluminescent echinoderms: Unityillumination. Science 208: 1286—1288. o f function in diversity o f expression? In: R.H. Emson, A.B. Smith & A.C. Campbell (eds), Echinoderm Research 1995. Rotterdam: A.A. Balkema.

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