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Bioluminescence in the ophiuroid Amphiura filiform is (O.E Miiller, 1776) is not temperature dependant - 37244 o. Bruggeman, S. Dupont, 1. Mallefet Laboratoire de Physiologie Animale & Centre de Recherche sur la Biodiversite, Universite catholique de Louvain, Louvain-Ia-Neuve, Belgium R. Bannister & M.C. Thomdyke Bourne Laboratory, Royal Halloway College, University of London, UK

ABSTRACT: Influence of temperature on the bioluminescence of the ophiuroid was experimentallytested.Amputatedarmsfromindividualsacclimatedat 6° or 14°Cwereincubatedat fivedif- ferent temperatures ranging from 6° to 14°C for 2 hours before KCI 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: Amphiurafiliformis, ophiuroid,bioluminescence, temperature.

INTRODUCTION natural environment of this , the aim of this work was to characterize the short-term (few hours) Amphiurafiliformis (O.P.Miiller, 1776)is a and long-term (a month) effect of temperature on the with a total diameter of 15cm. It lives in the mud of bioluminescence of A.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 ofbiomass per year (Nilsson & Sk61d1996).The species is also bioluminescent and arms emit blue SpecimensofAmphiurafiliformis were collectedin the light when individuals are mechanically stimulated Gullmar Fjord (Fiskebackskill, Sweden) by Petersen (Emson & Herring 1985; Mallefet pers. obs.). When mud grab at 40 meters depth and brought to the present, bioluminescence is always highly adaptive Kristineberg Marine Research Station. The mud was for the luminous organism (Hastings & Morin 1991; filtered on a sifter and non-regenerating specimens of Hastings 1995). Moreover,Herring (1995) suggested Amphiura filiformis were kept. These were that bioluminescence in is alwayslinked placed into the collected mud into deep water sup- to an anti-predatory function. Therefore, one might plied tanks. Brittle stars were taken to the laboratory suggest that luminous properties have an impact on of physiology (Louvain-Ia-Neuve, Belgium) the efficiency of the predation behavior. These prop- where they were kept into aquaria in aerated and fil- erties could be modulated by environmental condi- tered seawater at 14°C for three months. In the ophi- tions. For example, temperature is known to have an uroid natural environment, temperature varied from influence on bioluminescence (see Discussion, Table 6°C in winter to 14°Cin 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 intensity individuals 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 the a three minutes immersion in 3.5% w/w MgCI2 in

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JL 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 incubatedinASWat a giventemperature. crom6° to 14°C, fortwo hours beforestimulation with KCI 200 mM (fig. I). Light responses (fig. 2) were recorded during ~~t I minute using an FBI2 Berthold luminometer linked to a personal computer. Several parameters were measured on each curve: (I) Lmax, the maximal intensity of the luminous ~6°Cl,~-? ~1~Cresponse expressed in megaquanta per second and per mm of arm (in Mq/s.mm);and (2)Ltot (in Mq/mm) is the total amount of light divide by the length of Figure 1. Experimental set-up. arm. (3) TI is the time expressed in second (s) elaps. ing between the stimulation and the beginning of the luminous response; (4) Tlmax (s) is the time betweenthe beginningof the responseand the maximal ~ 15000 intensity. Statisticalanalyses(ANOVA)wereperformed using ~ SAS/STA~ software'scapabilities(SAS Institute 1nl', 10000 1990).

5000 3 RESULTS

0 Tables 1 and 2 show the mean values of the foul 0 0 0 =: ~ ~ (s) I parameters of the amputatedarms luminousresponsl" from specimens accIimated at 6° and 14°C respcr Figure 2. original recording ofluminous response induced tively. In both case, an important variability seems 10 by potassium chloride 200mM (arrow indicates the KCI be the rule for all the parameters and at all the teste.! stimulation, RLU/s: relative light unit per second). temperatures of incubation. However, no significant

Table I. Effect of temperature on the parameters of the luminous response of isolated arms ITom specimens Amphiura filijiJrmis acclimated at 6°C (mean :t standard error; n = 10).

Ltot (Mq/mm) Lmax (Mqls.mrn) TI (s) Tlmax (s)

6°C 31,16:t 10,76 4,30 :t 1,15 0,22 :t 0,06 9,08 :t 3,58 8°C 24,60 :t 9,48 3,19:t 1,00 0,18 :t 0,06 20,00 :t 5,90 10°C 29,77 :t 11,84 3,31:t 1,20 0,18:t 0,Q7 15,94 :t 4,06 12°C 30,67:t 12,94 3,27 :t 1,12 3,00 :t 1,76 16,20 :t 3,10 14°C 25,35 :t 6,31 3,89 :t 0,98 0,16 :t 0,05 21,38 :t 4,46

Table2. Effect of temperature on the parameters of the luminous response of isolatedanns ITomspecimens Amphiurafiliformis acclimated at 14°C (mean :t standard error; n = 10 for each treatment).

Ltot (Mqlmrn) Lmax (Mqls.mrn) Tl (s) Tlmax (s)

6°C 23,60 :t 5,26 4,56 :t 1,43 0,16 :t 0,06 11,12 :t 5,19 8°C 46,17 :t 24,45 3,35 :t 0,61 0,46 :t 0,22 14,18 :t 4,21 10°C 15,58 :t 2,25 2,14 :t 0,52 0,46 :t 021 27,22 :t 5,86 12°C 33,69 :t 9,62 1,98 :t 0,30 0,42 :t 0,21 21,30 :t 4,85 14°C 26,55 :t 4,76 2,23 :t 0,50 0,28 :t 0,18 21,08 :t 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 (NI, no information; 0, no effect; t, increase; ..t.,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 NI t Effect Young& Menscher Abralia trigoniura (Short term) (1980) (Cephalopod) Thysanoessaraschi in VIVO 0-20°C NI t NI Tett (1969) (Euphausiacea) (Short term) Ophiopsila californica in vitro 1O-35°C t ? NI Shimomura (1986) (Ophiuroidea) (photoprotein) (Short term) Amphipholis squamata in VIVO 8-20°C 0 0 NI Dubuisson (1995) (Ophiuroidea) (Short term) in vitro 8-20°C t&..t. t&..t. NI Dubuisson (1995) (dissociated cells) (Short term) in vitro 8-20°C 0 0 NI Dubuisson (1995) (dissociated cells) (15 days) Amphiurafiliformis 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 ofthe temper- selective advantage. ature onthe parameters of the light emission (Table3). As previously described within this species A short-tean incubationofthe 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. intensitywhen temperature increases from 17to 25°C This work constitutes a first step on the study of envi- (Eckert 1966). In cephalopods, short-tean incubation ronmental parameters on the bioluminescence in of the individuals has an effect on the kinetic and the A.filiform is. Further works are in progress in order to color of the emitted light (Young& Menscher 1980). understand this variability. " In the EuphausiaceaThysanoessaraschi,the rate of the light emission increase with temperature (Tett 1969). In the OphiuroidAmphtjJholissquamata, no effectof a ACKNOWLEDGEMENTS short-tean incubationwas observedin vivo (Dubuisson 1995).Nevertheless,in ophiuroids,a similartreatment We gratefully acknowledge support from European has variable effect in vitro on the light emission of Community Program Access to the Research dissociatedphotocytes(Dubuisson 1995)and onphoto- Infrastructure (ARI) at Kristinberg Marine Research proteins (Shimomura 1986). This effect disappeared Station. This research was supported by a FRIA grant whena long-tean acclimationwasappliedtodissociated to S. Dupont. 1. Mallefet is a research associate of photocytes (Dubuisson, 1995). FNRS (Belgium). Contribution to CIBIM. In A.filiformis, short-tean 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 Excitation in Noctiluca. In: confinned the observations carried out in vivo on Bioluminescence in Progress (Johnson & Haneda, eds), A. squamata. Princeton University Press, pp. 271-300.

179 Emson, R.H. & Herring, p.1.1985.Bioluminescence in deep SAS Institute Inc. 1990. SAS/STATUser's guide, Version 6, and shallow water brittlestars. Proc. Int. Echinoderm Fourth edition, Volume I. SAS Institute Inc., North Coni 5: 656. Rotterdam: Balkema. Carolina, United States. Hastings,1.W. 1995. Bioluminescence. In: Cell Physiology, Tett, P.B. 1969. The effect of temperature upon the flash- Academic Press, New York,pp.665--681. stimulated luminescence of the Euphasiid Thysanoessa Hastings, 1.W.& Morin, l.G. 1991. Bioluminescence. In: raschi. J. Mar.Bioi.Ass. u.K. 49: 245-258. Neural and Integrative Physiology (Prosser, C.L., ed.). Young, R.E. & Menscher, EM. 1980. Bioluminescence in Willey-Liss Inc, Chap. 3, pp. 131-170. mesopelagic squid: diet color change during counter- Herring, p.1. 1995. Bioluminescent echinoderms: Unity illumination. Science 208: 1286-1288. of function in diversity of expression? In: R.H. Emson, A.B. Smith & A.C. Campbell (eds), Echinoderm Research 1995. Rotterdam:A.A. Balkema.

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