International Journal for Parasitology 17 "0887# 0302Ð0313

Symbioses in Amphipholis squamata "Echinodermata\ Ophiuroidea\ Amphiuridae#] geographical variation of infestation and e}ect of symbionts on the host|s light production Dimitri Deheyna\"\ Nikki A[ Watsonb\ Michel Jangouxa\ c

aLaboratoire de Biologie marine "CP 059:04#\ Universite# Libre de Bruxelles\ 49 av[ F[D[ Roosevelt\ B!0949 Bruxelles\ Belgium bDivision of Zoology\ School of Biological Sciences\ University of New England\ Armidale\ NSW 1240\ Australia cLaboratoire de Biologie marine\ Universite# de Mons!Hainaut\ 08 av[ Maistriau\ B!6999 Mons\ Belgium

Received 16 March 0887^ received in revised form 02 May 0887^ accepted 02 May 0887

Abstract

Populations of the polychromatic and bioluminescent species Amphipholis squamata from eight locations were examined for internal and external symbionts[ At three locations "two in the United Kingdom and one in Papua New Guinea#\ no symbionts were present\ while four species were recovered from the remaining locations] Cancerilla tubulata and Parachordeumium amphiurae "copepods#\ Rhopalura ophiocomae "orthonectid# and an undescribed species of rhabdocoel turbellarian[ No ophiuroid individual hosted more than one symbiont species\ despite the presence of two or more within a population[ Symbiont presence and prevalence varied with location\ and with colour variety\ but with no apparent pattern or trends[ Light!production characteristics of the host were a}ected by the presence of all symbionts except C[ tubulata[ These e}ects\ however\ did not vary between colour varieties or between geographical locations\ but were speci_c to the symbiont species] the presence of P[ amphiurae resulted in enhanced intensity of light production\ while that of R[ ophiocomae and the turbellarian species resulted in reduced intensity[ The kinetics of light production "time until maximum output# were altered only by the presence of the turbellarian[ Changes in the light!production characteristics are discussed in relation to morphological\ energetical and physiological e}ects of the symbioses[ Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[

Keywords] Amphipholis squamata^ Echinoderm symbioses^ Geographical distribution^ Host luminescence^ Ophiuroid symbionts^ Physio! logical reaction

0[ Introduction of the nature of the hostÐsymbiont relationship ð0\ 1Ł[ One echinoderm species may host a parasite\ a Echinoderms act as hosts to a wide variety of commensal\ or both\ and most symbionts do not symbionts\ separately or simultaneously\ regardless appear to be mutually exclusive ð0\ 2Ł[ Echinoderm symbionts vary in degree of host speci_city from monoxenous symbionts\ such as copepods\ to polyxenous symbionts\ such as ciliates[ Multiple " Corresponding author[ Tel] "¦21# 1 549 11 23^ Fax] "¦21# hosts\ however\ are always of the same echinoderm 1 549 16 85^ e!mail] ddeheynÝulb[ac[be[ class ð1Ł[

9919!6408:87:,08[99 Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[ PII] S9919!6408"87#99008!1 0303 D[ Deheyn et al[ : International Journal for Parasitolo`y 17 "0887# 0302Ð0313

The cosmopolitan ophiuroid Amphipholis squa! mata has a wide range of tolerance towards symbionts\ with as many as 04 di}erent organisms reported in association with individuals[ These con! sist of copepods "seven species#\ ciliates "four species#\ polychaetes "two species#\ orthonectids "one species# and a turbellarian "one species\ _rst mentioned here#[ The associations have been reported from individuals of various seas and oce! ans in accordance with the wide distribution of this ophiuroid ð0\ 3Ł[ Symbiont life!cycles and the _ne morphological interface of the hostÐsymbiont relationship have been investigated for several spec! ies ð1\ 4Ł[ Impacts of infestation on the ophiuroid population structure have also been examined\ notably by investigating the in~uence of symbiont infestations on the ophiuroid life!cycle\ and its brooding and regenerative abilities ð5Ð09Ł[ E}ects on the ophiuroid physiology or metabolism\ however\ have been little studied as indicated by the fact that only one symbiont species has been assessed for its e}ect on the energetical activity of Fig[ 0[ Amphipholis squamata[ The eight geographical locations the ophiuroid ð6Ł[ that were investigated\ and occurrence of the four symbionts Amphipholis squamata is a polychromatic species considered] "0# Cancerilla tubulata "copepod#^ "1# Para! with various colour varieties present in distinct chordeumium amphiurae "copepod#^ "2# Rhopalura ophiocomae numbers and proportions at di}erent locations ð00\ "orthonectid#^ "3# the new species of rhabdocoel turbellarian^ "9# Absence of symbionts[ 01Ł[ Individuals have long been reported to produce light ð02\ 03Ł\ with light!production characteristics varying between colour varieties\ and between brooding and non!brooding individuals ð01Ł[ Other biological features\ such as concentration of neuro! from eight di}erent locations in various peptides\ types of neuroreceptors on photocytes\ geographical regions "Fig[ 0#\ i[e[ in France life!cycle\ reproductive pattern and genotype\ are "Langrune!sur!Mer and Rosco}#\ the U[K[ known to di}er between individuals of di}erent "Wembury\ Prawle Point\ Millport and varieties ð04Ł\ and also between individuals from New Haven#\ New Zealand "Portobello# and Papua widely distant locations "ð05Ł^ D[R[ Murray\ New Guinea "Laing Island#[ All locations were Macrogeographic genetic variation in the cosmo! politan brooding brittle star\ Amphipholis squa! intertidal "from mid! to upper!intertidal\ except for mata[ Master thesis\ Mount Allison University\ Prawle Point where specimens were collected from 0878#[ We aimed to determine whether symbioses tidal pools#\ and individuals were collected at low in A[ squamata varied with host colour variety or tide by hand using _ne forceps "the ophiuroid is geographic location of the population\ and what small\ adult specimens having a disc diameter of e}ect the di}erent symbionts had on the host light! approx[ 2 mm with arms approx[ 04 mm long#[ The production characteristics[ habitats consisted of muddy or detritic sand under boulders or mussels beds\ except for Prawle Point 1[ Materials and methods were the ophiuroid was found amongst algae Coral! lina of_cinalis[ All individuals found at each search Individuals of A[ squamata "Delle Chiaje\ 0717# were collected\ regardless of microhabitat\ size or were collected between March 0882 and June 0885 colour variety[ D[ Deheyn et al[ : International Journal for Parasitolo`y 17 "0887# 0302Ð0313 0304

1[0[ Measurement of infestation variety\ and examined within 01 h for the presence of the following four symbionts] "0# the copepod Collected individuals were transported to the Cancerilla tubulata Dalyell\ 0740 "Fig[ 1#\ which is laboratory in seawater\ sorted according to colour an ectosymbiont that clings to the oral base of the

Figs 1Ð4[ Scanning electron micrographs of the four symbionts from Amphipholis squamata "As#[ Fig[ 1[ Cancerilla tubulata "Ct# attached to the host[ Fig[ 2[ Parachordeumium amphiurae "isolated specimen#[ Fig[ 3[ Rhopalura ophiocomae "Ro# in the host[ Fig[ 4[ The new rhabdocoel turbellarian species "isolated specimen#[ 0305 D[ Deheyn et al[ : International Journal for Parasitolo`y 17 "0887# 0302Ð0313 ophiuroid arm ð06Ł^ "1# the copepod Para! 1[2[ Measurement of bioluminescence chordeumium amphiurae He#rouard\ 0895 "Fig[ 2#\ which lives in the bursa of infested individuals Only arms produce light in the ophiuroid^ there! that are then recognisable by an external swelling fore light production was measured from isolated and a pore that the symbiont induces on the ophi! arms of infested individuals[ "When the symbiont uroid disc\ and also by an orange tint of the sym! C[ tubulata occurred it was removed prior to light biont visible through the ophiuroid skin ð5Ł^ "2# measurement[# Light production was also measured the orthonectid Rhopalura ophiocomae Giard\ 0766 from 01 to 04 non!infested individuals of each "Fig[ 3#\ which infests the coelom and the coelom variety\ from each collection[ When locations were wall ð4Ł^ and "3# a new species of rhabdocoel tur! sampled several times\ results from the di}erent bellarian "in course of description# that occurs in collections were pooled[ Light production was the ophiuroid haemal system "Fig[ 4#[ There are always measured within 01 h of collection\ from no external signs of the presence of the orthonectid adult individuals with intact arms "i[e[ non!regen! and the turbellarian "ð4Ł^ Deheyn\ personal obser! erating# with disc diameter 1Ð2 mm[ Individuals vation^ respectively#[ The disc of every ophiuroid were anaesthetised by immersion for 2 min in a was therefore dissected to check for their presence\ 2[4) MgCl1 solution and the length of arms mea! which allowed simultaneous detection\ under the sured before separation from the disc using a _ne compound microscope\ of gonads and brooded scalpel[ The disc was dissected for symbionts\ and juveniles[ the presence of brooded juveniles and developed gonads determined\ while each isolated arm was measured for bioluminescence[ The luminous reac! 1[1[ Morphological observation tion was triggered using KCl 199 mM so that light production was maximal and unimodal\ and Infested ophiuroids were investigated by light occurred within 079 s following KCl addition ð01\ microscopy to precisely localize the symbionts and 19Ł[ Light was detected in a dark room at each to determine ophiuroid gonad development[ A research station\ using a portable system consisting qualitative assessment was made of gonad size rela! of a phototube IP10!S19 connected to a pho! tive to {{normal|| in uninfested adult individuals[ tomultiplier IL 659\ the signal then passing through Some degree of reduction was noted as partial cas! a radiometer IL 0699 before being graphically ana! tration\ while complete absence of testis or ovary lysed using a Servogor S recorder ð01Ł[ The exper! constituted total castration[ Isolated symbionts\ as imental device was calibrated once a week\ using a well as individuals in association with the ophi! tritium!phosphor light source of known intensity uroid\ were _xed in 2) glutaraldehyde in caco! within the spectrum of the ophiuroid luminescence dylate bu}er "9[0 M\ pH 6[7\ adjusted to ð01Ł[ Two parameters were used to characterise the 0929 mOsM with NaCl# at 3>C for 01 h\ dehydrated production\ "0# the maximum light intensity LMax\ in graded ethanol\ critical!point dried "using CO1 expressed in megaquanta produced per s per mm as transition ~uid#\ sputter coated with gold and of arm "Mq s−0 mm−0#\ which re~ects energy associ! observed with a JEOL JSM 5099 scanning electron ated with the maximal ~ux of produced photons microscope[ Specimens for histology were _xed in normalised per arm length\ and "1# the time TLMax 2) glutaraldehyde in cacodylate bu}er\ rinsed in to reach the maximum light intensity\ expressed bu}er\ post!_xed for 29 min in 0) osmium tet! in s\ which characterises kinetic e.ciency of the roxide in the same bu}er\ and rinsed again[ They biological pathway leading to the maximal light were then decalci_ed in the dark for 01Ð04 h at 3>C production[ When there were su.cient numbers of in 1) ascorbic acid in 9[2 M!NaCl ð07Ł\ dehydrated infested individuals to enable statistical analyses in graded ethanol\ and embedded in spurr resin[ regarding their variety and location\ di}erences in Semi!thin sections "9[2 mm# were stained with a 0]0 luminescence intensity and kinetics between methylene blueÐazur II mixture ð08Ł and examined infested vs non!infested individuals were tested for using a Leitz laborlux D light microscope[ signi_cance using nested analysis of variance D[ Deheyn et al[ : International Journal for Parasitolo`y 17 "0887# 0302Ð0313 0306

"Nested ANOVA\ arms being nested within indi! of 00 di}erent colour varieties "orange\ beige\ dark vidual# and multiple mean comparison test "Tukey# brown\ grey\ black\ spotted\ black!brown\ ochre\ "P³9[94 for all analyses#[ Brooding in~uences the speckled\ brown!grey and pale beige as listed in ophiuroid light!production performance ð01Ł\ so order of increasing light!production performance#[ results from brooding and non!brooding indi! The number of colour varieties present per location viduals were analysed separately[ ranged from one to six "Table 0#[ It was observed from locations sharing varieties "e[g[\ Langrune! sur!Mer and Rosco}\ or Prawle Point and Por! 2[ Results tobello# that the symbionts did not preferentially infest one or another variety[ The most heavily 2[0[ Geographical variation in infestation infested varieties at the di}erent locations were the grey one at Langrune!sur!Mer "03[8)#\ the black Table 0 summarises all data regarding A[ squa! one at Rosco} "05[4)#\ the beige one at Prawle mata infestations for the colour varieties in the vari! Point "03[4)#\ and the beige one at Portobello ous locations[ No symbionts were found in:on "00[2)#[ Some varieties were infested at one individuals of A[ squamata from three of the eight location but not at another "compare the black locations investigated\ i[e[ at New Haven "U[K[#\ variety at Langrune!sur!Mer and Rosco}#\ and Millport "U[K[# and Laing Island "Papua New Gui! individuals from the orange variety were never nea#[ At the remaining _ve locations\ infestation found to be infested while those of the dark brown among locations varied qualitatively "symbiont and grey varieties were infested at all locations[ species di}ered# and quantitatively "the same sym! Considering each location separately\ distribution biont infested di}erent numbers of individuals#[ of the symbionts was not homogenous among the Total infestation with all symbionts was highest at colour varieties\ as some were heavily infested and Wembury "U[K[# "04[0)#\ and lowest at Rosco} some lightly[ This was especially conspicuous for "France# "7[5)#[ Of the four symbionts found\ three the grey and the black varieties at Langrune!sur! occurred at more than one location\ while the Mer "03[8 and 9) of infestation\ respectively# and orthonectid R[ ophiocomae was found only at Lan! for the black and the beige ones at Rosco} "11[7 grune!sur!Mer "France#[ When the same symbiont and 4[0)\ respectively#[ occurred at several locations\ its prevalence varied with location\ e[g[\ P[ amphiurae infestation ranged 2[2[ Effect of infestation on light!production charac! from 9[6) at Portobello to 04[0) at Wembury[ teristics At two locations\ only one symbiont species was present\ namely P[ amphiurae\ at Wembury and Amphipholis squamata has the ability to produce Prawle Point[ At the other three locations\ two or light and the photocytes "i[e[ the light!producing three symbiont species were present in the popu! cells# occur in each arm segment at the periphery of lation but\ remarkably\ the di}erent species were the nerve ganglion that lies under each spine "there never found together in a single host individual[ are six spines and thus six spinal ganglia per arm The total intensity of infestation at a location was segment ð10Ł#[ All nerve ganglia are interconnected unrelated to the number of symbiont species occur! through the main radial nerve cord ð10\ 11Ł[ Since ring at that location[ For example\ the highest total the latter lies between two perihaemal sinuses and infestation "04[0) at Wembury# was due to a single the spinal ganglia are close to coelomic spaces\ it is symbiont species\ while a lower infestation "7[5) believed that both neuronal and endocrinal factors at Rosco}# was composed of two species[ may a}ect the light production of photocytes\ and thus there may be two avenues of in~uence resulting 2[1[ Variation in infestation with colour variety from the presence of a symbiont[ The light production was a}ected by three of Specimens of A[ squamata from the eight the four symbioses investigated[ The e}ect of each locations investigated were categorised into a total particular symbiont on host bioluminescence was 0307 D[ Deheyn et al[ : International Journal for Parasitolo`y 17 "0887# 0302Ð0313

Table 0 For the eight locations investigated\ number and percentage of Amphipholis squamata individuals that were non!infested and infested by the four symbionts considereda

Infested individuals

A[ squamata Non!infested C[ tubulatab P[ amphiurae R[ ophiocomae n[sp Station variety individuals "crustacean# "crustacean# "orthonectid# "turbellarian# Total

Laing Island Pale beige 276 "099)# * * * * 276 "Papua New Guinea# New Haven "U[K[# Beige 519 "099)# * * * * 519 Black!brown 320 "099)# * * * * 320 Total 0940 "099)# * * * * 0940 Millport "U[K[# Beige 516 "099)# * * * * 516 Black!brown 324 "099)# * * * * 324 Ochre 081 "099)# * * * * 081 Speckled 15 "099)# * * * * 15 Total 0179 "099)# * * * * 0179 Prawle Point "U[K[# Beige 386 "74[4)# * 73 "03[4)# * * 470 Black!brown 116 "80[4)# * 10 "7[4)# * * 137 Ochre 27 "81[6)# * 2 "6[2)# * * 30 Speckled 76 "099)# * * * * 76 Total 738 "77[6)# * 097 "00[2)# * * 846 Wembury "U[K[# Orange 5 "099)# * * * * 5 Beige 41 "70[4)# 01 "07[7)# * * * 53 Dark brown 65 "78[3)# 8 "09[5)# * * * 74 Grey 76 "75[0)# 03 "0[8)# * * * 090 Black 66 "66[7)# 11 "11[1)# * * * 88 Spotted 33 "80[6)# 3 "7[2)# * * * 37 Total 231 "73[8)# 50 "04[0)# * * * 392 Rosco} "France# Orange 5 "099)# * * * * 5 Beige 336 "83[8)# 1 "9[3)# 11 "3[6)# * * 360 Dark brown 377 "81[0)# 7 "0[4)# 23 "5[3)# * * 429 Grey 434 "89[3)# 07 "2[9)# 39 "5[5)# * * 592 Black 33 "66[1)# 3 "6[9)# 8 "04[7)# * * 46 Spotted 058 "75[4)# 5 "2[0)# 07 "8[2)# * * 082 Total 0588 "80[2)# 27 "1[9)# 012 "5[5)# * * 0759 Orange 00 "099)# * * * * 00 Beige 656 "78[7)# * * 58 "7[0)# 07 "1[0)# 743 Dark brown 286 "76[8)# * * 42 "00[6)# 1 "9[3)# 341 Grey 273 "74[0)# * * 52 "03)# 3 "9[8)# 340 Black 238 "099)# * * * * 238 Spotted 066 "76[5)# * * 14 "01[3)# * 191 Langrune:Mer "France# Total 1974 "78[8)# * * 109 "8[0)# 13 "0[9)# 1208 Beige 014 "77[6)# 00 "6[7)# * * 4 "2[4)# 030 Langrune:Mer "France# Black!brown 099 "78[2)# * 1 "0[7)# * 09 "7[8)# 001 Langrune:Mer "France# Brown!grey 07 "89[9)# 1 "09[9)# * * * 19 Portobello Total 132 "78[9)# 02 "3[7)# 1 "9[6)# * 04 "4[4)# 162 "New Zealand#

a Individuals in each location were distinguished by colour variety[ Numbers in bold are the total numbers "all varieties together# of investigated individuals[ b Identi_ed as Cancerilla neozelanica Stephensen in New Zealand "see ð37Ł#[ D[ Deheyn et al[ : International Journal for Parasitolo`y 17 "0887# 0302Ð0313 0308 similar across all locations and host colour varieties\ and kinetic parameters "LMax and TLMax\ but the di}erent symbionts had di}erent e}ects on respectively# were not signi_cantly di}erent the ophiuroid ability to produce light "Fig[ 5#[ Sym! between infested and non!infested individuals biont species also di}ered in their e}ects on host "Fig[ 5A\ B#[ reproduction\ through a}ecting the development Parachordeumium amphiurae induced partial cas! and:or occurrence of gonads and brooded juveniles tration of the host "testes and ovaries were always in the bursae[ reduced# and appeared to reduce its ability to brood Cancerilla tubulata did not alter gonad devel! juveniles "brooded juveniles were very few and con! opment or brooding ability of the ophiuroid] all sistently smaller#[ Multiple infestation with this infested individuals were found to be brooding\ symbiont was common[ As many as four of the 09 sometimes with well!developed gonads[ Most often bursae were found to be infested\ always with one only one copepod individual occurred per ophi! pair of symbionts "the large female and the dwarf uroid and the host brooding ability and light pro! male#[ Light!production intensity was enhanced by duction were una}ected^ light!production intensity the infestation "P³9[9990^ Fig[ 5C#\ while light!

Fig[ 5[ Intensity "LMax# and kinetic "TLMax# parameters of Amphipholis squamata luminescence] non!infested and brooding ophiuroids "BO#\ non!infested and non!brooding ophiuroids "NO#\ and ophiuroids infested "IO# by "A\ B# Cancerilla tubulata\ "C\ D# Para! chordeumium amphiurae\ "E\ F# Rhopalura ophiocomae\ or "G\ H# the new rhabdocoel turbellarian species "the number of stimulated arms are indicated in parentheses# "mean values284) con_dence limit^ "statistically di}erent#[ All ophiuroids were of the beige colour\ except those infested by C[ tubulata[ At Rosco}\ all non!infested individuals were brooding\ hence there was no measurement available for NO[ 0319 D[ Deheyn et al[ : International Journal for Parasitolo`y 17 "0887# 0302Ð0313 production kinetics "TLMax# did not vary signi_! ure Amphipholis squamata Della Chiaje cantly "Fig[ 5D#[ "Echinoderme#] e#tude du de#veloppement et donne#es Rhopalura ophiocomae was found by the hun! biologiques[ PhD thesis\ Paris VI University\ 0861Ł dreds infesting the host[ The symbiont clearly and Prawle Point] ð7\ 09Ł#[ Comparing those results caused total castration of the ophiuroid\ as neither with ours reveals wide ~uctuations in prevalence ovaries nor brooded juveniles were observed in the of symbionts\ suggesting that particular local and host "small testes were sometimes observed#[ Light temporal conditions may in~uence infestation[ production was decreased but only when compared The copepod C[ tubulata is an external symbiont\ with non!infested individuals that were brooding and at Rosco} it infested 1) of the ophiuroids[ "P³9[9990^ Fig[ 5E#[ Non!brooding individuals From the same location\ Bocquet ð12Ł reported 0) routinely exhibit lower light!production per! infestation and Carton ð06Ł mentioned 7)[ Such formance than brooding ones\ and the decreased variability may be explained in relation to the ecol! light!production intensity of infested individuals ogy of this symbiont\ as C[ tubulata may also exist was similar to that of non!infested ones when non! free living ð0\ 14Ł[ A stable {{odour|| of the ophiuroid brooding "Fig[ 5E#[ Kinetics of the light production is necessary for the C[ tubulata to detect and _nd were always similar between individuals\ whether the host for infestation\ but also to ensure durable or not they were infested or brooding "Fig[ 5F#[ linkage with the host ð2\ 15\ 16Ł[ Thus\ the low Individuals of the new species of rhabdocoel tur! infestation reported in the present study may be bellarian occurred in numbers usually ranging from indicative of particular environmental conditions[ four to 09 symbionts per ophiuroid[ The symbiont The copepod P[ amphiurae is an intrabursal sym! clearly reduced the host|s ability to brood "no biont that infested 5[5) of the individuals at Ros! brooded juveniles were ever observed in infested co} and 00[2) at Prawle Point\ while earlier individuals# with no obvious e}ects on the gonads surveys reported less than 0) ð12Ł and 02) "ð13Ł^ as they were always well developed[ The symbiont F[ Goudey!PerrieÁre\ PhD thesis\ 0861# at Rosco}\ strongly reduced the host|s intensity of light pro! and 09)\ 03[0) and 29) at Prawle Point "ð7Ð09Ł\ duction "P³9[9990^ Fig[ 5G#\ and signi_cantly respectively#[ Such variation in infestation between increased the time to maximum output "P³9[995^ years and locations is most likely related to environ! Fig[ 5H#[ mental and biological factors ð17Ł[ Success of infes! tation can be a}ected by seawater currents "ð13Ł^ F[ Goudey!PerrieÁre\ PhD thesis\ 0861#\ by tempera! 3[ Discussion ture and by photoperiod^ these factors determine the ophiuroid physiological condition ðV[ Alva\ 3[0[ Distribution and prevalence of symbionts among Reproduction\ de#veloppement\ incubation et dyna! locations mique de population de l|ophiure Amphipholis squamata "Echinodermata# en baie de seine[ PhD The four symbiont species were not present at all thesis\ Free University of Brussels "ULB#\ 0885Ł\ locations\ nor were all the species present at any which in turn in~uences the probability of infesta! one location[ The ophiuroid was free of symbionts tion\ which also depends on the ophiuroid size and at three locations\ and at the other _ve locations on whether or not it is brooding or already infested one\ two or three symbiont species were found[ The "ð5\ 8Ł^ F[ Goudey!PerrieÁre\ PhD thesis\ 0861#[ Vari! four symbionts considered here do not\ therefore\ ation in infestation could also be related to popu! have the same widespread geographical distribution lational organisation of the host "whether forming as does the host[ Two of the locations sampled have dense aggregates or not#\ since frequency of contact been the subject of earlier surveys investigating the between infesting larvae and host individuals could same symbionts "except for the newly discovered determine the level of infestation "ð13\ 18Ł^ F[ rhabdocoel turbellarian# "Rosco}] ð06\ 12\ 13Ł ðF[ Goudey!PerrieÁre\ PhD thesis\ 0861#[ Goudey!PerrieÁre\ Amphiurophilus amphiurae "He#r! The orthonectid R[ ophiocomae is an internal ouard#\ crustace# cope#pode endoparasite de l|ophi! symbiont found in the coelom and the coelom wall D[ Deheyn et al[ : International Journal for Parasitolo`y 17 "0887# 0302Ð0313 0310 of the ophiuroid disc[ It was found only at Lan! infested and some others not\ but varying from one grune!sur!Mer in 8[0) of the individuals[ This is location to another "e[g[\ compare the grey and less than the 07) minimal infestation reported at black varieties at Langrune!sur!Mer and Rosco}^ San Juan Island\ U[S[A[ ð6Ł\ and more than the Table 0#[ It is a common occurrence\ however\ that 5[4) maximal infestation mentioned from Rosco} frequency of infestation of a widely distributed ð29Ł[ The absence of the orthonectid from the other species of host varies from one location to another\ locations investigated is contrary to the reported the causes being related to environmental and:or worldwide distribution of the symbiont "ð29Ł\ for populational di}erences between locations\ and review#[ This discrepancy may re~ect the biology of sometimes also to genetic variance in the host R[ ophiocomae which is well adapted for rapid and and:or the symbiont between locations ð16\ 18Ł[ heavy infestation of hosts ð29\ 20Ł but apparently Amphipholis squamata from distinct geographical sensitive to ecological parameters\ making its abun! regions have been demonstrated to be genetically dance variable over time[ Little is known about the di}erent "D[R[ Murray\ Master thesis\ 0878#[ ecology of this symbiont and the environmental Genetic di}erences probably also exist between parameters that in~uence its infestation\ except that individuals of the di}erent colour varieties ð00\ 01Ł\ density and biological conditions of the host are which could result in di}erences in susceptibility: determinant ð6\ 29Ł[ Also\ the orthonectid causes tolerance to infestation between varieties even at noticeable e}ects on the host\ whose biological a given location[ Amphipholis squamata may thus activity\ growth rate and regeneration are reduced resemble another polychromatic ophiuroid "Ophio! ð6Ł\ and survival of infested individuals may then be coma echinata# in which {{colour morphs*from more threatened[ di}erent habitat*will eventually be recognised as The four investigated symbionts of A[ squamata distinct substrate!speci_c or host!speci_c taxa|| ð22Ł[ appear to be mutually exclusive\ since no host indi! vidual ever harboured more than one species of 3[2[ Effect of symbionts on the host light!production symbiont[ This was suggested earlier by J[ Johnson characteristics ðThe biology of Amphipholis squamata Delle Chiajei "Echinodermata] Ophiuroidea#[ PhD thesis\ Uni! The presence of the external symbiont C[ tubulata versity of Newcastle\ 0861Ł\ who found only two had negligible impact on the host[ Most often only cases of double!infestation out of 55 infested indi! one copepod was present\ and gonad development\ viduals at Northumberland "U[K[# where the two brooding ability and light!production parameters copepods and the orthonectid are present[ were all una}ected[ The three internal symbionts\ however\ were present in greater numbers\ and each 3[1[ Distribution of symbionts among colour var! caused some degree of reproductive impairment ieties and induced changes in light!production parameters[ Maximum light output was decreased The varieties of A[ squamata di}er in their innate when R[ ophiocomae or the rhabdocoel turbellarian ability to produce light ð01Ł[ Gotto ð21Ł suggested was present\ but increased in the presence of P[ that the distribution of symbionts could be cor! amphiurae[ Kinetics of the light production related with light!production intensity of the host\ "TLMax# were a}ected only by the presence of the with apparently the more luminous the individual\ rhabdocoel turbellarian[ the higher the frequency of infestation[ Such a Bioluminescence is a biological property associ! relationship was not substantiated in the present ated with considerable energetic cost ð23Ł\ and each study[ Symbionts occurred regardless of host of the symbionts also induces some energetic loss variety\ without any correlation with their bio! for the host "see ð6\ 7\ 24Ł^ F[ Goudey!PerrieÁre\ luminescence or their degree of pigmentation[ Yet\ PhD thesis\ 0861#[ The observed reductions in light! considering each location separately\ it is obvious production intensity in the presence of the ortho! that the symbionts were not distributed homo! nectid and the rhabdocoel turbellarian might there! geneously amongst the varieties\ some being heavily fore be interpreted as due simply to the energy cost 0311 D[ Deheyn et al[ : International Journal for Parasitolo`y 17 "0887# 0302Ð0313 of infestation[ The real explanation\ however\ is bioluminescence requires molecular oxygen ð23Ł\ so likely to be more complex[ Although nothing is an oxygen imbalance could also contribute to alter! known about the chemistry of bioluminescence in ation of the light!production performance in indi! A[ squamata\ light production is known to be under viduals infested by the rhabdocoel turbellarian[ the neurophysiological in~uence of brooding and It is also possible\ for any of the symbionts con! increases when at least one juvenile is brooded in the sidered\ that the variations of light!production per! bursae ð01Ł[ The orthonectid caused total castration formance from infested A[ squamata are related to and total absence of brooded juveniles in the the oxidative immune reaction of the ophiuroid[ bursae\ and the reduced light output was equivalent Bioluminescence ability is a}ected by oxygen reac! to that seen in non!infested\ non!brooding indi! tives\ such as superoxide ions\ free radicals\ or polar viduals[ The e}ect may therefore have been entirely steroids ð28\ 39Ł[ These reactives are toxic and pro! due to the absence of juveniles in the bursae[ On duced in response to infestation\ their production the other hand\ the copepod P[ amphiurae lives being part of the organism|s immune reaction ð30\ speci_cally in the bursae\ usually in reproductive 31Ł[ The molecules that are used in bioluminescence pairs\ inhabiting up to four of the 09 bursae[ The have a high a.nity for such oxygen reactives ð32\ signi_cant increase in light output observed from 33Ł and can be utilised by the host to neutralise the individuals infested with this symbiont may there! toxic\ oxidative e}ects of these reactives[ A lumi! fore also be due to a {{brooding|| e}ect\ the presence nous molecule that is used in this way for protection of the copepod stimulating the neurophysiological against the immune oxidative stress would then be responses normally due to the presence in the bur! unavailable for the production of light\ and the sae of brooded juveniles[ The stimulatory e}ect light!production performance thus would depend then exceeded any diminution due to energy cost of on the individual|s immune condition "see ð33Ł#[ The the infestation[ superoxide immune reaction has been dem! Individuals of the new species of rhabdocoel tur! onstrated in echinoderms ð34Ð36Ł\ although not bellarian caused the greatest alterations in the ophi! speci_cally in A[ squamata[ uroid light!production performance[ Intensity of the bioluminescence "LMax# was strongly reduced and the kinetics "time to reach maximal output^ Acknowledgements TLMax# signi_cantly increased by infestation[ The turbellarian develops long processes that underlie We thank directors of the following institutions the ophiuroid digestive epithelium "Deheyn\ per! for providing facilities] {{Le Centre de Recherche sonal observation#\ suggesting nutrient transfer et d|Etude CotieÁres|| "Luc!sur!Mer\ France#\ {{La "energy# from host to symbiont[ It also caused total Station Biologique de Rosco}|| "Rosco}\ France#\ absence of brooded juveniles in the bursae[ The the Marine Biological Laboratory "Plymouth\ decrease in light intensity in the presence of this U[K[#\ the University Marine Biological Station symbiont substantially exceeded that due to the "Millport\ U[K[#\ the Portobello Marine Lab! orthonectid\ and this implies an e}ect of the ener! oratory "Portobello\ New Zealand# and the King getic loss as well as an e}ect due to the absence of Le#opold III Biological Station "Laing Island\ Papua brooding[ Reduced intensity and increased kinetics New Guinea#[ We are especially grateful to Dr R[ could also be the result of interference with the Emson "U[K[# and Professor P[ Mladenov "New pathway of light production[ Symbiotic tur! Zealand# for providing access to their laboratories[ bellarians are known to induce alterations of cellu! Funds for this research were provided by an FRIA lar and sub!cellular functions of their hosts by grant to D[ Deheyn and an FRFC convention "ref[ abstracting molecular oxygen from host tissues\ 5[120[74#[ We also thank for _nancial support the utilising haemoglobin!like proteins ð25Ð27Ł[ The British Council\ the National Fund for Scienti_c turbellarian from A[ squamata is brown!red to Research "FNRS#\ the King Le#opold III Foun! orange in colour\ which suggests it may contain a dation\ and the {{MinisteÁre de la communaute# fran! haemoglobin!like protein ð25\ 26Ł[ The reaction of cžaise de Belgique||[ This study is a contribution to D[ Deheyn et al[ : International Journal for Parasitolo`y 17 "0887# 0302Ð0313 0312 the {{Centre Interuniversitaire de Biologie Marine|| trol in the brittle!star Amphipholis squamata] e}ects of chol! "CIBIM#[ inergic drugs[ Comp Biochem Physiol 0885^004C]64Ð71[ ð05Ł Emson RH\ Jones MB\ Whit_eld PJ[ Habitat and latitude di}erences in reproductive pattern and life!history in the cosmopolitan brittle!star Amphipholis squamata "Echino! References dermata#[ In] Ryland JS\ Tyler PA\ editors[ Reproduction\ genetics and distributions of marine organisms[ Fre! densborg] Olsen + Olsen\ 0878]64Ð70[ ð0Ł Barel CD\ Kramers PG[ A survey of the echinoderms ð06Ł Carton Y[ De#veloppement de Cancerilla tubulata Dalyell\ associates of the north!east Atlantic area[ Zool Verhand parasite de l|ophiure Amphipholis squamata Delle Chiajei[ "Rijksmus Nat Hist Leiden# 0866^045]0Ð048[ Crustaceana 0857^0]S00ÐS17[ ð1Ł Jangoux M[ Diseases of Echinodermata[ In] Kinne O\ ð07Ł Dietrich HF\ Fontaine AR[ A decalci_cation method for editor[ Diseases of marine animals\ vol[ III[ Hamburg] Bio! ultrastructure of echinoderm tissues[ Stain Technol logishe Anstalt Helgoland\ 0889]328Ð431[ 0864^49"4#]240Ð243[ ð2Ł Carton Y[ Comportement et spe#ci_cite# parasitaire chez les ð08Ł Richardson KC\ Jarret L\ Finke SEH[ Embedding in epoxy cope#podes parasites[ Essai d|analyse expe#rimentale[ Arch resins for ultrathin sectioning in electron microscopy[ Stain Zool Exp Ge#n 0857^098"3#]434Ð489[ Technol 0859^24]202Ð212[ ð3Ł Alva V\ Jangoux M[ On the symbiosis between polychaetes ð19Ł Mallefet J\ Vanhoutte P\ Baguet F[ Study of Amphipholis and ophiuroids with the description of a new case of com! squamata luminescence[ In] Scalera!Liaci L\ Canicatti C\ mensalism[ Vie Mar 0878^09]074Ð081[ editors[ Echinoderm research[ Rotterdam] Balkema AA\ ð4Ł Kozlo} EN[ The structure and origin of the plasmodium of 0881]014Ð29[ Rhopalura ophiocomae "Phylum Orthonectida#[ Acta Zool ð10Ł Deheyn D\ Alva V\ Jangoux M[ Fine structure of the photo! 0883^64]080Ð088[ genous areas in the bioluminescent ophiuroid Amphipholis ð5Ł Goudey!PerrieÁre F[ Modalite#s de l|infestation de l|ophiure squamata "Echinodermata\ Ophiuroidea#[ Zoomorphology Amphipholis squamata Delle Chiaje\ Echinoderme\ par le 0885^005]084Ð193[ crustace# cope#pode Amphiurophilus amphiurae "He#rouard# ð11Ł De Bremaeker N\ Deheyn D\ Thorndyke MC\ Baguet F\ et in~uence du parasite sur l|e#tat de gravidite# de l|ho¼te[ Mallefet J[ Localization of S0! and S1!like immu! 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