Ecologie et comportement
The symbiosis between Ophiomastix venosa and Ophiocoma scolopendrina on the
barrier reef of Toliara (Madagascar) : A case of Chemical associative learning in ophiuroids.
D. Fourgon *, I. Eeckhaut ^ and M. Jangoux
' Laboratoire de Biologie Marine (CP 160/15), Université Libre de Bruxelles, 1050 Bruxelles, Belgium ^ Laboratoire de Biologie marine. Université de Mons-Hainaut, 7000 Mons, Belgium
Submitted to: Invertebrate Biology
Abstract
The biology of the interophiuroid symbiosis between Ophiomastix venosa and its host
Ophiocoma scolopendrina is investigated through fîeld observations and laboratory
experiments. O. scolopendrina live on regularly emersed rocky dômes in the boulder tract of
the barrier reef of Toliara (Madagascar), while free O. venosa lie in adjacent tidal channels
that are always immersed. Only juvéniles O. venosa may inhabit the dômes providing they are
symbiont of adult O. scolopendrina. The formers are clung to their hosts’disk. There is rarely
more than one symbiont per host. Laboratory experiments showed that symbiotic (i.e.,
clinging) O. venosa is much more résistant to hot air exposure than non symbiotic ones. Such
résistance is due to the particular ability of O. scolopendrina to curl its arms above the disk
when emersed, a behaviour enabling to reduce water évaporation during hot air exposure.
Behavioural experiments showed that juvénile O. venosa living symbiotically were clearly
attracted both by adult O. scolopendrina and by water previously conditionned by adult O.
scolopendrina, while free-living juvéniles were almost not. This indicates that past expérience
of individuals may eondition their actual behaviour, and implies the capability for associative
Ecologie et comportement
Introduction
Echinoderms are frequently infested by various symbiotic organisms. Davenport
(1950) was the first to demonstrate the Chemical attraction of symbionts towards their host in
the case of asteroid or holothuroid hosting polychaetes. Chemical-mediated host récognition
was also put in evidence by various authors in symbionts of asteroids, echinoids and
holothuroids (see, e.g., Gage 1966, Gray et al. 1968, Dimock and Davenport 1971, Ache and
Davenport 1972, Van Meter and Ache 1974). More recently, it was demonstrated that
phoretic-like shrimps and crabs were chemically attracted by their crinoid host
(Vandenspiegel et al. 1998, Eeckhaut et al. 2000, respectively). Untill now such chemical-
mediated récognition has never been documented in any symbiosis involving ophiuroids as
hosts.
A particular interophiuroid symbiosis between Ophiomastix annulosa and Ophiocoma
scolopendrina was reported to occur in the intertidal zone of Sesoko Island (Okinawa, Japan)
(Hendler et al. 1999). These authors observed that O. annulosa individuals were clung to the
disk of their O. scolopendrina host. They also saw some juvéniles of O. annulosa within the
bursae of adult O. scolopendrina. A similar interophiuroid symbiosis was observed on the
barrier reef of Toliara (Madagascar) that lead us to question upon the possible protection
offered by the host species. The présent paper describes and deciphers the symbiosis from in
situ observations and laboratory experiments. It aims to détermine whether the symhiont can
recognize its host and what would be the possible advantage(s) it could gain from this
particular relationship.
Materials and methods
In situ observations
Samplings were made by hand-collecting at low tide on the barrier reef of Toliara
(Madagascar) (fig. 10 see Clausade et al., 1971, for a detailed description of the Toliara
barrier reef). The symbiose concems the species Ophiomastix venosa and Ophiocoma
Ecologie et eomportement
roeky dômes altemate with tidal channels, both being parallel to each other and perpendicular
to the beach border. Dômes measure 100 to 200m long and ca.lOm width. They consist in
accumulations of dead corals emerging at low tides, and are separated to each others by
permanently immersed tidal channels (water height at low tide: ea. 50cm). The bottom of the
latter is made of sand with living and dead corals.
43°40’E 43°45’E
B
Fig. 10; A. Location of the boulder tracts (A to E) on the Great Reef of Toliara where ophiuroids were sampled. B. Schematic transverse section of the study area (not to scale). c, channel; d, dôme;
FR, fringing reef; GRT, Great Barrier Reef of Toliara; wl; water level at low tide.
Transects were made at five different places along the barrier reef in February 2000 (A
to E, figure 10). Each of them consisted in four Im^ quadrats randomly placed in the dôme
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counted. The occurrenee of interophiurid symbiosis was recorded and its prevalence (i.e., the
percentage of hosts associated with at least one symbiont individual) was calculated. The site
with maximal prevalence was investigated monthly from February 2000 to April 2001 at low
tide in search of individuals either single or in pairs. Individuals were always found under
pièces of dead coral. Dôme and ehannel samplings were performed using sixteen Im^
quadrats placed either in the dôme (8 quadrats) or in the ehannel (8 quadrats). Quadrats in
each sériés were randomly placed along a line 2 m off the limit between the two biotopes.
The occurrenee of symbiotic O. venosa on O. scolopendrina or on other ophiuroids
was recorded for each quadrat. Symbionts and hosts were measured in the field (disk diameter
using a calliper) and the number of symbionts per host was noted. Since juvéniles of O.
venosa could also be found in the host's bursae, O. scolopendrina ffom two quadrats in each
sériés were dissected in the laboratory. The position of the symbionts on their host was
recorded on 180 venosa-scolopendrina pairs eollected in March 2002.
Behavioural approach
Ail behavioural experiments were done in aquarium containing natural filtered sea
water at ambient température (24 + 1°C). Two days before experiments, each group of
ophiuroids to be tested was kept separately in a 501 tank up to the time of experiments.
The first set of host-choice experiments were performed in a 3 0x3 0x10cm aquarium
filled with 41 of sea water. Experiments were done to test the possible attraction of two
individuals (one O. scolopendrina and one O. venosa) placed in aquarium opposite corners
towards a O. venosa juvénile placed in the aquarium centre.
The second set of host-choice experiments was made up to assess the ability of a O.
venosa juvénile to recognise water flows conditioned by either O. scolopendrina or O.
venosa. The experimental System used was a Y-tube System adapted ffom Davenport (1950;
see fig. 11). It consists in a Y-shaped glass tube of 3cm in diameter whose branches measure
each 10cm long. The paired branches were connected to two separate aquariums
(17x17x10cm; aquaria A and B, see fig. 11) while the unpaired one was closed by a
perforated plug crossed by a flexible pipe provided with a tap. Each aquarium was in tum
Ecologie et comportement
filled with either ‘neutral’ filtered sea water or filtered sea water eonditioned by one of the
tested ophiuroid species (water eonditioning consisted in placing 25 adults of the tested
ophiuroid species in 41 of sea water for 20 min). Water flowed from the upper aquarium to the
lower one, then to the paired branches and was evacuated through the flexible pipe. The
linearity of the current flow and the absence of turbulenees in the System was cheeked by
visualising the eurrent using to fluoresceine stain (50mg/l), and the tap of the flexible pipe
allowed to regulate the water speed within the tube (from 50 to lOOml/min).
J
Fig. 11 : A. Y-tube System adapted from Davenport (1950). Arrows indicate the direction of water flow. B. Detailed view of the place where tested juvéniles were introduced in the System.
Ecologie et comportement
Each experiment included thirty trials, the control experiment consisting in filling the
two aquaria with regular sea water. The hypothesis that individuals were chemically attracted
was investigated by comparing the number of time individuals started moving under a species
stimulation with the number of time they started moving when only seawater filled the two
aquaria, Le., the control. For each experiment, the statistical significance was assessed with a
Yates corrected Chi-Square test (Zar, 1996). The hypothesis that individuals preferred
ophiuroid-conditioned seawater from regular seawater was obtained by comparing the
frequency of choices that they made between the two paired aquaria of the System with the
frequency “50%”. For each experiment, the statistical significance was assessed with a Chi-
Square Goodness-of-Fit test (Zar, 1996).
Each tested individual was placed in the enlarged area of the unpaired branch whose
upper part is connected to a plug-equipped enlarged tube (fig. 11). Assays consisted in
recording individuals’behaviour for 15min when receiving a mixed water flows coming ffom
aquarium A and B. Between each assay, the Y-tube System was washed with detergent and
thoroughly rinsed with sea water. After 15 assays, the System was tumed over (the right
branch became the left, and vice versa) to avoid any other parameter that could hâve
influenced the choice of the animais.
Résistance to hot air exposure
Measurements of résistance to hot air (i.e., drying) exposure were done in placing
individuals in an oven (40°C) for up to four hours. The number of surviving individuals was
counted hourly. Investigated sériés accounted for 30 individuals either single (O.
scolopendrina, O. venosà) or in pair {O.venosa clung to O. scolopendrina).
Results
In situ observations
The average prevalence of the symbiosis between O. scolopendrina and O. venosa
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transects A, B, D and E, but was significantly higher in transect C (Mann-Whitney U Test,
p<0.05). The later was thus selected for the study of the interophiuroid symbiosis. The
investigated area comprises a channel and the Southern adjacent dôme; its surfaee measures
ca. 130m long and 60m wide (approximate wide of the dôme: 10m; approximate wide of the
channel: 50m).
Individuals of O. scolopendrina (free and symbiotic) were very abondant on the dôme
(124.4 ± 39.7indiv/m^, number of investigated quadrats [niq] = 112) from which non-
symbiotie O. venosa were almost absent (0.2 ± 0.5indiv/m^; niq = 112). On the eontrary,
individuals of the two species appear to be not, or very rarely, symbiotic in the channel where
O. scolopendrina occurred at a very low level (0.5 ± l.Oindiv/m^; niq = 228) and where O.
venosa was much more common (13.0 ± 9.3indiv/m^; niq = 228). Ail together the mean disk
diameter for O. venosa was 12.9 ± 4.7mm (n=3070), and it was 12.0 ± 4.0mm (n=3146) for
O. scolopendrina.
Ail dome-inhabiting O. venosa were both symbiotic (they clung on the disk of their
host; fig. 12) and juvéniles (their mean disk diameter was 6.5 + 1.3mm, n=180). Most of them
were loeated on the host's aboral side (93 out of 180 individuals), 55 being on the oral side,
and only 32 on the disk margin. Individuals on the aboral side were firmly attached to the disk
with their arms loeated in the host’s interradii, often extending up to their mouth. Those on
the oral side assumed the same attitude; their mouth, however, were facing that of the host. As
for those of the margin, they extended their arms to both the oral and aboral sides of the host’s
disk.
Fig. 12: Aboral view of a O. venosa symbiotic juvénile clung on the disk of a O. scolopendrina adult. Scale bar = 5 mm.
Ecologie et comportement
There was generally one symbiont per host (94.1 %), rarely two (5.4%), almost never
three (0.5%). The disk size of symbiotic O. venosa varied between 2 and 11mm in diameter,
and was correlated with the disk size of the host, the later varying between 5 and 24mm in
diameter (Spearman Rank Corrélation, N=305, r=0.47, p<0.001). Very small O. venosa (disk
diameter < 3mm) were also found in the host's bursae with a prevalence generally lower than
1% (a maximum prevalence of 2.4 % was observed in March 2000). Intrabursal juvéniles laid
in the bursal lumen and appeared harmless for their host.
Juvéniles of O. venosa hâve also been observed on the disk of Ophiocoma brevipes in
tidal chaimels. Of the 24 O. brevipes individuals found in the selected site during the 2000-
2001 survey, 14 were carrying firmly attached O. venosa juvéniles. Moreover, O.
scolopendrina juvéniles were also found on the disk of conspecific adults in the dôme area.
Yet they were not clung as O. venosa juvéniles were, and they could be removed easily. The
prevalence of this intraspecific association varied from 0.016 to 0.069% according to the
sampling period.
Behavioural approach
Two groups of O. venosa juvéniles were tested during these trials: ffee juvéniles
collected in the tidal channel, and symbiotic juvéniles collected on O. scolopendrina from the
rocky dôme. Symbiotic juvéniles were separated from the host and kept in aquaria two days
before to be tested.
Host choice experiments.
Experiments were performed in aquarium. One adult O. scolopendrina was placed
opposite to one adult O. venosa (disk diameter [dd] of used adults: 1.6 < dd < 2.0cm), the
juvénile O. venosa to be tested being placed in the very centre of the aquarium. Two sets of
experiments were performed using O. venosa juvéniles (i.e., individuals < 9mm in dd) from
either the dôme area (symbiotic juvéniles; 30 trials) or the channel area (free juvéniles; 30
trials). Each trial began at the end of the aftemoon (6 p.m.) and stopped the following
moming (9 a.m.). The position of the juvénile in relation to the co-occurring adults was then
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in loose eontact with any part of any of the two adults, or firmly clung to any part of any of
the two adults.) Different ophiuroid individuals were used for each of the 60 performed trials.
Results are gathered in Table 3. They indieate a clear différence in host récognition
according to the kind of tested individual, i.e. symbiotic versus free O. venosa juvéniles.
Indeed, 22 over 30 symbiotic juvéniles were found firmly attached to O. scolopendrina adults
at the end of the experiments, while only 2 over 30 free juvéniles were doing so {j2, df=l,
chi-square= 25.07, p<0.0001). On the contrary, free juvéniles appear to be more attracted by
eonspecific adults as 15 over 30 were found in loose contact with O. venosa adults at the end
of the experiments {yl, df=l, chi-square= 9.60, p=0.019).
free juvéniles symbiotic juvéniles alone 8 3 in contact with O. scolopendrina 3 2 clung to O. scolopendrina 2 22 in contact with O. venosa 15 3 clung to O. venosa 2 0
Table 3: Host choice experiment. Location of juvéniles of O.venosa, either free or symbiotic, at the end of the experiment (n=30).
Y-tube experiments
To be tested, eaeh individual was placed 3em deep in the unpaired branch of the Y-
tube System and their behaviour was recorded. Individuals were considered to be stimulated
when they started to move in the Y-tube. Individuals were considered to hâve chosen the A or
B aquaria when at least half of their disk entered one of these aquaria. For those remaining in
any part of the Y-tube after 15min, the trial was considered nul (no ehoiee).
When the aquaria were filled with ffesh filtered sea water (control test), both free and
symbiotie O. venosa either made no choice or shared equally between A and B aquaria (Table
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When submitted to water conditioned with adult individuals of O. scolopendrina, the
symbiotic juvéniles were, most of the time, stimulated since only one specimen stood
motionless (Table 4, experiment III, test I). Furthermore, these juvéniles preferred the host-
conditioned seawater from regular seawater since 24 of the 29 juvéniles that responded to the
stimulus hâve chosen the way leading to the conditioned aquaria (Tahle 4, experiment III, test
2). On the contrary, the free juvéniles submitted to the same experimental conditions were not
significantly attracted when compared to the control test (Table 4, experiment IV, test I).
Those that did move did not show any preference between host-conditioned seawater and
fresh filtered seawater (Table 4, experiment IV, test 2).
Experiments Aquarium A Aquarium B Results tests
Assay Nul A B 1 2
1 symbiotic juvénile sea water versus sea water 30 20 5 5 - Q II
.
c
T
II
II free juvénile sea water versus sea water 30 14 8 8 - x"=o, p=1
III symbiotic juvénile 0. scolopendrina versus sea water 30 1 24 5 X^=23,74, p<0,001 X"=12,44, p<0,001
IV free juvénile O. scolopendrina versus sea water 30 8 13 9 X^=1,79, p=0,18 X==0,73. p=0,39
V symbiotic juvénile 0. venosa versus sea water 30 16 8 6 X^=0,63. p=0,43 X"=0,28, p=0,59
VI free juvénile O. venosa versus sea water 30 6 13 11 X==3,67, p=0,055 x’=0,17. p=0,68
Table 4: Y-tube experiment. Chemical attraction of O. venoio juvéniles by
O. scolopendrina hosts in double choice experiments.
When submitted to water conditioned with adult individuals of O. venosa, the
symbiotic juvéniles were not significantly attracted nor did they show any preference between
conspecific-conditioned seawater and regular seawater (Table 4, experiment V, tests 1 and 2).
Similarly, free juvéniles were not significantly attracted by a Chemical eue from adult
conspecifics and those that moved did not show any preference between the two water flows
(Table 4, experiment VI, tests I and 2).
Résistance to hot air exposure
When O. scolopendrina individuals are exposed to air in the field at low tide, they
shelter under dead corals or in rock crevices and characteristically curl up their arms above
their disk. A similar posture was observed in specimens experimentally exposed to hot air
drying (40°C). Most of them (97%) survived up to 3h to such treatment while 27% survived
Ecologie et comportement
behaviour during similar treatment (no arm curling). Also they were much less résistant to hot
air exposure as most of them died after 2h (Fig. 13).
Time (mins)
Fig. 13: Survivorship curves of adults of O. scolopendrina and O. venosa, and of free and symbiotic juvéniles of O. venosa when exposed to hot air (40°). AdOs, O. scolopendrina adults;
AdOv, O. venosa adults; JuFOv, O. venosa free juvéniles; JuSOv, O. venosa symbiotic juvéniles.
Comparison of the loss of weight between individuals of the two species showed that
after Ih and 2h of hot air exposure, O. venosa lose significantly more water than O.
scolopendrina (ANOVA, 1 hour: df=58, F=240.89, p<0.0001; 2 hours: df=58, F=76.42,
p<0.0001). Considering these results and the survivorship curves (see Figs 13 and 14), one
may note that, although the two species did not resist the same time to the treatment {O.
scolopendrina being the more résistant), the highest mortality occurs after similar loss of body
weight. Indeed, the survivorship curves show a rapid decrease when the body weight lost
reached ca. 30% for the two species.
Figure 13 also shows that juvéniles of O. venosa reacted differently to the treatment
depending they were free or symbiotic. Ail of the 30 free juvéniles tested died after Ih hot air
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(Re: each symbiotic O. venosa was clung to a O. scolopendrina individual) (Fig. 13). Even
after 4h exposure, more than 25% of the symbiotic juvéniles were still alive, their
survivorship curve being very close to that of their host.
Fig. 14; Lost of body weight in O. scolopendrina and O. venosa adult individuals after hot air (40°C) exposure.
Discussion
The co-occurrence of Ophiomastix venosa and Ophiocoma scolopendrina in the
intertidal zone of Indo-Pacific coral reefs was already reported by varions authors {e.g., Clark
and Courtman-Stock 1976, Devaney 1978). Tidal channels are the usual habitat of O. venosa
where it generally lives on sand or under corals, rocks or algae (Macnae and Kalk 1962; Sloan
et al., 1979). As for O. scolopendrina it is frequently reported to occur in places submitted to
emersion, hence being exposed to air drying during low tides (Sloan et al. 1979, Chartock
1983).
O. scolopendrina is one of the commonest littoral ophiuroids of the whole tropical
Indo-west Pacific Océan where it often forms dense populations (Sloan et al., 1979; Chartock,
1983). The high density population of the species in the barrier reef of Toliara (almost 125
individuals/m^) could partly resuit from the occurrence of numerous natural shelters (dead
corals, rock crevices, etc.) that fits the high territorial behaviour characterizing O.
scolopendrina (Magnus, 1967). Moreover, the presence -at the air-water interface, during
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individuals occur)- of a coral-produced energy-rich detritic film on which individuals fed
should also favour the development of dense populations (see also Magnus 1967, James and
Pearse 1969, Chartock 1983). Last but not least, O. scolopendrina appears particularly well
adapted to live in such stressing environment (Re: individuals are emersed every low tide) as
individuals are able to protect themselves in curling up their arms over the disk when emersed
so forming a kind of water trap. Yet, O. venosa appear as tolérant as O. scolopendrina to high
water température : O. scolopendrina was reported to occur in water of up to 40°C (Sloan et