Table Of ContentReference:Biol. Bull 192:208-216.(April. 1997)
Uptake and Persistence of Homologous and
Heterologous Zooxanthellae in the Temperate Sea
Anemone Cereuspedunculatus (Pennant)
SIMON K. DAVY*. IAN A. N. LUCAS, AND JOHN R. TURNER
SchoolofOcean Sciences, Universityof\\'ales, Bangor. MarineScienceLaboratories.
Menai Bridge. Anglesey, LL595EY, UK
Abstract. The uptake and persistence ofsymbiotic di- were the same asthose in naturally infected C. peduncu-
noflagellates (zooxanthellae) were measured in the tem- latusofsimilarsize. The resultssuggest that zooxanthel-
perate sea anemone Cereus pedunculatus (Pennant). lae from a range of host species and environments can
Aposymbiotic specimens ofC. pedunculatus were inoc- establish symbioses with C. pedunculatus and that, over
ulated with zooxanthellae freshly isolated from a range long periods under laboratory conditions, heterologous
oftemperate and subtropical Anthozoa. Each inoculate zooxanthellaemaypopulateC.pedunculatustothesame
consisted ofzooxanthellae from asingle hostspeciesand extentas homologouszooxanthellae.
waseitherhomologous(zooxanthellae fromahostofthe
same species as the one being inoculated) or heterolo- Introduction
gous(from ahostofadifferentspeciesthantheonebeing
inoculated). The densities of zooxanthellae in host tis- Many benthic marineCnidariapossessendosymbiotic
sueswere determined at regular intervals. C. peduncula- dinoflagellates (zooxanthellae) ofthe genus Symbiodin-
tustook uphomologousand heterologouszooxanthellae iitni. These Cnidaria may lose zooxanthellae entirely
to similar degrees, except for zooxanthellae from the (i.e., become aposymbiotic) when under environmental
temperate Anlhopleura ballii, which were taken up to a stress (Williams and Bunkley-Williams, 1990; Glynn,
lesserextent. The densities ofall zooxanthellae declined 1991) or when zooxanthellae are not inherited mater-
between 4 hoursand 4days after uptake, indicatingthat nally (Trench, 1987; Shick 1991). Consequently, they
zooxanthellae were expelled, digested, or both during may be susceptible to reinfection and colonization by
this period. The densities ofall zooxanthellae increased zooxanthellae released from a range of host species
between 2 and 8 weeksafterinoculation, indicatingzoo- (Schoenberg and Trench. 1980c; Rowan and Powers,
xanthellagrowth. Overtheentire8-weekperiodafterup- 1991b; Buddemeierand Fautin, 1993). Despite this sus-
take,densitiesofhomologouszooxanthellaewerealways ceptibility and the high diversity ofhostsand symbionts
greater than those of heterologous zooxanthellae. Be- (Trench, 1987; Rowan and Powers, 1991b; Trench,
tween 8 and 36weeksafterinfection,densitiesofhomol- 1993; McNally et a/.. 1994), many species ofCnidaria
ogous zooxanthellae declined markedly and densities of havebeen reported to harborjustonestrain orspeciesof
some heterologous zooxanthellae increased further, re- Symbiodinium (Schoenberg and Trench, 1980a, b;
sulting in homologous and heterologous zooxanthella Rowan and Powers, 199la, b; Trench, 1993). Other
densities being the same at 36 weeks. These densities hosts have been found to contain more than one strain
or species of zooxanthella, but even in these cases the
algae are the same across the host's geographical range
Received 12January 1996;accepted9January 1997. (Trench and Winsor. 1987; Rowan and Powers, 1991b;
vis*ioPnreosfenMtaardidnreesSsc:ieDnecpea,rHtamrebnotrofBrSaynmcbhioOsciesaannodgrCaoprhailcBIinosltoigtyu.tiDoin-, Rowan and Knowlton, 1995).
5600U.S. 1 North,FortPierce,Florida34946. The uptakeand persistenceofzooxanthellaefollowing
208
PERSISTENCE OF ZOOXANTHELLAE IN SEA ANEMONES 209
inoculation ofaposymbiotic hosts has been investigated Materialsand Methods
in a range of tropical marine Cnidaria (Kinzie, 1974;
Kinzie and Chee, 1979; Schoenbergand Trench, 1980c; Collection andmaintenanceofsymbioticAnthozoa
Trench, 1981; Trench et ai. 1981; Colley and Trench, The sea anemones Cereus pedunculatus (Pennant),
1983;FittandTrench, 1983a,b; Bernerrttf/., 1993).The Anemonia viridis (Forskal), and Anthopleura ballii
zooxanthellae were either homologous (from a host of (Cocks), and the zoanthid Isozoanthus sulcatus (Gosse)
the same species of cnidarian as the one being inocu- were collected from Lough Hyne Marine Nature Re-
lated) or heterologous (from a host ofa different species serve, Eire(51 29' N;9 18' W). All fourspeciesareexclu-
than theone beinginoculated). In contrast, similarwork sively subtidal at this location. The anemones were all
has been performed usingjust one species oftemperate collected from a depth of 1 to 3 m, and the zoanthids
marine Cnidaria, the North American sea anemoneAn- from 5to9 m. SpecimensofA. viridiswerealsocollected
thopleura elegantissima (Trench, 1969, cited in Trench, fromtheintertidal zoneatShell Island, North Wales(52
1971;Muller-Parker, 1996; Weisand Levine, 1996). 47' N; 004 06' W). For comparative purposes, speci-
The temperate sea anemone Cereus pedunculatus mens of the subtropical sea anemone Aiptasia pallicla
(Pennant) (family Sagartiidae) is locally abundant (Verrill), cloned from one individual, were obtained
around the south and west coasts ofEurope, where it is from Dr. C- B. Cook, Bermuda Biological Station. The
found partially buried in sand and mud from the mid- A.pallidahadoriginallybeencollected fromamangrove
m
shore to a depth of25 (Manuel, 1981). C. peduncula- root in Walsingham Pond, Bermuda.
tus may reproduce by oviparity, but frequently it is her- AdultAnthozoawerekeptinseawaterfromthe Menai
maphroditic or parthenogenetic and viviparous (Rossi, Strait, North Wales. Theywere maintainedat 21C, illu-
1975;Schafer, 1981;Shick, 1991). The zooxanthellae of minated at 80 ^mol photons irT2 s~' on a cycle of 12 h
C. pedunculatus are located within the host's endoder- light:12 h dark, and fed twice weekly with Anemia sp.
mal cells and have been identified as Symbiodinium sp. (Bonneville Anemia International Inc.). The light and
(Davy et a/., in press). Oocytes may contain up to 300 temperature regimes were comparable to those experi-
zooxanthellae, suggesting that C. pedunculatus acquires enced during a warm summer in Lough Hyne (Turner,
its symbionts through maternal inheritance (Turner, 1988, and his pers. obs.).
pers. obs.). Brooded and recently released juvenile C.
pedunculatusalways harborzooxanthellae, but densities
Preparation ojaposymbioticAnthozoa
in recently released individuals may be less than 10% of
those in adult anemones (Darmayati, 1993; Davy et ai, Juvenile specimens ofC. pedunculatus were squeezed
C
1996). The fact that pedunculatus produces offspring from the enterons of brooding adult anemones and
that lack a full complementofzooxanthellae meansthat placed in a dark-box to render them aposymbiotic. Sea-
they maybesusceptibletoinfection byzooxanthellae re- waterfrom the Menai Strait, at theambient temperature
leased from otherAnthozoa. (10-18C), flowed through thedark-boxcontinuously. It
The sea anemones Anthopleura ballii (Cocks) (family is unlikelythat zooxanthellaewere present in thisseawa-
Actiniidae) and Anemonia viridis (Forskal) (family Ac- ter because Anthozoa-zooxanthella associations do not
tiniidae), and the zoanthid Isoioanthussulcatus(Gosse) occur locally. In addition, the examination of dark-
(family Parazoanthidae) are frequently found in the treated anemones suggested that infections had not oc-
same localitiesasC. pedunculatus, and also harborSym- curred underthese conditions(see below). Thejuveniles
biodinium sp. (Davy et al., in press). A ballii and A. vir- werecollected on two occasions, with thefirst batch (n =
idisregularlyexpelzooxanthellaeinlargeboluses,aswell 250) remaining in the dark for more than 3 years and
as in mucous strands (Davy, pers. obs.); it is also proba- the second batch (n = 120) remaining in the dark for
ble that /. sulcatus releases zooxanthellae, but this has 5 months. Aftercollection, theanemoneswere fedevery
not been observed. Thisexpulsion suggeststhat zooxan- 2 months with Anemia sp; the anemones also received
thellae from a variety ofsources are available to infect planktonic food viathe flow-through seawatersystem. It
newhosts. is likely that thejuvenilesweregenetically related toone
This research therefore aimed to determine whether anotherbecause all adult anemones were collected from
heterologous zooxanthellae can infect C. pedunculatus. the same site and spawning ofgametes has not been ob-
and to compare the persistence ofhomologous and het- served in thispopulation (Davyand Turner, pers. obs.).
erologouszooxanthellaeafterinfection. From thisinfor- Prior to use, 3-year dark-treated anemones (;; = 180)
mm
mation the potential for heterologous zooxanthellae to with oral discdiametersofabout 2 to 3 were placed
establish a lastingsymbiosis with C. pedunculatus in the in 100-mlcontainersfilledwith filtered(0.45 nm)seawa-
field could be inferred. ter(FSW). Fiveanemoneswereplacedineachcontainer.
210 S. K. DAVY ET AL.
These anemones were maintained at 21C, illuminated Persistenceofhomologousandheterologous
at 80 ^mol photons m : s ' on a cycle of 12 h light:12 h zooxanthellae
dark, and fed weekly with Anemia sp. for 2 weeks. The
anemones were ne\erobserved to change from white to To investigate theextentto which zooxanthellae from
brown over thi- ocriod and so were assumed to be apo- different host species could establish a symbiosiswith C.
symbiotic. Thisassumption waschecked by squashing a pedunculatus, theuptakeand persistenceofhomologous
subsample of five anemones (anemones sampled from and heterologous zooxanthellae following inoculation
the same container: nwaslimited byanemone availabil- were measured.
ity) and examining their tissues under bright-field and Threeoral discsplusattached tentaclesofC. peduncu-
epifluorescent illumination using a Leitz Orthoplan mi- latus, 5 tentacles from each of 10 individuals ofA. hallii
croscope. Neither zooxanthellae nor chlorophyll a were andA. viridis, 30 polyps of/, sii/catus, and 10 complete
detected, confirming that the 3-year dark-treated anem- A. pa/lida were collected for isolation ofzooxanthellae.
ones were aposymbiotic. Therefore, immediately after Host tissue for each host species was pooled, homoge-
being maintained in the light for 2 weeks, these anemo- nized in 10 ml FSW in a glass tissue-grinder, and centri-
nes were used for monitoring patterns oflong-term in- fuged for 10 min at 1200 rpm. The supernatant was dis-
fection byzooxanthellae. Non-inoculatedcontrol polyps carded,andthealgalpelletwasresuspendedin5 mlFSW
used in these experiments remained zooxanthella-free andcentrifuged fora further 5 min at 1200 rpm. Thesu-
overa period of36 weeks, reconfirmingthat theanemo- pernatant was again discarded and the algal pellet ad-
neswere aposymbiotic. justed with FSW togive a concentration of2 X 107 cells
In contrast, squashes made of 5-month dark-treated ml '. Isolates from different host specieswere kept sepa-
polyps (n = 5) before they were placed in the light re- rate.
vealed a small number of residual zooxanthellae (less Dark-treated specimens ofC. pedunculatus (n = 150
than 1000 zooxanthellae per polyp). These anemones and n = 90 for anemones maintained in darkness for
were therefore used for short-term monitoring ofinfec- 3 years and 5 months, respectively) with oral disc diam-
mm
tion patterns, where densities ofresidual zooxanthellae etersofabout2to3 weremaintainedin 100-mlcon-
were negligible in comparison to densities of phagocy- tainers (five anemones ofthe same dark-treatment per
tosed zooxanthellae. To keep residual zooxanthellae toa container)and inoculated with homologousor heterolo-
minimum, theseanemoneswere maintained in thedark gous zooxanthellae. Anemones in each container were
until inoculation. inoculatedwithzooxanthellae fromjustonesource, thus
preventing cross-infection by zooxanthellae from differ-
Injection ofaposymbioticCereus pedunculatus when co- ent sources. A 1-ml hypodermic syringe was used to de-
existing with symbioticC. pedunculatus posit 0.2 ml ofsuspension (i.e.. 4 X 10" zooxanthellae)
onto the oral disc ofeach aposymbiotic anemone. This
Under nonstressful conditions, C. pedunculatus regu- number of zooxanthellae was sufficient to saturate the
larly releases a small percentage (<0.3% per hour) ofits uptake sites ofthese anemones (Davy, 1994). Once the
zooxanthellae (Darmayati, 1993). A motile stage hasyet anemones had expanded again, 0.1 ml ofanAnemiasp.
to be observed in the life history ofthese zooxanthellae, suspension was pipetted onto each oral disc. This sus-
either following release or when in culture (Davy el a/., pension enhanced ingestion of zooxanthellae (Davy,
in press; Davy, pers. obs.). To establish whetherzooxan- 1994).ToremoveanyzooxanthellaeandAnemiasp.not
thellae released by one anemone could infect another captured by the anemones, the FSW was then changed.
anemone, 3-year dark-treated C. pedunculatus (n = 5) Uninoculated 3-year and 5-month dark-treated anemo-
were placed in acontainerwith symbiotic C. peduncula- nes (/; = 25 and n = 15, respectively; five anemones per
tus (n = 5). Three-year dark-treated C. pedunculatus (n 100-ml container) acted as controls for spontaneous in-
= 5) in a second containeracted as controls. All anemo- fection. All anemones were maintained at 2PC, illumi-
m
nes were illuminated at 80 ^mol photons irr: s ' on a nated with 80 ^mol photons - s ' on a cycle of 12 h
cycleof12 h light:12 hdark, maintainedat21 C, and fed light:12 h dark, and fed weekly with Anemia sp. The
weekly with Anemia sp. After 8 weeks, the presence or densities of zooxanthellae in 5-month dark-treated
absence ofzooxanthellae in the formerly aposymbiotic anemones were measured after 4 h. 2 days, and 4 days,
polyps (which were still distinct due to their lighter pig- and thedensitiesofzooxanthellae in 3-yeardark-treated
mentation)wasdetermined. Thisinvolvedsquashingthe anemoneswere measured after2, 4, 6, 8, and 36 weeks.
polyps and using bright-field and epifluorescent micros- Followingtheincubation period, polyps(/; = 4or5 for
copy to search their tissues. Densities of zooxanthellae each group; replicate anemones sampled from the same
were not quantified. container) were narcotized in 1.5% magnesium chloride
PERSISTENCE OF ZOOXANTHELLAE IN SEA ANEMONES 211
in FSW. Oral disc diameters were then measured using not homogeneous (as determined using Bartlett's statis-
an ocular micrometer, and oral disc areas were calcu- tic), datawere logarithmically transformed.
lated. This method (i) facilitated measurement oflarge
numbers of polyps; (ii) was repeatable; and (iii) gave Results
units(square millimetersoforaldisc)proportionaltothe
protein content of the polyp (r2 = 0.97 for anemones Infection ofaposymbioticCereuspedunculatus when
with disc diameters of 1.0 to 3.0 mm; Davy. 1994). Pol- co-e.\ixtinK with xymhioticC. pedunculatus
yps examined between 4 hours and 4 days after reinfec- All aposymbiotic Cereuspedunculatus maintained in
tion were cut longitudinally, and residual Artemia sp. the presenceofsymbiotic C. pedunculatusturned brown
and zooxanthellae werewashed from theirenterons; this after 2 months. Light microscopy revealed that this
step was not necessary for anemones examined after change in color resulted from the presence of zooxan-
2 weeks or more. Each polyp was then homogenized in thellae in the endodermal cells. In contrast, control
I ml FSW. and the number ofzooxanthellae in the ho- anemones remained white and free ofzooxanthellae af-
mogenate was counted using a Fuchs Rosenthal hema- ter2 months.
cytometer. The density ofzooxanthellae in the anemo-
ne's tissues was expressed as zooxanthellae per square
Short-termpersistenceofzooxanthellae
millimeteroforal disc.
The same method was used to determine the densities Figure 1 summarizes the patterns of infection of C.
ofzooxanthellaeinfivesimilarlysized, naturallyinfected pedunculatusby homologous zooxanthellaeand by zoo-
C.pedunculatus. Thesevalueswerecomparedwiththose xanthellae from Anthopleura ba/lii. Anemonia viridis,
from the infection experiments. Isoioantlnts sulcatus. and Aiptasiapa/lida overthe first
4 days(i.e.. overthe short-term).
The densities ofhomologous zooxanthellae were not
Statisticalanalysis
significantlydifferentfrom thedensitiesofanyofthehet-
Significantdifferences(P< 0.05)were identified using erologouszooxanthellaeat4 h (Bonferronipost-hocAN-
one-way analysis of variance (ANOVA) followed by OVA, P> 0.05); but note that the homologous zooxan-
Bonferroni pair-wisecomparisons. When varianceswere thellae were 2.5 times denser than the zooxanthellae
B
1
Time (days) Time (days)
Figure 1. Short-term persistence patternsofhomologousand heterologouszooxanthellae in infected
Cereuspedunculatus- All anemones dark-treated for 5 months prior to infection and unfed over period
shown,n= 5. Pointsat4daysoffsetforclarity.Allvaluesaremeans 1 SE.(A)Persistenceofhomologous
zooxanthellae from C. pedunculanm ( ). and heterologous zooxanthellae from Anthopleura hallii
(A AlandIsi>:<>antlnt\ >ulcalu.i(O- -O).D- -Drepresentszooxanthellaeinnon-inoculateddark-
treated controls. (B) Persistenceofheterologouszooxanthellae from Anemonia viridisfrom Lough Hyne
(4_ . _ *)andShell Island(V V),and.-liplasiapallidu( ).
212 S. K DAVY KT AL
B
Homologouszooxanthellae
/
468
H h
i
4 6 8 36 36
Time (weeks)
Figure 2. Long-term persistence patterns ofhomologousand heterologous zooxanthellae in infected
Cereuspedunculatus. All anemonesdark-treated for morethan 3yearspriortoinfection and fed weekly
overperiodshown,n= 5,exceptn =4forzooxanthellaefromAntlwpleurahallnandlaoioanlhussulcatus
at36weeks.Pointsat36weeksoffsetforclarity.Allvaluesaremeans 1 SE.(A)Persistenceofhomologous
zooxanthellae from C. pedunculatus( ). and heterologouszooxanthellae from A. balln (A A)
and / sulcatus(O- -O).D- -Drepresentszooxanthellaein non-inoculateddark-treatedcontrols,and
white diamond represents /ooxanthellae in naturally infected C pedunculatus (B) Persistence of
heterologous zooxanthellae from Anenwnia viridis from Lough Hyne (*--*) and Shell Island
(V V),andAiptasiapallida( ).
fromA. ballii(Fig. 1A). Subsequently, homologouszoo- andzooxanthellaefromA. ballii. A. viridis(Shell Island),
xanthellae persisted at the highest densities. Homolo- and/. sulcatusat8 weeks(Bonferronipost-hocANOVA,
gous zooxanthellae were present at significantly greater P<0.05). However, at 36 weeks, thedensitiesofhomol-
densities than any ofthe heterologous strains at 2 days ogous zooxanthellae, heterologous zooxanthellae, and
(Bonferronipost-hocANOVA, P< 0.01). zooxanthellaein naturallyinfected C. pedunculatuswere
Short-term infection patternsbyheterologouszooxan- notsignificantlydifferent (ANOVA, P> 0.05).
thellae were similarto one another, with the only major Zooxanthellae from A. pallida were present at signifi-
difference beingthe relatively low uptake ofzooxanthel- cantly lowerdensitiesthan zooxanthellae fromA. viridis
laefromA. bal/ii. ZooxanthellaefromA. balliiwerepres- (Lough Hyne)at4 weeks(Bonferronipost-hocANOVA,
ent at significantly lower densities than zooxanthellae P < 0.05). Zooxanthellae from A. viridis (Shell Island)
from /. sulcatus at 4 h (Bonferroni post-hocANOVA, P were present at significantly lower densities than any
<0.05). other heterologous zooxanthellae at 2 and 4 weeks, and
zooxanthellae fromA. balliiandA. viridis(Lough Hyne)
Long-termpersistenceofzooxanthellae at 6 weeks(Bonferronipost-hocANOVA, P< 0.02). No
other significant differences were evident between the
Figure 2 summarizes the densities ofzooxanthellae 2, densities of heterologous zooxanthellae (Bonferroni
4, 6, 8, and 36 weeksafterreinfection (i.e., overthe long- post-hocANOVA, P> 0.05).
term), and the density ofzooxanthellae in naturally in-
fected C. pedunculatus. Anemonesize
Homologous zooxanthellae persisted at significantly
greater densities than any of the heterologous zooxan- Prior to infection, anemones had oral disc diameters
thellae at 2 and 6 weeks (Bonferroni post-hoc ANOVA, ofabout 2 to 3 mm. At the end ofthe 36-week experi-
P < 0.05). Homologous zooxanthellae also persisted at mental period, all anemonesinfected with eitherhomol-
significantlygreaterdensitiesthanzooxanthellaefromA. ogous or heterologous zooxanthellae still had oral disc
mm
bal/ii, A. viridis(Shell Island), and A. pallida at 4 weeks. diametersof2 to 3 and were not significantly differ-
PERSISTENCE OF ZOOXANTHELLAE IN SEA ANEMONES 213
ent in size(averagediameter2.39 0.03 mm; ANOVA. is thought not to play a major role in the regulation of
P> 0.95). As oral discarea is proportional to polyp pro- populationsofzooxanthellae(ColleyandTrench, 1985).
tein content (see Materials and Methods), these results The elimination ofsome zooxanthellae more quickly
suggest that changes in anemone hiomass were minimal than others has also been observed in C. xamachana
during the experimental period, presumably reflecting (Colley and Trench, 1983). This trend may indicate
thelimited feedingregime(onceperweek). Thusthesub- differences in zooxanthella survivorship following isola-
stantial changes in zooxanthella densities were most tion ordiscriminationamonggenotypicallydistinctzoo-
likely due to changes in the number of zooxanthellae xanthellae by means of post-phagocytotic recognition
ratherthan inthenormalizingparameter(oraldiscarea). (Colley and Trench, 1983; Trench, 1988. 1992, 1993;
Markell et al., 1992; Markell and Trench, 1993). Ifzoo-
Discussion xanthellae were being discriminated against by post-
phagocytotic recognition, it iscurious that a small num-
The results reveal that zooxanthellae from a range of berofzooxanthellae always persisted. Perhaps there was
host species, localities, and environments can establish some variation within the source populations, with the
symbioses with Cereuspedunculutiis. In the short-term, retained zooxanthellae being the few that possessed cer-
homologous zooxanthellae persist to a greater extent tain appropriatecharacteristics.
than heterologouszooxanthellae, but overthe long-term
the two types ofzooxanthellae achieve similar densities. Long-termpersistenceofzooxanthellae
The possible reasons for these infection patterns and
their implicationswill bediscussed. Homologous zooxanthellae repopulated C. peduncu-
latus much more rapidly and achieved higher densities
Short-termpersistence than did heterologous zooCxanthellae. Similar patterns
have been observed in xamachana (Colley and
C peditncidatus took up zooxanthellae from An- Trench. 1983) andAiptasia tagetes(= pallida) (Schoen-
thopleiira hallii at a lower rate than zooxanthellae from berg and Trench, 1980c). In contrast, zooxanthellae
theotherhost species, perhapsindicatingdiscrimination fromAnemonia viridis from Shell Island repopulated C.
based upon thesurfacecharacteristicsofthezooxanthel- pednncidatus moreslowlythan did anyoftheotherzoo-
lae. However, uptake rates of zooxanthellae from the xanthellae. These repopulation trends may result from
other host speciesweresimilar. Thissuggeststhat during host-algal recognition and greaterexpulsion ordigestion
theinitial phasesofinfection C.pediinciilatus'wasunable ofsometypesofzooxanthellaethanothers. However,the
todiscriminatebetweenthesezooxanthellae,perhapsbe- repopulation patterns could also be determined by
cause ofthe similarity oftheir surface characteristics or differing growth rates ofzooxanthellae; changes in host
the host material contaminating their surfaces (Trench biomass (eithergrowth orshrinkage), which could cause
etai. 1981;ColleyandTrench, 1983). the 'dilution' or 'concentration' ofzooxanthella popula-
Following phagocytosis, all populations of zooxan- tions, were probably not responsible for the density
thellae declined, with the homologous populations de- changes. Comparison ofgrowth and expulsion rates of
clining less rapidly than most ofthe heterologous popu- homologous and heterologous zooxanthellae during re-
lations. Symbiont populations also declined following population ofaposymbiotic anemones would be an in-
infectionsofHydra viridissima(Jolleyand Smith, 1980) terestingtopic forfuturework.
and the jellyfish Cassiopeia xamachana (Colley and Several factors could influence the growth ofzooxan-
Trench. 1983), but did not do so following infections of thellae during repopulation. Firstly, the light and tem-
a number ofotherspecies ofCnidaria (Muscatine et al., perature regimes may favor growth by one type ofzoo-
1975; Schoenberg and Trench, 1980c; Berner el al., xanthellae overanother. However, this possibility is not
1993). The decline may represent the nonselective elim- supported by the similarity ofthe specific growth rates
ination ofa proportion ofthe phagocytosed zooxanthel- (0.25-0.29 d~') ofzooxanthellae from C. pedunculatus,
lae orthe selective elimination ofunhealthy and incom- A. ballii. andA. viridis, when cultured in vitro underthe
patible zooxanthellae. Elimination may occur viaexpul- same light and temperature regimes as those used here
sion or digestion. Regular expulsion ofzooxanthellae is (Davy. 1994). Secondly, the growth of zooxanthellae
a common feature ofAnthozoa-zooxanthella symbioses maydepend upontheabilityofaparticularstrainorspe-
(Steele, 1975; Heegh-Guldberget al., 1987; Stimson and cies to survive in the intracellular environment of the
Kinzie, 1991; McCloskey et al.. 1996). and the produc- host (Rahat and Reich, 1988). But this hypothesis is in-
tion of zooxanthella-containing boluses was observed consistent with evidence from analogous plant-micro-
during the course ofexperiments. In contrast, digestion bial symbioses (Trench, 1993). Thirdly, hosts may be
214 S. K. DAVY ET AL
able to control the growth of zooxanthellae directly unlikely toexplain the gradual increase in the density of
(Smithand Muscatine. 1996). Itisthereforepossiblethat zooxanthellae from A. pallida. This anemone is found
C. pedunculatus was able to exercise more control over at subtropical latitudes in the western Atlantic, and so
some types of / i.xanthellae than others. Finally, the probably contains zooxanthellae that are quite different
growth ofzo nellae may be limited by the intracel- from thosein C. pedunculatus. In fact, thezooxanthellae
lularspace ,iablewithin the host, with small zooxan- ofthisanemone arebelieved to bea single species, Sym-
thellaeachievinggreaterdensitiesthan largezooxanthel- biodinium bermudense(Banaszak et al., 1993). Further-
lae. It seemsunlikelythat this hypothesiscanexplain the more, evidence from carbon-flux studies suggests that
densities achieved by zooxanthellae from A. viridis the homologous and heterologous zooxanthella popula-
(Lough Hyne) and A. pallula (Fig. 2B), given that these tionswere not identical at 36 weeks(Davy, 1994).
zooxanthellae are similar in size to zooxanthellae from
C. pedunculatuswhen in theiroriginal hosts, after resid- Host-symbionl recombination in thefield
ingin reinfected C. pedunculatusfor36 weeks,andwhen
cultured in vitro (Davy, 1994; Davy et al., 1996, and in Moleculargenetictechniquesmustbeemployedtode-
press). It is unknown, though, whetherthesezooxanthel- termine the precise nature ofthe source populations of
laemaintained similarsizesthroughouttherepopulation zooxanthellae and whether heterologous zooxanthellae
process. actually establish symbioseswith C. pedunculatm in the
In spite ofprevious differences, at 36 weeksthe densi- field. The long-term infection patterns (Fig. 2) suggest
tiesofhomologousand heterologouszooxanthellae, and that host-symbiont recombination may be possible if,
zooxanthellae in naturally infected C. pedunculatus, that is, the new symbiosis is competitive under the pre-
were similar. The decline in the density ofhomologous vailing environmental conditions and the heterologous
zooxanthellae to a "normal" level is unique. Ifthe pop- zooxanthellae are not overgrown by homologous zoo-
ulation ofzooxanthellae was unialgal, then the decline xanthellae in the short-term (Schoenberg and Trench,
may result from the population being "brought under 1980c;ColleyandTrench, 1983). Theabilitytoestablish
control." However, why this occurred only several symbioses with zooxanthellae from a range of sources
months after the establishment ofthe symbiosis is un- may enable C. pedunculatus to adapt to different envi-
known. One possibility is that control mechanisms be- ronmental regimes, as has been suggested for corals
come repressed in aposymbiotic anemones and are re- (Buddemeier and Fautin, 1993; Rowan and Knowlton.
stored during the repopulation process. Alternatively, if 1995). Alternatively, it may simply increase the anemo-
the homologouspopulation contained a mixture ofzoo- ne's chances ofsurvival should it lose all ofits zooxan-
xanthella types, then the decline may result from an ini- thellae and have to acquire new symbionts. There are,
tially successful type becoming unstable and being re- however, no reports of "bleached" C. pedunculatus in
placed byatype bettersuited tothecultureconditions. the field. How zooxanthellae behave in mixed homolo-
The ultimate success of heterologous zooxanthellae gous-heterologous inoculations and the ecological ad-
that were initially slow to establish a symbiosis with C. vantagesofmaintaining heterologouszooxanthella pop-
pedunculatuscanalsobeinterpretedindifferentwaysde- ulations are important questions for future research,
pending upon the nature of the zooxanthella popula- both in temperateand tropical systems.
tions. Firstly, ifthe populationswere unialgal, then host-
symbiont adjustment over time may have enhanced the Acknowledgments
compatibility of the partners (Roughgarden, 1975;
Smith, 1980). It would be interesting to re-isolate heter- Wethank Drs. R. K. TrenchandC. B.Cook,andsev-
ologous zooxanthellae or make reinfected anemones eral anonymous reviewers for valuable comments on
aposymbioticagain, and determine whetherthe repopu- earlierdraftsofthepaper. Wealsothankthe Irish Wild-
lation process is faster when repeated. In a comparable life Service for permission to work and collect speci-
experiment, however, the growth of heterologous zoo- mens in Lough Hyne Marine Nature Reserve. This
xanthellae inA. tagetcswas not enhanced by theirprevi- work was carried out whilst SKD was in receipt of a
ous association with the same host species (Schoenberg SERCaward.
and Trench. 1980c). Alternatively, if the populations
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tshmeallllaenuatmb3e6rweoefkzsoomxaanythreelplraeeseindtentthiecaplrtoolitfheorsaetiuosnuaolflya Banasniosiv'zl.alak(,Pn'hAky.'tloTld.ai,cnRis.apl.eIsgnl)oe,vs.idai(snP-oePfrrliiadegilenoli,laalatenesd)sRay.nmbdKi.oGTnlrtoesenocodhfi.ntiw1uo9m93h.vydirsSomczrmoiapsnpps-.
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