Late Larval Development and Onset of Symbiosis in the Scleractinian Coral Fungia scutaria

10 Pages·1999·4.5 MB·English

by  J A Schwarz

Reference: Biol. Hull. 196: 70-79. (February, 1999)
Late Larval Development and Onset of Symbiosis in
the Scleractinian Coral Fungia scutaria
JODI A. SCHWARZ1 DAVE A. KRUPP2 AND VIRGINIA M. WEIS1'*
, ,
1 Department ofZoologv, Oregon State University, Comillis. Oregon 97331. and
2 Department ofNatural Sciences, Windward Community College, Kaneohe, Hawaii 96744
Abstract. Many corals that harbor symbiotic algae (zoo- eration ofhosts firstacquires its symbionts (Douglas, 1994).
xanthellae) produce offspring that initially lack zooxanthel- Symbionts may be acquired either vertically, whereby the
lae. This study examined late larval development and the symbiont is transmitted directly from parent to offspring, or
acquisition of zooxanthellae in the scleractinian coral Fun- horizontally, whereby the offspring must acquire symbionts
gin scutaria, which produces planula larvae that lack zoo- from the environment (Trench, 1987). Vertical transmission
xanthellae. Larvae reared under laboratory conditions de- ensures that offspring are provided with a complement of
veloped the ability to feed 3 days after fertilization; feeding symbionts, whereas horizontal transmission is more uncer-
behavior was stimulated by homogenized Artemia. Larvae tain; environmental variability may preventcontact between
began to settle and metamorphose 5 days after fertilization. symbiont and host, resulting in the failure of the host to
In laboratory experiments, larvae acquired experimentally become infected by its symbiont.
added zooxanthellae by ingesting them while feeding. Many members of the phylum Cnidaria (such as corals,
Zooxanthellae entered the gastric cavity and were phagocy- sea anemones, and jellyfish) harbor intracellular photosyn-
tosed by endodermal cells. As early as 1 h after feeding, thetic dinorlagellates (Symbiodinium spp.) in a mutually
zooxanthellae were observed in both endodermal and ecto- beneficial symbiotic association. The dinoflagellates, also
dermal cells. Larvae were able to form an association with known as zooxanthellae, contribute to host nutrition by
three genetically distinct strains of zooxanthellae. Both translocating photosynthetically fixed carbon, while the
zooxanthellate and azooxanthellate larvae underwent meta- hosts provide the zooxanthellae with nutrients and a pro-
morphosis, and azooxanthellate polyps were able to acquire tected, high-light environment. Many cnidarian host species
zooxanthellae from the environment. Preliminary evidence
are obligately symbiotic with zooxanthellae, thus vertical
suggests that the onset of symbiosis may influence larval transmission might be predicted to be the dominant mode of
development; in one study symbiotic larvae settled earlier
symbiont transmission. However, this is not the case, at
than aposymbiotic larvae. Protein profilesofeggs andlarvae least in scleractinian corals. Most scleractinian coral species
throughout development revealed a putative yolk protein
dwoausblaebtsetnhtatbwyasdaaybu6.ndTahnitsisnteugdygsisantdhe1f-idrsaty-tooledxlaamrivaneeatnhde tshpeallwane)ga(mFeadtleaslltahha.t a1r9e83a;zooBxaabnctohcelklaatned(i.He.e.ywlaacrkd,zoo1x9a8n6-;
onset of symbiosis between a motile cnidarian host and its Harrison and Wallace, 1990; Richmond and Hunter, 1990;
Richmond, 1997). The gametes are fertilized within the
algal symbiont.
water column and develop into azooxanthellate planula
larvae thatmustacquire zooxanthellae atsome stage oftheir
Introduction
development (Trench, 1987).
The life history ofsymbiotic associations between organ- Offsetting the uncertainty of infection via horizontal
isms necessarily includes a stage during which a new gen- transmission is the benefit that acquisition of symbionts
from the environment might allow the host to form an
association with genetically distinct symbionts that are
*ReTcoeiwvehdom30cAoprrrielsp1o9n9d8e;ncacecesphtoeudld25beSeapddtreemsbseerd.19E9-8m.ail: weisv@bcc. adapted to local conditions. Rowan and Knowlton (1995)
orst.edu found that the corals Montastraea faveolata and M. annn-
70
ONSET OF SYMBIOSIS IN FL/NGIA SCUTAKIA 71
laris naturally associate with several species of Synihio- quisition in the scleractinian coral Fungia scutaria. This
tliniiun that occur along an environmental gradient, and that solitary coral is gonochoric, and the females spawn azoo-
hosts can contain two species at one time. Many studies xanthellate eggs that are fertilized within the water column
have compared the uptake and influence of heterologous and develop into a/ooxanthellate larvae (Krupp, 1983).
and homologous /ooxanthellae on experimentally infected Krupp reported on theearly developmentofthis species and
cnidarian host polyps (literature summarized most recently observed that larvae reared in aquaria with adult corals
in Davy el /., 1997). Thus although vertical transmission acquired zooxanthellae 4 to 5 days after spawning. He
ensures that offspring are provided with zooxanthellae, hor- observed a "mouth opening" response to the addition of
i/ontal transmission may allow for the acquisition of sym- zooxanthellae obtained from homogenized tissues of adult
bionts that are adapted to the specific environment in which F. scutaria. but did not observe zooxanthellae entering the
the offspring ultimately settle. mouths of the larvae. In this paper we describe late larval
There are several mechanisms by which initially azoo- development and the process of symbiont acquisition (in-
xanthellate cnidarian hosts may acquire their algal symbi- fection) in F. scnttiria, including the developmental stages
onts from the environment and incorporate them into at which the host is competent to become infected by
endodermal cells, where they ultimately reside. First, zoox- zooxanthellae, the mechanisms by which the zooxanthella
anthellae may be incorporated into the embryo. Second, are acquired and incorporated into host tissue, the effect of
zooxanthellae may be incorporated into the host's ectoderm feeding behavior on the infection rate, the specificity ofthe
and then migrate intothe endoderm. Both these mechanisms host-symbiont relationship, and the protein profiles of
occurin the scyphozoanLinuche unguiculata', bothembryos azooxanthellate and zooxanthellate larvae through develop-
and young, nonfeeding planulae are capable of becoming ment.
infected by experimentally added zooxanthellae (Montgom-
ery and Kremer, 1995). Third, zooxanthellae may be incor- Materials and Methods
porateddirectly intoendodermal cells, as was firstdescribed
in scyphistomae (post-planula polyps) of Cassiopeia .\ain- Gamete collection and lan-al cultures
aclwna (Trench. 1980; Fitt and Trench. 1983a, b). In this About 75 adult specimens of Fungia scutaria are main-
third mode ofinfection, symbionts enter through the mouth tained year-round in running seawater tables at the Hawaii
ofthe host and are phagocytosed by endodermal cells lining Institute of Marine Biology on Coconut Island, Kaneohe
the gastric cavity (Colley and Trench, 1983; Fitt and Bay, Hawaii. For our experiments, the corals were rinsed
Trench, 1983a. b). with seawater and placed in standing seawater in individual
Although many studies have either documented in detail glass fingerbowls. Filtered (0.45 jum) seawaterwasused for
or anecdotally noted zooxanthella acquisition by naturally all cultures. This species generally spawns between 1700
azooxanthellate polyps (scyphozoans: Sugiura, 1964; and 1900 h, 2-4 days after the full moon during June
Trench, 1980; Colley and Trench. 1983: Fitt and Trench, through August. In August 1995, August 1996. and June
1983a. b; anthozoans: Kinzie, 1974; Babcock and Heyward, and July 1997. eggs were collected by removing the adults
1986; Benayahu el ai, 1989). little information exists about from the finger bowls and leaving the eggs in the bowl into
either the life-history events orthe mechanisms ofinfection which they were spawned. If the egg density was greater
associated with the onset of symbiosis in initially azooxan- than a single layer of eggs at the bottom of the dish, some
thellate planulae. Montgomery and Kremer (1995) found of the eggs were collected with a turkey baster and trans-
that young planulae ofthe scyphozoan Linuche unguiculata ferred to a new finger bowl. Within 30 min after spawning,
became infected (by an unknown mechanism) by experi- water from the dishes ofall spawning males was combined
mentally added zooxanthellae. Schwarz (1996) found that and a small volume was gently pipetted into the dishes
planulae ofthe temperate sea anemoneAntliopleura elegan- containing eggs. The dishes were left in a seawater table
tissimu acquired zooxanthellae via phagocytosis after feed- overnight forfertilization andearly larval development. The
ing on animal tissue that contained zooxanthellae recently following day, the water was changed. Larvae from all
isolated from a previous host. parental crosses were combined, and the larvae were main-
Given that most scleractinian corals produce azooxan- tained in large glass fingerbowls in filtered seawater. which
thellate planulae. it is likely that at least some acquire was changed every day.
zooxanthellae during the planula stage. Planulae are motile
and represent the dispersal stage ofcorals; the acquisition of
Preparation ofzooxanthella isolates
symbionts during this stage might therefore be advanta-
geous because it presents an opportunity for the planulae to Zooxanthellae were isolated from adult specimens of F.
acquire symbionts adapted to the environment in which the scutaria by using the spray from an oral hygiene device
larval hosts will settle live. (Water Pik) to remove and homogenize coral tissue; they
In this study we examined the process of symbiont ac- were then concentrated using a tabletop centrifuge at
72 J. A. SCHWARZ ET AL.
2000 X g. The zooxanthella pellet was rinsed twice in zooxanthellae were removed or 24 h later, to determine if
filtered seawater to partially clean it of animal tissue and they had become infected with zooxanthellae.
was again concentrated by centrifugation. Zooxanthella iso- For treatments Cl and C2 (Table I), we used the follow-
lateswereused within 2hofpreparation. The same methods ing method to determine the fraction of larvae that became
were used to isolate zooxanthellae from the sea anemone infected. Twenty-four h after the larvae were exposed to
Aiptasia pallida. except that whole animals were homoge- zooxanthellae, the water in the larval cultures was swirled
nized in a ground-glass tissue grinder. and one aliquot was removed from each replicate. Between
25 and 56 larvae per aliquot were examined under a com-
Preparation ofhomogenized Artemia sp. pound microscope to count how many contained zooxan-
thellae.
To stimulate feeding behavior in larvae, homogenized
Anemia sp. (brine shrimp) was added to larval cultures. A Lan'al development
small pinch of frozen Anemia was homogenized in a
ground-glass tissue grinder in about 1 ml of seawater and To observe and quantify the developmental progression
filtered through a 60-jam mesh to remove large paniculate of both azooxanthellate and zooxanthellate larvae, six rep-
matter. The resulting slurry was used within 15 min of licate cultures of each were maintained in plastic 6-well
preparation. culture dishes (300-500 larvae per well in 5 ml of filtered
seawater). Water was changed roughly once a day. Larval
Acquisition ofzooxanthellae development was monitored for about 2 weeks. Each rep-
To identify (a) the developmental stages at which F. clircoastceopwee,llanwdaswiptlhaicnetdhehafpiehladzoafrdvliyewu,ntdheernaumdibsesrecotfinlgarmvia-e
scutaria is competent to become infected and (b) the mech- at each developmental stage was counted.
anisms ofzooxanthella acquisition, larvae from four stages
of development (Table I) were exposed to zooxanthellae
Electron microscopy
from different sources, with or without homogenized Ar-
temia (afeeding stimulant). Homologous algae were freshly To follow the process ofzooxanthella incorporation into
isolated from adult F. scutaria, and heterologous algae were host tissue, larvae from treatment C2 were sampled and
either freshly isolated from the sea anemone Aiptasia pal- fixed forelectron microscopy 1 and 24h afterzooxanthellae
lida or taken from algal cultures originating from thejelly- were added to larval cultures. The larvae were placed in
fish Cassiopeia xamachana. Three replicates were estab- sampling cups, which were prepared by cutting off the
lished for all treatments. Larvae were concentrated in glass bottomsofmicrofuge tubes andaffixing 50-/j,m meshacross
finger bowls (>104 larvae per bowl), and an even layer of the bottom. The cups were placed inM1% glutaraldehyde in
zooxanthellae was pipetted along the bottom of the bowls. phosphate-buffered saline (PBS. 0.1 sodium phosphate.
Several drops of homogenized Anemia were added to the 0.45 Msodium chloride, pH 7.2) for 1 h; rinsed 3x10 min
appropriate treatments. Zooxanthellae and Anemia slurry in PBS: postfixed for I h in 1% osmium tetroxide in PBS:
were removed either 4 or 24 h later (Table I) by concen- rinsed 3 X 10 min in PBS; and dehydrated for 15 min each
trating larvae on a filter and placing them into clean filtered in 30%, 50%, and 70% ethanol, and then 1 h each in 80%,
seawater. Some larvae from each treatment were observed 95%, and 3 X 100% ethanol. Samples for scanning electron
under a compound microscope, either immediately after microscopy were dried for 15 min in hexamethyldisilane.
Table I
Experimental treatmentsofFungia scutaria lan-ae
Treatment: Developmental stage Source ofAlgae Anemia added? Exposure duration Infection determined
A: embryo-early planu'.a (0-12 h old)
ONSET OF SYMBIOSIS IN FUNGIA SCUTAR1A 73
mounted on stubs, coated with 60:40 Au:Pd, and viewed on Results
an Amray 3300FE scanning electron microscope. Samples
for transmission electron microscopy were infiltrated with Larval development
S2puXrr1's00re%sirnesiinn1f:1oret1hha.noaln:dres1i0n0f%orr2e.s5inh,ov1e:r3nmigihxtfaotr62.05Ch., (19L9a4r,val199d6e.vel1o99p7m)e.ntLawravsaeobfsreormveadlloyveearrsthrfeoellsowuemdmetrhse
TCwhiMtihn1u2sreatcnrtyailnosnamsciewstseairtoeenapenrldeepclaterraeoddnciomtinrcatraeon,scuaolnptdera.vmiiecwreodtoomne,asPthaiilniepsd ssFiawgmiuemrmepir1noAggraetnsodsidcoernteaeoipfliedndegvbeetloloopwsmeetitnnlteFadi.lgusTrtheaeg2e,sd,purraoasgtriielolsnussitonrfgatfeedraocimhn
developmental stage, however, was variable; for the later
Polyacrylamide gel electrophoresis stages it differed by up to several days both within and
We prepared one-dimensional SDS-PAGE protein pro- among replicates. Figure IB shows the time course of
files of both azooxanthellate and zooxanthellate larvae developmental events for zooxanthellate larvae from 1996.
through development (eggs through 6-day-old larvae). For All larvae progressed through the following series of
each sample, about 1000 larvae were counted, collected by stages. Within 12 h after fertilization, slowly moving, cili-
centrifugation, and frozen at 80C. Protein extracts were ated spherical planulae developed: within 24 h, barrel-
prepared by homogenizing frozen larvae over ice in a shaped planulae, roughly 100 /nm in length (shown in Fig.
groumndM-glass grinder imn MKM) p.\ of homogenization buffer 2A), had developed and were actively swimming at all
(40 Tris-HCl, 10 EDTA. protease inhibitor cock- depths in the culture dishes. By day 3, larvae had fully
tail (Sigma). pH 7.4). Homogenates were centrifuged for 10 formed mouths and functional gastric cavities, and were
min at 14.000 X g topellet zooxanthellae and animal debris. capable of feeding. Upon addition of food (homogenized
The protein concentration of the supernatant was deter- Anemia), larvae ceased swimming and dropped to the bot-
mined spectrophotometrically (Bradford. 1976); larvae con- tom of the dish. They extruded mucus, their oral ends
tained approximately 50-100 ng protein/larva. Larval pro- expanded, and they ingested whatever they landed on. in-
teins were resolved on 12.5% SDS-PAGE gels under cluding experimentally added zooxanthellae. As they fed,
reducing conditions (methods modified from Laemmli. their gastric cavities became filled with participate matter
1970). Gels were silver stained (methods modified from (Fig. 2B). Some larvae resumed swimming while trailing a
Heukeshoven and Dernick. 1986) and scanned on an Im- strand of mucus; the mucus trapped particulate matter that
agernaster desktop scanner (Pharmacia) and analyzed using slowly entered the mouth. Larvae continued to feed for
Irnagemaster software (Pharmacia). several hours and then resumed swimming. Except for
Figure 1. Progression ofdevelopmental events in Fttn^ut \citttirui larvae. (A) Schematic representation of
developmental stagesfromtheearlyplanulathrough metamorphosedpolyp. (B)Exampleofthe timecourseof
developmental events. Data shown are from zooxanthellate larvae in 1996. Larvae were infected with zooxan-
thellae on day 3 and then divided into six replicate dishes, which were monitored daily. Each point represents
data pooled from the six replicates. Larvae progressed from swimming to creeping to settling.
74 J. A. SCHWARZ ET AL.
Acquisition ofzooxanthellae and onset ofsymbiosis
Prior to the development ofa functional mouth on day 3,
planulae of F. sciituria did not become infected by experi-
mentally added zooxanthellae. Once the mouth was func-
tional, however, the planulae were able to acquire zooxan-
thellae. When stimulated to feed, larvae indiscriminately
ingested any particulate matter, including experimentally
added zooxanthellae. Zooxanthellae either were ingested as
part of a larger mass that was fully engulfed by the mouth,
or they adhered to mucous strands that were ingested by the
larvae. Figure 3A shows a zooxanthella adhered to a larval
mucous strand, and Figure 3B shows several zooxanthellae
surrounding and contained within the oral cavity ofa larva.
One hour after zooxanthellae were added, larvae were sam-
pled and fixed for transmission electron microscopy. Figure
4 shows a representative planula 1 h postfeeding. in longi-
tudinal section, with several algae resident in endodermal
figure 2. Light micrographs ofstages in the development ofFiingia
scularia larvae. (A)Two-day-old planula larva, priorto developmentofa
mouth. (B) Three-day-old feeding planula (m = mouth, mf = mucous
=
strand with food particles attached, z zooxanthella). (C) Polyp with
tentacles,6daysaftersettling.Zooxanthellaearevisibleasgoldenspheres.
Planula length and polyp diameter, approximately 100 /xm.
zooxanthellae, all ingested paniculate matter was digested
or expelled by the following day. When larvae were about
4 days old, they assumed a ball shape, ceased active swim-
ming, and began creeping slowly overthe substrate. Starting Figure3. Scanningelectronmicrographsdetailingzooxanthellaacqui-
on day 5, the ball-shaped larvae began to settle. They spread sziotoixoanntbhyel3l-adaayd-hoelrdedFitiongmiuacsocuusrasrtiraanpdla(numla=e.m(oAu)thF,eezdi=ngzopolxaannutlhaelwliat).h
out over the substrate and metamorphosed into volcano-
(B) Feeding planula. with multiple zooxanthellae entering the mouth.
shaped polyps, which began to develop tentacle buds sev- Larvae were fixed for electron microscopy 1 h after exposure to zooxan-
eral days after metamorphosis (Fig. 2C). thella isolates and homogenizedAnemia (see Methods). Bars = 10 ij,m.
ONSET OF SYMBIOSIS IN FUNGIA SCUTAKIA 75
days following showed that the zooxanthellae were retained
within the polyps throughout this period.
The proportion of larvae that became infected by zoo-
xanthellae isolated from adult F. scutaria (Treatment C)
depended on the strength of the feeding response. Feeding
was observed to be strongly stimulated (i.e.. virtually all
larvae began to feed) by the addition of homogenized Ar-
temia, but was also stimulated to a lesser extent (i.e., some
larvae began to feed) simply by the addition of zooxan-
thella-isolates, which contained residual animal host tissue.
We
quantified the effect of larval feeding strength on zoo-
xanthella acquisition for treatments Cl (zooxanthellae
alone) and C2 (zooxanthellae and Artemia). In the zooxan-
thellae alone treatment, 25.0% 0.02% ( = 2) of larvae
ec acquired zooxanthellae, whereas 96.8% 0.01% ( = 2)
became infected when exposed to both zooxanthellae and
Anemia. It was clearthat larvae in Treatments D and E also
Figure 4. Transmission electron micrograph ofa longitudinal section became infected at a higher rate when exposed to both
through a Fungia scutaria larva infected with zooxanthellae. Thickened zooxanthellae and homogenized Anemia than to zooxan-
oral end at lower left. Zooxanthellae appear in the endoderm as dark thellae alone, although the results were not quantified.
snpehmearteosc.ysLtisg.hetce=lliepcsteso,dermmo,stelny=inentdhoedeercmt,odzer=m,zoaorxeanptoheolrllay.pBraerse=rv2e0d An experiment in 1996 provided preliminary evidence
that symbiotic state may influence developmental events in
/im.
F. scutaria. Zooxanthellate larvae settled and metamor-
phosed earlier than azooxanthellate larvae, most of which
cells. Micrographs suggest that zooxanthellae are phagocy- became arrested in the "ball stage" andthen eventually died
tosed by endodermal cells lining the coelenteron (Fig. 5A, (Fig. 7). However, the same experiment repeated in 1997
B) and appear in both endodermal (Fig. 5C) and ectodermal showed low rates ofmetamorphosis for both zooxanthellate
tissue (Fig. 5D). Although zooxanthellae were still present and azooxanthellate larvae and no difference in the timing
in ectoderm 24 h later, we did not determine how long of metamorphosis.
zooxanthellae remained within the ectoderm or whether
they eventually migrated into the endoderm or were di- Lan'alprotein profiles
gested or expelled from the host.
Larvae were not limited to forming an association with a Protein profiles of larvae through development showed
specific strain of zooxanthellae; planulae were capable of changes with the age ofthe larvae. Two bands, at 84 and 79
becoming infected by zooxanthellae isolated from F. scu- kilodaltons (kDa), were abundant in eggs and 1-day-old
taria (Treatment C2) and Aiptasia pallida (Treatment D2), larvae (Fig. 8A). As shown in Figure 8B. this protein
as well as by cultured zooxanthellae from Cassiopeia xani- doublet comprised a significant proportion (36%) of total
acliana (Treatment E2) (see Table I). To determine whether protein in 1-day-old larvae, but was almost absent by day 6.
the host had retained zooxanthellae, larvae from Treatments The apparent depletion of this protein corresponds to the
C2 and D2 were observed over a period of 10 to 14 days. onset of settlement and metamorphosis. The abundance of
Larvae that had acquired zooxanthellae on day 3 remained the putative yolk protein did not differ between 6-day-old
infected as they progressed through development and meta- azooxanthellate and zooxanthellate larvae.
morphosis into polyps.
Infection by zooxanthellae was not required formetamor- Discussion
phosis: both zooxanthellate and azooxanthellate larvae suc- Lan'al development and acquisition ofzooxanthellae
cessfully settled and metamorphosed into polyps (Fig. 6).
Larvae infected with zooxanthellae from F. scutaria (Treat- Development in Fungia scutaria was similar to that re-
ment C2) and A. pallida (Treatment D2) both underwent ported in other broadcast-spawning species of coral (Bab-
metamorphosis (we did not monitor settlement for larvae cock and Heyward. 1986; review in Harrison and Wallace,
infected with zooxanthellae cultured from C. xamachana). 1990). Planula larvae had fully developed within 24 h after
Aposymbiotic polyps were able to ingest experimentally fertilization, which is within the rangeofoneto several days
added zooxanthellae via ciliary currents produced by the reported for other species. Larvae ofF. scutaria were about
polyps that swept paniculate matter, including zooxanthel- 100 /urn long, ciliated, and barrel-shaped; they exhibited
lae, over and into their mouths. Observations over the 6 active swimming behavior until they settled at an age of 5
76 J. A. SCHWARZ ET AL.
\ **
^,$Sr*
*** *
^M * ec
ec
,;,,'l
Figure 5. Transmission electron micrographsofonset ofsymbiosis between Fiing/n xcutariti planulae and
zooxanthellae. (A)Section throughendodermandgastriccavity ofaplanulashowing initialcontactbetweenan
endodermalcell and azooxanthella. Hostendodermal membranesare veryclosely associated with thealga. (B)
Endodermalcell partially surroundingazooxanthella, suggestingthatthealga isbeing ingestedbythehostcell.
(C)Zooxanthellaresident within avacuoleinanendodermal cell. (D)Twozooxanthellae in gastriccavity (one
is being phagocytosed) and one resident within a vacuole in an ectodermal cell, gc = gastric cavity ec =
ectoderm, en = endoderm, z = zooxanthella. Bars = S jum.
days to approximately 2 weeks. This appearance and be- xanthellate gametes that develop into azooxanthellate plan-
havior is typical for externally developed planula larvae. ulae (review in Richmond, 1997), little is known about how
Very little is known about the feeding ability or behavior planulae might acquire zooxanthellae from the environ-
of coral planulae. Although it appears that many species, ment. The results of this study support the idea that for
particularly brooding species, produce a nonfeeding larva, corals, competency for infection by zooxanthellae may gen-
the ability to feed has probably gone unrecognized in some erally depend on the development of a functional mouth.
species because rearing techniques generally do not expose We found that F. scitraria did not become infected by
larvae to a source of paniculate food. We found that the experimentally added zooxanthellae until after a mouth
feeding behavior of F. scuturui was very similar to that developed. Once the mouth was functional, all developmen-
reported for the temperate coral Caryophvllia smithi tal stages were competent to become infected. Reports of
(Tranter et al., 1982) and for the temperate sea anemones infection events in other species support this hypothesis
Anthopleura elegantissima and A. xanthogrammica species that are infected at the polyp stage appear to have a
(Siebert. 1974; Schwarz, 1996). Feeding consisted of a nonfeeding planula that does not develop a mouth until the
mouth-opening response to the addition of ground animal polyp stage (Kinzie, 1974: Babcock and Heyward, 1986;
tissue, as well as secretion of mucous strands that trapped Benayahu et ai, 1989). Studies of the feeding behavior of
participate matter for ingestion. planulae also support this hypothesis: planulae of both F.
Although most scleractinian coral species spawn azoo- scuttiria (this study) and A. elegantissima (Schwarz. 1996)
ONSET OF SYMBIOSIS IN FUNGIA SCVTARIA 77
2345
N V^?^fes*^ Pii--ii
43
30-
Figure 6. Light micrographs of newly settled polyps of Fungia scu- 20-
iiirin. (A) A/ooxanthellate polyp (m = mouth). (B)Zooxanthellate polyp. kDa
Zooxanthellae appear as brown spheres in the polyp. The two polyps
shown in this figureweresettledinthe samedish, adjacenttooneanother.
Contaminating diatoms appear as small ellipses around the polyps. Polyp
diameter = 100 jxm.
exhibit feeding behavior that leads to the ingestion of zoo-
xanthellae. It will be interesting to determine whether other
species that produce a feeding planula larva acquire zoo-
xanthellae in the same manner as shown for F. scutaria and
A. elegantissima. 34567
Both endodermal and ectodermal cells incorporated
zooxanthellae within 1 h after larvae were exposed to zoo-
xanthellae. The appearance of zooxanthellae in ectodermal
tissue was surprising because zooxanthellae phagocytosed Larval age(days)
bpoyrteenddoindteormtiaslsuecselwlshewroeutlhdeynodtobneoteuxlpteicmtaetedlytorebseidet.raWnse- FunFgiigaursecu8t.ariParolatreviane.pr(oAfi)leSsilavnerd-satbauinneddanIcDeSoDfSp-uptoaltyiavecryyollakmipdreotegienl oinf
did not determine how the zooxanthellae entered the ecto- total protein extracted from eggs (lane 2), 1-day-old larvae (lane 3),
derm. Future work will include long-term sampling of 6-day-old azooxanthellate larvae (lane 4), and 6-day-old zooxanthellate
newly infected larvae to investigate the fate ofthe ectoder- slatravnadeard(slanien l5a).neE1a.chArrlaonwehciognhtlaiignhetsda1.p2utfaitgivperoytoelikn.prMootleeincudloaurblweetig(h8t4
mal zooxanthellae. and79kDa)thatisabundant ineggsand 1-daylarvaebutabsentbyday6.
Horizontal transmission ofsymbionts would appearto be (BI Decline in abundanceofputative yolk protein through larval develop-
disadvantageous forobligately symbiotic species because of ment. The depletion ofputative yolk protein corresponded with the onset
ofsettlement on day 7. There was no difference in putative yolk protein
abundance in azooxanthellate and zooxanthellate larvae (days 5 and 6):
data shown represent the average ofthe two treatments.
100
the possibility that infection may not occur. However, for
planulae dispersed to areas with different environmental
conditions, the ability to acquire zooxanthellae from the
environment might confer a greater advantage to the host
than directly inheriting maternal zooxanthellae. This study
found that planulae ofF. scutaria were capable of forming
789 an association with members from three clades of zooxan-
thellae classified by Rowan and Powers (199la, b); zoo-
10 11 xanthellae from C. xamachanaare in groupA, thosefromA.
Larval age (days) pallida are in group B, and those from F. scutaria are in
group C. The degree to which zooxanthellae from different
Figure7. Effect ofsymbiotic state on larval settlement. Results from clades persist in F. scutaria remains to be investigated, but
s1e9t9t6lemeexnpterainmdentm.etaNmeoarrplhyos1i0s0b%yodfayzo1o0x.anwthheerlleaatsempolsatnualzaoeoxaunntdheerlwleantte our results suggest considerable flexibility in host-symbiont
planulae failed to settle. Each point represents data pooled from six specificity in this species. In contrast, planulae ofA. elegan-
replicate dishes for each treatment. tissima, although able to form an association with zooxan-
78 J. A. SCHWARZ ET AL
thellac recently isolated from a conspecific adult, were larval stage depends in part on the amount ofenergy avail-
unable to do so with cultured S. californium, which is the able for metabolism (Boidron-Metairon, 1995; Levin and
species reported to occur in A. elegantissima (Banaszak et Bridges, 1995). Larvae ofF. scutaria have several potential
til., 1993: Schwar/,. 1996). sources ofenergy that may allow them to extend the larval
The finding that a stronger larval feeding response re- stage sufficiently to explain their widespread occurrence
sulted in higher rates of infection indicates that larval feed- throughout the Pacific. First, larvae may initially obtain
ing behavior may play an important role in acquiring zoo- nutrition from yolk protein supplied through the egg. The
xanthellae from the ambient environment. Because so little presence and the pattern ofdecline oftwo abundant 84 kDa
is known about the distribution and abundance of zooxan- and 79 kDa proteins and the correlation between their
thellae in the natural environment, it is difficult to speculate depletion and the onset of settlement suggest that larvae
on potential sources of these symbionts. However, one may metabolize this protein over the course of their devel-
source thatis likelytooccurin abundance is mucusexpelled opment. Second, once the mouth has developed, larvae may
by corals. Cnidarian hosts regularly expel mucus containing obtain energy through feeding. Third, larvae that have ac-
high concentrations ofzooxanthellae (Steele, 1975; McClo- quired zooxanthellae may receive nutrition in the form of
skey et al., 1996; Schwarz. pers. obs.), and increased rates organic carbon translocated by zooxanthellae. Richmond
of expulsion have been reported to accompany spawning (1981, 1987) demonstrated that symbiotic planulae of the
(Montgomery and Kremer, 1995; D. Krupp, pers. obs.). coral Pocillopora damicornis received about 13%-27% of
Although this study did not examine whetherplanulae ofF. the carbon fixed by zooxanthellae. Each of these modes of
saitaria will feed on coral mucus, planulae of the sea nutrition may operate at different times in development, and
anemone Anthopleura elegantissima did feed on mucus each may function to extend the length of the dispersal
expelled by adults and became infected by the zooxanthel- stage.
lae within it (Schwarz, 1996). These results suggest that
ingestion ofzooxanthellae could occur either at the spawn-
Acknowledgments
ing site or at the sites in which the larvae ultimately settle,
allowing them to acquire symbionts adapted to different This work was supported by grants from the Office of
environments. Naval Research (NOOU149710101) to V. M. W.. and from
Sigma Xi and Oregon State University Zoology Department
Effect ofsymbiont acquisition on lan-al development to J. A. S. We thank P. Jokiel, B. Kinzie, F. Cox, and the
Zooxanthellae are known to affect the physiology oftheir staffofthe Hawaii Institute ofMarine Biology forfacilities,
adult hosts, and the acquisition of zooxanthellae by larval zooxanthella cultures (B. Kinzie), and support. We thank
hosts probably influences larval development. For example, the OSU Department of Botany and Plant Pathology Elec-
the acquisition ofsymbionts may act as a settlementcue. An tron Microscope facility staff for their assistance. Rick
experiment in 1996 demonstrated that zooxanthellate larvae Jones prepared the illustration for Figure 1. This is Contri-
settled earlier than azooxanthellate larvae (Fig. 6) indeed, bution #1043 from the Hawaii Institute of Marine Biology.
most azooxanthellate larvae failed to settle. However, the
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