Temperature Stress Causes Host Cell Detachment in Symbiotic Cnidarians: Implications for Coral Bleaching

9 Pages·1992·3.8 MB·English

by  R D Gates

Reference: Biol. Bull 182: 324-332. (June, 1992)
Temperature Stress Causes Host Cell Detachment
in Symbiotic Cnidarians: Implications
for Coral Bleaching
RUTH D. GATES, GAREN BAGHDASARIAN, AND LEONARD MUSCATINE
Department ofBiology, University ofCalifornia, Los Angeles, California 90024
Abstract. During the past decade, acute and chronic thellae to host biomass (Drew, 1972). During the past
bleaching of tropical reef corals has occurred with in- decade, however, ecologists have observed that relatively
creasing frequency and scale. Bleaching, i.e., the loss of small changes in the physical parameters ofthe marine
pigment and the decrease in population density ofsym- environment can dramatically influence the stability of
biotic dinoflagellates (zooxanthellae), is often correlated these symbioses (Glynn, 1990). Cnidarian bleaching and
with an increase or decrease in sea surface temperature. mortality have often been correlated with unusually high
Because little isknown ofthecellulareventsconcomitant or low sea surface temperatures in tropical oceans world-
with thermal bleaching, we have investigated the mech- wide (Brown and Suharsono. 1990; Coles and Fadlallah,
anism ofrelease ofzooxanthellae by the tropical sea ane- 1990; Glynn, 1990; Williams and Bunkley-Williams,
mone Aiptasia pulchella and the reef coral Pocillopora 1990). Bleaching hasbeen attributed toareduction in the
damicornis in response to cold and heat stress. Both spe- amount ofchlorophyll a(ColesandJokiel, 1977; Kleppel
cies released intact host endoderm cells containing zoox- ct a/.. 1989; Porter ct al., 1989; Szmant and Gassman,
anthellae. The majority of the released host cells were 1990) and accessory pigments (Kleppel el al, 1989) per
viable, butthey soondisintegrated in theseawaterleaving zooxanthella cell, a decline in the population density of
behind isolated zooxanthellae. The detachment and re- the zooxanthellae (Fisk and Done, 1985; Hoegh-Guldberg
leaseofintact hostcellssuggeststhatthermal stresscauses and Smith, 1989), or both (Glynn and D'Croz, 1990;
host cell adhesion dysfunction in these cnidarians. Lesserel al., 1990). Loss ofzooxanthellaepersehas been
Knowledge ofthecellularentity released bythe host dur- described extensively at the organismic level (Jaap. 1979;
ing bleaching provides insight into both the underlying Gates, 1990; Glynn and D'Croz, 1990; Goreau and Mac-
release mechanism and the way in which natural envi- farlane, 1990; Hayes and Bush. 1990; Jokiel and Coles,
ronmental stresses evoke a bleaching response. 1990; Lesser ct al., 1990; Szmant and Gassman, 1990),
yet few investigators have addressed the underlying cel-
Introduction lular mechanism (see O'Brien and Wyttenbach, 1980;
Sandeman. 1988; Lesser ct al.. 1990) or the morphology
Most tropical corals and sea anemones (Phylum Cni- ofthe cellular entity released. Insight into these features
daria) contain large populations ofsymbiotic dinoflagel- is essential for an understanding ofhow sea surface tem-
lates (zooxanthellae). The zooxanthellae are located in perature anomalies or other environmental stresses de-
vacuoles within the host endoderm cells (Glider ct a/.. stabilize zooxanthellae-cnidarian symbioses.
1980;Trench. 1987)wheretheymediatetheflux ofcarbon Zooxanthellae could be released by any offive mech-
and nutrients between the host and the environment anisms (Fig. 1), four ofthem resulting in the release of
(Muscatine, 1990). morphologically characteristic cellular entities. The five
Zooxanthellae-cnidarian symbiosesare normally stable; mechanisms are: (a)exocytosisofzooxanthellae from the
that is, they have a relatively constant ratio of zooxan- host cell, resulting in the release ofisolated algae (Steen
and Muscatine, 1987); (b) apoptosis (programmed cell
Received 17January 1992;accepted 17 March 1992. death) and (c) necrosis, both resulting in the release of
324
CELL DETACHMENT IN SYMBIOTIC CNIDARIANS 325
Host CellEndoderm Cellular Product Mechanism In this paper, we describe the cellular entity released
byA. piilclielld and the Hawaiian coral Pocillopora dam-
icornis immediately after a briefexposure to low or high
temperature. P. damicornis is one ofseveral coral genera
that have undergone extensive bleaching in the tropical
eastern Pacific during the elevated temperature ofthe El
EXOCYTOSIS
Nino-southern oscillationevent(Glynn, 1990),andduring
upwellingand seasonal low temperatures (see Discussion
in Glynn and D'Croz, 1990; seealsoWalkerela!., 1982).
Both species can be bleached in the laboratory, and
bleaching is due to a reduction in zooxanthellae popu-
APOPTOSIS
lationdensity(Glynnand D'Croz, 1990; Muscatineelal.,
1991). Observations of the cellular entities released at
hourly intervals during, and shortly after, both cold and
heat stress, showed clearly that temperature stress causes
detachment and releaseofintact endoderm cellscontain-
NECROSIS
ing zooxanthellae. Soon after release, the host cells dis-
integrate in the environment, leaving isolated zooxan-
thellae.
Materials and Methods
PINCHING OFF
Animal collection and maintenance
A.pulchellaand P. damicorniswerecollectedat 1 meter
HOST CELL depth on Checker Reefadjacent to the Hawaii Institute
DETACHMENT of Marine Biology (HIMB), Coconut Island, Oahu, Ha-
waii. Habitat temperatures rangeannually from 21-22C
Figure 1. A schematic representation offive potential mechanisms to 26-27C (Jokiel and Coles, 1977). P. damicornis col-
by which zooxanthellae could be released from the endoderm ofcni- onies were placed in running seawater and used for ex-
danans. and the cellular entities associated with each mechanism, m, periments at HIMB within three days of collection. A.
mesoglea;vm,hostvacuolarmembrane;hn,hostcellnucleus;zx,zoox- pulchella was transported to the University ofCalifornia
anthella(shaded forclarity ofpresentation). at Los Angeles, maintained in an aquarium at 25 C on
a 12 h light/dark regime, and fedtwiceaweekonAnemia
nauplii. Priorto experiments, theanemoneswere starved
zooxanthellae associated with remnants of the host cell for 24 h in an incubator (Precision Scientific Model 8)
(Searle a ai, 1982); (d) pinching offofthe distal portion at 25C on a 12 h light/dark cycle at 40 ^mole
ofthe host cell, resulting in the release ofzooxanthellae quanta irr2'S~'.
surrounded by the vacuolar and pinched off plasma
membrane (Glider, 1983); and (e) detachment ofendo- Temperature treatments
derm cells from the host and release ofthese intact cells
containing their complement ofzooxanthellae. All experiments were carried out in darkness following
Because cnidarians can be readily bleached in the lab- the protocol ofMuscatine et al. (1991).
oratory by briefexposure to low (Steen and Muscatine, Coldstress. Individuals ofA. pulchella were incubated
1987; Muscatine el ai, 1991) or high (Hoegh-Guldberg in Petri dishes (35 X 10 mm) containing 4 ml of0.45 ^m
and Smith, 1989; Glynn and D'Croz, 1990) seawater Millipore filtered seawater(MFSW) chilled to 12C. After
temperature, the mechanism ofbleaching and the mor- 2.5 h, thechilledseawaterwasremovedand replaced with
phology ofthe cellularentities released can be investigated seawater at 25C. Anemones were maintained at 25C
experimentally. This approach, together with scanning in an incubator in darkness for 14 h. The cellularentities
electron microscopy of endoderm of the Hawaiian sea released to the seawaterwere then collected and processed
anemone A. pulchella after experimental cold shock, re- as described below.
vealed profiles that were interpreted as evidence ofexo- Small branches ofP. damicornis (2-3 cm length) were
cytosisofzooxanthellae (Steen and Muscatine, 1987). In- removed from each coral colony and placed in beakers
deed, examination of the cellular entities released 24 h containing25 mlofMFSWchilledto 12 C(fortheprotein
aftercold stress revealed abundant isolated zooxanthellae. assay, corals were cold stressed at 14C). After 4 h, the
326 R. D. GATES ET AL
pipette and deposited onto coverslips coated with poly-
L-lysine(0.1% in distilled water). The entitieswerestained
forviabilitywith the fluorogenicdyefluorescein diacetate
(SigmaChemicalCo., stocksolution 15 mgM/ml inacetone;
working solution 0.04 ml in 9.96 ml 0.1 sodium phos-
phate, 3% sodium chloride, 0.004% calcium chloride, pH
7.4). The coverslipswere rinsedtwice in phosphate buffer,
mounted and viewed under epifluorescence with an
Olympus BH-2 microscope. Non-specific esterases in vi-
ablecellshydrolyze non-polarfluorescein diacetatetopo-
lar molecular fluorescein (Schupp and Erlandsen, 1987).
Additional coverslips were treated for 30 min with the
DNA fluorochrome Hoechst 33258 (Reynolds el
specific
ai. 1986; Sigma Chemical Co., stock solution 5 mg/ml
in distilled water; working solution 0.04 ml stock in 9.96
M
ml 0.1 sodium phosphate, 3%' sodium chloride, 0.004%
calcium chloride). The coverslips were dipped in phos-
phate buffer, mounted, and viewed with epifluorescence
microscopy.
Maceration and electron microscopy
Figure 2. Left panel: photomicrographsofthe hostcellsreleased to Thecellularentitiesreleasedaftercoldstresswerecom-
theseawaterbyAiptasiapulchellainresponsetocoldstress,stained for pared to isolated endoderm cells obtained by maceration
viability with fluorescein diacetate (X4000). Right panel: photomicro- ofcontrol anemones and corals. A. pulchella tissue was
giHnroaerpcehhssspotofn3ste3he2t5oh8ocs(otlXcd4e0lsl0ts0re)rs.esl,eassteadinteodthweitsheatwhaeteDrNbAyPsopceiclilfoipcnrfaludoarmoicchorronmies CmhaceemriactaeldCou.s)inagnd0.P.05d%amiccoolrlangiesnatsisesue(Twyapsedi1s,soSciiagtmead
using calcium-free artificial seawater (Gates and Musca-
tine, 1992). Endoderm cells released by maceration and
branches were immediately transferred to beakers con- the cellular entities released to the seawater as a result of
taining 25 ml of MFSW at ambient temperature (23- temperature stress were collected with a mouth pipette
24C). The beakerswereplaced in the seawatertables for and transferred onto poly-L-lysine coated coverslips. ThMe
ambienttemperaturecontrolandthecoraltissueandsea- coverslips were immersed in 3% glutaraldehyde in 0.1
water in the beakers was sampled after 12 h. Controls for sodium cacodylate buffer (pH 7.4) for 1 h, rinsed twice
both species were treated identically to experimental an- in 0.1 M sodium cacodylate buffer and post-fixed in 1%
M
imals but were maintained at ambient seawater temper- osmium tetroxide in 0.1 sodium cacodylate for 30
ature (25C for A. pulchella and 23-24C for P. dami- min. After dehydration in 30, 50, 70, 90. 95, and 100%
cornis) for 16-16.5 h. (X3) ethanol. the coverslips and attached cells were im-
Heat stress. Individuals ofA. pulchella were plMacFedSiWn mersed in hexamethyldisilazane (Applied Sciences, Inc.)
Petri dishes (35 X 10 mm) containing 4 ml of for 5 min (Nation, 1983), dried in air, and then mounted
warmed to 32 C. Small branches of P. damicornis (2-3 on aluminum stubs. The stubswerecoatedwithgold and
cm length) were placed in beakers containing 25 ml viewedonaCambridge 360scanningelectron microscope,
MFSW pre-heated to 32C. The animalswere maintained with an accelerating voltage of 10 kV.
at thistemperature for up to 16 h. Thewatersurrounding For transmission electron microscopy, the endoderm
experimental specimens was examined microscopically cells released by maceration ofP. damicornis tissue and
at hourly intervals and the cellular entity released to the the cellular entities released as a result of temperature
seawaterremoved and treated asdescribed below. Control stress were collected and centrifuged (Eppendorf model
animals ofboth species were maintained at ambient sea- 5414, full speed for 30 s) in microfuge tubes (Gilson, 1.5
water temperature (25C for A. pulchella and 23-24C ml). The pelletswere fixed asdescribed forscanningelec-
for P. damicornis) over the experimental time period. tron microscopy. After partial dehydration by sequential
30 min treatments in 30, 50, and 70% ethanol, the 70%
Staining andepijluorescence microscopy ethanol was drained from the tube and immediately re-
The cellular entities released during and after temper- placed with 2% agar. After the agar solidified, the tube
ature stresswere collected with a fine bore mouth suction was cut away from the agar plug containing either cold-
CELL DETACHMENT IN SYMBIOTIC CNIDARIANS 327
Figure3. Scanningelectron micrographsofindividual hostcellsreleased byAiplusiapulchella(A)and
Pocillopora damicornis (B) in response to cold stress, and those obtained from .-1 pulchella (C) and P.
damicornis(D) bytissue maceration. Bar = 1 urn.
stressed or macerated cells and dehydration completed (modified from McAuley, 1986). For P. damicornis a 4
through 90, 95,and 100% (X3)ethanol. Thepreparations ml sub-samplewasremoved from 25 ml seawatersamples,
were embedded via propylene oxide into epoxy resin homogenized, and treated with SDS as described above.
(Spurr). Thin sections were cut using a Sorvall 6000 ul- Each sample was incubated at room temperature for 45
tramicrotome, stained with lead acetate, and viewed on min to solubilize protein in the seawater and host cell
a JEOL transmission electron microscope. membranes associated with released algae. The algaewere
pelleted by centrifugation (Damon/IEC model HN-S for
Protein determination 4 min at 3000 rpm) and the supernatant put aside for
protein analysis as described below. Each algal pellet was
To investigate the lossofanimal protein totheseawater resuspended in a known volume ofMFSW and the total
asaresult oftemperature stress, theseawaterwasremoved number ofalgae assessed using a hemacytometer.
from the Petri dishes ofcold stressed and control A. pul- Two 1 mlsampleswereremoved fromeachsupernatant
chella and homogenized in a teflon-glass tissue grinder. and the amount ofprotein assessed spectrophotometri-
Sodiumdodecyl sulphate(SDS, 1%inseawater)wasadded cally using the method ofHartree (1972). To ensure that
to each homogenate to a final concentration of 0.05% protein in the seawatersamples was animal in origin and
328 R. D. GATES ET AL
* ,
V
Figure4. TransmissionelectronmicrographsofhostcellsreleasedbyPocillopuradamicornisinresponse
tocoldstress(A),andtissuemaceration(B).HN,hostcellnucleus;ZX,zooxanthella;VM.vacuolarmembrane;
PM. host cell plasma membrane; and M. mitochondria; Bar = 1 ^m.
not secreted by the algae during the 16.5-h experimental view them by epifluorescence microscopy. After staining
period, control algae were isolated from anemones using with fluorescein diacetate and Hoechst 33258, these cells
homogenization and centrifugation. The resulting algal were identical in profile to those released aftercold stress.
pellets were washed twice in MFSW and treated for 45 Host cells released in both cases appeared to be viable,
min with 0.05% SDS to solublize any animal protein as- with fluorescence restricted to the narrow compartment
sociated with the algal cells. After two more washes and ofthe host cell cytoplasm that surrounded from one to
re-suspension in MFSW, the numberofalgae present was fivezooxanthellae (Fig. 2, left panel). Fluoresceindiacetate
assessed using a hemacytometer. Algal suspensions were was either not taken up by the zooxanthellae, or it was
cold stressed (with controls) as described for whole ani- taken up but masked by the intense red autofluorescence
mals. SDS was added to a final concentration of0.05% ofthe zooxanthellae chlorophylls and the yellow autoflu-
and the samples were left at room temperature for 45 orescence of the zooxanthellae accumulation bodies.
min. The algae were removed by centrifugation and Staining with the bisbenzamide dye Hoechst 33258 re-
counted again to determine ifcells had lysed during the vealed a single nucleus within each ofthese cells (Fig. 2,
incubation. The remaining supernatant was assayed for right panel).
protein as before. When viewed with scanning electron microscopy, the
cellsreleased asaresultoflowtemperaturestressexhibited
Results a morphology that wassimilartoendoderm cellsreleased
from both P. damicornis andA. pulchella by maceration
Theentitiesreleasedduringandaftertemperaturestress (Fig. 3). In both cases, the host cell nucleus was visible
appeared to be intact host cells. Those released after cold underthe plasma membrane. This observation suggested
stress settled at the bottom ofthe container. In contrast, that entities released by thermal stress were intact cells
those released afterheat stressaccumulated at the surface and not "pinched off" products. Transmission electron
ofthe water. Unlike the former, the latterwereextremely microscopyconfirmed thesimilarity,and clearly revealed
difficult to collect and handle. They were too fragile to the host cell plasma membrane, the vacuolar membrane
manipulate for electron microscopy, but we were able to surroundingthe zooxanthellae, the host cell nucleus, and
CELL DETACHMENT IN SYMBIOTIC CNIDARIANS 329
Figure5. TransmissionelectronmicrographsshowingdegradationofthehostcellsreleasedbyPocillopora
damicomisinresponsetocoldstress. Afterdissociationfromtheepithelium,thehostcellplasmamembrane
ruptures (A) and the cytoplasmic constituents are free to disperse in the seawater (B). ZX, zooxanthella;
RPM.rupturedhostcellplasmamembrane;CC.hostcellcytoplasmicconstituents;IZX,isolatedzooxanthella.
Bar = I nm.
mitochondria (Fig. 4). Once released as a result oftem- profilesobserved by Steen and Muscatine (1987), and in-
perature stress, the host cells degraded rapidly. The host terpreted as exocytosis ofzooxanthellae, may have been
cell plasma membrane ruptured, the cytoplasmic com- incidentallyevokedbylowtemperature, orbyotherstim-
ponents dispersed, and the vacuolar membrane disap- uli, but neitherexocytosis. apoptosis. necrosis, norpinch-
peared completely, leaving isolated algae in the seawater ing off appear to be primary mechanisms of thermal
(Fig. 5). bleaching by the cnidarians observed in this investigation.
The release ofintact host cells by A. pu/c/ie/la and P. Loss ofhost cells may explain why investigators observe
damicomis after thermal stress was further indicated by loss of protein by bleached corals in excess of that ac-
a significant positive correlation between the number of counted for by loss ofzooxanthellae alone (Porter et al.,
algae released and the total soluble protein detected in 1989; Glynn and D'Croz, 1990; Szmant and Gassman,
thesurrounding medium afterthe host cellsdisintegrated 1990). Despite lossofcells, thehostssurvivethetreatment.
(Fig. 6). Zooxanthellae and soluble protein released by Release of zooxanthellae appears to be a two-phase
unstressed control animals was modest (A. pulc/iella) or process. Time-lapse video ofA.pulchelladuringandafter
negligible (P. damicomis). Protein released by isolated low temperature shock reveals that host cells containing
zooxanthellae was below the limits ofdetection, and cell zooxanthellae first dissociate from the endoderm and ac-
countsconfirmedthatisolated zooxanthellae had notlysed cumulate in the coelenteron where they form pellets or
during the incubation (data not shown). remain asloosecells. Then, duringtherewarmingperiod,
the pelletsand cellsareperiodically propelled byciliaand
Discussion muscles through the actinopharynx to the external me-
dium (Hoegh-Guldberg, 1989; Muscatine et al., 1991). A
Theresultsofthisinvestigation showthattransientlow protocol using gradual change in temperature also re-
and high temperature stressin darknesscausesa reduction vealed host cell detachment. However, this protocol was
in thepopulation density ofzooxanthellae inA. pu/chcl/a dismissed in favor ofthe precipitous change in tempera-
and P. damicomis. Quantitative aspects ofthis reduction ture because the former required a more lengthy and
are described elsewhere (Steen and Muscatine, 1987; complex sampling regime.
Muscatine et ai, 1991). This reduction is caused largely We speculate that the dissociation of host cells from
bydetachment ofhostcellscontainingzooxanthellae. The the endoderm iscaused by host cell adhesion dysfunction.
330 R. D. GATES ET AL.
ofthe former may cause dysfunction ofthe latter (Tak-
eichi, 1988). Alternatively, temperature stress may cause
denaturation ofproteinsinvolvedincelladhesion (Watson
and Morris, 1987; Suzuki and Choi, 1990).
Althoughwehavedescribedthecellularentitiesreleased
afterthermal stressin darkness, and a probable underlying
mechanism, low salinity (Goreau, 1964; Egana and Di-
salvo, 1982) and sedimentation (Acevedo and Goenaga,
1986) also evoke bleaching. Moreover, at high tempera-
ture, the bleaching response in some cnidarians isthought
to be exacerbated by high irradiance (Coles and Jokiel,
1978), ultraviolet radiation (Harriot, 1985; Jokiel and
York, 1982; Lesseret ai, 1990), andactive oxygen (Lesser
and Shick, 1990). These other types of stress cause de-
No.z1ooxanthellaer2eleased (x l(f)3 carneiassmedozfobolxeaancthhienlglaienpeoapcuhlaitnisotnandceensiistsyt,illbuutnkthneowmne.chI-t
450 may be fundamentally different from that observed in
400 Pocilloporadamicornis thermal bleaching. For example, we speculate that low
salinity may cause the cnidarians to lose zooxanthellae
350 by the mechanical disruption caused by hypoosmotic
I 3 shock (i.e., necrosis). Wesuggest that bleachingbedefined
3> more rigorously in termsofboth theenvironmental stress,
<D 250 -
and the morphology ofthe cellularentity released. Studies
are now under way to determine ifhost cell detachment
aQV. 15 after thermal stress is a general phenomenon or specific
02468 to selected cnidarian genera.
I 10
50 -i Acknowledgments
We thank Alicia Thompson and Birgitta Sjostrand for
No.ofzooxanthellaereleased (x 106) avsisdiisntginagnwietphifellueocrtersocnenmciecrmoisccroopsyc,oGpoeradtoHnIGMrBa,uafonrdprtoh-e
Figure6. Appearanceofsoluble protein in theincubation medium OfficeofNaval Research (Grant#NOOO14-89-J-3246 to
concomitant with release of zooxanthellae by Aipiasia pulchella and L.M.) and the National Science Foundation (Grant
Pocillopora damicornis. Control (open squares), cold stressed (closed #OCE-8723090 to L.M.) for research support.
squares). Line fit with linear regression (Zar, 1984), for A. pulchella,
r = 0.81 (y = 6.6012 + (2.71 10~5)x). For P. damicornis. r = 0.96
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