Heat Shock Protein Induction in Montastraea faveolata and Aiptasia pallida Exposed to Elevated Temperatures

7 Pages·1995·2.9 MB·English

by  N A Black

Reference: Bio/. Bull. 188: 234-240. (June, 1995)
Heat Shock Protein Induction in Montastraea
faveolata and Aiptasia pallida Exposed
to Elevated Temperatures
NANCY A. BLACK1 RICHARD VOELLMY2 AND ALINA M. SZMANT1 *
, ,
]Division ofMarine Biology andFisheries, RosenstielSchool ofMarine andAtmospheric Science,
University ofMiami, Miami, Florida 33149; and2Department ofBiochemistry andMolecular
Biology. School ofMedicine. University ofMiami, Miami, Florida 33136
Abstract. Frequentwidespreadepisodesofcoralbleach- phyla examined thus far, from bacteria and yeast to hu-
ing have made researchers aware ofthe sensitivity ofreef mans.
corals to moderately elevated temperatures and led us to Several functions have been proposed for hsps, includ-
investigate mechanismsoftemperaturestresstolerancein ing the transport ofnewly transcribed proteins to the en-
thisgroup. Onesuch mechanism maybetheinducedsyn- doplasmic reticulum and mitochondria and involvement
thesis of heat shock proteins (hsps), which have been in the insertion ofthese proteins into these organelles(re-
shown to play a role in thermotolerance in other organ- viewed in Gething and Sambrook, 1992; Parsell and
isms. However, induced synthesis ofhsps in scleractinian Lindquist, 1993). Hsps mayalso playarolein translation
corals was not reported until recently. and, during stress, may interact with unfolding (denatur-
Experiments were conducted in which Montastraea ing) proteins to either keep them in a refoldable confor-
faveolata was exposed to high temperatures (up to 35C) mation ortarget them fordegradation (Welch, 1992). The
for short periods (2 h). Under the conditions tested, the production ofhsps has also been correlated with the ac-
corals produced seven different hsps with approximate quisition ofenhanced thermotolerance(Parsell and Lind-
molecular weights of95, 90, 78. 74, 33, 28, and 27 kDa. quist. 1993). Because oftheir important functions in nor-
Anotherzooxanthellate species, the seaanemoneAiptasia mal metabolic and repair processes, moderate amounts
pallida, also synthesized hsps during temperature stress, ofmany hsps are present in unstressed cells at all times,
but fewer and with different molecular weights (82, 72, but synthesis increases after a stress. In some cases, this
68, and 48 kDa) than those produced by Montastraea. It increase in hsp synthesis isaccompanied bya decrease in
nowremainsto bedetermined whether hspsare involved the production of other non-hsp proteins (Parsell and
in differences in thermotolerance and susceptibility to Lindquist, 1993).
bleaching within and between the various species of Interest in the effects ofelevated temperature on scler-
Montastraea. and between species ofreefcnidarians. actinian reef-building corals and their ability to produce
heat shock proteins has increased with recent frequent,
Introduction widespread episodes of coral bleaching (Glynn. 1991;
Heatshock proteins(hsps)arealsoreferredtoas"stress Williams and Bunkley-Williams, 1990). Corals have a
proteins" because their expression can be induced by a complex symbiotic relationship with photosynthesizing
numberofstressful conditions, only one ofwhich is heat dinoflagellates, called zooxanthellae, that are contained
shock (reviewed in Craig, 1985). These proteins are highly within the gastrodermal cells ofcorals. Bleaching results
conserved evolutionarily: they have been found in all from the loss of the zooxanthellae, the degradation of
chlorophyll pigments within zooxanthellae, or a combi-
nation ofboth. Many conditions have been shown to in-
Received 8June 1994;accepted 17 March 1995.
* Author to whom correspondence and reprint requests should be duce bleaching or are implicated in its production (re-
sent. viewedin Williamsand Bunkley-Williams, 1990), butthe
234
HEAT SHOCK PROTEINS IN CORALS 235
condition most often correlated with recent bleaching nearJoultersCay(25 19'N, 78 05'W), north ofAndros
eventsisprolonged exposureto moderatelyelevatedtem- Island, Bahamas, in 1988 and 1989. A pneumatic drill
peratures. fitted with a hole-saw and powered by compressed air
Hsp induction has been reported in a number ofcni- from a scuba tank was used to obtain short cores, 5 cm
darians. Two thermotolerant species of the hydrozoan in diameter, with live tissue only on the top. The corals
Hydrawere found to increasethesynthesisofhsp60when were used in nondestructive and nonstressful laboratory
exposed to elevated temperature or to the chemical in- experimentsinvolvingtemperatureand lightexposureand
ducers sodium azide and cadmium chloride; six more then returned to uncontrolled ambient laboratory con-
thermosensitive species failed to produce this hsp (Bosch ditions for several months before the start ofthese exper-
el a/., 1988). In the scyphozoan jellyfish Aurelia aurita, iments.
sixhspswereidentifiedwhen variousdevelopmentalstages Sea anemones ofthe speciesAiptasiapallida were ob-
were exposed to elevated temperatures for a few hours tained from holding tanks at the Florida Keys Marine
(Blackand Bloom, 1984). An anemone,Anemonia viridis, Laboratory on Long Key in February 1993. They were
produced six different hsps in response to elevated tem- allowed one week to acclimate to laboratory conditions
peratures or copper chloride (Miller el a/., 1992). Hsp70 before experiments were started.
was found in the coral Goniopora djiboutiensis (Sharp el Specimens were maintained in flowing, sand-filtered
ai, 1994), and other reports of hsp production in zoo- seawater at ambient seawater temperature (ca. 28C for
20-24C
xanthellate cnidarians have been presented at recent sci- the corals used in experiment 1, for the cor-
entific meetings. als and anemones used in experiments 2-4, Table I)
The objective ofthis research wastodeterminewhether under cool-white fluorescent or metal halide lights (ca.
theCaribbean reefcoral Montastraeafaveolata (Ellisand 200/iEin m~-s"' of light) on a 14:10 hour light:dark
Solander) formerly Montastmea annularis (Knowlton schedule. Thawed frozen brine shrimp were provided as
el ai, 1992; Weil and Knowlton, 1994) produces hsps; food once a week.
and ifso, which ones. Individual coloniesand siblingspe-
cies ofthis coral have various degrees ofsusceptibility to Incubationprocedures
temperature-induced bleaching(Gates, 1990; Szmant and
Gassman. 1990; Black, 1993). Samplesofthiscoralwere For all experiments, the organisms were incubated in
subjected to acute exposure to higher temperature fol- filtered(Whatman GF/A)seawaterwith 5 /iCi/mlofeither
lowed by variable recovery periods at control tempera- 3H-leucine ( 156.0 or 179.6 Ci/mmol specific activity) or
ture and then tested for elevation ofhsp synthesis. Sea "S-methionine/cysteine (1 141.0 Ci/mmol specific activ-
anemones, Aiptasia pallida (Verrill), were also used to ity) purchased from New England Nuclear.
work out techniques and to illustrate hsp production by Incubationwaseitheringlassdisheswithgentleaeration
another zooxanthellate cnidarian species. The exposure in a dark incubator or in covered beakers in a shallow
conditions tested for each species are summarized in water bath. The specimens were placed in the containers
Table I. with the radiolabeled amino acid at the beginning ofthe
temperature exposure, left there for the duration ofthe
Methods and Materials recovery periods of each experiment, then rinsed three
times with filtered seawater (to remove excess radiolabel
Collection and maintenance ofthe animals before processing). Each treatment group contained two
Multiple cores from large colonies of Montastraea or three corals or four anemones, but anemones were
m
faveolata were collected from a depth of5-7 on a reef pooled for processing.
Table I
Summaryoftheanimals, conditions, andisotopes usedintheheatshockexperiments
Exp. no.
236 N. A. BLACK ET AL
Tissuepreparation wasincorporated within each treatment, but do not allow
us to compare rates ofsynthesis ofthe various hsps be-
Coral tissues were removed from the skeletons with a
M tween treatments.
jet of0.1 Tris (pH 6.8) sprayed through an airbrush;
resulting tissue slurry volumes ranged from 3 to 13 ml.
Densitometry
Preparationswerekepton icethroughoutprocessing. The
tissue slurry was homogenized and centrifuged (850 X g, The autoradiogram from experiment 4 (Fig. 2A). in
15 min. 4C) to separate the coral animal fraction (su- which specimens ofAiptasiapallida were incubated in a
pernatant) from the zooxanthellae (pellet). Anemones temperature series from 22 to 35C (Table I), was ana-
were ground in 2 ml ofcold Tris buffer (0.1 M, pH 6.8) lyzed with densitometry to compare quantitatively the
in a glass tissue grinder with a Teflon-tipped pestle. Zoo- densities ofthe hsp68 and hsp72 bands from animals ex-
xanthellae and unbroken cells were removed by centrif- posed tothe varioustemperatures. The densitometry im-
ugation (6600 X g. 20 min, 4C). age analysis system consisted ofa NEC single-chip color
In the first experiment, the supernatants were centri- video camera to produce the image, a Targa + 64 frame-
MOCHA
fugedagainat 12,000 Xg(15 min, 4C)toremovecellular grabber board to capture the image, and the
debris and larger organelles (Ford and Graham, 1991). software package(Jandel Scientific)todothedensityscan
To 100 n\ of the latter supernatants waMs added 50 n\ of ofthe image. Equal areas ofeach temperature lane were
2X protein loading buffer [PLB; 0.125 Tris, 20% glyc- scanned, and the density valuesassociated with each band
erol, 2% 2-mercaptoethanol, and 4% SDS (Gallagherand summed. Figure 3 includesthedensityscansforeach lane
Smith, 1991)].Theamount ofradioactivityinasubsample (A), and histograms that present the density and the per-
ofeach supernatant was measured with a Beckman model centage oftotal density ofeach hsp in each lane (B, C).
LSI80scintillationcounter. Theremainderofeachsample
was boiled for 1 min and frozen at -80C. Results
In the later experiments, proteins were precipitated Exposure to elevated temperature increased the syn-
fPrrootmeitnhseswuepreernaptelalnettsedwitbhy8c%enttrriicfhulgoartoiaocenti(c1a2c,i0d00(TCXA)g., tsheeasiasnoefmosenveesr.alEdaicschrestpeecpireostepirnosd(uhcspesd)iitnsboowtnh ccohraarlasctaenrd-
280%mTinC.A4aCn)d.tThrheee tpeilmleestswiwtehreacreitnosnede ttohrreeemtoivmeesTwCiAt-h istic set ofhsps. The results ofeach experiment are sum-
marized separately for each species.
soluble and acetone-soluble contaminants, and then re-
dissolved in 1 ml ofPLB. A subsample was removed for Montastraeafaveolata (Fig. 1)
scintillation counting and the rest was boiled and frozen
at -80C. 31 & 34Cvs. 2SC. Coralsexposedto 34Cexpressed
M
several hsps with of 33. 74, 78, 90, and 95 kDa. In
r
Gel electrophoresis andautoradiography this experiment, but not subsequent ones, there was re-
Proteins were separated by one-dimensional SDS- dpurceesdumseyndthteosibseoafcottihneranprdotaeninusnskucnhowasna9490kkDDaapprrootteeiinn
proulnynaicnrgylgealm;id3e%gsetlacekliencgtrgoepl)h.orEeqsuisal(aSmDoSu-nPtAsGEo)f r(ad1i0o%- (3Fi1gC. lwa)e.rTehseimpialtatrerannsdofdpirdotneoitnsinscylnutdheesaizneyddaits2ce8rniabnlde
activity per sample (approx. 400,000 dpm) were loaded hsp bands.
in each lane, and protein molecularweight standardswere 27-35C vs. 22C. Hsp synthesis was tested at five
0r.u0n25inMpaTrrailsl,el0..1T9h2eMelgelcytcrionpeh,oarensdis0.b1u%ffSerDSco(nLsaisetmemdlio,f attem2p7e,ra2tu9r,esorfr3o1mC27(nottos3h5owCn.).NTohhesposnlwyerhespssyntthahteswiezreed
1970). After electrophoresis, gels were fixed (50% meth- distinguishable in thisexperiment were hsp74 in the 33C
anol, 5%- glacial aceticacid, 30 min), rinsed in water(three (faint)and 35C (darker) treatmentsaswell astwobands
times, 10 min each), incubated in a solution of 10% sal- ofca. 27 and 28 kDa at 35C (Fig. Ib). A repeat ofthe
iaictnygl8iKc0oCadcai(dk2 ahX.n)-.dOAm3u%at>togrlAaydRcierofogilrlma(m2ts0omwtiehnre)e,drpiarenodddugveclaescdfubourymseexdvpreoirsea-dl 9303.3C748C,e.xapnodsur7e4 ekxDpaer(iFmige.ntIcp)rosidmuicleadr atopatthtatersneoefnhbsepfsoraet
at
daysat -80C. The approximate molecular weights (Mr)
of protein bands on the autoradiograms were estimated
Aiptasia pallida (Figs. 2. 3)
by comparison with the positions ofmolecular standards
on the gels. 27-35Cvs. 22C. In thisexperiment, anemoneswere
Because the gels were loaded with equal amounts of exposed to five different 2-h heat treatments (Fig. 2a). At
radiolabel ratherthanequal amountsofprotein,theresults 22 (control), 27,and 29C,theanemonesdidnotappear
provide us with a relative comparison ofhow radiolabel stressed. At 31 C, however, the anemones were partially
HEAT SHOCK PROTEINS IN CORALS 237
B
28 31 34 22
,95
-90
i
-78
74
-74
-
*
* -33
-28
27
^^fe ^^ife
f^rilMh
Figure 1. Autoradiograms showing proteins synthesized by Montastraeafaveolata after exposure to
varioustemperatures. (A)Synthesisafter 1 weekat 31 Cor2hat 34Ccompared tocontrols(28C). The
positions ofhsps produced at approximately 95, 90, 78, 74. and 33 kDa are indicated. (B & C) Synthesis
after2 hat33 or35Ccomparedtocontrols(22C).Thepositionsofhspsproducedatapproximately 74,
28. and 27 kDa (B) and 90. 78. and 74 kDa (C) are indicated. Each lane represents the proteins ofan
individual coral, with twoorthree replicatecoralspertemperature treatment, a = protein presumed tobe
actin.
contractedduringheatexposure, butrecoveredtonormal creased by 30% to 50% at the intermediate temperatures,
appearance during the 2-h recovery period at 22C. At (29, 31, and 33C: 2.6%, 2.4%, and 3.0% oftotal, re-
33 and 35C, the anemones were severely stressed: all spectively), and then increased dramatically by 280% at
weretotally contracted and some were lyingon theirsides. the highest temperature (35C: 7.25% oftotal). In some
The 33C anemones returned to normal appearance dur- experiments additional hsps were detected at 82 and
ing the recovery period, but the 35 C anemones did not. 48 kDa(Fig. 2B), thatwereapparently inducedtoalower
Only two hsps (68 and 72 kDa) could be distinguished extent than the hsps 68 and 72.
33C
visually in this experiment (Fig. 2A). The densitometry vs. 22 C. variable recoveryperiod. This experi-
was used to quantify the amounts ofradiolabel incorpo- ment examined the turnover ofhsps synthesized during
rated into each band with respect to the total amount of the temperature stress and post-stress recovery periods.
radiolabel in protein in each sample (Fig. 3). The density Increased amounts ofhsps 68 and 72 (and 48 and 82, to
scans for each lane are presented in Figure 3A and the aminorextent)weresynthesizedaftera2-hheattreatment
relative densities within the hsp68 and hsp72 bands in at33C(comparison oflanesCand in Fig. 2C;especially
Figures 3B and 3C, respectively. The 72 kDa hsp was noticetheincreaseddensityofthehsp68and hsp82bands
present as a faint band in the control (22C) and 27C in lane 0). Both ofthese hsps remained elevated for up
22C
anemones (2.8% oftotal density for each), with up to a to 24 h at (Fig. 2C), but were less evident in the 4-
32% increase in the 29, 31, and 33C animals (3.7%, h and 48-h samples. Because ofthe experimental design
3.2%, and 3.3% oftotal, respectively), and then declined (specimenswereincubatedwiththe radiolabelduringboth
at 35C (to 2.3% oftotal). Hsp68 was present at very low the stress and recovery periods), we cannot distinguish
levels (1.9% oftotal) at the lowest two temperatures, in- between radiolabeled hspsproduced duringthe stressitself
238 N. A. BLACK ET AL.
B C
A
22 27 29 31 33 35 22 33 C 1 2 4 24 48 C
ill*
72
-68
Figure 2. Autoradiogram showing proteins synthesized by Aiptasia palliila after exposure to various
temperatures. (A)Synthesisafter2 hat 27. 29. 31. or 35Ccompared tocontrols(22C). (B)Synthesis
after2hat33Ccomparedtocontrols(22C). (C)Synthesisafter2 h at 33Cwithvariablerecoverytimes
(for0. 1.2, 4, 24.or48 h)at 22Ccomparedtocontrols(22C). Each lanecontainsasampleconsistingof
pooled tissues from fourseaanemones, a = protein presumed to beactin. Thepositionsofhsps produced
atapproximately 72 and68 kDa(A)and 82. 72. 68. and 48 kDa(B, C) are indicated.
and subsequently during recovery. However, in other to be constitutive, with moderately increased synthesis
studies in which we have incubated reefcorals with ra- after moderate temperature shock, and down-regulation
diolabeled aminoacids, the medium wasdepleted ofmore at the highest temperature tested (35C). In Aiptasia,
than 80% oftheradiolabel within the first 3 h(FitzGerald hsp68 appears to be the major one produced with more
and Szmant. 1988, in prep.). Thus, it is likely that most extreme temperature stress. Given the near-background
oftheradiolabeled hsps(and otherproteins)inthesamples levels ofthis hsp in the control and 27C samples. hsp68
were synthesized during the stress period and the early may be inducible rather than constitutive.
hours ofthe recovery period. Comparison ofourresults with those reported forother
cnidarian species reveals a few common hsps and hsp
Discussion families (Table II). According to Craig (1985). nearly all
Several hspswere found in bothMontastraeafaveolata species produce hsps in the samethree sizeclasses: hsp90
andAiptasiapallidaafterbriefexposurestoelevatedtem- (80 to 90 kDa), hsp70 (68 to 74 kDa), and small hsps (18
perature. Overall, hsp74 for Montastraea, and hsps 68 to 30 kDa). Variability regarding the number and size of
and 72 forAiptasia were the dominant hsps produced in hsps is generally greater for small hsps. Five ofthe cni-
responsetoheatstress. In the latterspecies, hsp72appears darian species studied produced one hsp in the 80 to 90
HEAT SHOCK PROTEINS IN CORALS 239
200 be applied to determine whether the hsps detected are
members ofthe known hsp families. This approach has
been used recentlybySharpclal. (1994)toidentify mem-
bers of the hsp70 family in a coral and a sea anemone
(Table II).
Hsps in Aiptasia appear to have a moderate turnover
rate. Radiolabeled hsps synthesized during and after the
2-h heat shock were still present 24 h after temperature
100 200 300 400 500 exposure, but had apparentlybeen degraded by48 h after
DISTANCE
treatment. Experimentsshouldbeconductedtodetermine
22oC 27oC 23oC 31oC 33oC 36oC theduration ofheatstressduringwhich theseanimalscan
continue to synthesize hsps.
An attempt to determine whether zooxanthellae in
2000 8
Montastraea produce hsps failed because tissue homog-
5 enates of the zooxanthellae preparations contained too
6
little radiolabel (4% or less) to allow detection of hsps.
u.
_0 Although hsps have not yet been reported in zooxan-
thellae, the degradation and loss of pigments from the
chloroplasts of zooxanthellae in bleached corals (Glad-
felter, 1988; Kleppel etal.. 1989; Porteretal.. 1989;Glynn
and D'Croz, 1990; Szmant and Gassman, 1990; Black,
27 29 31 33 35 1993) would indicate that the zooxanthellae are heat
TEMPERATURE
stressed and could also benefit from hsps.
IfflBand Density %Total Density Inthisstudy, wehaveshown thatMontastraeafaveolata
2000 can produce hsps in response to short exposures (several
hours) to relatively highly elevated temperatures(>33C).
Ourexperimental conditionsdiffer from those associated
with natural bleaching, which is thought to be caused by
exposureto more moderately elevated temperatures(29-
30C)forlongerperiods(several weeksto months)(Glynn
and D'Croz, 1990; Black, 1993; Black and Szmant. in
prep.). Nohspproduction wasobserved incoralsexposed
to moderately elevated temperatures (27-31C) for
27 29 31 33 35 2 hours to one week. In addition, the duration ofour ex-
TEMPERATURE
periments was too short to expect any visible bleaching.
Figure 3. (A) Density profilealongthecentral portion ofeach lane Susceptibility of corals to bleaching varies between dif-
shown in Figure 2A, extending lengthwise about the mid-third ofeach ferent species (Williams and Bunkley-Williams, 1990).
lane. The scan width wasabout two-thirds the width ofeach lane. See The present results suggest that furtherworkiswarranted
texttorfurtherdetails. (B)Thetotaldensityassignedtothehsp68 band
ofeach sample, and asa percentage oftotal lane density. (C) Same as to determine whether hsps are involved in differences in
(B)but forthe hsp72 band. thermotolerance and susceptibility to bleaching within
and between reefcnidarian species.
range, and all but Hydra produced one or more hsps in Conclusions
the 68 to 74 range.
Ashasbeenobservedforotherspecies,the numberand (1) Both Montastraea faveolata and Aiptasia pallida
relativequantitiesofhspssynthesized in ourexperiments produce heat shock proteins in response to short periods
increased with increasingseverityofheattreatment. There ofextreme but sublethal thermal stress. Most ofthe hsps
was, however, variability between experiments in the produced appear to fall into a molecular weight range
amount and type of hsps produced, especially in Mon- similartothosefound in othercnidarian species. Western
tastraea. Some ofthisvariation may bedue to individual blots are needed to learn whether the hsps produced by
differences in thermotolerance or ability to turn on hsp Montastraea and Aiptasia belong to the same families as
synthesis.Thispossibilityshouldbeaddressedbyrepeating the hsps found in other groups. (2) The involvement of
these experiments with greater replication. In addition, heat shock proteins in coral bleaching remains to be de-
immunochemical techniques (i.e., Western blots) should termined.
240 N. A. BLACK ET AL
Table II
Comparison ofthehem v/jor/,-proteins ohwn'edinsixdifferent cnidarianspecies
Species