Galaxea, JCRS, 4: 33 - 42 (2002) RS Jc Japanese CoralReef S ociety

PHYSIOLOGICAL RESPONSES OF THE CORAL GALAXEA FASCICULARIS AND ITS ALGAL SYMBIONT TO ELEVATED TEMPERATURES

Bhagooli R. and M. Hidaka*

Department of Chemistry, Biology and Marine Sciences, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan.

Abstract: The purpose of this study was to examine the physiological responses of the coral host and their algal symbionts to elevated temperatures. Isolated polyps of the coral Galaxea fascicularis were exposed to three temperatures (24, 28 and 30•Ž) for 7 days and were then allowed to recover for 15 days at 24•Ž. Both zooxanthellae density and the maximum photosynthetic quantum yield of in hospite zooxanthellae, measured by pulse-amplitude-modulation (PAM) fluorometry, decreased after 7-day exposure to 28 and 30•Ž. In corals exposed to 28•Ž, both parameters returned to the initial level after 15-day recovery, but those exposed to 30•Ž died during the recovery period. A high re- lease of healthy-looking zooxanthellae was evident in the coral exposed to 30•Ž, while those exposed to 24 or 28•Ž mainly released degraded zooxanthellae particles. The mi- totic index of both retained and expelled zooxanthellae was highest at 30•Ž. The coral host appeared to lose the capacity of controlling algal cell division and of preferential expulsion of degraded algal cells after 7-day exposure to 30•Ž . The growth rate of corals was lowest at 30•Ž. The reduced growth rate, the low release of degraded zooxanthellae particles and high release of healthy-looking zooxanthellae, and increased mitotic index of zooxanthellae, in corals exposed to 30•Ž for 7 days, suggest that high temperature caused a physiological dysfunctioning at the level of host and that this change may be at least partly responsible for bleaching and the disruption of symbiosis.

Key words: coral, bleaching, zooxanthellae, symbiosis, thermal stress

*Corresponding author e-mail address hidaka@sci .u-ryukyu.ac.jp Tel. : 098-895-8547; Fax: 098-895-8576

INTRODUCTION 1989), or both (Porter et al. 1989, Glynn and D'Croz 1990, Brown et al. 1999, Fitt et al. 2000). Reef-building corals harbor symbiotic dinoflagellates A number of stresses, including unusually high Symbiodiniumspp. commonly referred to as zooxanthellae seawater temperatures (Cook et al. 1990, Gates (Trench 1987). These symbionts translocate a 1990, Glynn 1991, Brown et al. 1994, Goreau and large portion of their photosynthetically fixed Hayes 1994, Fitt and Warner, 1995), solar irra- carbon to the coral host to meet the latter's en- diation (Brown et al. 1994), high doses of ultra- ergy requirements (Muscatine 1990). Hermatypic violet light (Lesser et al. 1990, Gleason and corals respond to a variety of environmental Wellington 1993), changes in salinity (Hoegh- stresses through disruption of their symbiotic Guldberg and Smith 1989), bacterial infection relationship with the zooxanthellae, a process (Kushmaro et al. 1996), or a combination of these commonly referred to as bleaching. Bleaching factors (see reviews Brown 1997, Fitt et al. 2001), has been attributed to the reduction in density have been proposed to produce bleaching in cor- of zooxanthellae (Jaap 1985, Hoegh-Guldberg als. Of these environmental factors, both elevated and Smith 1989, Szmant and Gassman 1990, seawater temperature and solar irradiance have Fagoonee et al. 1999), the loss of chlorophyll a been mainly implicated in triggering large-scale per algal cell (Kleppel et al. 1989, Porter et al. coral bleaching (Coles and Jokiel 1978, Fitt and 34 Bhagooli R. and M. Hidaka

Warner 1995, Brown 1997, Hoegh-Guldberg 1999). 30% surface light and ambient temperature Several physiological responses of corals to ele- (24°C) for 3 days. Polyps (2-3 cm in length) were vated temperature have been reported. Responses isolated, using forceps, from the middle part of from field-based studies include loss of photo- the colonies. Isolation of polyps was carried out synthetic potential (Porter et al. 1989), a cessa- while the colonies were still immersed in seawater. tion or reduction of growth (Jokiel and Coles Thirty six polyps from each colony were mounted 1977, Porter et al. 1989, Goreau and MacFarlane vertically on acrylic plates (3 x 4 cm2) by insert- 1990) and a decrease in reproductive output ing the basal skeletal portion into a silicone tube (Szmant and Gassman 1990 Hirose and Hidaka fixed on the acrylic plate. Six plates each con- 2000). Recent experimental trials, using elevated taining 6 polyps were prepared from each col- temperature as stress, provided evidence for ony~ a total of 30 plates were prepared from 5 impairment of photosynthesis of algae in cul- colonies. Furthermore, 3 polyps were isolated ture (Iglesias-Prieto et al. 1992) and in intact from each of 4 colonies and were mounted on symbiosis (Warner et al. 1996, 1999, Jones et al. glass slides in a horizontal position with rubber 1998). On the other hand, Gates et al. (1992) bands. Isolated polyps mounted on glass slides reported that gastrodermal cells containing were used for chlorophyll fluorescence measure- zooxanthellae detach from the host tissue during ments. All the preparations were allowed to re- excessive temperature treatment. Thus there is cover from possible damage during isolation and still no consensus as to which of the symbiotic mounting under the running seawater system for partners is more affected by high temperature 5 days before commencement of the experiments. (Perez et al. 2001 Ralph et al. 2001). To maintain a healthy symbiosis, growth rates Experimental design of both partners must be in dynamic equilibrium. Polyps were exposed to 3 different tempera- To avoid the zooxanthellae outgrowing the host tures, 24, 28, and 30°C, for 7 days. Each tem- partner, host may control algal cell division, di- perature treatment included 2 sets of plates gest or release some of the zooxanthellae. Titlyanov from each colony. One set was used for measure- et al. (1996) have documented degradation of ment of growth rate. This set was also used to zooxanthellae from a number of coral species collect zooxanthellae released from the polyps. under normal conditions. They also reported that The other set was used for measurement of release of healthy zooxanthellae (Hz.) was ap- zooxanthellae density and mitotic index deter- proximately two orders of magnitude lower than mination. One polyp was sampled from each the release of degraded zooxanthellae particles plate after 7-day stress treatment and another (Dzp.). If corals expel more Hz. than Dzp. dur- polyp was sampled after 15-day recovery pe- ing bleaching, it may imply that coral hosts are riod. Since this set consisted of intact plates, more susceptible to thermal stress than algal that is, each plate holding six polyps as those partner. However, as far as we know, there has used in growth rate measurement, during the been no report on the rate of release of Hz. and first 7 days, plates in this set were also used Dzp. by corals during bleaching. for growth rate measurement during the first 7 The present study investigates physiological re- days. Zooxanthellae density of polyps prior to sponses of the coral Galaxea fascicularis to elevated stress treatment was measured using additional temperature. Changes in the host growth rate, polyps that were isolated and mounted on acrylic symbiont photosynthesis, and host control of algal plates as those used for stress treatment but population density (degradation of zooxanthellae, sampled before commencement of stress treatment. selective release of degraded zooxanthellae parti- Three water baths, two provided with tem- cles, and mitotic index of zooxanthellae) in re- perature control units, were set up in an indoor sponse to thermal stress were assessed. laboratory having glass ceiling. The two water baths, with temperature control units (REI- SEA, RZ-180), were adjusted to 28°C and 30°C. MATERIALS AND METHODS The third tank had a continuous flow of seawater Collection and maintenance of corals (running seawater). Data loggers (TidbiT) were Five colonies of Galaxea fascicularis Linnaeus installed in each tank to track temperature. 1767 (approximately 15-cm diameter) were col- Due to low capacity of the temperature control lected from a depth of 1-1.5 m at the northern units, the actual temperatures of the experi- reef of Sesoko Island, Okinawa, Japan. These mental tanks varied and were 27.8 ± 0.3 and colonies were kept in running seawater under 30.0 ± 0.2°C (± SD) for the 28°C and 30°C Responses of Galaxea fascicularis to elevated temperatures 35

designated tanks, respectively. The temperature polyps was calculated assuming that the corallites in the running seawater tank was 24.2 ± 0.7 were elliptical cylinders. °C . The range of within-container irradiance dur- The percentage of dividing cells was also ing the 22-day experimental period was 200-1200, counted using the haemocytometer prior to and 200-1600, and 130-640 ,i mol quanta m 2 s l for after 7-day exposure to stress. A cell was consid- the recording time 900, 1200 and 1500 h, re- ered to be dividing (i.e. undergoing cytokinesis) spectively. if it appeared as a doublet with a cell plate. The Each tank contained fourteen 1 1 containers number of dividing cells, out of a total of 230-500 with acrylic lids. In each of the ten containers, cells per sample, in three counts from the same acrylic plates mounted with polyps were placed. specimen were averaged and the resultant per- Two other containers had two-glass slides, each centage was taken as the mitotic index (Wilkerson slide with one mounted coral polyp. The remain- et al. 1983). All microscopic observations were ing two containers contained no polyps and were made under 400 X magnification using a Nikon used as control for zooxanthellae release. All OPTIPHOT-2 microscope. The dividing cell esti- the containers were filled with 800 ml filtered mates were done between 8 30 to 1000 h. (0.45 U m) seawater and were aerated. Every day, during one week, the seawater in the con- Zooxanthellae release rate tainers was replaced with filtered seawater ad- After 2, 5 and 7 days of stress, while polyps justed to respective temperatures. During this were removed from the container for changing process, polyps were kept in "holding tanks" , at seawater, the seawater, in which the corals had respective temperatures, for about two minutes. been exposed for 24 h, was stirred thoroughly and After 7-day exposure to elevated temperature 500 ml was sampled between 1100 -1300 h. The density and mitotic index determinations and sampled seawater was filtered, using an air pump, the remaining were returned to running seawater through Millipore filters (0.45 is m) to collect the tanks and left to recover for 15 days. released zooxanthellae. Zooxanthellae in the field of 1 mm diameter were counted under a light Zooxanthellae density and mitotic index microscope at a magnification of 200 X . The av- Five polyps each derived from different colonies erage number of zooxanthellae in 3-5 fields was were sampled 7 days after stress (n = 5) and 15 used to calculate the number of zooxanthellae on days after recovery (n = 5) for measurement of the whole filter. This value was then multiplied the zooxanthellae population density following by 815 to obtain the total number of released the method described in Muscatine et al. (1989). zooxanthellae in 800 ml medium per six polyps Coral tissues from the lateral surface of the pol- per 24 h. Counts were made for both healthy-looking yps were stripped off using a WaterPik (Johannes zooxanthellae (Hz.) and degraded zooxanthellae and Wiebe 1970). The coral blastate (40-90 ml) particles (Dzp.) as described by Titlyanov et al. produced was homogenized with a potter homogenizer (1996, 2001). In several species of corals, in- at 800-900 rpm. The volume of the homogenate cluding G. fascicularis, Hz. appear circular and was measured. Zooxanthellae in 10 ml of the ho- yellow brown in color whereas Dzp. have either a mogenate were pelleted by centrifugation at 1250 round or irregular shape, their diameters being g for 15 min. The pellet was suspended in a small less than 50% of Hz., and are orange to deep volume of filtered seawater. Then the volume brown in color (Titlyanov et al. 1996, 2001). was adjusted to 1 ml and resuspended thoroughly using a vortex. The samples were taken for deter- Chlorophyll a fluorescence measurements mining the number of zooxanthellae and the den- Chlorophyll fluorescence emission of the in sity was calculated as number of zooxanthellae hospite zooxanthellae was measured using a pulse- per unit surface area. amplitude-modulated (PAM) fluorometer (MINI To measure the surface area of polyps, draw- PAM, Heinz Walz GmbH). The minimum ings of the corallites were made using a camera fluorescence, K, and the maximum fluorescence, lucida under a dissecting microscope. The con- Fm, were recorded. The maximum quantum yield tours of the corallites were digitized using a scan- of PS II in dark-adapted corals was estimated ner (HP Scanjet IIcx) and the projected areas of by fluorescence ratio, Fv/Fm, where Fv = Fm - F0. the corallites were measured on a computer using The ratio of variable (F~) to maximal (Fm) fluo- NIH image analysis software. The lengths of the rescence in a dark-adapted sample is a conven- longer and shorter axes of calices were measured ient measure of the maximum quantum yield using an electric caliper. The surface area of the (Bjorkman and Demmig 1987). 36 Bhagooli R. and M. Hidaka

Coral polyps mounted on the glass slides were simultaneous pairwise mean comparisons and placed in a black box, designed to accommodate correlations statistics were performed. The null six samples. The black box allowed us to dark- hypothesis was rejected when the probability adapt five samples at the same time and to level was less than 0.05. make successive measurements of the Fv/Fm pa- rameter of the samples. The optical head of the fluorometer was fitted just on the lateral surface RESULTS of the coral polyp, the distance between optical Effect of elevated temperature on zooxanthellae den- head and sample being 13 mm. Measurements sity and chlorophyll a fluorescence ratio were done initially (day zero), 2, 5 and 7 days After 7-day exposure, zooxanthellae density after stress and 15 days after recovery. Measurements decreased in corals in all groups but the de- were done at the same respective treatment crease was more pronounced in corals exposed to temperatures, that is, 24, 28 and 30°C . All chlo- 30°C than those exposed to 24 or 28°C (SNK rophyll ratio measurements were done at the test, P < 0.01) (Fig. 1A). The 15-day recovery same time of the day, that is, 830 a.m, to avoid inteference by possible diel changes in Fv/Fm. A Growth measurement Buoyant weight of the plates each with 6 pol- yps was measured before and after the 7-day stress treatment, and after 15-day recovery pe- riod to estimate the average growth rate during the 7-day stress treatment and during the 15- day recovery period. An electric balance (FX- 300, A&D Corp., 310g range, O.OOlg readability) was used for weighing. Before weighing, air bubbles attached to the plates were removed using a small brush. The acrylic plate with six isolated polyps, while still immersed in seawater, was attached to the hook of the balance with a nylon monofilament line and weighed. The weight of the acrylic plates with silicone tubes B alone in seawater was measured at the beginning of the experiment, and was subtracted from the gross weight. Since the weight of the plate in seawater depends on the density of the seawater, a correction was made to compensate changes in the density of seawater as in Dodge et al. (1984). The weight of coral skeletons in air was calcu- lated using the formula of Jokiel et al. (1978). Density of seawater was measured by weighing an agate pestle, whose density was determined beforehand, in distilled water.

Statistical analyses The effects of temperature on the maximum quantum yield (Fv/Fm), zooxanthellae density, release rate of zooxanthellae and coral growth Fig 1. Density and the maximum quantum yield (F~/Fm) of zooxanthellae in Galaxea fascicularis after 7-day exposure to rates were analyzed using one-way analysis of temperature stress and after 15-day recovery. A, Zooxanthellae variance (ANOVA). Arcsine transformation was density. B, The maximum quantum yield of PSII. Isolated applied to percentage or proportion data prior to polyps of G. fascicularis were exposed to 24 (D), 28 (0), and ANOVA. Zooxanthellae density and release rate 30°C ( L\) for 7 days (hatched horizontal bar on the ab- of healthy and degraded zooxanthellae were scissa), and were allowed to recover at 24°C for 15 days (open bar). Means ± SD (n = 5 and 4 for zooxanthellae den- tested by ANOVA after logarithmic transforma- sity and F~/F,,, respectively, except for K/F~ after recovery tion. Student-Newman-Keuls (SNK) test for where n = 2). Responses of Galaxea fascicularis to elevated temperatures 37

period allowed the zooxanthellae density in the A 2d 28°C treated corals to return to initial levels. All the polyps treated at 30°C died within 15 days. The effect of elevated temperature on the chlorophyll a fluorescence ratio, Fv/Fm, of the in hospite zooxanthellae was not significantly dif- ferent among the three temperature exposures for the first 2 days (Fig. 1B). However, corals exposed to 28 and 30°C for 7 days showed sig- nificantly lower Fv/Fm than either their respec- tive initial values or the control polyps kept at 24°C (SNK test, P < 0.001). The polyps exposed to 28°C had regained their initial Fv/Fm values 15 days after the end of exposure to high tempera- S 5d ture, but all polyps exposed to 30°C died during the recovery period.

Expulsion of healthy-looking zooxanthellae and de- graded zooxanthellae particles The number of healthy-looking zooxanthellae (Hz.) released was significantly higher in corals exposed to 30°C than that of polyps kept at 24 °C after 5 or 7-day exposure (SNK test, P < 0.05) (Fig. 2). In addition, the release of degraded zooxanthellae particles (Dzp.) was lower at 30 °C th an at 24 or 28°C (SNK test, P < 0.001) and C 7d few Dzp. were released after 7-day treatment. Corals kept at 24°C and 28°C released much more Dzp. than Hz. (Fig. 2). There was no signifi- cant difference in the release rate of Hz, be- tween corals treated at 24 and 28°C while the release rate of Dzp. in 28°C-treated corals was significantly higher than that in 24°C -treated corals after 5 and 7 days (SNK test,P < 0.001 and P < 0.05, respectively) (Fig. 2).

Mitotic index and release rate of healthy looking- zooxanthellae The mitotic index (MI) of both retained and re- leased zooxanthellae was significantly higher in corals exposed to 30°C for 7 days than those ex- Fig 2. The number of healthy-looking zooxanthellae (Hz.) posed to 24°C or 28°C (Fig. 3A). The MI of re- and degraded zooxanthellae particles (Dzp.) released by pol- yps of Galaxea fascicularis exposed to temperature stress. tained zooxanthellae did not differ significantly Isolated polyps of G. fascicularis were exposed to 24, 28, and from those of the released ones at all the tempera- 30°C for 2 (A), 5 (B), and 7 (C) days. Means ± SD (n = 5). ture exposures. When algal release rate was plotted against their division rate using all the paired data points, no significant correlation was found between zooxanthellae release rate and mitotic index of the released zooxanthellae (P > 0.05) (Fig. 3B). and P < 0.01, respectively) (Fig. 4). The 15-day Effect of temperature on growth rate recovery period revealed no significant growth The weight increase after 7 days was signifi- rate difference between the 24 and 28°C treat- cantly higher in 28°C treated corals than those ments. The growth rate in 30°C treated corals exposed to 24 and 30°C (SNK test, P < 0.05 was not recorded due to death of the polyps. 38 Bhagooli R. and M. Hidaka

A

Fig 4. Growth rate of Galaxea fascicularis measured after 7- day treatment at 24, 28, and 30°C and after 15-day recov- ery at 24°C. Mean ± SD (n = 10 and 5 for after 7-day stress and after 15-day recovery, respectively). The growth rate of 30°C-treated polyps during the recovery period was B not shown as all polyps died during the period.

Correlation analysis of the parameters measured after 7d stress treatment While temperature showed positive correlation with mitotic index of in hospite zooxanthellae and release rate of healthy-looking zooxanthellae (Hz.), it was negatively correlated with zooxanthellae density and Fv/Fmof in hospite zooxanthellae after 7-day treatment (Table 1). Zooxanthellae den- sity was negatively correlated to the mitotic index of retained zooxanthellae, release rate of Hz. and temperature, but positively to in hospite Fv/Fm.The mitotic index of in hospite zooxanthellae showed negative correlation to Fv/Fm of in hospite zooxanthellae but a positive one to the release Fig 3. Mitotic index (MI) of Galaxea fascicularis zooxanthellae. rate of Hz. and temperature. In hospite Fv/Fm was A, MI of retained and released zooxanthellae from G. negatively correlated to both the release rate of fascicularis after 7-day exposure to 24, 28 and 30°C. Only MI of in hospite zooxanthellae is shown for the control pol- Hz. and temperature. yps before stress treatment (TNT). Means ± SD (n = 5). B, Relationship between release rate of zooxanthellae and mi- totic index of released zooxanthellae. Pooled data from pol- DISCUSSION yps exposed to 24, 28 and 30°C for 2(fl), 5(0), and 7(A)- Zooxanthellae density decreased significantly day exposure were plotted. at 28 and 30°C after 7-day exposure (Fig. 1A), indicating that bleaching occurred in the coral

Table 1. Correlation analysis among temperature and biological parameters measured. Correlation coefficients are shown when there was significant correlation (P < 0.05). Responses of Galaxea fascicularis to elevated temperatures 39

exposed to these temperatures. Significant de- Abnormal empty-looking zooxanthellae have cline in the maximum potential quantum yield been observed in bleached coral tissues (Hayes (Fv/Fm)of PS II was observed in in hospite zooxanthellae and Bush 1990 Szmant and Gassman 1990). exposed to 28 or 30°C for 7 days (Fig. 1B). The Brown et al. (1995) also described "degraded" z decreased Fv/Fm of in hospite zooxanthellae at 30 ooxanthellae in bleached corals as those "with a °C implies a decrease in PS II activity of the re- breakdown of integrity of cellular components tained zooxanthellae population indicating a high and an overall loss of circularity" . They also relative abundance of photosynthetically damaged noted increased vacuolation within degraded zooxanthellae. Fitt and Warner (1995) showed algal cells and in some cases enlargement of the that damage to PS II precedes decrease in cells. These characteristics of "degraded" zooxa zooxanthellae density. In the present study, nthellae within bleached coral tissues imply ne- zooxanthellae density and Fv/Fm appeared to de- crotic death of algae and are quite different from crease in parallel (r = 0.72, Table 1), but it was those of Dzp., which might be debris of digested not clear whether decrease in K/Fm preceded zooxanthellae. This suggests that the bleached decrease in zooxanthellae density. corals lost the ability to digest zooxanthellae. Healthy-looking zooxanthellae (Hz.) were re- Glynn et al. (1985) reported that zooxanthellae leased in significantly higher numbers at 30°C become degenerated only when the animal tissue than at 24 or 28°C (Fig. 2). In addition, release was necrotic and suggested that autolysis of the of degraded zooxanthellae particles (Dzp.) was coral cells might have caused necrosis of the algae. lowest at 30°C and declined to almost zero by 7- The mitotic index of zooxanthellae was highest day exposure. Corals kept at 24°C or 28°C released in the coral exposed to 30°C for 7 days. Corals suf- Dzp. approximately one order of magnitude more fering from bleaching have higher zooxanthellae than Hz. (Fig. 2). Titlyanov et al. (1996) found division frequencies (Fitt et al. 1993 Brown et al. that the number of Dzp. released from three 1995). Jones and Yellowlees (1997) also re- corals, Stylophora pistillata, Seriatopra caliendrum, ported highest mitotic index of zooxanthellae and Porites horizontalata, under normal condition when the zooxanthellae density was at its lowest, were two orders of magnitude more than that immediately after the bleaching event on Great of Hz. released. Dzp. have been suggested to re- Barrier Reef in January 1994. Other studies also sult from digestion or autodegradation of old and showed that elevated temperatures increase rates weak cells (Titlyanov et al. 1996). It is likely that of algal cell divisions (Suharsono and Brown the observed bleaching of G. fascicularis at 28°C 1992, Baghdasarian and Muscatine 2000). High occurred mainly through degradation of mitotic index of zooxanthellae at elevated tem- zooxanthellae and expulsion of resulting Dzp., perature may reflect loss of mitotic control by while at 30°C bleaching occurred through release host and hence host dysfunctioning. of healthy-looking zooxanthellae. There remains In the sea anemone Aiptasia pulchella and the the possibility that the low numbers of Dzp. at coral Pocillopora damicornis, dividing zooxanthellae 30°C was due to faster autolytic activity or bacte- are preferentially expelled from the host (Baghdasarian rial degradation of zooxanthellae at high tem- and Muscatine, 2000). Jones and Yellowlees perature. However, this is unlikely because the (1997) also reported that the mitotic index of collection of released zooxanthellae coincided zooxanthellae released from Acropora formosa was with the time of maximum release of Dzp. in two higher than those retained. However, preferen- corals, S. pistillata and S. caliendrum (Titlyanov et tial expulsion of dividing algal cells was not ob- al. 1996). The present results strongly suggest served in G. fascicularis (Fig. 3B). This is also that the coral host can selectively expel degraded the case with Porites compressa, Montipora verrucosa zooxanthellae at 24 or 28°C but lost the ability and Fungia scutaria (Baghdasarian and Muscatine, to expel degraded zooxanthellae selectively at 2000). 30°C. While the sea anemone Phyllactis flosculifera The range of temperature for optimal growth of expels pellets mostly consisting of zooxanthella corals is 27 - 31°C, although it may be species- debris (=Dzp.) (Steel and Goreau 1977), the sea dependent (Yap and Gomez 1981). In the pre- anemone Aiptasia tagetes expels pellets contain- sent study, the optimal growth temperature of G. ing mostly normal intact zooxanthellae (Steel fascicularis seemed to be 28°C (Fig. 4). The 30 1976). Although these anemones were not under °C temperature stre ss for 7 days led to decreased stressed condition, it is likely that slight differ- growth and resulted in death of all polyps, during ence in environmental condition affects the se- the 15-day recovery period. The reduced growth lectivity of zooxanthellae expulsion. rate of corals exposed to 30 °C also suggests 40 Bhagooli R. and M. Hidaka

physiological dysfunctioning of the host at ele- a high latitude coral reef the 1988 Bermuda event. vated temperature. Coral Reefs 9: 45-49 The present study indicates that the observed Dodge RE, Wyres SC, Frith HR, Knap AH, Smith SR, Cook CB, Sleeter TD (1984) Coral calcification rates decrease in zooxanthellae density of G. fascicularis by the buoyant weight technique effects of alizarin polyps at 28 and 30°C could be explained by staining. J Exp Mar Biol Ecol 75: 217-232 degradation of zooxanthellae and release of Fagoonee I, Wilson HB, Hassell MP, Turner JF healthy-looking zooxanthellae, respectively. At (1999) The dynamics of zooxanthellae populations elevated temperature, the coral may lose the a long-term study in the field. Science 283: 843- 845 capacity to degrade zooxanthellae and to se- Fitt WK, Spero HJ, Halas J, White MW, Porter JW lectively release degraded zooxanthellae parti- (1993) Recovery of the coral Montastrea annularis in cles. High mitotic index of zooxanthellae and the Florida Key after the 1987 Caribbean `bleaching reduced skeletal growth rate also suggest physio- event'. Coral Reefs 12: 57-64 logical dysfunctioning of host coral. Further in- Fitt WK, Warner ME (1995) Bleaching patterns of four species of Caribbean reef corals. Biol Bull 189: vestigation is necessary to understand whether 298-307 damage to algal photosynthetic machinery leads Fitt WK, McFarland FK, Warner ME and Chilcoat to physiological dysfunction of host or animal GC (2000) Seasonal patterns of tissue biomass and host is more susceptible to elevated temperature densities of symbiotic dinoflagellates in reef corals than algal symbionts. and relation to coral bleaching. Limnol Oceanogr 45: 677-685 Fitt WK, Brown BE, Warner ME, Dunne RP (2001) ACKNOWLEDGMENTS Coral bleaching interpretation of thermal tolerance limits and thermal thresholds in tropical corals. 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Mar Ecol 30℃ 処理群 のサ ンゴは 回復期 間中 に死 亡 した。 ス ト Prog Ser 220: 163-168 Steele RD (1976) Light intensity as a factor in the レス処理期 間中、30℃ 処 理群 では、 正常褐 虫藻が多 regulation of the density of symbiotic zooxanthellae く排 出 され てお り、28℃ 処理群 で は、 褐虫 藻の変性 in Aiptasia tagetes (Coelenterata, Anthozoa). J Zool した穎粒 が多 く排 出 され た。30℃ 処理 群で は、 褐 虫 179: 387-405 藻 の分裂指 数 も高 か った。30℃ 、7日 間 のス トレス Steele RD, Goreau NI (1977) The breakdown of 処理を受けた宿主サ ンゴは、褐虫藻の増殖をコントロー 42 Bhagooli R. and M. Hidaka

ル した り、 変性褐 虫藻を選択 的に排 出す る能 力を失 っ た と考 え られ る。サ ンゴ骨 格の成長 速度 は30℃ で低 下 した。 これ らの結果 は、 高温 ス トレスは宿主サ ンゴ の生理的変化を引き起 こしてお り、ス トレスによる褐 虫藻の損傷 のみでな く宿 主の生理 的変化が 白化 に関与 している可能性 が示 唆された。