Philippine Journal of Science 143 (1): 35-42, June 2014 ISSN 0031 - 7683 Date Received: 30 September 2013

Thermal Stress Affects Zooxanthellae Density and Chlorophyll-a Concentration of the Solitary Mushroom Coral, actiniformis

Senona A. Cesar1*,3, Homer Hermes Y. De Dios2, 3, Naomi B. Amoin3 and Danilo T. Dy3

1Visayas State University, Visca, Baybay City, Leyte 2Southern Leyte State University, Bontoc, Southern Leyte 3University of San Carlos, Cebu City

Corals lose their pigments or cell symbionts with prolonged exposure to temperatures higher by 1°C than the reef ambient temperature, leading to bleaching. , a top traded coral was subjected to lower (24-26°C), ambient (27-29°C), and higher (30-32°C) thermal conditions to determine the resilience of its zooxanthellae to thermal stress. The experiment followed a complete randomized design with three different temperatures and eight replicates per treatment level. Evaluations were done in terms of density of expelled zooxanthellae, biomass of expelled chlorophyll-a, and using a coral color reference card. Thermal stress had a

significant effect on the density (F2,21=3.691; p=0.042) and chlorophyll-a biomass (F2,21=10.711; p=0.001) of expelled zooxanthellae of H. actiniformis. Density and chlorophyll-a biomass of expelled zooxanthellae in the 30-32°C treatment doubled and tripled, respectively, compared to ambient conditions. However, these were still lower compared to published values for branching corals. The capability of H. actiniformis for downward migration to seek refuge, and its thick gastrodermis that harbors the zooxanthellae are possible adaptive mechanisms to survive the changing thermal conditions of tropical reefs.

Key Words: bleaching, color index card, live coral aquarium trade, Philippines, temperature

INTRODUCTION importance being among the top five traded aquarium in Indonesia (Knittweis et al. 2009; Knittweis & The ‘long tentacle mushroom coral’, Heliofungia Wolff 2010), information on its thermal stress response actiniformis is a large-polyped discoidal coral whose is still scanty. While optimal coral reef growth occurs tentacles are extended even during day time (Hoeksema between 25°S and 25°N corresponding at a 18°C and 30°C 1989; Gittenberger et al. 2011). It is a rich host to 23 temperature range (Hoegh-Guldberg 1999), and persists in associated fauna including 14 shrimp species (Hoeksema the Persian Gulf where temperature fluctuates from 13°C et al. 2012). Together with other free-living fungiid corals, in winter to 38°C in summer (Wells & Hannah 1992), they act as nuclei for the formation of new patch reefs temperature thresholds are species-specific (Berkelmans (Chadwick-Furman et al. 2000). Its high recruitment & van Oppen 2006). Studies of thermal tolerance in corals rate attributed to both sexual and asexual reproduction are very relevant since 70% of bleaching, the expulsion modes could be among the mechanisms that sustains of symbiotic zooxanthellae in the host coral or the loss of the Heliofungia fishery albeit its high exploitation rate photosynthetic pigment from the zooxanthellae following in the live coral aquarium trade in Indonesia. Despite its a dysfunction of the alga-coral symbiotic relationship, *Corresponding author: [email protected] point to temperature as the primary factor (Buddemeier

35 Philippine Journal of Science Cesar et al: Thermal Stress Affects Zooxanthellae Density Vol. 143 No. 1, June 2014 and Chlorophyll-a Concentration of H.actiniformis

& Fautin 1993; Goreau & Hayes 1994; Weis 2008). MATERIALS AND METHODS However, maintaining the right amount of zooxanthellae is necessary to support the nutritional exchange of the Free-living solitary mushroom corals Heliofungia host-symbiont relationship (Yellowless 2008; Weis 2008), actiniformis, were collected (ca. 10m depth) using and the algae-derived coloration is sought in the live coral SCUBA in the fringing reef off eastern Mactan Island aquarium industry (Olivotto et al. 2011), of which the of Cebu, Central Philippines. The 24 individuals of green colormorph demands a higher price. H. actiniformis which included both brown and green colormorphs (29%) were submerged in seawater while Anomalous fluctuations of sea surface temperature in transit to the laboratory. Acclimatization was done for (SST) had been coupled with bleaching events (Hoegh- eight hours with individual corals in the plastic containers Guldberg 1999; Hoeksema & Matthews 2010; Weis 2008; with seawater and provided with constant moderate Hoeksema et al. 2012). While some reefs were able to aeration from an aquarium pump. recover, catastrophic irreversible damage is attributed to recurrent massive bleaching events, the most potent threat The experiment followed a complete randomized design to maintenance of biodiversity in the marine tropical (CRD) with three different temperatures as treatment seas (Goreau & Hayes 1994; Baker et al. 2008). In situ levels, and eight replicates per treatment level. The 27- observations (Berkelmans & van Oppen 2006; Mattan- 29°C temperature was the control based on the ambient Moorgawa et al. 2012) and laboratory thermal induced temperature of the collection site while 24-26°C and 30- bleachings (Jones 1997; Bhagooli & Hidaka 2004; Mieog 32°C represented the lower and upper thermal stressors, et al. 2009) are employed to screen resilient corals, respectively. The experimental unit consisted of one that could withstand the predicted change in seawater of H. actiniformis, immersed in 4L plastic container temperature. H. actiniformis was among the bleach-resistant filled with filtered seawater from the collection reef (33-34 ppt). The corals used had a mean surface area of corals in Indonesian reefs where 2°C higher than ambient 2 temperature occurred (Hoeksema 1991; Loya et al. 2009) 109.87±8.06 cm (as measured using Image J software). and also in Koh Tao, Gulf of Thailand (Hoeksema & The two temperature levels (27-29°C, and 30-32°C) were Matthews 2011). While abrupt exposure but in shorter maintained using Odysea® thermostat heaters while the duration may not be the mechanism in the field (Bhagooli 24-26°C level was maintained manually using icewater & Hidaka 2004), since reefs bleached at least one month bags. The experiment was done in an indoor laboratory exposure to an elevated temperature of at least 1°C (Donner setting with constant lighting from two ceiling-mounted et al. 2005), such a scenario is prevalent in the live coral white flourescent lamps, 40 watts each. aquarium industry. Thermal studies are especially important The density of zooxanthellae and biomass of chl-a in the supply chain for internationally traded corals of expelled by mushroom corals were measured after 24 h which 90% are harvested from the wild (Borneman exposure to the thermal treatments. After exposure, 15 mL 2001). Once subjected to logistic processings, 80% is lost, of seawater samples from each of the 24 plastic containers starting with collections from a tropical reef to its final were kept in labeled amber bottles with a drop of Lugol’s destination in an aquarium of a hobbyist who probably live solution as preservative. The zooxanthellae of each polyp in a temperate country (Hoeksema 1989; Olivotto 2011). were counted (5 replicate counts) using a Neubauer The coral aquarium fishery, when regulated, may have a haemacytometer under 400x magnification. Density was lesser effect to coral reef degradation (Trautwein 2001), expressed as number of zooxanthellae cm-2 with the area of but the percentage of loss can be significantly reduced the polyp being factored in (Equation 1). For chlorophyll-a when information on optimal temperature along with other content as proxy to the biomass of the photosynthetic factors, are provided during shipment or freight (Borneman pigment (referred as biomass hereafter), 1L water sample 2001; Olivotto et al. 2011). was collected from the incubating media and was filtered We subjected specimens of H. actiniformis to three using Whatman GF/C filter (Aminot & Rey 2000). Each levels of thermal conditions and quantified the amount filter was cut into small pieces and grounded in the mortar of released zooxanthellae, Symbiodinium, in terms of with 10 ml 90% acetone. This was then centrifuged at 3000 cell density and biomass of photosynthetic chlorophyll-a rpm for 3 minutes to separate the residue from the filtrate. pigment. Qualitative assessment used a color reference The absorbance of the supernatant solution was read using card first introduced by Seibeck et al. (2006). The resulting a Spectrumlab 752S spectrophotometer. The equation information should be primarily relevant to the aquarium industry and secondarily, on the potential impact of thermal Equation 1. Determination of density of stress on coral species on top of the mesoscale events zooxanthellae of Heliofungia actiniformis. brought about by El Niño Southern Oscillation (ENSO) -2 -1 and global warming events resulting to anomalous seasonal Density of zooxanthellae (cm ) = (cell mL * volume -2 changes in seawater temperature (Donner et al. 2005). (mL) of incubating water)/ surface area of polyp (cm )

36 Philippine Journal of Science Cesar et al: Thermal Stress Affects Zooxanthellae Density Vol. 143 No. 1, June 2014 and Chlorophyll-a Concentration of H.actiniformis

of Jeffrey & Humphrey (1975) for trichromatic method

was used to calculate the chlorophyll-a concentration ) -2 -2 standardized to µg cm , with the surface area of the polyp 2E6 being factored in (Equation 2).

Color index, based on photographs of corals taken 1.5E6 before and after exposure to different temperatures, was determined using the coral health monitoring card of 1E6

Equation 2. Determination of the biomass of chlorophyll-a of Heliofungia actiniformis modified 5E6

from Aminot & Ray (2000). (cm Density of zooxanthellae

0 Biomass µg cm-2 = 11.85*(E664-E750)-1.54*(E647- )

E750)-0.08(E630-E750)] *Ve/L*Vf -2 4.5 -3 -2 the unit is in mg m so the need to convert to µg cm / 4.0 -2 the surface area (cm ) 3.5 a ( μ g.cm

3.0

Where : 2.5 Ve (extraction volume in ml)= 10mL 2.0 L(cuvette length path in cm) = 1cm Vf (filtered volume in Liter) =1L 1.5 1.0

0.5

Siebeck et al. (2006). The initial and final color indexes Biomass of chlorophyll- 0.0 were compared, wherein a change of color of 2 scale units 24-26 27-29 30-32 is indicative of bleaching. Temperature °C Figure 1. Density of zooxanthellae and biomass of Chl-a expelled by A model I one-way ANOVA with an a priori level of Heliofungia actiniformis, after 24 h exposure to different significance p=0.05 was used to examine the treatment levels of temperature. (Mean ± .95*SE) (temperature) effects on the chl-a biomass (µg cm-2) and density of zooxanthellae (cm-2) expelled by the organism. Test for homoscedasticity of the residuals indicated there was no significant difference (p=0.18) between that the data were homoscedastic and were normally the ambient and lower (457,446±218,130 cells cm-2) distributed. A Bonferroni test was used as a post hoc temperatures. The mean biomass (µg cm-2) of chl-a at comparison in case a significant effect was detected. The 30-32°C (3.38±0.59, mean±SE) was three times higher qualitative aspect using the difference of color scale was compared to the ambient condition (Figure 1). The subjected to Kruskall-Wallis test. mean biomass at lower and ambient temperatures did not vary significantly (p=0.56). There was a significant positive correlation (Pearson r=0.48; p=0.02) between density of expelled zooxanthellae and biomass in terms RESULTS of chlorophyll-a concentration (Figure 2). On the other The laboratory controlled thermal stress had significant hand, comparison of the change of color of at least 2 scale effect on the release of zooxanthellae in both density and units as indicative of bleaching (Siebeck et al. 2006) was biomass of chl-a. The mean number of zooxanthellae not significant between treatments (H=0.824, p=0.67). -2 -2 Contrary to the expected results, 50% of corals exposed cm (F2,21=3.691; p=0.042) and biomass (µg cm ) to both lower and ambient temperatures bleached while (F2,21=10.711; p=0.001) varied between treatments after 24 h exposure. At ambient temperature (27-29°C), the only 38% of those in the elevated temperature did. Corals mushroom coral released 777,548±264,021 (mean±SE) were returned back to the reef of origin after use. cells cm-2 (Figure 1). Highest density of zooxanthellae (1,517,844±353,137 (mean±SE) was expelled at higher temperature (30-32°C). The density of cells released was twice as much compared to the control. However,

37 Philippine Journal of Science Cesar et al: Thermal Stress Affects Zooxanthellae Density Vol. 143 No. 1, June 2014 and Chlorophyll-a Concentration of H.actiniformis

3.5E6 r=0.48; p=0.02 3E6 ) -2 2.5E6

2E6

1.5E6

1E6 Density of zooxanthellae (cm zooxanthellae of Density

5E5

0 0 1 2 3 4 5 6 7 Biomass of chlorophyll-a (ug.cm-2) Figure 2. Correlation between density of zooxanthellae and biomass of of Chl-a expelled algae of Heliofungia actiniformis after 24 h exposure to different levels of temperature. (Mean ± .95*SE)

DISCUSSION Guldberg et al. 1987). During bleaching, corals commonly lose 60-90% of their zooxanthellae (Glynn 1996). In this The density of released symbionts at ambient temperature study, the elevated temperature of 1-2°C was enough to (27-29°C), is within the average densities (0.5 and 5x106 -2 double the density of expelled symbionts in H. actiniformis cells cm ) of zooxanthellae symbionts in hermatypic after 24 h exposure. The doubling of density of expelled corals (Hoegh-Guldberg & Smith 1989). The branching 6 2 cells at 30-32°C can be considered bleaching. However, A. millepora contains ~1.4 × 10 zooxanthellae cm- H. actiniformis showed some resilience compared to (Mieog et al. 2009). However, Cervino et al. (2003) Stylophora spp., which after 7 h exposure at the same reported a lower density of 4,532 from H. actiniformis. thermal stress, resulted in specific expulsion rates of The collection of cells was confined with those released >1000 times that of controls (Hoegh-Guldberg & Smith from the gastrovascular cavity, in contrast to the collection 1989). The difference in the density and expulsion rate of of cells released by the entire polyp to the water column zooxanthellae between branching corals like Stylophora that yield higher value for this study. Release of symbiotic spp. and mushroom coral like H. actiniformis, reinforces zooxanthellae by corals is an innate immune response of the need to maintain diverse lifeforms in a reef or in an the host to a compromised symbiont (Weis 2008). In an aquarium. On early phase of elevated temperature stress, elevated temperature, the photosynthetic rate is enhanced zooxanthellae expelled from branching corals can fall off coupled with high oxygen level leading to formation of and colonize mushroom corals which in turn can evade reactive oxygen species (ROS). This high level of ROS the increasing temperature by moving to the deeper area inflicts oxidative stress to both zooxanthellae and coral with their flotation ability (Gittenberger et al. 2011). In (Weis 2008). Elimination of destroyed zooxanthellae future studies, it would be interesting to determine if could follow different mechanisms including in situ there will be shift of zooxanthellae clade composition to degradation, exocytosis and apoptosis (Steen & Muscatine the heat-tolerant clade D. Such studies will lend credence 1987; Jones & Yellowlees 1997; Weis 2008). The primary to the adaptation of this species to a 1-1.5°C increase in mechanism by which zooxanthellae are released involves temperature. The predicted temperature increase in the the exocytosis of algal cells from the epithelium into the upper ocean where coral reefs are found at the end of 2100 coelenteron (Steen & Muscatine 1987). In normal reef is 2°C (IPCC 2013). However, such elevated temperature- conditions, zooxanthellae released to the water column adaptation mechanism is compromised by the slower is less than 0.1% of the standing stock of cells (Hoegh-

38 Philippine Journal of Science Cesar et al: Thermal Stress Affects Zooxanthellae Density Vol. 143 No. 1, June 2014 and Chlorophyll-a Concentration of H.actiniformis

growth rate, both in situ and observed in the laboratory displacement result to extreme thermal limit, the use of a corals (Jones et al. 2008; Jones & Berkelmans 2010). closed Styrofoam box is better (Borneman 2001). Microspically, the expelled zooxanthellae were morphologically intact even at the highest temperature. Color reference card as indicator of bleaching At 30-32°C, the release of morphologically intact Predicting occurrence of massive bleaching at global coupled with photosynthetically normal cells by Galaxea scale with 95% accuracy is now possible with access fascicularis, showed the susceptibility of the host rather to NOAA’s Coral Reef Watch Satellite Bleaching Alert than the symbiont zooxanthellae to thermal stress System (Goreau & Hayes 1994; Hoegh-Guldberg 1999). (Bhagooli & Hidaka 2004). However, sensitivity of However, at local scale and in the aquarium trade, less Symbiodinium had been observed in the impairment of expensive and technical ground truth surveys had been its photosynthetic function above 32°C (Hoegh-Guldberg introduced (e.g direct visual inspection of color hue/ 1999). The host’s innate response is to get rid of the intensity, use of coral reference card). impaired symbionts thru apoptosis (Weis 2008). In mushroom corals, the change in coloration was most The 24 h incubation period already covered the diel distinct at the tip of the tentacles, the mouth parts and pattern of cell release (Jones 1997). Corals during massive periphery of the polyps (Hoeksema 1991). The detected bleaching event tend to have intact tissues but with change of color intensity/hue could be the result of the decreased zooxanthellae population, with or without loss expulsion of zooxanthellae as initial response to handling of pigments (Hoegh-Guldberg & Smith 1989). Loss of stress coupled with the secretion of mucus (Cervino et al. photosynthetic pigments can be 50-80% (Glynn 1996). 2003). The inherent fleshy tentacles of these corals upon In this study, release of cells is coupled with release of retraction, thus covering the polyp’s surface, could have pigments as seen in the positive correlation of density of masked the decrease brightness, the immediate visual cells and biomass of chl-a. indicator of bleaching. Brown et al. (1994) opined that retraction of tentacles, can expose the naked skeleton At ambient temperature, H. actiniformis released 1.34 without necessarily loosing cells but could be mistaken -2 µg cm of chlorophyll-a to the water (Fig. 1). Buchheim as bleaching. However, in H. actiniformis, this retraction (1998) reported 2-10 pg of chlorophyll-a per zooxanthella and subsequent covering of the skeleton made the polyp of which together with other major zooxanthellar appeared darker. Darker color and engulfed in mucus was pigments could be 9-34 times higher in non-bleached also observed in cyanide exposed H. actiniformis (Cervino corals (Kleppel et al. 1989). The lower amount of et al. 2003). Thus, the masking effect of tentacle retraction chlorophyll-a expelled by those exposed to both lower of this large tentacle coral is crucial in color-based and ambient temperatures indicated that this species can qualitative evaluation. Corals were exposed to laboratory tolerate the lower range of temperature. Actually, what induced thermal stress for four days in the development of is considered as lower temperature in this study could this color reference card (Siebeck et al. 2006) compared be ambient condition in Japan reefs (Bhagooli & Hidaka to the 24 h exposure in this study. Despite the shorter 2004). Although bleaching is more common in elevated exposure to elevated temperature, most of the corals used temperature, it also occurs at lower temperature (Goreau in the experiment did not immediately recover even when & Hayes 1994; Hoegh-Guldberg et al. 2005). Corals returned to the reef. However, such demise can be avoided from cooler regions are proned to bleaching due to low by H. actiniformis with their downward migration through temperature (Hoegh-Guldberg 1999). Compared to the flotation (Hoeksema 1988), a mobility mechanism to find 24 h exposure, bleaching might also occur with longer refuge in the deeper portion with optimal temperature. duration of exposure as when corals are kept in marine This may explain the highest density of fungiids at depths aquaria. The resilience of this mushroom coral to lower of 3-9 m (Hoeksema 1991) and 7-15 m (Knittweis et al. temperature needs further validation since the study 2009) although they can occur in shallower area down to was done within short exposure duration. Although the 21 m (Hoeksema 1991). corals used in this study tolerated the lower temperature treatment, low temperature could be more deleterious than high temperature. A decrease in the natural water temperature was found to be more harmful than a SUMMARY AND CONCLUSION temperature increase of the same magnitude in Hawaiian reefs (Jokiel & Coles 1977). In the transport of coral In summary, H. actiniformis exposed to thermal stress for the aquarium trade, displaced icepack resulting to at a level for the predicted temperature increase in coral 190C temperature was a probable factor leading to 50% reefs, bleached within 24 hours of exposure. However, bleaching mortality of corals during transit (Borneman some resilient traits could enable this traded aquarium 2001). Instead of relying on heatpacks or icepacks whose species to adapt when ambient condition changes in

39 Philippine Journal of Science Cesar et al: Thermal Stress Affects Zooxanthellae Density Vol. 143 No. 1, June 2014 and Chlorophyll-a Concentration of H.actiniformis the near future, on top of the 0.7°C increase in tropical REFERENCES waters (IPCC 2007). Although the density and biomass of chl-a of expelled zooxanthellae subjected to 30- AMINOT A, REY F. 2000. Standard procedure for the 32°C, doubled and tripled respectively, this is still lower determination of chlorophyll a by spectroscopic compared to the response of branching corals subjected to methods. Copenhagen K Denmark: International similar thermal stressor. It is recommended that possible Council for the Exploration of the Sea. Palaegade 2–4 shift of composition of Symbiodinium from the usual C DK-1261. 1-17p. clade dominated to the heat tolerant clade D (Jones et al. BAKER AC, GLYNN PW, RIEGL B. 2008. Climate 2008), be investigated as a coping mechanism to elevated change and coral reef bleaching: An ecological temperature. The capability of mushroom coral to seek assessment of long-term impacts, recovery trends refuge in the deeper and cooler area and to shift to heat and future outlook. Estuar Coast and Shelf Sci 80(4): tolerant Symbiodinium clade D are added advantages 435–471. (Berkelmans & van Oppen 2006). Furthermore, the BHAGOOLI R, HIDAKA M. 2004. Release of thick gastrodermis that harbors the algae and the green zooxanthellae with intact photosynthetic activity by flourescent pigments, both provide protection mechanism the coral Galaxea fascicularis in response to high against intense sunlight by reducing the UV and light temperature stress. Mar Biol 145 (2): 329-337. flux that is associated with bleaching events (Mattan- Moorgawa et al. 2012). BERKELMANNS R, VAN OPPEN MJH. 2006. The role of zooxanthellae in the thermal tolerance of corals: A On the other hand, resilience of this coral to lower ‘nugget of hope’ for coral reefs in an era of climate temperature should be taken with caution. The sublethal change. Proc Roy Soc B 273: 2305–12. effect of lower temperature could just be due to limited exposure in time since lower temperature is found to be BORNEMAN EH. 2001. Aquarium Corals. Neptune City, more deleterious to reefs (Jokiel & Coles 1977). This NJ: TFH Publication Inc. 464 p. warrants further verification especially in the aquarium BROWN BE, LE TISSIER MDA, DUNNE RP. 1994. industry. The severe retraction and subsequent attachment Tissue retraction in the scleractinian coral Coeloseris to the skeleton of the inherent large tentacles of H. mayeri, its effect upon coral pigmentation, and actiniformis, may masks the supposedly decrease in preliminary implications for heat balance. Mar Ecol color brightness, the immediate visual sign of bleaching. Prog Ser 105: 209-218. Thus, along with large and fleshy tentacles species like euphyllids and bubble corals, the color reference card BUCHHEIM J. 1998. Coral Reef Bleaching. Odyssey should be coupled with close observations to include Expeditions - Tropical Marine Biology Voyages. http:// cloudy water due to excessive mucus production, www.marinebiology.org/coralbleaching.htm. retrieved detachment of pale tissues in the oral area and complete 01 June 2013 retraction of the tentacles. BUDDEMEIER RW, FAUTIN DG. 1993. as an adaptive mechanism a testable hypothesis. Bio Sci 43 (5): 320-326. ACKNOWLEDGMENT CERVINO JM, HAYES RL, HONOVICH M, GOREAU TJ, JONES S, RUBEC PJ. 2003. Changes in We thanked the University of San Carlos, Marine Biology zooxanthellae density, morphology, and mitotic index Section for logistics support. Special thanks to Benjamin in hermatypic corals and anemones exposed to cyanide. Pangatungan for assisting in the collection of specimens, Mar Pollut Bull 246 (5): 573-86. and to Desiderio Asane for assistance during the laboratory activities. The scholarship grants given by the Department CHADWICK-FURMAN NE, GOFFREDO S, LOYA of Science and Technology-Philippine Council for Y. 2000. Growth and population dynamic model of Agriculture, Aquatic and Natural Resources Research and the reef coral granulosa Klunzinger, 1879 Development (DOST-PCAARRD) to Senona A. Cesar at Eilat, northern Red Sea. J Exp Mar Biol and Ecol and Homer Hermes Y. De Dios are hereby acknowledged. 249: 199-218. The study is a joint marine science contribution of VSU, DONNER SD, SKIRVING WJ, LITTLE CM, SLSU, and USC, and is dedicated to the late PCAARRD OPPENHEIMER M, HOEGH-GULDBERG, O. 2005. deputy executive director for aquatic resources, Cesario Global assessment of coral bleaching and required rates R. Pagdilao. of adaptation under climate change. Glob Change Biol 11 (12): 2251-65.

40 Philippine Journal of Science Cesar et al: Thermal Stress Affects Zooxanthellae Density Vol. 143 No. 1, June 2014 and Chlorophyll-a Concentration of H.actiniformis

GITTENBERGER A, REINEN BT, HOEKSEMA BW. Policymakers. In: Climate Change 2013: The Physical 2011. A molecularly based phylogeny reconstruction Science Basis. Contribution of Working Group I to of mushroom corals (: ) with the Fifth Assessment Report of the Intergovernmental taxonomic consequences and evolutionary implications Panel on Climate Change. Stocker TF, Qin D, Plattner for life history traits. Contrib Zool 80:107-132. G-K, Tignor M, Allen SK, Boschung J, Nauels A, Xia Y, Bex V, Midgley PM eds. Cambridge, United GLYNN PW. 1996. Coral reef bleaching: facts, hypotheses Kingdom and New York, NY, USA: Cambridge and implications. Glob Change Biol 2: 495–509. University Press. 953-1028p. GOREAU TJ, HAYES RM. 1994. Coral bleaching and [IPCC] INTERGOVERNMENTAL PANEL ON ocean ‘hot spots’. Ambio 23: 176-180. CLIMATE CHANGE. 2007. Climate Change 2007: HOEGH-GULDBERG O. 1999. Climate change, coral The Physical Science Basis. Contribution of Working bleaching and the future of the world’s coral reefs. Mar Group I to the Fourth Assessment Report of the Freshwater Res 50:839-866. Intergovernmental Panel on Climate Change, Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB, HOEGH-GULDBERG O. FINE M, SKIRVING W, Tignor M, Miller HL, Eds. Cambridge, UK and New JOHNSTONE R, DOVE S. STRONG A. 2005. Coral York, NY, USA: Cambridge University Press. 1-17p. bleaching following wintry weather. Limnol Oceanogr 50 (1) : 265-271. JOKIEL PL, COLES SL. 1977. Effects of temperature on the mortality and growth of Hawaiian reef corals. Mar HOEGH-GULDBERG O, Mc CLOSKEY LR, Biol 43: 201-208. MUSCATINE L. 1987. Expulsion of zooxanthellae by symbiotic cnidarians from the Red Sea. Coral Reefs JONES A, BERKELMANS R. 2010. Potential costs 5: 201-204. of acclimatization to a warmer climate: Growth of a reef coral with heat tolerant vs. sensitive symbiont HOEGH-GULDBERG O, SMITH GJ. 1989. The effect of types. PLoS ONE 5(5): e10437. doi:10.1371/journal. sudden changes in temperature, light and salinity on the pone.0010437 population density and export of zooxanthellae from the reef corals Stylophora pistillata and Seriatopora JONES AM, BERKELMANS R, VAN OPPEN MJH, hystrix. J Exp Mar Biol Ecol 129 (3): 279–303. MIEOG JC, SINCLAIR W. 20O8. A community change in the algal endosymbionts of a scleractinian HOEKSEMA BW. 1988. Mobility of free-living fungiid coral following a natural bleaching event: field corals (Scleractinia), a dispersion mechanism and evidence of acclimatization. Proc R Soc B 275:1359- survival strategy in dynamic reef habitats. Proceedings 1365 6th International Coral Reef Symposium, Townsville, Australia 2: 715-720. JONES RJ. 1997. Zooxanthellae loss as a bioassay for assessing stress in corals. Mar Ecol Prog Ser 149: HOEKSEMA BW. 1989. , phylogeny and 163-171. biogeography of mushroom corals (Scleractinia: Fungiidae). Zool Verb 254: 1-295. JONES RJ, YELLOWLEES D. 1997. Regulation and control of intracellular algae (= zooxanthellae) in hard HOEKSEMA BW. 1991. Control of bleaching in corals. Philos Trans R Soc Lond B Biol Sci 352(1352): mushroom coral populations (Scleractinia: Fungiidae) 457–68. in the Java Sea: stress tolerance and interference by life history strategy. Mar Ecol Prog Ser 74: 225-37. KLEPPEL GS, DODGE RE, REESE CJ. 1989. Changes in pigmentation associated with the bleaching of stony HOEKSEMA BW, MATTHEWS JL. 2011. Contrasting corals. Limnol Oceanogr 34 (7): 1331-1335. bleaching patterns in mushroom coral assemblages at Koh Tao, Gulf of Thailand. Coral Reefs 30: 95. KNITTWEIS L, JOMPA J, RICHTER C, WOLFF M. 2009. Population dynamics of the mushroom HOEKSEMA BW, MATTHEWS JL, YEEMIN T. 2012. coral Heliofungia actiniformis in the Spermonde The 2010 coral bleaching event and its impact on the Archipelago, South Sulawesi, Indonesia. Coral Reefs mushroom coral fauna of Koh Tao, Western Gulf of 28 (3): 793-804. Thailand. Phuket. Mar Biol Cent Res Bull 71: 71–81. KNITTWEIS L, WOLFF M. 2010. Live coral trade impacts HOEKSEMA BW, van der MEIJ SET, FRANSEN CHJM. on the mushroom coral Heliofungia actiniformis in 2012. The mushroom coral as a habitat. J Mar Biol Indonesia: Potential future management approaches. Assoc U K 92: 647-663. Biol Conserv 143: 2722-2729. [IPCC] INTERGOVERNMENTAL PANEL ON CLIMATE CHANGE. 2013: Summary for

41 Philippine Journal of Science Cesar et al: Thermal Stress Affects Zooxanthellae Density Vol. 143 No. 1, June 2014 and Chlorophyll-a Concentration of H.actiniformis

LOYA Y, SAKAI K, HEYWARD A. 2009. Reproductive patterns of fungiid corals in Okinawa, Japan. Galaxea J Coral Reef Studies 11(2):119-129. MATTAN-MOORGAWA SM, BHAGOOLI R, RUGHOOPUTH S. 2012. Thermal stress physiology and mortality responses in scleractinian corals of Mauritius. In: Proceedings of the 12th International Coral Reef Symposium; 9-13 July 2012; Cairns, Australia: James Cook University, Townsville Quenzland, Australia. 1-6p. MIEOG JC, van OPPEN MJH, BERKELMANS R, STAM WT, OLSEN JL. 2009. Quantification of algal endosymbionts (Symbiodinium) in coral tissue using real-time PCR. Mol Ecol Res 9: 74–82. OLIVOTTO I, PLANAS M, SIMOES N, HOLT GJ, CALADO R. 2011. Advances in breeding and rearing marine ornamentals. J World Aquacult Soc 42: 135–166. SIEBECK UE, MARSHALL NJ, KLUTER A, HOEGH- GULDBERG O. 2006. Monitoring coral bleaching using a colour reference card. Coral Reefs 25: 453–460. STEEN RG, MUSCATINE L. 1987. Low temperature evoke rapid exocytosis of symbiotic algae by a . Biol Bull 172:246– 263. TRAUTWEIN SE. 2001. Advances in coral husbandry: Propagation for the future. Drum and Croaker: A Highly Irregular Journal for the Public Aquarist. 32: 29-34 p. WEIS V. 2008. Cellular mechamisms of Cnidarian bleaching: stress causes the collapse of symbiosis. J Exp Biol 2011: 3059-3066. WELLS S, HANNAH N. 1992. The Greenpeace Book of Coral Reefs. New York: Sterling. 160p. YELLOWLESS D, REES TAV, LEGGAT W. 2008. Metabolic interactions between algal symbionts and invertebrate hosts. Plant Cell Environ 31: 679-694.

42