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, Heliofungia 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. Heliofungia actiniformis, 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 species 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 polyp 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 4.0 the unit is in mg m so the need to convert to µg cm / g.cm -2 μ the surface area (cm ) ( 3.5 a 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.