Journal of Coastal Development ISSN : 1410 - 5217 Volume 9, Number 3, June 2006 : 155-162 Accredited : 23a/Dikti/Kep/2004

ESTIMATING CONTRIBUTION OFZOOXANTHELLAE TO RESPIRATION (CZAR) AND TO ANIMAL GROWTH (CZAG) OF GIANT Tridacna maxima

Ambariyanto *) Marine Science Department, Diponegoro University, Kampus Tembalang, Semarang, 50275, Indonesia.

Received : April, 20, 2006 ; Accepted : May 25, 2006

ABSTRACT Zooxanthellae are symbiotic which live in association with marine invertebrates including giant . These algae are capable of translocating part of their photosynthetic products to the host. This translocation is one of the nutrition sources of the host. The present study aims to calculate the contribution of zooxanthellae on the energy requirements of adult (Tridacna maxima) during their respiration and growth processes. The result showed that zooxanthellae are capable of contributing 260.67% and 452.54% energy required by giant clams for respiration and growth during summer and 171.51% and 273.51 % during winter, respectively. It suggests that all the energy required for these two processes can be supplied by zooxanthellae.

Key words : giant clams, zooxanthellae, energy, CZAR, CZAG *Corespondence : Phone / fax : 024. 7474 698 ; Email : [email protected],

INTRODUCTION

Giant clams are marine bivalves which live have been degrading rapidly including in in ecosystems in the Indo-Pacific Indonesia. region. There are two genera and eight Giant clams are also known as species of giant clams around the world, and marine invertebrate which live in seven of them can be found in Indonesian association with single cell dinoflagelate waters, except Tridacna tevoroa which only algae commonly known as zooxanthellae. Lives in South Pacific countries These algae which can be found in the (Romimohtarto et al., 1987; Knop, 1996). mantle of the clams has an important role as Traditionally, these clams were used by a source of energy for the host. These algae coastal people as a source of meat, building are capable of translocating part of their materials, and other purposes such as house photosynthetic products to the host which hold equipments. Recently, these clams have then be used for biological processes become important export commodities by (Lucas, 1994). several countries mainly for aquarium So far there is no study available (Lucas, 1994). Consequently, the especially, on the estimation of the role of condition of giant clams natural populations zooxanthellae on energy requirement by the clams. This study aims at estimating the

Estimating Contribution of Zooxanthellae to Animal Respiration (CZAR) and to Animal Growth (CZAG) of Giant Clam, Tridacna maxima 155

Journal of Coastal Development ISSN : 1410 - 5217 Volume 9, Number 3, June 2006 : 155-162 Accredited : 23a/Dikti/Kep/2004

contribution of zooxanthellae to animal energy requirements for respiration (CZAR) Carbon content of zooxanthellae was and for growth (CZAG). calculated using an equation that relates cell volume and elemental carbon (Strathmann, MATERIALS AND METHODS 1967). Cell volume of zooxanthellae was calculated base on the mean size of 100

zooxanthellae isolated from the giant clams. Giant clams, Tridacna maxima were The equation to calculate carbon content is collected from intertidal areas on the reef as follows: crest at One Three Island lagoon, Great log carbon = -0.314+0.712 log cell Barrier Reef, Australia using permit at the volume (Strathmann, 1967) depth of approximately 150 cm. These clams were grown in microatolls during winter and summer months. Isolation of zooxanthellae Carbon required for zooxanthellae growth. was done by using the method used by Carbon used by zooxanthellae for growth Ambariyanto and Hoegh-Guldberg (1997). can be calculated by multiplying In order to calculate CZAR and zooxanthellae specific growth rate and CZAG several parameters are required. carbon content, as the following: These are the specific growth rates of C = µ x Carbon per cell zooxanthellae, the carbon used for Zg zooxanthellae growth and respiration, and Carbon translocated by zooxanthellae the carbon used for animal growth and respiration (Muscatine et al., 1981; Carbon translocated can be calculated by Ambariyanto, 2002). the following formula, T = P - R - G Z Z Z where : Specific growth rate of zooxanthellae. PZ = carbon gained from Zooxanthellae specific growth rate zooxanthellaephotosynthesis was calculated by using an equation first RZ = carbon used for zooxanthellae used by Wilkerson et al. (1983). This respiration formula is based on the mitotic index (f = the GZ = carbon used for zooxanthellae percent of dividing cells in a population) and growth the duration of paired cell stage (td). Mitotic index was calculated based Estimating CZAR and CZAG on the number of dividing zooxanthellae in 1000 isolated zooxanthellae times 100 CZAR can be calculated by the following percent. While the value of td used in this formula : CZAR = T / RA, where calculation is 0.46 d based on several studies T = carbon translocated and (Wilkerson et al., 1983; Muscatine et al., RA = carbon required by the animal 1984; Hoegh-Guldberg et al., 1986). It is for respiration also noted that variation in td has only small While CZAG can be calculated by the impacts on CZAR (Muscatine et al., 1981). following formula : µ = 1/td ln(1+fmax) CZAG = (T - RA.)/Carbon growth where µ = zooxanthellae specific growth rate t = 0.46 (Wilkerson et al., 1983; Muscatine d et al., 1984). fmax= mitotic index.

Carbon content of the zooxanthellae. RESULTS AND DISCUSSION

Estimating Contribution of Zooxanthellae to Animal Respiration (CZAR) and to Animal Growth (CZAG) of Giant Clam, Tridacna maxima 156

Journal of Coastal Development ISSN : 1410 - 5217 Volume 9, Number 3, June 2006 : 155-162 Accredited : 23a/Dikti/Kep/2004

Specific growth rate of zooxanthellae. Pteraeolidia ianthina; Hoegh-Guldberg et al., 1986), and 88.85 and 86.5 pg cell-1 The result of this study showed that (zooxanthellae from light and shade adapted maximum value of mitotic index is 0.025, coral Stylophora pistillata respectively; thus the specific growth is : Muscatine et al., 1984). Using the average -1 µ = 1/0.46 ln (1+0.025) = 0.054 day .... (1) value (77.7 pg cell-1), the protein content in zooxanthellae is Carbon content of the zooxanthellae. = 3.8 108 cell x 77.7 pg cell-1 = 29.53 mg The results of the present study showed that the cross-sectional area of zooxanthellae in The total protein content of zooxanthellae the clams was 47.9 µm2. The cell radius (r) is and the animal host (giant clam protein hence: content) was calculated by multiplying the r = √47.9/π = 3.9 µm, and the 3 protein content per gram mantle (5.329 mg Cell volume = 4/3 x π x r = -1 3 g ) by the total wet tissue weight (47.311 g; 248.47 µm Ambariyanto, 1996). It is assumed that Carbon content of zooxanthellae can be protein content within clam mantle and calculated as follows other internal parts is similar. Hence, the log carbon = -0.314+0.712 log cell volume -1 total protein content in zooxanthellae and Carbon per cell = 24.628 pg C cell ...... (2) animal is = 5.329 mg g-1 x 47.311 g = 252.12 mg Carbon required for zooxanthellae growth. -1 The percent of the total respiration due to CZg = µ x Carbon. cell = 0.0054 day-1 x 24.628 pgC zooxanthellae was -1 =29.53 mg/252.12 = 0.117, or = 11.7 % cell

-1 -1 = 1.3299 pgC cell day The mean value of total respiration by intact Carbon required for growth of the entire clam (RC) in this model was 8 -1 -1 population of zooxanthellae (=3.8 10 cell RC= 0.172 mmol O2 clam h clam-1) is (Ambariyanto, 1996), which is 8 -1 - 1 CZg = 3.8 10 cell x 1.3299 pgC = 0.172 x 32 =5.504 mg O2 clam h = -1 -1 -1 -1 cell day 5504 µg O2 clam h -1 -1 - = 0.502 mg C day ...... (3) = 5.504 x 24 = 132.09 mg O2 clam day 1

Respiration of giant clam and This value can be converted into zooxanthellae. carbon using RQA (respiratory quotient for the animal = 0.8; Muscatine et al., 1981) The respiration due to the and the ratio of the masses of C and O2 (ie. zooxanthellae within a clam is a fraction of 12/32 or 0.375), as the following the total respiration. In previous studies the ratio of zooxanthellae and animal biomass -1 -1 RC = 5504 µg O2 clam h x 0.375 x RQ has been used to calculate the portion of the = 5504 µg O clam-1 h-1 x 0.375 x 0.8 total respiration due to zooxanthellae 2 = 1651.2 µg C clam-1 h-1 (Muscatine et al., 1984; Hoegh-Guldberg et -1 -1 al., 1986). = 39.63 mg C clam day ...... (4) Protein content per zooxanthellae is The carbon required for 77.7 pg cell-1. This value is based on an zooxanthellae respiration (RZ) was average of values reported from the literature calculated by multiplying (4) by the for similar size of cells. These were 58.1 pg proportion on the total respiration due to the cell-1 (zooxanthellae from nudibranch zooxanthellae (that is 0.117, see above) : Estimating Contribution of Zooxanthellae to Animal Respiration (CZAR) and to Animal Growth (CZAG) of Giant Clam, Tridacna maxima 157

Journal of Coastal Development ISSN : 1410 - 5217 Volume 9, Number 3, June 2006 : 155-162 Accredited : 23a/Dikti/Kep/2004

= 96.9 % -1 -1 -1 RZ = 39.63 mg C clam day x 0.117 = b). Twinter = 60.01 mg C day - 0.502 mg 4.64 mg C day-1 ...... (5) C day-1 - 2.32 mg C day-1 =57.19 mg C day-1...... (10) Therefore, carbon required for animal %Twinter= (57.19/60.01) x 100 % = 95.3 % respiration (RA): -1 RA = 39.63 - 4.64 = 34.99 mg C day .. (6) Contribution of zooxanthellae to animal respiration (CZAR) The carbon translocated (T) by zooxanthellae to the host was calculated by subtracting the CZAR was calculated by dividing the carbon used for zooxanthellae respiration carbon translocated by the zooxanthellae to (RZ) and growth (GZ) from the carbon the host during summer and winter (T; 9 and gained from zooxanthellae photosynthesis 10) by the carbon required by the animal for (PZ) This is : respiration (RA; 6). T = PZ - RZ - GZ CZAR = T / RA -1 CZAR summer = (91.21 mg C day / -1 PZ is based on the oxygen output resulted 34.99 mg Cday ) x 100 % from the gross photosynthesis (Pg) of = 260.67 % -1 zooxanthellae within the clam host. PZ is CZAR winter = (60.01 mg C day / -1 calculated using the relationship between Pg 34.99 mg Cday ) x 100 % and irradiance and light data collected every = 171.51 % 10 minutes for 24 h at One Tree Island at twice i.e. in the summer and winter. Contribution of zooxanthellae to animal max -αI/Pgmax PZ = Pg (1-e ) growth (CZAG) -1 a. PZ during summer= 221.12 mg O2 clam day -1 CZAG was calculated by first determining Converted into carbon, PZ = 221.12 mg the carbon used for clam growth. This -1 -1 -1 O2 clam day x 0.375 x PQ calculation is based on the average total -1 -1 = 221.12 mg O2 clam day x 0.375 x carbon content of the soft tissue of the 1.1 model giant clam (that is 3.689 g clam-1) = 91.21 mg C clam-1 day -1 ...... (7) and the percent change in buoyant weight per day (0.32 % in summer and 0.22 % in -1 b. PZ during winter= 145.48 mg O2 clam winter; Ambariyanto, 1996). day -1 Converted into carbon, PZ = 145.48 mg a). CZAG for the model clam growing in the -1 -1 -1 O2 clam day x 0.375 x PQ summer was -1 -1 -1 = 145.48 mg O2 clam day x 0.375 x Carbon growth = 3.689 g clam x (0.32 1.1 % / 100%) = 11.8 mg C day -1 -1 -1 = 60.01 mg c. clam day ...... (8) CZAG = (T - RA.)/Carbon growth = (88.39 mg C day-1 - 34.99 mg The carbon translocated (T) by zooxanthellae Cday-1) / 11.8 mg C day-1 x 100% to the host was calculated by subtracting (3) = 452.54 % and (5) from (7) for summer and (8) for b). CZAG for the model clam growing winter respectively. That was in winter was Carbon growth = 3.689 g -1 -1 a). Tsummer = 91.21 mg C day - 0.502 mg clam x (0.22 % / 100%) = 8.12 mg C C day-1 - 2.32 mg C day-1 day -1 -1 = 88.39 mg C day ...... (9) CZAG = (T - RA)/Carbon growth %Tsummer = (88.39/91.21) x 100 %

Estimating Contribution of Zooxanthellae to Animal Respiration (CZAR) and to Animal Growth (CZAG) of Giant Clam, Tridacna maxima 158

Journal of Coastal Development ISSN : 1410 - 5217 Volume 9, Number 3, June 2006 : 155-162 Accredited : 23a/Dikti/Kep/2004

= (57.19 mg C day-1 - 34.99 mg The result of this analysis is summarized in Cday-1) / 8.12 mg C day-1 x 100% Table 1. = 273.40 %

Table 1. Summary results of CZAR and CZAG calculation on untreated giant clam, Tridacna maxima, for summer and winter periods.

-1 Period PZ (mg O2 R (mg O2d P/R ratio Translocat CZAR CZAG d-1 clam-1) clam-1) ion (%) Summer 221.12 132.09 1.67 96.9 % 260.67 % 452.54 % Winter 145.48 132.09 1.10 95.3 % 171.51 % 273.40 %

As can be seen in Table 1., there is seasonal (summer) and 273.40% (winter). It is influence on the estimated values of CZAR interesting that CZAG both during summer and CZAG. This result is in agreement with and winter are above 100% suggesting that Hoegh-Guldberg et al. (1986) who predicted zooxanthellae are able to contribute all the that the contribution of zooxanthellae to the requirements of animal growth. The CZAR host, Pteraeolidia ianthina (Nudibranchia) values of giant clams calculated during this was also substantially influenced by season. study were higher than those reported by Hoegh-Guldberg et al. (1986) predicted that Trench et al., (1981). These authors in animals with high zooxanthellae reported that the contribution of population densities, the symbionts zooxanthellae to the respiratory carbon contribute to the host’s respiratory needs requirements of T. maxima range from 62% during winter, spring and summer at rate of on cloudy days to 84% (assuming 40% 79%, 121% and 173%, respectively. Hoegh- translocation) on sunny days. The Guldberg et al. (1986) suggested that these differences in CZAR values was probably differences is primarily due to differences on due to differences in percentage the light availability during different seasons. translocation used by Trench et al. (1981). The decreasing light with depth also The CZAR values from the present study influences CZAR as shown by the variation are comparable to those from larger clams in values for Stylophora pistillata at different Tridacna gigas. Fisher et al. (1985) reported depth (78% at 35 m and 157% at 3 m depth; that CZAR of T. gigas was 83% and 197% McCloskey and Muscatine, 1984). Light by using the values of 40% and 95% history can also have photoadaptive translocation respectively. Similarly Fitt et influences as shown for CZAR in S. al. (1986) found that CZAR value in pistillata. Light adapted corals had higher Hippopus hippopus, was between 7% and CZAR than shade-adapted corals, 143% and 137% using translocation values of 40% and 58% respectively (Muscatine et al., 1984). 98% respectively. The results of the present study show It should be kept in mind, however, that CZAR values for Tridacna maxima that the organic carbon lost through range from 260.67% in summer to 170.51% calcification and mucus production is not in winter. The percent translocation remains included in this calculation, since no similar at 96.9 % and 95.3 % for winter experiment was conducted during the and summer respectively (see Table 1.). The present study to quantify the amount of contribution of zooxanthellae to animal carbon used for these processes. Secondly, growth (CZAG) ranged between 452.54% the values of percent translocation, CZAR

Estimating Contribution of Zooxanthellae to Animal Respiration (CZAR) and to Animal Growth (CZAG) of Giant Clam, Tridacna maxima 159

Journal of Coastal Development ISSN : 1410 - 5217 Volume 9, Number 3, June 2006 : 155-162 Accredited : 23a/Dikti/Kep/2004

and CZAG of nutrient treated clams are It is not clear, however, whether the td value probably different from the values calculated of zooxanthellae in giant clams is also 0.46. for the control clams here. Differences due to Moreover, during this calculation the carbon the fact that zooxanthellae isolated from content of the zooxanthellae was not ammonium treated clams reduced measured, but was estimated using the zooxanthellae size and starch accumulation equation proposed by Strathmann (1967). within the cell are likely to have significant This equation, however, may underestimate effect on the energy budgets calculated here. the carbon content of zooxanthellae (Hinde The calculation of CZAR which was pers. Comm). Finally, in this calculation it proposed by Muscatine et al. (1981) has been was also assumed that there are no changes widely used for corals (Muscatine and Porter, in the photosynthetic and respiratory rates of 1977; McCloskey and Muscatine, 1984; giant clams in different seasons Hoegh-Guldberg et al., 1986 etc) and giant clams (Fisher et al., 1985; Mingoa, 1988; CKNOWLEDGEMENTS Klumpp et al., 1992; Klumpp and Lucas A 1994 etc). The results of this method, however, have to be interpreted cautiously I would like to thank Prof. Ove Hoegh- since some assumptions, and estimated Guldberg and Prof. David Yellowlees for values of some parameters, have been used in allowing me to join the ENCORE Project. the calculation. The first assumption is that This paper is part of the author PhD Thesis. respiration rate in the dark and in the light are the same, while the second assumption is REFERENCES that the respiration rate of zooxanthellae and their host is proportional to their biomass. By Ambariyanto, 1996. Effects of nutrient using these assumptions, the result of the enrichment in the field on the giant CZAR calculation may not represent the real clam, Tridacna maxima. PhD contribution of zooxanthellae to animal Thesis. The University of Sydney, respiration. For example, the respiration rate Sydney Australia. 267 p. of zooxanthellae within the clams is Ambariyanto and Hoegh-Guldberg, O, impossible to measure, although it can be 1997. The effects of nutrient measured in vitro after isolation. The enrichment on the biomass, growth respiration rate of isolated algae, however, is and calcification of giant clam, not necessarily similar to the respiration rate Tridacna maxima. Mar. Biol 129 (4): of zooxanthellae within the host. Changes in 635-642 zooxanthellar morphology and physiology Ambariyanto and Hoegh-Guldberg, O, 1999 occur soon after isolation (Trench, 1979). The influence of field-based nutrient Secondly, the respiration rates per biomass of enrichment on the photobiology of zooxanthellae and the clams may not be the the giant clam, Tridacna maxima. same. Hoegh-Guldberg and Hinde (1986) Mar Biol. 133: 659-664 estimated that the respiration rate of Ambariyanto, 2002. Calculating the zooxanthellae is higher than that of the host, contribution of zooxanthellae to giant based on their protein biomasses. In addition, clams respiration energy the calculation of the specific growth rate of requirements. A review. J. Coast.Dev. the zooxanthellae is based on the duration of 5(3): 101-110 the paired cell stage (td) and the mitotic Fisher, C.R., Fitt, W.K., Trench, R.K, 1985 index of the zooxanthellae. Since the value Photosynthesis and respiration in of td is difficult to measure, the value of 0.46 Tridacna gigas as a function of d has been used (see Wilkerson et al., 1983).

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