World Applied Sciences Journal 26 (Natural Resources Research and Development in Sulawesi Indonesia): 112-118, 2013 ISSN 1818-4952 © IDOSI Publications, 2013 DOI: 10.5829/idosi.wasj.2013.26.nrrdsi.26020

Mitotic Index of Algae Zooxanthellae from Sea Anemone from

12M. Ahsin Rifa’I and Kamaruzaman Jusoff

1Faculty of F isheries and Marine Science, Lambung Mangkurat University, Jl. A. Yani Km 36 Banjarbaru, Banjarmasin, 70713South Kalimantan, Indonesia 2Department of Forest Production, Faculty of Forestry, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Submitted: Oct 1, 2013; Accepted: Dec 3, 2013; Published: Dec 20, 2013 Abstract: Zooxanthellae is symbiotic algae that can form mutualism relation to endodermis tissue of sea anemone. The dynamics of zooxanthellae algae are well known in the anemone asexual reproduction. The objective of this paper was to determine the rate of zooxanthellae cells bisection that form a with sea anemone resulted from asexual reproduction with the technique of fragmentation. Research was conducted from November 2010 to July 2011 in the coral reef of Teluk Tamiang village, South Kalimantan Province. The experiment used zooxanthellae from sea anemone resulted from asexual reproduction and natural sea anemone. The mitotic index of zooxanthellae that form the symbiosis with sea anemone after body fragmentation was measured. Zooxanthellae and sea anemone were cultured for five months in the natural waters. Results showed that the rate of zooxanthellae cells bisection (mitotic index) have a significant difference between anemone which were resulted from asexual reproduction and natural sea anemone.

Key words: Sea Anemone Mitotic Index Zooxanthellae Asexual Reproduction

INTRODUCTION anemone can conduct sexual and asexual reproduction. Sea anemone (Stichodactyla gigantean) can be bred Zooxanthellae is dinoflagellata symbiotic that can through asexual reproduction with the technique of body form mutualism relation to various of sea invertebratae fragmentation. Asexual reproduction can produce quicker [1, 2]. They supply energy to their host in the form of seed with a 100% survival rate [12, 13]. carbon compound bounded [3, 4] and its importance for Nevertheless, until now the dynamics of growth and survival of host under limited nutrient zooxanthellae especially its rate of cell bisection condition [5]. Zooxanthellae contributed to the health of (index mitotic) which is important to the growth rate of the hosts [6] and play an important role in successfulness zooxanthellae in the host after asexual reproduction is still reef-building corals [7]. Zooxanthellae symbiosis as unknown. Therefore, the objective of the paper was to intracellular symbion in endodermis cells of sea anemone determine the rate of zooxanthellae cells bisection that in tropical areas [3, 8]. There is a tendency that symbiosis with sea anemone (Stichodactylagigantea) by zooxanthella becomes the controlling factor in the asexual reproduction using fragmentation technique. abundance distribution of sea anemone (Stichodactylagigantea) in the Indonesian waters [9, 10] MATERIALS AND METHODS and have a higher density of zooxanthellae than Tridacnasquamosaand coral branch Acroporasamoensis Collection and Culture of Host: The host used in this [11]. The existence of zooxanthellae symbion is important study is the sea anemone Stichodactylagigante for viability and growth of sea anemone. Naturally sea (Figure 1). It was collected during month of September -

Corresponding Author: M. AhsinRifa’i, Faculty of Fisheries and Marine Science, Lambung Mangkurat University, Jl. A. Yani Km 36 Banjarbaru, Banjarmasin, 70713, South Kalimantan, Indonesia. Tel: +6281342232186. 112 World Appl. Sci. J., 26 (Natural Resources Research and Development in Sulawesi Indonesia): 112-118, 2013

Fig. 1: A sample of sea anemone, Stichodactyla gigantea

Fig. 2: A map of Teluk Tamiang Village showing the location of study sites (red dots)

October 2010 around the Tamiang Bay Coral Reef, South reproduction process. During the acclimatization process, Kalimantan (Figure 2). Anemone samples were brought to the water in the aquarium was aerated and anemone was the mini hatchery for acclimatization in an aquarium glass fed up with an additional feed Artemianaupliand which has a capacity of 500L for 1 week before the asexual Tetraselmis sp.

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Fig. 4: A. Dredging a zooxanthellae sample from sea anemone using a scapel B. Zooxanthellae alga screening result

03.00, 06.00, 09.00, 12.00, 15.00, 18.00, 21.00 and 24.00 for 24 hours and repeated for five times. In order to determine Fig. 3: The process of longitudinal body fragmentation the anemone mitotic index difference of zooxanthellae from (A-B), seed of fragmented anemone of two parts asexual reproduction, a Kruskal-Wallis test from SPSS (C), seed of fragmented anemone of four parts (D) version 11.5 program was performed [15] as below:

12 Rj2 Fragmentation and Culture of Anemone: The anemone Hn= +∑ −+31[] asexual reproduction by fragmentation technique was n( n+ 1)  nj performed after the acclimatization process. Body where, fragmentation was conducted by splitting longitudinally n = Total number of data into two and four parts (Figure 3). The 30 fragmented nj = The number of data each variable anemones parts were then placed into the bottom of the Rj = Total range of all variables cage. The size of the cage was 3 m x 2 m x 0.75 m with an opened design at the upper section whereas the ruble RESULTS AND DISCUSSION coral (dead coral) was stored at the bottom of the cage. The dead coral acts as a substrate for anemone‘s walking The highest and the lowest mitotic index were found base once they were placed into the cage. There were two at 03.00 – 09.00 and 12.00 – 18.00, respectively (Figure 5). simulated coral reef conditions marked as bad (BC) and Based on the fragmentation technique, the result showed good condition of coral reef (GC). The depth of waters for that the highest mitotic index was found from the BC and GC ranged from 1 – 2.5 m and 2 – 5 m, non-fragmented anemone. The lowest mitotic index was respectively. The selected criterion of bad and good found in the four fragmented anemone (Figure 6). corals (BC/GC) selection was based on the percentage of Statistically, there was a significant difference of mitotic coral covered according to UPMSC. The anemone was index between non and fragmented anemone (Sig asymp cultured for five months effective June to October 2008. = 0,007<0.05). Figure 7 showed that the highest mitotic index was found at 2011 July and the lowest mitotic index Sample Collection of Zooxanthellae: Sampling was was on 2011 January. However there is no significant conducted three times that is on June, August and difference of mitotic index between periods of anemone October. Samples of Zooxanthellae were analyzed using culture (Sig asymp = 0.701> 0.05). homogenization method [11, 14]. Zooxanthellae and its Based on the location of anemone culture, the result host were disassociated by dredging with scalpel and showed that the highest mitotic index was found in the later suspended by clean saltwater. The sample was anemone that was cultured in the good coral reef filtered with a filter size of 250, 175 – 150 µm to condition (GC) (Figure 8). However, there was no disassociate zooxanthellae from the dirts (Figure 4). significant difference of mitotic index between locations Mitotic index was counted from 500 zooxanthellae samples of anemone culture (Sig asymp = 0.184> 0.05). The rate of that were in the process of cytokinesis or caryokinesis. zooxanthellae cell bisection was represented as the mitotic The cell was observed as a twin cell under microscope index expressed in percentage of cell bisection occurred at magnification of 400 times. Samples were observed at in the population of zooxanthellae [16]. Mitotic index was

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Fig. 5: Mean zooxanthellae mitotic index ( ± SE = 6.273 ± 0.076, N = 240) with sea anemone for a 24 hr observation. AA: Non fragmented sea anemone, AF2: Two fragmented sea anemone parts, AF4: Four fragmented sea anemone parts

Fig. 6: Mean zooxanthellae mitotic index ( ± SE = 6.273 ± 0.076, N = 240) with sea anemone from AA: Non fragmented sea anemone, AF2: Two fragmented sea anemone parts, AF4: Fours fragmented sea anemone parts

Fig. 7: Mean zooxanthellae mitotic index ( ± SE = 6.273 ± 0.076, N = 240) that found at anemone which is resulted from asexual reproduction on Nop-10, Jan-11, Mar-11, May-11 and Jul-11. AA: Non fragmented sea anemone, AF2: Two fragmented sea anemone parts, AF4: Fours fragmented sea anemone parts

perceived the growth rate of zooxanthellae in their host with the highest 06.00 accounted for 11.47% and the and their influenced on the symbion host [14]. Mitotic lowest (3.5%) at noon till evening (12.00 – 18.00) [17]. index measured on November 2010 to July 2011 had the However, the highest and the lowest mitotic index of sea same pattern with the time of the observed mitotic index anemone Anthopleura elegantissima occurred at 22.00 such as the highest mitotic index at 06.00 (7.70 – 8.78%) (6%) and 16.00 (2%), respectively [18]. Meanwhile, mitotic and the lowest at 12.00 (3.87 – 4.46%). index of sea anemone Anemonia viridis had quite a The above result was similar with other findings similar pattern with others, i.e the highest mitotic index at where the sea anemone Heteractis malu had mitotic index 24.00 – 05.00 and the lowest 16.00 [19]. The results of this

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Fig. 8: Mean zooxanthellae mitotic index ( ± SE = 6.273 ± 0.076, N = 240) that found at anemone that cultured at difference coral reef area. AA: Non fragmented sea anemone, AF2: Two fragmented sea anemone parts, AF4: Four fragmented sea anemone parts, GC: Good coral Reef area, BC: Bad coral reef area study clearly indicated that mitotic index of sea that 45 – 48% photosynthesis was translocated to sea anemone was the highest at mid night to early morning anemone A. viridishost [24]. The difference of (22.00 – 06.00). On the other hand, the lowest mitotic index zooxanthellae mitotic index that come from AA, AF2 and was from noon to late afternoon (12.00 – 18.00). It was AF4 anemone (Figure 6) indicated that asexual assumed that the significant difference of water reproduction with the technique of body fragmentation temperature led to the difference of mitotic index between influenced significantly to speed up the rate of early morning and daytime. During this study, water zooxanthellae cells bisection. The lowest mitoticin index temperature at early morning till morning (22.00 – 06.00) was found in zooxanthellae that comes from anemone ranged from 27°C – 28°C, whereas in the day time which was resulted from four body fragmentation parts (12.00 – 18.00) it ranged from 30°C – 31°C. The (AF4) and the highest wasa found in the non- measurement of mitotic index alga based on cell bisection fragmentated anemone (AA). Low percentage of has been proven very sensitive to several environment zooxanthellae mitotic index that come from AF4anemone pressures, such as temperature changes [20]. The highest is possibly due to changes of morphology and mitotic index at early morning (22.00 - 06.00) in this study physiology as a consequence of body fragmentation. was possible due to the low water temperature of 26.4°C It was even worse compared to AF2 and AA anemone. – 28.6°C. Low water temperature forced the host to The change of morphology and physiology affects the conduct metabolism process (catabolism) that releases nutrition supply and protection of zooxanthellae by free energy, such as heat to stabilize its body anemone. Coral and anemone could provide protection, temperature [11]. Heat that was produced by host cell is shelter and supply of carbon dioxide constantly for exploited by zooxanthellae cells to conduct metabolism photosynthesis activity [25, 26]. Zooxanthellae population process to reduce complex molecules from in the host tissue was limited by availability of nutrient, photosynthesis in the day time. This complex molecules sunlight and cell excess. Whereas zooxanthellae has the was kept in the extra fluid cell, later converted to become character of autotrof and can provide energy for host from energy which is used to speed up the process of carbon compound transformation as a result of zooxanthellae cells bisection which responded to photosynthesis activity The host usually provide some temperature fluctuations [21]. protective measures and metabolisms such as carbon Low mitotic index in the day time is possibly due to dioxide and possibilities of some nutrients for all zooxanthellae activity during photosynthesis process zooxanthellae [27].. Zooxanthellae also can utilize the to produce organic compound. When zooxanthellae excretions such as phosphorus, sulphur and nitrogen- undergo photosynthesis activity, a lot of carbons were compound coming from host. produced, enabling its host to form glycerol, glucose and other organic matter [22]. Autoradiographic analysis CONCLUSION result in Anemonia viridis (A. Sulcata) tissue showed 60% of carbon that bound by zooxanthellae when It can be concluded that there is a mitotic index photosynthesis occurred was transferred to the host percentage difference of zooxanthellae in the duration tissue [23]. Using radioisotope technique, it was estimated of culture even though it is not significant different.

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The longer anemone culture resulted in the higher 5. Bé, A.W.H., H.J. Spero and O.R. Anderson. 1982. percentage mitotic index probably due to the improved Effect of Simbiont Elimination and Reinfection physiological and morphological conditions of the on the Life Processes of the Planktonic anemone after fragmentation. Consequently, the nutrient Foraminifer Globigerinoides sacculifer. Mar. Biol., supply by anemone as a host to zooxanthellae symbion or 70: 73-86. vise versa is back to normal conditions after a certain 6. Rinkevich, B., 1989. The Contribution of period of time. Sea anemone did not have a protective Photosynthetic Product to Coral Reproduction. layer as others and is very highly sensitivity to sudden Mar. Biol., 101: 259-263. environmental change. To overcome this situation, sea 7. Heckel, P.H., 1974. Carbonate Buildup in the Geologic anemone required physiological adaptations that have an Record: A Review, In: L.F. Laporte, (ed) Reefs in Time effect on their metabolism process with their symbion and Space. Spec. Publs Soc. Econ. Palont Miner organism. Zooxanthellae mitotic index in sea anemone that Tulsa, Oklahoma, pp: 90-154. was cultured at bad coral reef location (BC) and good 8. Fautin, D.G. and Allen, 1997. Field Guide to Anemone coral reef location (GC) showed that coral reef condition Fishes and Their Host Sea Anemones. 2nd ed. did not have an effect to increase the rate of zooxanthellae Western Australian Museum, Perth Australia, cells bisection. This study implies that it is very important pp: 160. to conserve the coral reefs in the management of anemone 9. Muscatine, L. and V. Weis. 1992. Productivity of resources for future conservation program although some Zooxannthellae and Biogeochemical Cycles. In: P.G. studies discovered that the current environmental Falkowski and A.D. Woodhead, (eds). Primary pressure could give an impact to speed up the bisection Productivity and Biogeochemical Cycles. Plenum, of zooxanthellae cells. New York, pp: 257-271. 10. Dunn, D.F., 1981. The Clownfish Sea Anemones: ACKNOWLEDGEMENT Stichodactylidae (Coelenterata: Actiniaria) and Other Sea Anemones Symbiotic with Pomacentrid Fishes. The authors extend their gratitude to the Directorate Transactions of the American Philosophical Society, General of Higher Education, Ministry of Education and 71(1): 1-115. Culture the Republic of Indonesia for providing research 11. Niartiningsih, A., 2001. Analisis Mutu Zooxanthellae grant under Fundamental program in the fiscal year 2011 dari Berbagai Inang dan Pengharuhnya Terhadap to support this research. Sintasan dan Pertumbuhan Juvenil Kima Sisik (Tridacna squamosa). Disertasi Program REFERENCES Pascasarjana Universitas Hasanuddin. Makassar. 168 hal (in Indonesian). 1. Taylor, D.L., 1974. Symbiotic Marine Algae: and Biological Fitness. In: W.B. Vernberg, 12. Rifa’i, M.A., P. Ansyari, H. Kudsiah and Dan A. (Ed). Symbiosis in the sea. University of South Naparin, 2003-2005. Rekayasa Fragmentasi Anemon Carolina Press, Columbia, pp: 245-262. Laut Jenis Stichodactyla gigantea untuk Restocking 2. Trench, R.K., 1987. The Physiology and dan Rehabilitasi Kawasan Terumbu Karang non Biochemestry of Zooxanthellae Symbiotic with Produktif. Laporan Penelitian Hibah Bersaing XI Marine Coelenterata I. The Assimilation of tahun 2003 -2005. Lembaga Penelitian Unlam Photosynthetic Product of Zooxanthellae by Two Banjarmasin (in Indonesian). Marine Coelenterates. Proc. R. Soc. Lond., B., 13. Rifa’i, M.A. and Dan H. Kudsiah, 2007. 177: 225-235. Reproduksi Aseksual Anemon Laut 3. Muscatine, L., C.F. Pages, A. Blackburn, R.D. Gates, Stichodactyla gigantea (Forsskal, 1775) dengan G. Baghdasarian, D. Allemand. 1998. Cell-Specific Teknik Fragmentasi dan Habitat Penumbuhan Density of Symbiotic in Tropical Berbeda. J. Sains and Teknologi. Augustus, Anthozoans. Coral Reefs, 17: 329-337. 7(2): 65-76 (in Indonesian). 4. Klumpp, D.W., B.L. Bayne and A.J.S. Hawkins. 1992. 14. Ambariyanto, 1996. Effect of Nutrient Enrichment Nutrition of the Giant Clam Tridacna gigas (L). I. in the Field on the Giant Clam Tridacna maxima. Contribution of Filter Feeding and Photosynthates Ph.D. Thesis School of Biological Science and to Respiration and Growth. J. Exp. Mar. Biol. Ecol., Marine Studies Centre the University of Sidney 155: 105-122. Australia, pp: 269.

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