Bionatura-Jurnal Ilmu-ilmu Hayati dan Fisik ISSN 1411 - 0903 Vol. 13, No. 2, November 2011: 209 - 216 STUDY ON SEVERAL REPRODUCTIVE ASPECTS OF SOFT CORAL SINULARIA FLEXIBILIS QUOY & GAIMARD IN BARRANG LOMPO ISLAND, SPERMONDE ARCHIPELAGO, MAKASSAR CITY Haris, A1., and Rani, C.1 1 Faculty of Marine Science and Fishery, Hasanuddin University, Jl. Perintis Kemerdekaan km.10, Makassar 90245 E-mail: haris_pagala@yahoo.co.id ABSTRACT Sinularia lexibilis is one species of soft coral having high economic value due to its pharmacologically active contents. This research was aimed to analyze several reproductive aspects of this soft coral using direct observation in its natural habitat, periodically collecting samples of body tissues, and then preparing and observing its histological preparation. Reproductive aspects found in this research were analyzed descriptively. Results of this research showed that the sexuality type of soft coral S. lexibilis was simultaneous hermaphrodite that reproduced by spawning, its reproductive pattern was hermaphrodite followed by spawning. It spawned from May-September, and the development of its sperm and oocyt was divided into four stages with different characters between one to another, particularly differed in shape, presence of organelles, and organelle colors. Key words: Sinularia lexibilis, reproduction, Barrang Lompo Island STUDI BEBERAPA ASPEK REPRODUKSI KARANG LUNAK SINULARIA FLEXIBILIS QUOY & GAIMARD DI PULAU BARRANG LOMPO, KEPULAUAN SPERMONDE, KOTA MAKASSAR ABSTRAK Sinularia lexibilis adalah salah satu jenis karang lunak yang memiliki nilai ekonomis karena mengandung senyawa bersifat farmakologik. Penelitian ini bertujuan untuk menganalisis beberapa aspek reproduksi karang lunak tersebut dengan metode pengamatan secara langsung di alam, mengambil sampel jaringan tubuhnya secara berkala, kemudian membuat dan mengamati sediaan histologiknya. Aspek-aspek reproduksi yang didapatkan pada penelitian ini dianalisis secara deskriptif. Hasil penelitian menunjukkan bahwa tipe seksualitas karang lunak S. lexibilis adalah hermafrodit simultan yang bereproduksi dengan cara spawning, pola reproduksinya adalah hermafrodit yang diikuti oleh spawning, memijah pada bulan Mei-September, dan perkembangan sperma dan oositnya terbagi atas empat tahap dengan karakter yang berbeda antara satu dengan yang lainnya, terutama perbedaan bentuk, keberadaan organel-organel, dan warna organel-organelnya. Kata kunci: Sinularia lexibilis, reproduksi, Pulau Barrang Lompo INTRODUCTION Sinularia lexibilis is one species of soft corals having a high economical value from coral reef ecosystem. Secondary metabolites contained in this soft coral have been known to have pharmaceutical characters. Compounds produced by this soft coral are sinulariolyde, sinularin, dihydrosinularin (Tursch et al., 1978; Michalek and Bowden, 1997), sandensolide monoacetate, lexibolide (Anjaneyulu et al., 1997), cembranoid diterpenes, sinulexolide, dihydrosinulexolide, sinulexibilin (Duh et al., 1998), diterpenes lexibilide (Aceret et al., 1998), 11-epi-sinulariolide acetate, 11dehydrosinulariolide, sinulariolide (Hsieh et al., 2003). These compounds have activity as antimicrobe, cytotoxic character, and potential to be used as anticancer compounds. Beside its signiicant economical potency to be developed, its population has been threatened worldwide, because most of its habitats i.e. coral reefs have experienced a serious degradation and in terriic condition. Haris, A., and Rani, C. According to Wilkinson (1993) in Tomascik et.al. (1997) approximately 10% of global coral reefs have been damaged, and about 30% of these damaged coral reefs will loss within 10-40 years, if level of anthropogenic pressures, population and association of the causal factors are continuously increase, while in Indonesia, according to Suharsono (1999), from 416 locations in 43 areas of Indonesian waters, there were only 6.49% in very good condition, 24.28% in good condition, 28.61% in moderate condition and 40.62% in bad condition. In order to control high rate of habitat destruction and overexploitation, particularly for utilization as a source of bioactive compounds and production of particular bioactive compounds from technological engineering, it is urgent to provide controlling efforts, especially those related to their culture development. This culture development is focused to produce raw extract and large-scale or industrial-scale of extract fraction and to supply seed for restocking at damaged coral reef areas. Culture development for production of raw extract and extract fraction is only achieved if reproductive biology aspects of this soft coral have been well understood. Aspects of reproductive biology that should be intensively studied are: sexuality, reproductive style, reproductive pattern, sperm and development stages. The objective of this research was to analyze several reproductive aspects of soft coral Sinularia lexibilis i.e. sexuality, reproductive style, reproductive pattern, sperm and development stages. MATERIALS AND METHODS Study location and time overall stages in this study were conducted approximately nine months from March to November 2006. Observation and sample collection were done in the vicinity of coral reef of Barrang Lompo Island, Makassar City following lunar cycle. Observation of reproductive behavior was done based on one lunar cycle, while collection of histological samples for determination of sexuality, method and pattern of reproduction, stages of gonad development were done in two lunar cycles. For determination of peak of 210 reproductive season, collection of histological samples was done for ive lunar cycles. Materials and Tools Materials and tools in this research were used in the ield, laboratory and hatchery Listed in Table 1. Table 1. Materials and tools used in this research. No. Materials/Tools 1. Soft Coral Sinularia lexibilis 2. 3. 4. Formalin 5-10% Sample bottles 12 N HCl 10% 5. Alcohol 70%-100% 6. NaOH 10% 7. 8. Xylol Parain Microtom and microtom blade Preservative solution Store tissue samples Decalciied solution Preservative solution and dehidration Solution for whitening the skeleton of coral’s larvae Clearing solution Make tissue blocks Incise the tissue (coral polyp) 10. Object and cover glasses Stick the histological incision 11. Hematoxylin and Eosin Coloring solution for histological preparation 12. Light microscope Observe the coral gonads 13. Photomicroscope and ilm negative Take photomicrograph 14. Box glass object 15. Scuba diving Set 16. Water Quality Checker 9. Utility Tested organism Box to store the histological preparation For diving during observation coral spawning time Measure temperature and salinity Research Procedure Observation to know sexuality, reproductive style and strategy and level of gonad development were done based on lunar cycle (Glynn et al. 1994) for two months (two lunar cycles). This observation was done twice for each lunar phase started from the 1st of this phase and 3-4 next days. Sample collection was commenced and ended with new month. The schedule of sample collection was presented in Table 2. Fragments of sampled branches were ixated by putting them into ixative solution (formalin 5-10% in seawater) for a week, then Study on Several Reproductive Aspects of Soft Coral Sinularia lexibilis Quoy 211 were decalciied with 12N HCl 10% solution (solved in aquadest) for 4-6 hours or more (Wallace 1985, Glynn et al., 1991; 1994). Polyps that had been decalciied were stored in speciic container (tissue cassette) and were rinsed at tap water for 24 hours to discharge HCl at the tissue surface. These polyps then were temporarily stored in 70% alcohol solution (Fadlallah & Pearse 1982, Glynn et al., 1994) before doing histological preparation. This step followed process of standard tissue technique (Wallace 1985, Kiernan 1990, Glynn et al. 1991; 1994). into four stages, as follows: I, II, III, and IV stages based on criterion of Glynn et al. (1991, 1994); and Stoddart (1984a, b) in Ward (1992). Table 2. Schedule of ield observation and collection of histological samples for observation of sexuality, reproductive style, reproductive pattern, and characteristics of sperm and oocyt development according to the lunar months. Sexuality, Reproductive Style and Pattern Results of histological observation showed that sexuality of S. lexibilis was simultaneous hermaphrodite, where eggs and sperms developed at the same polyp or at different polyp but in the same colony, and achieved maturity at the same time. This result was supported by observation of spawning both in natural condition and in hatchery where the mature eggs and sperms were spawned simultaneously within the same colony. Based on this fact, it can be inferred that sexuality type of soft coral S. lexibilis is simultaneous hermaphrodite, its reproductive style is spawning, its reproductive pattern is hermaphrodite followed by spawning, and spawned during May-September. Hermaphrodite sexuality type and spawning reproductive pattern as found at S. lexibilis in Barrang Lompo Island is different from those observed at soft coral Briareum asbestinum (Brazaeu and Lasker, 1990 in Seo et al., 2008) in Panama, Corallium rubrum (Allemand, 1992 in Seo et al., 2008) in Mediterranean, Pseudoplexaura porosa (Kapela and Lasker, 1999) in Caribbean, Fannyella spinosa (Orejas et al., 2007) in Antarctic whose sexuality is gonochoric brooding reproductive style. Similar hermaphrodite sexuality type was also found at stony corals from families Acroporiidae, Faviidae, Merulinidae, Mussidae, Pectinidae, and Pocilloporidae as studied by Harrison and Wallace (1990). Soft coral S. lexibilis in Barrang Lompo Island spawned from May-September, while stony corals Porites lobata, Acropora squarrosa, and Montipora foveola in Central Paciic Monthly week/day Research activity collection of histological samples for observation of sexuality, reproductive style, reproductive pattern, and characteristics of sperm and oocyt development Moon cycle I Moon cycle II 1 2 3 4 1 2 3 4 x x x x x x x x Determination of sexuality type of corals followed Richmond & Hunter (1990); Harrison & Wallace (1990); & Richmond (1997), reproductive style of corals was analyzed based on the results of the ield survey when the corals were reproducing and during the experiment of artiicial spawning, or from results of observation of the histological preparation. Reproductive style of corals followed Harrison & Wallace (1990); Richmond & Hunter (1990); dan Richmond (1997), whereas, reproductive pattern was determined from data of observation on sexuality type and style. Cell size and morphology were used to determine stages of sperm and oocyt development (Szmant, 1986). Color character occurred after ixation of Hematoxylin and Eosin was also used to determine these stages. Development of sperm and oocyt was divided Data Analysis Sexuality, reproductive style, reproductive pattern and characters of each sperm and oocyt development stage of soft coral S. lexibilis were presented as tables and igures to be analyzed descriptively. RESULTS AND DISCUSSION Haris, A., and Rani, C. spawned during July (Richmond and Hunter, 1990), Goniastrea palauensis, Goniastrea retiformis, and Platygyra lamellina in Great Barrier Reef spawned during October-August (Babcock et al.,1986 in Richmond et al., 1990), Pavona varians and Pavona maldivensis in Hawaii spawned during June (Kolinski and Cox, 2003), and Madracis senaria, M. mirabilis M. decactis and M. pharensis in Curaçao spawned from April-December (Vermeij et al. (2003). Characteristics of Sperm Development Characteristics of each sperm development stage of soft coral S. lexibilis could be seen at Table 4, whereas, characters of each stage could be viewed from photomicrograph of histological preparation at Figure 1. From the results of histological observation to the colony of the soft coral S. helexibilis, its sperm type could be determined as well. Soft coral S. lexibilis has sperm type pier-like or ovoid at head and middle parts. Its center shape was ovoid or bullet-like, and its upper end had a zone with less compact material. At the midpiece of sperm, there was mitochondrial sheet or group of single mitochondria, lipid body and complex centriol structure, and cytoplasm at neck of front sperm. This type was similar to that introduced by (Harrison 1985 in Harrison and Wallace 1990) explaining that sperm type like this was commonly found at scleractinian corals having hermaphrodite sexuality type. Further, Benayahu (1991); Gutiérrez-Rodríguez and Lasker (2004) described that at soft corals, S.I S.III 212 sperm was developed within gastrodermal mesentery, particularly in ventral and lateral parts, and attached to the mesentery during maturation process. Sperm diameter of S. lexibilis at all stages (I-IV) was varied in size ranges and mean size. Sperm had size ranges and mean size 25-100 µm and 66.531 µm (Stage I). 62.5200 µm and 118.218 µm (Stage II), 150-350 µm and 197.290 µm (Stage III), and 225500 µm and 327.437 µm (Stage IV) (Table 4). Sperm diameter differences between soft coral S. lexibilis and species A. dimorpha was caused by difference in polyp size and difference in size of gastrovascular cavity at mesentery between these two soft corals. Characteristics of Oocyt Development Based on modiication of Glynn et. al (1994), characteristics of oocyt development of soft coral S. lexibilis was divided into four oocyt development stages as presented at Table 5 with structure as shown at Figure 2. Oocyt character I of soft coral S. lexibilis found during this research (Table 4) was relatively different from oocyt characters I, II, III, and IV of coral Caulastrea furcata and Lobophyllia corymbosa as studied by Kawaroe et.al. (2007). According to Kawaroe et.al. (2007), oocyt characteristics I of coral C. furcata and L. corymbosa had relatively large nucleus and the oocyt tend to occur singly. Cortical wall was still very thin, and contain less S.II S.IV Figure 1. Sperm structure at soft coral S. lexibilis obtained from photomicrograph of histological preparation Study on Several Reproductive Aspects of Soft Coral Sinularia lexibilis Quoy 213 Table 3. Characteristics of each sperm development stage of soft coral S.lexibilis based on Photomicrograph of Histological preparation Sperm Maturation Stage Characteristics of S. lexibilis Sperm S.I Sperm Diameter (µm) Range Mean ± (SE) Like a dumbbell, small and clumped, illed by interstitial cells in mesoglea, dark bluish red color 25 - 100 66.531 ± 1.626 S.II Its shape started to prolong and spherical, showed a distinct sperm border, extended to mesoglea, bluish violet color 62.5 - 200 118.218 ± 2.616 S.III Prolonged, there was distinct lumen and tail, more solid spermatocyt nuclei with dark color (blue/red) 150 - 350 197.290 ± 5.061 S.IV Prolonged (spherical oval), there was solid lumen and decreased cytoplasm volume and had distinct tail, dark blue/red color 225 - 500 327.437 ± 8.583 Table 4. Oocyt Maturation Stage Oocyt I Oocyt II Oocyt III Oocyt IV Characteristics of Oocyt Development at soft Coral S. lexibilis Based on Results of Histological Observation Charac-teristics of S. lexibilis Oocyt Originated from extension of interstitial cells, rounded and small, red and pale in color, unclear wall and nucleus Rounded (oval), larger, red, unclear nucleus, thick wall had been formed Rounded (tend to ellipse), there were more solid fatty granules, distinct nucleus and wall, bluish red in color More oval in shape, larger and more solid size, its position moved to the edge, dark red in color, there were sub-nucleus within several nucleus Oocyt Diameter (µm) Nucleus Diameter (µm) Diameter ratio (µm) RanMean ± ge SE Mean ± SE Range Mean ± SE 25 - 87.5 38.6±1.4 0 0±0 0 0 37.5 - 150 84.0± 3.5 0 0±0 0 0 100 - 275 150.3 ± 6.9 25 - 62.6 40.6 ± 10.3 3.7 1 187.5 - 625 277.0 ±12.5 37.5 - 150 52.1 ± 8.4 5.3 1 Range cytoplasm and fatty granules. Oocyt character II had increased cytoplasm volume and egg size. Sub nucleus has been differentiated from the nucleus. Vitellogenesis has occurred, however, numbers of fatty granules was still limited with relatively large diameter. Oocyt character III had high cytoplasm volume and high content of fatty granules; however, cortical membrane has not well developed yet. Oocyt character IV was mature oocyt. Cortical membrane has compressed and clearly observed compact and solid fatty granules. Oocyt development of soft coral was also developed at mesentery at polyp autozoid calyx, similar to sperm development. After maturation, oocyt moved from mesentery to gastrovascular cavity. Immature eggs hanged at mesentery, and gradually mature eggs detached from the Haris, A., and Rani, C. mesentery (Kawaroe et,al., 2007; Seo et al., 2008). Autozoid or fertile polyps had separate gonads (male and female). Gonad was located at each mesentery. Reproductive processes included egg and sperm releasing by each polyp into water column. External fertilization occurred outside the coral body. Larvae formed had cilia, and then freely swim to look for suitable hard substrates for attachment before forming polyp or new colony (Manuputty, 2002). Oocyt diameter of S. lexibilis at various stages (I-IV) had different size ranges and mean size. Oocyt stage I had size range between 2587.5 µm and mean size was 38.6 µm. Oocyt stage II had size range between 37.5-150 µm and mean size was 84.0 µm. Oocyt stage III had size range between 100-275 µm and mean size was 150.3 µm, and Oocyt stage IV had size range between 187.5-625 µm and mean size was 277.0 µm (Table 5). Besides difference in size range and mean diameter, oocyt of S. lexibilis had also difference in size range and mean size of nucleus, especially at oocyt stage 214 III and IV, while oocyt I and II have no clear nucleus. As a comparison, oocyt diameter of soft coral A. dimorpha as studied by Seo et al. (2008) had relatively different size ranges and mean size compared to S. lexibilis at all stages of oocyt development. Seo et al. (2008) found oocyt I of coral A. dimorpha had size <51 µm and mean diameter of 43 µm. Oocyt II had diameter range of 51-175 µm and mean diameter 114 µm. Oocyt III had diameter range between 176-275 µm and mean diameter was 211 µm. Oocyt IV had diameter range between >276 µm and mean diameter was 359 µm, while Kawaroe et.al. (2007) studied oocyt development of coral Caulastrea furcata and found mean diameter of oocyt I by 0.275 µm, oocyt II by 0.533 µm, oocyt III by 1.508 µm, oocyt IV by 2.424 µm, and at species Lobophyllia corymbosa, they found mean diameter of oocyt I, II, III and IV as much as 0.185 µm, 0.456 µm, 1.092 µm, and 1.558 µm, respectively. Oosit II Oocyt Oosit IIII Oocyt Oosit III Oocyt III Oosit IV Oocyt IV Figure 2. Oocyt structure (I-IV) of soft coral S. lexibilis obtained from photomicrograph of histological preparation 215 Study on Several Reproductive Aspects of Soft Coral Sinularia lexibilis Quoy CONCLUSION Sexuality type of soft coral S. lexibilis was simultaneous hermaphrodite reproducing spawning, and its reproductive pattern was hermaphrodite followed by spawning, while sperm and oocyt maturity was divided into four stages each, where each stage has different characters compared to others, particularly in shape and presence of organelles. AKNOWLEDGEMENT This work was funded by Hibah Bersaing DP2M DIKTI. I wish to thank M. Rajab and Nasruddin for their help to carry out the ield and laboratory work. spawning in Paracyathus stearnsii. Mar Biol., 71: 233-239. Glynn, P. W., Colley, S. B., Eakin, C. M., Smith, D. B., Cortés, J., Gassman, N. J., Guzmán, H. M., Del Rosario, J. B., & Feingold, J. S. 1994. 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