Rearing Cuttings of the Soft Coral Sarcophyton Glaucum (Octocorallia, Alcyonacea): Towards Mass Production in a Closed Seawater System

Home , Alcyonacea, Artificial seawater, Octocorallia, Sarcophyton (coral)

Aquaculture Research, 2010, 41,1748^1758 doi:10.1111/j.1365-2109.2009.02475.x

Rearing cuttings of the soft coral Sarcophyton glaucum (Octocorallia, Alcyonacea): towards mass production in a closed seawater system

Ido Sella & Yehuda Benayahu Department of Zoology,George S.Wise Faculty of Life Sciences,Tel-Aviv University,Tel-Aviv, Israel

Correspondence: I Sella, Department of Zoology,George S.Wise Faculty of Life Sciences,Tel-Aviv University,Tel-Aviv 69978, Israel. E-mail: [email protected]

Abstract for diverse natural products with pharmaceutical or cosmetic value (e.g., Blunt, Copp, Munro, Northcote & The octcoral Sarcophyton glaucum has a wide Indo- Prinsep 2005; Slattery, Gochfeld & Kamel 2005; Sip- Paci¢c distribution and is known for its diverse con- kema, Osinga, Schatton, Mendola,Tramper & Wij¡els tent of natural products.The aim of the current study 2005), as well as for the reef-aquarium trade (Wab- was to establish a protocol for rearing miniature cut- nitz,Taylor, Grenn & Razak 2003). The increased de- tings of S. glaucum in a closed seawater system. In or- mand for these organisms has led to their massive der to determine the optimal conditions for rearing, harvesting (Castanaro & Lasker 2003) and has raised the survival, average dry weight, percentage of or- the need for e⁄cient farming methodologies (Ellis & ganic weight and development of the cuttings were Ellis 2002; Mendola 2003). monitored under di¡erent temperature, light, salinity Coral propagation has been commonly used for the and feeding regimes. At 26 1C, the highest dry weight production of daughter colonies, rather than harvest- was obtained, and at 20 1C, the highest percentage of ing naturally grown ones (e.g., Soong & Chen 2003; organic weight. The dry weight of the cuttings in- Fox, Mous, Pet, Muljadi & Caldwell 2005). This prac- creased with the light intensity, while under 35^ tice has been based on the ability of corals to repro- 130 mEmÀ 2 s À 1, survival was high. Salinity did not duce asexually (e.g, Delbeek 2001; Ellis & Ellis 2002). a¡ect any of the colonies’ features. Feeding intervals Captive-grown colonies, or ¢eld-collected ones, are of 7 and 30 days yielded a better result than of 2 days. used for the production of cuttings or fragments. The A comparison of the colonies derived from the closed latter are usually glued or attached by various means system with the colonies reared in a £ow-through to bases or stubs, until natural attachment is system, those reared in the sea and with ¢eld-col- achieved (Delbeek 2001). lected colonies revealed the importance of environ- Over the last decade, coral propagation in closed mental conditions in determining the features of the arti¢cial seawater systems has been commonly used, colonies. The study emphasizes the advantages of a making the process more simple and cost e¡ective closed seawater system in controlling the conditions (Borneman & Lowrie 2001; http://www.reefkeeping. needed for rearing cuttings of S. glaucum for targeted com). The advantage of these systems lies in their farming. ability to control abiotic and biotic parameters (Wheeler 1996; Borneman & Lowrie 2001; Abramo- Keywords: soft corals, closed seawater system, vitch-Gottlib, Katoshevski & Vago 2002). Such sys- Sarcophyton glaucum, coral farming, Red-Sea tems have been proposed as a source by which to obtain corals and other invertebrates with commercial value (e.g., Wabnitz et al. 2003; Sipkema et al. Introduction 2005), as well as for restoration and conservation A large variety of reef invertebrates, including soft purposes (Becker & Mueller 1999; Petersen, Laterv- corals (Octocorallia), have long been used as a source eer, Van Bergen, Hatta, Hebbinghaus, Janse, Jones,

r 2010 The Authors 1748 Aquaculture Research r 2010 Blackwell Publishing Ltd Aquaculture Research, 2010, 41, 1748^1758 Rearing cuttings of the soft coral S. glaucum I Sella & Y Benayahu

Richter, Ziegler,Visser & Schuhmacher 2006; Okuza- Experimental closed seawater system wa, Maliao, Quinitio, Buen-ursua, Lebata, Gallardo, We constructed a closed seawater system at Tel Aviv Garcia & Primavera 2008). University (TAU). The system comprised three 1m3 Species of the soft coral genus Sarcophyton (Octo- PVC tanks, each ¢lled with arti¢cial seawater (Red corallia) have a wide Indo-Paci¢c distribution and are Sea saltr) and 200 kg of live rocks obtained from Ei- known for the diverse content of their natural pro- lat (Gulf of Aqaba, northern Red Sea). Each tank was ducts (e.g., Look, Fenical, Matsumoto & Clardy 1986; provided with 40 Trochos dentatus snails for algal Tanaka, Yoshida & Benayahu 2005; Nguyen, Tran, grazing. Every 10 days, their faeces were removed Phan, Chau, Eun & Young 2008). S. glaucum (Quoy & from the bottom of the tanks, together with ca.5% of Gaimard, 1833) is the most common species of the the water volume, which was then replaced. A set of genus, also on the northern Red Sea reefs, where it 24 glass aquaria (30 L each) was connected to the was found to be a dioecious broadcaster with the on- tanks by 26-mm-diameter pipes. The aquaria were il- set of reproduction at the age of 6^10 years (Benaya- luminated by neon bulbs (22 W,400^800 nm, JEBO hu & Loya 1986). S. glaucum is zooxanthellate and daylight,12L:12D,35mEmÀ 2 s À 1 on the colony sur- extremely rich in natural products, which have been face). The water circulated from the aquaria to the studied extensively (e.g., Fridkovsky,Rudi, Benayahu, tanks, which were equipped with protein skimmers Kashman & Schleyer1996; Badria, Guirguis, Perovic, (JEBO 3500), a chiller (JEBO 2000, Zhongshan Zhen- Ste¡en, Muller & Schroder1998;Tanaka et al. 2005). hua Aquarium Accessories, Zhongshan, China) and The aim of the current study was to establish the a 5000 L per hour circulation pump (JEBO). The scienti¢c ground for a protocol suitable for rearing water temperature in each aquarium was controlled miniature cuttings of S. glaucum in a closed seawater using an individual heater (60 W). Salinity, tempera- system. Our objectives were to study the e¡ect of tem- ture and pH were monitored daily, and the nutrient perature, light intensity, salinity and feeding regimes levels weekly (nitrite o0.05 ppm, nitrate o10 p p m, on the survival, average dry weight, percentage of or- ammonia 0 ppm and 8.1^8.3 pH) (see Delbeek 2001). ganic weight and development of the cuttings. In addition, colonies reared in a closed seawater system were compared with those reared in a £ow-through sea- Collection of colonies, preparation of cuttings water system in the sea and with ¢eld-collected ones. and experimental set-up

Colonies of S. glaucum with a polypary diameter of 5^ 7 cm (the polyp-bearing part of the colony) were col- Materials and methods lected by SCUBA from three reef sites in Eilat (5^7 m, In a preliminary work, we examined attachment of August 2004^January 2007). For each experiment, the S. glaucum cuttings to stubs made of ceramic and we collected 10^15 colonies, growing at least 5 m plastic materials, using a cyanoacrylate adhesive apart in order to avoid the use of asexually produced (3M) and rubber bands.We also examined the mini- colonies (Benayahu & Loya1986). In order to prevent mal size of cuttings yielding the highest survivorship possible bias in the study, only colonies that did not (10^400 mm 2) (Sella 2007). Consequently,in the cur- contain gonads were used for formation of the cut- rent study, we decided to use cuttings measuring tings (see Okubo, Motokawa & Omori 2007). At the 36 mm 2 (6 Â 6 mm) with an average dry weight of Interuniversity Institute for Marine Science (IUI), 0.0077 Æ 0.0024 g and 8.864 Æ 2.461 average per- the colonies were placed in a £ow-through seawater centage of organic weight (n 5 30 random cuttings system for 3 days and all epibiotic organisms were re- from colonies used in the experiments; see‘Collection moved.The colonies were £own toTAU in plastic con- of colonies, preparation of cuttings and experimental tainers ¢lled with seawater and placed in thermally setup’). The cuttings were glued using a cyanoacry- isolated boxes. They were then maintained in the ex- late adhesive to cylindrical ceramic (Fig. 1a) stubs perimental system for a 1-month quarantine period, that we produced ourselves (clay WBB Fuchfs GmbH under conditions resembling the average ambient Ei- &CoR2502,burntat12001C for 12 h) at a cost of lat ones: salinity 40 ppt, pH 8.2 and 26 1C(IUIData 4 US cents (without labour). The stubs measured Base, http://www.iui-eilat.ac.il/NMP/Default.aspx). 5 cm in length and 1cm in diameter, with a hexago- For preparation of the cuttings, the colonies of nal indentation (ca. 3 mm deep and 7^8 mm wide) at S. glaucum were handled with latex gloves. The peri- one end, into which the cuttings were attached. meter of the polypary was removed using a scalpel

r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,1748^1758 1749 Rearing cuttings of the soft coral S. glaucum I Sella & Y Benayahu Aquaculture Research, 2010, 41, 1748–1758

(see section ‘General observations’ ahead), and the http://www.iui-eilat.ac.il) (10^15 cuttings per aquaria: remaining part was then kept for future regeneration March^May 2005). Seawater at 20 1C was circulated (Sella 2007). The perimeter of the polypary was thor- from the tanks to all the aquaria. The 23,26 and 29 1C oughly rinsed with arti¢cial seawater for removal of seawater temperatures were obtained using 60^300 W mucus and cellular debris, and 36 mm2 (6 Â 6mm) heaters with thermostats. The temperature was routi- cuttings were prepared using autoclaved scissors, nely monitored using an alcohol thermometer. scalpels and forceps and paper blotted. An adhesive To determine the e¡ect of illumination on the cut- was applied to the cuttings’ lower epidermal surface, tings, they were reared at light intensities of 20, 35, and they were mounted on the stubs and kept in the 130 and 250 mE(mEmÀ 2 s À 1), as measured on their air for 30 s to dry. Notably, the cuttings retrieved for surface, falling within the prevailing Eilat values the di¡erent treatments of each experiment were along the depth gradient (Winters, Loya & Beer randomly derived from the same parent colonies and 2006) (20^30 cuttings per aquaria: December 2005^ therefore represented similar genotypes. The stubs January 2006). In order to supply 20 mE, a PVC ¢lter were then inserted into test tube stands (Fig.1a), and (PLASON UV1) was placed between the light source kept in arti¢cial seawater for 20 min in order to wash and the surface of the respective aquaria, while for away any adhesive residue before introduction into 130 and 250 mE, one or two additional neon bulbs the aquaria. During the following week, if mortality were added. Light intensity was monitored daily did not exceed15%, dead cuttings were replaced with using an underwater-light photometer (Bio-sciences, new ones and only then the 60-day experiments Light 250L Li-CO). were started; otherwise, all cuttings were discarded To determine the e¡ect of salinity on the cuttings, and a new experiment was set up as it occurred once. the aquaria were disconnected from the tanks. The In all the experiments, each treatment comprised cuttings were reared under salinities of 30,34, 37 and three aquaria and a control, which was maintained 40 ppt, representing the salinity within the geographic under Eilat average environmental conditions. Un- distribution of the species (Levitus 1982) (15^25 cut- less stated otherwise, the aquaria were supplied tings per aquaria: February^March, 2006). Each aqua- every 25 days with 5000 nauplii of Artemia salina rium had a 60 W heater with a thermostat, a ¢lter (Sorgeloos & Persoone1975; Sella 2007). (JEBO Eco) and an air stone.Water from the1m3 tanks Development and transformation of the cuttings (40 ppt) was diluted with distilled water in order to ob- into small colonies with a stalk and a polypary was tain the lower salinities. Ten litres of seawater was monitored weekly by digital photography and their changed daily in each aquarium and the salinity was survival was calculated at the end of the experiments monitored using a light refractometer and an electro- (day 60). The cuttings were then removed with a scal- nic conductivity meter (MRC Salinity 1, Lutron YK- pel from the stubs and placed individually in alumi- 315A, Lutron Electronic Enterprise,Taipei,Taiwan). nium dishes (5 cm in diameter). Their dry weight was Todetermine the e¡ect of feeding, cuttings were fed determined bydrying (Binder oven,60 1C) for 24 h and at intervals of 2, 7 and 30 days, where a 2-day inter- then weighing to an accuracy of four decimal points val is commonly used by aquarists (http://www.reef (Mettler balance, Toledo AB54-S, Mettler-Toledo, keeping.com) (12^20 cuttings per aquaria: April^ Columbus, OH, USA). Subsequently, they were burnt May 2006). Five thousand nauplii were supplied to (Carbolite furnace,450 1C) for 6 h and their inorganic each aquarium for each feeding episode, which lasted weight was determined. The organic weight of each 3 h, and was terminated by replacing the water. cutting was calculated by subtracting the inorganic from the dry weight, and the result was used to cal- Organic weight in ¢eld-collected colonies culate the percentage of organic weight of each cutting. The stubs were reused after burning (800 1C, To examine the percentage of organic weight in 4 h) for removal of fouled material. S. glaucum colonies of di¡erent sizes, samples were removed from colonies of three polypary diameters: 5^7,10^15 and 20^30 cm (n 5 6 for each size group: E¡ect of environmental parameters August 2006). Each sample constituted a longitudi- In order to determine the e¡ect of temperature on the nal section comprising ca. 10% of each colony, and cuttings of S. glaucum, they were reared at 20, 23, 26 included respective portions of the polypary and the and 29 1C, falling within the range representing Eilat stalk.Their dry weight, inorganic weight and percen- annual seawater temperatures Æ 1 1C (IUI Data Base, tage of organic weight were determined.

r 2010 The Authors 1750 Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,1748^1758 Aquaculture Research, 2010, 41, 1748^1758 Rearing cuttings of the soft coral S. glaucum I Sella & Y Benayahu

Comparison between cuttings reared in the (60 days, January 2007). Fifty cuttings from each set- reef, £ow-through seawater system and closed ting were then randomly retrieved and their dry, in- seawater system organic and percentage of organic weight were determined. Fifteen colonies of S. glaucum were collected from the IUI reef and placed in a £ow-through seawater system at the IUI for 3 days for acclimatization (Novem- Statistics ber 2006). Following this,320 mounted cuttings were produced, divided into three groups and reared in In each experiment, results from aquaria of the same three settings. The ¢rst setting consisted of 90 cut- treatment were tested using one-way ANOVA, and be- tings placed on a table-like structure (galvanized cause no signi¢cant di¡erences were found, the results steel, 220 Â 80 Â 80 cm) at the IUI reef (5 m depth). were pooled. Between treatments, the average dry The second setting consisted of 110 cuttings placed weight and percentage of organic weight were tested in three £ow-through seawater tanks at the IUI with using one-way ANOVA and post hoc Tukey test. Log and 20 complete water changes per 24 h (each tank arcsinus transformations were used to normalize dis- 80 Â 80 Â 40 cm, 35^38 cuttings per tank) and tribution when needed. Survival of the cuttings at the 2 shaded by a net reducing 40% sun radiation. For the end of each experiment was tested by w .The deviation third setting, 120 cuttings were placed in the TAU around means is presented by standard error. closed seawater system (six aquaria, 20 cuttings per aquaria). Based on the results obtained from the en- Results vironmental parameter experiments (see ‘Results’), the following conditions were chosen for the latter During the experiments, the miniature cuttings of setting: 26 1C, 250 mE and weekly feeding intervals S. glaucum regenerated and acquired the structure of with 5000 nauplii as well as Eilat salinity of 40ppt. naturally growing juvenile colonies with a mush- The cuttings were monitored weekly and their survi- room-shaped morphology (Fig. 1). Between days 5 voral was determined at the end of the experiment and 18 of the experiments, the rectangular cuttings

(a) (b)

Figure 1 Rearing cuttings of Sarcophyton glaucum in a closed seawater system: (a) cuttings of 36 mm2 attached to ceramic stubs by a cyanoacrylate adhesive, and placed in PVC test tube holders at day one of the experiment and (b) cuttings at day 60.

r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,1748^1758 1751 Rearing cuttings of the soft coral S. glaucum I Sella & Y Benayahu Aquaculture Research, 2010, 41, 1748–1758 became rounded and natural attachment occurred. Table 1 Survivorship of Sarcophyton glaucum cuttings in a A distinct stalk then developed and by days 30^37, closed seawater system under di¡erent environmental para- the colonies had attained the typical S. glaucum meters and in di¡erent settings on day 60 of the experi- shape. ments

Treatments Initial # of cuttings % survivorship on day 60

E¡ect of environmental parameters Environmental parameters Temperature ( 1C) There were signi¢cant di¡erences in the average dry 20 18 66 (12) weight and percentage of organic weight of S. glau- 23 35 65 (23) 26 42 88 (37) cum cuttings reared under the tested temperatures 29 35 88 (31) (Fig. 2, one-way ANOVA, Po0.05). The average dry Light (mE) weight of cuttings at 26 1C was signi¢cantly higher 20 65 62 (42) than those at 23, 20 and 29 1C(Tukeytest,Po0.05). 35 65 89 (58) Cuttings reared at 20 1C showed higher average per- 130 80 94 (75) 250 65 62 (42) centage of organic weight than those at 23, 26 and Salinity (ppt) 29 1C(Tukeytest,Po0.05), while no signi¢cant dif- 30 40 80 (32) ferences were noted among the other temperatures 34 42 78 (33) (P40.05). The survival of cuttings at the end of this 37 40 100 (40) experiment ranged between 65% and 88%. There 40 40 67 (27) Feeding intervals (days) were no signi¢cant di¡erences in the survival of the 2 60 25 (15) 2 cuttings among the tested temperatures (Table 1, w 7 43 44 (19) 0.05, d.f. 3, P40.05). 30 36 44 (16) Signi¢cant di¡erences in the average dry weight of Settings the cuttings were found among the di¡erent light Reef 90 96 (87) Flow-through 110 69 (76) Closed system 120 96 (116)

Number of cuttings on day 60 is given in parentheses. 0.018 (a) 0.016 0.014 0.012 intensities tested (Fig. 3a, one-way ANOVA, Po0.05). 0.01 Under the highest intensity of 250 mE, the dry weight 0.008 was signi¢cantly higher compared with all the other 0.006 treatments, and under 20 mE, it was the lowest (Tu- Dry weight (g) 0.004 key test, Po0.05). The average percentage of organic 0.002 weight signi¢cantly di¡ered among treatments (Fig. 0 3b, one-way ANOVA, Po0.05). Cuttings reared under 70 a light intensity of 130 mE had a signi¢cantly higher (b) 60 average percentage of organic weight than in all the 50 other treatments (Tukey, test Po0.05), while under 40 250 mE, it was the lowest (Tukey test, P40.05). The 30 survival of cuttings at the end of the experiment ran- 20 ged between 62% and 94% (Table 1). Under both the lowest (20 E) and the highest (250 E) light intensity % Organic weight 10 m m tested, a signi¢cantly lower survival was obtained 0 20 23 26 29 than under the intermediate intensities (35 and Temperature°C 130 mE) (w2 0.05, d.f. 4, Po0.05). Figure 2 Rearing cuttings of Sarcophyton glaucum in a No signi¢cant di¡erences were found in the aver- closed seawater system: (a) average dry weight (g) and (b) age dry weight (Fig.4a) and average percentage of or- average percentage of organic weight at di¡erent tempera- ganic weight (Fig. 4b) in cuttings reared under the tures on day 60 of the experiment (Æ SE, number of cut- di¡erent salinities (one-way ANOVA, P40.05). The sur- tings indicated in Table1). vival of cuttings at the end of this experiment ranged

r 2010 The Authors 1752 Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,1748^1758 Aquaculture Research, 2010, 41, 1748^1758 Rearing cuttings of the soft coral S. glaucum I Sella & Y Benayahu between 67% and 100%. There were no signi¢cant There were no signi¢cant di¡erences in the aver- di¡erences in the survival of the cuttings under the age dry weight of cuttings obtained from the di¡erent 2 di¡erent salinities (Table 1, w 0.05, d.f. 3, P40.05). feeding intervals (Fig. 5a, one-way ANOVA, P40.05), Natural attachment of the cuttings did not occur si- whereas the average percentage of their organic multaneously and was observed during days 5 and 6. weight did signi¢cantly di¡er among them (Fig. 5b, one-way ANOVA, Po0.05). Under the two-day feeding interval, there was a signi¢cantly lower average percentage of organic weight than under the 7- and 30- 0.03 (a) day intervals (Tukey test, P 0.05). There was a simi- 0.025 o lar average percentage of organic weight in cuttings 0.02 0.015 reared under the latter two intervals (one-way ANOVA, 0.01 P40.05). Survival of cuttings at the end of the experi-

Dry weight (g) 0.005 ment ranged between 25% (2-day feeding interval) 2 0 and 44% (7- and 30-day intervals) (Table 1, w 0.05, d.f. 3, P40.05) and was lower than that in all the 60 (b) other experiments (Table1). 50 40 30 20 Organic weight in ¢eld-collected colonies 10 % Organic weight 0 Signi¢cant di¡erences were found in the average per- 20 35 130 250 centage of organic weight among the S. glaucum co- µE m–2 s–1 lonies of di¡erent size groups (Fig. 6, one-way ANOVA, Figure 3 Rearing cuttings of Sarcophyton glaucum in a Po0.05). The smallest colonies had a lower percen- closed seawater system: (a) average dry weight (g) and (b) tage of organic weight compared with the larger ones average percentage of organic weight under di¡erent light (Tukey test, Po0.05). intensities on day 60 of the experiment (Æ SE, number of cuttings indicated in Table1).

0.025 (a) 0.016 (a) 0.014 0.02 0.012 0.015 0.01 0.008 0.01 0.006 0.004 Dry weight (g) 0.005 Dry weight (g) 0.002 0 0

70 (b) 70 (b) 60 60 50 50 40 40 30 30 20 20 % Organic weight

% Organic weight 10 10 0 0 30 34 37 40 2730 ppt Feeding intervals (days)

Figure 4 Rearing cuttings of Sarcophyton glaucum in a Figure 5 Rearing cuttings of Sarcophyton glaucum in a closed seawater system: (a) average dry weight (g) and (b) closed seawater system: (a) average dry weight (g) and (b) average percentage of organic weight under di¡erent sali- average percentage of organic weight under di¡erent feed- nities on day 60 of the experiment (Æ SE, number of cut- ing regimes on day 60 of the experiment (Æ SE, number of tings indicated in Table1). cuttings indicated in Table1).

r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,1748^1758 1753 Rearing cuttings of the soft coral S. glaucum I Sella & Y Benayahu Aquaculture Research, 2010, 41, 1748–1758

35 0.08 (a) 30 0.07 25 0.06 20 0.05 15 0.04 10 0.03 % Organic weight 5 Dry weight (g) 0.02 0 0.01 5-7 cm 10-15 cm 20-23 cm 0 Disc diameter 45 Figure 6 Samples removed from reef-collected Sarcophy- (b) 40 ton glaucum colonies of di¡erent size classes (disc dia- 35 meter) average percentage of organic weight (Æ SD, n 5 6 30 samples for each size group). 25 20 15 10 Comparison between cuttings reared in the % Organic weight 5 reef, £ow-through seawater system and closed 0 ﬂow-through system in the sea closed system seawater system Rearing settings

Signi¢cant di¡erences were found in the average dry Figure 7 Rearing of cuttings of Sarcophyton glaucum in a weight of S. glaucum cuttings among the three set- closed seawater system, £ow-through seawater system tings (Fig. 7a, one-way ANOVA, Po0.01). The average and in the sea (a) average dry weight (g) and (b) average dry weight of cuttings reared in the reef was the high- percentage of organic weight on day 60 of the experi- est, while that of those reared in the £ow-through ments (Æ SE, n 5 50 colonies for each group). seawater system was the lowest (Tukey test, Po0.01). The average percentage of organic weight 12-h period that took place during days14^16. In the of the cuttings signi¢cantly di¡ered among the three salinity experiment, natural attachment of the cut- settings (one-wayANOVA, Po0.01).Those reared in the tings already started on day 5 and ended on day 8. reef had a lower average percentage of organic Low water temperature (201^23 1C) during the ¢rst weight than in the other two systems (Fig. 7b,Tukey 4^5 days of the experiment minimized necrosis test, Po0.01). A similar average percentage of organ- among the cuttings. Foraging of the T. dentaus snails ic weight was obtained in cuttings reared in the £ow- larger than 1cm disturbed the freshly introduced through system and the closed system (P40.05). Sur- cuttings and prevented their attachment, and there- vival of cuttings at the end of this experiment ranged fore only small snails were used. Nonetheless, after between 69% and 96%. Both cuttings reared in the natural attachment of the cuttings had taken place, reef and in the closed seawater system had a signi¢- larger snails were successfully used. cantly higher survival rate than those reared in the Cuttings reared in the reef achieved a hemispheric £ow-through system (Table1, w2 0.05, d.f. 2, Po0.05). polypary, short polyps and were more rigid than those in the closed system. The cuttings reared in the closed system were characterized by a soft £at- General observations tened polypary, long polyps and an elongated stalk. Those reared in the £ow-through seawater system During the course of preliminary work, we noted developed both the above-mentioned morphologies, that cuttings derived from the periphery of the polyp- with some intermediate shapes. ary survived better than those taken from its inner part. Although the cuttings from both areas were similar in size (36mm2), the former had more polyps Discussion per unit area. We noted that cuttings that consisted of 45 polyps survived better than those with fewer The current study examined the practice and condi- polyps. It is interesting to note that in all the experi- tions required in order to develop a protocol for rearing ments, except the salinity one, natural attachment of miniature cuttings removed from colonies of S. glau- cuttings was synchronized among the aquaria over a cum in a closed seawater system. We compared the

r 2010 The Authors 1754 Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,1748^1758 Aquaculture Research, 2010, 41, 1748^1758 Rearing cuttings of the soft coral S. glaucum I Sella & Y Benayahu derived colonies with those reared in a £ow-through but the lowest percentage of organic weight (Fig. 3). seawater system, with those reared in the seas and These results further indicate the trade-o¡ between with ¢eld-collected colonies. The ability of these cut- calci¢cation rate and percentage of organic weight tings to successfully regenerate and form juvenile colo- of the cuttings. At a high light intensity, photosynth- nies was facilitated by examining the e¡ect of various esis increased and enhanced calci¢cation (e.g., Alle- environmental parameters on their development. mand, Tambutte, Girard & Jaubert 1998; Tentori & The ceramic stubs enabled the natural attachment Allemand 2006), yielding cuttings with higher dry of the cuttings and their further development (see weight and lower percentage of organic weight. Cut- Sella 2007). The durability of clay (ceramic) in the tings reared under intermediate light intensities (35^ sea is well known from archaeological ¢ndings (Hal- 130 mE) had considerably higher survival than those dane1993), and its malleability,low cost, tendency to reared under the extreme intensities. It can be be fouled and to support coral growth (see Baine assumed that photo-inhibition at the high light inten- 2001) made it suitable for the purpose of the present sity (see Smith, Suggett & Baker 2005) and photo- study. Ceramic can endure high temperatures and adaptation at the low intensity (see Muller-Parker & therefore can be sterilized and reused. The current D’Elia 1997) caused high mortality during the ¢rst 14 study is the ¢rst to use clay-made stubs for rearing days of the experiment, and that only those cuttings soft coral cuttings, and to suggest clay as a suitable that managed to adapt to these intensities survived. material for large-scale soft coral farming. We suggest that gradual light acclimatization should be performed in order to increase survival. Salinity did not a¡ect the survival, average dry weight or organic weight of the S. glaucum cuttings. E¡ect of environmental parameters This ¢nding is economically important, because salt Notably,there were signi¢cant di¡erences in the aver- is considered to constitute a signi¢cant expense for age dry weight and percentage of organic weight of S. coral farming in closed seawater systems. (Borne- glaucum cuttings reared under the di¡erent tempera- man & Lowrie 2001; Calfo 2003). Interestingly,in this tures (Fig. 2a). Cuttings reared at 26 1C showed the experiment, natural attachment of the cuttings was highest average dry weight while cuttings at 20 1C not synchronized and occurred earlier compared showed the highest percentage of organic weight with the other experiments (see ‘Results’). Notably, (Fig. 2b). Many studies have indicated that the calci¢- in the salinity experiment, the cuttings were reared cation rate in corals is enhanced with increased tem- in 30 L aquaria that were disconnected from the perature (Carricart-Ganivet 2004). Thus, it is 1m3 tanks. Therefore, we suggest that by maintain- suggested that the di¡erences in the average dry ing the cuttings in a small water volume, natural at- weight between treatments are a result of di¡erent tachment was achieved earlier than in the other calci¢cation rates. In addition, it seems that at low experiments of this study,possibly due to a particular temperature, there is some kind of a trade-o¡, as cal- substance released by the cuttings, whose greater ci¢cation decreased, and the percentage of organic concentration in the water was consequently more weight increased (see also Rodolfo-Metalpa, Richard, e¡ective. Benthic marine organisms such as mol- Allemand & Ferrier-Pages 2006). This ¢nding is valu- lusks, echinoderms and arthropods synchronized able for the farming of soft corals for natural product processes such as courtship and mating, aggregation purposes, which requires maximizing the yield of or- behaviour and habitat selection through the secre- ganic weight. We also noted that, at the low water tion of certain chemicals (Zimmer & Butman 2000). temperature, necrosis of the cuttings took place only Undoubtedly, future studies are still needed in order during the ¢rst sevendays of the experimentandwas to verify our suggestion, which has obvious advan- minimal compared with all the other experiments, tages for commercial coral farming. most probably due to inhibition of bacterial spread The cuttings of S. glaucum that were fed most fre- (Bourne, Lone,Webster, Swan & Hall 2006). It is sug- quently (every 2 days) had the lowest percentage of gested that the use of anti microbial methods, such as organic weight while those fed every 7 or 30 days an Ozone reactor or UV,may further decrease necro- had the highest one.The digestion process of A. salina sis in the system (Delbeek 2001). nauplii by Sarcophyton sp. may take up to 24 h (Lewis S. glaucum cuttings reared under the highest light 1982).We suggest that the time required for digestion intensity (250 mE) had a signi¢cantly higher dry may determine the rate of food capture. Furthermore, weight than at all the other light intensities tested, the low number of polyps of the developed small

r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,1748^1758 1755 Rearing cuttings of the soft coral S. glaucum I Sella & Y Benayahu Aquaculture Research, 2010, 41, 1748–1758 colonies limited their food-capturing abilities. There- Comparison between cuttings reared in the fore, frequent feeding does not necessarily bene¢t the reef environment, closed and £ow-through cuttings and may even harm them. The higher mor- seawater systems tality of cuttings in this experiment can be explained This comparison revealed that environmental condi- as a result of the introduction of microbial fauna as- tions determine the percentage of organic weight and sociated with theA. salina nauplii, and the decompo- morphology of S. glaucum colonies. Because the cut- sition of organic debris in the aquaria, as the water tings used for each rearing system were derived from change after each feeding episode did not completely the same parent colonies and were genetically identi- remove debris from the colonies. It is suggested that cal (see ‘Materials and methods’), those reared in the the use of decapsulating methods for the A. salina closed seawater system revealed a higher percentage cysts (Lim, Cho, Dhert, Wong, Nelis & Sorgeloos of organic weight than those in the reef, possessed 2002), with anti-microbial water ¢ltration as indi- long polyps, a long stalk and a £at polypary and dif- cated above, may decrease mortality. Venn, Loram fered from those in the reef. These ¢ndings provide and Douglas (2008) indicated that a large number of further evidence that the conditions in the closed soft corals possess a high density of zooxanthellae in rearing system can determine the particular features their tissues and obtain most of their energetic re- of the developed cuttings, as desired for the reef aqua- quirements from photosynthetic assimilates. It ria trade or for the extraction of natural products seems that plankton feeding plays only a partial role (large and robust colonies vs. high percentage of or- in the energy demands of the cuttings. Minimizing ganic weight). food quantityand optimizing its sterilitycan increase We noted that cuttings derived from the perimeter survival and development of the cuttings. This ¢nd- of the polypary survived better than those taken from ing has practical implications as live food is a costly its inner part (Sella 2007). Although all cuttings were component in aqua farming (Kyewalyanga 2003). of the same size, the former possessed more polyps per unit area. Oren, Benayahu and Loya (1997) found that lesions in the stony coral Favia favus with a rela- Organic weight in ¢eld-collected colonies tively long perimeter obtained a higher energetic allocation from the colony, probably due to the larger The percentage of organic weight in all three size colony portion associated with their recovery. In ad- groups of S. glaucum colonies was signi¢cantly lower dition, regeneration from injury in corals was found compared with those reared in the closed seawater to be faster when more tissue was available to supply system. Interestingly,the lowest percentage of organ- the damaged area (Henry & Hart 2005). It is sug- ic weight (10%) was found in the small-size group gested, therefore, that the cuttings from the periph- (5^7 cm polypary diameter), which was used for the ery of the colonies supplied the energy needed for preparationofthecuttings.Attheendofthedi¡erent the regeneration and growth of these cuttings, either experiments conducted in the closed system, the re- by photosynthesis of zooxanthellae harvested in the sulting colonies had three to four times more organic polyps (Muller-Parker & DiElia 1997) or by capture of weight (Fig. 6). Further studies are still needed in or- zooplankton (Lewis1982). der to determine the percentage of organic weight in colonies reared in a closed seawater system beyond the 60-day period studied by us. It is likely that the large reef colonies might possess more calcareous Conclusions sclerites, which are required to contend with conditions of strong water currents, wave action and pre- The results of the current study emphasize the ad- dation pressure, as was found for the octocorals vantages of a closed seawater system for rearing cut- Junceella fragilis (see Chang, Chi, Fan, & Dai 2007) tings of S. glaucum for various purposes. Our ¢ndings and Renilla muelleri (see Clavico, De Souza, Da Gama indicate those conditions of the rearing system that & Pereira 2007). We suggest that colonies of S. glau- will determine the desired particular features of the cum quickly adapt to a new suite of environmental developed cuttings, as required for purposes such as conditions by changing their relative organic vs. in- the reef aquaria trade and restoration or for the ex- organic weight. This ¢nding may also explain the traction of natural products (large and robust colo- wide distribution of this species in avarietyof reef ha- nies vs. colonies with a high percentage of organic bitats (Benayahu & Loya1986;Tanaka et al. 2005). weight).

r 2010 The Authors 1756 Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,1748^1758 Aquaculture Research, 2010, 41, 1748^1758 Rearing cuttings of the soft coral S. glaucum I Sella & Y Benayahu

Throughout the entire rearing period, water qual- roprotective compound from the soft coral Sarcophyton ity must be maintained, with the following nutrient glaucum. Toxicology 131,133^143. Baine M. (2001) Arti¢cial reefs: a review of their design, ap- concentration values : NO2 0^0.05ppm, NO3 0^ plication, management and performance. Ocean & Coastal 10 p p m, N H 3 0 ppm and under a pH of 8.2. During the ¢rst week, the cuttings should be maintained at Management 44, 241^259. Becker L. & Mueller E. (1999) The culture, transplantation, and 20 1C, light intensities of 35^130 mE (400^800 nm), storage of Montastraea faveolata, Acropora cervicornis, salinityof 30^37ppt and no feeding.Water circulation and A. palmata: what we learned so far. NCRI International in the rearing aquaria should be moderate in order to Conference on Scienti¢c Aspects of Coral Reef Assess- avoid detachment of the cuttings from the stubs. A ment, Monitoring, and Restoration, Ft. Lauderdale, FL, routine removal of tissue debris and decayed cuttings USA,53. from the aquaria during the ¢rst week is important in Benayahu Y. & Loya Y. (1986) Sexual reproduction of a soft order to reduce the spread of necrosis to other cut- coral ^ synchronous and brief annual spawning of Sarco- tings. After the ¢rst week, it is possible to adjust the phyton glaucum (Quoy and Gaimard,1833). Biological Bul- rearing conditions for the di¡erent purposes. For the letin170,32^42. aquarium trade and restoration purposes, cuttings Blunt J.W., Copp B.R., Munro M.H.G., Northcote P.T. & Prin- sep M.R. (2005) Marine natural products. Natural Product should be maintained at 26^29 1C, a light intensity Reports 22,15^61. of 250 mE and salinity of 30^37ppt. Weekly feeding Borneman E.H. & Lowrie J. (2001) Advances in captive hus- by A. salina nauplii should be conducted. 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r 2010 The Authors 1758 Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,1748^1758