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Rearing cuttings of the soft coral Sarcophyton glaucum

(Octocorallia, Alcyonacea): towards mass production

in a closed seawater system

Ido Sella & Yehuda BenayahuDepartment 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

The octcoral Sarcophyton glaucum has a wide Indo-Paci¢c distribution and is known for its diverse con-tent of natural products.The aimof the current studywas to establish a protocol for rearing miniature cut-tings of S. glaucum in a closed seawater system. In or-der to determine the optimal conditions for rearing,the survival, average dry weight, percentage of or-ganic weight and development of the cuttings weremonitored under di¡erent temperature, light, salinityand feeding regimes. At 26 1C, the highest dry weightwas obtained, and at 20 1C, the highest percentage oforganic weight. The dry weight of the cuttings in-creased with the light intensity, while under 35^130 mE m�2 s�1, survival was high. Salinity did nota¡ect any of the colonies’ features. Feeding intervalsof 7 and 30 days yielded a better result than of 2 days.A comparison of the colonies derived from the closedsystem with the colonies reared in a £ow-throughsystem, those reared in the sea and with ¢eld-col-lected colonies revealed the importance of environ-mental conditions in determining the features of thecolonies. The study emphasizes the advantages of aclosed seawater system in controlling the conditionsneeded for rearing cuttings of S. glaucum for targetedfarming.

Keywords: soft corals, closed seawater system,Sarcophyton glaucum, coral farming, Red-Sea

Introduction

A large variety of reef invertebrates, including softcorals (Octocorallia), have long been used as a source

for diverse natural products with pharmaceutical orcosmetic value (e.g., Blunt, Copp, Munro, Northcote &Prinsep 2005; Slattery, Gochfeld & Kamel 2005; Sip-kema, Osinga, Schatton, Mendola,Tramper &Wij¡els2005), as well as for the reef-aquarium trade (Wab-nitz,Taylor, Grenn & Razak 2003). The increased de-mand for these organisms has led to their massiveharvesting (Castanaro & Lasker 2003) and has raisedthe need for e⁄cient farming methodologies (Ellis &Ellis 2002; Mendola 2003).Coral propagation has been commonly used for the

productionof daughter colonies, rather than harvest-ing naturally grown ones (e.g., Soong & Chen 2003;Fox, Mous, Pet, Muljadi & Caldwell 2005). This prac-tice has been based on the ability of corals to repro-duce asexually (e.g, Delbeek 2001; Ellis & Ellis 2002).Captive-grown colonies, or ¢eld-collected ones, areused for the production of cuttings or fragments. Thelatter are usually glued or attached by various meansto bases or stubs, until natural attachment isachieved (Delbeek 2001).Over the last decade, coral propagation in closed

arti¢cial seawater systems has been commonly used,making the process more simple and cost e¡ective(Borneman & Lowrie 2001; http://www.reefkeeping.com). The advantage of these systems lies in theirability to control abiotic and biotic parameters(Wheeler 1996; Borneman & Lowrie 2001; Abramo-vitch-Gottlib, Katoshevski & Vago 2002). Such sys-tems have been proposed as a source by which toobtain corals and other invertebrates with commer-cial value (e.g., Wabnitz et al. 2003; Sipkema et al.2005), as well as for restoration and conservationpurposes (Becker & Mueller 1999; Petersen, Laterv-eer, Van Bergen, Hatta, Hebbinghaus, Janse, Jones,

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

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Richter, Ziegler,Visser & Schuhmacher 2006; Okuza-wa, Maliao, Quinitio, Buen-ursua, Lebata, Gallardo,Garcia & Primavera 2008).Species of the soft coral genus Sarcophyton (Octo-

corallia) have awide Indo-Paci¢c distributionand areknown for the diverse content of their natural pro-ducts (e.g., Look, Fenical, Matsumoto & Clardy 1986;Tanaka, Yoshida & Benayahu 2005; Nguyen, Tran,Phan, Chau, Eun & Young 2008). S. glaucum (Quoy &Gaimard, 1833) is the most common species of thegenus, also on the northern Red Sea reefs, where itwas found to be a dioecious broadcaster with the on-set of reproduction at the age of 6^10 years (Benaya-hu & Loya 1986). S. glaucum is zooxanthellate andextremely rich in natural products, which have beenstudied extensively (e.g., Fridkovsky, Rudi, Benayahu,Kashman & Schleyer1996; Badria, Guirguis, Perovic,Ste¡en, Muller & Schroder1998;Tanaka et al. 2005).The aim of the current study was to establish the

scienti¢c ground for a protocol suitable for rearingminiature cuttings of S. glaucum in a closed seawatersystem. Our objectives were to study the e¡ect of tem-perature, light intensity, salinity and feeding regimeson the survival, average dry weight, percentage of or-ganicweight and development of the cuttings. Inaddi-tion, colonies reared in a closed seawater systemwerecompared with those reared in a £ow-through sea-water system in the sea and with ¢eld-collected ones.

Materials and methods

In a preliminary work, we examined attachment ofthe S. glaucum cuttings to stubs made of ceramic andplastic materials, using a cyanoacrylate adhesive(3M) and rubber bands.We also examined the mini-mal size of cuttings yielding the highest survivorship(10^400mm2) (Sella 2007). Consequently, in the cur-rent study, we decided to use cuttings measuring36mm2 (6 � 6mm) with an average dry weight of0.0077 � 0.0024 g and 8.864 � 2.461 average per-centage of organic weight (n530 random cuttingsfrom colonies used in the experiments; see‘Collectionof colonies, preparation of cuttings and experimentalsetup’). The cuttings were glued using a cyanoacry-late adhesive to cylindrical ceramic (Fig. 1a) stubsthat we produced ourselves (clayWBB Fuchfs GmbH& Co R2502, burnt at 1200 1C for 12 h) at a cost of�4 US cents (without labour). The stubs measured5 cm in length and1cm in diameter, with a hexago-nal indentation (ca.3mm deep and 7^8mmwide) atone end, into which the cuttings were attached.

Experimental closed seawater system

We constructed a closed seawater system at Tel AvivUniversity (TAU). The system comprised three 1m3

PVC tanks, each ¢lled with arti¢cial seawater (RedSea saltr) and 200 kg of live rocks obtained from Ei-lat (Gulf of Aqaba, northern Red Sea). Each tank wasprovided with 40 Trochos dentatus snails for algalgrazing. Every 10 days, their faeces were removedfrom the bottom of the tanks, together with ca.5% ofthe water volume, which was then replaced. A set of24 glass aquaria (30 L each) was connected to thetanks by 26-mm-diameter pipes. The aquariawere il-luminated by neon bulbs (22W, 400^800 nm, JEBOdaylight,12 L:12 D,35 mE m�2 s�1on the colony sur-face). The water circulated from the aquaria to thetanks, which were equipped with protein skimmers(JEBO 3500), a chiller (JEBO 2000, Zhongshan Zhen-hua Aquarium Accessories, Zhongshan, China) anda 5000 L per hour circulation pump (JEBO). Thewater temperature in each aquarium was controlledusing an individual heater (60W). Salinity, tempera-ture and pH were monitored daily, and the nutrientlevels weekly (nitrite o0.05 ppm, nitrate o10 ppm,ammonia 0 ppm and 8.1^8.3 pH) (see Delbeek 2001).

Collection of colonies, preparation of cuttingsand experimental set-up

Colonies of S. glaucumwith a polypary diameter of 5^7 cm (the polyp-bearing part of the colony) were col-lected by SCUBA from three reef sites in Eilat (5^7m,August 2004^January 2007). For each experiment,we collected 10^15 colonies, growing at least 5mapart in order to avoid the use of asexually producedcolonies (Benayahu & Loya1986). In order to preventpossible bias in the study, only colonies that did notcontain gonads were used for formation of the cut-tings (see Okubo, Motokawa & Omori 2007). At theInteruniversity Institute for Marine Science (IUI),the colonies were placed in a £ow-through seawatersystem for 3 days and all epibiotic organisms were re-moved.The colonies were £own toTAU in plastic con-tainers ¢lled with seawater and placed in thermallyisolated boxes. They were then maintained in the ex-perimental system for a 1-month quarantine period,under conditions resembling the average ambient Ei-lat ones: salinity 40 ppt, pH 8.2 and 26 1C (IUI DataBase, http://www.iui-eilat.ac.il/NMP/Default.aspx).For preparation of the cuttings, the colonies of

S. glaucum were handled with latex gloves. The peri-meter of the polypary was removed using a scalpel

Aquaculture Research, 2010, 41, 1748^1758 Rearing cuttings of the soft coral S. glaucum I Sella & YBenayahu

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(see section ‘General observations’ ahead), and theremaining part was then kept for future regeneration(Sella 2007). The perimeter of the polypary was thor-oughly rinsed with arti¢cial seawater for removal ofmucus and cellular debris, and 36mm2 (6 � 6mm)cuttings were prepared using autoclaved scissors,scalpels and forceps and paper blotted. An adhesivewas applied to the cuttings’ lower epidermal surface,and they were mounted on the stubs and kept in theair for 30 s to dry. Notably, the cuttings retrieved forthe di¡erent treatments of each experiment wererandomly derived from the same parent colonies andtherefore represented similar genotypes. The stubswere then inserted into test tube stands (Fig.1a), andkept in arti¢cial seawater for 20min in order to washaway any adhesive residue before introduction intothe aquaria. During the following week, if mortalitydid not exceed15%, dead cuttings were replacedwithnew ones and only then the 60-day experimentswere started; otherwise, all cuttings were discardedand a new experiment was set up as it occurred once.In all the experiments, each treatment comprisedthree aquaria and a control, which was maintainedunder Eilat average environmental conditions. Un-less stated otherwise, the aquaria were suppliedevery 25 days with 5000 nauplii of Artemia salina(Sorgeloos & Persoone1975; Sella 2007).Development and transformation of the cuttings

into small colonies with a stalk and a polypary wasmonitored weekly by digital photography and theirsurvival was calculated at the end of the experiments(day 60). The cuttings were then removed with a scal-pel from the stubs and placed individually in alumi-nium dishes (5 cm in diameter). Their dry weight wasdetermined bydrying (Binder oven,60 1C) for 24 handthen weighing to an accuracy of four decimal points(Mettler balance, Toledo AB54-S, Mettler-Toledo,Columbus, OH, USA). Subsequently, they were burnt(Carbolite furnace,450 1C) for 6 hand their inorganicweight was determined. The organic weight of eachcutting was calculated by subtracting the inorganicfrom the dry weight, and the result was used to cal-culate the percentage of organic weight of each cut-ting. The stubs were reused after burning (800 1C,4 h) for removal of fouled material.

E¡ect of environmental parameters

In order to determine the e¡ect of temperature on thecuttings of S. glaucum, they were reared at 20, 23, 26and 29 1C, falling within the range representing Eilatannual seawater temperatures � 1 1C (IUI Data Base,

http://www.iui-eilat.ac.il) (10^15 cuttings per aquaria:March^May 2005). Seawater at 20 1C was circulatedfrom the tanks to all the aquaria. The 23, 26 and 29 1Cseawater temperatureswere obtainedusing 60^300Wheaters with thermostats. The temperature was routi-nely monitored using an alcohol thermometer.To determine the e¡ect of illumination on the cut-

tings, they were reared at light intensities of 20, 35,130 and 250 mE (mE m�2 s�1), as measured on theirsurface, falling within the prevailing Eilat valuesalong the depth gradient (Winters, Loya & Beer2006) (20^30 cuttings per aquaria: December 2005^January 2006). In order to supply 20 mE, a PVC ¢lter(PLASON UV1) was placed between the light sourceand the surface of the respective aquaria, while for130 and 250 mE, one or two additional neon bulbswere added. Light intensity was monitored dailyusing anunderwater-light photometer (Bio-sciences,Light 250L Li-CO).To determine the e¡ect of salinity on the cuttings,

the aquaria were disconnected from the tanks. Thecuttings were reared under salinities of 30,34,37 and40 ppt, representing the salinitywithin the geographicdistribution of the species (Levitus 1982) (15^25 cut-tings per aquaria: February^March, 2006). Each aqua-rium had a 60W heater with a thermostat, a ¢lter(JEBO Eco) and an air stone.Water from the1m3 tanks(40 ppt) was diluted with distilled water in order to ob-tain the lower salinities. Ten litres of seawater waschanged daily in each aquarium and the salinity wasmonitored using a light refractometer and an electro-nic conductivity meter (MRC Salinity 1, Lutron YK-315A, Lutron Electronic Enterprise,Taipei,Taiwan).To determine the e¡ect of feeding, cuttingswere fed

at intervals of 2, 7 and 30 days, where a 2-day inter-val is commonly used by aquarists (http://www.reefkeeping.com) (12^20 cuttings per aquaria: April^May 2006). Five thousand nauplii were supplied toeachaquarium for each feeding episode, which lasted3 h, and was terminated by replacing the water.

Organic weight in ¢eld-collected colonies

To examine the percentage of organic weight inS. glaucum colonies of di¡erent sizes, samples wereremoved from colonies of three polypary diameters:5^7,10^15 and 20^30 cm (n56 for each size group:August 2006). Each sample constituted a longitudi-nal section comprising ca. 10% of each colony, andincluded respective portions of the polypary and thestalk.Their dry weight, inorganicweight and percen-tage of organic weight were determined.

Rearing cuttings of the soft coral S. glaucum I Sella & Y Benayahu Aquaculture Research, 2010, 41, 1748–1758

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Comparison between cuttings reared in thereef, £ow-through seawater system and closedseawater system

Fifteen colonies of S. glaucumwere collected from theIUI reef and placed in a £ow-through seawater sys-tem at the IUI for 3 days for acclimatization (Novem-ber 2006). Following this,320mounted cuttings wereproduced, divided into three groups and reared inthree settings. The ¢rst setting consisted of 90 cut-tings placed on a table-like structure (galvanizedsteel, 220 � 80 � 80 cm) at the IUI reef (5m depth).The second setting consisted of 110 cuttings placedin three £ow-through seawater tanks at the IUI with20 complete water changes per 24 h (each tank80 � 80 � 40 cm, 35^38 cuttings per tank) andshaded by a net reducing 40% sun radiation. For thethird setting, 120 cuttings were placed in the TAUclosed seawater system (six aquaria, 20 cuttings peraquaria). Based on the results obtained from the en-vironmental parameter experiments (see ‘Results’),the following conditions were chosen for the lattersetting: 26 1C, 250 mE and weekly feeding intervalswith 5000 nauplii as well as Eilat salinity of 40 ppt.The cuttings were monitored weekly and their survi-voral was determined at the end of the experiment

(60 days, January 2007). Fifty cuttings from each set-ting were then randomly retrieved and their dry, in-organic and percentage of organic weight weredetermined.

Statistics

In each experiment, results from aquaria of the sametreatment were tested using one-way ANOVA, and be-cause no signi¢cant di¡erenceswere found, the resultswere pooled. Between treatments, the average dryweight and percentage of organic weight were testedusing one-wayANOVA and post hocTukey test. Log andarcsinus transformations were used to normalize dis-tributionwhen needed. Survival of the cuttings at theend of each experimentwas tested by w2.The deviationaroundmeans is presented by standard error.

Results

During the experiments, the miniature cuttings ofS. glaucum regenerated and acquired the structure ofnaturally growing juvenile colonies with a mush-room-shaped morphology (Fig. 1). Between days 5and 18 of the experiments, the rectangular cuttings

(b)(a)

Figure 1 Rearing cuttings of Sarcophyton glaucum in a closed seawater system: (a) cuttings of 36mm2 attached to cera-mic stubs bya cyanoacrylate adhesive, and placed in PVC test tube holders at dayone of the experiment and (b) cuttings atday 60.



became rounded and natural attachment occurred.A distinct stalk then developed and by days 30^37,the colonies had attained the typical S. glaucumshape.


There were signi¢cant di¡erences in the average dryweight and percentage of organic weight of S. glau-cum cuttings reared under the tested temperatures(Fig. 2, one-way ANOVA, Po0.05). The average dryweight of cuttings at 26 1C was signi¢cantly higherthan those at 23, 20 and 29 1C (Tukey test, Po0.05).Cuttings reared at 20 1C showed higher average per-centage of organic weight than those at 23, 26 and29 1C (Tukey test, Po0.05), while no signi¢cant dif-ferences were noted among the other temperatures(P40.05). The survival of cuttings at the end of thisexperiment ranged between 65% and 88%. Therewere no signi¢cant di¡erences in the survival of thecuttings among the tested temperatures (Table 1, w2

0.05, d.f.3, P40.05).Signi¢cant di¡erences in the average dry weight of

the cuttings were found among the di¡erent light

intensities tested (Fig. 3a, one-way ANOVA, Po0.05).Under the highest intensity of 250 mE, the dry weightwas signi¢cantly higher compared with all the othertreatments, and under 20 mE, it was the lowest (Tu-key test, Po0.05). The average percentage of organicweight signi¢cantly di¡ered among treatments (Fig.3b, one-way ANOVA, Po0.05). Cuttings reared undera light intensity of 130 mE had a signi¢cantly higheraverage percentage of organic weight than in all theother treatments (Tukey, test Po0.05), while under250 mE, it was the lowest (Tukey test, P40.05). Thesurvival of cuttings at the end of the experiment ran-ged between 62% and 94% (Table 1). Under both thelowest (20 mE) and the highest (250 mE) light intensitytested, a signi¢cantly lower survival was obtainedthan under the intermediate intensities (35 and130 mE) (w20.05, d.f. 4, Po0.05).No signi¢cant di¡erences were found in the aver-

age dry weight (Fig.4a) and average percentage of or-ganic weight (Fig. 4b) in cuttings reared under thedi¡erent salinities (one-wayANOVA, P40.05). The sur-vival of cuttings at the end of this experiment ranged

0

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0.018 (a)

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Figure 2 Rearing cuttings of Sarcophyton glaucum in aclosed seawater system: (a) average dry weight (g) and (b)average percentage of organicweight at di¡erent tempera-tures on day 60 of the experiment (� SE, number of cut-tings indicated inTable1).

Table 1 Survivorship of Sarcophyton glaucum cuttings in aclosed seawater system under di¡erent environmental para-meters and in di¡erent settings on day 60 of the experi-ments

Treatments Initial # of cuttings % survivorship on day 60

Environmental parameters

Temperature ( 1C)

20 18 66 (12)

23 35 65 (23)

26 42 88 (37)

29 35 88 (31)

Light (mE)

20 65 62 (42)

35 65 89 (58)

130 80 94 (75)

250 65 62 (42)

Salinity (ppt)

30 40 80 (32)

34 42 78 (33)

37 40 100 (40)

40 40 67 (27)

Feeding intervals (days)

2 60 25 (15)

7 43 44 (19)

30 36 44 (16)

Settings

Reef 90 96 (87)

Flow-through 110 69 (76)

Closed system 120 96 (116)

Number of cuttings on day 60 is given in parentheses.



between 67% and 100%. There were no signi¢cantdi¡erences in the survival of the cuttings under thedi¡erent salinities (Table 1, w2 0.05, d.f. 3, P40.05).Natural attachment of the cuttings did not occur si-multaneouslyandwas observed during days 5 and 6.

There were no signi¢cant di¡erences in the aver-age dry weight of cuttings obtained from the di¡erentfeeding intervals (Fig. 5a, one-way ANOVA, P40.05),whereas the average percentage of their organicweight did signi¢cantly di¡er among them (Fig. 5b,one-way ANOVA, Po0.05). Under the two-day feedinginterval, there was a signi¢cantly lower average per-centage of organic weight than under the 7- and 30-day intervals (Tukey test, Po0.05). There was a simi-lar average percentage of organic weight in cuttingsreared under the latter two intervals (one-wayANOVA,P40.05). Survival of cuttings at the end of the experi-ment ranged between 25% (2-day feeding interval)and 44% (7- and 30-day intervals) (Table 1, w2 0.05,d.f. 3, P40.05) and was lower than that in all theother experiments (Table1).


Signi¢cant di¡erences were found in the average per-centage of organic weight among the S. glaucum co-lonies of di¡erent size groups (Fig. 6, one-way ANOVA,Po0.05). The smallest colonies had a lower percen-tage of organicweight compared with the larger ones(Tukey test, Po0.05).

00.0050.01

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Figure 3 Rearing cuttings of Sarcophyton glaucum in aclosed seawater system: (a) average dry weight (g) and (b)average percentage of organicweight under di¡erent lightintensities on day 60 of the experiment (� SE, number ofcuttings indicated inTable1).

00.0020.0040.0060.0080.01

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010203040506070

30 34 37 40ppt

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Figure 4 Rearing cuttings of Sarcophyton glaucum in aclosed seawater system: (a) average dry weight (g) and (b)average percentage of organic weight under di¡erent sali-nities on day 60 of the experiment (� SE, number of cut-tings indicated inTable1).

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Figure 5 Rearing cuttings of Sarcophyton glaucum in aclosed seawater system: (a) average dry weight (g) and (b)average percentage of organicweight under di¡erent feed-ing regimes on day 60 of the experiment (� SE, number ofcuttings indicated inTable1).



Comparison between cuttings reared in thereef, £ow-through seawater system and closedseawater system

Signi¢cant di¡erences were found in the average dryweight of S. glaucum cuttings among the three set-tings (Fig. 7a, one-way ANOVA, Po0.01). The averagedryweight of cuttings reared in the reef was the high-est, while that of those reared in the £ow-throughseawater system was the lowest (Tukey test,Po0.01). The average percentage of organic weightof the cuttings signi¢cantly di¡ered among the threesettings (one-wayANOVA, Po0.01).Those reared in thereef had a lower average percentage of organicweight than in the other two systems (Fig.7b,Tukeytest, Po0.01). A similar average percentage of organ-ic weight was obtained in cuttings reared in the £ow-through systemand the closed system (P40.05). Sur-vival of cuttings at the end of this experiment rangedbetween 69% and 96%. Both cuttings reared in thereef and in the closed seawater system had a signi¢-cantly higher survival rate than those reared in the£ow-through system (Table1, w20.05, d.f. 2, Po0.05).

General observations

During the course of preliminary work, we notedthat cuttings derived from the peripheryof the polyp-ary survived better than those taken from its innerpart. Although the cuttings from both areas were si-milar in size (36mm2), the former had more polypsper unit area.We noted that cuttings that consistedof 45 polyps survived better than those with fewerpolyps. It is interesting to note that in all the experi-ments, except the salinity one, natural attachment ofcuttings was synchronized among the aquaria over a

12-h period that took place during days14^16. In thesalinity experiment, natural attachment of the cut-tings already started on day 5 and ended on day 8.Low water temperature (201^23 1C) during the ¢rst4^5 days of the experiment minimized necrosisamong the cuttings. Foraging of theT. dentaus snailslarger than 1cm disturbed the freshly introducedcuttings and prevented their attachment, and there-fore only small snails were used. Nonetheless, afternatural attachment of the cuttings had taken place,larger snails were successfully used.Cuttings reared in the reef achieved a hemispheric

polypary, short polyps and were more rigid thanthose in the closed system. The cuttings reared inthe closed system were characterized by a soft £at-tened polypary, long polyps and an elongated stalk.Those reared in the £ow-through seawater systemdeveloped both the above-mentioned morphologies,with some intermediate shapes.

Discussion

The current study examined the practice and condi-tions required in order to develop a protocol for rearingminiature cuttings removed from colonies of S. glau-cum in a closed seawater system. We compared the

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Figure 6 Samples removed from reef-collected Sarcophy-ton glaucum colonies of di¡erent size classes (disc dia-meter) average percentage of organic weight (� SD, n56samples for each size group).

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1015202530354045

flow-through system in the sea closed system

Rearing settings

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Figure 7 Rearing of cuttings of Sarcophyton glaucum in aclosed seawater system, £ow-through seawater systemand in the sea (a) average dry weight (g) and (b) averagepercentage of organic weight on day 60 of the experi-ments (� SE, n550 colonies for each group).



derived colonies with those reared in a £ow-throughseawater system, with those reared in the seas andwith ¢eld-collected colonies. The ability of these cut-tings to successfully regenerate and form juvenile colo-nies was facilitated by examining the e¡ect of variousenvironmental parameters on their development.The ceramic stubs enabled the natural attachment

of the cuttings and their further development (seeSella 2007). The durability of clay (ceramic) in thesea is well known from archaeological ¢ndings (Hal-dane1993), and its malleability, low cost, tendency tobe fouled and to support coral growth (see Baine2001) made it suitable for the purpose of the presentstudy. Ceramic can endure high temperatures andtherefore can be sterilized and reused. The currentstudy is the ¢rst to use clay-made stubs for rearingsoft coral cuttings, and to suggest clay as a suitablematerial for large-scale soft coral farming.


Notably, therewere signi¢cant di¡erences in the aver-age dry weight and percentage of organicweight of S.glaucum cuttings reared under the di¡erent tempera-tures (Fig. 2a). Cuttings reared at 26 1C showed thehighest average dry weight while cuttings at 20 1Cshowed the highest percentage of organic weight(Fig. 2b). Many studies have indicated that the calci¢-cation rate in corals is enhanced with increased tem-perature (Carricart-Ganivet 2004). Thus, it issuggested that the di¡erences in the average dryweight between treatments are a result of di¡erentcalci¢cation rates. In addition, it seems that at lowtemperature, there is some kind of a trade-o¡, as cal-ci¢cation decreased, and the percentage of organicweight increased (see also Rodolfo-Metalpa, Richard,Allemand & Ferrier-Pages 2006). This ¢nding is valu-able for the farming of soft corals for natural productpurposes, which requires maximizing the yield of or-ganic weight. We also noted that, at the low watertemperature, necrosis of the cuttings took place onlyduring the ¢rst seven days of the experiment andwasminimal compared with all the other experiments,most probably due to inhibition of bacterial spread(Bourne, Lone,Webster, Swan & Hall 2006). It is sug-gested that the use of anti microbial methods, suchasan Ozone reactor or UV, may further decrease necro-sis in the system (Delbeek 2001).S. glaucum cuttings reared under the highest light

intensity (250 mE) had a signi¢cantly higher dryweight than at all the other light intensities tested,

but the lowest percentage of organic weight (Fig. 3).These results further indicate the trade-o¡ betweencalci¢cation rate and percentage of organic weightof the cuttings. At a high light intensity, photosynth-esis increased and enhanced calci¢cation (e.g., Alle-mand, Tambutte, Girard & Jaubert 1998; Tentori &Allemand 2006), yielding cuttings with higher dryweight and lower percentage of organic weight. Cut-tings reared under intermediate light intensities (35^130 mE) had considerably higher survival than thosereared under the extreme intensities. It can beassumed that photo-inhibition at the high light inten-sity (see Smith, Suggett & Baker 2005) and photo-adaptation at the low intensity (see Muller-Parker &D’Elia 1997) caused high mortality during the ¢rst 14days of the experiment, and that only those cuttingsthat managed to adapt to these intensities survived.We suggest that gradual light acclimatization shouldbe performed in order to increase survival.Salinity did not a¡ect the survival, average dry

weight or organic weight of the S. glaucum cuttings.This ¢nding is economically important, because saltis considered to constitute a signi¢cant expense forcoral farming in closed seawater systems. (Borne-man & Lowrie 2001; Calfo 2003). Interestingly, in thisexperiment, natural attachment of the cuttings wasnot synchronized and occurred earlier comparedwith the other experiments (see ‘Results’). Notably,in the salinity experiment, the cuttings were rearedin 30 L aquaria that were disconnected from the1m3 tanks. Therefore, we suggest that by maintain-ing the cuttings in a small water volume, natural at-tachment was achieved earlier than in the otherexperiments of this study, possibly due to a particularsubstance released by the cuttings, whose greaterconcentration in the water was consequently moree¡ective. Benthic marine organisms such as mol-lusks, echinoderms and arthropods synchronizedprocesses such as courtship and mating, aggregationbehaviour and habitat selection through the secre-tion of certain chemicals (Zimmer & Butman 2000).Undoubtedly, future studies are still needed in orderto verify our suggestion, which has obvious advan-tages for commercial coral farming.The cuttings of S. glaucum that were fed most fre-

quently (every 2 days) had the lowest percentage oforganic weight while those fed every 7 or 30 dayshad the highest one.The digestionprocess of A. salinanauplii by Sarcophyton sp. may take up to 24 h (Lewis1982).We suggest that the time required for digestionmaydetermine the rate of food capture. Furthermore,the low number of polyps of the developed small



colonies limited their food-capturing abilities. There-fore, frequent feeding does not necessarily bene¢t thecuttings and may even harm them. The higher mor-tality of cuttings in this experiment can be explainedas a result of the introduction of microbial fauna as-sociated with theA. salina nauplii, and the decompo-sition of organic debris in the aquaria, as the waterchange after each feeding episode did not completelyremove debris from the colonies. It is suggested thatthe use of decapsulating methods for the A. salinacysts (Lim, Cho, Dhert, Wong, Nelis & Sorgeloos2002), with anti-microbial water ¢ltration as indi-cated above, may decrease mortality. Venn, Loramand Douglas (2008) indicated that a large number ofsoft corals possess a high density of zooxanthellae intheir tissues and obtain most of their energetic re-quirements from photosynthetic assimilates. Itseems that plankton feeding plays only a partial rolein the energy demands of the cuttings. Minimizingfood quantityand optimizing its sterilitycan increasesurvival and development of the cuttings. This ¢nd-ing has practical implications as live food is a costlycomponent in aqua farming (Kyewalyanga 2003).


The percentage of organic weight in all three sizegroups of S. glaucum colonies was signi¢cantly lowercompared with those reared in the closed seawatersystem. Interestingly, the lowest percentage of organ-ic weight (10%) was found in the small-size group(5^7 cm polypary diameter), which was used for thepreparation of the cuttings. At the end of the di¡erentexperiments conducted in the closed system, the re-sulting colonies had three to four times more organicweight (Fig. 6). Further studies are still needed in or-der to determine the percentage of organic weight incolonies reared in a closed seawater system beyondthe 60-day period studied by us. It is likely that thelarge reef colonies might possess more calcareoussclerites, which are required to contend with condi-tions of strong water currents, wave action and pre-dation pressure, as was found for the octocoralsJunceella fragilis (see Chang, Chi, Fan, & Dai 2007)and Renilla muelleri (see Clavico, De Souza, Da Gama& Pereira 2007).We suggest that colonies of S. glau-cum quickly adapt to a new suite of environmentalconditions by changing their relative organic vs. in-organic weight. This ¢nding may also explain thewide distributionof this species inavarietyof reef ha-bitats (Benayahu & Loya1986;Tanaka et al. 2005).

Comparison between cuttings reared in thereef environment, closed and £ow-throughseawater systems

This comparison revealed that environmental condi-tions determine the percentage of organicweight andmorphology of S. glaucum colonies. Because the cut-tings used for each rearing systemwere derived fromthe same parent colonies andwere genetically identi-cal (see ‘Materials and methods’), those reared in theclosed seawater system revealed a higher percentageof organic weight than those in the reef, possessedlong polyps, a long stalk and a £at polypary and dif-fered from those in the reef. These ¢ndings providefurther evidence that the conditions in the closedrearing system can determine the particular featuresof the developed cuttings, as desired for the reef aqua-ria trade or for the extraction of natural products(large and robust colonies vs. high percentage of or-ganic weight).We noted that cuttings derived from the perimeter

of the polypary survived better than those taken fromits inner part (Sella 2007). Althoughall cuttings wereof the same size, the former possessed more polypsper unit area. Oren, Benayahu and Loya (1997) foundthat lesions in the stony coral Favia favus with a rela-tively long perimeter obtained a higher energetic al-location from the colony, probably due to the largercolony portion associated with their recovery. In ad-dition, regeneration from injury in corals was foundto be faster when more tissue was available to supplythe damaged area (Henry & Hart 2005). It is sug-gested, therefore, that the cuttings from the periph-ery of the colonies supplied the energy needed forthe regeneration and growth of these cuttings, eitherby photosynthesis of zooxanthellae harvested in thepolyps (Muller-Parker & DiElia1997) or by capture ofzooplankton (Lewis1982).

Conclusions

The results of the current study emphasize the ad-vantages of a closed seawater system for rearing cut-tings of S. glaucum for various purposes. Our ¢ndingsindicate those conditions of the rearing system thatwill determine the desired particular features of thedeveloped cuttings, as required for purposes such asthe reef aquaria trade and restoration or for the ex-traction of natural products (large and robust colo-nies vs. colonies with a high percentage of organicweight).



Throughout the entire rearing period, water qual-ity must be maintained, with the following nutrientconcentration values : NO2 0^0.05 ppm, NO3 0^10 ppm, NH3 0 ppm and under a pH of 8.2. Duringthe ¢rst week, the cuttings should be maintained at20 1C, light intensities of 35^130 mE (400^800 nm),salinityof 30^37 ppt and no feeding.Water circulationin the rearing aquaria should be moderate in order toavoid detachment of the cuttings from the stubs. Aroutine removal of tissue debris and decayed cuttingsfrom the aquaria during the ¢rst week is important inorder to reduce the spread of necrosis to other cut-tings. After the ¢rst week, it is possible to adjust therearing conditions for the di¡erent purposes. For theaquarium trade and restoration purposes, cuttingsshould be maintained at 26^29 1C, a light intensityof 250 mE and salinity of 30^37 ppt. Weekly feedingbyA. salina nauplii should be conducted. For naturalproducts’ oriented rearing, cuttings should be main-tained at 20^23 1C, a light intensity of130 mE, salinityof 30^37 ppt and a feeding regime as stated above.The ability to control the environmental condi-

tions undoubtedly makes such a system an e⁄cientmeans for targeted soft coral farming. Our study hasestablished the scienti¢c ground for a protocol suita-ble for large-scale soft coral farming, and presents aplatform for future joint endeavours by researchersand the commercial sector engaged in coral farming.

Acknowledgments

We thank the Interuniversity Institute at Eilat for as-sistance and use of facilities, the Eilat-Ashkelon Pipe-line Company (EAPC) for allowing the research attheir premises, N. Sharon for technical assistance,N. Paz for her editorial assistance, I. Glerenter for sta-tistical advice and the Israeli Nature and NationalParks Protection Authority for their cooperation.

References

Abramovitch-Gottlib L., Katoshevski D. & Vago R. (2002) Acomputerized tank system for studying the e¡ect of tem-perature on calci¢cation of reef organisms. Journal of Bio-chemical and Biophysical Methods 50, 245^252.

Allemand D.,Tambutte E., Girard J.P. & Jaubert J. (1998) Or-ganic matrix synthesis in the scleractinian coral Stylo-phora pistillata: role in biomineralization and potentialtarget of the organotin tributyltin. Journal of ExperimentalBiology 201, 2001^2009.

Badria F.A., Guirguis A.N., Perovic S., Ste¡en R., MullerW.E.G. & Schroder H.C. (1998) Sarcophytolide: a new neu-

roprotective compound from the soft coral Sarcophytonglaucum.Toxicology131,133^143.

Baine M. (2001) Arti¢cial reefs: a review of their design, ap-plication,management and performance.Ocean&CoastalManagement 44, 241^259.

Becker L. &Mueller E. (1999) The culture, transplantation, andstorage of Montastraea faveolata, Acropora cervicornis,and A. palmata: what we learned so far. NCRI InternationalConference on Scienti¢c Aspects of Coral Reef Assess-ment, Monitoring, and Restoration, Ft. Lauderdale, FL,USA,53.

BenayahuY. & LoyaY. (1986) Sexual reproduction of a softcoral ^ synchronous and brief annual spawning of Sarco-phyton glaucum (Quoy and Gaimard,1833). Biological Bul-letin170,32^42.

Blunt J.W., Copp B.R., Munro M.H.G., Northcote P.T. & Prin-sep M.R. (2005) Marine natural products. Natural ProductReports 22,15^61.

Borneman E.H. & Lowrie J. (2001) Advances in captive hus-bandry and propagation: an easily utilized reef replenish-ment means from the private sector? Bulletin of MarineScience 69,897^913.

Bourne D.G., Lone H., Webster N.S., Swan J. & Hall M.R.(2006) Bio¢lm development within a larval rearing tankof the tropical rock lobster, Panulirus ornatus. Aquaculture260, 27^38.

Calfo A. (2003) Book of Coral Propagation, Vol. 1. ReadingTrees Publication, Monroeville, PA, USA, p.450.

Carricart-Ganivet J.P. (2004) Sea surface temperature andthe growth of theWest Atlantic reef-building coral Mon-tastraea annularis. Journal of Experimental Marine Biologyand Ecology 302, 249^260.

Castanaro J. & Lasker H.R. (2003) Colony growth responsesof the Caribbean octocoral, Pseudopterogorgia elisabethae,to harvesting. Invertebrate Biology122, 299^307.

Chang W.-L., Chi K.-J., Fan T.-Y. & Dai C.-F. (2007) Skeletalmodi¢cation in response to £ow during growth in colo-nies of the sea whip, Junceella fragilis. Journal of Experi-mental Marine Biology and Ecology 347,97^108.

Clavico E.E.G., De Souza A.T., Da Gama B.A.P. & Pereira R.C.(2007) Antipredator defense and phenotypic plasticity ofsclerites from Renilla muelleri, aTropical Sea Pansy. Biolo-gical Bulletin 213,135^140.

Delbeek J. (2001) Coral farming: past, present and futuretrends. Aquarium Sciences and Conservation 3,171^181.

Ellis S. & Ellis E. (2002) Recent advances in lagoon-based farm-ing practices for eight species of commercially valuable hardand soft corals. Technical Report for Tropical and Subtro-pical Aquaculture Center, Centre for Tropical and Subtro-pical Aquaculture, Hawaii, USA,147,59pp.

Fox H.E., Mous P.J., Pet J.S., Muljadi A.H. & Caldwell R.L.(2005) Experimental assessment of coral reef rehabilita-tion following blast ¢shing. Conservation Biology 19,98^107.

Fridkovsky E., Rudi A., BenayahuY., KashmanY. & SchleyerM. (1996) Sarcoglane, a newcytotoxic diterpene from Sar-cophyton glaucum.Tetrahedron Letters 37,6909^6910.



Haldane C. (1993) Direct evidence for organic cargoes in thelate bronze-age.World Archaeology 24,348^360.

Henry L.-A. & Hart M. (2005) Regeneration from injury andresource allocation in sponges and corals ^ a review. In-ternational Review of Hydrobiology 90, 125–158.

KyewalyangaM. (2003) Assessment of types andabundanceof live food for ¢sh farming in Makoba Earthen Ponds,Zanzibar,Tanzania.Western Indian OceanJournal ofMarineScience 2, 45^56.

Levitus S. (1982) Climatological atlas of the world ocean.NOAA/ERLGFDL Professional Paper 13, Princeton, NJ,USA,173pp.

Lewis J. (1982) Feeding behavior and feeding ecology of theOctocorallia (Coelenterata: Anthozoa). Journal of Zoology(London) 196,371^384.

Lim L.C., ChoY.L., Dhert P.,Wong C.C., Nelis H. & Sorgeloos P.(2002) Use of decapsulated Artemia cysts in ornamental¢sh culture. Aquaculture Research 33,575^589.

Look S., Fenical W., Matsumoto G. & Clardy J. (1986) Thepseudopterosins: a newclass of antiin£ammatoryandan-algesic diterpene pentosides from the marine sea whipPseudopterogorgia elisabthae (octocorallia). The Journal ofOrganic Chemistry 51,5140^5145.

Mendola D. (2003) Aquaculture of three phyla of marine in-vertebrates to yield bioactive metabolites: process devel-opments and economics. Biomolecular Engineering 20,441^458.

Muller-Parker G. & DiElia C. (1997) Interactions betweencorals and their symbiotic algae. In: Life and Death of CoralReefs (ed. by C. Birkeland), pp. 96^113. Chapman & Hall,NewYork, NY, USA.

Nguyen X.C., Tran A.T., Phan V.K., Chau V.M., Eun M.C. &Young H.K. (2008) New cembranoid diterpenes from thevietnamese soft coral Sarcophyton mililatensis stimulateosteoblastic di¡erentiation in MC3T3-E1 cells. Chemicaland Pharmaceutical Bulletin 56,988^992.

Okubo N., Motokawa T. & Omori M. (2007) When fragmen-ted coral spawn? E¡ect of size and timing on survivorshipand fecundity of fragmentation in Acropora formosa.Mar-ine Biology151,353^363.

Okuzawa K., Maliao R.J., Quinitio E.T., Buen-Ursua S.M.A.,Lebata M.J.H.L., GallardoW.G., Garcia L.M.B. & PrimaveraJ.H. (2008) Stock enhancement of threatened species inSoutheast Asia. Reviews in Fisheries Science16,394^402.

OrenU., BenayahuY. & LoyaY. (1997) E¡ect of lesion size andshape on regeneration of the Red Sea coral Favia favus.Marine Ecology Progress Series146,101^107.

Petersen D., Laterveer M.,Van Bergen D., Hatta M., Hebbin-ghaus R., JanseM., Jones R., Richter U., ZieglerT.,Visser G.& Schuhmacher H. (2006) The application of sexual coralrecruits for the sustainable management of ex situ popu-lations in public aquariums to promote coral reef conser-vation ^ SECORE Project. Aquatic Conservation ^ Marineand Freshwater Ecosystems16,167^179.

Rodolfo-Metalpa R., Richard C., Allemand D. & Ferrier-PagesC. (2006) Growth and photosynthesis of two Mediterra-

nean corals, Cladocora caespitosa and Oculina patagonica,under normal and elevated temperatures. Journal of Ex-perimental Biology 209, 4546^4556.

Sella I. (2007) Development of a propagation protocol forcuttings of the soft coral Sarcophyton glaucum. MSc thesis,Ecology and Environmental Quality, Tel Aviv University,72pp.

SipkemaD., OsingaR., SchattonW., Mendola D.,Tramper J. &Wij¡els R.H. (2005) Large-scale production of pharma-ceuticals by marine sponges: sea, cell, or synthesis? Bio-technology and Bioengineering 90, 201^222.

Slattery M., Gochfeld D.J. & Kamel H.N. (2005) Hybrid softcoral aquaculture to produce novel ‘‘pharmaceutical fac-tories’’. Global AquacultureAdvocate 8, 42–43.

Smith D., Suggett D. & Baker N. (2005) Is photoinhibition ofzooxanthellae photosynthesis the primary cause of ther-mal bleaching in corals? Global Change Biology11,1^11.

Soong K. & Chen T.A. (2003) Coral transplantation: regen-eration and growth of Acropora fragments in a nursery.Restoration Ecology11,62^71.

Sorgeloos P. & Persoone G. (1975) Technological improve-ments for cultivation of invertebrates as food for ¢shesand crustaceans. 2. Hatching and culturing of brineshrimp, Artemia salina L. Aquaculture 6,303^317.

Tanaka J.,Yoshida T. & BenayahuY. (2005) Chemical diver-sity of Sarcophton soft corals in Okinawa. Galaxea (JCRS)7,1^9.

Tentori E. & Allemand D. (2006) Light-enhanced calci¢ca-tion and dark decalci¢cation in isolates of the soft coralCladiella sp during tissue recovery. Biological Bulletin211,193^202.

Venn A.A., Loram J.E. & Douglas A.E. (2008) Photosyntheticsymbioses in animals. Journal of Experimental Botany 59,1069^1080.

Wabnitz C.,Taylor M., Grenn E. & RazakT. (2003) From oceanto aquarium the global trade in marine species. UNEPWorldConservation Monitoring Centre, Cambridge, UK,65pp.

Wheeler J. (1996) The marine aquarium trade: a tool for coralreef conservation. A report for the Sustainable Develop-ment and Conservation Biology Program, University ofMaryland, College Park, MD, USA,47pp.

Winters G., LoyaY. & Beer S. (2006) In situ measured seaso-nal variations in F-v/F-m of two common Red Sea corals.Coral Reefs 25,593^598.

Zimmer R.K. & Butman C.A. (2000) Chemical signaling pro-cesses in the marine environment. Biological Bulletin198,168^187.

Web References

Available at http://www.iui-eilat.ac.il/NMP/Default.aspx(accessed 2005-2009)

Available at http://www.reefkeeping.com/issues/2002-07/eb/index.php (accessed 2005-2009)



http://www.iui-eilat.ac.il/NMP/Default.aspx

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