ANNUAL REPORT
Project: C14-015 January 2000 - December 2000 Principal Investigator: B. Rinkevich J--
ANNUAL REPORT
Covering Period: January-December 2000
Submitted to the u.s. Agency for International Development;
Bureau for Global Programs, Field Support and Research;
Center for Economic Growth
Costa Rican coral reefs under siltation stress
Principle Investigator: Baruch Rinkevich
Grantee Institution: IOLR
Collaborator: Jorge Cortes
Institution: Universidad de Costa Rica
Project Number: C14-015
Grant Number: TA-MOU-97-Cl4-015
Project Officer: Y. Dishon
Project Duration: May 1, 1998 to April 30, 2003 3
Table of Contents
Page
Executive Summary 4
Section I parts A and B 5
part C 60
partD 60
partE 61
part F 61
Section II 62 4
Executive Summary
The purpose of this collaborative project is to evaluate the effects of siltation stress to Costa Rican coral reefs, to determine stress phenomena, to restore denuded reef areas and to train young Costa Rican scientists in reefmanagement, restoration protocols ofcoral reefs, modem techniques in coral reefbiology and in the biology ofreeforganisms. During this last year, several lines ofresearch have been employed in the field and in the laboratory. In the field, both groups, (the Costa Rican group head by Dr. 1. Cortez, and the Israeli group, headed by Dr. B. Rinkevich) continued the collaborative monitoring studies in the reef on the Pacific Coast (once a month) and on the Caribbean Coast (every 3-4 months). Long-term monitoring observations of specific selected stations are continued at the same pace as before, in addition to field studies on variety of stress phenomena, such as the appearance of tumors in coral tissues and skeletons, bleaching events in Costa Rican reefs, interspecific and intraspecific competitions, and more. We have further analysed the implementation of a small-scale "no use zone" policy in the reef ecosystem and developed further steps towards the evaluation of coral reef restoration by using small branch fragments. In the laboratory, we have characterized polymorphism of soft coral larvae by amplified fragment length polymorphism markers, aquarium maintenance ofreef octocorals (soft corals) raised from field collected larvae and are in the development ofprotocols and studies on ex situ cultures ofcolonial marine invertebrates. As in previous years, both groups mutually carried out the study. To further facilitate the work, two Israeli scientists (Dr. D. Gateno and Dr. Y. Barki) are continuing to reside in Costa Rica, participating in all field trips and other scientific activities involved in the project. The Israeli PI has visited Costa Rica twice during this period for tight collaboration and discussions with the Costa Rican group. Two Costa Rican scientists (a Ph.D. student and a technician) have visited this year the laboratory in Haifa, went to Eilat and were trained by the Israeli PI (B. Rinkevich) and two of the students in a variety ofaspects on reefrestoration. 5
Section I
A-B Published Data
Below are the detailed results of 4 manuscripts that were published during the fiscal year of 2000 X ~eplacing Sections A-B). These manuscripts were supported by the AID-CDR project. Reprints of those papers are also appended:
Rinkevich, B. Steps towards the evaluation of coral reef restoration by using small branch fragments. Mar. BioI. 136,807-812,2000. Barki, Y., J. Douek, D. Graur, D. Gatefio and B. Rinkevich. Polymorphism in soft coral larvae revealed by amplified fragment-length polymorphism (AFLP) markers. Mar. BioI. 136, 37-41, 2000. Rinkevich, B. and S. Shafir. Ex situ culture of colonial marine ornamental invertebrates: Concepts for domestication. Aquarium Sci. Conservation 2,237-250,2000. Gatefio, D., Barki, Y. and B. Rinkevich. Aquarium maintenance ofreef octocorals raised from field collected larvae. Aquarium Sci. Conservation 2,227-236,2000.
Four subjects conclude the material presented in Parts A-B.
SUBJECT 1 - REEF RESTORATION BY SMALL BRANCH FRAGMENTS
INTRODUCTION The emerging discipline of restoration ecology has already shown to provide a powerful suite of tools for speeding the recovery of degraded terrestrial habitats (Dobson et al. 1997; Montalvo et ai. 1997). The methodologies for restoration of degraded marine habitats, however, particularly for coral reefs, are still in their preliminary stages, addressing a variety of theoretical and practical problems for experimentalists and practitioners (Rinkevich 1995). The urgent need for restoration strategies to be applied to reef habitats, not only stems from the generalization that ecosystems do not usually recover from anthropogenic stress without some manipulations (Pratt 1994), but also emerges from the recognition that leaving restoration to natural processes may take prolonged periods, measured in decades or centuries (Rinkevich 1995; Dobson et al. 1997). A previous study (Rinkevich 1995) has discussed the potential of active restoration by coral sexual and asexual recruits ("gardening" reefareas) in rehabilitating degraded coral reefhabitats. 6
The approach for reef restoration by asexual recruits (either by colony fragments or by transplantation of whole colonies) has been studied by several authors working on coral reefs worldwide (Shinn 1966, 1976; Maragos 1974; Birkeland et al., 1979; et al. 1981; Kojis and Quinn 1981; Alcala et al. 1982; Auberson 1982; Fucik et al. 1984; Yap and Gomez, 1985; Plucer-Rosario and Randall 1987; Harriott and Fisk 1988; Guzman 1991, 1993; Yap et al. 1992; Yates and Carlson 1993; Clark and Edwards 1995; Bowden-Kirby 1997; Hudson and Goodwin 1997; Munoz-Chagin 1997; van Treeck and Schuhmacher 1997). Several restoration experiments (all with an eye for large-scale operations) have revealed that the use of coral fragments may serve as a good tool for reef rehabilitation. One of the first large-scale rehabilitation attempts (Guzman 1993) was carried out in two shallow Costa Rican reefhabitats. Approximately 80% ofcoral fragments, imported from a nearby reef and transplanted onto dead reef frameworks, were still alive after three years. During this study over 3,500 fragments were transplanted, more than 43,000 other fragments were replenished, and 32,500 m2 of reefs were restored (Guzman 1993). In Cozurnel Island, Mexico (Munoz-Chagin 1997), almost 2,500 coral and octocoral specimens were transplanted to denuded reefs using underwater containers. Only 3% mortality was recorded 1 month after the operation. Bowden-Kerby (1997) further demonstrated that transplanting unattached coral fragments on Caribbean reef flat rubble resulted in an impressive survivorship (97%) and in the formation ofreef structure within 3 years. An overall survivorship of 51 %, 28 months after transplantation has been recorded in the Maldives (Clark and Edwards 1995) and >87% within 7 months in the northern Red Sea area (van Treed: and Schuhmacher 1997). The above studies and others (Rinkevich 1995) further revealed that local conditions, the type of substrate and coral species chosen may significantly affect the results. We have studied the rehabilitation of Red Sea reefs by using both sexual and asexual recruits. The common branching hermatypic coral Stylophora pistillata (Fig. 1) is the studied species. Colonies of this species exhibit an axially rod-like growth form. New up-growing branches (UGBs) are primarily added by dichotomous fission at the tip of a branch. In addition to these UGBs, many lateral (inside and outside) branches are formed. The lateral branches facing outside the colony elongate similarly to the up-growing branches, so the resulting symmetry of a typical S. pistillata colony approximates a sphere (Loya 1976). This pattern formation is maintained even after symmetry is lost through partial branch breakage. The lateral branches facing the internal volume of the colony (internal-side branches = ISBs; Fig. 1) are developed from the middle and bases of the UGBs and it could be predicted that after prolonged elongation, ISBs would encounter and fuse with 7
UGBs. However, such fusion was never observed in unharmed colonies growing in the reef (Rinkevich and Loya 1985). While fragmentation may serve as an important tool for coral reefrestoration (Rinkevich 1995), the intra-colony variation in growth modes and the documented variation in fragment size vs. survival (Highsmith 1982; Lewis 1991; Bruno 1998) may obscure the effectiveness ofany applied approach used. Additionally, there should be a calculated tradeoff measurement for either adapting the protocol of pruning many small fragments from a single colony or subcloning a few large ones. Subcloning large fragments from colonies may be maladaptive due to the stress inflicted on these colonies. Subcloning small fragments may reduce the stress inflicted on the colonies but may increase ramet's mortality. We evaluate here variations in growth rates and the choice of small branch fragments to be sampled from S. pistillata for restoration purposes. Although this species is not known to reproduce asexually under natural conditions, previous studies on allorecognition and physiological characterization of S. pistillata were successfully conducted on small isolated fragments.
METHODS AND MATERIALS Coral sampling The study was carried out in front of the Marine Biological Laboratory at Eilat, Gulf of Eilat, Red Sea. Mature and healthy specimens of S. pistillata were chosen in shallow (5 m depth) and in deep water (25-30 m). A hammer and chisel were used to detach the colonies from the substratum. The colonies were carefully transferred underwater and placed in a new site where they were tied with plastic cords to concrete plates, at the same depth as they had been naturally growing. Specimens harmed by this procedure were excluded. All underwater work was carried out by SCUBA diving.
Alizarin-vital staining S. pistillata colonies were incubated in situ with alizarin Red S solution for 24 h (l0 ppm; Barnes 1970) by enclosing them within plastic bags. After a postlabeling acclimation period of 24 h, branches were removed by wire cutters and were held vertically on 20 x 20 cm concrete plates with preglued plastic clothespins. The tip of the branch, the essential site of calcification, is marked by the red dye during incubation. New skeleton is then added as white calcium carbonate above the red area. After one year, the plates with the branches were brought to the laboratory. Tissues were removed by H20 2 (Rinkevich and Loya 1984) and the branches were sun-dried for at least 24 h. Each dried skeleton was photographed, weighed (to the nearest 0.1 g) and measured (to the nearest 8
0.1 mm). Thereafter, the branches were trimmed by wire cutters above the alizarin-marked zones and measured again. The net growth of each branch and the net weight were calculated. We termed "tips" as the ~0.5 em terminal portion ofthe branch. Branch bases are not (in most cases) the actual bases since sampled Stylophora branches in this study were usually <4 cm in length, representing only the upper part ofthe branches.
45Ca_ labelled branches 1 Up-growing dichotomous single tip branches were marked in situ with 4S CaCh (0.01 Ilci mr ) within transparent tanks for 24 h. Following incubation, tip branches were removed and tissues were digested (H202). The calcareous fragments were washed under tap water (0.5 h), transferred into scintillation minivials and dissolved with concentrated HCI containing a few drops of butyl alcohol to retard foaming. The vials were placed on a hot plate to evaporate the acid, cooled and then 1.5 ml ofdistilled water was added to each vial to dissolve the calcium salt, followed by 2.5 ml ofInstagel scintillation cocktail (Packard). Samples were counted in a Tri-Carb liquid scintillation counter (packard). The specific activity of calcium in each experimental tank was calculated from 3 water samples. Calcification rates of branch tips (upper 0.5 em) were expressed as DPM. mm-2 estimated surface area (ESA) during 24 h. ESA was calculated from the length (1) width (w) and height (h) of each fragment using calipers (accuracy of0.1 mm), by applying the formula for a sphere sector, i.e., 2 2 2 S = n(h + r ) or S = n[h + (1 + wi/16]. When the sampled tip approximated a half sphere, h = r, then S = 2nr2 (Rinkevich and Loya 1983). The amount of calcium in the seawater was taken to be 0.49 g 1-1 (Rinkevichand Loya 1984).
RESULTS UGB tip calcification: intracolony variability Five intermediate-sized colonies (geometric mean radius, r [Loya 1976] = 3.2-3.9 em) were chosen in shallow water to elucidate the variation in calcification rates between different UGBs. The colonies were labeled in situ by 4sCaCh and all major UGB tips (n=18-27) were sampled. The results (Table 1) show high variability (ranged 33.2%-71.6%) in calcification rates between different UGBs within the same colony.
Dichotomous - branch calcification: within-pair variability Up-growing dichotomous branches were sampled from 880 S. pistillata shallow and deep water colonies (one dichotomous branch/colony). Growth rate comparison between the two tips in each 9 branch were measured in short-term experiments (24 h, 45CaCh method) and in long-term studies (up to I year, alizarin method). Dichotomous branches (Fig. 1), were divided into 2 group sizes; those that have just started to divide «0.5 em in length), and large dichotomous branches (0.5-2.0 em in length from point of deviation). A tip ratio for each dichotomous branch was calculated by dividing the higher calcification value obtained for one of the tips by the lower one. The results (Table 2) reveal that tips ratios in the newly formed dichotomous branches are significantly lower than in older dichotomous branches which may be regarded as compatible to regular UGBs.
Growth ofisolated small branches Five large S. pistillata colonies (i' >10 em) were chosen. Alizarin Red S labelled UGBs (17-32/colony; Table 3) were sampled and left in the field for 1 year. Two types of branches were studied: single tip and dichotomous tip (all <0.5 em each) branches. All sampled branches were <6 em in length (most <4 em). After 1 year, most (68.7%; range 50.0 - 89.3% in different colonies; Table 3) of the 131 sampled branches survived. The vast majority of branch deaths were recorded following strong winter storms. Significantly longer branches were originally sampled from colonies 1 and 4 (4.3 and 3.9 cm on the average, respectively) than from colonies 2, 3 and 5 (2.5, 2.1 and 2.9 em respectively; P<0.05, Duncan's Multiple Range Test). This outcome, however, is not reflected in the "within genet analyses" results (Table 4). For example, dichotomous branches of colony 1 grew significantly faster than single tips using the 3 criteria studied (total added weight, percentage of weight added and length added per tip). This trend was reversed in colony 4 (Table 4) indicating genet (colony) specific patterns rather than to an impact of the branch lengths. With the exclusion of colony 4, the total length added in dichotomous branches (for both tips) was at least twice that recorded for single tips of a specific genet. For one-year of growth, up to 25% and 30% weight were added on the average, for single and dichotomous tips, respectively. This was reflected in average length addition ofup to 2.3 em per tip (Table 4.). Between colony analyses (Table 5) clearly reflects the variability in growth rates characteristic of S. pistillata colonies. Both characters studied (mm length added/tip; total weight added/branch, gr CaC03) differed significantly between the 5 genets studied and also between single tip and dichotomous branches. For example, while in the case ofdichotomous branches, colonies 3, 4 and 5 were grouped together for length added/tip and for total weight added/branch (in the last case, colony I was also grouped together). In the single tip branches, no such grouping was achieved. 10
DISCUSSION The present study aimed at evaluating the potential for restoration using small branch fragments (mostly <4 em length) from the common Red Sea branching coral Stylophora pistillata. Several sources of branch types were studied: UGBs (both dichotomous and single tip branches) and side growing branches. Dichotomous branches, in most cases, revealed faster growths than single branch tips. The results also show that survivorship as well as growth rates are genet specific. Variation for survivorship, although ranging between 50% and almost 90% within one year after sampling, clearly showed that these small-sized branches are potentially suitable for restoration purposes (also see the high survivorship of S. pistillata fragments in a recent study carried out for 7 months in the Red Sea; van Treeck and Shuhmacher 1997). Similar results were obtained for Madracis mirabilis by Bruno (1998), where again there was no apparent increase in survivorship with fragment size. Consequently, we may choose either to sacrifice S. pistillata colonies by sampling many branches from each colony or alternatively to prune a few small branches per genet, producing minimal damage that does not affect survivorship or reproduction (Rinkevich and Loya 1989) of the colonies. Ecological restoration, one ofthe plethora of ill-defmed normative conservation concepts (Callicott et al. 1999) is engaged with the process of returning, as close to possible, the biotic community or the habitat to the primeval condition ofbiological integrity. While actions for restoration are usually regarded as responses to the pervasive effects of human actions and not to naturally evolved phenomena (such as hurricanes, floods, etc.; Anderson 1991; Hunter 1996), degradation ofso many coral reefs has come to the point where any additional natural disturbance or man-made detrimental effect should be treated equally with regard to restoration. Coral reefs that are in advanced, degrading states, should be regarded as potential subjects for active restoration regardless of whether degradation phenomena are man-made or natural. The major question "is which protocol should be employed. Due to the high expenses involved in restoration actions and the variety ofpractices that should be studied, we need innovative but general templates that will guide us in restoring damaged habitats (Montalvo et al. 1997). The gardening of coral reefs with sexual and asexual recruits after their mariculture in situ within special protected areas (or "nurseries"; Rinkevich 1995) may serve as such framework for restoration protocols. However, local conditions may shape methodologies in accordance with the coral species present, reef topography, environmental conditions and more (Yap et al. 1992; Clark and Edwards 1995; Bruno 1998). In some places, almost all loose coral branches will survive, grow and will form a reef structure within 3 years (Bowden-Kerby 1997). 11
This methodology, however, cannot be applied to the northern Red Sea coral reefs where the vast majority of scattered coral branches will die within a few months (unpublished observations). Moreover, since pruning the branches of coral colonies may cause stress to both, the parental colonies and the subcloned branches (profoundly decreasing survivorship and/or retarding calcification and reproduction; Buddemeier and Kinzie 1976; Hudson 1982; Hudson 1982; Yap and Gomez 1985; Rinkevich and Loya 1989; Yap et al. 1992), it is plausible to search for methodologies producing minimal damage to "donor" colonies and isolated branches. Growth rates of isolated branches are also of paramount importance for the production of new colonies. Intrinsic biological factors (such as the specific physiological-metabolic state of the coral-genet), the species-specific growth patterns (UGBs vs. side-growing branches, single tip vs. dichotomous tip branches, etc.) and extrinsic stochastic environmental biological events (Clark and Edwards 1995; van Treeck and Schuhmacher 1997; Bruno 1998) have significant effects on the growth rates. However, several studies (e.g., Clark and Edwards 1995; Bowden-Kerby 1997) have clearly shown that large colonies can be grown from relatively small fragments within about 3 y. The present results on S. pistillata reveal a longer period. The average 20-30% growth in one year (up to 2.3 em length added tip -ly.l, on the average) may lead, ifgrowth rates in successive years are similar, to a "nursery period" ofabout 10 years to form medium-sized colonies. On the other hand, if reef rehabilitation through small asexual recruits is established as a long and permanent project, the benefit of using these small fragments is clearly evident. Their sampling has almost no toll inflicted on large colonies, minimizing environmental stress involved with restoration protocols.
REFERENCES Alcala AC, Gomez ED, Alcala LC (1982) Survival and growth of coral transplants in central Philippines. Kalikasan, Philippines J BioI 11: 136-147. Anderson JE (1991) A conceptual framework for evaluating and quantifying naturalness. Conservation BioI. 5: 347-352. Auberson B (1982) Coral transplantation: an approach to the re-establishment of damaged reefs. Kalikasan, Philippines J Bioi 11 : 158-172. Barnes DJ (1970) Coral skeletons: an explanation of their growth and structure. Science 170: 1305-1308. Birkland C, Randall RH, Grimm G (1979) Three methods ofcoral transplantation for the purpose of re-establishing a coral community in the thermal effluent area ofthe Tanguisson Power Plant. Technical report 60. University ofGuam Marine Laboratory, Guam. 12
Bouchon C, Jaubert J, Bouchon-Navaro Y (1981) Evolution of a semi-artificial reef built by transplanting coral heads. Tethys 10: 173-176. Bowden-Kerby A (1997) Coral transplantation in sheltered habitats using unattached fragments and cultured colonies. Proc 8th Int Coral ReefSymp 2: 2063-2068. Bruno JF (1998) Fragmentation in Madracis mirabilis (Duchasssaing and Michelotti): how common is size-specific fragment survivorship in corals? J Exp Mar BioI Ecol 230: 169-181. Buddemeier RW, Kinzie RA (1976) Coral growth. Oceanog Mar BioI Ann Rev 14: 183-225. Callicott JB, Crowder LB, Mumford K (1999) Current normative concepts in conservation. Conservation BioI 13: 22-35. Clark S, Edwards AJ (1995) Coral transplantation as an aid to reef rehabilitation: evaluation of a case study in the Maldive Islands. Coral Reefs 14: 201-213. Dobson AP, Bradshaw AD, Baker AJM (1997) Hopes for the future: restoration ecology and conservation biology. Science 277: 515-522. Fucik KW, Bright TJ, Goodman KS (1984) Measurements of damage, recovery, and rehabilitation ofcoral reefs exposed to oil. In: Caime J, Buikema AL (eds) Restoration ofhabitats impacted by oil spills. Buttorworth, Boston, pp. 115-153. Guzman HM (1993) Transplanting coral to restore reefs in the eastern Pacific. Rolex Awards Book, New York, pp. 409-411. Harrigan JF (1972) The planula larva of Pocillopora damicornis: lunar periodicity of swarming and substratum selection behaviour. PhD thesis: University ofHawaii, Honolulu. Harriot VJ, Fisk DA (1988) Coral transplantation as a reef management option. Proc 6th Intemat Coral ReefSym 2: 375-379. Highsmith RC (1982) Reproduction by fragmentation in corals. Mar Ecol Prog Ser. 7: 207-226. Hudson JR (1982) Response of Montastrea annularis to environmental change in the Florida keys: Proc 4th Int Coral ReefSym 2: 233-240. Hudson JR, Goodwin WB (1997) Restoration and growth rate ofhurricane pillar coral (Dendrogyra cylindrus) in the Key Largo National Marine Sanctuary, Florida. Proc 8th Int Coral Reef Sym 1: 567-570. Hunter M (1996) Benchmarks for managing ecosystems: are human activities natural? Conservation BioI 10: 695-697. Kojis BL, Quinn NJ (1981) Factors to consider when transplanting hermatypic corals to accelerate regeneration of damaged coral reefs. In: Conference on Environmental Engineering, Townsville, Australia, pp. 183-189. 13
Lewis JB (1991) Testing the coral fragment size-dependent survivorship hypothesis for the calcareous hydrozoan Millepora complanata. Mar Ecol Prog Ser 70: 101-104. Loya Y (1976) Skeletal regeneration in a Red Sea scleractinian coral population. Nature 261: 490-491. Maragos JE (1974) Coral transplantation: a method to create, preserve and manage coral reefs. Sea grant advisory report 74-OJ-COR-MAR-14, University ofHawaii, Honolulu. Montalva AM, Williams SL, Rice KJ, Buchmann SL, Cory C, Handel SN, Nabhan GP, Primack R, Robichaux RH (1997) Restoration biology: a population biology perspective. Restoration Ecol 5: 277-290. Muiioz-Chagin RF (1997) Coral transplantation program in the Paraiso coral reef, Cozumel Island, Mexico. Proc 8th Int Coral Reef Sym 2:2075-2078. Plucer-Rosario GP, Randall RH (1987) Preservation of rare coral species by transplantation: an examination oftheir recruitment and growth. Bull Mar Sci 4: 585-593. Pratt JR (1994) Artificial habitats and ecosystem restoration: management for the future. Bull Mar Sci 55: 268-275. Rinkevich B (1995) Restoration strategies for coral reefs damaged by recreational activities: the use ofsexual and asexual recruits. Restoration Ecol 3: 241-251. Rinkevich B, Loya Y (1983) Short-term fate ofphotosynthetic products in a hermatypic coral. J Exp Mar BioI Ecol 73: 175-184. Rinkevich B, Loya Y (1984) Does light enhance calcification in hermatypic corals? Mar BioI 80: 1-6. Rinkevich B, Loya Y (1985) Coral isomone: a proposed chemical signal controlling intraclonal growth patterns in a branching coral. Bull Mar Sci 36: 319-324. Rinkevich B, Loya Y (1989) Reproduction in regenerating colonies of the coral Stylophora pistillata. In: Spanier E, Steinberger Y, Luria M (eds) IVB Environment quality and ecosystem stability. ISEEQS Publ., Jerusalem, Israel, pp. 259-265. Shinn EA (1966) Coral growth rate, an environmental indicator. J Paleontology 40: 233-242. Shinn EA (1976) Coral reefrecovery in Florida and the Persian Gulf. Environ Geol 1: 241-254. Van Treeck, P, Schuhmacher H (1997) Initial survival ofcoral nubbins transplanted by a new coral transplantation technology-options for reefrehabilitation. Mar Ecol Prog Ser 150: 287-292. Yap HT, Gomez ED (1985) Growth of Acropora pulchra. III. Preliminary observations on the effects oftransplantation and sediment on the growth and survival oftransplants. Mar BioI 87: 203-209. 14
Yap HT, Almo MP, Gomez ED (1992) Trends in growth and mortality of three coral species (Anthozoa: Scleractinia), including effects oftransplantation. Mar Ecol Prog Ser 83: 91-1Ol. Yates KR, Carlson BE (1993) Corals in aquariums: how to use selective collecting and innovative husbandry to promote coral preservation. Proc 7th Internat Coral ReefSym 2: 1091-1095. 15 Table 1. Mean calcification rates of all major up-growing branch (DGB) tips from S. pistil/ata colonies Colony r (em) Number of Mean calcification rates Within colony variation 2 No. UGB tips (DPM mm- ± SD) (%)
1 3.2 18 244 ± 82 33.6
2 3.4 22 162 ± 116 71.6
3 3.5 18 231 ± 108 46.8 4 3.7 24 190 ± 63 33.2 5 3.9 27 328 ± 136 41.5 16 Table 2. Tip-ratios for calcification rates in dichotomous branches
Locality, methodology N Length of Dichotomous branches: Level of dichotomous pair-ratio distribution (%) significance* tips (em) <1.5 1.5-2.0 >2.0
Shallow. 134 up to 0.5 73.9 19.4 6.7 P < 0.001 45Camethod 230 0.5 - 2.0 54.3 21.7 24.0
Deep, 170 up to 0.5 64.1 20.0 15.9 P <0.05 45Camethod 211 0.5 - 2.0 52.6 22.7 24.7
Shallow, 84 up to 0.5 96.4 3.6 0.0 P < 0.02 alizarin method 51 0.5 - 2.0 84.3 15.7 0.0
*Testing equality oftwo percentages. The statistic was performed on the percentages of "pair ratios" - up to a ratio of 1.5 vs. higher ratios, ~ 1.5. 17 Table 3. Survivorship ofsmall single and dichotomous tip branches, one year after isolation Colony No. Experimental No. ofbranches Total no. ofbranches Phase single tip dichotomous (% survivorship) tips 1 commencement 12 5 17 termination 8 3 11 (64.7%)
2 commencement 14 14 28 termination 12 13 25 (89.3%)
3 commencement 16 14 30 termination 12 9 21 (70.0%)
4 commencement 20 12 32 termination 11 5 16 (50.0%)
5 commencement 12 12 24 termination 9 8 17 (70.8%) l8
Table 4. Growth and length added values for S. pistillata small isolated branches, after one year
Colony no. Branch type Weight added Signifi- Weight Added Signifi- Length added Signifi- (gr. branch-I) eanee (%) eanee (per tip, mm) cance *
single tip 0.44 ± 0.21 (8) ** 12.26 ± 5.11 (8) * 11.59 ± 5.98 (8) * dichotomous 1.48 ± 0.23 (3) 21.00 ± 2.92 (3) 23.38 ± 12.28 (6)
2 single tip 2.08 ± 0.74 (9) ** 24.99 ± 8.44 (9) ns 11.31 ± 8.03 (12) ns dichotomous 0.95 ± 0.83 (9) 30.09 ± 13.14 (9) 13.86 ± 8.37 (18)
3 single tip 0.23 ± 0.23 (8) ns 10.43 ± 8.66 (8) ns 4.74 ± 4.89 (12) ns dichotomous 0.40 ± 0.38 (7) 10.53 ± 8.38 (7) 5.24 ± 5.47 (18)
4 single tip 2.14 ± 0.39 (10) * 24.95 ± 5.36 (10) ** 13.75 ± 2.66 (11) ** dichotomous 1.45 ± 0.14 (3) 10.57±3.21 (3) 2.09 ± 1.17 (10)
5 single tip 0.28 ± 0.09 (6) ns 13.60 ± 3.30 (6) ns 6.49 ± 3.95 (8) ns dichotomous 0.32 ± 0.18 (4) 14.93 ± 9.61 (4) 6.85 ± 4.68 (16)
*p <0.05; ** p Table 5 Stylophora pistil/ata. I year growth (between colonies Statistical Duncan grouping for colonies Duncan's mUltiple range tests) - analysis expressed as length per tip and 2 3 4 s total weight added. For each specific test. colonies that share Length added Single-tip branches the same horizontal line have per tip means that are not significantly different (P > 0.05) Dichotomous branches Total weight Single-tip branches added per branch 1-=--1 Dichotomous branches 20 Fig. 1 Typical shape of a Stylophora pistil/ala colony, schematically drawn from a live specimen growing at 5 m depth (0 up-growing branch tips: b dichotomous branch tips: c lateral. inside and outside growing branches) BEST AVAILABLE COfY 21 SUBJECT 2 - LARVAL GENETICS INTRODUCTION Population genetic structures of sessile marine invertebrates may be affected by modes of asexual replication, type of mating, larval dispersal potentiality and by settlement and substratum exploitation of larvae (Knowlton and Jackson 1993). For example, limited dispersal of gametes or larvae with the ability to reproduce clonally can give rise to genetic subdivisions as a result of isolation-by-distance (Jackson 1986; Knowlton and Jackson 1993; McFadden and Aydin 1996). These, and other studies revealed that incorrect interpretations may result in cases where scientists are not fully versed on population genetic portraits of sessile marine invertebrates, even on well-studied species. This problem may also arise in cases where methodologies are not well established. One interesting case study is the Red Sea encrusting soft coral Parerythropodium fulvum fulvum (Benayahu and Loya 1983, 1984). This coral, commonly found between 3 and 40 m depth in the reefs of Eilat, is a dioecious species. However, more females than males are found in the shallow water populations (3-5 m), whereas the deep reef zone populations (27-30 m) are characterized by a 1: 1 sex ratio. These differences were assumed to be the outcome of asexual reproductive activities characteristic of shallow water populations (Benayahu and Loya 1983). Colonies ofthis species are equipped with a gastrovascular system that actively transports symbiont and coral cells (Gatefio et a1. 1998) and by self-nonself recognition capabilities (Frank et aI., 1996). It is an oviparous surface brooder species. Fertilization presumably takes place inside the polyp cavities, followed by spawning that occurs from the end ofJune till the beginning ofAugust, at dusk, a few days after the new moon and a few days preceding the last quarter (Benayahu and Loya 1983). Embryogenesis takes place on the surface offemale colonies within a mucoid suspension. The nonsymbiotic larvae complete their development within six days after fertilization and tend to settle immediately upon leaving the maternal colonies. Larvae crawl and adhere to the substratum, forming an aggregated pattern ofsettlement (Benayahu and Loya 1983). The mode of surface brooding in larvae is, therefore, an important feature for the spatial pattern of P. f fulvum which should be further explored. Although sexual reproduction is presumably the method for juvenile production in this species (Benayahu and Loya 1983), ameiotic (asexual) reproduction in P. f fulvum has not yet been convincingly excluded, especially since a variety ofsea anemones (Black and Johnson 1979; Ayre 1984; Edmands 1995; Edmands and Potts 1997; and literature therein) and hard corals (Stoddart 1983; Ayre and Resing 1986; Ayre et a1. 1997) are known to produce asexually-derived planulae. Moreover, most of the P. f fulvum colonies in the 22 shallow waters ofEitat are females (Benayahu and Loya 1983). This phenomenon coupled with the sessile mode ofthe life of this species, may favor the system ofproducing both sexual and asexual planulae where the production ofnew juveniles is retained even in the absence ofmale colonies. The present study describing the first step in the analyses of the genetic population structure and polymorphism of P. f fulvum larvae, by using amplified fragment-length polymorphism (AFLP) markers (Vos et a1. 1995). One ofthe main questions asked is to elucidate if larvae of this species are also produced asexually. This novel DNA fingerprinting technique is based on selective peR amplification of restriction fragments from a total digest of any genomic DNA. Typically, 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels, without any necessary knowledge ofnucleotide sequences. MATERIALS AND METHODS Sample collection. P. f fulvum embryos were collected from the surface of 4 maternal colonies (colonies A, B, C and D) at depths between 4 and 6 m, in front of the H. Steinitz Marine Biology Laboratory at Eilat, Red Sea. Colonies A and B were chosen from a dense population (0.5 m from each other). Colonies C and D were chosen from a less dense population (15 m from each other), with no other colonies between them. Both were situated 100 m south ofcolonies A and B. Several hundreds of brooded embryos were collected from each one of the 4 mother colonies by using Pasteur pipettes. They were washed from their mucous sheets with natural filtered seawater and transferred to the laboratory at the National Institute ofOceanography in Haifa in plastic boxes (0.4 I) with filtered seawater. The embryos were kept in plastic boxes with sterile seawater (0.22 11m) at 25°C and water was changed every 48 hours. Within five days, all embryos developed into mature larvae. Ten larvae from each colony were randomly chosen for AFLP analysis. DNA extraction. The preparation of genomic DNA was in accordance with the protocols of Graham (1978) and ten Lohuis et al. (1990). Larvae were lysed with 100 III lysis buffer (1 M Trisborate pH 8.2, 0.5 M EDTA, 10% SDS, 5 M NaCl) and homogenized gently. 20 III ofNaCI and 120 III ofphenol was then added, the solution was mixed and the aqueous phase was collected after 1 minute ofcentrifugation. The aqueous phase was extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1 v/v/v). Genomic DNA was precipitated with absolute alcohol, washed twice with alcohol 70%, dried and dissolved in sterile water. DNA quality was checked on 0.8% agarose TAB gels. 23 Preparation of DNA template and AFLP reactions. AFLP analysis was performed using AFLP Analysis System 1 (GibcoBRL, Life Technologies, Paisley UK) based on the protocol described by Vos et al. (1995). MseI was used as the nfrequent l1 cutter and EeaRl as the "rare" cutter. Restriction enzymes digests were performed in a total volume of 25 p,l for 2 h at 37°C. Following heat inactivation of the restriction endonucleases, the genomic DNA fragments were ligated to EeoRl adapter 5'-CTCGTAGACTGCGTACC. CTGACGCATGGTTAA-5' and to MseI adapter 5'-GACGATGAGTCCTGAG, TACTCAGGACTCAT-5' to generate template DNA for amplification (http://carnegiedpb.stanford.edu/methods/aflp.html). PCR was performed in two consecutive reactions: in the preamplification step, genomic DNA fragments were amplified with a single EeaRl -Oligo primer (5'-CTCGTAGA CTGCGTACC -3'). The PCR products of the preamplification reaction were diluted and used as a template for the selective amplification using three combinations of AFLP primers each containing three different nucleotides (EeaRl primer: 5'-AGACTGCGTACCAATTCACA -3' and Msel primer: 5'-GATGAGTCCTGA GTAACAT-3'; E-AGG and M-CAG; E-ACG and M-CTG. E and M refers to additional selective nucleotides at the 3'-end of the primer). The EeaRl selective primer was 33P-labeled before amplification. Products from the selective amplification were separated on a 7.5% denaturing polyacrylamide (sequencing) gel. The resultant banding pattern (nfmgerprintn) was visualized by autoradiography and analyzed manually. Nei's Genetic Distance (1972, 1978) was calculated using Tools for Population Genetic Analyses (TFPGA) Program, Version 1.3 (M.P. Miller 1997; http://herb.bio.nau.edu/~mil1er/tfpga.htm ). RESULTS A total of39 aposymbiontic larvae from 4 mother colonies were analyzed (DNA ofone larvae from colony A was damaged and excluded from the experiment). Amplified fragment sizes was 100 to 400 bp (Fig. 1). Several ofthe larvae analysed in each primer combination (Fig. 1, Table 1) resulted in unclear band patterns and were excluded from the specific set of analyses. Depending on the primer combination (E-ACA, M-CAT; E-AGG, M-CAG; E-ACG, M-CTG) totals of 61, 63, 63 bands (markers), respectively, were analyzed. Most of the bands (55.7%-88.9%, Table 1) were polymorphic. All genetic distances were calculated by using only the polymorphic markers. With regard to the fingerprint profiles, all 39 larvae exhibited different banding patterns from one another. Only two sister larvae from colony A presented identical band patterns under a single primer combination (E-ACA, M-CAT) but they differed in the other two primer combinations. All 39 larvae therefore were genetically distinct from each other. 24 Mean genetic distances (Nei 1972, 1978) for all 12 possible combinations of larva origin (among sister larvae [A-A, B-B. ..] versus larvae of different origins [A-B, A-C....]), calculated for each primer combination, are shown in Table 2. The values between sister larvae were usually (34 out of 36 cases; except in combinations B-B vs. B-C and B-B vs. B-D for one primer combination) smaller than those between unrelated larvae. In most combinations (7/12 to 10112, depending on the primer combination; Table 2), the mean genetic distances among sister larvae were significantly lower than those calculated for between-colonies larvae. These results are consistent for all primer combinations as shown by the low coefficient of variability among the 3 calculated mean genetic distances (Table 3). When analysing within-group variations, the genetic distances among sister larvae varied with colony origin. Larvae from colony C showed the lowest values, followed by colonies D, A and B (Table 3). Moreover, the mean genetic distances of the 4 colonies larvae may be grouped into 3 statistical clusters (ANOVA, Duncan's multiple range test; P<0.05) in which the genetic distances of colonies A, B are not significantly different from each other but differ from colonies C and D which are grouped separately from each other. The MGDs average values, grouped for the dense population (A-A, A-B, B-B), are unexpectedly higher than those calculated for the less dense population (C-C, C-D, D-D; 0.347 vs. 0.257, respectively). DISCUSSION Our results reveal extensive polymorphism among larvae from the same mother colony (each one of the primer combinations used revealed 61-63 scored bands of which 55.7%-88.9% were polymorphic). Band analyses revealed that all 39 larvae studied were genetically distinct from each other indicating only sexual production of larvae in the gonochoric surface brooder soft coral Parerythropodium fulvum fulvum, as suggested (Benayahu and Loya 1983). Furthermore, the mean genetic distances among sister larvae are, as expected, lower than the genetic distances among larvae from different maternal colonies. The differences recorded among sister larvae are probably the outcome of the genetic distances between the parents (the mother colonies and sperm donors) and may also be affected by the number of males fertilizing a specific female colony. This is probably the reason why the combined average of MGDs in the dense population is unexpectedly higher than in the less dense population. More sperm donor colonies will raise the polymorphism of the produced larvae. Larvae ofcolonies A and B, which are about 0.5 m from each other and which are located within a dense P. f fulvum population, have similar, significantly higher mean genetic distances (which again reflected fertilization by more than a single sperm donor). Colonies C and D were chosen from a less crowded population (15 m from each other without any other P. f fulvum 25 colony between them) about 100 m further south to the dense population. Larvae originating from colonies C and D exhibited significantly lower mean genetic distances as compared to colonies A and B larvae, which, in addition, are significantly different from each other. All these outcomes coupled with the result for low coefficient of variability recorded between the 3 pairs of primer combinations, further emphasize the efficiency ofusing AFLP. Any population genetic analysis requires the accurate identification of individual genotypes. AFLP is one ofthe best recently developed genetic techniques that identify variations with high resolution at the level of the individual's DNA (Parker et al. 1998). In the phylum Cnidaria, several other techniques have already been employed such as DNA fmgerprints (Coffroth et al. 1992; Coffroth 1997; Edmands and Potts 1997), randomly amplified polymorphic DNA (RAPDs; Coffi'oth and Mulawka 1995; Lasker et al. 1996; Brazeau et al. 1998) and'even AFLP on a scleractinian coral (Lopez and Knowlton 1997). Unfortunately, we could not use this or either one of the above techniques to screen the maternal colonies or other nearly settled P, f fulvum colonies. The symbiotic zooxanthellae DNA may interact and add additional uncontrolled factors. The larvae of this species which lack symbiotic algae are, on the other hand, a suitable candidate for such molecular approaches. It is well documented that the dispersal of larvae in benthic marine invertebrates constitutes the primary sources ofcontinuing gene flow (Knowlton and Jackson 1993; McFadden and Aydin 1996; Yu et al. 1999). Broad dispersal of propagules should genetically homogenize populations. In contrast, geographically restricted gene flow enhances differentiation among populations (Yu et al. 1999). New recruits arriving from mother colonies or from nearby populations is not the only factor that shape genetic population structures ofPI fulvum in Eilat. Random selection acting on patches of larvae (Burnett et al. 1994), brooding oflarvae and the limited dispersal ofthe sexually produced propagates (Benayahu and Loya 1983, 1984), clonal propagation (unpubl.) and the skewed sex ratios (Benayahu and Loya 1983) which may potentially reduce the numbers of sperm donors, all probably contribute to the small-scale patchiness in the genetic structure of shallow water P. f fulvum populations in Eilat. Genetic differentiation among a subpopulation is therefore envisioned. Further work on the genetic structure of within and between population is therefore needed to establish this prediction. REFERENCES Ayre DJ (1984) The effects of sexual and asexual reproduction on geograhic variation in the sea anemone Actinia tenebrosa. Oecologia 62: 222-229 26 Ayre DJ, Resing 1M (1986) Sexual and asexual production of larvae in reef corals. Mar BioI 90: 187-190 Ayre DJ, Hughes TP, Standish RJ (1997) Genetic differentiation, reproductive mode, and gene flow in the brooding coral Pocillopora damicornis along the Great Barrier Reef, Australia. Mar Ecol Prog Ser 159: 175-187 Benayahu Y, Loya Y (1983) Surface brooding in the Red Sea soft coral Parerythropodiumfulvum fulvum (Forskal). BioI Bull 165: 353-369 Benayahu Y, Loya Y (1984) Substratum preferences and planulae settling of two Red Sea alcyonaceans: Xenia macrospiculata Gohar and Parerythropodiumfulvumfulvum (Forskal) J Exp Mar BioI Eco183: 249-261 Black R, Johnson MS (1979) Asexual viviparity and population genetics ofActinia tenebrosa. Mar Bio1S3: 27-31 Brazeau DA, Gleason DF, Morgan ME (1998) Self-fertilization in brooding hermaphroditic Caribbean corals: Evidence from molecular markers. J Exp Mar BioI Eco1231: 225-238 Burnett WJ, Benzie JAR, Beardmore JA, Ryland JS (1994) High genetic variability and patchiness in a common Great Barrier Reefzoanthid (Palythoa eaesia). Mar BioI 121: 153-160 Coffroth MA (1997) Molecular approaches to the study of clonal organisms: deciphering the alphabet soup. Proc 8th Int Coral Reef Symp 2: 1603-1608 Coffroth MA, Mulawka 1M (1995) Identification of marine invertebrate larvae by means of PCR-RAPD species-specific markers. Limnol Oceanogr40: 181-189 Coffroth MA, Lasker RR, Diamond ME, Bruenn JA, Berminhgam E (1992) DNA fingerprints of a gorgonian coral: a method for detecting clonal structure in a vegetative species. Mar Bioi 114: 317-325. Edmands S (1995) Mating systems in the sea anemone genus Epiactis. Mar BioI 123: 723-733 Edmands S, Potts DC (1997) Population genetic structure in brooding sea anemones (Epiactis spp.) with contrasting reproductive modes. Mar Bioi 127: 485-498 Frank U, Bak RPM, Rinkevich B (1996) Allorecognition responses In the soft coral Parerythropodiumfulvumfulvum from the Red Sea. J Exp Mar BioI Eco1197: 191-201 Gatefio D, Israel A, Barki Y, Rinkevich B (1998) Gastrovascular circulation in an octocoral: evidence ofsignificant transport ofcoral and symbiont cells. BioI Bull 194: 178-186. Graham DE (1978) The isolation of high molecular weight DNA from whole organisms or large tissue masses. Analytical Biochem 85: 609-613 Jackson JBC (1986) Modes of dispersal of clonal benthic invertebrates: consequences for species 27 distributions and genetic structure oflocal populations. Bull Mar Sci 39: 588-606 Knowlton N, Jackson JBC (1993) Inbreeding and outbreeding in marine invertebrates. In: Thornhill NW (ed) The natural history of inbreeding and outbreeding. University of Chicago Press, pp 200-249 Lasker HR, Kim. K, Coffroth MA (1996) Reproductive and genetic variation among Caribbean gorgonians: the differentiation ofPlexaura lama, new species. Bull Mar Sci 58: 277-288 Lopez N, Knowlton N (1997) Discrimination of species in the Montastraea annularis complex using multiple genetic loci. Proc 8th Int Coral Reef Symp 2: 1613-1618 McFadden CS, Aydin KY (1996) Spatial autocorrelation analysis of small-scale genetic structure in a clonal soft coral with limited larval dispersal. Mar Bioi 126: 215-224 Nei M (1972) Genetic distance between population. Amer Nat 106: 283-292 Nei M (1978) Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89: 583-590 Parker PG, Snow AA, Schug MD, Booton GC, Fuerst PA (1998) What molecules can tell us about populations: choosing and using a molecular marker. Ecology 79: 361-382 Stoddart JA (1983) Asexual production oflarvae in the coral Pocillopora damicornis. Mar BioI 76: 279-284 ten Lohuis M, Alderslade P, Miller DJ (1990) Isolation and cloning ofDNA from somatic tissue of soft corals (Cnidaria: Octocorallia). Mar BioI 104: 489-492 Vos P, Rogers R, Bleeker M., Reijans M, van de Lee T, Homes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP: a new teclmique for DNA fingerprinting. Nucleic Acids Res 23: 4407-4414 Yu JK, Wang RY, Lee SC, Dai CF (1999) Genetic structure of a scleractinian coral, Mycedium elephantotas, in Taiwan. Mar BioI 133: 21-28 28 Table 1. Summary oflarvae tested, number ofAFLP bands scored and markers used for each ofthe primer combinations employed. Primer Combination E-ACA, M-CAT E-AGG, M-CAG E-ACG, M-CTG No. oflarvae, Colony A 6 8 9 No. oflarvae, Colony B 8 8 9 No. oflarvae, Colony C 8 10 9 No. oflarvae, Colony D 6 9 10 No. ofbands scored 61 63 63 No. ofpolymorphic bands 34 (55.7%) 56 (88.9%) 56 (88.9%) 29 Table 2. Mean genetic distance (Nei 1972, 1978) analyses between larvae from the same mother colony (sister larvae) vs. larvae from different mother colonies (unrelated larvae). Duncan grouping outcomes: * = p<0.05; NS = not significant. ANOVA values are: df = 368, F = 12.84, P <0.001; df= 551, F = 21.3, P <0.001; df= 656, F = 24.29, P <0.001, respectively for each primer combination. Primer Combination Analysis E-ACA, M-CAT E-AGG, M-CAG E-ACG, M-CTG Combination Mean genetic Significance Mean genetic Significance Mean genetic Significance distances distances distances A-A vs. A-B 0.302 vs. 0,404 * 0.302 vs. 0.332 NS 0.359 vs. 0,443 * A-A vs. A-C 0.302 vs. 0.344 NS 0.302 vs. 0.360 * 0.359 vs. 0,410 * A-A vs. A-D 0.302 vs. 0.395 * 0.302 vs. 0,417 * 0.359 vs. 0,420 * B-B vs. A-B 0.364 vs. 0,404 NS 0.313 vs. 0.332 NS 0.294 vs. 0.443 * B-B vs. B-C 0.364 vs. 0.344 NS 0.313 vs. 0.362 * 0.294 vs. 0.311 NS B-B vs. B-D 0.364 vs. 0.358 NS 0.313 vs. 0.369 * 0.294 vs. 0.335 NS C-C vs. A-C 0.198 vs. 0.344 * 0.232 vs. 0.360 * 0.194 vs. 0,410 * C-C vs. B-C 0.198 vs. 0.344 * 0.232 vs. 0.362 * 0.194vs.0.311 * C-C vs. C-D 0.198 vs. 0.275 * 0.232 vs. 0.278 * 0.194 vs. 0.343 * D-D vs. A-D 0.265 vs. 0.395 * 0.277 vs. 0,417 * 0.247 vs. 0.420 * D-Dvs. B-D 0.265 vs. 0.358 * 0.277 vs. 0.369 * 0.247 vs. 0.335 * D-Dvs. C-D 0.265 vs. 0.275 NS 0.277 vs. 0.278 NS 0.247 vs. 0.343 * 30 Table 3. Average of the mean genetic distances (MGDs ± SD) and the coefficient of variability (CV = 100 SD/Mean) between sister larvae for the 3 primer combinations. Colonies Average MGDs ± SD CV (%) A-A 0.321 ± 0.033 10.2 B-B 0.323 ± 0.036 11.2 C-C 0.208 ± 0.021 10.0 D-D 0.263 ± 0.015 5.6 A-B 0.393 ± 0.056 14.3 A-C 0.371 ± 0.034 9.3 A-D 0.410 ± 0.014 3.3 B-C 0.339 ± 0.026 7.6 B-D 0.354 ± 0.017 4.9 C-D 0.299 ± 0.038 12.9 J 1 --- _.~----- _ ~ -..«1'..... 'II'J - -- ,..... - - -...... --~ --, ~ "'-.;;, ~-~~ - ..~:::: .... ~~-;~~ -~ ...... ,;l;,.Sli>'il.- ...... ~~ \'!II!IIIta"":';"- Fig. 1 PQre:·ylhropodir...rn jttlvwr. fl.llvum. Part of a representat1\ e --\FLP tamplified fragment~length polymorphjsm) p;ofi1e carried out on 14 larvae. Each larva IS deSignated by a capital letter (C, 0) for tbe ;nother ongm. followed by a number. 1\\/0 replicate samples were taken from larva D 1 depictlng the reproducibility of the band pattern. Scored bands 3.re marked on the left. Larva D4 reproduced weak bands and \vas deleted from the analysIs. The pniTler combination E-AGG and ;vi-CAG was used. D?'iA fragment Sizes ranged from 100 :0 .+00 bp..--\. C, G, T t.right columns) are the nucleotlde sequences BESTAVAILABLE COpy 32 SUBJECT 3: CONCEPTS FOR DOMESTICATION THE RATIONALE The marine aquarium industry is a fast developing commercial sector, targeting in supplying ornamental organisms to millions of households worldwide (in the US alone, at least 600,000 households maintain marine aquaria; Lewbart et aI., 1999), public exhibitors (universities, zoos, etc.; Atkinson et aI., 1995; Carlson, 1999), laboratories and research institutions (Davies, 1995). However, contrary to the more established sector of the freshwater aquarium industry, marine aquarium trade is mostly dependent on specimens captured from the wild (Heslinga, 1996). As a result of the intensive exploitation of tropical marine ornamental organisms (especially reef fishes, molluscs, hermatypic and soft corals), for many years, many wild populations diminished practically to the point of no return and whole ecosystems were destroyed in many places (Lubbock and Polunin, 1975; Mamonov, 1980; Grigg, 1984; Ross, 1984; Wells and Alcala, 1987; Sadovy, 1992; Best, 1997; Pet-Soede et aI., 1999; and literature therein). For example, some of the popular Tridacna species, the giant reef-dwelling clams, have been essentially eliminated from vast areas of Indonesia, the Philippines, Papua, New Guinea, Micronesia and Okinawa (Heslinga and Fitt, 1987). Major ecological impacts may also be imposed indirectly. One such example is the suggestion that population explosions ofthe coral-eating starfish, Acanthaster planci, in Sri Lanka, could have been caused by the removal of fish that eat its larvae (literature cited in Sadovy, 1992). With the increasing global awareness for conservation and rehabilitation of endangered habitats (like coral reefs), international legal framework regulations (such as CITES; Best, 1997) and local/national legislation, continuous collections from the wild become more problematic. It is evident, therefore, that sustainable aquaculture techniques for ornamental marine organisms are a critical prerequisite to the future ofthe trade (Heslinga, 1996). Marine animal husbandry may be a crucial source ofa live material not only for aquaria hobbists but also an eminent organismal stock for education (Yates and Carlson, 1993; Evans, 1997), for scientific research (Kinne, 1977; Emschermann, 1987; Atkinson et al., 1995; Davies, 1995; Tambutte et aI., 1995; Ritchie et aI., 1997; Rinkevich and Shapira, 1998; Gatefio et aI., in press) and for conservation purposes (Lubbock and Polunin, 1975; Mamonov, 1980; Yates and Carlson, 1993; Carlson, 1999; Gatefio et aI., in press). Moreover, at least for marine ornamental fishes, it was noted that "the traffickers themselves admit that the live-to-dead ratio is more than 1 to 100", a note which further imposes for the concept of breeding in captivity (Lubbock and Polunin, 1975). Similar estimations, although varied from species to species may be drawn for ornamental marine invertebrates (Sadovy, 1992; Best, 1997). As a matter of fact, the culture methodologies employed 33 for marine organisms are relatively simple and are based on proven results (Kinne, 1977; Sykes, 1997). With regard to marine invertebrates, long-term ex-situ cultures were established in a variety of organisms from different phyla including cnidarians (Worthman, 1974; Kinne, 1977; Ritchie et aI., 1997; Carlson, 1999; Gatefio et aI., in press), bryozoans (Hunter and Hughes, 1993), chaetognath worms (Goto and Yoshida, 1997), molluscs (Heslinga and Fitt, 1987; Carroll and Kempf, 1990), protochordates (Nakauchi et aI., 1979; Kawamura and Nakauchi, 1986; Rinkevich and Shapira, 1998), and other groups. An emerging issue, not yet discussed in detail in the literature, is the concept of domesticating reef dwelling organisms. Domestication (Webster: To adapt an organism to life in intimate association with, and to the advantage ofman's use) is the way that by modifying growth and life history traits (by provisions of food, protection from enemies, etc.) and by selective breeding protocols through generations, specific species are adapted for high yield, sustainable production. Domestication may therefore be an attractive procedure from economic, social and ecological perspectives (Heslinga and Fitt, 1987; Carlson, 1999). Several facets for the domestication of reef-dwelling organisms, based on field-collection constrains are discussed below. The rationale is to corne to a point where most (if not all) marine ornamental trade will be based on ex-situ propagation and not on field collections. Although the proposed strategies may be adapted to marine fish as well, the discussion below will concentrate mainly on colonial marine invertebrates with examples from animals belonging to two major phyla, cnidarians and urochordates (tunicates). Sexual propagules Larger numbers of sexually-derived propagules may be formed by single invertebrate specimens through broadcasting ofgametes into the water column or by larval brooding, the two most common sexual reproduction pathways. One ofthe dramatic examples for the reproductive potentiality is the yearly mass-spawning event in the Great Barrier Reef, Australia where immense amounts of germ cells are simultaneously released into the water by hundreds ofcoral species during just one or two nights (Harrison et aI., 1984; Alina and Call, 1989). Only a minute fraction of propagules develop successfully into coral colonies. For example, Weil (1990) estimated a yield of 1-3 x 106 released larvae from Litophyton arboreum colonies (a Red Sea soft coral) within a distinctive reef area in Eilat. Detailed field observations have documented only a single recruited young 1. arboreum colony. Similar results of very low numbers of recruits were also obtained in other soft and hard corals in different reef areas worldwide (Farrant, 1987; Alino and CoIl, 1989). On the other hand, ex-situ maintenance of coral larvae may result in up to 100% metamorphosis and settlement (Ben 34 David-Zaslow and Benayahu, 1998) and survivorship rates may exceed 30% following >200 days of aquaria maintenance (Gatefio et aI., in press). In another study (SWesinger, 1985) up to 1000 planula larvae per coral head were collected from the branching coral Seriatopora caliendrum. They usually settled 6-8 h after release. More than 84% survived the following 3 days. Out of 1,900 settle planulae studied, >30% survived for 1 month, 3.5% for 3 months and 0.35% for 1 year, in non-optimal mariculture conditions. Ex-situ survivorship rates, therefore, exceed in situ rates by several orders ofmagnitude. Spawning among captive coral species has been observed in many cases although colony formation following these spawning events has not yet been reported (Atkinson et aI., 1995; Carlson, 1999). We therefore still are dependent on organisms grown in situ. Collections of reproductive products from these organIsms may be carried out through several previously established, ecologically-friendly protocols. In the field, collections are made by plankton nets placed over gravid coral colonies (Rinkevich and Loya, 1979). Inland collections (Sh1esinger, 1985; Yates and Carlson, 1993; Gatefio et aI., in press) are performed by transferring gravid colonies from the field into aquaria just before they spawn gametes or shed the planula larvae. There are many similar studies that document high rates of settlement of coral larvae in ex-situ conditions (for example: Harrigan, 1972; Rinkevich and Loya, 1979; Goreau et aI., 1981; Sato, 1985; Shlesinger, 1985; Harrison and Wallace, 1990; Wei!, 1990; Gatefio et aI., in press) or other marine invertebrates (Kinne, 1997; Carroll and Kempf, 1990; Goto and Yoshida, 1997; Rinkevich and Shapira, 1998). A major benefit of the ex-situ settlement approach is based on the higher yield of larvae settlement than under natural conditions and higher survival rates. As a result, significantly greater numbers of adults are obtained from the same initial stock of larvae. Improved culture/settlement conditions (such as the provision of ameliorate substrate for settlement; Rinkevich, 1995; Gatafio et aI., in press) has resulted in further augmenting the rates ofsuccess. The approach ofsupplying the marine ornamental industry with sexually-derived propaguIes further preserves the genetic diversity of the species of interest. Moreover, "genetic diversity of some species may be maintained in thousands of public aquariums, home aquariums of research laboratories" (Carlson, 1999). This is essential for reef conservation purposes, where some of the developed animals may be returned to the reefs to restore denuded reef areas. On the other hand, high genetic variation does not coincide with customary aquaria conditions where different genotypes require diverse rearing constrains. 35 Nubbins The term "nubbins" has been coined by Birkeland (1976) for small coral pieces and has been adapted by several authors (reviewed in Davis, 1995) to characterize isolated branch tips. Nubbins (Webster: something that is small of its kind) is further used here to describe an isolated, minute portion of an organism (for example: in corals, a single polyp or a group of a few polyps; also see AI-Mograhbi et aI., 1993) which has the capabilities to regenerate to a whole organism. Fragmentation, the creation of several to many ramets subcloned (naturally or man-made) from a specific genet is a common and fruitful procedure that is often used to increase the number of available organisms (summarised in Highsmith, 1982; Rinkevich, 1995; Tambutte et al., 1995; Rinkevich and Shapira, 1998; Carlson, 1999). When using fragmentation protocols for ex-situ culture purposes, various biological and economical tradeoff evaluations should be taken into consideration. Thoughtful attention is given to the contrasting outcome of either pruning many small fragments from a single colony or subc10ning a few large ones. SubcIoning large fragments from colonies may be a maladaptive process to the colonies due to the possible stress inflicted upon fragmentation. On the other hand, subcloning of small fragments may reduce the stress inflicted on the colonies but may increase the mortality of the ramets. From a commercial point of view, however, the first approach may reveal short-term cultivation periods before the subc10nes are amenable for trade, while the second approach, although associated with long culturing periods, may result in more material. In most cases, the decision between the two subc10ning approaches is based on economical equations. A third approach, the use of numerous numbers of minute fragments from a single colony (nubbins) for the development of vast numbers of new colonies is discussed below. This novel approach is illustrated below by the findings from two different invertebrate phyla. Colonies of the cosmopolitan encrusting sea squirt Botrylloides are very common members of benthic assemblages in temperate to tropical zones, including coral reefs. In this group of organisms, zooids are arranged symmetrically in groups embedded within translucent organic matrix (the tunic) and are connected to each other via a ramified network of blood vessels. From this blood system, many pear-shaped vascular termini (called ampullae) are extended all along the peripheral margins of the colony. Light microscopy observations reveal that the ampullae are very delicate structures with walls essentially one cell thick (Milkman, 1967). In some species of this genus, any minute fragment of peripheral blood vessel containing a limited number (l00-300) of blood cells has the capacity to regenerate to fully functional organisms (including gonads) within a very short period of about 1-2 weeks (Rinkevich et aI., 1995, 1996; Fig. la-d). Regeneration 36 resulted from a small number of totipotent stem cells circulating in the blood system. The developmental process starts from the formation of chaotic masses of blood cells through blastula-like structures which develop rapidly to normal zooids possessing all 3 embryonic layers. By employing the ampullae regeneration protocol (Rinkevich et aI., 1995, 1996), any single genet may be the source for production ofhundreds oframets. The branching Red Sea hermatypic coral Stylophora pistillata serves as a model coral species for a variety of purposes, including in situ restoration activities (Rinkevich, 1995). Colonies of this species exhibit an axially rod-like growth form with up-growing branches that are primarily added by dichotomous fission of the branch and laterally growing branches, some growing on the inside and others on the outside orientations (Rinkevich and Loya, 1985). The typical pattern formation of an S. pistil/ata colony approximates the symmetry ofa sphere. In a recent experiment, we employed the protocol of subcloning from adult colonies many nubbins, in the size of a single or only a few polyps each (a polyp is approximately 2 mm in diameter). Nubbins were pruned from branch tips and bases and under this protocol, a single colony may completely be divided into several thousands of different sized nubbins, down to lor 2 polyp-sized. Survivorship ofnubbins is very high (almost 100%) and an immediate regeneration process followed by fast growth rates (Fig. 2a-c) has been observed during the first few months after pruning the colonies (nubbins at the average size of 5 polyps grew to the size of45 polyps within 90 days; S. Shafir, unpublished). Both examples describe cases where hundreds and thousands ofnew organisms (colonies) per single colony may be produced by subcloning minute fragments from adults. Although this protocol is based on a long-term cultivation approach, private enterprises may benefit from it as a result ofthe production ofalmost unlimited replicated material readily available for a steady supply in the trade of a specific species. A routine protocol of nubbin subcloning is expected to support sustainable aquaculture for the marine aquarium trade and laboratory ecotoxicological studies with the minimal need for field collections of new material. This procedure may be performed on other colonial marine organisms such as sponges (Osinga et aI., 1998; Wilkinson and Vacelet, 1979), bryozoans and more. Viable minute pieces isolated from hermatypic corals can also be produced by pinching off small pieces of coral tissues without skeletal material with a forceps. In one experiment, separated tissue fragments were organized into flattened, ciliated, motile bodies and within a few days to one month, they differentiated into polyps, settled and began to secrete skeletons (Ritchie et aI., 1997). Colonies produced by this procedure do not exhibit any genetic variation between ramets ofthe same genet and are amenable for standardized maintenance conditions. 37 Replicates- and inbred-lines Improved ex-situ maintenance and culturing methods for marine invertebrates is one of the goals in the domestication of these species (Yates and Carlson, 1993). Improved husbandry methods are crucial prerequisites for two approaches ofmass production ofanimals: clonal replicates and inbred lines. Each approach has its own benefits and deficiencies; each approach is targeted for different purposes. Clonal replicates (the formation ofnumerous nubbins or subcloning a colony to few/many ramets) is an excellent approach to reduce the impact of animal collections on natural habitats (coral reefs, mangrove swamps, etc.). There are species that are more amenable for fragmentation and yet others that are less adapted (Yates and Carlson, 1993; Atkinson et al., 1995; Ritchie et al., 1997; Rinkevich and Shapira, 1998; Carlson, 1999). Therefore, the approach should be applied to species that are expected to yield good results. The employment of this technique provides large numbers of genetically identical ramets which may be reared ex-situ under identical conditions, eliminating, for example, variation in growth rate imposed by the highly polymorphic genetic properties characteristic of many marine invertebrates (for example, Rinkevich and Shapira, 1998). Such duplicate clones could be very valuable also in biological studies (physiology, biochemistry, etc.) and are a good source of material for coral trade. Uniform genetic material, on the other hand, can be a problem when, for example, a disease susceptible or a senescent clone is reared. Then, all or most replicates may be lost within a short period oftime. The consideration of the use of inbred lines (Rinkevich and Shapira, 1998), a novel approach in ex-situ invertebrate cultures involves a considerable amount of time and effort. In this approach, conspecifics are self-crossed (where applicable) or defined-crossed, under controlled aquarium conditions, to produce generations of homozygotes organisms on specific morphological (such as color morphs) physiologicallbiochemical (like specific temperature resistance), or any other biological trait the breeder may choose. A successful establishment of long-term, defined genetic stocks of selected ornamental saltwater invertebrates may be considered as a breakthrough in the marine aquarium industry. Currently, collection protocols from the wild may suffer from seasonal availability of certain species, from natural genetic variations, from problems in ex-situ acclimatization and with no efficient laboratory methodologies to completely circumvent the need for sporadic collections from the field (Rinkevich and Shapira, 1998). Inbred lines established from key species in the aquarium trade may overcome the above-mentioned obstacles. It is therefore encouraging that in at least one case, the development of a sea squirt inbred line has been documented (Rinkevich and Shapira, 1998). In this study, an 38 inbred line of colonies from the tunicate Botryllus schlosseri, an important model species in the study of the evolution of immunity, has been cultured for over 7 years through 5 successive generations ofself-crossed offspring. Inbred lines and other laboratory established species may also be a key issue in resolving conceptual points in conservation genetics such as inbreeding-outbreeding depressions, genetic adaptation to captivity and its affect on reintroduction success, mutational accumulation, and more (Frankham, 1999). Marine invertebrates are characterized by high variability in life history patterns (Kinne, 1977). For example, in the case of B. schlosseri, colonies originating from the same hatch ofa specific mother colony , under the same ex-situ culturing conditions and even within the same aquarium, showed differences in growth rates which exceeded one order of magnitude or showed high, intensive reproductive states versus sterility (Rinkevich and Shapira, 1998). Success in ex-situ cultivation of such physiologically variable taxa depends on the ability to develop critical maintenance protocols which are not affected by high genetic heterogeneity. Inbred lines of organisms adapted to aquaria conditions and characterized by different morphological/physiological traits, are therefore, one of the best solutions for this problem. There are however some drawbacks and costs to the production ofinbred lines. Inbred-line animals will probably manage poorly in natural conditions and therefore may not be considered as a potential source for coral reef restoration. Inbred colonies may become sterile, a phenomenon which is not related to poor conditions but which results from genetic disorders (Rinkevich and Shapira, 1998) or from a general inbreeding depression (Sabbadin, 1971). Perspectives In addition to the destruction imposed on the coral reef as the result of animal collections, the high losses between capture and final retail distribution has its own significant toll (Lubbock and Polunin, 1975; Best, 1997). For example, thousands of dead coral colonies per shipment were confiscated by the Dutch authorities during 1992-1994 interception actions (Best, 1997). These shipments originated from the Philippines, Indonesia, Mexico, Miami and other places, may reveal the global impact of the issue for animal losses during collections. The perceived expansion in animal trade further augments the potential threat to the fragile coral reefs. Minimizing the damages to areas under collection pressures can therefore be achieved through collections in a nondestructive manner (such as planula larvae collections by plankton nets; Rinkevich and Loya, 1979), by returning animals that are raised ex-situ to in situ conditions (Gatefio et aI., in press), or alternatively, by raising material for the trade independently with field organisms (Yates and Carlson, 1992). Domestication of many reef invertebrates for the purposes outlined in this 39 manuscript is therefore a feasible task, an attractive approach from economic, social, educational and ecological perspectives. Three concepts for the domestication approach were outlined: the use of sexual products, asexual propagation through numerous of nubbins and the development of inbred lines versus replicated lines (by applying the two concepts above, respectively). As previously discussed, each rationale bears its own benefits, costs and pitfalls. The combined use of the above mentioned concepts will help in the basic idea of limiting, to a minimum, all field collections. Unfortunately, much of the information regarding ex-situ maintenance of marine ornamental organisms is anecdotal and reported in hobby magazines and popular books, instead ofscientific journals (Carlson, 1999). There are important additional concepts which were not yet discussed in detail since they were involved in only limited experimental approaches. One of the most promising avenues is the employment ofcryopreservation. Cryopreservation has been widely used for long-term conservation of a variety ofvertebrate cells and organs, cell lines and insect lines and has been tested in marine inveltebrate cell and tissue cultures as of 1992 (Rinkevich, 1999). Cryopreservation, as is rationalized here, may be applied on two major sources: the germ cells and embryos of ornamental marine invertebrates. It is supposed to provide a continuous, year-round supply of various material with similar qualifications. Cryopreservation of gametes and embryos is also critical for supporting and developing inbred lines from ornamental marine organisms. Single cryopreserved collections of huge numbers of larvae, eggs and sperm bundles that have been shed from marine organisms (such as corals) will be used by breeders year-round, independent of reproductive seasons, under favorable, controlled conditions and with expected increased yields. A few studies have already examined the feasibility of whole cryopreserved embryos from marine invertebrates. This was performed on bivalve oocytes, trochophores and veligers (Odintsova and Tsal, 1995; Nadienko, 1997), on polychaete larvae (Olive and Wang, 1997), on sponge parenchymella larvae (Rinkevich et aI., 1998) and others. These studies have been performed for the production ofproliferating cell culture from marine organisms, for developing live organisms or for live garnets. With bivalve larvae (Odintsova and Tsal, 1995), the veligers showed high cell viability (>90%) while only <35% of the trocophore cells survived the freezing/thawing protocol. In sponge larvae (Rinkevich et aI., 1998) also, >70% of embryonic cells survived cryopreservation. The cryopreservation approach therefore deserves further consideration. Conceptions for husbandry methods should also be considered as important figures in the rationale for the marine aquarium industry. Topics such as spacing between marine invertebrates maintained within the same aquarium and co-culturing of different animals of the sarne species, different 40 species of the same taxon or organisms from different taxa (which raise the ecological issues of allelopathy, interspecific and intraspecific competitions, predation, chimerism and parasitism) were not yet discussed in detail with regard to the tropical marine aquarium trade (but see Lubbock and Polunin, 1975). Another important concept is the threat of introduction of non-indigenous marine organisms by aquarists. More than 30 years ago, Torchio (1968) already reported the appearance of an Indo-Pacific fish in Italian Mediterranean waters. This problem of invasion of non-indigenous organisms is currently envisaged as carrying potentially significant economic, aquacultural and health issues (Jousson et al., 1998). This problem, however, is not strictly related to ex-situ cultures but is also relevant to the whole trade, especially for the sectors where animals are directly collected from the field. The development and the use of inbred lines of ornamental organisms, adapted specifically for aquaria conditions, may reduce the threats from uncontrolled invasions of marine organisms through marine aquarium trade. Coral reefs as other tropical habitats worldwide are continuously deteriorated from a variety of human activities, despite national and international legislation and rules (Rinkevich, 1995). This reef destruction is partly inflicted by animal collections for the trade of ornamental organisms (Lubbock and Polunin, 1995; Mamonov, 1980; Heslinga and Fitt, 1987; Yates and Carlson, 1993; Gateno et a1., in press). It is also evident that the international trade of saltwater ornamental organisms is going to expand. 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(1968) Sulla eventua1e presenza in acque mediterranee di individui dei generi Cephalopholis Bl. Schn, e Chaetodon L. Natura, Milano 59, 210-212. Weil, D. (1990) Life history of the Alcyonacean Litophyton arboreum in the Gulf of Eilat: sexual and asexual reproduciton. M.Sc. Thesis, Department of Zoology, Tel Aviv University (Hebrew with English summary), 142 pp. Wells, S.M. and Alcala, A.C. (1987) Collecting of corals and shell. In: Salvat, B. ed., Human Impacts on Coral Reefs: Facts and Recommendations. Antenne Museum E.P.H.E., French Polynesia: pp. 13-27. Wilkinson, c.R. and Vacelet, J. (1979) Transplantation of marine sponges to different conditions of light and current. Journal ofExperimental Marine Biology and Ecology 37,91-104. Worthman, S.G. (1974) A method of raising clones of the hydroid Phialidiuim gregarium (A. Agassiz, 1862) in the laboratory. American Zoologist 14,821-824. Yates, K.R. and Carlson, B.E. (1993) Corals in aquariums: how to use selective collecting and innovative husbandry to promote coral conservation. In Richmond, R.H. ed., Proceedings ofthe Seventh International Coral ReefSymposium, Mangilao, University ofGuam 2, 1091-1095. 45 FIGURE LEGENDS Fig. 1. Regeneration of a whole urochordate from isolated peripheral ampullae. a: A group of ampullae embedded within the tunic matrix are isolated from a Botrylloides colony, b. 3 days later, arrangements of the ampullae within the nubbin center, c: one week thereafter, formation of a bud within the opaque center, d: formation of a functional zooid with a typical set of peripheral ampullae, resembling a young developed oozooid (the first zooid metamorphosed from the settling tadpole larva). a = ampulla, b = bud, s = exhalant siphon, t =tunic matrix, z =zooid. Fig. 2. Regeneration of a small Stylophora pistillata nubbin a = Size of 3 polyps, 19 days after separation of a single polyp, b = 54 days after separation, the regenerated nubbin has started to spread over the substrate (10 polyps), c = 91 days after separation, a small colony of 22 polyps. Polyps that are in the focus plane of photographing are marked (P). An asterisk refers to a small piece of broken skeleton which was covered with tissue only at day 19 without being cemented to the nubbin but became an integral part ofthe nubbin at day 91. 46 i a b c d Flg!lre 1. Regeneration of a whole urocnordate (Born/lOidesl from Isolated penpheral ampullae. 1a: Day I. Group of ampullae embedded wnhm the tumc matnx. Isolated from a BorryllOiJes colony. 1b: Day 4. Arrangements of the ampullae wlthlO the nubbm centre. Ie: Day 11. Formation of J bud wlthm the opaque centre Id: Day :ZO. Fonnanon of a funcHol1a! zooid with a typical set of peripheral ampullae. resembling a young developed oozoOid !the fir,t ZOOid metamorphosed from the settlmg tadpole larval. Legend: a = ampulla. b ;;;: bud. s :=;; exhalant 'ilphon. t :=;; tume m.mn. Z := ZOOid. Each ampulla IS ca. 0.1 mm m length. 47 Figure 2. Regeneration of a small Srylophora pistil/ala nubbin. ::!a = 5 polyp~. ::! days after ~eparatlon Area' = [207 mm:. ::!b = 11 polyps. 33 days after separation. Area' = 2-1-.91 mm:. The regenerated nubbm has started to spread over the substrate. 2c = 19 polyps. 5'+ days after ~eparatlOn. Area' =45.35 mm;. Legend: p =polyp. Scale bar = 1mm.•Area measured using the top projection Image of the nubbtn. BEST AVAILABLE COpy 48 SUBJECT 4: EXSITU MAINTENANCE INTRODUCTION The ex-situ maintenance of reef dwelling invertebrates, although still in its early stages, is an important prerequisite for the supply of ornamental marine species for the aquarium industry (Helsinga, 1996), as well as for educational purposes (Evans, 1997) and for marine conservation (Mamonov, 1980). The current uncontrolled collections of a variety ofreef invertebrates and fishes may result in the extinction of species and the destruction of marine habitats (Wells and Alcala, 1987; Sadovy, 1992; Best, 1997). Additionally, the ability to breed and maintain aquatic invertebrates in aquaria may significantly contribute to our knowledge ofthe life histories ofthese organisms (Kinne, 1977; Emschermann, 1987; Rinkevich and Shapira, 1998). A variety of anthozoans have been reared under laboratory conditions (earlier studies are summarized in Kinne, 1977). Some have been cultivated with considerable success, over several years. However, most published studies on anthozoans concentrated on the maintenance of scleractinian forms (Kinne, 1977; Ritchie et aI., 1997) and only very few earlier studies (references cited in Kinne, 1977) have studied octocorallians as candidates for aquarium culture. Moreover, reef dwelling alcyonarians have not been raised for extended periods in aquaria under controlled conditions although many aquarists have successfully reared a variety of alcyonarians (unpublished). Given the increasing impact ofthe international trade in reef organisms on the marine environment (Wells and Alcala, 1987; Sadovy, 1992; Helsinga, 1996; Best, 1997) the aquaculture of such organisms ex-situ to supply the trade may be preferable to coral reef harvesting (Mamanov, 1980; Helsinga, 1996; Sykes, 1997). Furthermore, aquaculture may also provide a means for restocking impoverished reef habitats (Maroz and Fishelson, 1997). This culture should ideally be performed using established protocols, and with the application of sustainable mariculture techniques (Helsinga, 1996). Material for the above needs can be obtained as coral fragments subcloned from mature organisms/colonies or as larvae collected either in situ or from animals reared in the laboratory. Here, we present aquarium results on the successful rearing of 3 Red Sea alcyonarians originating from planula larvae collected from gravid colonies growing in the field. MATERIALS AND METHODS Species used and larvae collections 1. Clavularia hamra Gohar 1948 CClavulariidae). An encrusting, soft coral that inhabits shallow waters on the Red Sea. It is a dioecious surface brooder where embryogenesis takes place on 49 the surface of female colonies within a mucoid suspension during short periods in the summer. The nonsymbiotic larvae complete their development within six days after fertilization (Benayahu, 1989). Embryos for this study were collected with a pipette from the surface of one colony on July 31, 1996, at a depth of 0.5 m, situated north of the H. Steinitz Marine Biology Laboratory (MBL) in Eilat. Embryos (~300) developed into mature larvae within 6 days after collection. 2. Nephthea sp. (Nephthidae). An arborescent soft coral, found in Eilat's shallow waters on the reef table only. This species is a dioecious brooder that releases zooxanthellate larvae during the summer (Ben-David-Zaslow, 1994). In the present study, small coral fragments (5-10 em in length) were collected during the reproductive seasons in 1994 and 1995, from Big Routa, Sinai (N 29°11'00", E 34°43'45") and from the Coral Reef Nature Reserve at Eilat. Each branch was held separately overnight in an aerated tank (3.3 1) at the MBL and released larvae (-700) were collected with Pasteur pipettes during the next morning. The coral fragments were returned thereafter to the area from which they were collected. 3. Litophyton arboreum Forskal 1775 (Nephtheaidae). An arborescent soft coral, similar in morphology to Nephthea sp. that inhabits deeper waters, down to 18 m in the Northern Gulf of Eilat. It is a dioecious brooder that releases zooxanthellate larvae during the summer and autumn. Spawning occurs usually between June and September (Weil, 1990), although we also collected larvae later on, between October and November 1996. For this study, small branches (5-10 cm in length) were collected from gravid colonies in front of the MBL on November 6, 1996 and held overnight in an aerated tank (3.3 1). The released larvae (n=138) were collected the following morning with a Pasteur pipette and adult fragments were returned to the sea. Laboratory conditions After collection, all larvae and embryos were washed with natural seawater several times to eliminate excess mucus and placed in 50 ml tubes or 400 m1 plastic containers, depending on the number of larvae. Collected material was placed in a thermally insulated cool-box and transported to the National Institute of Oceanography in Haifa, Israel, approximately 500 km north ofEilat. Upon their arrival at the laboratory, larvae were placed in open petri dishes (plastic; 60 or 90 mm in diameter). In some dishes, small stones or hard coral skeleton fragments (natural substrates), freshly collected from the sea were added. Different numbers of larvae (C hamra: ~100; Nephthea sp.: 12 to 100; L. arboreum: 33 to 35) were placed in each dish. Larvae were kept in a temperature 50 controlled room at 25°C with constant light. Water was changed every 48 hours with fresh 20j.Lm filtered, non-sterilized seawater. After larval settlement and metamorphosis, dishes were transferred to 17 I aerated aquaria filled with running (1 I per min.), filtered (20 j.Lm) seawater at 24±1 DC. Photosynthetic photon flux (PPF) was provided by 500 W halogen lamps at 12:12h (light: dark) regimen. PPF on the surface of the aquaria, measured with a quantum meter (LI-COR, North 2 Carolina, USA), averaged 150 !lmol photon m- S-l, which is approximately the average irradiance at 10 m depth (Gateno et al., 1998). Corals were fed twice a day with either 50 !lm microcapsules artificial plankton (Argent, Redmond, USA), three times a week with live (1-2 days old) Artemia nauplii (Neptune Industries, Salt Lake City, USA) or once a week with lyophilized rotifers and live unicellular algae (Nannochloropsis sp.). Dishes, stones and coral skeletons were periodically cleaned from fouling organisms (mainly filamentous algae) using razor blades, forceps and fine paintbrushes. We documented coral survivorship and growth rates. Growth was measured in terms ofadditional polyps per unit of time. To test the effect of water flow on the coral development, we placed a small 12 v electric water pump (ITT Jabosco) in one 1. arboreum aquarium. A plastic barrier has divided this aquarium (17 1) into two equal longitudinal parts to allow circular water flow ofabout 6.71/min. RESULTS Larvae of the three soft coral species clearly preferred to settle on the natural substrates covered with organic materials. In all dishes, where freshly collected natural substrates were provided, larvae settled almost exclusively on them. Settlement rate was also faster as compared to that in dishes without natural substrates. 1. Clavularia hamra. Embryos developed into mature red larvae within 6 days after collection. Larvae were transferred to three petri dishes (90 mm diameter; ~ 100 larvae/dish) containing coral fragments. Four days thereafter, 106 planula larvae were settled (103 on the natural substrates, 3 on the petri dishes). Metamorphosis was completed within 6 days. Immediately after metamorphosis, the pale white primary polyps started to develop white spiculae. The spiculae colour changed from white to red 17-27 days after metamorphosis. At the age of one month, zooxanthellae began to appear and by the end of the second month, all polyps contained dense amounts of zooxanthellae. At this age, polyps were brown with red spiculae, typically characteristic of adult colonies. Most larvae (85%) settled as aggregates of 2 to 17 polyps, fused and formed chimeras. 51 Out ofthe 106 larvae that settled on the coral fragments, almost 18% survived for at least 307 days when the experiment was terminated (Fig. 1). Most mortality (75%) was recorded in the first 115 days. The main reasons for mortality were coral overgrown by algae (61%), detachment from substrates during cleaning (9.9%), sampling for histological studies (6.9%) and resorption by other partners in the chimeras (4.0%). To reduce the negative impacts ofthe overgrowing filamentous algae, the aquaria were placed in the dark at day 252 of the experiment for a period of 5 days and then returned to former conditions. This procedure eliminated overgrowing algae and since then, no further mortalities were documented (Fig. 1). From the 19 primary polyps that survived the entire observation period (307 days), only 4 developed an additional, secondary polyp. Our culturing conditions clearly do not sustain the growth ofyoung C. hamra colonies. 2. Nephthea sp. Planulae were placed in petri dishes with or without natural substrates. In the dishes lacking natural substrate, larvae started to settle only after 10 days, and completed settlement 20 days thereafter. Planulae in dishes with natural substrates started to settle within a few hours after introduction and within two weeks all larvae were settled. When natural substrate was presented, no settlement was recorded on the walls of the petri dishes. Larvae metamorphosed within 24 hours after settlement (n=258), developing into primary polyps with open mouths and tentacles. These primary polyps started to bud additional polyps after 2 weeks. Figure 2 depicts Nephthea sp. survivorship ofalmost 17% (n=43) after a period of 16 months. Mortality was high during the first three months (65.1 %). A breakdown of the water temperature control system between days 56 and 65 (water temperature increased to 41°C) was considered to be responsible for an additional 37.2% mortality. After the age of 4.5 months, no further mortalities were recorded over the following 12-month period. The number of polyps per colony (Fig. 2) depicts a logarithmic growth pattern. At age 225 days, the average colony size was 54.9±27.7 (n=8) polyps, while after 460 days, the average number of polyps per colony reached the size of324.5±21 0.1 (n=8) polyps. These colonies were up to 20 em high (Barki, 1999). 3. Litophvton arboreum. From the 138 larvae collected (Table 1) only 60 (43.5%) were settled either on the plastic petri dishes (18.9% within 7 days) or on the natural substrates provided (67.2% within 24h). They immediately metamorphosed into primary polyps. Larvae settled significantly faster (p high during the first month (43.3%) but stabilized at age 108 days (65.0%), until the end of observations (Figure 3). At age 66 days, the 28 remaining colonies were divided into two groups, one group was held in static water (n=18) and the other held in flowing water (n=IO). Both groups were maintained in 17 I aquaria. Mean colony sizes in both aquaria was initially equal (p>O.05; t test). Figure 3 depicts the mean number ofpolyps for the colonies grown with and without water flow. Water flow increased growth rates. Colonies in the circular flow aquarium grew faster than those in the static seawater tank and over the following 19 days (age 85 days) they became significantly larger (p DISCUSSION The results ofthe present study not only show that soft coral mariculture in aquaria is amenable but that different alcyonarian species require different maintenance conditions (such as water movement, circulating current aquaria vs. static water aquaria). The planula larvae of these sessile forms are able to metamorphose in relatively high numbers without the need for special externally added food. The ample numbers of metamorphosed larvae and the low mortality during prolonged periods ofcultivation for more than 1 year (as compared to mortality expected in the field) provide a potential tool for mass cultivation of alcyonarian colonies in captivity. The results of the present study also indicate an improved yield when compared to other aquarium culture studies. For example, Weil (1990) reported lower rates of 1. arboreum larvae settlements (in 6 out of his 10 experiments settlement rates ranged from 10.3%-29.5%) and survivorship (7.5% after 1 year). Higher settlement rates of L. arboreum larvae (up to 60-80%) have been recorded after 100 days (Ben-David-Zaslow and Benayahu, 1998), a much longer free-swimming period than the present study (100% settlement, two weeks in dishes with natural substrates). Additionally, young 1. arboreum colonies under the conditions reported here grew faster than in an earlier study by Wei! (1990) where primary polyps bud secondary polyps only 6 months after settlement and reached sizes of4-9 polyps only at the age of 11 months. In most published cases (Kinne, 1977), the methodologies employed for anthozoan mariculture were relatively simply since within the Cnidaria food specialists are rare, and most reef dwelling cnidarians reared in aquaria are symbiotic organisms. For such organisms, general improved maintenance protocols are easily available, and are known to aquarium hobbyists and to the aquarium industry (Adey and Loveland, 1991; Delbeek and Sprung, 1994; Sprung and Delbeek, 53 1997). Aspects of developmental phases, sexual reproductive modes and seasonalities, morphological differentiations, and different life history parameters of many anthozoans are already well studied and available in the literature. The time is ripe, therefore, for experimentalists to establish more defined, controlled experiments for the facilitation and the improvement of culture conditions in order that high numbers of reef dwelling invertebrates can be successfully reared in aquaria. This would serve a variety ofpurposes such as supplying material for the coral trades; for education; reseeding depleted natural stocks; conservation, etc. (Mamonov, 1980; Wells and Alcala, 1987; Sadovy, 1992; Best, 1997; Helsinga, 1996; Evans, 1997; Maroz and Fishelson, 1997; Sykes, 1997). The 3 alcyonarian species were chosen primarily because of the availability of larvae and not because we had previous information that they thrive under aquarium conditions. This study is a preliminary, introductory approach which clearly indicates that the applications of sustainable aquaculture techniques are highly amenable to coral culture. For example, our results on L. arboreum revealed survivorship rate of approximately 30% following 207 days of continuous aquaria maintenance. Weil (1990) recorded in a span of six months 100% mortality of 3,680 primary L. arboreum polyps settled in the laboratory and transferred to the field. In the study by Weil (1990) ofestimated 1-3.106 larvae released within a distinctive reef area in the Red Sea, only one established as a young L. arboreum colony. A very low survivorship rate after one year (0.26%) has also been recorded in a soft coral (Capnella gaboensis) under field conditions (Farrant, 1987) and in 6 other soft corals from the Great Barrier Reef, Australia following 2-4 months after settlement (Alino and ColI, 1989). By contrast, in the present study, our results further indicate the potential conservation benefit of raising coral larvae under ex-situ conditions. In our study many more young colonies survived and can be raised from the same number ofreleased larvae. It is also evident, however, that each stage in the development ofa young alcyonarian organism may raise the needs for developing different rearing conditions (such as accessibility of natural hard substrates for increasing settlement/metamorphosis rates, the development ofcirculating water tanks for improving growth rates, improving light and nutrient conditions for the reduction of algae development, the establishment of better colony cleaning methodologies in order to reduce young specimen mortalities. As culture conditions become more improved and refmed it should be possible to realise high success rates in the culture ofoctocorals using experimental protocols. REFERENCES Adey, W.H. and Loveland, K. (1991) Dynamic Aquaria. Building Living Ecosystems, Academic 54 Press, San Diego, 643 pp. Alino, P.M. and ColI, lC. (1989) Observations of the synchronized mass spawning and post settlement activity of octocorals on the Great Barrier Reef, Australia: biological aspects. Bulletin ofMarine Science 45, 697-707. Barki, Y. (1999) Aspects in the development, allorecognition responses and genetics in Red Sea soft corals. Ph.D. Dissertation, Tel Aviv University (Hebrew with English summary), 97 pp. Benayahu, Y. (1989) Reproductive cycle and developmental processes during embryogenesis of Clavularia hamra (Cnidaria, Octocorallia). Acta Zoologica (Stockholm) 70, 29-36. Ben-David-Zaslow, R. (1994) Longevity and competency ofplanulae ofOctocorallia. M.Sc. Thesis, Tel Aviv University (Hebrew with English summary), 67 pp. Ben-David-Zaslow, R. and Benayahu, Y. (1998) Competence and longevity in planulae of several species ofsoft corals. Marine Ecology Progress Series 163, 235-243. Best, M.B. (1997) Trade in corals and living stones. Proceedings ofthe 6th International Conference on Coelenterate Biology, pp. 47-52. Delbeek, lC. and Sprung, 1 (1994) The Reef Aquarium. Ricordea Publishing, Coconut Grove, Florida. Vol. 1, 544 pp. Emschermann, P. (1987) A circulating water tank for culturing sessile or hemisessile aquatic organisms in a continuous water current. Archives in Hydrobiology 108, 425-438. Evans, K.L. (1997) Aquaria and marine environmental education. Aquarium Sciences and Conservation 1, 239-250. Farrant, P.A. (1987) Population dynamics of the temperate Australian soft coral Capnella gaboensis. Marine Biology 96, 401-407. Gatefio, D., A. Israel, Y. Barki and B. Rinkevich (1998) Gastrovascular circulation in an octocoral: Evidence of significant transport of coral and symbiont cells. Biological Bulletin 194, 178-186. Helsinga, G.A. (1996) Sustainable aquaculture in the marine aquarium industry. Proceedings ofthe Pacon Conference on Sustainable Aquaculture: 174. Kinne, O. (1977) Cultivation of animals. In: Kinne, O. ed., Marine Ecology. Vol. III. Cultivation, Part 2, John Wiley & Sons, New York: pp. 579-668. Mamonov, G.A. (1980) Aquarium hobby and nature conservation. Przeglad Zoologiczny Zoology 24,241-247. Maroz, A. and Fishelson, L. (1997) Juvenile production of Amphiprion bicinctus (pomacentridae, Telostei) and rehabilitation of impoverished habitats. Marine Ecology Progress Series 151, 55 295-297. Rinkevich, B. and Shapira, M. (1998) An improved diet for inland broodstock and the establishment of an inbred line from Botryllus schlosseri, a colonial sea squirt (Ascidiacea). Aquatic Living Resources 11, 163-171. Ritchie, R.J., Eltringham, K., Salih, A, Grant, Al, Withers, KJ.T. and Hinde, R. (1997) Plesiastrea versipora (Lamarck, 1816). The white rat for coral research? Proceedings ofthe 6th International Conference on Coelenterate Biology: pp. 403-408. Sadovy, y. (1992) A preliminary assessment of the marine aquarium export trade in Puerto Rico. Proceedings of the i h International Conference on Reef Symposium, Guam, Vol. 2, pp. 1014-1022. Sprung, 1. and Delbeek, J.C. (1997) The ReefAquarium. Ricordea Publishing, Coconut Grove, Florida. Vol. 2, 546 pp. Sykes, G.R. (1997) Coral aquaculture. An alternative to coral reef harvests. Aquartist and Pondkeeper 61, 6-9. Weil, D. (1990) Life history of the Alcyonacean Litophyton arboreum in the Gulf of Eilat: sexual and asexual reproduction. M.Sc. Thesis, Department of Zoology, Tel Aviv University (Hebrew with English summary), 142 pp. Wells, S.M. and Alcala, AC. (1987) Collecting of corals and shell. In: Salvat, B. ed., Human Impacts on Coral Reefs: Facts and Recommendations. Antenne Museum E.P.H.E., French Polynesia: pp. 13-27. Table 1. Settlement preference of Litophyton arboreum larvae with or without the addition of freshly collected natural substrates. Petri dish Natural No. oflarvae settled no. larvae % Settled Substrate 1 + 35 23 65.7 2 + 35 24 68.6 3 35 9 25.7 4 33 4 12.1 56 FIGURE LEGENDS Fig. 1. Survivorship (%) of Clavularia hamra colonies from metamorphosis (day 0) until the age of307 days. Fig. 2 Survivorship (%; dots) and average number of polyps (triangles) ofNephthea sp. colonies from metamorphosis (day 0) until the age of460 days. Bracket depicts mortality caused by the water temperature system failure. Fig. 3 Survivorship (%) and average number of polyps of Litophyton arboreum colonies from metamorphosis (day 0) until the age of 203 days. An arrow indicates the age at which the circular water flow aquarium has been activated. 57 100 90 80 70 C- 60 ~ iii ...Q 50 0:;> ... 4Q en= 30 20 10 0 0 60 120 180 240 300 Age (days) F'eJ 1 58 100 350 90 300 80 70 250 III Co >- 60 'E ~ Co .g- 200 '0.. Ui Ql .. 50 ,Q 0 E ~ ~ 'S; .. 150 ~ :: 01 Vl 40 ..0:1 ::-OJ 20 50 10 0 0 0 60 120 180 240 300 360 420 480 Age (days) 59 • 100 16 90 -Sumvcr.dup (%) 14 -iIt- A'JU"Ilum WIth slaUe water ao - Aquanum WIth wau:r flow 12 10 10 .. ;? 60 ::..,. :E f c 50 a "0 Q .. III .a ..:l ~ E :I :I til 40 I: 6 • eo III 30 «> 4 20 2 10 0 I) a 30 6l'l 90 120 150 lao 210 Ago {days) 60 c. Scientific Collaboration Dr. D. Gateno and Dr. Y. Barki, two Israeli scientists (two postdoctoral fellows) are now residing in Costa Rica working together with the Costa Rican team and are in direct collaboration contacts with • the Costa Rican PI. An Israeli Ph.D. student is now working on the same questions in the Red Sea coral reefs and is analyzing some ofthe data collected from Costa Rican coral reefs (N. Epshtein). During the fiscal year of 2000, 2 field trips were conducted by the Israeli principal investigator to Costa Rica's reefs (on both sides, the Pacific and the Caribbean coasts). These trips which were conducted collaboratively by both teams of scientists, continued the framework of mutual collaboration that was established in the specific areas studied. Two Costa Rican scientists (a Ph.D. student and a senior technician) visited the laboratory at Haifa this year, went for 2 weeks to the Red Sea for diving in the studied sites and were trained with techniques, the rationale and the aspects for reefrestoration as developed in the laboratory at Haifa. We have selected areas in the Pacific (Bahia Culebra) and the Caribbean (Cahuita) coasts that will be studies over the next years. From the Costa Rican side, two students have been selected to develop their thesis within the framework of the project. A Costa Rican technician who visited Israel is collecting basic physical and biological data at the Pacific site. All studies/field trips will be mutually inspected by both PIs. D. Description ofthe Project impact As mentioned above, (sections A, B), the results ofthe projects have already been published in the 6 manuscripts (2 during 1999, 4 during fiscal year 2000). Detailed evaluation of "reef management policies", the concepts for reefdomestication, reefrestoration policies as developed from the studies and the other results obtained from the collaborative studies will help the decision making authorities for choosing better protocols in reef management. We have also started to evaluate genetic structures of coral populations. This opens novel routes and opportunities for reef conservation. Detailed monitoring data received, will help us in understanding the year-to-year changes in Costa Rican coral reefs. This will establish a "data bank" of physical-chemical reef conditions. 61 E. Strengthening ofdeveloping country institutions The mutual work of the two Israeli scientists residing in Costa Rica with the local scientific group • and the recent visit of the Costa Rican team to Israel further strengthen the CIMAR scientific .. activities. Close studies that are done provide the opportunity to transfer ideas, data, knowledge and specific scientific issues to local students. Several meetings with other scientists from CIMAR were already conducted, for the purpose ofinvolving additional scientists in the project. F. Future work No changes in future studies. The work will proceed as scheduled. 62 Section II A. Managerial Issues Not applicable • B. Budget No major changes C. Special concerns none D. Collaboration, publication, training, travel Collaboration has been detailed in part C. of Section 1. Six manuscripts were published under the AID-CDR project: 1. Tom, M., Douek, J., Yankelevich, 1., Bosch, T.C.G. and Rinkevich, B. Molecular characterization of the first heat shock protein 70 (HSP70) from a reef coral. Biochem. Biophys. Res. Commun. 262, 103-108, 1999. 2. Epstein, N., R.P.M. Bak and B. Rinkevich. Implementation of small scale "no-use zone" policy in a reef ecosystem: Eilat's reef-lagoon six years later. Coral Reefs 18, 327-332, 1999. 3. Barki, Y., J. Douek, D. Graur, D. Gatei'io and B. Rinkevich. Polymorphism in soft coral larvae revealed by amplified fragment-length polymorphism (AFLP) markers. Mar. BioI. 136,37-41, 2000. 4. Gatei'io, D., Barki, Y. and B. Rinkevich. Aquarium maintenance ofreef octocorals raised from field collected larvae. Aquarium Sci. Conservation 2,227-236,2000. 5. Rinkevich, B. and S. Shafrr. Ex situ culture of colonial marine ornamental invertebrates: • Concepts for domestication. Aquarium Sci. Conservation 2,237-250,2000. 6. Epstein, N., R.P.M. Bak and B. Rinkevich. Toxicity of 3rd generation dispersants and dispersed Egyptian crude oil on Red Sea coral larvae. Mar. PolIut. Bull. 40, 497-503, 2000. 63 Five ofthe manuscripts are appended (ms. 1 was appended to fiscal year 1999 report). Travel and training: Trips to and from Costa Rica were recently activated, where the Israeli PI visited the Costa Rican colleagues, 1 week for each visit, for discussions and trips to the field. As stated before, a tight collaboration has been established on a day-to-day basis, as the result ofthe 2 • Israeli postdoctoral fellows residing in Costa Rica. This is in addition to the direct contacts through e-mail, faxes and fast mails between the two laboratories. E. Request for American Embassy, Tel Aviv None.