Ex situ culture of colonial marine ornamental invertebrates: concepts for domestication BARUCH RINKEVICH AND SHAI SHAFIR National Institute of Oceanography, Tel Shikmona, P.O. Box 8030, Haifa 31080, Israel Accepted 19 February 2000 Key words: aquarium industry, conservation, coral reef, domestication, marine ornamental invertebrates ABSTRACT The worldwide market for ornamental saltwater invertebrates supplies the needs of millions of aquarium hobbyists, as well as for public exhibition (zoos, aquaria), universities, and research institutions. The large-scale continuous collection of marine organisms is responsible, in many places, for the destruction of habitats, including coral reefs. The perceived expansion of the animal trade further threatens these fragile habitats. In the present paper, several concepts for the domestication of marine ornamental invertebrates (mainly colonial species) are discussed, offering an alternative commercial approach. The major rationale is based on future ex situ propagation, not field collections; a strategy aimed to circumvent the need for wild-harvested animals. This strategy is based on: (1) collection, settlement and metamorphosis of large numbers of larvae from marine organisms or of naturally shed germ cells under aquarium conditions, where survivorship exceeds several orders of magnitude than that in nature; (2) fragmentation of very small pieces (such as the size of a single polyp in colonial corals or blood vessel ampullae in tunicates) for the production of new colonies; (3) the development of replicates and inbred-lines from chosen ornamental species; (4) the use of cryopreservation of larvae and germ cells which will support the supply of material year-round; (5) several concepts for husbandry methods. Some benefits and deficiencies associated with the strategy for ex situ cultures are discussed, revealing its importance to the future of the trade.

THE RATIONALE

The marine aquarium industry is a fast developing commercial sector (Green and Shirley, 1999) that supplies ornamental organisms to millions of households worldwide (in the US alone, at least 600,000 households maintain marine aquaria; Lewbart et al., 1999). It also supplies stock for public exhibition (zoos, aquaria; Atkinson et al., 1995; Carlson, 1999), and for university laboratories and research institutions (Davies, 1995). However, in contrast to the more established sector of the freshwater aquarium industry, the marine aquarium trade is mostly dependent on specimens captured from the wild (Heslinga, 1996). As a result of the intensive exploitation of wild ornamental tropical marine organisms (especially reef fishes, molluscs, hermatypic and soft corals) over many years, many wild populations have diminished practically to the point of no return and whole ecosystems have been destroyed in many areas (Lubbock and Polunin, 1975; Mamonov, 1980; Grigg, 1984; Ross, 1984; Wells and Alcala, 1987; Sadovy, 1992; Best, 1997; Franklin et al., 1998; Pet-Soede et al., 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, Aquarium Sciences and Conservation 2: 237–250, 1998. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Micronesia and Okinawa (Heslinga and Fitt, 1987). The trade in marine organisms may also have had indirect effects on the marine ecology. For example it has been suggested that population explosions of the coral-eating starfish, Acanthaster planci, in Sri Lanka, could have been caused by the removal of ornamental fish that eat its larvae (literature cited in Sadovy, 1992). The increasing global awareness of the need to conserve and restore endangered habitats (e.g., coral reefs), together with international legal regulations (such as CITES; Best, 1997) and local/national legislation (such as in Australia; Oliver and McGinnity, 1985), makes it problematic to continue collecting marine organisms from the wild. The high mortalities associated with wild caught stocks must also be considered. In the case of marine ornamental fishes it was noted that the traders themselves admit that ‘the live-todead ratio is more than 1 to 100’ (Lubbock and Polunin, 1975). Similar mortality estimations, although varying from species to species, may be drawn for ornamental marine invertebrates (Sadovy, 1992; Best, 1997). It is evident, therefore, that sustainable aquaculture techniques for ornamental marine organisms will become critical to the future of the trade (Heslinga, 1996). Marine animal husbandry may provide an important source of live material not only for aquarium hobbyists but also to supply stock for educational needs (Yates and Carlson, 1993; Evans, 1997), for scientific research (Kinne, 1977; Emschermann, 1987; Atkinson et al., 1995; Davies, 1995; Tambutt´e et al., 1995; Ritchie et al., 1997; Rinkevich and Shapira, 1998; Gate˜no et al., in press) and for conservation purposes (Lubbock and Polunin, 1975; Mamonov, 1980; Yates and Carlson, 1993; Carlson, 1999; Gate˜no et al., in press). The culture methodologies employed 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 have been established for a variety of organisms from different phyla including cnidarians (Worthman, 1974; Kinne, 1977; Ritchie et al., 1997; Carlson, 1999; Gate˜no et al., 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 al., 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 live in intimate association with, and to the advantage of man’s use) is the practice by which a given species is adapted for high yield, sustainable production, by modifying its growth and life history traits (by provision of food, protection from enemies, etc.) and by selective breeding protocols through generations. Domestication may, therefore, be an attractive procedure from economic, social and ecological perspectives (Heslinga and Fitt, 1987; Franklin et al., 1998; Carlson, 1999). Several aspects of the domestication of reef-dwelling organisms, based on field-collected material, are discussed below. The rationale is to reach a situation where most (if not all) of the marine ornamental trade will be based on ex situ propagated stocks, and not on field-collected material. Although the

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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

Large numbers of sexually-derived propagules may be produced by an individual invertebrate specimen through broadcasting of gametes into the water column or by larval brooding, the two most common sexual reproduction strategies practiced by colonial marine invertebrates. One of the dramatic examples for reproductive potential is the yearly mass-spawning event in the Great Barrier Reef, Australia, where vast quantities of germ cells are simultaneously released into the water by hundreds of coral species during just one or two nights (Harrison et al., 1984; Alino and Coll, 1989). Only a minute fraction of these propagules develop successfully into coral colonies. For example, Weil (1990) estimated a yield of 1–3 × 106 released larvae from Litophyton arboreum colonies (a Red Sea soft coral) within a distinctive reef area in Eilat, yet detailed field observations documented only a single recruited young L. arboreum colony. Similar results of very low numbers of recruits were also obtained for other soft and hard corals in different reef areas worldwide (Farrant, 1987; Alino and Coll, 1989). On the other hand, ex situ maintenance of coral larvae may result in up to 100% metamorphosis and settlement (Ben David-Zaslow and Benayahu, 1998) and survivorship rates may exceed 30% following > 200 days of aquarium maintenance (Gate˜no et al., in press). In another study (Shlesinger, 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 1900 settled planulae studied, > 30% survived for 1 month, 3.5% for 3 months and 0.35% for 1 year, under suboptimal mariculture conditions. Ex situ survivorship rates in captivity, therefore, can exceed in situ rates by several orders of magnitude. 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 al., 1995; Carlson, 1999). We are, therefore, still largely dependent on organisms grown in situ. Collections of reproductive products from these organisms may be carried out by 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 (Shlesinger, 1985; Yates and Carlson, 1993; Gate˜no et al., 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 under ex situ conditions (e.g., Harrigan, 1972; Rinkevich and Loya, 1979; Goreau et al., 1981; Sato, 1985; Shlesinger, 1985; Harrison and Wallace, 1990; Weil, 1990; Franklin et al., 1998; Gate˜no et al., in press) or other marine invertebrates (Kinne, 1997; Carroll and Kempf, 1990; Goto and Yoshida, 1997;

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Rinkevich and Shapira, 1998). A major benefit of the ex situ settlement approach is the higher yield of larval settlement and higher survival rates as compared with that under natural conditions. As a result, significantly greater numbers of adults can be obtained from the same initial stock of larvae. Improved culture/settlement conditions (such as the provision of suitable substrates for settlement; Rinkevich, 1995; Gate˜no et al., in press) has resulted in further increasing the rates of success. The approach of supplying the marine ornamental industry with sexually-derived propagules 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 or 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, standardized aquaria conditions do not support high genetic variation since different genotypes may require distinct rearing conditions (Rinkevich and Shapira, 1998).

NUBBINS

The term ‘nubbins’ was coined by Birkeland (1976) to describe small coral pieces and has been adapted by several authors (reviewed in Davies, 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 Al-Moghrabi et al., 1993) which has the capability to regenerate to a whole organism. Fragmentation, the creation of several to many ramets that are subcloned (either naturally or by man) from a specific genet, is a common and fruitful procedure that is often used to increase the number of available organisms (summarized in Highsmith, 1982; Rinkevich, 1995; Tambutt´e et al., 1995; Rinkevich and Shapira, 1998; Carlson, 1999). When using fragmentation protocols for ex situ culture purposes, various biological and economical trade-off evaluations should be taken into consideration. Careful consideration should be given to the contrasting outcomes of either pruning many small fragments from a single colony or subcloning a few large ones. Pruning of large fragments from colonies may be a detrimental to the colonies due to the possible stress inflicted upon fragmentation. On the other hand, subcloning of small fragments may reduce fragmentation stress but may increase the mortality of the fragments. From a commercial point of view, however, the first approach could achieve suitable material for the trade within a short cultivation period, while the second approach, although associated with long culturing periods, may ultimately yield more material. In most cases, the decision between the two propagation approaches is based on economical equations. A third approach, the harvesting of numerous minute fragments from a single colony (nubbins) for the development of vast numbers of new colonies is discussed below, using examples from two different invertebrate phyla, the Urochordates and the Cnidarians.

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Urochordata Colonies of the cosmopolitan encrusting sea squirt Botrylloides sp. are very common members of benthic assemblages in temperate to tropical zones, including coral reefs. In this group of organisms, the 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 (100–300) of blood cells has the capacity to regenerate to a fully functional organism (including gonads) within a very short period of about 1–2 weeks (Rinkevich et al., 1995, 1996; Figure 1a–d). Thus, regeneration can arise from a small number of totipotent stem cells circulating in the blood

a

b

c

d

Figure 1. Regeneration of a whole urochordate (Botrylloides) from isolated peripheral ampullae. 1a: Day 1. Group of ampullae embedded within the tunic matrix, isolated from a Botrylloides colony. 1b: Day 4. Arrangements of the ampullae within the nubbin centre. 1c: Day 11. Formation of a bud within the opaque centre. 1d: Day 20. 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). Legend: a = ampulla, b = bud, s = exhalant siphon, t = tunic matrix, z = zooid. Each ampulla is ca. 0.1 mm in length.

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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 three embryonic layers. By employing the ampullae regeneration protocol (Rinkevich et al., 1995, 1996), any single genet may yield hundreds of ramets. Cnidaria The branching hermatypic Red Sea coral, Stylophora pistillata serves as a model coral species for a variety of purposes, including in situ regeneration studies (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 (Rinkevich and Loya, 1979). The typical structural formation of an S. pistillata colony approximates the symmetry of a sphere. In a recent experiment (S. Shafir, unpublished), we employed the protocol of subcloning many nubbins from adult colonies, each nubbin comprising just one or a few polyps (a polyp is approximately 2 mm in diameter). Nubbins were pruned from branch tips and bases and, using this protocol, a single colony could yield several thousands of different sized nubbins, each as small as one or two polyps in size. Survivorship of nubbins is very high (almost 100%) and an immediate regeneration process followed by fast growth rates (Figure 2a–c) has been observed during the first few months after pruning the colonies (nubbins at an average size of 5 polyps grew to 45 polyps within 90 days). Both of the above examples describe cases where subcloning minute fragments from a single adult colony may produce hundreds and thousands of new organisms (colonies). Although this protocol is based on a long-term cultivation strategy, it lends itself to commercial exploitation because it can yield almost unlimited amounts of replicated material which is readily available in a steady supply for the trade of the species in question. This approach however, has yet to be economically tested. A routine protocol of nubbin subcloning could therefore support sustainable aquaculture for the marine aquarium trade as well for various laboratory studies (e.g., ecotoxicology), with minimal need for field collections of new material. This procedure may be applicable to other colonial marine organisms such as sponges (Osinga et al., 1998; Wilkinson and Vacelet, 1979), urochordates (Rinkevich et al., 1996), hydrozoans and bryozoans. In the case of hermatypic corals, the technique of pinching off (using forceps) small pieces of coral tissue without skeletal material can also produce viable minute pieces. In one experiment, using Pleisastrea versipora, the separated tissue fragments became 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 al., 1997). Colonies produced by this procedure do not exhibit any genetic variation between ramets of the same genet and are, therefore, amenable for precisely standardized maintenance conditions.

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Figure 2. Regeneration of a small Stylophora pistillata nubbin. 2a = 5 polyps, 2 days after separation. Area∗ = 12.07 mm2 . 2b = 11 polyps, 33 days after separation. Area∗ = 24.91 mm2 . The regenerated nubbin has started to spread over the substrate. 2c = 19 polyps, 54 days after separation. Area∗ = 45.35 mm2 . Legend: p = polyp. Scale bar = 1 mm. ∗ Area measured using the top projection image of the nubbin. 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 protocols are crucial prerequisites for two methods of mass production of marine invertebrtes: clonal replicates and inbred-lines. Each approach has its own benefits and deficiencies; each approach is targeted for different purposes. The production of clonal replicates (the formation of numerous nubbins or subcloning a colony to few/many ramets) to supply the livestock industry is an excellent method for reducing 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

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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 (e.g., Rinkevich and Shapira, 1998). Such replicate clones could be very valuable in biological research (physiology, biochemistry, etc.) and are also a good source of material for the coral trade. Uniform genetic material, on the other hand, can have drawbacks, when, for example, the clone is sensecent or exhibits susceptibility to disease. In such cases, all or most of the clone replicates may be lost within a short period of time. The use of inbred-lines (Rinkevich and Shapira, 1998), a novel approach in ex situ invertebrate culture, 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 homozygotic organisms that are identical in terms of, for example, specific morphological features (e.g., colour morphs), or physiological/biochemical properties (e.g., specific temperature resistance), or any other biological trait that the breeder may choose. The successful establishment of long-term, defined genetic stocks of selected ornamental marine invertebrates would constitute 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 the lack of 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 the development of an inbred-line has been established for a marine invertebrate on at least one occasion, in the case of the sea squirt (Rinkevich and Shapira, 1998). In this study, an 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 seven years through five successive generations of self-crossed offspring. The production of inbred-lines and other laboratory established species may also be important for resolving certain aspects of conservation genetics such as inbreeding–outbreeding depressions, genetic adaptation to captivity and its affect on reintroduction success, and mutational accumulation (Frankham, 1999). Marine invertebrates are characterized by high variability in life history patterns (Kinne, 1977). This variation can manifest even within a species. For example, colonies of B. schlosseri originating from the same hatch of a 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 that 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

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to the production of inbred-lines. Inbred-line animals will probably manage poorly in natural conditions and, therefore, may not be suitable candidates 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 caused to coral reefs as the result of live animal collections, the high losses of livestock 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 a programme of interceptions between 1992 and 1994 (Best, 1997). These shipments, which originated from the Philippines, Indonesia, Mexico, Miami and other places, may reflect the global situation in terms of invertebrate animal mortalities between capture and final destination. The perceived expansion in marine animal trade further augments the potential threat to the fragile coral reefs. The damage caused to marine environments that are under collection pressure could be minimized in several ways by switching to non-destructive collecting techniques (such as planula larvae collections by plankton nets; Rinkevich and Loya, 1979), by returning animals that are raised ex situ to in situ conditions (Gate˜no et al., in press), or alternatively, by raising material for the trade using domesticated specimens (Yates and Carlson, 1992). Domestication of many reef invertebrates for the purposes outlined in this manuscript is a feasible task, and an attractive approach from economic, social, educational and ecological perspectives. An additional approach not discussed here, is the use of ranching techniques for colonial organisms, as applied successfully in the case of Tridacna (Heslinga and Fitt, 1987). However, ranching, in which the organisms are cultured outdoors (e.g., within a fenced-off area of the coast) cannot match the precisely controlled conditions that can be attained under aquarium conditions. Unfortunately, much of the information regarding ex situ maintenance of marine ornamental organisms is anecdotal and reported in hobby magazines and popular books, instead of scientific journals (Carlson, 1999). Other important concepts for ex situ culture of marine invertebrates have not so far been considered in this paper, due to limited studies. For example, one of the most promising of the applied techniques is cryopreservation. Cryopreservation has been widely used for the long-term conservation of a variety of vertebrate cells and organs and insect cell lines. This technique was evaluated for marine invertebrate cell and tissue culture as from 1992 (Rinkevich, 1999). Cryopreservation, as considered here, may be applied to the germ cells and embryos of ornamental marine invertebrates. It has the potential to provide a continuous, year-round supply of various materials. Cryopreservation of gametes and embryos is also critical for supporting and developing inbred-lines from ornamental marine organisms.

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Single cryopreserved collections of huge numbers of larvae, eggs and sperm bundles that have been shed from marine organisms (such as corals) could be used by breeders year-round, independent of reproductive seasons, under favourable, controlled conditions and with expected increased yields. A few studies have already examined the feasibility of cryopreserving whole embryos of marine invertebrates. These investigations were performed on a variety of organisms, including bivalve oocytes, trochophores and veligers (Odintsova and Tsal, 1995; Nadienko, 1997), polychaete larvae (Olive and Wang, 1997) and sponge parenchymella larvae (Rinkevich et al., 1998). The aim of such studies was to sustain cell cultures of marine organisms or for developing live organisms. With bivalve larvae (Odintsova and Tsal, 1995), the veligers showed high cell viability (> 90%) while only < 35% of the trochophore cells survived the freezing/thawing protocol. High viability was also observed in cryopreserved sponge larvae (Rinkevich et al., 1998), with over 70% of embryonic cells surviving cryopreservation. The cryopreservation approach, therefore, deserves further consideration. Careful attention to husbandry techniques should also be considered, as this area has not so far received detailed attention by aquaculturists with regard to the tropical marine aquarium trade (but see Lubbock and Polunin, 1975). These husbandry considerations include the appropriate spacing between marine invertebrates maintained within the same aquarium, and the co-culturing of different animals of the same species, different 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). Another important concept is the threat of introduction of non-indigenous marine organisms by aquarists. More than 30 years ago, Torchio (1968) reported the appearance of an Indo-Pacific fish in Italian Mediterranean waters and recent observations have documented the appearance of non-indigenous ornamental fish in Pearl Harbor, Hawaii waters (J.C. Delbeek, Waikiki Aquarium, Honolulu; personal communication). This problem of invasion of non-indigenous organisms (i.e. wild-collected organisms that may be dumped or escape into new habitats) is currently envisaged as carrying potentially deleterious 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 utilization of inbred-lines of ornamental marine organisms that are adapted specifically for aquarium conditions, could in some cases reduce the risks of exotic introductions of aquarium stocks (i.e., these inbred organisms may be unfit to survive and/or propagate in the wild). Coral reefs, in common with other tropical habitats worldwide, are subjected to continuous deterioration due to a variety of human activities, despite national and international legislation and rules (Rinkevich, 1995). 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;

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Gate˜no et al., in press). It is also evident that the international trade of saltwater ornamental organisms is going to expand (reviewed in Green and Shirley, 1999). The ex situ culture of ornamental marine invertebrates (and fish) is probably the best solution for environmental conservation with respect to this trade. This approach is deemed preferable to alternative strategies (Lubbock and Polunin, 1975; Yates and Carlson, 1993) that have concentrated on measures such as selective collection protocols, improved shipping methodologies, efficient husbandry methods and imposition of strict regulations. Banning most field oriented trade by the replacement of available material originating from ex situ cultures will most probably increase the price of animals. However, it will not only be an environmentally-friendly approach but rather improve the quality of material available to aquarists.

ACKNOWLEDGEMENTS

This study is part of the research carried out at the Minerva Center for Marine Invertebrates Immunology and Developmental Biology and was also supported by the EC-INCO Program and the US-AID-CDR.

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