Metamorphosis in the Cnidaria1 - CiteSeerX

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In many instances the final stimulus that triggers settlement and metamorphosis derives from substrate- borne bacteria or other biogenic cues which can be ...
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REVIEW/SYNTHÈSE

Metamorphosis in the Cnidaria1 Werner A. Müller and Thomas Leitz

Abstract: The free-living stages of sedentary organisms are an adaptation that enables immobile species to exploit scattered or transient ecological niches. In the Cnidaria the task of prospecting for and identifying a congenial habitat is consigned to tiny planula larvae or larva-like buds, stages that actually transform into the sessile polyp. However, the sensory equipment of these larvae does not qualify them to locate an appropriate habitat from a distance. They therefore depend on a hierarchy of key stimuli indicative of an environment that is congenial to them; this is exemplified by genera of the Anthozoa (Nematostella, Acropora), Scyphozoa (Cassiopea), and Hydrozoa (Coryne, Proboscidactyla, Hydractinia). In many instances the final stimulus that triggers settlement and metamorphosis derives from substrateborne bacteria or other biogenic cues which can be explored by mechanochemical sensory cells. Upon stimulation, the sensory cells release, or cause the release of, internal signals such as neuropeptides that can spread throughout the body, triggering decomposition of the larval tissue and acquisition of an adult cellular inventory. Progenitor cells may be preprogrammed to adopt their new tasks quickly. Gregarious settlement favours the exchange of alleles, but also can be a cause of civil war. A rare and spatially restricted substrate must be defended. Cnidarians are able to discriminate between isogeneic and allogeneic members of a community, and may use particular nematocysts to eliminate allogeneic competitors. Paradigms for most of the issues addressed are provided by the hydroid genus Hydractinia. Résumé : Chez les organismes sédentaires, les stades libres sont des adaptations qui permettent aux espèces immobiles d’exploiter des niches écologiques fragmentées ou temporaires. Chez les cnidaires, la recherche et la reconnaissance d’un habitat convenable sont restreintes aux minuscules larves planula ou aux bourgeons larvaires, stades qui donneront éventuellement des polypes sessiles. Cependant, ces larves ne possèdent pas les structures sensorielles nécessaires au repérage à distance d’un habitat approprié. Elles dépendent donc de toute une hiérarchie de stimulus clés propres à leur indiquer qu’elles sont en présence d’un habitat qui leur convient. On trouve des exemples de cette situation chez certains anthozoaires (Nematostella, Acropora), scyphozoaires (Cassiopea) et hydrozoaires (Coryne, Proboscidactyla, Hydractinia). Dans plusieurs cas, le dernier stimulus qui déclenche l’établissement et la métamorphose vient de bactéries transportées par le substrat ou d’autres signaux biogéniques qui peuvent être explorés par les cellules sensorielles sensibles aux stimulus mécaniques et chimiques. Au moment de la stimulation, les cellules libèrent ou facilitent la libération des signaux internes, tels que des neuropeptides, qui peuvent envahir tout le corps et déclencher la décomposition des tissus larvaires et l’acquisition des cellules adultes. Les cellules progénitrices peuvent être pré-programmées pour s’adapter rapidement à leurs nouvelles fonctions. Les établissements en groupes facilitent les échanges d’allèles, mais peuvent aussi entraîner des guerres civiles. Il s’agit de défendre un substrat rare et restreint. Les cnidaires sont capables de faire la distinction entre les membres allogènes et les membres isogènes de leur communauté et peuvent utiliser des nématocystes spécialisés pour éliminer leurs compétiteurs allogènes. Dans la plupart des questions examinées ici, les paradigmes sont fournis par l’étude de l’hydroïde Hydractinia. [Traduit par la Rédaction]

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Introduction Cnidarians are primarily members of communities that occupy benthic habitats. “Primarily” in this context means that

during their embryonic phase of life cnidarians develop a planula larva which settles on a substrate and transforms into a benthic phenotype or morph known as a polyp. This morph most probably reflects the basic organization of the

Received 6 September 2001. Accepted 16 May 2002. Published on the NRC Research Press Web site at http://cjz.nrc.ca on 19 November 2002. W.A. Müller.2 Institute of Zoology, University of Heidelberg, Im Neuenheimer Feld 230, D 69120 Heidelberg, Germany. T. Leitz. Animal Developmental Biology, Faculty of Biology, University of Kaiserslautern, Building 13/1, Erwin-Schroedinger Straße, D 67661 Kaiserslautern, Germany. 1

This review is one of a series dealing with aspects of the biology of the phylum Cnidaria. This series is one of several virtual symposia on the biology of neglected groups that will be published in the Journal from time to time. 2 Corresponding author (e-mail: [email protected]). Can. J. Zool. 80: 1755–1771 (2002)

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DOI: 10.1139/Z02-130

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ancient precursors in the evolution of the phylum (Miller and Ball 2000). Even if the life cycle of a species includes a free-swimming planktonic morph known as a medusa or jellyfish, this swimming phase is generated by a process of asexual propagation from a preceding sessile polyp. Remarkably, although asexual, uniparental reproduction such as strobilation (Fig. 1) is a type of natural cloning, the offspring released from the parent develop a phenotype different from that of the parent: one and the same genotype includes information for constructing different phenotypes! This aspect will not be discussed further here. Rather, this review will focus on the settlement and metamorphosis of planulae and planula-like swimming buds: the ecophysiological aspects of the triggering of metamorphosis, signals that orchestrate the internal processes of transformation, a short description of metamorphic transformation at the cellular level, and finally, selected aspects of cnidarian population biology related to settlement. In cnidarians, as in many marine invertebrates with a larval stage, metamorphosis is triggered not by autonomously rising or falling hormone levels but by external, environmental cues (Müller et al. 1976; Leitz 1997). Moreover, each transition from one developmental stage to the next in the individual life history of a representative cnidarian has to be considered a “checkpoint” at which external physical, chemical, or biological factors elicit, or inhibit, a particular pattern of development and behaviour (Hofmann et al. 1996).

General ecophysiological aspects How do cnidarians find and select an appropriate habitat? The life cycle of all sedentary organisms includes a freeliving stage, which has two predominant assignments: (1) to exploit a scattered or transient ecological niche and (2) to promote genetic exchange throughout the various populations of a given species. Colonists arriving at appropriate sites from different home populations will most probably contribute different alleles to the common gene pool of the colony, thus augmenting its genetic diversity and flexibility and enhancing its chances of long-term survival. In cnidarians the task of prospecting for and identifying a congenial habitat is not assigned to the elaborate planktonic swimming phase known as the medusa or jellyfish, a morph that is lacking in the Anthozoa anyway, but to the tiny planula larvae, that is, those multicellular spindle-like or elliptical bodies which in sexual reproduction arise from fertilized eggs (Fig. 1; see also Fig. 5); or the task is assigned to planula-like buds called propagules, which are asexually produced and released from polyps or, in rare instances (trachyline Hydrozoa), from medusae. Even when the metagenetic life cycle has provided the species with a large medusa capable of brooding the eggs throughout development until the settling stage, as in the genus Cassiopea (see below), the larvae are eventually set free and it is their job to find a suitable substrate on which to settle. Except in sea anemones, which remain mobile, the polyp arising from the larva is permanently bound to a substrate and cannot correct a wrong choice. Careful habitat selection, therefore, is essential for the survival of a population. But how can the tiny larvae carry out such a task? Their sensory equipment is lim-

Can. J. Zool. Vol. 80, 2002 Fig. 1. Paradigm of a (metagenetic) cnidarian life cycle: Aurelia aurita (Scyphozoa).

ited. It consists of mechanosensitive or chemosensitive neurosensory cells fitted with a sensory cilium. This equipment qualifies the larvae to explore some physical and chemical properties of a substrate and its microenvironment but does not enable them to locate an adequate habitat from a distance; this is discussed in the following section. As a rule, the larvae of sedentary organisms depend on a hierarchy of key stimuli that are indicative of their adult environment and lead them to their destination stage by stage. In terms of behaviour, larvae often display searching activities until they are presented with a specific stimulus that triggers settlement and metamorphosis. Are navigation and chemotaxis possible from a distance? Chemotaxis from a distance, guided by soluble and diffusing target-borne molecules under natural conditions of water turbulence, is probably not a sufficiently reliable means of detecting a distant preferred substrate or precisely locating a specific site in the habitat, such as a particular species of alga. The presence of abundant diffusible molecules such as NH3 /NH+4 , H2S, amino acids, and other conventional organic compounds can indicate a favourable or inappropriate largescale habitat like a mangrove swamp. But more specific diffusible factors are likely to be present in perceptible quantities only in the viscous boundary layer adjacent to the substrate or directly on its surface. Moreover, the minuscule cnidarian larvae are subjected to flow characterized by low Reynolds numbers and are more apt to be carried along by the mass of water than to travel through it. How do pelagic larvae find a substrate? The task of prospecting for a congenial substrate appears to present different levels of difficulty for pelagic (planktonic) larvae compared with benthic larvae, since the latter move over the sea floor. Pelagic larvae, or the medusae and jellyfish that release them into the expanse of the ocean, are © 2002 NRC Canada

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Müller and Leitz Fig. 2. Patterns of vertical movement of planktonic larvae in the water column.

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surfaces of the leaves of the sacred lotus) that certain regular nanostructures at surfaces prevent any adhesion of fluids or solid particles (Barthlott and Neinhuis 1997). Thus, some marine organisms might avoid being occupied by fouling organisms by exposing such nanostructures on their surface. widely dispersed and transported by currents far from the place where their parental polyps reside; the larvae also have to travel through a large column of water until they reach the bottom. However, pelagic larvae commonly move up and down the water column in a daily rhythm, presumably in response to an internal circadian clock and guided by light and (or) gravity, or they are passively shifted by thermally driven convections that arise and fade daily (Fig. 2). At the end of their sinking phase the larvae may approach the bottom, so they have the opportunity to explore the environment near the sea floor at daily intervals. If a larva enters an appropriate habitat, it may gradually be guided to an appropriate site by a cascade of cues. The hierarchy of cues may include unspecific chemical or physical parameters, such as the NH3 /NH+4 content or viscosity of the water, microcirculation properties close to the surface of solids, surface energies such as the tension of wetting, the thermal capacity of the substrate, and other parameters indicative of the presence of a particular substrate and its quality (e.g., soft versus hard). At this point at the latest, pelagic and benthic larvae are now confronted with the following problem, which is common to all larvae below a critical size. How do larvae establish physical contact with a substrate? Tiny larvae can find it difficult to establish intimate contact with a solid surface. They have to overcome several physical barriers such as shear stress, forces of surface tension, and repulsive electrostatic potentials. Of particular importance is the liquid/solid-surface energy known as tension of wetting (Müller et al. 1976). If the surface of the substrate and the surface of the larva both display hydrophilic properties, high capillary forces strongly bind a water film at the larva–substrate interface, and this film is not easily dislodged by the larva. On the other hand, if both the surface of the substrate and the surface of the larva are hydrophobic, the larva is attracted to the substrate as a a drop of oil would be (Fig. 3). Adhesion through holdfasts and cements is also strongly influenced by the physical properties of the substrate. This applies not only to animal larvae but also to microbes. Only in recent years has it been recognised (by botanists using a scanning electron microscope to look at the ever-clean

Levels of specificity of settlement: a case study As a rule, different species have different final destinations. Patterns of abundance may reflect both passive deposition of larvae that sink as particles under the influence of local hydrodynamic conditions and successful, active substrate selection. A substrate covering areas whose size surpasses the average spatial scale of passive larval dispersal could be colonized simply by random deposition of larvae. The more specific the destination, the more specific the cues that are needed in order to restrict settlement to particular sites within the habitat. The following examples represent increasing demands of specificity. Nematostella vectensis: a soft-bottom-colonizing cnidarian Nematostella vectensis is a small euryhaline sea anemone found in estuaries along the European and American coasts of the North Atlantic Ocean, along the shores of the Gulf of Mexico, and along the Pacific coast of Mexico (Hand and Uhlinger 1992 and references therein). The sea anemone occurs in soft sediments, in plant debris, and among living plants in permanent pools and tidal creeks in salt marshes. In the laboratory the animals are sexually active throughout the year, showing no sign of seasonality in their reproduction, although in nature reproduction may depend on favourable external conditions. Male and female sea anemones preferentially spawn in the early evening. The eggs are discharged in masses embedded in gelatinous material. They develop into planulae, which are equipped with an apical tuft of long cilia. Most likely this tuft is a sensory organ. However, metamorphosis of the planulae appears not to depend on specific environmental cues and is even initiated before settlement. In the laboratory the pear-shaped larvae have already developed tentacles by day 5 post insemination, i.e., before settlement; they cease swimming, sink to the bottom, and complete their transformation into juveniles about 1 mm in length. As the larvae spend only a short time in the plankton, their dispersal is mostly restricted to the area of their birth. Moreover, the small sea anemone that arises from the larva © 2002 NRC Canada

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is not bound to a particular place. During the first few days after metamorphosis, the juveniles glide over the bottom with the aboral end forward. (After a few days the direction of movement reverses.) If the moving juveniles find a soft sediment, they burrow into it by digging their peduncle into the sediment. The independence of metamorphosis from specific inducing external cues reflects the wide distribution of the metamorphosed animals on the substrate, and their mobility; they remain able to change their position if local conditions prove not to be favourable. In favourable places the animals start cloning themselves by transverse fission. Thus, a pond can be colonized quickly and the quantity of eggs produced rises. The coral Acropora millepora: a hard-bottom dweller In marine environments, hard substrata are much less extensive, and far more patchily distributed, than sedimentary deposits. Therefore, selective pressure for a high degree of site-specific settlement among hard-bottom dwellers is more intense. Besides Nematostella, the coral Acropora millepora has been proposed as a new model organism representing the class Anthozoa in particular and the phylum Cnidaria in general (Miller and Ball 2000). In the area of the Great Barrier Reef, many corals, including A. millepora, practice synchronous mass spawning once a year. The conventional embryonic development of the floating eggs leads to spindle-shaped planula larvae, the typical dispersal stage for most sedentary cnidarians. The planulae contain large amounts of lipids, enabling them to remain in the water column for months. The larvae are passively dispersed by currents and convections in the water column but also display active swimming behaviour that is driven by cilia. They have a well-developed nervous system, a mouth opening into a gastrovascular cavity, and nematocysts. When the planulae are ready to settle, they sink to the bottom, showing a characteristic rotating swimming pattern as they repeatedly test the substrate for a place to settle. Only after having settled do the larvae undergo metamorphosis and form tentacles. Since the planulae have been reported to test the substrate actively and repeatedly, we can infer that they are probably exploring it in search of some characteristic properties. Is one of these properties the presence of substrate-bound bacteria, as in case of the hydrozoan genus Hydractinia (described below)? In recent experimental investigations it was concluded that bacteria are among the sources of inductive signals (Negri et al. 2001). In future studies, attention should also be given to a physical phenomenon not hitherto taken into account. In areas with hard substrata, particularly along rocky shorelines and reefs, surf produces air bubbles and foam, and such conditions have been found to induce sinking and the onset of metamorphosis in the scyphozoan Aurelia aurita (Kroiher and Berking 1999). Another case: Cassiopea and bioorganic cues Cassiopea (e.g., C. andromeda, C. xamachana) is a jellyfish genus that differs in its habits from other genera of the typically holopelagic scyphomedusae, as it prefers to live in shal-

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low tropical lagoons and mangrove swamps, often lying upside down on a sandy bottom instead of swimming around like other jellyfish. The adult females incorporate sperm released into the surrounding water by the males. They envelop the internally fertilized eggs in mucus and wrap this mass around the base of sex-specific vesicles, thus protecting the embryos until the ciliated planula larvae hatch from the egg envelopes. In addition to sexually generated planulae, Cassiopea also produces planula-like swimming bodies by asexual budding. Both types of larvae, whether produced sexually or asexually, have to find a hard substrate on which to settle and transform into a polyp (called a scyphistoma). There is experimental evidence that the larvae avoid settling on clean, sterile substrata. Instead, they attach only to substrata covered with a microbial film to undergo metamorphosis (Hofmann et al. 1996). The presence of a surface film of microorganisms has long been recognised as a prerequisite for the settlement of many fouling invertebrates (Pawlik 1992 and references therein). The supporting effect of microbial films has commonly been attributed to their physical properties. The bio-organic film may affect patterns of larval settlement by virtue of the altered tension of wetting it confers on a hard substrate. However, bacteria in the environment also provide soluble cues: by decomposing mangrove leaves they liberate proline-rich peptides that cause the larvae to terminate larval life and enter metamorphosis (Fitt and Hofmann 1985; Fitt et al. 1987; Fleck and Fitt 1999; Fleck et al. 1999). Preference for a substrate covered with a bio-organic film is also displayed by other scyphozoan planulae. The planulae of Cyanea have been reported to settle on aged mollusc shells rather than on freshly vacated shells, and this preference has been attributed to the decreased wettability of filmcovered surfaces (Brewer 1984). However, as will be shown with larvae of Hydractinia (“Metamorphosis of Hydractinia, the best studied cnidarian model organism” below), the influence of substrate-bound bacteria can be more specific and indicative of a particular substrate. Proboscidactyla flavicirrata, Coryne uchidae, and others: living on biogenic substrata If the preferred substrate is biogenic, the cues mediating specificity or enhancement of settlement are believed to be chemical in nature. However, in no case has the chemical stimulus been identified with certainty, although pioneering investigations made in Japan (Nishihira 1968; Kato et al. 1975) were promising. The hydroid Coryne uchidai settles on brown algae of the family that includes the genus Sargassum. Boiled aqueous extracts (Nishihira 1968) as well as hexane extracts from dried Sargassum tortile (Kato et al. 1975) caused larvae to cease swimming, and a varying fraction of the motionless larvae underwent metamorphosis Several diterpenoid chromanols have been isolated, among them δ-tocotrienol epoxide (Fig. 4), which caused larvae to metamorphose. However, only a few larvae were available for bioassays, and it is not known whether the identified diterpenoids are exposed on the surface of the algae. Any chemical that interferes with internal triggering mechanisms could initiate metamorphosis without being the natural inducer. This became evident in our studies on Hydractinia: the planulae of this species can be induced to metamorphose with the use of tumour-promoting phorbol © 2002 NRC Canada

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Müller and Leitz Fig. 4. Chemical structure of a substance derived from an alga (Sargassum sp.), δ-tocotrienol epoxide, which causes settlement and metamorphosis of the planulae of the hydroid Coryne uchidae.

esters (Müller 1985), and these compounds are derived not from marine plants but from terrestrial plants (of the family Euphorbiaceae). Phorbol esters are highly active without being natural inducers. The particular processes that are activated by phorbol esters will be discussed in the following section (“Metamorphosis of Hydractinia, the best studied cnidarian model organism”). Epiphytic associations are common in several species of marine organisms. For instance, the scleractinian coral Agaricia humilis prefers to settle on certain crustose coralline algae. A compound has been extracted from these algae that induces the settlement and metamorphosis of A. humilis larvae (Morse et al. 1988, 1994; Morse and Morse 1991). The compound appears to be a macromolecule containing sulfated glucosaminoglycan. Proboscidactyla flavicirrata is found solely on the tube rims of sabellid polychaetes. The planulae establish preliminary physical attachment to a substrate by extruding the sticky threads of nematocysts. Nematocysts of the particular type borne by the planulae of this species were specifically stimulated to discharge on contact with the feeding appendages or body surface of sabellids, resulting in larval attachment to the worm (Donaldson 1974). Threads of particular nematocysts (atrichous isorhizas) are also used by the actinula larvae of Tubularia mesembryanthemum (Yamashita et al. 1998) and planulae of Hydractinia (below) to attach to a solid surface.

Metamorphosis of Hydractinia, the best studied cnidarian model organism The organism The colonial marine hydroid Hydractinia has in recent years progessively acquired the status of a cnidarian model organism (Leitz 1998; Frank et al. 2001). Hydractinia occurs in the North Atlantic Ocean as two sibling species, Hydractinia echinata along the coasts of northern Europe and Hydractinia symbiolongicarpus along the coast of North America (Buss and Yund 1989). The large-scale habitat of these two closely related species comprises shallows with a sandy or granular bottom. Most colonies are found encrusting the outside of gastropod shells inhabited by pagurids (hermit crabs). Colonies are occasionally found on other substrata, such as the shells of living snails and bivalves, piles, or simply stones that are exposed to strong tidal currents, so that sand is not deposited permanently and planktonic food frequently passes with the tidal currents. Young colonies derived from metamorphosing planula larvae (Fig. 5) consist of feeding polyps connected with each other through a network of gastrovascular

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channels formed by tubular stolons that run along the substrate. Colonies grow by peripheral extension of the tubular stolons, which ramify and anastomose, forming a dense network. This network becomes increasingly dense and eventually acquires the structure of a closed mat. Asexual budding of polyps from this stolonal network adds new clonal members to the colony. In the central mat, particular sexual polyps, called gonozooids or blastostyles, emerge. These develop gonophores, which are medusae that remain sessile and are morphologically reduced to ball-shaped containers which serve as gonads (Hertwig and Hündgen 1984). Sexual reproduction In Hydractinia colonies the sexes are separate. Under standard conditions in the laboratory, mature colonies spawn synchronously and almost daily 1–2 h after the onset of illumination in the morning. The gametes are released into the surrounding seawater, where fertilization occurs. After 3 days the fertilized eggs develop into planulae, which are competent to metamorphose. However, in clean water and under sterile conditions the larvae do not enter metamorphosis. In the laboratory they can be kept (at 6°C) for months until they starve to death, but they will not undergo metamorphosis without being stimulated by natural or artificial key stimuli or chemical inducers. In nature, the best place for Hydractinia planulae to select for settling is a shell inhabited by a hermit crab. The benefits of a mobile home On shoals and mud flats exposed to strong tides with large lifts, a mobile home carried by a hermit crab provides many benefits for an epiphytic symbiont such as Hydractinia, while the crab benefits because the dangerous nematocysts of its symbiotic partner, the hydroid, discourage its potential predators (Brooks and Gwaltney 1993 and references therein). The following list summarizes the unpublished observations of one of the present authors (W.A.M.). • The hermit crab frequently roams about in search of food. While the shell is dragged along the bottom, the large feeding polyps found along the lower edge of the shell’s mouth can fish for and collect the many small nematodes, annelids, and crustaceans that live in the superficial layers of sandy bottoms. The crab also ferrets out small animals hidden in the grooves of hard bottoms or in bunches of algae. • Shells exposed to tidal currents in sandy areas are frequently covered with sand. Crabs can quickly dig themselves and their home out. • On shoals, particularly around the North Sea in Europe, large areas become dry during low tide. Crabs retreat into remaining tidal ponds or protect themselves under bundles of wet algae. • During stormy and rainy periods and during the winter, the crabs retreat into deeper waters. • From time to time crabs assemble at common locations, lured either by a rich source of food or by sexual partners. Such aggregations favour not only fertilization of the crab’s own eggs but also encounters between the gametes released by symbiotic colonies of Hydractinia. © 2002 NRC Canada

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Fig. 5. Life cycle of the hydroid Hydractinia echinata (and Hydractinia symbiolongicarpus).

Finding a mobile home How can a planula detect a gastropod shell carried around by a hermit crab, and how can it climb onto the shell? In fact, the planulae of Hydractinia are not able to locate shells occupied by crabs, nor do they climb onto shells. The spindleshaped planula larva of Hydractinia is equipped with neurosensory cells located at or near the anterior pole and also on its tapered posterior tip. Here in the posterior region of the larva, in addition, numerous nematocysts are found (Weis et al. 1985; Weis and Buss 1986). This equipment enables the larvae to display a behaviour that enables it to attach to a quickly moving shell (Müller et al. 1976). The planulae are positively phototactic and glide,

through the beating of their cilia, to an elevated location, be it a grain of sand or a stone, and adhere to its surface by means of mucus secreted at their anterior, blunt end. Here they remain standing in a vertical posture until a solid object is dragged past. Quick movements of the object, in fits and starts, stimulate competent planulae to attach to its surface by discharging the sticky threads of nematocysts (atrichous isorhizas and desmonemes) that are found only in the larvae and are clustered in their tapered posterior region (Weis and Buss 1986). Thus the larvae are transferred to the moving object, which, hopefully, is a gastropod shell. Discharge of the nematocysts is facilitated by the velocity gradient in the fluid between the larva and the passing sur© 2002 NRC Canada

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face. The drag of the moving solid is transmitted to the liquid, so that the liquid layer close to the surface is also set in motion. Thus, a velocity gradient is set up in the shear plane surrounding the object (Fig. 6). The change in velocity along the larva, in addition to the impact of the collision, constitutes the stimulus for the larva to fire its lasso or grapnel. Likewise, when planulae are drawn into a pipette, the velocity gradient in the fluid along the walls of the pipette causes them to attach to the wall, but when the relative movement stops, the larvae eventually expel their nematocysts and resume crawling about. When larvae are transferred to moving shells, the anterior region bends toward the substrate. Here the larvae encounter a film of bacteria, and these will most probably provide the desired metamorphosis-inducing principle. Natural cues that trigger metamorphosis Used shells are colonized by bacteria, as are almost all surfaces of solid objects in the marine environment (e.g., Corpe 1970; Dang and Lovell 2000). These bacteria attach to the surface of solid objects by means of particular fimbriae or pili (species of Pseudomonas) or by secreting sticky capsular polysaccharides or other cementing substances. In the marine environment, bacteria prefer to attach to solid objects because the surface binds and accumulates nutritive molecules such as amino acids dissolved in seawater. Bacteria that colonize mollusc shells, in addition, find the organic layer of the periostracum, which they may attack and use as a source of nutrients. In the marine environment, microbiologists interested in ecology find bacteria predominantly from the genera Alteromonas and Pseudoalteromonas on solid surfaces (e.g., Acinas et al. 1999; Dang and Lovell 2000), and species of these genera are among those that have been identified as colonists of shells occupied by hermit crabs, and as the source of metamorphosis-inducing activity. In a pioneering study, it was shown that planulae of H. echinata can be induced to undergo metamorphosis by presenting them with a film of such bacteria on filter membranes (Müller 1969). Several bacterial species and strains isolated from the microenvironment of settling planulae were found to be effective, but these amounted to only a small fraction of the strains tested with a standard assay protocol (Müller 1969, 1973a, 1973b; Wittmann 1977). In subsequent studies, effective bacterial strains were identified as Alteromonas haloplanctis and Alteromonas macleodii, as well as some belonging to the genus Oceanospirillum (W.A. Müller, unpublished data). Another bacterial species isolated from shells colonized by Hydractinia was identified as Alteromonas espejiana (Leitz and Wagner 1993), now called Pseudoalteromonas espejiana. Bacteria with metamorphosis-inducing capacity are found on many substrata (Kroiher and Berking 1999), and frequently belong to the genera Alteromonas or Pseudoalteromonas (Acinas et al. 1999; Dang and Lovell 2000). These genera are known to be frequently associated with higher organisms and to produce biologically active extracellular agents (Holmström and Kjelleberg 1999). The presence of such bacteria on solid substrata easily explains the occasional settlement of Hydractinia on substrata other than gastropod shells carried around by hermit crabs. Interestingly, all bacteria tested so far in our laboratory are

1761 Fig. 6. Transfer of a planula larva of Hydractinia to a moving object such as a gastropod shell carried around by a hermit crab. The impact of collision and the velocity gradient of the water around the moving object cause the discharge of the sticky threads of nematocysts (atrichous isorhiza).

effective only if they are attached to a solid object, not when grown in conventional breeding media and presented in a suspension. The bacteria do not release a metamorphosis-inducing substance into the medium; rather, the larvae must come into close contact with the bacterial layer, and the bacteria are particularly effective if they have been starved for some hours. This observation suggests that the metamorphosis-inducing cue is a component of the outer bacterial wall or of the fimbriae produced by the adhering bacteria. When starved, marine bacteria produce specific exopolysaccharides, which confer upon the previously free-swimming bacteria the ability to adhere to surfaces (in this case the filter membrane) (for example, see Wrangstadh et al. 1990). By applying an osmotic shock, the metamorphosis-inducing principle could be released from living and surviving bacteria (Müller 1973a, 1973b), but its chemical nature has not yet been definitively identified. The metamorphosis-inducing principle appears to be a macromolecule, as it does not pass through ultrafilters with exclusion limits of about 100 kilodaltons. At present, our working hypothesis is focused on hydrophobic proteins or lipopolysaccharides (Bläß 1997). On the other hand, whereas the larvae of the bryozoan genus Bowerbankia can be caused to settle and metamorphose by providing them with a hydrophobic solid alone (Müller et al. 1976), hydrophobic surfaces per se do not induce settlement and metamorphosis of Hydractinia planulae. The bacterial inducer is effective not solely by virtue of its physical properties. Using the initiation of metamorphosis as the criterion, it is to be expected that in nature these bacteria are present at high density and in effective condition on mollusc shells and occasionally on other large solid objects, but not on sand grains. The characteristic vibrating movements of the shell carried by a hermit crab might be another stimulus that induces metamorphosis, as proposed by Cazaux (1961). However, as he was unaware of the significance of the bacteria on the shells, his experiments were not conducted under sterile conditions. Artificial metamorphosis inducers and the primary mechanism of induction Metamorphosis in Hydractinia can be induced artificially and conveniently with great efficiency by either (i) bathing the larvae for some hours in seawater enriched with lithium © 2002 NRC Canada

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Fig. 7. Depolarization of an excitable cell by cations, illustrating the explanation given in the text. The resting potential is the distribution of ions in a typical resting excitable cell such as a nerve cell. Depolarization occurs when cesium ions are transported into the interior of the cell by the ion pump known as Na+,K+-ATPase or they squeeze through potassium leak channels to enter the interior, driven by the concentration difference. In the interior the positively charged cesium ions occupy anionic sites, neutralize their charge, and thus depolarize (diminish) the electrical potential between the interior and the exterior of the cell.

ions (Spindler and Müller 1972) or potassium, rubidium, or cesium ions, Cs+ being the most potent (Müller 1973a; Müller and Buchal 1973), or (ii) treating the larvae with activators of protein kinase C (PKC), such as tumour-promoting phorbol esters or certain diacylglycerols like dioctanoylglycerol (diC8) (Müller 1985; Leitz and Müller 1987; Leitz 1993; Schneider and Leitz 1994). Subsequent to these findings, many other (less effective) inducers have been found, among them NH+4 (Berking 1988a). Our interpretations of these findings converge in a common hypothesis about the primary mechanism of induction. We think that all inducing agents act by stimulating excitable neurosensory cells such as are found near the anterior pole of the larva (anterior with respect to the direction of its movement when gliding over the substrate). The ions used to trigger metamorphosis are known to cause depolarization, and thus stimulation, of excitatory cells. Elevated levels of external K+ directly cause depolarization according to the Nernst equation:

Uvolt =

R × T [K + ]outside ln Z × F [K + ]inside

This classical equation describes the dependence of the electric membrane potential on the difference in concentrations of cations, here K+, between the exterior and interior of a cell with a semipermeable membrane. For details see textbooks of neurophysiology (Fig. 7). Cs+, Rb+, Li+, and likewise NH+4 , on the other hand, are known to be transported into excitable cells via the membrane-associated ion pump known as Na+,K+-ATPase. This pump does not discriminate well between the rare monovalent cations Cs+, Rb+, Li+, and NH+4 and its natural-load K+. From time to time potassium ions must be pumped into the cell to replace those lost by diffusion through the electrogenic K+-leak channels. Owing to the activity of this pump and its low specificity, with time any excitable cell will accumulate Cs+, Rb+, Li+, or NH+4 if they are present. (If Na+,K+-ATPase is blocked with ouabain, Rb+ and Li+ are © 2002 NRC Canada

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Müller and Leitz Fig. 8. Transplantations documenting the transmission of an internal metamorphosis-triggering signal (putatively neuropeptides) from the anterior part of the larva to the posterior part. (a) Control: a posterior larval fragment grafted onto a noninduced anterior part does not undergo metamorphosis. (b) Only the anterior part was induced (and can be induced) to initiate metamorphosis by an external stimulus. The grafted posterior part did not receive an external inducing stimulus; nevertheless it participated in metamorphosis because it received an internal stimulus that travelled from the anterior part of the larva to the posterior part. (c) Posterior fragments are not able to respond to external stimulation by bacteria or cesium ions. (d) Posterior fragments respond to neuropeptides of the GLWamide class by metamorphosing.

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more, while a highly effective bacterial inducer must be present for merely 20–30 min. The supporting influence of mechanical stimuli The sensory cells of Cnidaria frequently respond to combinations of chemical and mechanical stimuli. The supporting influence of mechanical shearing forces is indicated by the dependence of dose-response curves not only on the concentration of cesium ions but also on the degree of hydrophobicity of the substrate (Berking and Walter 1994; Berking 1998). Hydrophobic surfaces have a promoting action and shift the apparent KM of the dose-response curves for cesium to lower values. Hydrophobic adhesion provides shearing forces that act upon sensory cilia. On the other hand, as stated above, hydrophobic surfaces per se do not induce metamorphosis. Accordingly, the action of the bacterial inducer cannot merely be attributed to the hydrophobicity that it may bestow upon a substrate. Activation of PKC The activation of PKC is an event frequently associated with excitation. PKC plays a key role in the PI-PKC signal transduction cascade (Leitz and Müller 1987 and references therein, 1991; Leitz and Klingmann 1990; Schneider and Leitz 1994; Hassel et al. 1996). This cascade is triggered by an external chemical signal. The external signal becomes the ligand of molecular receptors, and we think that a component of the bacterial cell wall is the ligand, and that the molecular receptor in the larva is associated with the cilium of neurosensory cells. The cascade causes the opening of channels for Na+ and (or) Ca++ in the cell membrane of the neurosensory cell. The influx of these cations leads to depolarization, and thus to stimulation of the cells (Leitz 1993, 1997). In addition, activation of PKC precedes and mediates the exocytosis of transmitters or neurohormones at the axonal terminals of neurosensory cells. (This interpretation does not exclude the possibility that activation of PKC, in addition, mediates downstream processes of metamorphosis).

almost ineffective, but very high doses of Cs+ still induce metamorphosis (Müller 1973a). Driven by high concentration potentials, Cs+ presumably penetrates the cell membrane also through leak channels.) In the interior of the cells the infiltrating cations cause depolarization through the combination of two effects: (1) They occupy anionic sites and thus neutralize the surplus of electronegative charge that exists inside resting excitable cells. (2) They enter and block the electrogenic K+-leak channels from inside the cells; blocking these channels prevents repolarization because no positive charge can leave the cell (Fig. 7). Depolarization by these indirect mechanisms takes time, so the larvae must be bathed in Cs+ solutions for 4 h or

The internal orchestration of metamorphosis If sensory cells perceive a distinct external cue, their function is to inform the other cells in the larval body. They have to emit the following message: it is time to start the program that leads to the decomposition of specific larval characters and the development of adult ones. When planulae of Hydractinia are cut into anterior and posterior fragments, only the anterior fragments can be stimulated to start metamorphic transformation (Fig. 8). The posterior part remains in the larval state, but when posterior fragments are grafted onto anterior fragments previously induced to metamorphose shortly before transplantation (Fig. 8), the posterior fragments undergo metamorphosis too. They receive a message from the anterior region inviting the posterior region of the larval body to participate in metamorphosis (Müller et al. 1976; Schwoerer-Böhning et al. 1990). A hypothesis concerning the existence of internal signals of neuronal origin has also been proposed, based on histological, cytochemical, and pharmaceutical studies in other hydrozoans, e.g., Halycordyle disticha (Kolberg and Martin 1988; Martin and Archer 1997). Efforts to isolate and identify the molecules that transmit this internal signal in Hydractinia culminated in the identification of a novel class of neuropeptides (Leitz et al. 1994b; © 2002 NRC Canada

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Leitz and Lay 1995; Gajewski et al. 1996, 1998; Leitz 1998, 2000). These share the carboxy terminal consensus sequence -Gly-Leu-Trp-NH2 (GLWamide) and cause not only anterior fragments but also posterior fragments of planulae to undergo metamorphosis. From Hydractinia complementary DNA (cDNA) clones the presence of a precursor protein that includes repetitive sequences of two peptides ending with the GLWamide has been deduced. Both these peptides, pERPPGLW-NH2 and KPPGLW-NH2, are effective, as are also peptides from heterologous sources, if they share this terminal consensus sequence (Schmich et al. 1998a). In situ hybridization with anti-sense RNA and immunocytochemistry with antibodies generated against PPGLWamide (or the GLWamide terminus alone) stained cells with the expected properties (Fig. 9). That is, they were neurosensory cells located in a belt near the anterior pole with their axonal fibres extending along the mesogloea to the posterior pole (Schmich et al. 1998b). This organization is suggestive. We think that these fibres convey the message to the remaining body parts. The neuropeptides may be released along the length of the fibres and (or) at their terminal. Our interpretation does not aim to completely account for all biochemical processes associated with metamorphosis. Berking and co-workers propose triggering mechanisms based on elevated internal levels of NH+4 , the presence of methylation potentials, and synthesis of polyamines (Berking 1991, 1998; Berking and Walther 1994). We agree with those authors in assuming that entering metamorphosis presupposes the release of internal blockades (see below). Putative additional signals synchronizing metamorphosis, such as calcium transients Experience with other developing systems suggests that nature often operates not with one single signalling molecule or signalling system but with several parallel signal-transmission systems to ensure a reliable result. The cDNA coding the GLWamide peptides contains one more sequence for a putative peptide. This has not yet been synthesized and tested. Supporting functions in eliciting particular downstream responses might be fulfilled by other types of molecules, particularly lysophosphatidylcholine (Leitz and Müller 1991) and arachidonic acid and derivatives of this versatile putative signalling substance, collectively known as eicosanoids (Leitz et al. 1993, 1994a). All these substances do not trigger metamorphosis by themselves, at least not efficiently, but may support induction by cesium ions. In addition to chemical transmitters of messages, another signalling system must be taken into consideration. Using highly sensitive photomultipliers Freeman and Ridgeway (1987, 1990) detected waves of emitted light travelling along the body of planula larvae induced to metamorphose by exposure to bacteria or CsCl. The planulae were not Hydractinia but Eutonia victoria, Mitrocomella polydidemata, and Phialidium gregarium. In contrast to the planulae of Hydractinia, the planulae of these species not only contain an endogenous photoprotein but also are translucent. The waves of light are preceded by epithelial action potentials travelling along the surface of the larva. The potentials are probably carried not by sodium ions but by calcium ions, and are propagated from cell to cell through gap junctions (like those in vertebrate heart muscle). Calcium entering the epithelial cells from the

Can. J. Zool. Vol. 80, 2002 Fig. 9. Planula larva with neurosensory cells labelled with antibodies against neuropeptides ending with the aa sequence GLWamide or RFamide.

outer medium or released from internal stores interacts with photoproteins, and this interaction results in emission of light. When the calcium transients were inhibited with calcium channel blockers, metamorphosis was also blocked. The calcium transients did not occur during phorbol esterinduced metamorphosis, indicating that the transients play a role upstream of PKC activation. On the other hand, transients can be released with various treatments that do not induce metamorphosis. Therefore, we think that calcium transients have a preparatory and supporting function but do not ultimately trigger metamorphosis. Abandoning the juvenile state and the putative role of taurine and methyl betaines Since nonstimulated Hydractinia larvae remain in the larval state for weeks until they eventually disintegrate, the presence of substances that have a juvenile hormone-like function or stabilize the larval state by blocking biochemical pathways needed for development is suggested. One such compound may be taurine. It is present in planulae in large quantities, is released into the surrounding medium upon induction of metamorphosis, and prevents larvae from entering metamorphosis if applied externally (Berking 1988b). Three other metamorphosis-inhibiting compounds isolated from planulae share the potential to provide methyl groups in transmethylation pathways (Berking 1986a, 1986b, 1987). All these substances are members of the family of betaine compounds: N-methylpicolinic acid (homarine), N-methylnicotinic acid (trigonelline), and N-trimethylglycine (glycine betaine). Like taurine, they are present in large quantities (several millimoles overall concentration in oocytes and larval tissue), and in micromole concentrations they reversibly inhibit metamorphosis. During metamorphosis the internal content of these betaines declines. Berking (1986a) ascribes the inhibitory activity of these substances to their potential to methylate as yet unknown targets. An extension of the hypothesis assigns a second role to these methyl donors during post-metamorphic development. They might control spacing of polyps along the stolons of colonial hydroids such as Eirene viridula (Berking 1986a). Patterning in the primary polyp The Cnidaria possess only one axis of asymmetry: the anterior–posterior axis of the planula larva, from which the aboral–oral axis of the polyp is derived. The posterior end of the larva corresponds to the oral pole of the primary polyp. The body axis of the future primary polyp has already been specified during early oogenesis (Freeman 1981). In oocytes the nucleus is shifted from the centre of the cell into a peripheral position. It becomes visible through the translucent wall of the gonad (gonophore) as a large “germi© 2002 NRC Canada

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nal vesicle”. The site on the egg surface below which the nucleus resides is conventionally designated the animal pole. It is the site of polar-body formation during maturation and the only place where the sperm can enter the egg cell. It is also the site where the first cleavage furrow starts (Fig. 5). The animal pole corresponds to the tapered posterior pole of the larva, which during metamorphosis gives rise to the oral pole of the primary polyp. However, the early specification of the body axis does not result in early irreversible determination. Centrifugation and cutting experiments revealed a high capacity to regulate (Freeman 1981, 1987, 1990). In larvae competent to metamorphose, cell proliferation and cell differentiation are almost at a standstill. They resume when the larvae enter metamorphosis (Plickert and Kroiher 1988; Plickert et al. 1988). These events follow an internal program that is specified during embryogenesis. Although the planula larva lacks overt longitudinal organization in its external appearance, the body pattern of the primary polyp is generated during metamorphosis by the determining influence of a covert anterior–posterior prepattern that is encoded in the internal organization of the larva and based in part on stored mRNA (Eiben 1982). When larvae are stimulated to enter metamorphosis and are cut into pieces immediately after induction, the most anterior fragment forms only stolons, whereas the most posterior fragment forms only the head (Fig. 8; Kroiher et al. 1990; Schwoerer-Böhning et al. 1990). The development of this prepattern in gastrulation and the realization of the final pattern in metamorphosis are accessible to experimental interference (Berking 1984, 1987) and can be dramatically changed by a low molecular weight factor of unknown chemical structure, termed the proportion-altering factor. This factor also stimulates the formation of nerve cells in developing adults (Plickert 1987, 1989, 1990; Kroiher and Plickert 1992). The primary signals controlling patterning along the anterior–posterior axis in larvae and the apex–base axis in polyps are likely to be the same, but the interpretation of these primary signals by the individual cells changes during metamorphosis (Kroiher 2000). A hypothetical, hierarchical model of pattern formation has been put forward by Berking (1998). Patterns of asymmetrical morphogen distribution provide positional information. The density of these sources along the body column, or their content of stored morphogen precursors, constitutes a tissue property known in developmental biology as positional value. With respect to the actual processes occurring at the cellular level, the distribution pattern of morphogens may merely provide global positional cues to which the various cell types respond very differently according to their previously specified fate. Exchange of cellular inventory Occurrence of interstitial stem cells Microscopic examination of hydroid embryos, larvae, and metamorphosing specimens has mainly focused on the origin of the interstitial cells, the founder cells that give rise to neurosensory cells, ganglionic nerve cells, nematocytes, and germ cells (for a review see Thomas and Edwards 1997). In the embryos of the Hydrozoa, interstitial cells generally arise as a central core of cells in the endoderm and eventually migrate through the mesogloea to the ectoderm. Interstitial cells and nematoblasts undergo extensive long-distance mi-

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gration. Nematoblasts of the various types enter the ectoderm and colonize distinct locations in the larval body. The distribution pattern may be the result of selective migration of predetermined cells to their destination (Pennaria tiarella and Halocordyle disticha, Martin and Thomas 1980; Martin and Archer 1986; Martin 1988a, 1988b, 1992; P. gregarium, Thomas et al. 1987; Hydractinia, McFadden et al. 1984; Weis et al. 1985). In Hydractinia, interstitial cells appear to be moving through the mesogloea beginning 4 h post insemination and continuing throughout the later stages of metamorphosis. Once in the ectoderm, the interstitial cells reside at the base of the ectoderm along the mesogloea, often occurring in clusters of 3 or 4 cells (Van de Vyver 1964; Weis and Buss 1986). During the final stages of metamorphosis, the interstitial cells move from the basal region of the primary polyp into the developing stolons. Apoptosis and extrusion of larval cell types By definition, metamorphosis comprises the decomposition of larval structures and the elaboration of adult structures. The planula of Hydractinia is characterized by five ectodermal cell types (Weis and Buss 1986): (1) epitheliomuscular cells, which in larvae bear a cilium, (2) gland cells, (3) neurosensory cells, (4) ganglionic nerve cells, and (5) nematocytes. Only during metamorphosis does the ectoderm acquire interstitial cells from the endoderm. Larval cells that fulfill transitory functions in presettlement behaviour and metamorphosis are (i) the neurosensory cells found clustered at the tapered end of the competent planula; these probably perceive the impact of collision with a passing shell; (ii) the nematocytes, also distributed in the posterior region and used to anchor the larva onto the shell; (iii) the gland cells, which are concentrated at the anterior end and secrete a cement at the onset of metamorphosis to permanently fix the larval body to the substrate, and which disappear in late metamorphosis; (iv) metamorphosis also includes the disappearance of those ectodermal GLWamide-positive neurosensory cells that are located near the anterior end and were probably the recipients of the external triggering signal (Schmich et al. 1998a), as well as the disappearance of RFamide-positive cells (Plickert 1989). A similar reorganization of the nervous system has been described for P. tiarella (Martin 2000). Considerable parts of the larval cellular inventory are removed by apoptosis followed by cell shedding and probably phagocytosis. Apoptosis can be recognised as early as 20 min after induction of metamorphosis, before the larvae reach the point at which development of adult features is initiated (Seipp et al. 2001). Acquisition of adult characteristics The ectodermal epithelial muscle cells lose their cilia, while the endodermal cells acquire a cilium with associated microvilli. The endoderm is completed by two types of gland cells; these are concentrated in the hypostome. Unexpectedly, several of the endodermal cells in the young hypostome transitorily contain transcripts for the GLW-amide precursor protein and display GLWamide immunoreactivity (Schmich et al. 1998a, 1998b; Gajewski et al. 1996). The nerve net becomes restructured. While nerve cells are absent in the young primary polyp GLWamide-positive, adult feeding polyps possess many putative nerve cells displaying © 2002 NRC Canada

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GLWamide precursor transcripts; they are scattered in the ectoderm along the body column except in the upper hypostomal region (Gajewski et al. 1996). This region is occupied by nerve cells exhibiting immunoreactivity against neuropeptides with the carboxy terminus Arg-Phe-amide (RFamide) (Grimmelikhuijzen 1985). At the late stages of metamorphosis, both nematoblasts and nematocytes, complete with cnidocil, begin to move into the emerging tentacles. When secondary polyps are formed on the elongating stolons, they are colonized by nematocytes immigrating from the stolonal compartment where their stem cells reside. Gene expression Studies on gene expression in cnidarians in general, and metamorphosing Hydractinia in particular, are still fragmentary. Using the methods of reverse genetics, attention was paid to homologues of selector genes known to control development and patterning in other systems. Remarkably, a gene indicative of “head” in various animal phyla, ems, is also expressed in the “head” of Hydractinia polyps (Mokady et al. 1998). The Hox gene Cnox-2 is expressed in the lower region of polyps and the stolonal compartment (Cartwright and Buss 1999).

A comparative review of metamorphosis in other Cnidaria Hydractinia has attained the status of a model organism in research on the metamorphosis of marine invertebrates (Hadfield 1998). We restrict our review to research on Cnidaria. With respect to other organisms we refer to the reviews by Chia and Bickel (1978), Pawlik (1992), Rodriguez et al. 1993, and Hadfield (1998). The common denominators of comparative studies are the inductive role of environmental bacteria and stimulation by depolarization of sensory cells used to recognise bacteria-borne or other environmental cues. Induction of metamorphosis by bacteria Hydrozoa Following the pioneering study on Hydractinia (Müller 1969), distinct populations of substrate-borne bacteria were shown to induce metamorphosis in a variety of hydrozoans, including E. victoria, M. polydiademata, P. gregarium (Freeman 1981; Thomas et al. 1987, 1997; Freeman and Ridgeway 1990), and H. disticha (Edwards et al. 1987). Scyphozoa Planulae and propagules of some Scyphozoa have also been successfully induced to enter metamorphosis using bacteria found in the environment of settled polyps. In particular, larvae of Cassiopea andromeda responded to a species of Vibrio alginolyticus (Neumann, 1979; Hofmann et al. 1984, 1996; Fitt and Hofmann 1985; Fitt et al. 1987; Hofmann and Brand 1987), the propagules (pedal stolons) of Aurelia aurita to a species of Micrococcaceae (Schmahl 1985a, 1985b), and planulae of Cyanea capillata and A. aurita were reported to settle on solid objects covered with a film of (unidentified) bacteria (Brewer 1976, 1978, 1984; Schmahl 1985a, 1985b). However, A. aurita does not respond to only one external stimulus. The water–air interface of the air bubbles that occur

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in foamy surf along rocky coasts induces sinking and the onset of metamorphosis before actual settlement takes place (Kroiher and Berking 1999). Anthozoa From the Anthozoa, too, examples of settlement and metamorphosis in response to bacterial films covering the substrate can be adduced. One of these two representative anthozoans is the octocoral Heteroxenia fuscescens (Henning et al. 1991, 1993) and the other is Acropora willisae. The bacteria were isolated from crustose algae and belong, once more, to the genus Pseudoalteromonas (Negri et al. 2001). Moreover, induction of metamorphosis by environmental bacteria is a phenomenon now known from many sedentary invertebrate species (for a comprehensive but no longer complete review see Pawlik 1992). Physiological mechanisms Artificial inducers and the mechanism of induction In most species known to respond to bacteria, and also in species that perhaps respond to other, as yet unknown environmental cues, the effects of natural inducers could be duplicated in the laboratory by substituting potassium or cesium ions, or activators of PKC (for a review see Leitz 1997). Only the swimming buds of Cassiopea failed to respond to ionic stimuli with transformation (Müller at al. 1976), but they responded to treatment with the PKC activators TPA and diC8 (Siefker et al. 2000). Besides potassium ions, PKC activators might be the most universal artificial inducers. These experiences reveal a common strategy used by invertebrate marine larvae to cope with the demands of their life cycles: all these larvae have to detect a key environmental stimulus by means of sensory cells, and most sensory cells can be artificially stimulated by elevated levels of external potassium or by activators of key enzymes in signaltransduction systems. Internal signals Of course, not all sensory cells use the same transmitter to transmit their message to target larval cells. In H. disticha, but not in Hydractinia, catecholamines trigger metamorphosis (Edwards et al. 1987). Fluorophores indicative of the presence of catecholamines produced blue–green fluorescence in the anterior region of the planulae (Kolberg and Martin 1988). In P. gregarium serotonin appears to trigger and synchronize the internal processes of metamorphosis (McCauley 1997). Although serotonin is not among the inducers of metamorphosis in Hydractinia, a serotonergic mechanism might be involved in downstream secondary processes, since an inhibitor of serotonin synthesis retarded metamorphosis (Walther et al. 1996). Release from inhibition In the swimming buds of Cassiopea a particular mechanism that controls development is operating. The buds enter metamorphosis without being stimulated by external cues when their anterior part, which in normal development gives rise to the basal disk and stalk of the scyphistoma (polyp), is removed. The posterior fragment will differentiate into a free-swimming scyphistoma (Müller et al. 1976). The anterior portion of the body is apparently the source of activity © 2002 NRC Canada

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Müller and Leitz Fig. 10. A stolon tip contacting the flank of an established stolon in Hydractinia. Nematocytes of a special type (microbasic mastigophores) migrate to and assemble at the contact site. In encounters between histoincompatible colonies the nematocytes are discharged in order to kill and eliminate the competitor for living space.

that inhibits further development. Application of cholera toxin (Wolk et al. 1985), known to interfere with G protein-coupled signal transduction, ammonium ions (Berking and Schüle 1987), and the protein phosphatase inhibitor cantharidin (Kehls et al. 1999) mimics the removal of this source of inhibition.

Benefits and costs of gregarious settlement: mutual support versus civil war Advantages versus disadvantages of gregarious settlement Benthic sedentary invertebrates such as barnacles, mussels, echiurids, sabellariid polychaetes, and ascidians are most frequently found in dense clusters. Most cnidarians also prefer to live with conspecifics. In fact, the largest community of related animal species, and the largest residential premises ever constructed by living organisms, is the Great Barrier Reef along the eastern coast of Australia. Although large reefs are composed of many species, the distribution of individual members and their kinship in the reef community are not random. Colonies composed of individuals that are genetically related or identical (clones) are formed by asexual division of an initial settler, as in the growth of corals, or by the settlement of short-term larvae near their mothers. Aggregations of genetically different individuals may also be formed by the settlement of planktonic larvae on or near adult conspecifics. This last condition is particularly prevalent among hard-bottom, sessile intertidal organisms, including sea anemones and hydrozoans. Epibiotic invertebrates may become aggregated as a result of larval preferences for settling on particular living substrata. Gregarious settlement has several advantages, but is not without costs. There are clear trade-offs between the advantages and disadvantages, but the prevalence of gregariousness in hard-substrate marine communities suggests that the benefits outweigh the costs. The benefits are manifold: • When larvae settle on or near adult conspecifics, they choose a habitat that is more likely to support postlarval growth than if they had settled indiscriminately. • Proximity increases the chances of successful fertilization for both internally fertilizing and freely spawning species. Synchronization of spawning can be improved by the release of pheromones.

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• Gregarious settlement of non-isogeneic individuals favours exchange of alleles and rapid distribution of those alleles that are favourable under the particular local environmental conditions. Disadvantages include the following: • Predators conveniently find a richly laid table, and infections spread readily through the community. • Aggregated adults must compete for food, which may reduce individual fitness. • On spatially restricted substrata the growth of colonial species, and likewise the expansion of communities that clone themselves by asexual reproduction, soon reach a limit. The availability of habitable space controls the size, reproductive output, and survival of colonies. The larger the colony or clone, the more offspring bearing the genomic inheritance of the parental organism can be produced. Competition for space may cause settling larvae to exhibit spacing-out or avoidance behaviour, but examples are not known from the Cnidaria. Therefore, sessile cnidarian species living on specific, spatially limited substrata will sooner or later come into conflict with allogeneic neighbours and must be eager to eliminate such competitors. This is the case in Hydractinia, therefore we return to this model organism once more, while not forgetting parallel phenomena in other genera. Allorecognition responses and elimination of competitors Various taxa of sedentary marine invertebrates, including cnidarians such as sea anemones, colonial corals, and colonial Hydrozoa, are known to possess a complex array of responses, primed to nonself tissue contacts (reviewed by Grosberg 1988). Members of cnidarian populations are able to discriminate nonself from self and can efficiently react against various allogeneic and xenogeneic challenges. Sea anemones, which are able to change their position, are caused to leave their place of residence by intolerant neighbours when the latter discharge nematocysts mounted on knob-like protrusions of the body wall, called acrorhagi, or sling nematocysts-bearing filamentous acontia out of their mouths or through openings in the body wall towards unwanted competitors for living space. Corals may try to harm and weaken their allogeneic neighbours during toxic interactions and to overgrow them (Frank and Rinkevich 1994; Rinkevich et al. 1994; Frank et al. 1995, 1997). However, all these interactions occur long after metamorphosis has taken place. In Hydractinia, the ability to discriminate between self and nonself matures during metamorphosis, and defence against allogeneic competitors is possible as soon as metamorphosis is completed (Fuchs et al. 2002). When two primary polyps or colonies come into contact by way of their extending stolons, either the stolons fuse, in which case a chimæra results, or they do not fuse, in which case one eliminates the neighbouring competitor for living space. The competitor is attacked by means of nematocytes of a particular type, classified as microbasic mastigophores. These are only found in the stolonal compartment of the colony, patrolling permanently along the stolons. The nematocytes accumulate at contact sites (Fig. 10), direct the cnidocils toward the neighbour, and suddenly discharge their poisons into the foreign tissue. Upon repeated and mutual attacks, one of the two competitors loses © 2002 NRC Canada

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and becomes paralyzed (Müller 1964; Buss et al. 1984; Lange et al. 1989; Buss 1990; Buss and Shenk 1990; Lange et al. 1992). Incompatibility and attacks are always observed in colonies derived from different parents but less frequently among close kin (and never when the two colonies belong to the same clone, a situation that occurs only in the laboratory). Based on the outcome of crosses, it has been proposed that allorecognition is genetically controlled by a single, yet highly polymorphic genetic locus with codominantly expressed alleles. Sharing at least one allele at this locus enables two colonies to fuse, whereas rejection results when no allele is shared (Hauenschild 1954, 1955; Grosberg et al. 1996; Mokady and Buss 1996, 1997). Sharing an identical allele is unlikely in distantly related conspecifics. The phenomenon has received two different interpretations, which, however, are not contradictory but complementary. (1) Nonfusion prevents the invasion of foreign primordial germ cells, which in fact can colonize the neighbouring tissue and displace the germ cells in the gonads (Müller 1964, 1967). This is tolerable only when the two colonies are siblings and therefore share many alleles at other genetic loci also. The reduction of fitness in one partner might be compensated for by avoiding the costs of war and through the benefits of fusion. Upon fusion, the enlarged common colony can occupy the valuable substrate faster than a single colony could do. (2) If, however, the two competitors are allogeneic and share only a few alleles, each colony must take care to ensure its own fitness (Yund et al. 1987; Hart and Grosberg 1999). Because hard substrata in general, and shells inhabited by hermit crabs in particular, are very rare in the habitat of Hydractinia, an allogeneic competitor is better eliminated than tolerated. Only in H. symbiolongicarpus can incompatible allogeneic colonies coexist, in that they deposit noncellular material to form a barrier between them (Buss et al. 1984). Hence, the question arises as to whether competition is less severe in North American habitats than in European habitats. Thus, we return to ecological aspects: metamorphosis in cnidarians can be understood only when these are taken into account.

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